# Patent

## Title
Microrna profiling for diagnosis of dysplastic nevi and melanoma

## Abstract

Provided herein are methods for miRNA profiling for the diagnosis, prognosis, and management of melanoma and differentiation of melanoma from nevi.

## Background
FIELD OF THE INVENTION
The invention generally relates to miRNA profiling for the diagnosis, prognosis, and management of melanoma and differentiation of melanoma from nevi.
BACKGROUND OF THE INVENTION
The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
Skin cancer is the most common of all cancers, afflicting more than a million Americans each year, a number that is rising rapidly. It is also the easiest to cure, if diagnosed and treated early. If allowed to progress to the point where it spreads to other sites (metastasizes), the prognosis (forecast) is very poor. More than 8,000 melanoma deaths now occur per year.
Melanoma most often appears as an asymmetrical, irregularly bordered, multicolored or tan/brown spot or growth that continues to increase in size over time. It may begin as a flat spot and become more elevated. In rare instances, it may not be pigmented.
Dysplastic nevi (atypical moles) are unusual or benign moles that may resemble melanoma. People who have them are at increased risk of developing single or multiple melanomas. The higher the number of these moles someone has, the higher the risk; those who have 10 or more have 12 times the risk of developing melanoma compared to the general population. Dysplastic nevi are found significantly more often in melanoma patients than in the general population.
Melanoma is distinguished from nevi, other forms of cancer, and normal skin on the basis of clinical presentation and histopathological examination of a skin biopsy, usually a formalin fixed, paraffin embedded (FFPE) sample. Considerable expertise is required to reliably distinguish between nevi and melanoma.
This application describes novel microRNA biomarkers with microRNA array and RT-PCR to better characterize dysplastic nevi, malignant melanoma and metastatic melanoma, miRNA can therefore serve as an adjunct to histopathology for correct classification of melanoma, nevi and other conditions, especially where there is doubt as to the diagnosis.
SUMMARY OF THE INVENTION
The present invention is based on the discovery that melanoma can be distinguished from nevi by measuring changes in the levels of as little as two miRNAs.
In one aspect, the invention provides a method for differentially diagnosing melanoma from nevi, by (a) measuring the level of two or more miRNAs selected from the group consisting of miR-132, miR-150, miR-339-5p, miR-15b, miR-342-3p, miR-572, miR-155, miR-425, miR-1202, miR-1268, HBII-382_s, miR-1225-5p, miR-30c, miR-106b-star, miR-125a-5p, mgU6-53B, miR-25, miR-149-star, miR-939, miR-92b-star, miR-500-star, miR-22, HBII-142_x, miR-181b, HBII-142, U38B, miR-663, miR-1224-5p, miR-23a, HBII-85-6_x, miR-1207-5p, miR-1301, miR-1228-star, miR-345, miR-30a-star, ENSG00000199411, ENSG00000202327, miR-92a, miR-127-3p, HBII-85-26, miR-1308, miR-31, miR-921, miR-146b-5p, miR-768-3p, miR-708, miR-139-5p, ACA24_x, miR-501-3p, miR-502-3p, miR-923, and miR-191; and (h) diagnosing the skin sample as containing melanoma when a difference in the level of the two or more miRNAs compared to a reference level indicates melanoma in the sample. The level of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more, miRNAs are measured.
In specific embodiments, the miRNAs to be measured include the following combinations (i) miR-150 and miR-149-star; (ii) miR-150, miR-149-star and miR-1308; (iii) miR-150, miR-149-star, miR-1308, and miR-191; (iv) miR-150, miR-149-star, miR-1308, miR-191, and miR-1228-star; (v) miR-150, miR-149-star, miR-1308, miR-191, miR-1228-star, and ENSG00000199411; (vi) miR-150, miR-149-star, miR-1308, miR-191, miR-1228-star, ENSG00000199411, and miR-1268; (vii) miR-150, miR-149-star, miR-1308, miR-191, miR-1228-star, ENSG00000199411, miR-1268, and miR-923; (viii) miR-150, miR-149-star, miR-1308, miR-191, miR-1228-star, ENSG00000199411, miR-1268, miR-923, and miR-23a; (ix) miR-150, miR-149-star, miR-1308, miR-191, miR-1228-star, ENSG00000199411, miR-1268, miR-923, miR-23a, and miR-132; (x) miR-150, miR-149-star, miR-1308, miR-191., miR-1228-star, ENSG00000199411, miR-1268, miR-923, miR-23a, miR-132, and miR-1207.5p; (xi) miR-150, miR-149-star, miR-1308, miR-191, miR-1228-star, ENSG00000199411, miR-1268, miR-923, miR-23a, miR-132, miR-1207.5p, and miR-342.3p; (xii) miR-150, miR-149-star, miR-1308, miR-191, miR-1228-star, ENSG00000199411, miR-1268, miR-923, miR-23a, miR-132, miR-1207.5p, miR-342.3p, and U38B; (xiii) miR-150, miR-149-star, miR-1308, miR-191, miR-1228-star, ENSG00000199411, miR-1268, miR-923, miR-23a, miR-132, miR-1207.5p, miR-342.3p, U38B, and miR-155.
In specific embodiments, melanoma is diagnosed in the skin sample by alterations in the level of an miRNA compared to a reference level, with the following alterations observed in at least two miRNA selected from the group consisting of: (i) miR-150 increase; (ii) miR-149-star decrease; (iii) miR-1308 decrease; (iv) miR-191 increase; (v) miR-1228-star decrease; (vi) ENSG00000199411_s decrease; (vii) miR-1268 decrease; (viii) miR-923 decrease; (ix) miR-23a increase; (x) miR-132 increase; (xi) miR-1207.5p decrease; (xii) miR-342.3p increase; (xiii) U38B decrease; and (xiv) miR-155 increase.
In one aspect, the invention provides a method for differentially diagnosing melanoma from nevi, by (a) measuring the level of two or more miRNAs selected from the group consisting of: miR-1268, miR-1228-star, miR-92b-star, miR-155, miR-345, miR-425, miR-132, miR-1207-5p, miR-1301, miR-663, miR-339-5p, miR-149-star, miR-150, miR-18a, miR-103, miR-191, miR-296-3p, miR-31, miR-107*, miR-93*, miR-1275*, miR-181B*, miR-921*, miR-1225-5p, miR-1202, and miR-342-3p and (b) diagnosing the skin sample as containing melanoma when a difference in the level of the two or more miRNAs compared to a reference level indicates melanoma in the sample.
In specific embodiments, melanoma is diagnosed in the skin sample by alterations in the level of an miRNA compared to a reference level, with the following alterations observed in at least two miRNA selected from the group consisting of: miR-1268 decrease, miR-1228-star decrease, miR-92b-star decrease, miR-155 increase, miR-345 increase, miR-425 increase, miR-132 increase, miR-1207-5p decrease, miR-1301 increase, miR-663 decrease, miR-339-5p increase, miR-149-star decrease, miR-150 increase, miR-18a increase, miR-103 increase, miR-191 increase, miR-296-3p decrease, miR-31 increase, miR-107* increase, miR-93* increase, miR-1275* decrease, miR-181B* increase, miR-921* decrease, miR-1225-5p increase, miR-1202 decrease, and miR-342-3p increase.
The method of the invention may further include internal controls, such as measuring the level of an miRNA selected from miR-27b, miR-195, miR-199b-3p, and miR-199a-3p.
In yet further embodiments, the level of two or more miRNAs are used to distinguish melanoma from normal skin, and nevi from normal skin. Additional miRNA levels may be assayed for this purpose.
The level of miRNA in the sample can be determined by microarray and/or quantitative real-time PCR. The method of the invention may be performed on a fresh skin sample, on a fixed and/or paraffin-embedded sample. In one embodiment the skin sample is formalin-fixed and paraffin-embedded.
The method of the invention may further comprise other steps in the diagnosis of melanoma, and the differentiation between nevi and melanoma, including histopathological assessment, and clinical assessment. In related embodiments, the clinical and/or histopathological evaluations may be converted into a score that can be combined with a score derived from miRNA levels, resulting in a diagnostic score that reflects the likelihood of melanoma.
The method of the invention may further include a step of isolating nucleic acids from the sample. An additional step may include amplification of the nucleic acid.
In further embodiments, the invention comprise a kit. In one embodiment, a kit for differentially diagnosing between melanoma and nevus in a skin sample comprises primers for the amplification of at least two miRNA selected from the group consisting of: miR-150, miR-149-star, miR-1308; miR-191.; miR-1228-star; ENSG00000199411_s; miR-1268; miR-923; miR-23a; miR-132; miR-1207.5p; miR-342.3p; U38B; and miR-155.
In another embodiment, the kit comprises primers for the amplification of at least two miRNAs selected from the group consisting of: miR-1268, miR-8-star, miR-92b-star, miR-155, miR-345, miR-425, miR-132, miR-1207-5p, miR-1301, miR-663, miR-339-5p, miR-149-star, miR-150, miR-18a, miR-103, miR-191, miR-296-3p, miR-31, miR-107*, miR-93*, miR-1275*, miR-181B*, miR-921*, miR-1225-5p, miR-1202, and miR-342-3p.
The kit may also primers for amplification of controls, such as miR-27b, miR-195, miR-199b-3p, and miR-199a-3p.
The kit may also include suitable buffers, reagents for isolating nucleic acid, and instructions for use Kits may also include a microarray for measuring miRNA levels.



BRIEF DESCRIPTION OF THE FIGURE
FIGS. 1-10 show, respectively, the best 2-10 miRNA combinations for differentiating melanoma (MM) from normal skin (NS), and the relevant error rates and AUC, as determined by different statistical algorithms.
FIGS. 11-23 show, respectively, the best 2-14 miRNA combinations for differentiating melanoma (MM) from nevi (NV), and the relevant error rates and AUC as determined by different statistical algorithms.
FIG. 24 shows the log 2 signal for three miRNA: miR-150 (miR-150), miR-149-star (miR-149-star), and hsa-miR-1308 (miR-1308) across 137 samples. Samples 1-19 are normal skin (NS), 20-57 nevi (NV), 58-115 melanoma (MM) and 116-137 metastatic melanoma (Mets). As can be appreciated, miR-150, miR-149-star and miR-1308 distinguish normal skin and nevi from melanoma and metastatic.
FIG. 25 shows an example of error rate and AUC from 9 programs analyzing the ability of groups of miRNA analytes to differentiate between melanoma and nevi.



DETAILED DESCRIPTION OF THE INVENTION
The present inventors have discovered that the levels of miRNAs in skin samples is a powerful tool to differentiate melanoma from non-tumorous nevi and, thereby, replace or supplement traditional clinical and histological methods of diagnosis.
DEFINITIONS
The present technology is described herein using several definitions, as set forth throughout the specification. As used herein, unless otherwise stated, the singular forms “a,” “an,” and “the” include plural reference. Thus, for example, a reference to “a nucleic acid” is a reference to one or more nucleic acids.
As used herein, “about” will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art, given the context in which it is used, the term “about” in reference to quantitative measurements or values will mean up to plus or minus 10% of the enumerated value.
The term “amplification” or “amplify” as used herein means one or more methods known in the art for copying a target nucleic acid, thereby increasing the number of copies of a selected nucleic acid sequence. Amplification may be exponential or linear. A target nucleic acid may be either DNA or RNA. The sequences amplified in this manner form an “amplicon.” While the exemplary methods described hereinafter relate to amplification using the polymerase chain reaction (“PCR”), numerous other methods are known in the art for amplification of nucleic acids (e.g., isothermal methods, rolling circle methods, etc.). The skilled artisan will understand that these other methods may be used either in place of, or together with, PCR methods. See, e.g., Saiki, “Amplification of Genomic DNA” in PCR Protocols, Innis et al., Eds., Academic Press, San Diego, Calif. 1990, pp. 13-20; Wharam et al., Nucleic Acids Res., 2001, 29(11):E54-E54; Hafner et al., Biotechniques 2001, 30(4):852-6, 858, 860; Zhong et al., Biotechniques, 2001, 30(4):852-6, 858, 860.
The terms “determining,” “measuring,” “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative measurement, and include determining if a characteristic, trait, or feature is present or not. Assessing may be relative or absolute. “Assessing the presence of” includes determining the amount of something present, as well as determining whether it is present or absent.
The term “clinical factors” as used herein, refers to any data that a medical practitioner may consider in determining a diagnosis of melanoma. Such factors include, but are not limited to, the patient's medical history, age, gender, skin color, a physical examination of the patient, and histopathology.
The term “complement” used herein means the complementary sequence to a nucleic acid according to standard Watson/Crick base pairing rules. A complement sequence can also be a sequence of RNA complementary to the DNA sequence or its complement sequence, and can also be a cDNA. The term “substantially complementary” as used herein means that two sequences hybridize under stringent hybridization conditions. The skilled artisan will understand that substantially complementary sequences need not hybridize along their entire length. In particular, substantially complementary sequences comprise a contiguous sequence of bases that do not hybridize to a target or marker sequence, positioned 3′ or 5′ to a contiguous sequence of bases that hybridize under stringent hybridization conditions to a target or marker sequence.
As used herein, the term “diagnosis” means detecting melanoma or the presence of melanoma cells. Usually, a diagnosis of a disease or disorder is based on the evaluation of one or more factors and/or symptoms that are indicative of the disease. That is, a diagnosis can be made based on the presence, absence or amount of a factor which is indicative of presence or absence of the disease or condition. Each factor or symptom that is considered to be indicative for the diagnosis of a particular disease does not need be exclusively related to the particular disease; i.e. there may be differential diagnoses that can be inferred from a diagnostic factor or symptom. Likewise, there may be instances where a factor or symptom that is indicative of a particular disease is present in an individual that does not have the particular disease. The term “diagnosis” also encompasses determining the therapeutic effect of a drug therapy, or predicting the pattern of response to a drug therapy. The diagnostic methods may be used independently, or in combination with other diagnosing and/or staging methods known in the medical art for a particular disease or disorder, particularly melanoma.
As used herein, the phrase “difference of the level” refers to differences in the quantity of a particular marker, such as a nucleic acid or a protein, in a sample as compared to a control or reference level. For example, the quantity of a particular biomarker may be present at an elevated amount or at a decreased amount in samples of patients with a neoplastic disease compared to a reference level. In one embodiment, a “difference of a level” may be a difference between the quantity of a particular biomarker present in a sample as compared to a control of at least about 1%, at least about 2%, at least about 3%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50%, at least about 60%, at least about 75%, at least about 80% or more. In one embodiment, a “difference of a level” may be a statistically significant difference between the quantity of a biomarker present in a sample as compared to a control. For example, a difference may be statistically significant if the measured level of the biomarker falls outside of about 1.0 standard deviations, about 1.5 standard deviations, about 2.0 standard deviations, or about 2.5 stand deviations of the mean of any control or reference group.
By “isolated”, when referring to a nucleic acid (e.g., an oligonucleotide such as RNA, DNA, or a mixed polymer) is meant a nucleic acid that is apart from a substantial portion of the genome in which it naturally occurs and/or is substantially separated from other cellular components which naturally accompany such nucleic acid. For example, any nucleic acid that has been produced synthetically (e.g., by serial base condensation) is considered to be isolated. Likewise, nucleic acids that are recombinantly expressed, cloned, produced by a primer extension reaction (e.g., PCR), or otherwise excised from a genome are also considered to be isolated. In some embodiments, the nucleic acid is isolated from the skin sample before further processing, such as PCR.
The term “label” as used herein, refers to any physical molecule directly or indirectly associated with a specific binding agent or antigen which provides a means for detection for that antibody or antigen. A “detectable label” as used herein refers any moiety used to achieve signal to measure the amount of complex formation between a target and a binding agent. These labels are detectable by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluoresence, or chemiluminescence, electrochemiluminescence or any other appropriate means. Suitable detectable labels include fluorescent dye molecules or fluorophores.
As used herein interchangeably, a “microRNA,” “miR,” or “miRNA” refers to the unprocessed or processed RNA transcript from a miRNA gene. MicroRNAs (miRNAs) are non-coding RNAs of 19-25 nucleotides in length that regulate gene expression by inducing translational inhibition or cleavage of their target mRNA through base pairing to partially or fully complementary sites. The unprocessed miRNA gene transcript is also called a “miRNA precursor,” and typically comprises an RNA transcript of about 70-100 nucleotides in length. The miRNA precursor can be processed by digestion with an RNAse (for example, Dicer, Argonaut, or RNAse III) into an active 19-25 nucleotide RNA molecule. This active 19-25 nucleotide RNA molecule is also called the “processed” miRNA gene transcript or “mature” miRNA.
As used herein, “nucleic acid” refers broadly to segments of a chromosome, segments or portions of DNA, cDNA, and/or RNA. Nucleic acid may be derived or obtained from an originally isolated nucleic acid sample from any source (e.g., isolated from, purified from, amplified from, cloned from, or reverse transcribed from sample DNA or RNA).
As used herein, the term “oligonucleotide” refers to a short polymer composed of deoxyribonucleotides, ribonucleotides or any combination thereof. Oligonucleotides are generally between about 10 and about 100 nucleotides in length. Oligonucleotides are typically 15 to 70 nucleotides long, with 20 to 26 nucleotides being the most common. An oligonucleotide may be used as a primer or as a probe. An oligonucleotide is “specific” for a nucleic acid if the oligonucleotide has at least 50% sequence identity with a portion of the nucleic acid when the oligonucleotide and the nucleic acid are aligned. An oligonucleotide that is specific for a nucleic acid is one that, under the appropriate hybridization or washing conditions, is capable of hybridizing to the target of interest and not substantially hybridizing to nucleic acids which are not of interest. Higher levels of sequence identity are preferred and include at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity.
As used herein, a “primer” for amplification is an oligonucleotide that specifically anneals to a target or marker nucleotide sequence. The 3′ nucleotide of the primer should be identical to the target or marker sequence at a corresponding nucleotide position for optimal primer extension by a polymerase. As used herein, a “forward primer” is a primer that anneals to the anti-sense strand of double stranded DNA (dsDNA). A “reverse primer” anneals to the sense-strand of dsDNA.
As used herein, the term “reference level” refers to a level of a substance which may be of interest for comparative purposes. In one embodiment, a reference level may be the miRNA levels expressed as an average of the level of miRNA from an area of normal skin or skin containing nevi and not melanoma. Nucleic acid samples may also be normalized relative to an internal control nucleic acid.
As used herein, the term “sample” refers to a skin biopsy from the subject, such as would typically be used for histopathological examination, or any section derived from such a sample. That is, a suspected melanoma may be entirely excised from the skin, but the biopsy is fixed and embedded in paraffin, and sectioned for further examination.
As used herein, the term “subject” refers to a mammal, such as a human, but can also be another animal such as a domestic animal (e.g., a dog, cat, or the like), a farm animal (e.g., a cow, a sheep, a pig, a horse, or the like) or a laboratory animal (e.g., a monkey, a rat, a mouse, a rabbit, a guinea pig, or the like). The term “patient” refers to a subject who is, or is suspected to be, afflicted with melanoma.
The phrase “substantially the same as” in reference to a comparison of one value to another value for the purposes of clinical management of a disease or disorder means that the values are statistically not different. Differences between the values can vary, for example, one value may be within 20%, within 10%, or within 5% of the other value.
As used herein, the term “diagnostic score” refers to a single number or score, based on a statistical analysis of the measured level of one or more biomarkers that reflects a relationship of a specific subject to any one particular group of individuals, such as normal individuals or individuals having a disease or any progressive state thereof. In some embodiments, the diagnostic score is derived from a quantitative multivariate analysis, which reflects the overall statistical assessment of an individual patient's clinical condition based upon an integrated statistical calculation of a plurality of qualitatively unique factors, e.g., levels of diagnostic miRNA, combined with clinical presentation, etc.
Melanoma and Nevi
Nevus (or naevus, plural nevi or naevi, from nævus, Latin for “birthmark”) is the medical term for sharply-circumscribed and chronic lesions of the skin. These lesions are commonly named birthmarks and moles. Nevi are benign by definition.
A melanocytic nevus (nevomelanocytic nevus, nevocellular nevus) is a benign proliferation of melanocytes, and are very common; almost all adults have at least one, usually more. A melanocytic nevus may be congenital or acquired.
A dysplastic nevus usually an acquired melanocytic nevus with abnormal features making it difficult to distinguish from a melanoma. It can be a marker for an individual at risk for developing melanomas.
Melanoma is a malignant tumor of melanocytes. Melanocytes predominantly occur in skin, between the outer layer of the skin (the epidermis) and the next layer (the dermis), but are also found in other parts of the body, including the bowel and the eye (see uveal melanoma). Melanoma can occur in any part of the body that contains melanocytes. Melanoma is less common than other skin cancers but is much more dangerous and causes the majority (75%) of deaths related to skin cancer.
Melanoma arises from DNA damage to melanocytcs. The early stage of the disease is called the radial growth phase, and the tumour is less than 1 mm thick. Next is the invasive radial growth phase, when individual cells start to acquire invasive potential. The Breslow's depth of the lesion is usually less than 1 mm (0.04 in), the Clark level is usually 2. The following step is invasive melanoma, “vertical growth phase” (VGP). The tumour attains invasive potential, growing into the surrounding tissue and can spread around the body through blood or lymph vessels to form metastases. The tumour thickness is usually more than 1 mm (0.04 in), and the tumour involves the deeper parts of the dermis.
An immunological reaction against the tumour during the VGP may be judged by the presence and activity of the tumour infiltrating lymphocytes (TILs). These cells sometimes completely destroy the primary tumour, this is called regression, which is the latest stage of the melanoma development. In certain cases, the primary tumour is completely destroyed and only the metastatic tumour is discovered.
Melanoma may also have a genetic predisposition. Mutations in CDKN2A, CDK4, MC1R, MDM2 SNP309 and those associated with xeroderma pigmentosum (XP) predispose one to melanoma. Familial melanoma is genetically heterogeneous,[10] and loci for familial melanoma have been identified on the chromosome arms 1p, 9p and 12q. Multiple genetic events have been related to the pathogenesis (disease development) of melanoma.
Clinical and Pathological Diagnosis
Melanoma is usually first detected by visual examination of the skin, notably (A) asymmetry, (B) a border that is uneven, ragged, or notched, (C) coloring of different shades of brown, black, or tan and (D) diameter that had changed in size. Normal moles are symmetrical, have an even border, even color, and no change in diameter. The main concern is distinguishing between a benign nevus, a dysplastic nevus, and a melanoma. Moles that are irregular in color or shape are often treated as candidates of melanoma. Following a visual examination and a dermatoseopic exam, or in vivo diagnostic tools such as a confocal microscope, a sample (biopsy) of the suspicious mole may be obtained.
Sample Preparation
When an atypical mole has been identified, a skin biopsy takes place in order to best diagnose it. Local anesthetic is used to numb the area, then the mole is biopsied. The biopsy material is then sent to a laboratory to be evaluated by a pathologist. A skin biopsy can be a punch, shave, or complete excision. The complete excision is the preferred method, but a punch biopsy can suffice if the patient has cosmetic concerns (i.e. the patient does not want a scar) and the lesion is small. A scoop or deep shave biopsy is often advocated, but should be avoided due to risk of a recurrent nevus, which can complicate future diagnosis of a melanoma, and the possibility that resulting scar tissue can obscure tumor depth if a melanoma is found to be present and re-excised.
Most dermatologists and dermatopathologists use a system devised by the NIH for classifying melanocytic lesions. In this classification, a nevus can be defined as benign, having atypia, or being a melanoma. A benign nevus is read as (or understood as) having no cytologic or architectural atypia. An atypical mole is read as having architectural atypia, and having (mild, moderate, or severe) cytologic (melanocytic) atypia. Usually, cytologic atypia is of more important clinical concern than architectural atypia. Usually, moderate to severe cytologic atypia will require further excision to make sure that the surgical margin is completely clear of the lesion.
The most important aspect of the biopsy report is that the pathologist indicates if the margin is clear (negative or free of melanocytic nevus), or if further tissue (a second surgery) is required. If this is not mentioned, usually a dermatologist or clinician will require further surgery if moderate to severe cytologic atypia is present—and if residual nevus is present at the surgical margin.
miRNA Markers to Distinguish Nevi from Melanoma
Distinguishing nevi from melanoma requires a high degree of skill. Misdiagnosis of a melanoma as a nevus can result in delay in treatment, which can be lethal because melanoma is an aggressive cancer that requires prompt intervention. Conversely, incorrectly identifying a nevus as a melanoma may subject a patient to aggressive treatment that is unnecessary and harmful. The present inventors have established that melanoma may be distinguished from nevi by monitoring the levels of select miRNA.
The methods described herein can distinguish melanoma, normal skin, nevi and malignant melanoma. Most importantly, the method is suitable for differentiating nevi from melanoma, and therefore fill a need for diagnosis that it not fully met by histology. As such, the methods of the invention can replace, supplement, or confirm histology. A particular advantage of the methods of the invention is that they provide independent objective evidence.
An additional advantage of the invention is that they can be performed on formalin fixed paraffin embedded (FFPE) tissue, and therefore can be used on the same samples that are processed for standard histopathological examination, and thus do not require a separate sample, or special handling. Another advantage is that the inventors have found that the miRNAs are stable in FFPE tissue and can be detected some time after fixing and embedding.
Another advantage of the invention is that the mRNA's chosen do not require a relatively pure sample of melanoma cells, and can detect melanoma in a sample that also contains normal skin, nevi and other skin cells. Thus, the miRNA assay is not overly sensitive to contamination nor require special handling beyond that which is normally used for preparation of FFPE tissue for regular histology.
The inventors have identified 50 miRNAs that can readily distinguish melanoma from nevi. Through statistical modeling and analysis, groups of the best 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14 markers have been identified. An increase in the number of markers used may improve sensitivity and accuracy, but comes with increased cost and complexity.
Additional diagnostic markers may be combined with the miRNA measurements to further aid diagnosis. For example, the clinical and/or histopathological results can be converted into a score, which is then combined with a score derived from the miRNA data. The combination of scores can be used to obtain a single “diagnostic score” that reflects the likelihood of melanoma.
Nucleic Acid Extraction and Detection
The level of a miRNA gene product in a sample can be measured using any technique that is suitable for detecting RNA expression levels in a biological sample. Suitable techniques for determining RNA expression levels in a biological sample are well known to those of skill in the art. These include, for example, Northern blot analysis, RT-PCR, and in situ hybridization.
The nucleic acid to be detected may be from a biological sample such as a tissue sample and the like. Various methods of extraction are suitable for isolating the DNA or RNA. Suitable methods include phenol and chloroform extraction. See Maniatis et al., Molecular Cloning, A Laboratory Manual, 2d, Cold Spring Harbor Laboratory Press, pp. 16-54 (1989). Numerous commercial kits also yield suitable DNA and RNA including, but not limited to, QIAampTM mini blood kit, Agencourt GenfindTM, Roche Cobas® Roche MagNA Pure® or phenol:chloroform extraction using Eppendorf Phase Lock Gels®, and the NucliSens extraction kit (Biomerieux, Marcy l'Etoile, France). In an illustrative embodiment, RNA is isolated from patient serum on the NucliSens easyMAG system (Biomeriux SA, France) according to the manufacturer's protocol.
In one embodiment, the level of at least one miRNA gene product is detected using Northern blot analysis. For example, total RNA can be purified from a sample in the presence of nucleic acid extraction buffer, followed by centrifugation. Nucleic acids are precipitated, and DNA is removed by treatment with DNase and precipitation. The RNA molecules are then separated by gel electrophoresis on agarose gels according to standard techniques, and transferred to nitrocellulose filters. The RNA is then immobilized on the filters by heating. Detection and quantification of specific RNA is accomplished using appropriately labeled DNA or RNA probes complementary to the RNA in question. See, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook et al., eds., 2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapter 7.
Suitable probes (e.g., DNA probes or RNA probes) for Northern blot hybridization of a given miRNA gene product can be produced from the known nucleic acid sequences and include, but are not limited to, probes having at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% complementarity to a miRNA gene product of interest, as well as probes that have complete complementarity to a miRNA gene product of interest. Methods for preparation of labeled DNA and RNA probes, and the conditions for hybridization thereof to target nucleotide sequences, are described in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., eds., 2nd edition. Cold Spring Harbor Laboratory Press, 1989, Chapters 10 and 11.
For example, the nucleic acid probe can be labeled with, e.g., a radionuclide, such as 3H, 32P, 33P 14C, or 35S; a heavy metal; a ligand capable of functioning as a specific binding pair member for a labeled ligand (e.g., biotin, avidin or an antibody); a fluorescent molecule; a chemiluminescent molecule; an enzyme or the like. Probes can be labeled to high specific activity by either the nick translation method or by the random priming method. Autoradiographic detection of hybridization can then be performed by exposing hybridized filters to photographic film. Densitometric scanning of the photographic films exposed by the hybridized filters provides an accurate measurement of miRNA levels. Using another approach, miRNA gene transcript levels can be quantified by computerized imaging systems.
In one embodiment, the miRNA is detected using a nucleic acid amplification process. Nucleic acid extracted from a sample can be amplified using nucleic acid amplification techniques well known in the art. By way of example, but not by way of limitation, these techniques can include the polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), nested PCR, ligase chain reaction. See Abravaya, K., et al., Nucleic Acids Research, 23:675-682, (1995), branched DNA signal amplification, Urdea, M. S., et al., AIDS, 7 (suppl 2):S11-S14, (1993), amplifiable RNA reporters, Q-beta replication, transcription-based amplification, boomerang DNA amplification, strand displacement activation, cycling probe technology, isothermal nucleic acid sequence based amplification (NASBA). See Kievits, T. et al., J Virological Methods, 35:273-286, (1991), Invader Technology, or other sequence replication assays or signal amplification assays may also be used.
Some methods employ reverse transcription of RNA to cDNA. The method of reverse transcription and amplification may be performed by previously published or recommended procedures. Various reverse transcriptases may be used, including, but not limited to, MMLV RT, RNase H mutants of MMLV RT such as Superscript and Superscript II (Life Technologies, GIBCO BRL, Gaithersburg, Md.), AMV RT, and thermostable reverse transcriptase from Thermus thermophilus. For example, one method which may be used to convert RNA to cDNA is the protocol adapted from the Superscript II Preamplification system (Life Technologies, GIBCO BRL, Gaithersburg, Md.; catalog no. 18089-011), as described by Rashtchian, A., PCR Methods Applic., 4:S83-S91, (1994).
In a suitable embodiment, PCR is used to amplify a target sequence of interest, PCR is a technique for making many copies of a specific template DNA sequence. The reaction consists of multiple amplification cycles and is initiated using a pair of primer sequences that hybridize to the 5′ and 3′ ends of the sequence to be copied. The amplification cycle includes an initial denaturation, and typically up to 50 cycles of annealing, strand elongation and strand separation (denaturation). In each cycle of the reaction, the DNA sequence between the primers is copied. Printers can bind to the copied DNA as well as the original template sequence, so the total number of copies increases exponentially with time. PCR can be performed as according to Whelan et al., J of Clin Micro, 33(3):556-561 (1995). Briefly, a PCR reaction mixture includes two specific primers, dNTPs, approximately 0.25 U of Taq polymerase, and 1×PCR Buffer.
The skilled artisan is capable of designing and preparing primers that are appropriate for amplifying a target or marker sequence. The length of the amplification primers depends on several factors including the nucleotide sequence identity and the temperature at which these nucleic acids are hybridized or used during in vitro nucleic acid amplification. The considerations necessary to determine a preferred length for an amplification primer of a particular sequence identity are well-known to a person of ordinary skill. For example, the length of a short nucleic acid or oligonucleotide can relate to its hybridization specificity or selectivity.
In some embodiments, the amplification may include a labeled primer or probe, thereby allowing detection of the amplification products corresponding to that primer or probe. In particular embodiments, the amplification may include a multiplicity of labeled primers or probes; such primers may be distinguishably labeled, allowing the simultaneous detection of multiple amplification products. Oligonucleotide probes can be designed which are between about 10 and about 100 nucleotides in length and hybridize to the amplified region. Oligonucleotides probes are preferably 12 to 70 nucleotides; more preferably 15-60 nucleotides in length; and most preferably 15-25 nucleotides in length. The probe may be labeled.
In one embodiment, a primer or probe is labeled with a fluorogenic reporter dye that emits a detectable signal. While a suitable reporter dye is a fluorescent dye, any reporter dye that can be attached to a detection reagent such as an oligonucleotide probe or primer is suitable for use in the invention. Such dyes include, but are not limited to, Acridine, AMCA, BODIPY, Cascade Blue, Cy2, Cy3, Cy5, Cy7, Edans, Eosin, Erythrosin, Fluorescein, 6-Fam, Tet, Joe, Hex, Oregon Green, Rhodamine, Rhodol Green, Tamra, Roz, and Texas Red.
In yet another embodiment, the detection reagent may be further labeled with a quencher dye such as Tamra, Dabcyl, or Black Hole Quencher® (BHQ), especially when the reagent is used as a self-quenching probe such as a TaqMan® (U.S. Pat. Nos. 5,210,015 and 5,538,848) or Molecular Beacon probe (U.S. Pat. Nos. 5,118,801 and 5,312,728), or other stemless or linear beacon probe (Livak et al., 1995, PCR Method Appl., 4:357-362; Tyagi et al, 1996, Nature Biotechnology, 14:303-308; Nazarenko et al., 1997, Nucl. Acids Res., 25:2516-2521; U.S. Pat. Nos. 5,866,336 and 6,117,635).
Nucleic acids may be amplified prior to detection or may be detected directly during an amplification step (i.e., “real-time” methods). For example, amplified fragments may be detected using standard gel electrophoresis methods. In some embodiments, amplified fractions are separated on an agarose gel and stained with ethidium bromide by methods known in the art to detect amplified fragments. In some embodiments, the target sequence is amplified using a labeled primer such that the resulting amplicon is detectably labeled. In some embodiments, the primer is fluorescently labeled.
In one embodiment, detection of a miRNA, such as a nucleic acid from an a miR-16 or miR-199a, is performed using the TaqMan® assay, which is also known as the 5′ nuclease assay (U.S. Pat. Nos. 5,210,015 and 5,538,848). The TaqMan® assay detects the accumulation of a specific amplified product during PCR. The TaqMan® assay utilizes an oligonucleotide probe labeled with a fluorescent reporter dye and a quencher dye. The reporter dye is excited by irradiation at an appropriate wavelength, it transfers energy to the quencher dye in the same probe via a process called fluorescence resonance energy transfer (FRET). When attached to the probe, the excited reporter dye does not emit a signal. The proximity of the quencher dye to the reporter dye in the intact probe maintains a reduced fluorescence for the reporter. The reporter dye and quencher dye may be at the 5′ most and the 3′ most ends, respectively or vice versa. Alternatively, the reporter dye may be at the 5′ or 3′ most end while the quencher dye is attached to an internal nucleotide, or vice versa. In yet another embodiment, both the reporter and the quencher may be attached to internal nucleotides at a distance from each other such that fluorescence of the reporter is reduced.
During PCR, the 5′ nuclease activity of DNA polymerase cleaves the probe, thereby separating the reporter dye and the quencher dye and resulting in increased fluorescence of the reporter. Accumulation of PCR product is detected directly by monitoring the increase in fluorescence of the reporter dye. The DNA polymerase cleaves the probe between the reporter dye and the quencher dye only if the probe hybridizes to the target-containing template which is amplified during PCR.
TaqMan® primer and probe sequences can readily be determined using the nucleic acid sequence information of the miRNA of interest. A number of computer programs, such as Primer Express (Applied Biosystems, Foster City, Calif.), can be used to rapidly obtain optimal primer/probe sets. It will be apparent to one of skill in the art that such primers and probes for detecting the target nucleic acids are useful in diagnostic assays for neoplastic disorders, such as HCC, and can be readily incorporated into a kit format. The present invention also includes modifications of the TaqMan® assay well known in the art such as the use of Molecular Beacon probes (U.S. Pat. Nos. 5,118,801 and 5,312,728) and other variant formats (U.S. Pat. Nos. 5,866,336 and 6,117,635).
In an illustrative embodiment, real time PCR is performed using TaqMan® Assays in combination with a suitable amplification/analyzer such as the ABI Prism® 7900HT Sequence Detection System. The ABI PRISM® 7900HT Sequence Detection System is a high-throughput real-time PCR system that detects and quantitates nucleic acid sequences. Real-time detection on the ABI Prism 7900HT or 7900HT Sequence Detector monitors fluorescence and calculates Rn during each PCR cycle. The threshold cycle, or Ct value, is the cycle at which fluorescence intersects the threshold value. The threshold value is determined by the sequence detection system software or manually. The Ct can be correlated to the initial amount of nucleic acids or number of starting cells using a standard curve.
In one embodiment, TaqMan® MicroRNA Assays are used to detect the miRNA. TaqMan® MicroRNA Assays are predesigned assays that are available for the majority of content found on the miRBase miRNA sequence repository. In another embodiment, the mirVanaTM qRT-PCR miRNA Detection Kit (Ambion) is a used to detect and quantify the miRNA. This is a quantitative reverse transcription-PCR (qRT-PCR) kit enabling sensitive, rapid quantification of miRNA (miRNA) expression from total RNA samples.
As a quality control measure, an internal amplification control may be included in one or more samples to be extracted and amplified. The skilled artisan will understand that any detectable sequence that is not typically present in the sample can be used as the control sequence. A control sequence can be produced synthetically. If PCR amplification is successful, the internal amplification control amplicons can then be detected. Additionally, if included in the sample prior to purification of nucleic acids, the control sequences can also act as a positive purification control.
Statistical Methods
Statistical methods can be used to set thresholds for determining when the level in a subject can be considered to be different than or similar to a reference level. In addition, statistics can be used to determine the validity of the difference or similarity observed between a patient's circulating miRNA level and the reference level. Useful statistical analysis methods are described in L. D. Fisher & G. vanBelle, Biostatistics: A Methodology for the Health Sciences (Wiley-Interscience, NY, 1993). For instance, confidence (“p”) values can be calculated using an unpaired 2-tailed t test, with a difference between groups deemed significant if the p value is less than or equal to 0.05. As used herein a “confidence interval” or “CI” refers to a measure of the precision of an estimated or calculated value. The interval represents the range of values, consistent with the data that is believed to encompass the “true” value with high probability (usually 95%). The confidence interval is expressed in the same units as the estimate or calculated value. Wider intervals indicate lower precision; narrow intervals indicate greater precision. Preferred confidence intervals of the invention are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%. A “p-value” as used herein refers to a measure of probability that a difference between groups happened by chance. For example, a difference between two groups having a p-value of 0.01 (or p=0.01) means that there is a 1 in 100 chance the result occurred by chance. Preferred p values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and 0.0001. Confidence intervals and p-values can be determined by methods well-known in the art. See, e.g., Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983.
On linear model for assessing differential expression in microarray experiments: Smith G K (2004) “Linear models and empirical bayes method for assessing differential expression in microarray experiments” Statistical Applications in Genetics and Molecular Biology. For AUC calculation: Mason S J and Graham N E (1982) “Areas beneath the relative operating characteristics (ROC) and relative operating levels (ROL) curves: Statistical significance and interpretation,” Q. J. R. Meteorol. Soc. textbf30 291-303. Multiple algorithms program for marker combination selection: An R based program with nine algorithms including random forest, ada boosting, svm, bagging, logistic regression, lasso, matt, cart, ctree is available, for example, as open-source software from the R Foundation. Random forests were also conducted according to Breiman, L. (2001), Random Forests, Machine Learning 45(1), 5-32. Sec also Breiman, L (2002), “Manual On Setting Up, Using, And Understanding Random Forests V3.1.
In connection with miRNA used to diagnose melanoma, one may seek levels that are lower or higher than a control. The term “elevated levels” or “higher levels” as used herein refers to levels of an miRNA that are higher than what would normally be observed in a comparable sample from control or normal subjects or normal tissue from the patient (i.e., a reference value). Similarly, “reduced levels” or “lower levels” as used herein refer to levels of that are lower than what would normally be observed in a comparable sample from control or normal subjects, or normal tissue from the patient (i.e., a reference value). In some embodiments, “control levels” (i.e., normal levels) refer to a range of miRNA levels that would be normally be expected to be observed in nevi, or normal skin. A control level may be used as a reference level for comparative purposes. The ranges accepted as outside “control levels” are dependent on a number of factors. For example, one laboratory may routinely determine the level of circulating miRNA in a sample that is different than the miRNA obtained for the same sample by another laboratory. Also, different assay methods may achieve different value ranges. Value ranges may also differ in various sample types, for example, different body fluids or by different treatments of the sample. One of ordinary skill in the art is capable of considering the relevant factors and establishing appropriate reference ranges for “control values” and “elevated/reduced values” of the present invention. For example, a series of samples from control subjects and subjects diagnosed with melanoma can be used to establish ranges that are “normal” or “control” levels and ranges that are “elevated” or “reduced” than the control range.
The level of one or more miRNAs measured in the test sample is normalized, such as by comparison to an internal reference nucleic acid, e.g., U44 or small RNA U6. The levels of the one or more miRNAs may then be compared to a reference value to determine if the levels of the one or more miRNAs are elevated or reduced relative to the reference value. Typically, the reference value is the level measured in a comparable sample from one or more healthy individuals. An increase or decrease in the level of the one or more miRNAs may be used in conjunction with clinical factors to diagnose melanoma.
In some embodiments, the level of one or more miRNAs is combined with one or more additional markers to improve diagnostic sensitivity and specificity. Exemplary markers include, but are not limited to, any useful diagnostic marker associated with melanoma including those which may be assessed by fluorescence in-situ hybridization (FISH) and/or comparative genomic hybridization (CGH).
Kits
A kit may be used for conducting the diagnostic and prognostic methods described herein. Typically, the kit should contain, in a carrier or compartmentalized container, reagents useful in any of the above-described embodiments of the diagnostic method. The carrier can be a container or support, in the form of e.g., bag, box, tube, rack, and is optionally compartmentalized. The carrier may define an enclosed confinement for safety purposes during shipment and storage. In one embodiment, the kit includes one or more PCR primers capable of amplifying miRNA selected from miR-132, miR-150, miR-339-5p, miR-15b, miR-342-3p, miR-572, miR-155, miR-425, miR-1202, miR-1268, HBII-382_s, miR-1225-5p, miR-30c, miR-106b-star, miR-125a-5p, mgU6-53B, miR-25, miR-149-star, miR-939, miR-92b-star, miR-500-star, miR-22, HBII-142_x, miR-181b, HBII-142, U38B, miR-663, miR-1224-5p, miR-23a, HBII-85-6_x, miR-1207-5p, miR-1301, miR-1228-star, miR-345, miR-30a-star, ENSG00000199411_s, ENSG00000202327, miR-92a, miR-127-3p, HBII-85-26, miR-1308, miR-31, miR-921, miR-146b-5p, miR-768-3p, miR-708, miR-139-5p, ACA24_x, miR-501-3p, miR-502-3p, miR-923, and miR-191.
In further embodiments, the invention comprise a kit. In one embodiment, a kit for differentially diagnosing between melanoma and nevus in a skin sample comprises primers for the amplification of at least two miRNA selected from the group consisting of miR-150, miR-149-star, miR-1308; miR-191; miR-1228-star; ENSG00000199411_s; miR-1268; miR-923; miR-23a: miR-132; miR-1207.5p; miR-342.3p; U38B; and miR-155.
In another embodiment, the kit comprises primers for the amplification of at least two miRNAs selected from the group consisting of: miR-1268, miR-1228-star, miR-92b-star, miR-155, miR-345, miR-425, miR-132, miR-1207-5p, miR-1301, miR-663, miR-339-5p, miR-149-star, miR-150, miR-18a, miR-103, miR-191, miR-296-3p, miR-31, miR-107*, miR-93*, miR-1275*, miR-181B*, miR-921*, miR-1225-5p, miR-1202, and miR-342-3p.
The kit may also primers for amplification of controls, such as miR-27b, miR-195, miR-199b-3p, and miR-199a-3p.
The kit may also include suitable buffers, reagents for isolating nucleic acid, and instructions for use. Kits may also include a microarray for measuring miRNA level.
The primers may be labeled with a detectable marker such as radioactive isotopes, or fluorescence markers. Instructions for using the kit or reagents contained therein are also included in the kit.
EXAMPLES
The present methods and kits, thus generally described, will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present methods and kits. The following is a description of the materials and experimental procedures used in the example.
Example 1
137 samples were examined: (1) 19 normal skin; (2) 38 nevi, of which 20 were intradermal, where the nevus cells are located in the dermis only, and 18 were compound nevus, which is a mixture of junctional and intradermal proliferation and are slightly raised and brown to black; (3) 58 primary melanoma; (4) 22 metastatic melanoma.
Total RNA, including low molecular weight RNA, was isolated from ten 10 μm-FFPE sections with RecoverAll RNA extraction kit (Applied Biosystems), according to the manufacturer's protocol.
The Affymetrix GeneChip® miRNA array was used, according to the manufacturer's protocol, to evaluate miRNA expression in FFPE samples. The miRNA array covers 71 organisms, including human, mouse, rat, and monkey, and contains 1801 sets of human miRNA, snoRNAs and scaRNAs. Samples were labeled using the Genisphere FlashTagTM Biotin Labeling Assay, which utilizes the 3DNATM technology. The 3DNATM dendrimer was ligated to samples to allow multiple biotin molecules ( ̃15) to bind to each poly-A tailed RNA molecule. Following FlashTagTM ligation, samples were hybridized on the Affymetrix GeneChip® miRNA array overnight. The hybridized chips were washed and processed to scan in an Affimatrix GeneChip Scanner 3000 7G.
Statistical Analyses
The raw microarray data was analyzed using the miRNA QC tool which performed an RNA normalization and extracted signals for data analysis. Log 2 values were used for miRNA expression in each group. The log 2 fold change (log 2 FC) was calculated by subtracting the mean of log 2 of group 1 from the mean log 2 of group 2. Student's t test was used to compare the miRNA expression level of each miRNA between normal skin, nevi, melanoma, and metastatic melanoma. P<0.05 was used as statistical significance. The area under ROC (AUC) was calculated to reflect the separation between each group. The default AUC value is 0.5 meaning no separation, and the maximum possible value is 1.0, meaning complete separation between each group.
A screen on probe signal detection in samples was performed having of 137 samples at least >30 samples with signal detectable. There are 729 of 1801 human probes satisfying such condition. These 729 candidate markers were analyzed using multiple algorithm programs.
Two or three markers completely separate these melanoma from nevi, as confirmed by multiple algorithms. The best 15 miRNA markers from randomForest share 14 markers overlap with the best 15 markers from boosting. In addition, these 14 best overlap markers include 6 of 7 best markers from previous analyses of 40 melanoma vs 20 nevi. These results therefore show consistency across different assays at different times.
Example 2
Additional experiments were performed to identify miRNA that can distinguish between different skin conditions.
For melanoma vs nevus, specimens (n=380) included a training set of 20 paraffin-embedded blocks of normal skin, 60 paraffin-embedded blocks of skin biopsies with benign nevus, 60 paraffin-embedded blocks of skin biopsies with malignant melanoma. Next, a validation set of 100 paraffin-embedded blocks of skin biopsies with benign nevus, 100 paraffin-embedded blocks of skin biopsies with malignant melanoma and 50 paraffin-embedded blocks of dysplastic nevus.
To distinguish between primary melanoma and metastatic melanoma, 180 study specimens included a training set of 60 paraffin-embedded blocks of skin biopsies with malignant melanoma and 30 paraffin-embedded blocks of metastatic melanoma. The validation set comprised 60 paraffin-embedded blocks of skin biopsies with malignant melanoma and 30 paraffin-embedded blocks of metastatic melanoma.
The subject population targeted all ethnicities and was approximately 50% male and 50% female. Specimens of adults 18 year and younger, and all subjects 89 years or older (“>90”) were discarded.
Tissues (normal benign, nevi and indeterminate nevus, malignant melanoma and metastatic melanoma) were collected at DermaPath. 240 nevi and 40 melanoma FFPEs were purchased from BioTheme.
Total RNA, including low molecular weight RNA, was isolated from ten 10 μm-FFPE sections with RecoverAll RNA extraction kit from Applied Biosystems according to the manufacturer's protocol.
The Affymetrix GeneChip® miRNA array was used to evaluate miRNA expression in FFPE samples. The miRNA array covers 71 organisms, including human, mouse, rat, and monkey, and contains 1801 sets of human miRNA, snoRNAs and scaRNAs.
The experiment procedure was according to the Affymetrix miRNA expression analysis manual. Samples were labeled with the Genisphere FlashTagTM Biotin Labeling Assay, which utilizes the 3DNATM technology. The 3DNATM dendrimer was ligated to samples to allow multiple biotin molecules ( ̃15) to bind to each poly-A tailed RNA molecule. Following FlashTagTM ligation, samples were hybridized on the Affymetrix GeneChip® miRNA array overnight. The hybridized chips were washed and processed to scan in an Affimatrix GeneChip Scanner 3000 7G.
Statistical Analyses
Log 2 values were used for miRNA expression in each group. The log 2 fold change (log 2 FC) was calculated by subtracting the mean of log 2 of group 1 from the mean log 2 of group 2. Student's t test was used to compare the miRNA expression level of each miRNA between normal skin, nevi, melanoma, and metastatic melanoma. P<0.05 was used as statistical significance. The area under ROC (AUC) was calculated to reflect the separation between each group. The default AUC value is 0.5 meaning no separation, and the maximum possible value is 1.0, meaning complete separation between each group.
Semi-Quantitative Reverse-Transcriptase PCR Analysis of miRNA
Two-step TaqMan reverse-transcriptase PCR analysis was performed for analysis of miRNAs. Reverse transcription was performed in a 15-μl reaction volume using specific primers for each miRNA contained in the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, Calif.) by sequentially incubating at 16° C. for 30 min, 42° C. for 30 min, and 85° C. for 5 min. Real-time PCR was done using the standard TaqMan MicroRNA assay protocol on an Applied Biosysytems 7900 system (Applied Biosystems). Each PCR mixture (20 μl) included the reverse transcription products, TaqMan 2× Universal PCR Master Mix without UNG Amperase, miRNA-specific TaqMan probes, and primers supplied by Applied Biosystems. The reactions were incubated in a 96-well plate with an initial denaturation at 95° C. for 10 min, followed by 40 cycles of 95° C. for 15 s and 60° C. for 1 min. The level of miRNA expression was measured using the threshold cycle (Ct), the fractional cycle number at which the fluorescence of each sample passes a fixed threshold. miRNA expression levels were normalized using an endogenous small RNA control U44 (Applied Biosystems). The expression of miRNA relative to small RNA U44 is reported as ΔCt, which was calculated by subtracting the Ct of U44 RNA from the Ct of target miRNA.
4. Results
MiRNA expression was analyzed between each group, Table 1 shows 523 miRNAs which have significant expression level, either over or less expression, between melanoma and nevi groups (p<0.05). Table 2 showed 50 miRNAs with the most significant expression between the two groups. Table 3 showed 378 miRNAs which have significant expression differences between melanoma and normal skin groups (p<0.05). Table 4 showed 50 miRNAs with the most significant expression differences between the two groups. Table 5 showed 174 miRNAs which have significant expression differences between melanoma and metastatic melanoma groups (p<0.05). Table 6 showed 50 miRNAs with the most significant expression differences between the two groups. Table 7 showed 442 miRNAs which have significant expression differences between nevi and normal skin groups (p<0.05). Table 8 showed 50 miRNAs with the most significant expression differences between the two groups.







TABLE 1







miRNAs significantly expressed between melanoma and nevi













Log2-



#Sample


Probe Name
FC
P value
AUC
ProbeType
Detected















miR-132
2.98
 1.2728E−32
0.994
miRNA
96


miR-150
3.27
5.83192E−30
1.000
miRNA
117


miR-339-5p
2.73
1.69388E−27
0.988
miRNA
75


miR-15b
3.02
6.41784E−27
0.978
miRNA
125


miR-342-3p
2.23
7.03798E−26
0.993
miRNA
136


miR-572
−2.78
1.00008E−25
0.970
miRNA
116


miR-155
4.14
1.28454E−25
0.981
miRNA
123


miR-425
2.74
8.13135E−25
0.975
miRNA
114


miR-1202
−2.63
2.97795E−23
0.986
miRNA
57


miR-1268
−2.68
6.05905E−23
0.997
miRNA
133


HBII-382_s
−1.71
6.58931E−22
0.971
scaRna
126


miR-1225-5p
−2.36
1.05361E−21
0.953
miRNA
90


miR-30c
2.39
2.58594E−21
0.980
miRNA
125


miR-106b-star
2.24
3.48507E−21
0.961
miRNA
72


miR-125a-5p
2.39
6.90609E−21
0.967
miRNA
128


mgU6-53B
−1.51
1.74782E−20
0.973
CDBox
99


miR-25
2.57
1.52769E−19
0.956
miRNA
118


miR-149-star
−2.03
1.64858E−19
1.000
miRNA
135


miR-939
−2.21
 1.9129E−19
0.973
miRNA
57


miR-92b-star
−2.30
2.05085E−19
0.969
miRNA
111


miR-500-star
2.38
3.11256E−19
0.956
miRNA
97


miR-22
2.69
 3.8844E−19
0.965
miRNA
120


HBII-142_x
−1.22
4.93318E−19
0.981
CDBox
135


miR-181b
2.41
1.00639E−18
0.960
miRNA
130


HBII-142
−1.33
1.18794E−18
0.988
CDBox
135


U38B
−1.95
1.35964E−18
0.975
CDBox
134


miR-663
−2.10
2.04672E−18
0.984
miRNA
134


miR-1224-5p
−2.54
2.91169E−18
0.946
miRNA
78


miR-23a
1.18
2.99523E−18
0.967
miRNA
137


HBII-85-6_x
−1.73
4.78418E−18
0.939
CDBox
137


miR-1207-5p
−2.14
4.98991E−18
0.995
miRNA
133


miR-1301
2.36
5.22389E−18
0.931
miRNA
54


miR-1228-star
−2.41
5.51513E−18
0.997
miRNA
134


miR-345
2.45
6.43552E−18
0.942
miRNA
75


miR-30a-star
2.32
6.92165E−18
0.932
miRNA
64


ENSG00000199411_s
−1.97
7.23325E−18
0.991
snoRNA
135


ENSG00000202327
−1.40
8.75839E−18
0.938
snoRNA
54


miR-92a
1.67
9.49427E−18
0.979
miRNA
136


miR-127-3p
2.43
9.84615E−18
0.936
miRNA
89


HBII-85-26
−2.07
1.50619E−17
0.951
CDBox
136


miR-1308
−2.14
1.99888E−17
0.999
miRNA
135


miR-31
3.29
2.46697E−17
0.913
miRNA
103


miR-921
−1.46
2.61089E−17
0.928
miRNA
49


miR-146b-5p
2.37
6.10422E−17
0.918
miRNA
83


miR-768-3p
−1.05
6.66153E−17
0.949
miRNA
137


miR-708
2.29
6.86187E−17
0.929
miRNA
102


miR-139-5p
2.23
2.78962E−16
0.922
miRNA
80


ACA24_x
1.33
3.37973E−16
0.925
HAcaBox
98


miR-501-3p
1.83
4.76751E−16
0.923
miRNA
80


miR-502-3p
2.16
5.19509E−16
0.925
miRNA
110


miR-923
−1.86
5.55191E−16
0.996
miRNA
135


U94
−1.17
6.40118E−16
0.926
CDBox
109


miR-574-3p
2.32
8.68105E−16
0.946
miRNA
122


miR-135a-star
−1.90
1.03062E−15
0.914
miRNA
44


ENSG00000207098_x
−1.03
1.25346E−15
0.924
snoRNA
86


U38B_x
−1.55
2.54255E−15
0.958
CDBox
133


miR-423-3p
1.83
2.74508E−15
0.932
miRNA
107


miR-198
−1.79
3.57486E−15
0.909
miRNA
31


ACA16
1.43
4.07871E−15
0.919
HAcaBox
47


ACA25
1.06
4.62028E−15
0.910
HAcaBox
73


miR-769-5p
1.63
4.79679E−15
0.920
miRNA
56


ENSG00000212523_x
−1.38
6.22717E−15
0.927
snoRNA
135


mgU6-53B_x
−0.99
6.32629E−15
0.919
CDBox
121


Z17B
0.97
 1.1349E−14
0.903
CDBox
117


U81_x
1.22
1.25935E−14
0.913
CDBox
120


miR-532-5p
2.10
1.27686E−14
0.933
miRNA
113


ENSG00000200879
1.27
1.49096E−14
0.917
snoRNA
120


HBII-419
−1.07
2.02184E−14
0.938
CDBox
133


U58B_x
0.96
9.25535E−14
0.898
CDBox
132


ENSG00000201619
−1.66
1.14582E−13
0.911
snoRNA
62


miR-28-3p
2.15
1.17402E−13
0.900
miRNA
94


miR-1300
−1.86
1.21666E−13
0.892
miRNA
32


miR-191
1.18
 1.2767E−13
0.983
miRNA
135


miR-181a-2-star
1.93
2.91245E−13
0.898
miRNA
99


U38A
−1.52
3.04032E−13
0.971
CDBox
134


U59A
−1.01
3.86379E−13
0.948
CDBox
135


ENSG00000212397
−1.21
4.58111E−13
0.902
snoRNA
134


HBII-85-26_x
−1.13
4.73092E−13
0.897
CDBox
135


miR-638
−1.62
5.93273E−13
0.967
miRNA
135


miR-421
1.84
6.09941E−13
0.881
miRNA
68


miR-21
2.62
6.26336E−13
0.888
miRNA
79


miR-24-2-star
1.81
8.95923E−13
0.879
miRNA
63


U36C
−0.92
 1.1248E−12
0.931
CDBox
135


miR-92b
1.43
1.15239E−12
0.901
miRNA
91


miR-199a-5p
1.69
1.22362E−12
0.901
miRNA
129


ACA24_s
1.36
1.86137E−12
0.880
HAcaBox
128


ACA9
1.36
1.92813E−12
0.888
HAcaBox
82


ENSG00000199411_x
−0.87
2.22903E−12
0.868
snoRNA
132


ENSG00000199435
−0.94
2.45067E−12
0.903
snoRNA
63


miR-182
1.80
3.73608E−12
0.905
miRNA
113


miR-1275
−1.56
3.97629E−12
0.888
miRNA
127


miR-150-star
−1.70
4.59839E−12
0.897
miRNA
48


ACA48_x
1.08
5.05927E−12
0.881
HAcaBox
130


HBII-85-8_x
−1.09
5.47576E−12
0.877
CDBox
135


miR-99b
1.33
5.48764E−12
0.907
miRNA
129


U74_x
−1.00
6.23625E−12
0.941
CDBox
135


miR-1271
1.71
 6.2815E−12
0.885
miRNA
70


ENSG00000206637_x
−0.86
6.43179E−12
0.878
snoRNA
59


miR-20b
2.16
1.13294E−11
0.892
miRNA
99


ENSG00000200652
−0.93
1.18064E−11
0.876
snoRNA
40


U13
−0.94
1.19458E−11
0.899
CDBox
135


ENSG00000201660
−1.13
1.27245E−11
0.903
snoRNA
132


HBII-85-23_x
1.18
1.36679E−11
0.883
CDBox
58


miR-149
1.85
1.38908E−11
0.896
miRNA
100


ACA9_x
1.14
2.05352E−11
0.877
HAcaBox
77


miR-1234
−1.10
2.16855E−11
0.856
miRNA
68


miR-1180
1.50
2.61371E−11
0.878
miRNA
37


miR-30a
1.80
3.39494E−11
0.861
miRNA
113


U44_x
1.13
3.71768E−11
0.953
CDBox
135


miR-181c
1.54
4.41605E−11
0.861
miRNA
59


ENSG00000202498_x
−1.07
4.69703E−11
0.855
snoRNA
137


miR-940
−1.22
5.55177E−11
0.861
miRNA
53


miR-500
1.76
6.23662E−11
0.854
miRNA
86


ENSG00000212627
−0.78
6.92771E−11
0.871
snoRNA
69


ENSG00000207027
−0.84
8.23933E−11
0.879
snoRNA
36


miR-27a-star
1.63
8.66657E−11
0.856
miRNA
40


miR-128
1.66
9.46068E−11
0.858
miRNA
44


snR38C
−1.01
9.50785E−11
0.926
CDBox
134


ENSG00000212266
−0.93
 9.6633E−11
0.848
snoRNA
93


miR-185
1.76
1.06661E−10
0.922
miRNA
132


ACA64
−0.97
1.08821E−10
0.858
HAcaBox
33


miR-151-3p
1.71
1.37187E−10
0.884
miRNA
117


miR-130b
2.12
1.55732E−10
0.870
miRNA
90


miR-27b-star
1.70
1.72122E−10
0.849
miRNA
51


miR-665
−1.43
1.78835E−10
0.853
miRNA
47


miR-18a
2.01
1.91068E−10
0.854
miRNA
77


ACA36_x
−1.03
 2.0756E−10
0.875
HAcaBox
88


ENS00000212432_s
−0.94
2.08962E−10
0.882
snoRNA
75


ENSG00000202093_x
1.01
4.04338E−10
0.873
snoRNA
125


miR-487b
1.41
4.22069E−10
0.848
miRNA
58


U65
−0.98
4.31315E−10
0.847
HAcaBox
128


miR-532-3p
1.19
4.73885E−10
0.881
miRNA
95


U101
−0.79
5.89958E−10
0.845
CDBox
134


miR-138
2.09
6.68933E−10
0.839
miRNA
82


HBII-99
1.01
7.89663E−10
0.847
CDBox
122


miR-222
1.12
9.21171E−10
0.913
miRNA
135


miR-652
1.46
9.82734E−10
0.862
miRNA
119


miR-10a
1.82
9.97674E−10
0.843
miRNA
69


miR-625
1.46
1.06062E−09
0.833
miRNA
59


ENSG00000212182
−0.89
1.13997E−09
0.828
snoRNA
59


miR-331-3p
1.47
1.22124E−09
0.834
miRNA
64


miR-1281
−1.46
1.31027E−09
0.843
miRNA
137


miR-28-5p
1.53
1.35526E−09
0.863
miRNA
105


ACA23
0.76
2.45172E−09
0.837
HAcaBox
113


U27
1.04
2.55698E−09
0.874
CDBox
134


miR-339-3p
1.12
 2.9234E−09
0.836
miRNA
60


U68_x
1.00
3.99716E−09
0.861
HAcaBox
133


miR-92a-2-star
−1.06
4.67209E−09
0.830
miRNA
46


miR-107
1.08
5.05068E−09
0.946
miRNA
135


U104
−0.79
5.07142E−09
0.918
CDBox
135


miR-671-5p
−1.16
6.05404E−09
0.877
miRNA
69


miR-193b
1.22
6.27171E−09
0.852
miRNA
130


miR-1272
−1.13
6.29028E−09
0.826
miRNA
67


miR-221
1.23
 6.5716E−09
0.911
miRNA
135


14qll-14
1.27
 6.8516E−09
0.828
CDBox
56


miR-1288
−0.92
6.95468E−09
0.804
miRNA
43


ENSG00000212458
−0.80
7.62772E−09
0.853
snoRNA
95


miR-584
1.61
7.63622E−09
0.815
miRNA
57


U106
0.64
8.00299E−09
0.821
CDBox
112


miR-152
1.58
8.26552E−09
0.885
miRNA
122


ENSG00000212551
−0.72
9.08023E−09
0.841
snoRNA
73


U96a_x
−0.89
1.00143E−08
0.863
CDBox
134


ENSG00000212139_x
−0.74
1.22887E−08
0.811
snoRNA
124


miR-409-3p
1.14
1.25923E−08
0.832
miRNA
92


miR-214-star
1.59
1.34912E−08
0.830
miRNA
47


ACA20
0.91
1.36416E−08
0.873
HAcaBox
131


HBII-82
−0.73
1.52532E−08
0.823
CDBox
90


U49A
−0.86
1.55769E−08
0.856
CDBox
135


miR-362-5p
1.63
1.67441E−08
0.824
miRNA
94


ACA40_x
1.17
1.68127E−08
0.872
HAcaBox
134


U63
−0.98
1.83203E−08
0.916
CDBox
135


HBII-436
0.76
1.85498E−08
0.810
CDBox
102


miR-15a
1.74
 1.8639E−08
0.826
miRNA
80


U103_s
0.83
2.02002E−08
0.809
CDBox
111


miR-1185
−0.96
2.35573E−08
0.822
miRNA
42


ACA15_s
0.74
2.84086E−08
0.812
HAcaBox
116


miR-30b
1.37
3.22874E−08
0.843
miRNA
128


spike_in-control-21
−0.70
3.25874E−08
0.832
Oligonucleotide
42






spike-in







controls



U15B
0.71
3.51612E−08
0.825
CDBox
129


miR-143
1.99
3.72999E−08
0.820
miRNA
126


U67_x
0.79
4.12783E−08
0.813
HAcaBox
50


miR-10b
1.41
4.29563E−08
0.821
miRNA
106


U99
−0.65
4.50576E−08
0.817
HAcaBox
137


miR-194
1.27
5.18787E−08
0.813
miRNA
54


miR-324-5p
1.14
5.23291E−08
0.804
miRNA
102


miR-125b-2-star
1.19
5.54514E−08
0.819
miRNA
58


HBII-52-37_x
−0.76
8.93997E−08
0.805
CDBox
37


ACA48
0.72
9.13165E−08
0.825
HAcaBox
121


miR-103
0.97
9.25296E−08
0.931
miRNA
135


miR-342-5p
1.32
9.27998E−08
0.827
miRNA
98


miR-93
1.08
9.93604E−08
0.896
miRNA
134


miR-296-3p
−1.00
1.10115E−07
0.818
miRNA
90


miR-200b-star
1.30
1.13507E−07
0.791
miRNA
74


U44
0.87
1.17791E−07
0.897
CDBox
134


ENSG00000207016_x
−0.63
1.23122E−07
0.803
snoRNA
48


spike_in-control-31
−0.14
1.24357E−07
0.798
Oligonucleotide
137






spike-in







controls



U68
0.83
1.72086E−07
0.856
HAcaBox
130


U24
−0.55
2.39582E−07
0.822
CDBox
133


U83
0.62
2.42808E−07
0.846
CDBox
135


ENSG00000212273_x
−0.80
2.45728E−07
0.811
snoRNA
126


14qll-14_x
1.03
2.59298E−07
0.797
CDBox
56


miR-660
1.26
2.65402E−07
0.788
miRNA
71


14qll-7
−0.70
2.89281E−07
0.762
CDBox
77


U78_s
1.01
3.07849E−07
0.836
CDBox
135


miR-744
−1.03
3.11141E−07
0.818
miRNA
133


U22
−0.54
3.14269E−07
0.793
CDBox
135


ACA11
0.70
3.50307E−07
0.793
HAcaBox
48


ENSG00000212553_x
−0.69
3.51184E−07
0.794
snoRNA
60


U67
0.76
3.78503E−07
0.796
HAcaBox
44


miR-382
1.35
3.83783E−07
0.787
miRNA
47


miR-212
1.58
3.89653E−07
0.854
miRNA
62


miR-93-star
1.26
4.02443E−07
0.808
miRNA
48


U18C_x
0.74
 4.1571E−07
0.794
CDBox
74


miR-1274a
1.22
 4.1577E−07
0.788
miRNA
48


ACA25_x
0.77
4.42948E−07
0.799
HAcaBox
114


U15A
−0.74
4.57833E−07
0.821
CDBox
128


14ql-1
−0.77
5.08646E−07
0.784
CDBox
70


ENSG00000201816
−0.60
5.51521E−07
0.789
snoRNA
49


miR-145
1.56
6.30644E−07
0.775
miRNA
135


HBII-420
−0.75
7.96309E−07
0.828
CDBox
131


ACA2b
−0.66
8.56351E−07
0.787
HAcaBox
47


miR-1273
−0.94
1.01224E−06
0.768
miRNA
47


miR-641
−0.85
 1.0165E−06
0.765
miRNA
39


ENSG00000202216
−0.60
1.02726E−06
0.778
snoRNA
53


U97
−0.72
1.06795E−06
0.794
CDBox
133


EN8G00000200706_x
−0.66
1.06937E−06
0.778
snoRNA
43


miR-363
1.33
1.20135E−06
0.806
miRNA
44


ENSG00000201348
−0.48
1.23741E−06
0.779
snoRNA
94


U83A
0.75
1.32496E−06
0.818
CDBox
127


miR-197
1.27
 1.3431E−06
0.807
miRNA
103


miR-106b
1.23
1.49024E−06
0.805
miRNA
127


HBII-180A_x
0.60
1.62491E−06
0.784
CDBox
123


snR38B
−0.76
1.68966E−06
0.803
CDBox
126


snR38A
−0.87
1.99208E−06
0.838
CDBox
130


miR-134
1.15
2.09717E−06
0.774
miRNA
61


miR-29b-1-star
1.41
2.20964E−06
0.764
miRNA
46


mgh18S-121
−0.66
2.37986E−06
0.833
CDBox
134


mgU6-77
0.59
2.38866E−06
0.742
CDBox
129


HBII-52-25_x
−0.60
2.44738E−06
0.767
CDBox
57


U48
0.83
2.51688E−06
0.777
CDBox
129


14qll-19
−0.73
2.52816E−06
0.770
CDBox
43


miR-552
−0.66
2.70576E−06
0.760
miRNA
34


miR-424-star
1.31
2.79344E−06
0.762
miRNA
41


HBII-316
−0.60
3.06016E−06
0.795
CDBox
130


U35B
−0.64
3.21695E−06
0.811
CDBox
128


miR-30d
1.02
3.25088E−06
0.817
miRNA
131


miR-1248
−0.72
3.27937E−06
0.746
miRNA
39


ENSG00000212579_x
−0.65
3.55239E−06
0.758
snoRNA
100


hsa-let-7b
0.90
3.76667E−06
0.919
miRNA
136


miR-29b-2-star
−0.78
3.86015E−06
0.760
miRNA
126


miR-148a
1.04
4.16979E−06
0.773
miRNA
41


miR-629
1.16
4.35623E−06
0.776
miRNA
64


HBII-295
−0.58
4.36224E−06
0.767
CDBox
128


miR-877
−1.00
4.43293E−06
0.794
miRNA
126


ACA62
−0.61
4.43469E−06
0.774
HAcaBox
88


ENSG00000207100_x
−0.50
4.49659E−06
0.765
snoRNA
72


HBII-180C
0.62
4.78509E−06
0.780
CDBox
115


HBII-115
−0.57
5.03448E−06
0.768
CDBox
117


miR-192
1.14
5.73989E−06
0.763
miRNA
32


miR-505-star
1.23
5.74055E−06
0.762
miRNA
58


spike_in-control-7
−0.80
5.89851E−06
0.790
Oligonucleotide
54






spike-in







controls



miR-422a
1.17
5.96437E−06
0.782
miRNA
84


U83B
−0.56
6.55847E−06
0.842
CDBox
135


ENSG00000212134_x
−0.65
6.92676E−06
0.752
snoRNA
88


U50B_x
−0.67
7.13703E−06
0.783
CDBox
135


miR-193a-3p
0.91
8.82408E−06
0.747
miRNA
42


ENSG00000201848
−0.62
9.39369E−06
0.759
snoRNA
34


miR-148b
0.93
9.94696E−06
0.757
miRNA
32


miR-483-5p
−1.10
 1.1191E−05
0.756
miRNA
48


ACA58_x
0.62
1.17327E−05
0.766
HAcaBox
89


miR-1260
0.94
1.21296E−05
0.776
miRNA
120


U108_x
−0.50
1.23481E−05
0.763
HAcaBox
87


ACA61
−0.60
1.27104E−05
0.839
HAcaBox
135


14q-0
−0.58
1.39356E−05
0.767
CDBox
49


ACA16_x
0.55
1.40972E−05
0.763
HAcaBox
98


miR-30b-star
1.22
1.61096E−05
0.755
miRNA
61


ENSG00000201848_x
−0.58
2.11059E−05
0.740
snoRNA
40


U3-2_s
−0.57
2.19611E−05
0.800
CDBox
135


spike_in-control-2
−0.31
2.25868E−05
0.747
Oligonucleotide
137






spike-in







controls



miR-933
−0.56
2.89505E−05
0.776
miRNA
100


miR-181d
1.03
2.90988E−05
0.750
miRNA
61


HBII-239
0.45
2.92435E−05
0.744
CDBox
135


miR-497
1.17
2.92719E−05
0.730
miRNA
104


HBII-251
−0.48
3.04823E−05
0.797
CDBox
135


U51
−0.73
3.10371E−05
0.809
CDBox
134


miR-211
1.45
3.41149E−05
0.759
miRNA
84


miR-29a
1.10
3.68833E−05
0.777
miRNA
128


ENSG00000212538
−0.54
3.71864E−05
0.746
snoRNA
35


miR-886-3p
1.11
4.28603E−05
0.711
miRNA
110


miR-361-5p
0.58
4.51572E−05
0.859
miRNA
135


U51_x
−0.48
 4.6523E−05
0.773
CDBox
130


U46
0.59
4.67792E−05
0.744
CDBox
133


HBII-85-11
0.73
4.72933E−05
0.743
CDBox
31


ENSG00000207410_x
−0.51
4.76931E−05
0.731
snoRNA
55


ENSG00000200492
−0.51
5.13688E−05
0.756
snoRNA
60


miR-503
1.05
5.22927E−05
0.730
miRNA
41


HBII-85-21_x
0.68
5.24419E−05
0.749
CDBox
39


miR-506
1.27
5.49222E−05
0.750
miRNA
76


ENSG00000212206_x
−0.55
5.68573E−05
0.730
snoRNA
74


U64
0.49
 5.7913E−05
0.755
HAcaBox
101


miR-629-star
1.07
5.89793E−05
0.745
miRNA
46


ENSG00000212284
−0.62
6.56826E−05
0.740
snoRNA
89


ENSG00000212423_x
−0.60
6.59628E−05
0.728
snoRNA
115


U14B_x
0.58
6.71139E−05
0.714
CDBox
53


miR-324-3p
0.78
6.71385E−05
0.766
miRNA
100


miR-1287
0.79
7.37546E−05
0.733
miRNA
40


ENSG00000200897
−0.49
7.55587E−05
0.730
snoRNA
38


miR-498
−0.68
8.15427E−05
0.730
miRNA
56


U80
0.53
8.25012E−05
0.750
CDBox
132


14ql-8_x
−0.55
8.60271E−05
0.740
CDBox
74


miR-181a
0.82
8.99722E−05
0.766
miRNA
134


HBII-166
−0.46
9.07119E−05
0.782
CDBox
134


miR-181a-star
1.03
9.30754E−05
0.727
miRNA
35


U17b
0.85
9.69173E−05
0.854
HAcaBox
135


U55
0.61
0.000102241
0.749
CDBox
135


ENSG00000207407
−0.43
0.000115453
0.706
snoRNA
56


ENSG00000212206
−0.50
0.000123184
0.701
snoRNA
46


miR-489
0.86
0.000125451
0.724
miRNA
58


hsa-let-7g
1.09
0.000128044
0.820
miRNA
127


ENSG00000199363
0.63
0.000135904
0.722
snoRNA
42


miR-27a
0.72
0.000140934
0.687
miRNA
136


ACA32
−0.40
0.000144838
0.746
HAcaBox
135


ENSG00000207022
−0.51
0.000149628
0.733
snoRNA
36


miR-371-5p
−0.86
0.000152576
0.706
miRNA
64


miR-559
−0.73
0.000161415
0.728
miRNA
72


miR-193a-5p
0.92
0.000163281
0.777
miRNA
121


U23
0.46
0.00016459
0.724
HAcaBox
127


miR-196a-star
−0.69
0.000167433
0.695
miRNA
37


U52
0.47
0.00016782
0.770
CDBox
135


ACA38
0.50
0.000170509
0.713
HAcaBox
42


miR-30e-star
0.82
0.000173654
0.736
miRNA
35


U49A_x
−0.61
0.000183863
0.785
CDBox
135


U49B_x
−0.54
0.000189603
0.729
CDBox
122


U71a
0.50
0.000191925
0.721
HAcaBox
50


HBII-95
0.42
0.000194646
0.724
CDBox
78


ENSG00000212587
−0.55
0.000201583
0.711
snoRNA
45


ENSG00000212401
−0.45
0.000208666
0.714
snoRNA
41


HBII-296A
−0.45
0.000214033
0.713
CDBox
78


ACA67_x
0.49
0.000214513
0.730
HAcaBox
54


miR-494
0.79
0.000221314
0.725
miRNA
135


miR-17
0.77
0.00024803
0.782
miRNA
134


HBII-85-4_x
−0.49
0.000264241
0.714
CDBox
125


miR-16
0.67
0.000270072
0.819
miRNA
135


miR-135b-star
−0.68
0.000271929
0.710
miRNA
38


ACA37_x
0.47
0.00028645
0.718
HAcaBox
84


miR-126
0.82
0.000326027
0.780
miRNA
133


miR-200b
0.93
0.000339327
0.714
miRNA
109


miR-320d
0.89
0.000373646
0.811
miRNA
134


U100
0.41
0.000374728
0.702
scaRna
80


HBII-85-27_x
−0.40
0.000383642
0.706
CDBox
56


HBII-85-15_x
0.60
0.000397454
0.706
CDBox
49


miR-885-3p
−1.11
0.000407264
0.692
miRNA
73


miR-23b-star
0.76
0.000423736
0.723
miRNA
52


ENSG00000206909_x
−0.46
0.000424153
0.710
snoRNA
47


U92
−0.50
0.000428521
0.750
scaRna
129


miR-628-3p
0.86
0.000458761
0.702
miRNA
38


miR-27b
0.70
0.00047114
0.718
miRNA
133


U88
−0.43
0.000500023
0.708
scaRna
69


miR-130a
1.00
0.000518644
0.729
miRNA
122


ACA4
0.42
0.00055437
0.673
HAcaBox
117


miR-151-5p
0.60
0.00055594
0.790
miRNA
135


ENSG00000200394
0.46
0.000561813
0.692
snoRNA
119


ACA5
0.48
0.000585113
0.708
HAcaBox
85


miR-1285
−0.74
0.000607741
0.698
miRNA
54


miR-664-star
−0.82
0.000652475
0.686
miRNA
96


U56
−0.57
0.000672557
0.787
CDBox
135


ENSG00000200307
−0.44
0.000679263
0.686
snoRNA
86


miR-18b
0.81
0.000708619
0.688
miRNA
32


U76
0.38
0.000723275
0.712
CDBox
135


miR-378
0.75
0.000726542
0.769
miRNA
131


ENSG00000200307_x
−0.43
0.000739867
0.716
snoRNA
50


miR-513b
1.02
0.000775353
0.701
miRNA
64


U56_x
0.54
0.000779671
0.716
CDBox
134


miR-642
−0.59
0.000808799
0.695
miRNA
69


miR-106a
0.66
0.000811882
0.788
miRNA
135


miR-379
0.94
0.000829873
0.702
miRNA
57


miR-570
−0.61
0.000842848
0.697
miRNA
88


ACA3-2
0.46
0.000855145
0.720
HAcaBox
135


U55_x
0.50
0.000899835
0.746
CDBox
135


ACA7_s
−0.34
0.000995798
0.703
HAcaBox
133


miR-1228
−0.67
0.001047707
0.730
miRNA
114


miR-199a-3p
0.75
0.001282519
0.729
miRNA
134


ENSG00000212508
−0.59
0.001303596
0.687
snoRNA
122


ENSG00000200961
−0.41
0.001335597
0.699
snoRNA
41


miR-23b
0.45
0.00142683
0.817
miRNA
137


ENSG00000207177
−0.34
0.001464718
0.697
snoRNA
59


ACA50
0.41
0.001642667
0.674
HAcaBox
88


miR-550-star
0.75
0.001687411
0.692
miRNA
58


miR-617
−0.56
0.001827038
0.676
miRNA
64


U75
−0.57
0.001850734
0.729
CDBox
134


HBII-85-2_x
−0.41
0.001956673
0.706
CDBox
135


miR-508-5p
0.77
0.001978936
0.711
miRNA
92


miR-200a
0.66
0.002095909
0.676
miRNA
33


miR-574-5p
0.79
0.002104165
0.749
miRNA
103


ACA49
−0.37
0.00221029
0.692
HAcaBox
127


miR-1826
0.46
0.002224183
0.799
miRNA
137


ENSG00000212377
−0.45
0.002249473
0.676
snoRNA
31


U45A
−0.44
0.002283826
0.686
CDBox
74


miR-34a-star
0.61
0.002356105
0.682
miRNA
39


miR-378-star
0.71
0.002495647
0.710
miRNA
82


miR-509-5p
0.98
0.002612774
0.705
miRNA
81


miR-34a
0.79
0.00270219
0.676
miRNA
132


ACA2a
0.41
0.002741773
0.690
HAcaBox
55


miR-98
0.63
0.002795533
0.698
miRNA
47


miR-337-3p
−0.59
0.002924904
0.709
miRNA
94


U58A
−0.37
0.002957231
0.667
CDBox
129


miR-30e
0.66
0.003008812
0.673
miRNA
57


miR-196a
0.99
0.003061353
0.686
miRNA
77


miR-17-star
0.87
0.003088295
0.675
miRNA
56


HBII-429
−0.26
0.003238494
0.681
CDBox
137


HBII-85-5_x
0.38
0.003305314
0.678
CDBox
31


miR-191-star
−0.50
0.003313493
0.701
miRNA
54


U102
−0.32
0.003411715
0.662
CDBox
124


ACA57
−0.35
0.003594842
0.737
scaRna
135


HBII-234_x
−0.33
0.003636016
0.676
CDBox
102


hsa-let-7i
0.62
0.003881627
0.748
miRNA
135


spike_in-control-23
−0.30
0.003884651
0.673
Oligonucleotide
137






spike-in







controls



HBII-276
0.37
0.003895578
0.676
CDBox
132


mgU6-53
−0.37
0.004017934
0.667
CDBox
94


miR-575
−0.55
0.004110263
0.666
miRNA
41


miR-486-5p
1.06
0.004302169
0.672
miRNA
65


HBII-85-20_x
0.37
0.004413582
0.662
CDBox
42


U43
0.49
0.004416752
0.847
CDBox
135


miR-199b-3p
0.64
0.00460804
0.711
miRNA
134


U50B
−0.47
0.004646908
0.678
CDBox
135


HBII-202
−0.37
0.004720422
0.711
CDBox
135


U32A_x
0.30
0.004742469
0.634
CDBox
136


U28_x
0.43
0.005077583
0.733
CDBox
133


miR-223
0.54
0.005512872
0.635
miRNA
83


U36A
0.26
0.005677159
0.654
CDBox
132


miR-548a-3p
−0.65
0.005852929
0.669
miRNA
65


U82
−0.43
0.005931718
0.686
CDBox
131


ACA13
−0.40
0.005934282
0.697
HAcaBox
134


ENSG00000208308_x
0.48
0.005982879
0.755
snoRNA
129


14ql-8
−0.41
0.006020932
0.647
CDBox
61


ACA41_x
0.38
0.006458897
0.687
HAcaBox
122


U20
0.34
0.006504359
0.665
CDBox
117


HBII-210
−0.33
0.006967845
0.684
CDBox
135


ACA53
0.33
0.007009476
0.656
HAcaBox
105


miR-188-5p
0.59
0.00730559
0.668
miRNA
51


miR-653
−0.48
0.007396776
0.652
miRNA
50


ACA18_x
0.33
0.007506763
0.656
HAcaBox
135


ENSG00000202440_x
−0.29
0.007646916
0.654
snoRNA
91


HBII-180C_x
0.33
0.007676115
0.666
CDBox
131


ENSG00000200235_x
−0.29
0.007854884
0.650
snoRNA
59


HBII-85-17_x
0.43
0.008991412
0.650
CDBox
57


miR-205
0.39
0.009048012
0.665
miRNA
135


ACA27_x
0.24
0.009084132
0.676
HAcaBox
130


ACA52
0.31
0.009338308
0.664
HAcaBox
121


U8_x
0.37
0.00950871
0.638
CDBox
135


ENSG00000201229
0.35
0.009710558
0.662
snoRNA
32


miR-423-5p
−0.49
0.009884603
0.704
miRNA
132


U72_x
0.27
0.009902439
0.642
HAcaBox
103


U50
0.37
0.010130893
0.679
CDBox
134


miR-34b
0.95
0.010634429
0.647
miRNA
32


U60
0.33
0.010937468
0.653
CDBox
103


ACA26
−0.38
0.011152582
0.688
scaRna
125


ACA21
−0.44
0.011353671
0.661
HAcaBox
133


U47
0.30
0.01143234
0.651
CDBox
74


miR-26a
0.40
0.011704306
0.793
miRNA
136


miR-21-star
0.54
0.012900809
0.648
miRNA
64


U43_x
0.42
0.013355042
0.808
CDBox
135


HBII-55
0.27
0.013604568
0.636
CDBox
135


ACA19
−0.29
0.013694584
0.634
HAcaBox
118


ENSG00000200897_x
−0.34
0.013964134
0.628
snoRNA
70


ENSG00000207118
0.42
0.01420542
0.652
snoRNA
93


U31
−0.42
0.014421405
0.684
CDBox
132


miR-204
0.70
0.015040931
0.620
miRNA
53


miR-195
0.59
0.01545596
0.646
miRNA
132


miR-432
0.70
0.015655418
0.649
miRNA
51


hsa-let-7c
0.48
0.015958151
0.801
miRNA
135


ACA10_s
0.33
0.015973764
0.658
HAcaBox
113


miR-1825
−0.60
0.016002367
0.679
miRNA
132


14qll-1
−0.53
0.016171959
0.621
CDBox
128


14qll-21_x
0.32
0.017134601
0.670
CDBox
51


U34
0.24
0.017406393
0.609
CDBox
137


miR-138-1-star
0.64
0.017790103
0.611
miRNA
91


miR-1257
−0.40
0.018052873
0.633
miRNA
31


ACA51_x
−0.23
0.018226343
0.656
HAcaBox
135


14qll-1_x
−0.50
0.018262482
0.626
CDBox
131


ACA43
0.31
0.018272258
0.620
HAcaBox
114


hsa-let-7d
0.38
0.01921824
0.855
miRNA
135


14qll-12_x
0.40
0.020581734
0.652
CDBox
85


14qll-28_x
0.34
0.02070105
0.641
CDBox
51


spike_in-control-30
−0.36
0.02072199
0.657
Oligonucleotide
53






spike-in







controls



U49B_s
−0.38
0.021350114
0.648
CDBox
129


U45B_x
−0.32
0.022203975
0.615
CDBox
46


miR-125b
0.42
0.022489574
0.718
miRNA
135


ENSG00000202252
−0.23
0.023928736
0.654
snoRNA
135


miR-1324
−0.37
0.024126574
0.638
miRNA
57


HBI-61
−0.34
0.024172419
0.639
HAcaBox
134


miR-99b-star
0.34
0.024194238
0.626
miRNA
74


miR-20a
0.51
0.024441924
0.640
miRNA
134


U54
0.36
0.02454759
0.749
CDBox
135


ENSG00000200932
−0.31
0.024839208
0.656
snoRNA
47


ENSG00000212326
0.34
0.024965859
0.611
snoRNA
104


U95
−0.35
0.026080784
0.723
CDBox
135


miR-193b-star
0.57
0.028801534
0.684
miRNA
77


ACA5_x
0.29
0.0293861
0.662
HAcaBox
103


ACA1_x
−0.30
0.029505966
0.627
HAcaBox
51


mgU6-53_x
−0.26
0.03076806
0.618
CDBox
113


U26
−0.36
0.030778322
0.608
CDBox
135


miR-24
0.36
0.031162316
0.771
miRNA
135


spike_in-control-29
−0.24
0.03150161
0.615
Oligonucleotide
137






spike-in







controls



miR-19b
0.57
0.03221182
0.612
miRNA
124


miR-513c
0.78
0.032937767
0.632
miRNA
73


U42B_x
0.27
0.033688384
0.613
CDBox
109


miR-20a-star
−0.90
0.033742183
0.639
miRNA
61


U25
−0.30
0.035318167
0.655
CDBox
135


miR-455-3p
0.34
0.036186611
0.643
miRNA
136


U109
0.32
0.036189693
0.634
scaRna
69


miR-938
−0.32
0.037572276
0.639
miRNA
45


ENSG00000207503
−0.27
0.037791965
0.622
snoRNA
42


HBII-289
−0.24
0.041306388
0.652
CDBox
135


U17b_x
0.34
0.041402099
0.728
HAcaBox
135


ACA8_x
−0.28
0.042136422
0.624
HAcaBox
125


ACA44
0.29
0.043557966
0.693
HAcaBox
135


U45C_x
−0.33
0.043838427
0.586
CDBox
72


ENSG00000201133
−0.27
0.045035669
0.645
snoRNA
35


ACA55
−0.26
0.045152107
0.606
HAcaBox
85


miR-451
0.53
0.046730942
0.628
miRNA
72


14qll-26
−0.32
0.047726134
0.619
CDBox
32


miR-141
0.44
0.049534195
0.627
miRNA
69
















TABLE 2







50 miRNAs most significantly expressed between melanoma and nevi












Probe Name
Log2-FC
P value
AUC
ProbeType
#Sample Detected















miR-132
2.98
 1.2728E−32
0.994
miRNA
96


miR-150
3.27
5.83192E−30
1.000
miRNA
117


miR-339-5p
2.73
1.69388E−27
0.988
miRNA
75


miR-15b
3.02
6.41784E−27
0.978
miRNA
125


miR-342-3p
2.23
7.03798E−26
0.993
miRNA
136


miR-572
−2.78
1.00008E−25
0.970
miRNA
116


miR-155
4.14
1.28454E−25
0.981
miRNA
123


miR-425
2.74
8.13135E−25
0.975
miRNA
114


miR-1202
−2.63
2.97795E−23
0.986
miRNA
57


miR-1268
−2.68
6.05905E−23
0.997
miRNA
133


HBII-382_s
−1.71
6.58931E−22
0.971
scaRna
126


miR-1225-5p
−2.36
1.05361E−21
0.953
miRNA
90


miR-30c
2.39
2.58594E−21
0.980
miRNA
125


miR-106b-star
2.24
3.48507E−21
0.961
miRNA
72


miR-125a-5p
2.39
6.90609E−21
0.967
miRNA
128


mgU6-53B
−1.51
1.74782E−20
0.973
CDBox
99


miR-25
2.57
1.52769E−19
0.956
miRNA
118


miR-149-star
−2.03
1.64858E−19
1.000
miRNA
135


miR-939
−2.21
 1.9129E−19
0.973
miRNA
57


miR-92b-star
−2.30
2.05085E−19
0.969
miRNA
111


miR-500-star
2.38
3.11256E−19
0.956
miRNA
97


miR-22
2.69
 3.8844E−19
0.965
miRNA
120


HBII-142_x
−1.22
4.93318E−19
0.981
CDBox
135


miR-181b
2.41
1.00639E−18
0.960
miRNA
130


HBII-142
−1.33
1.18794E−18
0.988
CDBox
135


U38B
−1.95
1.35964E−18
0.975
CDBox
134


miR-663
−2.10
2.04672E−18
0.984
miRNA
134


miR-1224-5p
−2.54
2.91169E−18
0.946
miRNA
78


miR-23a
1.18
2.99523E−18
0.967
miRNA
137


HBII-85-6_x
−1.73
4.78418E−18
0.939
CDBox
137


miR-1207-5p
−2.14
4.98991E−18
0.995
miRNA
133


miR-1301
2.36
5.22389E−18
0.931
miRNA
54


miR-1228-star
−2.41
5.51513E−18
0.997
miRNA
134


miR-345
2.45
6.43552E−18
0.942
miRNA
75


miR-30a-star
2.32
6.92165E−18
0.932
miRNA
64


ENSG00000199411_s
−1.97
7.23325E−18
0.991
snoRNA
135


ENSG00000202327
−1.40
8.75839E−18
0.938
snoRNA
54


miR-92a
1.67
9.49427E−18
0.979
miRNA
136


miR-127-3p
2.43
9.84615E−18
0.936
miRNA
89


HBII-85-26
−2.07
1.50619E−17
0.951
CDBox
136


miR-1308
−2.14
1.99888E−17
0.999
miRNA
135


miR-31
3.29
2.46697E−17
0.913
miRNA
103


miR-921
−1.46
2.61089E−17
0.928
miRNA
49


miR-146b-5p
2.37
6.10422E−17
0.918
miRNA
83


miR-768-3p
−1.05
6.66153E−17
0.949
miRNA
137


miR-708
2.29
6.86187E−17
0.929
miRNA
102


miR-139-5p
2.23
2.78962E−16
0.922
miRNA
80


ACA24_x
1.33
3.37973E−16
0.925
HAcaBox
98


miR-501-3p
1.83
4.76751E−16
0.923
miRNA
80


miR-502-3p
2.16
5.19509E−16
0.925
miRNA
110


miR-923
−1.86
5.55191E−16
0.996
miRNA
135
















TABLE 3







miRNAs significantly expressed between melanoma and normal skin













Log2-



#Sample


Probe Name
FC
P value
AUC
ProbeType
Detected















miR-146a
4.6
9.43624E−20
0.983
miRNA
129


miR-509-3p
5.2
5.61558E−17
0.969
miRNA
116


14qll-14
−2.6
1.16829E−14
0.978
CDBox
56


miR-25
2.4
  2.08E−14
0.949
miRNA
118


miR-138
3.2
2.51219E−13
0.954
miRNA
82


miR-509-3-5p
3.6
2.94532E−13
0.963
miRNA
107


miR-506
3.4
6.99753E−13
0.954
miRNA
76


14qll-14_x
−2.2
8.36026E−13
0.970
CDBox
56


14ql-4
−2.1
 8.9267E−13
0.964
CDBox
80


miR-30b
2.2
2.43822E−12
0.957
miRNA
128


miR-513a-5p
3.5
3.17158E−12
0.939
miRNA
105


miR-21
3.3
4.58391E−12
0.926
miRNA
79


Z17B
−1.0
6.92137E−12
0.956
CDBox
117


U33
−0.9
8.19977E−12
0.968
CDBox
137


miR-20b
2.8
2.24496E−11
0.939
miRNA
99


hsa-let-7i
2.3
3.27742E−11
0.973
miRNA
135


HBII-239
−1.0
 3.9295E−11
0.920
CDBox
135


miR-146b-5p
2.3
8.71488E−11
0.910
miRNA
83


14qll-26_x
−1.8
9.17329E−11
0.914
CDBox
41


miR-155
2.8
1.12361E−10
0.940
miRNA
123


miR-151-3p
1.8
 1.7141E−10
0.922
miRNA
117


HBII-289
−1.1
1.76712E−10
0.963
CDBox
135


14qll-12_x
−1.7
 1.9475E−10
0.907
CDBox
85


miR-1274a
1.9
3.28343E−10
0.928
miRNA
48


HBII-180A_x
−0.8
4.68853E−10
0.933
CDBox
123


miR-1301
2.0
7.22101E−10
0.896
miRNA
54


14qll-1_x
−1.7
7.72545E−10
0.965
CDBox
131


miR-193b
−1.4
 8.7852E−10
0.974
miRNA
130


miR-510
2.7
1.03096E−09
0.914
miRNA
82


miR-126
2.1
1.35297E−09
0.959
miRNA
133


miR-24-2-star
1.8
1.36986E−09
0.895
miRNA
63


miR-106b
2.1
1.91606E−09
0.930
miRNA
127


HBII-276
−1.0
2.06051E−09
0.908
CDBox
132


miR-532-5p
1.8
 2.9654E−09
0.887
miRNA
113


14qll-12
−1.7
3.71443E−09
0.859
CDBox
58


miR-19b
2.1
3.71682E−09
0.908
miRNA
124


miR-30a
1.6
6.61873E−09
0.894
miRNA
113


HBII-85-26_x
−1.1
7.76845E−09
0.895
CDBox
135


miR-150
1.7
1.08172E−08
0.894
miRNA
117


14qll-26
−1.5
1.16704E−08
0.869
CDBox
32


miR-324-5p
1.6
1.24724E−08
0.880
miRNA
102


14ql-8
−1.2
1.39067E−08
0.894
CDBox
61


miR-185
1.6
1.44139E−08
0.952
miRNA
132


miR-194
1.8
1.72961E−08
0.883
miRNA
54


14qll-1
−1.5
2.76507E−08
0.928
CDBox
128


HBII-202
−1.0
2.89826E−08
0.946
CDBox
135


miR-768-5p
−1.6
 3.1365E−08
0.986
miRNA
135


miR-421
1.6
4.42916E−08
0.872
miRNA
68


miR-28-5p
1.7
5.45027E−08
0.877
miRNA
105


miR-151-5p
1.2
5.48542E−08
0.968
miRNA
135


miR-15a
2.0
5.77662E−08
0.889
miRNA
80


miR-26a
1.3
 6.8712E−08
0.968
miRNA
136


U25
−1.0
7.35643E−08
0.976
CDBox
135


HBII-180C
−0.9
7.47464E−08
0.836
CDBox
115


miR-584
1.9
 8.0812E−08
0.863
miRNA
57


hsa-let-7f
2.2
1.05873E−07
0.924
miRNA
130


miR-1268
−1.7
1.09503E−07
0.954
miRNA
133


miR-572
−1.4
1.12523E−07
0.886
miRNA
116


ENSG00000200897
−0.9
1.38587E−07
0.843
snoRNA
38


miR-508-5p
1.8
1.57345E−07
0.873
miRNA
92


hsa-let-7g
1.8
1.57818E−07
0.916
miRNA
127


miR-20a
1.7
1.58351E−07
0.926
miRNA
134


miR-1225-5p
−1.3
 6.2291E−07
0.890
miRNA
90


U55
−0.8
 1.8721E−07
0.942
CDBox
135


miR-509-5p
2.3
1.95354E−07
0.864
miRNA
81


HBII-85-6_x
−1.1
 2.0107E−07
0.872
CDBox
137


U99
−0.7
2.22915E−07
0.872
HAcaBox
137


miR-501-3p
1.4
2.27557E−07
0.858
miRNA
80


miR-29a
1.8
2.42514E−07
0.907
miRNA
128


miR-1274b
1.6
 2.6096E−07
0.863
miRNA
112


miR-210
−1.2
2.81748E−07
0.895
miRNA
130


HBII-85-26
−1.4
 3.5949E−07
0.896
CDBox
136


miR-199a-5p
−0.8
3.93185E−07
0.870
miRNA
129


miR-149
−1.6
5.63545E−07
0.920
miRNA
100


miR-1307
−0.9
 5.6459E−07
0.907
miRNA
97


miR-744
−1.1
5.78243E−07
0.971
miRNA
133


miR-92b-star
−1.4
6.45948E−07
0.907
miRNA
111


ACA11
0.8
7.08497E−07
0.842
HAcaBox
48


miR-27a
1.0
7.17204E−07
0.887
miRNA
136


miR-34a-star
1.4
8.89848E−07
0.842
miRNA
39


U46
−0.8
 9.2248E−07
0.884
CDBox
133


miR-214
−1.2
1.02234E−06
0.930
miRNA
135


miR-132
1.2
1.15319E−06
0.808
miRNA
96


miR-18a
2.0
 1.3554E−06
0.866
miRNA
77


miR-125a-5p
−1.0
1.86884E−06
0.934
miRNA
128


14ql-8_x
−0.9
1.95428E−06
0.831
CDBox
74


miR-663
−1.3
 1.9913E−06
0.910
miRNA
134


miR-16
1.1
2.78721E−06
0.955
miRNA
135


HBII-142_x
−0.8
2.95334E−06
0.909
CDBox
135


U103_s
−0.8
 3.6466E−06
0.825
CDBox
111


U55_x
−0.8
3.84586E−06
0.958
CDBox
135


HBII-142
−0.8
3.88875E−06
0.917
CDBox
135


miR-130b
1.9
4.19812E−06
0.858
miRNA
90


miR-339-3p
1.1
4.25751E−06
0.796
miRNA
60


miR-30d
1.2
4.40714E−06
0.902
miRNA
131


miR-196a
2.0
4.53608E−06
0.833
miRNA
77


miR-199a-3p
1.4
5.28457E−06
0.889
miRNA
134


miR-211
2.1
 5.7974E−06
0.859
miRNA
84


miR-30e
1.2
5.88414E−06
0.839
miRNA
57


ACA54
−0.6
7.09713E−06
0.861
HAcaBox
133


miR-106b-star
1.1
7.86756E−06
0.720
miRNA
72


14qll-3
−1.1
8.39354E−06
0.832
CDBox
95


HBII-55
−0.6
8.41728E−06
0.860
CDBox
135


miR-486-5p
−2.2
1.02088E−05
0.829
miRNA
65


miR-145
−1.3
1.03604E−05
0.831
miRNA
135


miR-34a
1.4
1.04763E−05
0.868
miRNA
132


miR-106a
1.2
1.06568E−05
0.919
miRNA
135


miR-1271
1.3
1.06608E−05
0.829
miRNA
70


miR-500
1.3
1.07278E−05
0.816
miRNA
86


miR-625
1.4
1.08708E−05
0.800
miRNA
59


14qll-9_x
−0.8
1.14849E−05
0.821
CDBox
36


14qll-21_x
−0.9
1.17647E−05
0.797
CDBox
51


U83
−0.6
1.19481E−05
0.889
CDBox
135


miR-638
−1.3
1.34963E−05
0.914
miRNA
135


HBII-180C_x
−0.6
1.39632E−05
0.841
CDBox
131


ENSG00000212139_x
−0.6
1.67825E−05
0.831
snoRNA
124


miR-628-3p
1.4
1.76296E−05
0.811
miRNA
38


U104
−0.8
1.84466E−05
0.886
CDBox
135


miR-10b
1.2
2.02886E−05
0.829
miRNA
106


U34
−0.4
2.06445E−05
0.807
CDBox
137


miR-127-3p
−1.1
2.16929E−05
0.799
miRNA
89


14qll-28_x
−0.9
2.21554E−05
0.815
CDBox
51


miR-1207-5p
−1.2
2.25272E−05
0.917
miRNA
133


miR-199b-3p
1.3
2.29577E−05
0.863
miRNA
134


U35A
−0.7
2.50745E−05
0.900
CDBox
135


U91_s
−0.8
2.92768E−05
0.806
scaRna
132


spike_in-control-36
0.6
2.95775E−05
0.865
Oligonucleotide
137






spike-in







controls



U32A_x
−0.4
3.05381E−05
0.827
CDBox
136


miR-21-star
1.2
3.24139E−05
0.808
miRNA
64


miR-149-star
−1.1
4.06943E−05
0.888
miRNA
135


miR-193b-star
−1.4
4.09065E−05
0.831
miRNA
77


U67_x
0.8
4.09255E−05
0.792
HAcaBox
50


ACA20
−0.8
4.42355E−05
0.839
HAcaBox
131


miR-1287
1.2
4.69003E−05
0.775
miRNA
40


ACA64
−0.7
4.84198E−05
0.802
HAcaBox
33


U57
−0.8
5.04018E−05
0.944
CDBox
135


miR-193a-3p
1.1
5.08442E−05
0.766
miRNA
42


miR-371-5p
1.1
5.19682E−05
0.775
miRNA
64


HBII-85-8_x
−0.7
5.46471E−05
0.782
CDBox
135


miR-503
1.4
 5.4991E−05
0.794
miRNA
41


miR-138-1-star
1.5
5.60721E−05
0.877
miRNA
91


ACA67_x
0.7
5.78125E−05
0.790
HAcaBox
54


miR-1228-star
−1.3
5.81565E−05
0.895
miRNA
134


U41
−0.8
5.85491E−05
0.875
CDBox
135


miR-193a-5p
−1.2
5.98826E−05
0.897
miRNA
121


ENSG00000202252
−0.5
6.64141E−05
0.839
snoRNA
135


14ql-4_x
−1.0
 6.7457E−05
0.760
CDBox
51


miR-508-3p
1.7
6.91432E−05
0.801
miRNA
74


miR-200b
1.4
7.07093E−05
0.779
miRNA
109


miR-660
1.2
7.13979E−05
0.791
miRNA
71


U67
0.8
7.16134E−05
0.774
HAcaBox
44


U50B
−0.8
 7.249E−05
0.869
CDBox
135


miR-513c
1.9
7.46404E−05
0.771
miRNA
73


14ql-9_x
−0.7
7.67649E−05
0.772
CDBox
34


U60
−0.6
8.34902E−05
0.779
CDBox
103


U102
0.5
8.47602E−05
0.772
CDBox
124


EN8G00000200235_x
−0.5
 9.4306E−05
0.793
snoRNA
59


miR-17
1.0
9.79941E−05
0.910
miRNA
134


U36C
−0.6
0.000107866
0.854
CDBox
135


miR-491-5p
0.9
0.000121664
0.753
miRNA
92


miR-425
1.0
0.000138461
0.771
miRNA
114


miR-500-star
1.0
0.000148221
0.761
miRNA
97


U78_x
1.0
0.000150233
0.872
CDBox
134


ENSG00000202327
−0.6
0.000153478
0.783
snoRNA
54


miR-181c
1.1
0.000156102
0.772
miRNA
59


ENSG00000212523_x
−0.8
0.000165203
0.772
snoRNA
135


mgU6-53B_x
−0.5
0.00016649
0.784
CDBox
121


miR-99b-star
0.8
0.00017311
0.743
miRNA
74


U53
−0.7
0.000175804
0.763
CDBox
126


miR-296-3p
−1.0
0.000180884
0.771
miRNA
90


U50B_x
−0.7
0.000181435
0.829
CDBox
135


miR-30a-star
1.2
0.00018331
0.766
miRNA
64


U46_x
−0.8
0.000183742
0.819
CDBox
131


ENSG00000207410_x
−0.5
0.000198904
0.779
snoRNA
55


miR-99a
−0.8
0.000204711
0.829
miRNA
135


miR-513b
1.4
0.000214575
0.781
miRNA
64


miR-23a
0.6
0.000218135
0.908
miRNA
137


miR-31
1.7
0.00023269
0.721
miRNA
103


miR-345
1.2
0.000250483
0.782
miRNA
75


miR-212
1.5
0.000254534
0.831
miRNA
62


ENSG00000212266
−0.6
0.000255339
0.777
snoRNA
93


miR-152
1.1
0.000260732
0.813
miRNA
122


U19
0.6
0.000277432
0.757
HAcaBox
118


miR-886-3p
1.0
0.000298839
0.768
miRNA
110


U73a
−0.6
0.000338569
0.846
CDBox
135


miR-423-3p
−0.7
0.000347496
0.848
miRNA
107


mgh28S-2411
−0.6
0.00034785
0.901
CDBox
135


miR-103
0.8
0.000430411
0.930
miRNA
135


miR-629
1.1
0.000473215
0.769
miRNA
64


U65
−0.6
0.000562655
0.778
HAcaBox
128


14ql-7
−0.7
0.000569692
0.803
CDBox
51


miR-933
−0.6
0.000572446
0.769
miRNA
100


miR-205
−0.6
0.000653671
0.767
miRNA
135


U105
−0.4
0.000692027
0.749
CDBox
126


miR-378-star
1.1
0.000731339
0.769
miRNA
82


miR-939
−0.9
0.000748523
0.822
miRNA
57


hsa-let-7d
0.8
0.000759497
0.978
miRNA
135


miR-362-5p
1.2
0.000775756
0.762
miRNA
94


ACA9
0.7
0.000834583
0.739
HAcaBox
82


miR-10a
1.1
0.000844868
0.759
miRNA
69


miR-877
−0.9
0.000845569
0.789
miRNA
126


miR-128
1.1
0.000865867
0.762
miRNA
44


miR-27b
0.8
0.000876954
0.799
miRNA
133


miR-17-star
1.2
0.000905238
0.748
miRNA
56


mgh18S-121
−0.5
0.000964406
0.789
CDBox
134


ENSG00000200394
0.5
0.000968073
0.772
snoRNA
119


ENSG00000212315
0.6
0.000971189
0.757
snoRNA
117


ENSG00000212615_x
−0.5
0.000998331
0.707
snoRNA
102


miR-148a
0.9
0.001014157
0.733
miRNA
41


miR-923
−0.9
0.001032772
0.826
miRNA
135


ACA20_x
−0.6
0.001087206
0.830
HAcaBox
134


ENSG00000212579_x
−0.6
0.001126846
0.765
snoRNA
100


miR-589-star
0.5
0.001225281
0.703
miRNA
43


miR-107
0.7
0.001260426
0.932
miRNA
135


ENSG00000199363
0.6
0.001425983
0.750
snoRNA
42


ACA55
0.5
0.001430114
0.746
HAcaBox
85


U54
−0.7
0.001511402
0.868
CDBox
135


U36A
−0.4
0.001519955
0.759
CDBox
132


U95
−0.7
0.001551616
0.929
CDBox
135


14qll-22_x
−0.7
0.001591836
0.701
CDBox
50


U58B_x
−0.5
0.001603166
0.726
CDBox
132


miR-720
0.7
0.001622509
0.760
miRNA
137


ENSG00000200394_x
0.5
0.001742151
0.711
snoRNA
115


HBII-166
−0.5
0.001874456
0.769
CDBox
134


miR-29b-1-star
1.3
0.001938943
0.736
miRNA
46


HBII-436
−0.5
0.001978078
0.731
CDBox
102


U23
−0.5
0.002014594
0.748
HAcaBox
127


miR-346
−0.9
0.002054981
0.774
miRNA
55


U58A
−0.5
0.002095349
0.735
CDBox
129


HBII-95
0.5
0.00219824
0.703
CDBox
78


miR-339-5p
0.7
0.002357824
0.693
miRNA
75


ACA25
0.4
0.002440171
0.724
HAcaBox
73


miR-363
1.1
0.002716266
0.735
miRNA
44


miR-432
−1.1
0.002752773
0.738
miRNA
51


miR-424-star
1.1
0.002765097
0.718
miRNA
41


U27
−0.6
0.002765603
0.792
CDBox
134


miR-30c
0.6
0.002778453
0.770
miRNA
125


miR-93
0.7
0.002813996
0.863
miRNA
134


hsa-let-7a
0.8
0.002856983
0.911
miRNA
136


miR-181d
1.0
0.002954008
0.748
miRNA
61


miR-99b
−0.5
0.00296227
0.757
miRNA
129


miR-550-star
0.9
0.003086147
0.740
miRNA
58


U43
−0.7
0.00327637
0.865
CDBox
135


U52
−0.5
0.003332494
0.783
CDBox
135


miR-502-3p
0.8
0.003628993
0.775
miRNA
110


spike_in-control-29
−0.4
0.003663934
0.723
Oligonucleotide
137






spike-in







controls



14qll-17
−0.7
0.003689916
0.703
CDBox
71


ENSG00000212302_x
−0.4
0.003876466
0.676
snoRNA
56


miR-574-3p
−0.7
0.003955226
0.842
miRNA
122


miR-320d
−0.8
0.004248663
0.868
miRNA
134


miR-1246
1.0
0.004275913
0.772
miRNA
131


ACA7_s
−0.4
0.004475709
0.719
HAcaBox
133


miR-15b
0.6
0.004714973
0.792
miRNA
125


HBII-52-37_x
−0.5
0.004773282
0.714
CDBox
37


gi555853_copy0
−0.6
0.004784885
0.799
5.8s rRNA
137


miR-30e-star
0.8
0.00500785
0.717
miRNA
35


miR-18a-star
0.8
0.005024206
0.723
miRNA
40


ENSG00000201847_x
−0.4
0.005059414
0.688
snoRNA
48


gi555853_copy5
−0.5
0.005220274
0.809
5.8s rRNA
137


U24
−0.4
0.005634443
0.708
CDBox
133


miR-1280
−0.4
0.005689807
0.740
miRNA
134


miR-1231
−0.8
0.005810993
0.676
miRNA
79


ENSG00000202498_x
−0.5
0.005898128
0.701
snoRNA
137


gi555853_copy7
−0.5
0.006325433
0.804
5.8s rRNA
137


miR-28-3p
0.9
0.006506887
0.711
miRNA
94


ENSG00000199435
0.4
0.006656491
0.706
snoRNA
63


gi555853_copy2
−0.5
0.00671481
0.800
5.8s rRNA
137


U43_x
−0.6
0.006913554
0.854
CDBox
135


ENSG00000212273_x
−0.5
0.007104981
0.725
snoRNA
126


ENSG00000200961
−0.4
0.007247826
0.716
snoRNA
41


U38A
−0.7
0.007390068
0.814
CDBox
134


E3_x
−0.4
0.007704052
0.779
HAcaBox
135


gi555853_copy8
−0.5
0.00776022
0.799
5.8s rRNA
137


HBII-234_x
−0.3
0.007814833
0.703
CDBox
102


U28
−0.5
0.008026327
0.771
CDBox
135


ENSG00000212587
−0.5
0.008211191
0.685
snoRNA
45


ENSG00000212149_x
−0.5
0.008233504
0.678
snoRNA
77


HBII-420
−0.5
0.00828299
0.771
CDBox
131


gi555853_copy1
−0.5
0.008395769
0.791
5.8s rRNA
137


HBII-85-21_x
0.5
0.008439987
0.717
CDBox
39


gi555853_copy4
−0.5
0.008541113
0.793
5.8s rRNA
137


gi555853_copy6
−0.5
0.008815947
0.792
5.8s rRNA
137


ENSG00000200897_x
−0.5
0.009024011
0.686
snoRNA
70


ENSG00000207002
−0.4
0.009046282
0.706
snoRNA
35


ENSG00000212627
−0.4
0.009345896
0.714
snoRNA
69


gi555853_copy3
−0.5
0.009628403
0.777
5.8s rRNA
137


HBII-85-17_x
0.5
0.009677702
0.685
CDBox
57


miR-92b
0.5
0.009817789
0.672
miRNA
91


HBII-210
−0.4
0.009959111
0.756
CDBox
135


ACA3
−0.5
0.010166004
0.740
HAcaBox
134


miR-769-5p
0.7
0.010207709
0.622
miRNA
56


U100
0.4
0.010232137
0.645
scaRna
80


U13
−0.4
0.010247574
0.720
CDBox
135


miR-181b
0.6
0.010316868
0.816
miRNA
130


ACA46
0.4
0.010365242
0.699
HAcaBox
124


gi555853_copy9
−0.5
0.010460125
0.788
5.8s rRNA
137


U70_x
−0.5
0.011265988
0.704
HAcaBox
53


HBII-85-15_x
0.5
0.011411104
0.695
CDBox
49


miR-203
0.7
0.01182754
0.822
miRNA
132


miR-320c
−0.6
0.011901129
0.845
miRNA
135


miR-191-star
−0.6
0.012197187
0.680
miRNA
54


ACA18_x
−0.4
0.012258626
0.694
HAcaBox
135


miR-27a-star
0.8
0.013060406
0.702
miRNA
40


miR-192
0.7
0.013123545
0.703
miRNA
32


U74_x
0.5
0.013418084
0.804
CDBox
135


ACA15_x
−0.3
0.013532632
0.691
HAcaBox
124


miR-198
−0.6
0.013839541
0.687
miRNA
31


U28_x
−0.5
0.013863106
0.711
CDBox
133


ACA28
0.4
0.013886424
0.685
HAcaBox
120


U97
−0.4
0.013970203
0.692
CDBox
133


ACA4
0.3
0.014571497
0.655
HAcaBox
117


miR-423-5p
−0.6
0.015447652
0.754
miRNA
132


ACA25_x
0.4
0.015584731
0.701
HAcaBox
114


HBII-382_s
−0.4
0.016066802
0.670
scaRna
126


miR-34b
1.2
0.016083233
0.693
miRNA
32


miR-181a-2-star
0.7
0.016427521
0.646
miRNA
99


U15B
−0.3
0.017584552
0.689
CDBox
129


miR-361-5p
0.4
0.017617483
0.783
miRNA
135


ACA9_x
0.5
0.017621407
0.669
HAcaBox
77


ACA15_s
−0.3
0.017633335
0.738
HAcaBox
116


miR-23b-star
0.7
0.018210894
0.672
miRNA
52


ACA60
−0.3
0.018634054
0.702
HAcaBox
129


14q-0
−0.4
0.019666039
0.681
CDBox
49


HBII-85-22_x
0.5
0.019999179
0.673
CDBox
42


miR-30b-star
0.9
0.020172985
0.686
miRNA
61


miR-125b
−0.5
0.020376572
0.707
miRNA
135


ENSG00000199411_s
−0.6
0.020504379
0.779
snoRNA
135


HBII-251
−0.3
0.020538747
0.679
CDBox
135


miR-483-3p
0.9
0.020547545
0.666
miRNA
40


ACA23
−0.3
0.021199129
0.685
HAcaBox
113


ENSG00000212432_s
−0.4
0.021742945
0.649
snoRNA
75


miR-25-star
0.7
0.022483461
0.671
miRNA
52


ENSG00000200932
−0.3
0.023007003
0.687
snoRNA
47


U49B_s
0.5
0.023157964
0.726
CDBox
129


ENSG00000212214_x
0.4
0.023504965
0.687
snoRNA
88


U83B
−0.4
0.024471226
0.745
CDBox
135


miR-320b
−0.6
0.025686249
0.790
miRNA
135


miR-489
0.6
0.025963923
0.680
miRNA
58


miR-26b
0.7
0.026201573
0.682
miRNA
45


U14B
−0.4
0.027046414
0.632
CDBox
51


HBII-115
−0.3
0.027115569
0.730
CDBox
117


ENSG00000207027
−0.4
0.028391297
0.621
snoRNA
36


miR-494
0.5
0.029570695
0.689
miRNA
135


miR-181a-star
0.7
0.030787798
0.668
miRNA
35


U49A_x
0.4
0.03088655
0.750
CDBox
135


ACA14b_x
−0.3
0.030912946
0.693
HAcaBox
105


U21
−0.4
0.031731894
0.691
CDBox
134


HBII-135_x
0.6
0.032076241
0.663
CDBox
132


miR-382
0.8
0.032245385
0.651
miRNA
47


miR-532-3p
0.4
0.032767036
0.697
miRNA
95


miR-214-star
0.8
0.032818193
0.670
miRNA
47


mgU6-53B
−0.3
0.033293267
0.663
CDBox
99


miR-200c
−0.7
0.033609817
0.659
miRNA
134


miR-575
0.5
0.034489882
0.657
miRNA
41


mgU6-53
−0.3
0.034658143
0.674
CDBox
94


miR-422a
0.8
0.035772116
0.674
miRNA
84


ENSG00000201848
−0.3
0.036190647
0.652
snoRNA
34


ACA45
0.3
0.036575475
0.653
scaRna
53


miR-559
−0.5
0.036810259
0.647
miRNA
72


ACA61
−0.4
0.037187144
0.716
HAcaBox
135


snR38C
−0.4
0.037656608
0.721
CDBox
134


HBII-85-23_x
0.5
0.037660456
0.650
CDBox
58


miR-551b-star
−0.5
0.037971736
0.645
miRNA
61


miR-27b-star
0.7
0.041289539
0.663
miRNA
51


U50
−0.4
0.041514005
0.696
CDBox
134


ACA58_x
0.3
0.041793061
0.653
HAcaBox
89


ACA53
−0.3
0.0421874
0.635
HAcaBox
105


U49A
0.4
0.042284609
0.694
CDBox
135


ACA16
−0.4
0.042968027
0.658
HAcaBox
47


ACA13
−0.4
0.043493352
0.722
HAcaBox
134


miR-320a
−0.5
0.043683231
0.755
miRNA
135


ENSG00000200969
−0.4
0.043904585
0.663
snoRNA
67


miR-642
−0.4
0.04542452
0.640
miRNA
69


miR-148b
0.5
0.046811178
0.645
miRNA
32


U84
−0.2
0.046963896
0.683
CDBox
131


U56_x
0.4
0.047943262
0.701
CDBox
134


miR-451
0.7
0.049304242
0.632
miRNA
72


miR-195
0.6
0.049397144
0.698
miRNA
132
















TABLE 4







50 miRNAs most significantly expressed between


melanoma and normal skin













Log2-



#Sample


Probe Name
FC
P value
AUC
ProbeType
Detected















miR-146a
4.6
9.43624E−20
0.983
miRNA
129


miR-509-3p
5.2
5.61558E−17
0.969
miRNA
116


14qll-14
−2.6
1.16829E−14
0.978
CDBox
56


miR-25
2.4
  2.08E−14
0.949
miRNA
118


miR-138
3.2
2.51219E−13
0.954
miRNA
82


miR-509-3-5p
3.6
2.94532E−13
0.963
miRNA
107


miR-506
3.4
6.99753E−13
0.954
miRNA
76


14qll-14_x
−2.2
8.36026E−13
0.970
CDBox
56


14ql-4
−2.1
 8.9267E−13
0.964
CDBox
80


miR-30b
2.2
2.43822E−12
0.957
miRNA
128


miR-513a-5p
3.5
3.17158E−12
0.939
miRNA
105


miR-21
3.3
4.58391E−12
0.926
miRNA
79


Z17B
−1.0
6.92137E−12
0.956
CDBox
117


U33
−0.9
8.19977E−12
0.968
CDBox
137


miR-20b
2.8
2.24496E−11
0.939
miRNA
99


hsa-let-7i
2.3
3.27742E−11
0.973
miRNA
135


HBII-239
−1.0
 3.9295E−11
0.920
CDBox
135


miR-146b-5p
2.3
8.71488E−11
0.910
miRNA
83


14qll-26_x
−1.8
9.17329E−11
0.914
CDBox
41


miR-155
2.8
1.12361E−10
0.940
miRNA
123


miR-151-3p
1.8
 1.7141E−10
0.922
miRNA
117


HBII-289
−1.1
1.76712E−10
0.963
CDBox
135


14qll-12_x
−1.7
 1.9475E−10
0.907
CDBox
85


miR-1274a
1.9
3.28343E−10
0.928
miRNA
48


HBII-180A_x
−0.8
4.68853E−10
0.933
CDBox
123


miR-1301
2.0
7.22101E−10
0.896
miRNA
54


14qll-1_x
−1.7
7.72545E−10
0.965
CDBox
131


miR-193b
−1.4
 8.7852E−10
0.974
miRNA
130


miR-510
2.7
1.03096E−09
0.914
miRNA
82


miR-126
2.1
1.35297E−09
0.959
miRNA
133


miR-24-2-star
1.8
1.36986E−09
0.895
miRNA
63


miR-106b
2.1
1.91606E−09
0.930
miRNA
127


HBII-276
−1.0
2.06051E−09
0.908
CDBox
132


miR-532-5p
1.8
 2.9654E−09
0.887
miRNA
113


14qll-12
−1.7
3.71443E−09
0.859
CDBox
58


miR-19b
2.1
3.71682E−09
0.908
miRNA
124


miR-30a
1.6
6.61873E−09
0.894
miRNA
113


HBII-85-26_x
−1.1
7.76845E−09
0.895
CDBox
135


miR-150
1.7
1.08172E−08
0.894
miRNA
117


14qll-26
−1.5
1.16704E−08
0.869
CDBox
32


miR-324-5p
1.6
1.24724E−08
0.880
miRNA
102


14ql-8
−1.2
1.39067E−08
0.894
CDBox
61


miR-185
1.6
1.44139E−08
0.952
miRNA
132


miR-194
1.8
1.72961E−08
0.883
miRNA
54


14qll-1
−1.5
2.76507E−08
0.928
CDBox
128


HBII-202
−1.0
2.89826E−08
0.946
CDBox
135


miR-768-5p
−1.6
 3.1365E−08
0.986
miRNA
135


miR-421
1.6
4.42916E−08
0.872
miRNA
68


miR-28-5p
1.7
5.45027E−08
0.877
miRNA
105


miR-151-5p
1.2
5.48542E−08
0.968
miRNA
135
















TABLE 5







miRNAs significantly expressed between metastatic melanoma and melanoma













Log2-



#Sample


Probe Name
FC
P value
AUC
ProbeType
Detected















miR-31
−2.4
6.23649E−09
0.891
miRNA
103


miR-150
−1.2
4.84574E−06
0.783
miRNA
117


miR-203
−2.0
2.77555E−05
0.764
miRNA
132


ENSG00000212139_x
0.6
2.83257E−05
0.799
snoRNA
124


mgU6-53
0.6
 3.8628E−05
0.828
CDBox
94


U72_x
0.5
4.47709E−05
0.783
HAcaBox
103


U94
0.6
7.34024E−05
0.776
CDBox
109


HBII-85-15_x
0.8
0.000176472
0.767
CDBox
49


HBII-85-29_x
0.8
0.000274959
0.734
CDBox
85


HBII-55
0.5
0.000331716
0.806
CDBox
135


snR38B
0.8
0.000374886
0.750
CDBox
126


miR-200c
−1.6
0.00042063
0.718
miRNA
134


U61
0.7
0.000440758
0.785
CDBox
116


HBII-316
0.6
0.000548042
0.744
CDBox
130


U32A_x
0.4
0.000553879
0.748
CDBox
136


U81_x
0.6
0.000598062
0.720
CDBox
120


U15A
0.7
0.000649777
0.785
CDBox
128


ACA14b_x
0.4
0.000764147
0.748
HAcaBox
105


miR-182
−0.9
0.000868427
0.762
miRNA
113


miR-455-3p
−0.7
0.000874654
0.718
miRNA
136


miR-532-5p
−0.8
0.000883675
0.752
miRNA
113


mgU6-53_x
0.5
0.001038416
0.783
CDBox
113


ACA46
0.4
0.001076758
0.746
HAcaBox
124


miR-1234
−0.5
0.001112272
0.756
miRNA
68


U53
0.6
0.001130502
0.769
CDBox
126


HBII-85-29
0.7
0.001192467
0.702
CDBox
82


U47
0.5
0.001218674
0.702
CDBox
74


U42B_x
0.5
0.001412559
0.735
CDBox
109


miR-155
−1.3
0.001425842
0.716
miRNA
123


U64
0.5
0.001493551
0.728
HAcaBox
101


ACA28
0.5
0.001565276
0.692
HAcaBox
120


U13
0.6
0.001630341
0.748
CDBox
135


U84
0.4
0.001673776
0.769
CDBox
131


U22
0.5
0.001739769
0.714
CDBox
135


U46_x
0.6
0.001758635
0.755
CDBox
131


ACA17_x
0.6
0.001827243
0.705
HAcaBox
44


ENSG00000200897
0.4
0.001857043
0.735
snoRNA
38


U36A_x
0.4
0.00192703
0.728
CDBox
133


HBII-180C
0.4
0.001987135
0.757
CDBox
115


HBII-251
0.4
0.0020213
0.741
CDBox
135


ACA49
0.4
0.002175278
0.744
HAcaBox
127


U19
0.6
0.00221226
0.696
HAcaBox
118


miR-940
−0.6
0.002377881
0.713
miRNA
53


U76
0.6
0.002408733
0.755
CDBox
135


HBII-13_x
0.7
0.002521867
0.730
CDBox
60


HBII-382_s
0.6
0.002554678
0.770
scaRna
126


miR-205
−1.1
0.002610442
0.603
miRNA
135


U21
0.7
0.002613305
0.774
CDBox
134


miR-342-3p
−0.5
0.002621512
0.706
miRNA
136


ENSG00000212326
0.5
0.002879558
0.728
snoRNA
104


U82
0.7
0.002979246
0.828
CDBox
131


HBII-234_x
0.3
0.003087142
0.734
CDBox
102


HBII-85-24_x
0.4
0.003172633
0.703
CDBox
34


U101
0.5
0.003644287
0.672
CDBox
134


U46
0.5
0.003690792
0.747
CDBox
133


U58A
0.5
0.003778891
0.735
CDBox
129


HBII-210
0.5
0.003785668
0.767
CDBox
135


U102
0.4
0.00401337
0.705
CDBox
124


HBII-85-3_x
0.5
0.004030857
0.694
CDBox
72


U92
0.4
0.004575813
0.712
scaRna
129


spike_in-control-31
0.1
0.004779896
0.705
Oligonucleotide
137






spike-in






controls


snR38C
0.6
0.005413855
0.779
CDBox
134


HBII-99
0.5
0.005634509
0.714
CDBox
122


ACA47
0.5
0.006095831
0.696
scaRna
77


HBII-420
0.6
0.006404916
0.824
CDBox
131


ENSG00000212423_x
0.5
0.006424898
0.684
snoRNA
115


HBII-85-17_x
0.6
0.006577979
0.678
CDBox
57


ACA27_x
0.3
0.006580858
0.725
HAcaBox
130


U79
0.6
0.007498133
0.748
CDBox
134


U107
0.4
0.00761608
0.707
HAcaBox
129


HBI-43
0.3
0.007872874
0.691
CDBox
122


U49B_x
0.5
0.008037057
0.736
CDBox
122


miR-548i
−0.5
0.008242048
0.684
miRNA
36


miR-500-star
−0.6
0.008434274
0.697
miRNA
97


HBII-85-2_x
0.4
0.008511275
0.708
CDBox
135


ACA41
0.5
0.008991193
0.723
HAcaBox
115


ACA3
0.5
0.009231708
0.737
HAcaBox
134


U49A
0.6
0.009381377
0.745
CDBox
135


HBII-85-20_x
0.5
0.00942941
0.672
CDBox
42


ENSG00000212508
−0.5
0.009471802
0.712
snoRNA
122


U38B_x
0.7
0.009547747
0.837
CDBox
133


ACA51_x
0.3
0.009599762
0.686
HAcaBox
135


ENSG00000200879
0.5
0.009860049
0.697
snoRNA
120


miR-181a
−0.6
0.009890715
0.730
miRNA
134


ENSG00000200932
0.4
0.010075893
0.740
snoRNA
47


miR-589-star
−0.4
0.010240715
0.650
miRNA
43


ENSG00000199262
0.4
0.010309025
0.694
snoRNA
59


miR-1324
−0.5
0.010648472
0.724
miRNA
57


U91_s
0.5
0.010903574
0.695
scaRna
132


U3-2_s
0.5
0.011269742
0.729
CDBox
135


U51_x
0.4
0.011519055
0.713
CDBox
130


ACA9
0.5
0.012046848
0.703
HAcaBox
82


miR-1825
−0.8
0.01292318
0.738
miRNA
132


mgU6-47
0.4
0.013536315
0.710
CDBox
47


miR-1281
−0.7
0.014086281
0.701
miRNA
137


U71d_x
0.5
0.01413005
0.678
HAcaBox
99


ACA42
0.4
0.014283243
0.786
HAcaBox
117


ACA34
0.3
0.014399902
0.637
HAcaBox
109


U48
0.4
0.014650847
0.654
CDBox
129


ENSG00000202093
0.5
0.014675533
0.689
snoRNA
97


miR-665
0.5
0.014853517
0.678
miRNA
47


miR-141
−0.7
0.015316578
0.647
miRNA
69


U23
0.4
0.015408258
0.701
HAcaBox
127


U15B
0.4
0.015684928
0.719
CDBox
129


miR-1274b
0.6
0.01695735
0.679
miRNA
112


U31_x
0.5
0.017818548
0.771
CDBox
135


ENSG00000200394_x
0.4
0.017859341
0.682
snoRNA
115


ACA67_x
0.4
0.018242565
0.663
HAcaBox
54


U80
0.4
0.019116778
0.683
CDBox
132


HBII-85-18_x
0.4
0.019329528
0.661
CDBox
48


U106
0.3
0.019924806
0.653
CDBox
112


U99
0.3
0.01993828
0.674
HAcaBox
137


U24
0.3
0.020062119
0.673
CDBox
133


miR-20a-star
1.0
0.020518991
0.694
miRNA
61


U16
0.4
0.0213066
0.669
CDBox
131


U49B_s
0.5
0.0213349
0.716
CDBox
129


miR-885-3p
0.7
0.021367498
0.694
miRNA
73


U36B
0.4
0.021413004
0.669
CDBox
121


HBII-295
0.3
0.021589703
0.678
CDBox
128


miR-148b
0.6
0.021711762
0.661
miRNA
32


U38B
0.7
0.022451358
0.834
CDBox
134


U31
0.6
0.023045818
0.738
CDBox
132


ACA8_x
0.4
0.023508719
0.697
HAcaBox
125


HBI-6_x
0.5
0.02438732
0.675
HAcaBox
115


mgU6-53B
0.3
0.024830223
0.687
CDBox
99


ACA62
0.4
0.024948935
0.714
HAcaBox
88


miR-23b
−0.5
0.025010006
0.658
miRNA
137


mgh18S-121
0.4
0.02717981
0.773
CDBox
134


miR-134
−0.6
0.027498011
0.648
miRNA
61


ENSG00000202216
−0.3
0.02787169
0.654
snoRNA
53


snR38A
0.5
0.028058616
0.706
CDBox
130


ACA54
0.4
0.028079033
0.741
HAcaBox
133


U83
0.4
0.028849489
0.733
CDBox
135


U56_x
0.5
0.03056515
0.673
CDBox
134


ENSG00000207118
0.5
0.030731527
0.642
snoRNA
93


U71b_x
0.3
0.031331331
0.639
HAcaBox
117


U104
0.5
0.031533136
0.750
CDBox
135


miR-26b
0.6
0.031546885
0.666
miRNA
45


ENSG00000201129
0.4
0.031647545
0.658
snoRNA
54


HBII-180C_x
0.3
0.031765824
0.686
CDBox
131


miR-1257
−0.4
0.032490122
0.676
miRNA
31


miR-1285
0.6
0.032817723
0.656
miRNA
54


ACA6
0.4
0.033332825
0.706
HAcaBox
126


HBII-166
0.3
0.033479679
0.715
CDBox
134


U20
0.3
0.033581135
0.659
CDBox
117


U49A_x
0.5
0.034235707
0.734
CDBox
135


HBII-85-11
0.5
0.034401945
0.626
CDBox
31


miR-198
0.5
0.035071443
0.673
miRNA
31


miR-1273
0.4
0.035126423
0.649
miRNA
47


U8_x
0.4
0.035369399
0.661
CDBox
135


miR-127-3p
−0.5
0.035478749
0.646
miRNA
89


U36C
0.3
0.036400622
0.690
CDBox
135


miR-502-3p
−0.5
0.036440109
0.696
miRNA
110


U97
0.4
0.036556318
0.713
CDBox
133


HBII-13
0.5
0.038037592
0.639
CDBox
52


miR-148a
0.6
0.038972124
0.647
miRNA
41


hsa-let-7b
−0.7
0.040092161
0.713
miRNA
136


U55
0.3
0.041929175
0.687
CDBox
135


HBII-85-5_x
0.3
0.04234391
0.645
CDBox
31


U105
0.3
0.043199784
0.658
CDBox
126


U90
0.3
0.043617609
0.701
scaRna
114


ENSG00000202252
0.3
0.044418964
0.647
snoRNA
135


ENSG00000201467_x
−0.3
0.04502792
0.628
snoRNA
31


ACA21
0.5
0.045481211
0.754
HAcaBox
133


U52
0.4
0.045645643
0.708
CDBox
135


U18C_x
0.4
0.046127928
0.637
CDBox
74


HBII-296B
0.3
0.046365102
0.657
CDBox
98


ENSG00000200307
0.3
0.048211492
0.613
snoRNA
86


miR-509-3p
−1.2
0.048257749
0.574
miRNA
116


miR-1202
0.5
0.048318042
0.661
miRNA
57


ACA36_x
0.4
0.048990385
0.670
HAcaBox
88


ENSG00000207016_x
0.3
0.049618219
0.660
snoRNA
48


miR-30c
0.3
0.049698
0.709
miRNA
125


ACA19
0.3
0.049825075
0.655
HAcaBox
118
















TABLE 6







50 miRNAs most significantly expressed between metastatic melanoma and melanoma













Log2-






Probe Name
FC
P value
AUC
ProbeType
#Sample Detected















miR-31
−2.4
6.23649E−09
0.891
miRNA
103


miR-150
−1.2
4.84574E−06
0.783
miRNA
117


miR-203
−2.0
2.77555E−05
0.764
miRNA
132


ENSG00000212139_x
0.6
2.83257E−05
0.799
snoRNA
124


mgU6-53
0.6
 3.8628E−05
0.828
CDBox
94


U72_x
0.5
4.47709E−05
0.783
HAcaBox
103


U94
0.6
7.34024E−05
0.776
CDBox
109


HBII-85-15_x
0.8
0.000176472
0.767
CDBox
49


HBII-85-29_x
0.8
0.000274959
0.734
CDBox
85


HBII-55
0.5
0.000331716
0.806
CDBox
135


snR38B
0.8
0.000374886
0.750
CDBox
126


miR-200c
−1.6
0.00042063
0.718
miRNA
134


U61
0.7
0.000440758
0.785
CDBox
116


HBII-316
0.6
0.000548042
0.744
CDBox
130


U32A_x
0.4
0.000553879
0.748
CDBox
136


U81_x
0.6
0.000598062
0.720
CDBox
120


U15A
0.7
0.000649777
0.785
CDBox
128


ACA14b_x
0.4
0.000764147
0.748
HAcaBox
105


miR-182
−0.9
0.000868427
0.762
miRNA
113


miR-455-3p
−0.7
0.000874654
0.718
miRNA
136


miR-532-5p
−0.8
0.000883675
0.752
miRNA
113


mgU6-53_x
0.5
0.001038416
0.783
CDBox
113


ACA46
0.4
0.001076758
0.746
HAcaBox
124


miR-1234
−0.5
0.001112272
0.756
miRNA
68


U53
0.6
0.001130502
0.769
CDBox
126


HBII-85-29
0.7
0.001192467
0.702
CDBox
82


U47
0.5
0.001218674
0.702
CDBox
74


U42B_x
0.5
0.001412559
0.735
CDBox
109


miR-155
−1.3
0.001425842
0.716
miRNA
123


U64
0.5
0.001493551
0.728
HAcaBox
101


ACA28
0.5
0.001565276
0.692
HAcaBox
120


U13
0.6
0.001630341
0.748
CDBox
135


U84
0.4
0.001673776
0.769
CDBox
131


U22
0.5
0.001739769
0.714
CDBox
135


U46_x
0.6
0.001758635
0.755
CDBox
131


ACA17_x
0.6
0.001827243
0.705
HAcaBox
44


ENSG00000200897
0.4
0.001857043
0.735
snoRNA
38


U36A_x
0.4
0.00192703
0.728
CDBox
133


HBII-180C
0.4
0.001987135
0.757
CDBox
115


HBII-251
0.4
0.0020213
0.741
CDBox
135


ACA49
0.4
0.002175278
0.744
HAcaBox
127


U19
0.6
0.00221226
0.696
HAcaBox
118


miR-940
−0.6
0.002377881
0.713
miRNA
53


U76
0.6
0.002408733
0.755
CDBox
135


HBII-13_x
0.7
0.002521867
0.730
CDBox
60


HBII-382_s
0.6
0.002554678
0.770
scaRna
126


miR-205
−1.1
0.002610442
0.603
miRNA
135


U21
0.7
0.002613305
0.774
CDBox
134


miR-342-3p
−0.5
0.002621512
0.706
miRNA
136


ENSG00000212326
0.5
0.002879558
0.728
snoRNA
104
















TABLE 7







miRNAs significantly expressed between nevi and normal skin













Log2-



#Sample


Probe Name
FC
P value
AUC
ProbeType
Detected















14qll-14
−3.87
 3.6592E−27
1.000
CDBox
56


14qll-14_x
−3.21
 3.815E−24
1.000
CDBox
56


U74_x
1.46
3.89068E−23
1.000
CDBox
135


miR-509-3p
5.78
1.67661E−22
0.997
miRNA
116


miR-768-5p
−1.46
5.23441E−22
1.000
miRNA
135


ENSG00000199411_s
1.36
1.56796E−21
1.000
snoRNA
135


Z17B
−1.94
2.89347E−20
0.997
CDBox
117


miR-149
−3.46
1.55425E−19
1.000
miRNA
100


miR-125a-5p
−3.44
1.86526E−19
1.000
miRNA
128


U43
−1.19
3.32514E−19
1.000
CDBox
135


miR-1308
1.55
 8.1184E−19
0.964
miRNA
135


U59A
1.13
9.99768E−18
0.993
CDBox
135


miR-146a
4.44
3.16232E−17
0.975
miRNA
129


miR-513a-5p
3.77
1.05679E−16
0.972
miRNA
105


ACA20
−1.74
1.56297E−16
0.993
HAcaBox
131


U43_x
−1.05
1.04459E−15
1.000
CDBox
135


U44_x
−1.37
5.54123E−15
0.994
CDBox
135


miR-127-3p
−3.56
7.53549E−15
0.988
miRNA
89


miR-193b
−2.57
7.81928E−15
1.000
miRNA
130


U83
−1.25
9.30493E−15
0.993
CDBox
135


miR-768-3p
0.98
2.39925E−14
0.972
miRNA
137


U33
−1.02
2.44194E−14
0.997
CDBox
137


miR-574-3p
−3.05
 3.0433E−14
0.974
miRNA
122


ACA16
−1.86
3.35128E−14
0.968
HAcaBox
47


14ql-4
−2.43
3.96317E−14
0.979
CDBox
80


hsa-let-7c
−1.01
4.99111E−14
0.986
miRNA
135


miR-342-3p
−2.06
1.67074E−13
0.996
miRNA
136


hsa-let-7b
−1.01
2.25904E−13
0.964
miRNA
136


miR-423-3p
−2.52
2.97967E−13
1.000
miRNA
107


HBII-239
−1.41
 3.2162E−13
0.975
CDBox
135


U38B_x
1.52
3.80613E−13
0.968
CDBox
133


miR-320b
−0.63
6.31909E−13
0.971
miRNA
135


miR-923
0.93
6.48332E−13
0.990
miRNA
135


U54
−1.06
1.17197E−12
0.975
CDBox
135


U38B
1.80
1.18885E−12
0.976
CDBox
134


U44
−1.07
1.28887E−12
0.981
CDBox
134


miR-139-5p
−2.70
2.74261E−12
0.940
miRNA
80


miR-1826
−0.71
2.83317E−12
0.958
miRNA
137


ENSG00000199435
1.38
2.88249E−12
0.954
snoRNA
63


miR-26a
0.89
2.88328E−12
0.960
miRNA
136


U58B_x
−1.47
3.60008E−12
0.940
CDBox
132


HBII-180A_x
−1.43
4.25614E−12
0.992
CDBox
123


14qll-12_x
−2.09
4.30741E−12
0.949
CDBox
85


miR-509-3-5p
3.46
5.53398E−12
0.953
miRNA
107


U55_x
−1.35
 6.7836E−12
0.996
CDBox
135


miR-149-star
0.93
 9.6022E−12
0.940
miRNA
135


HBII-276
−1.41
1.19464E−11
0.964
CDBox
132


miR-23b
−0.75
1.72105E−11
0.968
miRNA
137


miR-15b
−2.42
 1.7823E−11
0.968
miRNA
125


miR-921
1.73
1.93285E−11
0.954
miRNA
49


miR-191
−0.85
 2.0881E−11
0.972
miRNA
135


U103_s
−1.60
2.57483E−11
0.943
CDBox
111


miR-486-5p
−3.27
2.90873E−11
0.942
miRNA
65


U27
−1.61
3.28995E−11
0.960
CDBox
134


miR-1202
2.18
4.44562E−11
0.945
miRNA
57


U49A
1.25
4.51441E−11
0.970
CDBox
135


ACA24_x
−1.50
5.61856E−11
0.922
HAcaBox
98


HBII-382_s
1.30
5.89071E−11
0.953
scaRna
126


U52
−0.92
1.05668E−10
0.976
CDBox
135


miR-99b
−1.84
1.06853E−10
0.988
miRNA
129


U38A
0.84
1.30902E−10
0.936
CDBox
134


U46
−1.44
1.33613E−10
0.965
CDBox
133


U57
−1.10
1.66897E−10
0.967
CDBox
135


U3-2_s
0.70
1.79537E−10
0.925
CDBox
135


14qll-26_x
−1.60
1.92804E−10
0.922
CDBox
41


ENSG00000207098_x
1.21
2.17044E−10
0.956
snoRNA
86


miR-320c
−0.84
2.33286E−10
0.994
miRNA
135


miR-199a-5p
−2.53
2.85196E−10
0.994
miRNA
129


HBII-180C
−1.55
3.80017E−10
0.924
CDBox
115


U63
0.94
4.46838E−10
0.945
CDBox
135


HBII-436
−1.22
4.98357E−10
0.929
CDBox
102


U81_x
−1.25
6.27975E−10
0.928
CDBox
120


HBII-419
1.10
6.92884E−10
0.932
CDBox
133


U17b
−1.32
8.76094E−10
0.996
HAcaBox
135


miR-506
2.11
1.46252E−09
0.931
miRNA
76


miR-1268
1.02
1.54826E−09
0.909
miRNA
133


miR-671-5p
1.38
1.60795E−09
0.914
miRNA
69


miR-339-5p
−2.03
1.92249E−09
0.907
miRNA
75


ACA48_x
−1.25
1.98889E−09
0.945
HAcaBox
130


14qll-21_x
−1.20
2.04198E−09
0.907
CDBox
51


ENSG00000202093_x
−1.37
2.13676E−09
0.902
snoRNA
125


U73a
−0.76
3.11274E−09
0.924
CDBox
135


miR-1228-star
1.09
3.32612E−09
0.925
miRNA
134


miR-193b-star
−1.99
3.54373E−09
0.925
miRNA
77


miR-145
−2.87
3.67076E−09
0.986
miRNA
135


miR-1207-5p
0.91
3.82606E−09
0.936
miRNA
133


miR-23a
−0.62
4.38608E−09
0.950
miRNA
137


14qll-3
−1.37
5.04275E−09
0.938
CDBox
95


U56
0.82
5.34057E−09
0.910
CDBox
135


miR-320a
−0.50
5.87182E−09
0.917
miRNA
135


HBII-289
−0.82
6.53343E−09
0.975
CDBox
135


mgU6-53B
1.17
6.66322E−09
0.946
CDBox
99


miR-222
−1.42
6.66965E−09
1.000
miRNA
135


miR-135a-star
1.78
6.83122E−09
0.914
miRNA
44


HBII-142
0.50
6.91032E−09
0.911
CDBox
135


14qll-12
−1.90
 7.3584E−09
0.896
CDBox
58


ACA23
−1.07
9.00722E−09
0.922
HAcaBox
113


miR-1224-5p
2.00
9.14764E−09
0.902
miRNA
78


U17b_x
−0.78
9.16941E−09
0.917
HAcaBox
135


U41
−0.89
 1.0052E−08
0.928
CDBox
135


U55
−1.40
1.07645E−08
0.988
CDBox
135


U101
1.01
1.22325E−08
0.911
CDBox
134


miR-193a-5p
−2.09
1.43928E−08
0.954
miRNA
121


miR-720
0.87
1.45059E−08
0.918
miRNA
137


miR-205
−1.04
1.53388E−08
0.956
miRNA
135


mgh28S-2411
−0.86
1.81685E−08
0.965
CDBox
135


miR-1300
1.84
1.83075E−08
0.911
miRNA
32


miR-22
−2.31
1.87992E−08
0.943
miRNA
120


ACA40_x
−1.14
1.89306E−08
0.935
HAcaBox
134


miR-92a
−1.48
1.96388E−08
1.000
miRNA
136


14qll-28_x
−1.20
2.08991E−08
0.911
CDBox
51


U23
−0.93
2.35134E−08
0.913
HAcaBox
127


snR38C
0.62
2.69211E−08
0.902
CDBox
134


miR-939
1.33
 2.8172E−08
0.947
miRNA
57


miR-214
−0.88
2.85157E−08
0.921
miRNA
135


U94
1.03
3.14773E−08
0.898
CDBox
109


miR-510
2.20
3.61106E−08
0.899
miRNA
82


miR-181b
−1.85
5.28195E−08
0.961
miRNA
130


U15B
−1.06
5.57521E−08
0.898
CDBox
129


gi555853_copy5
−0.51
7.13438E−08
0.910
5.8s rRNA
137


miR-432
−1.83
7.49361E−08
0.902
miRNA
51


HBII-142_x
0.47
8.53374E−08
0.886
CDBox
135


snR38B
1.11
8.92959E−08
0.899
CDBox
126


ACA36_x
1.12
9.60154E−08
0.885
HAcaBox
88


miR-320d
−1.66
1.03365E−07
0.997
miRNA
134


hsa-let-7i
1.72
1.07923E−07
0.947
miRNA
135


miR-125b-2-star
−1.66
1.09644E−07
0.881
miRNA
58


miR-572
1.35
1.13617E−07
0.906
miRNA
116


U102
0.87
 1.2385E−07
0.874
CDBox
124


gi555853_copy8
−0.51
1.25699E−07
0.903
5.8s rRNA
137


miR-371-5p
1.93
1.26302E−07
0.886
miRNA
64


miR-125b
−0.97
1.35947E−07
0.988
miRNA
135


ENSG00000201619
1.40
 1.4629E−07
0.892
snoRNA
62


U78_x
1.11
1.62568E−07
0.921
CDBox
134


ENSG00000212397
0.90
1.65415E−07
0.898
snoRNA
134


ENSG00000212182
1.09
1.80486E−07
0.884
snoRNA
59


hsa-let-7f
1.99
 2.0615E−07
0.909
miRNA
130


HBII-55
−0.82
2.17315E−07
0.940
CDBox
135


gi555853_copy2
−0.49
2.37022E−07
0.893
5.8s rRNA
137


ACA15_s
−1.09
2.48572E−07
0.875
HAcaBox
116


U28_x
−0.90
 2.5645E−07
0.896
CDBox
133


gi555853_copy0
−0.50
2.65495E−07
0.889
5.8s rRNA
137


gi555853_copy4
−0.48
2.85254E−07
0.898
5.8s rRNA
137


ENSG00000201816
0.83
3.19058E−07
0.878
snoRNA
49


gi555853_copy1
−0.48
3.26181E−07
0.886
5.8s rRNA
137


U60
−0.97
3.26261E−07
0.871
CDBox
103


gi555853_copy6
−0.47
3.40126E−07
0.900
5.8s rRNA
137


miR-132
−1.77
3.42625E−07
0.870
miRNA
96


U68_x
−1.05
3.51611E−07
0.910
HAcaBox
133


gi555853_copy7
−0.48
4.04469E−07
0.892
5.8s rRNA
137


ENSG00000201660
1.12
4.27296E−07
0.896
snoRNA
132


miR-150-star
1.63
4.51511E−07
0.874
miRNA
48


miR-665
1.55
5.14634E−07
0.871
miRNA
47


miR-150
−1.60
 6.5278E−07
0.884
miRNA
117


miR-210
−1.24
6.58793E−07
0.911
miRNA
130


miR-664-star
1.38
6.79224E−07
0.864
miRNA
96


U49A_x
1.03
7.09035E−07
0.911
CDBox
135


miR-30c
−1.75
7.10279E−07
0.911
miRNA
125


ENSG00000212458
0.92
7.71477E−07
0.871
snoRNA
95


ENSG00000200879
−1.08
7.74449E−07
0.868
snoRNA
120


hsa-let-7d
0.39
8.11829E−07
0.886
miRNA
135


14qll-1_x
−1.24
9.36723E−07
0.899
CDBox
131


14qll-26
−1.14
1.46915E−06
0.827
CDBox
32


hsa-let-7a
0.51
1.65358E−06
0.841
miRNA
136


U28
−0.75
1.71566E−06
0.874
CDBox
135


U68
−0.88
1.83186E−06
0.892
HAcaBox
130


HBII-180C_x
−0.95
2.06263E−06
0.882
CDBox
131


gi555853_copy3
−0.45
2.14632E−06
0.863
5.8s rRNA
137


miR-2001b-star
−1.70
2.20935E−06
0.861
miRNA
74


miR-1234
1.03
 2.2168E−06
0.861
miRNA
68


U95
−0.36
2.37891E−06
0.852
CDBox
135


U46_x
−0.98
2.42909E−06
0.873
CDBox
131


spike_in-control-21
0.74
2.57369E−06
0.827
Oligonucleotide
42






spike-in






controls


miR-1248
1.03
3.51695E−06
0.874
miRNA
39


U36A
−0.61
3.61256E−06
0.882
CDBox
132


U13
0.50
4.36581E−06
0.834
CDBox
135


miR-92a-2-star
1.02
4.99387E−06
0.859
miRNA
46


miR-663
0.77
5.24563E−06
0.859
miRNA
134


miR-409-3p
−1.42
5.63777E−06
0.837
miRNA
92


U34
−0.68
5.88695E−06
0.852
CDBox
137


U25
−0.71
6.25404E−06
0.896
CDBox
135


U32A_x
−0.75
6.51615E−06
0.882
CDBox
136


U96a_x
0.68
6.70342E−06
0.849
CDBox
134


gi555853_copy9
−0.42
 7.0704E−06
0.863
5.8s rRNA
137


ENSG00000199411_x
0.77
7.82781E−06
0.846
snoRNA
132


spike_in-control-7
1.00
7.92241E−06
0.848
Oligonucleotide
54






spike-in






controls


miR-198
1.24
8.11341E−06
0.846
miRNA
31


14ql-9_x
−0.81
8.29164E−06
0.820
CDBox
34


miR-425
−1.72
8.48515E−06
0.846
miRNA
114


HBII-82
0.77
8.59528E−06
0.839
CDBox
90


miR-1180
−1.22
9.75327E−06
0.828
miRNA
37


ENSG00000206637_x
0.74
1.15035E−05
0.850
snoRNA
59


miR-502-3p
−1.41
1.18784E−05
0.831
miRNA
110


4qll-22_x
−0.97
1.49319E−05
0.792
CDBox
50


ACA18_x
−0.72
1.77602E−05
0.837
HAcaBox
135


miR-1274b
1.66
1.82625E−05
0.813
miRNA
112


HBII-99
−0.94
1.84996E−05
0.837
CDBox
122


U35B
0.78
1.93006E−05
0.837
CDBox
128


ACA20_x
−0.84
2.02452E−05
0.882
HAcaBox
134


miR-143
−2.21
 2.0661E−05
0.864
miRNA
126


ENSG00000200652
0.81
2.09666E−05
0.824
snoRNA
40


HBII-202
−0.59
2.32067E−05
0.886
CDBox
135


miR-708
−1.81
2.34758E−05
0.816
miRNA
102


miR-182
−1.64
2.56273E−05
0.791
miRNA
113


miR-345
−1.28
 3.1705E−05
0.816
miRNA
75


ACA62
0.82
3.35475E−05
0.817
HAcaBox
88


miR-1273
1.12
3.38999E−05
0.827
miRNA
47


ACA24_s
−1.10
3.44078E−05
0.823
HAcaBox
128


miR-1272
0.97
3.51756E−05
0.820
miRNA
67


14ql-4_x
−1.11
3.64993E−05
0.796
CDBox
51


14qll-9_x
−0.89
3.72476E−05
0.820
CDBox
36


U106
−0.62
3.79813E−05
0.817
CDBox
112


miR-1285
1.22
4.16925E−05
0.796
miRNA
54


ENSG00000200492
0.77
4.61258E−05
0.838
snoRNA
60


ENSG00000202327
0.77
4.61736E−05
0.812
snoRNA
54


miR-126
1.24
4.86215E−05
0.845
miRNA
133


U49B_x
0.62
4.92521E−05
0.795
CDBox
122


ENSG00000207016_x
0.74
 5.4547E−05
0.794
snoRNA
48


HBII-52-25_x
0.68
5.50793E−05
0.827
CDBox
57


U49B_s
0.90
5.94839E−05
0.848
CDBox
129


U53
−0.97
6.07982E−05
0.809
CDBox
126


miR-1246
1.19
7.10577E−05
0.799
miRNA
131


ENSG00000212615_x
−0.67
7.76933E−05
0.798
snoRNA
102


miR-575
1.09
8.31223E−05
0.794
miRNA
41


snR38A
0.69
8.40239E−05
0.807
CDBox
130


14ql-7
−0.78
8.70579E−05
0.825
CDBox
51


miR-29b-2-star
0.86
 8.9139E−05
0.802
miRNA
126


U80
−0.60
9.75089E−05
0.825
CDBox
132


miR-1225-5p
1.04
9.99249E−05
0.830
miRNA
90


miR-92b-star
0.94
0.000102055
0.809
miRNA
111


ACA53
−0.61
0.000103826
0.814
HAcaBox
105


ACA55
0.79
0.000105025
0.805
HAcaBox
85


U51
0.58
0.000112048
0.802
CDBox
134


U108_x
0.55
0.000112181
0.798
HAcaBox
87


U48
−1.07
0.000118819
0.875
CDBox
129


miR-221
−1.06
0.00013613
0.911
miRNA
135


HBII-13
0.74
0.000139215
0.798
CDBox
52


miR-19b
1.53
0.0001409
0.795
miRNA
124


ACA3-2
−0.76
0.000143105
0.819
HAcaBox
135


HBII-85-23_x
−0.73
0.000143482
0.787
CDBox
58


14ql-8
−0.75
0.000167587
0.802
CDBox
61


miR-138-1-star
0.88
0.000190948
0.798
miRNA
91


spike_in-control-28
0.63
0.000200982
0.802
Oligonucleotide
76






spike-in






controls


miR-500-star
−1.36
0.000204792
0.803
miRNA
97


ENSG00000212423_x
0.75
0.000232739
0.789
snoRNA
115


ENSG00000201348
0.50
0.000243216
0.778
snoRNA
94


miR-1275
1.09
0.000244874
0.821
miRNA
127


miR-30a-star
−1.14
0.000247652
0.758
miRNA
64


miR-93-star
−1.16
0.000248272
0.777
miRNA
48


ACA25
−0.64
0.000248534
0.802
HAcaBox
73


U50
−0.73
0.000255129
0.853
CDBox
134


14qll-1
−1.00
0.000255606
0.807
CDBox
128


U14B_x
−0.79
0.000258746
0.788
CDBox
53


miR-638
0.35
0.000261809
0.823
miRNA
135


U51_x
0.57
0.000299812
0.820
CDBox
130


miR-99a
−0.87
0.000321188
0.832
miRNA
135


miR-491-5p
1.04
0.000341416
0.756
miRNA
92


miR-508-3p
1.23
0.000350578
0.778
miRNA
74


miR-508-5p
1.02
0.000377448
0.783
miRNA
92


miR-641
0.88
0.000391728
0.774
miRNA
39


miR-885-5p
−1.23
0.00039874
0.801
miRNA
100


miR-497
−1.48
0.00041742
0.780
miRNA
104


U31_x
−0.65
0.000420569
0.795
CDBox
135


miR-498
0.85
0.000424439
0.787
miRNA
56


miR-20a
1.21
0.000429079
0.807
miRNA
134


miR-509-5p
1.35
0.000476275
0.776
miRNA
81


miR-331-3p
−1.11
0.000481237
0.777
miRNA
64


HBII-295
0.53
0.000526137
0.785
CDBox
128


ACA27_x
−0.45
0.000528795
0.778
HAcaBox
130


miR-155
−1.29
0.000530869
0.751
miRNA
123


miR-652
−1.30
0.000593692
0.774
miRNA
119


ENSG00000208308_x
−0.58
0.000595144
0.758
snoRNA
129


miR-1281
0.98
0.000606821
0.766
miRNA
137


E3_x
−0.53
0.000619964
0.819
HAcaBox
135


miR-27a-star
−0.88
0.000656179
0.773
miRNA
40


miR-92b
−0.90
0.0006705
0.773
miRNA
91


miR-570
0.93
0.000670846
0.776
miRNA
88


miR-181a-2-star
−1.19
0.000676824
0.753
miRNA
99


miR-1185
0.76
0.000680122
0.752
miRNA
42


ACA33
0.57
0.000718022
0.780
HAcaBox
133


ENSG00000212401
0.57
0.000773614
0.799
snoRNA
41


U20
−0.51
0.000816772
0.758
CDBox
117


U19
0.84
0.00082895
0.759
HAcaBox
118


miR-203
0.69
0.000839225
0.878
miRNA
132


ENSG00000212508
0.83
0.000873066
0.801
snoRNA
122


ACA54
−0.54
0.000880465
0.817
HAcaBox
133


ACA9_x
−0.68
0.001025389
0.752
HAcaBox
77


14ql-1
0.61
0.001136212
0.729
CDBox
70


miR-940
0.87
0.001164857
0.737
miRNA
53


U14B
−0.69
0.001178164
0.738
CDBox
51


mgU6-77
−0.62
0.001238112
0.760
CDBox
129


spike_in-control-30
0.54
0.001290622
0.753
Oligonucleotide
53






spike-in






controls


miR-629-star
−0.92
0.00138462
0.756
miRNA
46


ACA49
0.56
0.001394857
0.791
HAcaBox
127


miR-483-5p
0.96
0.001461794
0.738
miRNA
48


ENSG00000212214_x
0.51
0.001505107
0.748
snoRNA
88


miR-574-5p
−0.99
0.00156768
0.766
miRNA
103


miR-28-3p
−1.29
0.001658961
0.747
miRNA
94


U36C
0.34
0.001694833
0.849
CDBox
135


miR-1288
0.73
0.001697813
0.738
miRNA
43


ACA37_x
−0.59
0.001721183
0.742
HAcaBox
84


miR-1280
−0.72
0.001869035
0.777
miRNA
134


miR-487b
−0.96
0.001988497
0.731
miRNA
58


miR-532-3p
−0.80
0.00199381
0.766
miRNA
95


miR-346
−0.91
0.0020172
0.749
miRNA
55


miR-27b-star
−1.04
0.00209985
0.742
miRNA
51


ACA32
0.34
0.002161552
0.785
HAcaBox
135


ENSG00000212206_x
0.58
0.002337296
0.740
snoRNA
74


ACA51_x
0.43
0.002449336
0.742
HAcaBox
135


ENSG00000201847_x
−0.53
0.002461896
0.729
snoRNA
48


miR-513c
1.13
0.002510473
0.749
miRNA
73


miR-455-3p
−0.62
0.00258213
0.785
miRNA
136


miR-188-5p
−0.90
0.002825937
0.730
miRNA
51


ENSG00000207027
0.48
0.002903474
0.704
snoRNA
36


ENSG00000202498_x
0.56
0.002908782
0.735
snoRNA
137


HBII-85-26
0.66
0.003001767
0.741
CDBox
136


miR-337-3p
0.80
0.003015225
0.740
miRNA
94


U35A
−0.47
0.003204612
0.723
CDBox
135


miR-106b-star
−1.10
0.003210131
0.717
miRNA
72


ENSG00000202216
0.55
0.003292357
0.758
snoRNA
53


HBII-316
0.63
0.003333247
0.705
CDBox
130


miR-31
−1.63
0.003410838
0.720
miRNA
103


U22
0.43
0.003510102
0.699
CDBox
135


miR-34a-star
0.77
0.003765001
0.715
miRNA
39


HBII-85-6_x
0.62
0.003870576
0.735
CDBox
137


ACA15_x
−0.50
0.003952375
0.734
HAcaBox
124


ENSG00000200706_x
0.48
0.00407636
0.762
snoRNA
43


ENSG00000212627
0.41
0.00409948
0.726
snoRNA
69


14qll-7
0.59
0.004220389
0.719
CDBox
77


ENSG00000212432_s
0.52
0.004352612
0.745
snoRNA
75


miR-214-star
−0.82
0.004514439
0.708
miRNA
47


miR-106a
0.58
0.004682448
0.724
miRNA
135


ACA42
−0.54
0.004759271
0.720
HAcaBox
117


mgU6-53B_x
0.46
0.0048197
0.720
CDBox
121


ACA19
0.38
0.005153495
0.733
HAcaBox
118


ACA50
−0.53
0.005300761
0.738
HAcaBox
88


miR-138
1.07
0.005618175
0.716
miRNA
82


U47
−0.47
0.005755719
0.729
CDBox
74


ENSG00000212551
0.48
0.005885716
0.740
snoRNA
73


miR-769-5p
−0.93
0.005951319
0.687
miRNA
56


miR-1228
0.82
0.006114287
0.724
miRNA
114


miR-30b
0.87
0.00634299
0.748
miRNA
128


ENSG00000212315
0.54
0.006470787
0.752
snoRNA
117


ENSG00000212538
0.48
0.006558923
0.735
snoRNA
35


ACA2b
0.39
0.007073038
0.756
HAcaBox
47


ENSG00000212523_x
0.59
0.007115341
0.716
snoRNA
135


miR-196a
1.04
0.007138032
0.705
miRNA
77


HBII-429
0.22
0.007262326
0.720
CDBox
137


miR-16
0.46
0.007360456
0.749
miRNA
135


mgh28S-2409
0.27
0.007642791
0.741
CDBox
135


ENSG00000200307_x
0.54
0.007656242
0.695
snoRNA
50


U76
−0.27
0.008469646
0.734
CDBox
135


14qll-17
−0.54
0.008809376
0.698
CDBox
71


miR-324-3p
−0.67
0.009111464
0.726
miRNA
100


ACA48
−0.49
0.009193835
0.798
HAcaBox
121


miR-200c
−0.31
0.009670586
0.625
miRNA
134


miR-1260
−0.78
0.010502121
0.726
miRNA
120


miR-130a
−0.94
0.010557093
0.687
miRNA
122


U17a
−0.52
0.010593144
0.702
HAcaBox
105


ACA52
−0.45
0.010843862
0.708
HAcaBox
121


miR-197
−0.79
0.011117393
0.724
miRNA
103


U66
−0.54
0.011246742
0.705
HAcaBox
119


ENSG00000202252
−0.30
0.011273976
0.699
snoRNA
135


ACA61
0.22
0.011490951
0.730
HAcaBox
135


miR-151-5p
0.56
0.01153084
0.830
miRNA
135


ACA16_x
−0.47
0.011881173
0.727
HAcaBox
98


U18C_x
−0.45
0.012073238
0.691
CDBox
74


U15A
0.45
0.012634875
0.706
CDBox
128


miR-1274a
0.71
0.012698242
0.684
miRNA
48


ACA
−0.62
0.01271037
0.691
HAcaBox
82


U91_s
−0.54
0.012949986
0.733
scaRna
132


ENSG00000207002
−0.52
0.013212929
0.702
snoRNA
35


miR-129-3p
−0.54
0.013303701
0.705
miRNA
59


spike_in-cortrol-17
0.48
0.013556785
0.705
Oligonucleotide
33






spike-in






controls


spike_in-control-2
0.19
0.014699249
0.684
Oligonucleotide
137






spike-in






controls


ENSG00000212206
0.49
0.014866744
0.670
snoRNA
46


miR-107
−0.39
0.015131674
0.669
miRNA
135


ACA63
0.42
0.01519582
0.695
HAcaBox
66


ENSG00000212553_x
0.45
0.015776333
0.684
snoRNA
60


U50B
−0.37
0.016500241
0.704
CDBox
135


ENSG00000207100_x
0.36
0.016825837
0.666
snoRNA
72


U78_s
−0.55
0.016840855
0.702
CDBox
135


miR-551b-star
−0.65
0.016907402
0.686
miRNA
61


ACA47
0.45
0.017340107
0.666
scaRna
77


U92
0.45
0.017435918
0.760
scaRna
129


miR-342-5p
−0.78
0.01779742
0.695
miRNA
98


miR-128
−0.56
0.017820912
0.699
miRNA
44


U17a_x
−0.46
0.018370462
0.720
HAcaBox
115


ENSG00000206909_x
0.44
0.018373221
0.706
snoRNA
47


U30
−0.35
0.018607695
0.729
CDBox
135


miR-98
−0.57
0.019510485
0.677
miRNA
47


miR-21-star
0.62
0.019769732
0.686
miRNA
64


miR-135b-star
0.57
0.0198019
0.704
miRNA
38


ACA2a
−0.42
0.019986353
0.677
HAcaBox
55


U88
0.44
0.020351208
0.672
scaRna
69


HBII-85-29_x
0.39
0.020664805
0.666
CDBox
85


14qll-19
0.47
0.02089152
0.695
CDBox
43


U83B
0.19
0.020956657
0.677
CDBox
135


miR-134
−0.86
0.020986754
0.681
miRNA
61


U83A
−0.40
0.021328386
0.698
CDBox
127


ENSG00000202370
0.53
0.02172281
0.651
snoRNA
88


U75_x
−0.35
0.021862862
0.697
HAcaBox
103


ACA38
−0.38
0.023364662
0.688
HAcaBox
42


ENSG00000200394_x
0.42
0.023498919
0.661
snoRNA
115


miR-1324
0.50
0.023518344
0.684
miRNA
57


miR-106b
0.85
0.023747964
0.670
miRNA
127


miR-589-star
0.56
0.023792146
0.675
miRNA
43


ACA6
−0.36
0.024014637
0.673
HAcaBox
126


ACA28
0.36
0.02403059
0.687
HAcaBox
120


ACA57
0.36
0.024679323
0.748
scaRna
135


HBII-85-4_x
0.36
0.024717662
0.711
CDBox
125


ENSG00000200897
−0.41
0.024775335
0.662
snoRNA
38


U16
0.42
0.024969966
0.712
CDBox
131


ENSG00000200418
−0.41
0.025790882
0.668
snoRNA
31


U105
−0.31
0.026442326
0.673
CDBox
126


spike_in-control-34
0.44
0.026481739
0.665
Oligonucleotide
58






spike-in






controls


spike_in-control-23
0.24
0.027477358
0.645
Oligonucleotide
137






spike-in






controls


miR-196a-star
0.61
0.028243677
0.677
miRNA
37


HBII-296A
0.33
0.02845867
0.659
CDBox
78


U82
0.43
0.028925335
0.681
CDBox
131


ENSG00000212558_x
−0.35
0.029963606
0.662
snoRNA
31


miR-21
0.67
0.030060673
0.668
miRNA
79


ACA31
0.37
0.030929941
0.702
HAcaBox
123


miR-1257
0.55
0.031016296
0.676
miRNA
31


ACA14b_x
−0.37
0.031018594
0.688
HAcaBox
105


ACA33_x
0.38
0.033957171
0.687
HAcaBox
129


miR-199b-3p
0.64
0.034530062
0.737
miRNA
134


miR-100
−0.61
0.034553062
0.681
miRNA
133


U64
−0.38
0.034971581
0.633
HAcaBox
101


miR-10a
−0.67
0.037624151
0.666
miRNA
69


miR-30e
0.56
0.037770496
0.683
miRNA
57


miR-199a-3p
0.62
0.038490813
0.704
miRNA
134


HBII-85-11
−0.37
0.038507691
0.687
CDBox
31


miR-505-star
−0.62
0.039242017
0.647
miRNA
58


hsa-let-7f-1-star
0.44
0.042251211
0.658
miRNA
64


spike_in-control-29
−0.19
0.043064083
0.684
Oligonucleotide
137






spike-in






controls


miR-20b
0.64
0.043397678
0.655
miRNA
99


ACA5
−0.40
0.044405124
0.693
HAcaBox
85


miR-628-3p
0.57
0.044447799
0.652
miRNA
38


HBII-85-8_x
0.40
0.045108021
0.681
CDBox
135


HBII-85-27_x
0.29
0.045678744
0.651
CDBox
56


miR-99b-star
0.49
0.049531821
0.651
miRNA
74
















TABLE 8







50 miRNAs most significantly expressed between nevi and normal skin













Log2-






Probe Name
FC
P value
AUC
ProbeType
#Sample Detected















-14
−3.87
 3.6592E−27
1.000
CDBox
56


14qll-14_x
−3.21
 3.815E−24
1.000
CDBox
56


U74_x
1.46
3.89068E−23
1.000
CDBox
135


miR-509-3p
5.78
1.67661E−22
0.997
miRNA
116


miR-768-5p
−1.46
5.23441E−22
1.000
miRNA
135


ENSG00000199411_s
1.36
1.56796E−21
1.000
snoRNA
135


Z17B
−1.94
2.89347E−20
0.997
CDBox
117


miR-149
−3.46
1.55425E−19
1.000
miRNA
100


miR-125a-5p
−3.44
1.86526E−19
1.000
miRNA
128


U43
−1.19
3.32514E−19
1.000
CDBox
135


miR-1308
1.55
 8.1184E−19
0.964
miRNA
135


U59A
1.13
9.99768E−18
0.993
CDBox
135


miR-146a
4.44
3.16232E−17
0.975
miRNA
129


miR-513a-5p
3.77
1.05679E−16
0.972
miRNA
105


ACA20
−1.74
1.56297E−16
0.993
HAcaBox
131


U43_x
−1.05
1.04459E−15
1.000
CDBox
135


U44_x
−1.37
5.54123E−15
0.994
CDBox
135


miR-127-3p
−3.56
7.53549E−15
0.988
miRNA
89


miR-193b
−2.57
7.81928E−15
1.000
miRNA
130


U83
−1.25
9.30493E−15
0.993
CDBox
135


miR-768-3p
0.98
2.39925E−14
0.972
miRNA
137


U33
−1.02
2.44194E−14
0.997
CDBox
137


miR-574-3p
−3.05
 3.0433E−14
0.974
miRNA
122


ACA16
−1.86
3.35128E−14
0.968
HAcaBox
47


14ql-4
−2.43
3.96317E−14
0.979
CDBox
80


hsa-let-7c
−1.01
4.99111E−14
0.986
miRNA
135


miR-342-3p
−2.06
1.67074E−13
0.996
miRNA
136


hsa-let-7b
−1.01
2.25904E−13
0.964
miRNA
136


miR-423-3p
−2.52
2.97967E−13
1.000
miRNA
107


HBII-239
−1.41
 3.2162E−13
0.975
CDBox
135


U38B_x
1.52
3.80613E−13
0.968
CDBox
133


miR-320b
−0.63
6.31909E−13
0.971
miRNA
135


miR-923
0.93
6.48332E−13
0.990
miRNA
135


U54
−1.06
1.17197E−12
0.975
CDBox
135


U38B
1.80
1.18885E−12
0.976
CDBox
134


U44
−1.07
1.28887E−12
0.981
CDBox
134


miR-139-5p
−2.70
2.74261E−12
0.940
miRNA
80


miR-1826
−0.71
2.83317E−12
0.958
miRNA
137


ENSG00000199435
1.38
2.88249E−12
0.954
snoRNA
63


miR-26a
0.89
2.88328E−12
0.960
miRNA
136


U58B_x
−1.47
3.60008E−12
0.940
CDBox
132


HBII-180A_x
−1.43
4.25614E−12
0.992
CDBox
123


14qll-12_x
−2.09
4.30741E−12
0.949
CDBox
85


miR-509-3-5p
3.46
5.53398E−12
0.953
miRNA
107


U55_x
−1.35
 6.7836E−12
0.996
CDBox
135


miR-149-star
0.93
 9.6022E−12
0.940
miRNA
135


HBII-276
−1.41
1.19464E−11
0.964
CDBox
132


miR-23b
−0.75
1.72105E−11
0.968
miRNA
137


miR-15b
−2.42
 1.7823E−11
0.968
miRNA
125


miR-921
1.73
1.93285E−11
0.954
miRNA
49









Example 4
The data from Example 3 was further analyzed to identify the combination of markers best able to distinguish (A) melanoma from normal skin and (B) melanoma from nevi. Each miRNA (“analyte”) was examined by svm, random forest, boosting, lasso, baggin, cart, matt, logistic regression, and ctree analyses. The use of multiple statistical method demonstrates that the best combination of markers identified by one statistical method is validated by every other method, making the choice of marker combinations less subject to the specific weaknesses of any one statistical algorithm.
FIGS. 1-10 show, respectively, the best 2-10 miRNA combinations for differentiating melanoma (MM) from normal skin (“NS”) and the relevant error rates, as determined by different statistical algorithms.
FIGS. 11-23 show, respectively, the best 2-14 miRNA combinations for differentiating melanoma (MM) from nevi (NV), and the relevant error rates, as determined by different statistical algorithms.
FIG. 24 shows the log 2 signal for three miRNA: miR-150 (R-150), miR-149-star (miR-149-star), and hsa-miR-1308 (miR-1308) across 137 samples. Samples 1-19 are normal skin (NS), 20-57 nevi (NV), 58-115 melanoma (MM) and 116-137 metastatic melanoma (Mets). As can be appreciated, miR-150, miR-149-star and miR-1308 distinguish normal skin and nevi from melanoma and metastatic melanoma.
Example 5
In a follow up study, a total of 78 melanoma and 98 nevi (Intradermal-20, Compound-18, Junctional-20, Blue-20, Spitz-20) were studied on microarray. The candidate microRNAs were chosen based on AUC, p value, log 2 fold change, and 9 analysis programs, as before. A example of error rate and AUC from 9 programs is set forth in FIG. 25. The candidate microRNAs identified from 2 programs (random forest and boosting) are listed in Table 9, below.







TABLE 9







MicroRNA candidates from 78 melanoma and 98 nevi











microrna
adj.P.Val (FDR)
AUC
Log2FC
Order in program














miR-1268
1.45E−19
0.89
−1.80
5


miR-1228-star
2.87E−19
0.91
−1.77
2


miR-92b-star
4.62E−18
0.88
−1.73
3


miR-155
5.56E−18
0.86
2.85
4


miR-345
1.19E−16
0.85
1.83
7


miR-425
5.11E−16
0.85
1.80


miR-132
5.24E−16
0.85
1.75
10


miR-1207-5p
1.18E−15
0.86
−1.40


miR-1301
2.17E−15
0.83
1.70


miR-663
2.26E−15
0.85
−1.43


miR-339-5p
2.26E−15
0.85
1.60
9


miR-149-star
5.28E−15
0.85
−1.32


miR-150
5.25E−14
0.82
2.10
6


miR-18a
5.80E−13
0.8
0.84


miR-103
1.52E−11
0.86
0.93
8


miR-191
7.32E−13
0.9
0.85
1


miR-296-3p
1.05E−09
0.79
−0.85
11


miR-31
1.39E−12
0.79
2.46


miR-107*
3.27E−10
0.85
0.86


miR-93*
1.05E−09
0.85
1.14


miR-1275*
4.59E−11
0.8
−1.16


miR-181B*
3.69E−12
0.83
1.69


miR-921*
7.21E−12
0.81
−0.89


miR-1225-5p
7.63E−10
0.78
−1.3


miR-1202
1.08E−09
0.76
−1.34


miR-342-3p
1.55E−10
0.78
1.29


Internal control


candidates


miR-27b
0.72
0.07
0.501


miR-195
0.95
0.01
0.506


miR-199b-3p
0.78
0.05
0.509


miR-199a-3p
0.86
0.03
0.511









REFERENCES


    1. Leidinger P, Keller A, Borries A, Reichrath J, Rass K, Jager S U, Lenhof H P, Meese E. High-throughput miRNA profiling of human melanoma blood samples. BMC Cancer. 2010 Jun. 7; 10:262.
    2. Demetra Philippidou, Martina Schmitt, Dirk Moser, Christiane Margue, Petr V Nazarov, Arnaud Muller, Laurent Vallar, Dorothee Nashan, Iris Behrmann and Stephanie Kreis Signatures of MicroRNAs and Selected MicroRNA Target Genes in Human Melanoma. Cancer Res 2010 May 70(10); 4163-4173
    3. Segura F, Ilana Belitskaya-Lévy I, Rose A, Zakrzewski J, Gaziel A. Melanoma MicroRNA Signature Predicts Post-Recurrence Survival Clinical Cancer Research 2020 March 16(5); 1577-1586


The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.
The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

## Claims

1. A method for distinguishing melanoma cells from dysplastic nevi cells in a skin lesion sample from a subject comprising:
(a) performing reverse transcriptase polymerase chain reaction (RT-PCR) to amplify nucleic acids from a skin sample isolated from a skin lesion, wherein the skin sample is previously identified as containing melanoma cells and/or dysplastic nevi cells;
(b) assaying the expression level of two or more miRNAs in the skin sample by detecting the amplified nucleic acids of (a), wherein the two or more miRNAs are selected from the group consisting of:
i. miR-150;
ii. miR-149-star;
iii. miR-1308;
iv. miR-191;
v. miR-1228-star;
vi. ENSG00000199411_s;
vii. miR-1268;
viii. miR-923;
ix. miR-23a;
x. miR-132;
xi. miR-1207.5p;
xii. miR-342.3p;
xiii. U38B; and
xiv. miR-155,

wherein assaying the level of expression comprises using a detectably labeled probe specific for each miRNA assayed;
(c) quantitatively comparing the levels of the two or more miRNAs in the skin lesion sample as assayed in (b) to the levels of each corresponding miRNA in a normal skin or nevus reference sample; and
(d) identifying the skin sample as containing:
(i) melanoma cells if the levels of each of the two or more miRNAs in the skin sample differs from the normal skin or nevus reference sample, wherein miR-150, miR-191, miR-23a, miR-132, miR-342.3p, and miR-155 are increased and miR-149-star, miR-1308, miR-1228-star, ENSG00000199411_s, miR-1268, miR-923, miR-1207.5p, and U38B are decreased relative to the normal skin or nevus reference sample; or
(ii) dysplastic nevi cells and/or no melanoma cells if the levels of each of the two or more miRNAs in the skin sample do not differ from the normal skin or nevus reference sample.

2. The method of claim 1, comprising measuring the level of:
i. miR-150; and
ii. miR-149-star.

3. The method of claim 1, comprising measuring the level of:
i. miR-150;
ii miR-149-star, and
iii miR-1308.

4. The method of claim 1, comprising measuring the level of:
i. miR-150;
ii. miR-149-star;
iii. miR-1308; and
iv. miR-191.

5. The method of claim comprising measuring the level of:
i. miR-150;
ii. miR-149-star;
iii, miR-1308;
iv. miR-191; and
V. miR-1228-star.

6. The method of claim 1, comprising measuring the level of:
i. miR-150;
ii. miR-149-star;
iii. miR-1308;
iv. miR-191;
v. miR-1228-star; and
vi. ENSG00000199411_s.

7. The method of claim 1, comprising measuring the level of:
i. miR-150;
ii. miR-149-star;
iii. miR-1308;
iv. miR-191;
v. miR-1228-star;
vi. ENSG00000199411_s; and
vii. miR-1268.

8. The method of claim 1, comprising measuring the level of:
i. miR-150;
ii. miR-149-star;
iii. miR-1308;
iv. miR-191;
V. miR-1228-star;
vi. ENSG00000199411_s;
vii. miR-1268; and
viii. miR-923.

9. The method of claim 1, comprising measuring the level of:
i. miR-150;
ii. miR-149-star;
iii. miR-1308;
iv. miR-191;
v. miR-1228-star;
vi. ENSG00000199411_s;
vii. miR-1268;
viii. miR-923; and
ix. miR-23a.

10. The method of claim 1, comprising measuring the level of:
i. miR-150;
ii. miR-149-star;
iii. miR-1308;
iv. miR-191;
v. miR-1228-star;
vi. ENSG00000199411_s;
vii. miR-1268;
viii. miR-923;
ix. miR-23a; and
x. miR-132.

11. The method of claim 1, comprising measuring the level of:
i. miR-150;
ii. miR-149-star;
iii. miR-1308;
iv. miR-191;
v. miR-1228-star;
vi. ENSG00000199411_s;
vii. miR-1268;
viii. miR-923;
ix. miR-23a;
x. miR-132; and
xi. miR-1207.5p.

12. The method of claim 1, comprising measuring the level of:
i. miR-150;
ii. miR-149-star;
iii. miR-1308;
iv. miR-191;
v. miR-1228-star;
vi. ENSG00000199411_s;
vii. miR-1268;
viii. miR-923;
ix. miR-23a;
x. miR-132;
xi. miR-1207.5p; and
xii. miR-342.3p.

13. The method of claim 1, comprising measuring the level of:
i. miR-150;
ii. miR-149-star;
iii. miR-1308;
iv. miR-191;
v. miR-1228-star;
vi. ENSG00000199411_s;
vii. miR-1268;
viii. miR-923;
ix. miR-23a;
x. miR-132;
xi. miR-1207.5p;
xii. miR-342.3p; and
xiii. U38B.

14. The method of claim 1, comprising measuring the level of:
i. miR-150;
ii. miR-149-star;
iii. miR-1308;
iv, miR-191;
V. miR-1228-star;
vi. ENSG00000199411_s;
vii. miR-1268;
viii. miR-923;
ix. miR-23a;
x, miR-132;
xi. miR-1207.5p;
xii. miR-342.3p;
xiii. U38B; and
xiv. miR-155.

15. The method of claim 1, further comprising measuring the level of at least one miRNA as an internal control.

16. The method of claim 15, wherein the internal control is selected from the group consisting of:
i. miR-27b,
ii. miR-195,
iii. miR-199b-3p, and
iv. miR-199a-3p.

17. The method of claim 1, further comprising isolating the nucleic acid from the skin sample.

18. The method of claim wherein the skin sample is formalin fixed paraffin-embedded.

19. The method of claim 1, further comprising histopathological assessment of the skin lesion sample.

20. The method of claim 1, further comprising
(a) scoring (i) clinical observations of the subject (ii) histopathological assessment of the skin lesion sample and (iii) expression levels of the two or more miRNAs; and
(b) combining (i), (ii) and (iii) to obtain a diagnostic score, that indicates the likelihood of melanoma in the subject.