# Patent

## Title
Methods and kits useful for the identification of astrocytoma, it's grades and glioblastoma prognosis

## Abstract

The present invention relates to novel primers for identification of astrocytoma, it's grades and glioblastoma prognosis. Further, disclosed is a method of diagnosing the presence of different grades of diffuse astrocytoma and glioblastoma, in a human subject, which involves detection of the expression levels of said genes in tumor tissue samples in comparison to normal brain. Also disclosed is a method of distinguishing between the two types of Glioblastoma—the progressive and de novo types. Also disclosed is a method of prognosis of glioblastoma based on the expression of the gene PBEF1, wherein the higher level of expression of the gene in the tumor sample, indicates poorer survival of the human subject. The disclosed compositions are useful, for example, in the diagnosis, prevention, treatment and/or prognosis of astrocytoma. The invention further provides kits for the detection and prognosis of the said diseases.

## Background
This application is a national phase application under 35 U.S.C. §371 of International Application No. PCT/IN2009/000017 filed 6 Jan. 2009, which claims priority to Indian Application No. 0054/DEL/2008 filed 7 Jan. 2008 and Indian Application No. 0055/DEL/2008 filed 7 Jan. 2008. The entire text of each of the above-referenced disclosures is specifically incorporated herein by reference without disclaimer.
FIELD OF THE INVENTION
The present invention relates to novel markers for identification of astrocytoma, it's grades and glioblastoma prognosis. The invention further provides novel markers and methods for diagnosing the presence of astrocytoma, it's grades and glioblastoma prognosis in a human subject and also provides a kit for characterization thereof. The present invention also relates to a method for diagnosing the presence of malignant astrocytoma followed by a kit for its characterization. The present invention further relates to a method for diagnosing glioblastoma in human subject and provides a kit for identifying the various types of glioblastoma followed by the characterization thereof in a human subject.
BACKGROUND AND PRIOR ART OF THE INVENTION
Gliomas are the most common primary brain tumors and occur at an incidence of almost 12 per 100,000 people (Landis et al., 1999). Diffuse astrocytoma may be classified (as per WHO classification) as low-grade diffuse (DA; Grade II), anaplastic (AA; Grade III) and glioblastoma (Grade IV; GBM), in the order of increasing malignancy (Mischel et al., 2001). Currently, these classifications are based on the observed histopathological characteristics of the tumor, which are sometimes subjective and inconsistent. GBM constitutes more than 80% of malignant gliomas (DeAngelis et al., 2001) and patients with GBM have a median survival of less than one year. Current treatments, including surgery, radiation therapy, and chemotherapy, unfortunately have not changed the natural history of these incurable neoplasms; and the prognosis of patients with GBMs has not improved significantly in the past 30 years (Davis et al., 1998). To find new diagnostic and therapeutic strategies, a better understanding of the biological pathway(s) leading to glial tumorigenesis is warranted.
Astrocytoma development is known to involve accumulation of a series of genetic alterations (Nagane et al., 1997) similar to other cancers. Identification of many of the genes involved in astrocytoma development, using standard molecular approaches, has helped to understand the process of astrocytoma genesis and progression (Louis and Gusella, 1995). Frequent amplification of epidermal growth factor receptor (EGFR) (Hill et al., 1999; Brock and Bower, 1997), platelet derived growth factor receptor (PDGFR) (Hermanson et al., 1992; Hermanson et al., 1996; Maxwell et al., 1990; Westermark et al., 1995; Fleming et al., 1992), amplification of chromosome 12q region, which carries the cdk4 gene (Nagane et al., 1997; Hill et al., 1999) and alterations in chromosomes 1p, 9p, 10, 17p, 19q, and 22q have frequently been found in these tumors. In addition, mutations in the tumor suppressor gene p53 were found to be associated with chromosome Yip alterations in low grade and progressive astrocytoma (Maher et al., 2001; Phatak et al., 2002). Inactivation of the cdk inhibitor p16 INK4a residing in chromosome 9p, is very common in sporadic astrocytoma, occurring in 50-70% of high-grade gliomas and 90% of GBM cell lines (James et al., 1991; Olopade et al., 1992). LOH in chromosome 10 is one of the most frequent alterations in GBM and is accompanied by the loss of PTEN/MMAC gene (Hill et al., 1999; Li et al., 1997).
GBMs are of two types: primary GBM (de novo type), which manifests in older patients (mean age: 55 yrs) as an aggressive, highly invasive tumor, usually without any evidence of prior clinical disease after a short clinical history of less than 3 months; secondary GBM (progressive type) is usually seen in younger patients (mean age: 40 yrs) and develops more slowly by malignant progression from diffuse (WHO grade II) or anaplastic astrocytoma (WHO grade III). Although some differences in the genetic lesions between these two GBMs have been identified, they are not sufficient enough to be used as differentiating markers considering the fact that the two types of GBMs have comparable clinical, genetic and biological characteristics (Kleihues et al., 2002). However, it is likely that these subtypes would respond differently to specific novel therapies as they are developed in the future (Kleihues and Ohgaki, 1999).
Despite all this information about astrocytoma, our understanding of astrocytoma development is not sufficient enough to improve prognosis for GBM patients. A more global, systematic understanding of expression patterns of various genes and their downstream gene products in astrocytoma will hopefully provide new diagnostic and therapeutic targets. Towards this, a number of studies have reported the gene expression profile of astrocytoma (Liau et al., 2000; Sallinen et al., 2000; Rickman et al., 2001; Ljubimova et al., 2001; Watson et al., 2001; Tanwar et al., 2002; Fathallah-Shaykh et al., 2002; Nutt et al., 2003; Wang et al., 2003; Godard et al., 2003).
It is also desirable to be able to target specific therapeutic modalities to pathogenetically distinct tumor types to maximize efficacy and minimize toxicity to the patient. (Golub et al., 1999; Kudoh et al., 2000). Previously, cancer classification has been based primarily on the morphological appearance of tumor cells. But this has serious limitations, because tumors with similar histopathgological appearance can follow significantly different clinical courses and show different responses to therapy. For example, based on histopathological appearance, astrocytoma grade IV cannot consistently be distinguished from astrocytoma grade III. Immunophenotyping for brain tumors has defined and refined diagnosis, e.g., distinguishing oligoastrocytoma from astrocytomas, and high-grade from low-grade astrocytomas. However, differential protein expression (GFAP, vimentin, synaptophysin, nestin) has not helped to improve therapeutic approaches. Prediction of transitions from low- to high-grade astrocytomas is difficult to make with currently available markers (De Girolami et al., 1994).
Tews and Nissen reported that immunohistochemical detection of various cancer-associated markers failed to reveal significant differential expression patterns among primary and secondary glioblastomas and precursor tumors; there was also no intra-individual constant expression pattern during glioma progression or correlation with malignancy. (Tews and Nissen, 1998-99). In contrast, class prediction for leukemia has been described based on monitoring gene expression profiles with DNA microarrays. (Golub et al., 1999).
But no class prediction capability, based on gene expression profiles, has been available heretofore for classifying high-grade gliomas to allow for optimizing treatment regimens. Zhang et al. (US Patent 20040053277) have identified a number of gene sets whose expression can accurately classify a glioma as glioblastoma (GBM), anaplastic astrocytoma (AA), anaplastic oligodendroglioma (AO) or oligodendroglioma (OL). However, these and other molecular markers currently in use are not capable of unambiguously identifying the subtypes of GBM. Mutations in p53 gene are reported to be associated with about 50% of grade WILL astrocytomas and secondary glioblastomas, but are seen only in 10-20% of primary glioblastoma (Campomenosi et al., 1996; Watanabe et al., 1997; Schmidt et al., 2002). Similarly, Epidermal growth factor receptor (EGFR), another marker routinely used in the classification of GBMs is found to be amplified in only 40% of all primary GBM cases and is rarely reported in secondary GBMs (Frederick et al., 2000). Microarray gene expression profiling of glioma allows simultaneous analysis of thousands of genes and is likely to identify molecular markers associated with tumor grade, progression and survival. Through cDNA microarray experiments, and subsequent validation with real-time quantitative PCR and/or immunohistochemistry, we have identified several distinct gene categories of transcripts over expressed in different set of astrocytoma. In addition, we have identified genes which characterize GBMs in general and primary GBMs in particular. Furthermore, we have also established the correlation between treatment response and the expression of the genes identified. Therefore, it is also a desideratum to be able to predict the presence of astrocytoma, type of glioblastoma and subtype of glioblastoma in the context of prognosis and, thus, to be able to administer appropriate treatment. These and other benefits are provided by the present invention.
OBJECTS OF THE INVENTION
The first object of the present invention is to provide a method for diagnosing the presence of astrocytoma in a human subject.
Another object of the present invention is to provide a kit for characterizing astrocytoma in a human subject.
Another object of the present invention is to provide a method for characterizing malignant astrocytoma (AA, GBM) in a human subject.
Another object of the present invention is to provide a kit for characterizing malignant astrocytoma (AA, GBM) in a human subject.
Another object of the present invention is to diagnose the presence of glioblastoma in human subject.
Another object of the present invention is to provide a kit for characterizing glioblastoma (GBM) in a human subject.
Still another object of the present invention is to provide a method for identifying the type of glioblastoma in human subject.
Another object of the present invention is to provide a kit for distinguishing primary and secondary glioblastoma in a human subject.
Another object of the present invention relates to a method for the prognosis of glioblastoma in human subjects.
Further, another object of the present invention relates to a kit for the prognosis of glioblastoma in human subjects.
SUMMARY OF THE INVENTION
The present invention relates to a method of diagnosing astrocytoma from a normal sample in a human subject based on the expression level of a single gene in a test sample of brain tissue cells obtained from the said human subject and in a control sample of known normal brain tissue cells wherein higher or lower level of expression of the mentioned genes in the test sample as compared to the normal sample in the said human subject indicates astrocytoma. It also relates to a kit to identify astrocytoma in a human subject.
The present invention relates to a method of diagnosing malignant astrocytoma (AA, GBM) in a human subject based on the expression level of a single gene in a test sample of brain tissue cells obtained from the said human subject and in a control sample of known normal brain tissue cells wherein higher level of expression of the gene in the test sample as compared to the normal sample, indicates malignant astrocytoma in the human subject from which the test sample has been obtained. It also relates to a kit to distinguish malignant astrocytoma from the benign astrocytoma in a human subject.
The present invention relates to a method of diagnosing glioblastoma from low-grade astrocytoma in a human subject based on the expression level of a single gene in a test sample of brain tissue cells obtained from the said human subject and in a control sample of known normal brain tissue cells wherein higher level of expression of the gene in the test sample as compared to the normal sample, indicates glioblastoma (GBM) in the human subject from which the test sample has been obtained. It also relates to a kit to distinguish glioblastoma from low-grade astrocytoma in a human subject.
The present invention relates to a method of distinguishing primary glioblastoma from secondary glioblastoma in a human subject based on the expression level of a single gene, in a test sample of brain tissue cells obtained from the said human subject and in a control sample of known normal brain tissue cells wherein higher level of expression of the above mentioned genes in the test sample as compared to the normal sample, indicates primary glioblastoma in the human subject from which the test sample has been obtained. It also relates to a kit to distinguish primary and secondary glioblastoma in a human subject.
Further, another object of the present invention also relates to a method for the prognosis of glioblastoma in human subjects. The present invention relates to a method of prognostication of the survival of human subject with glioblastoma based on the expression of the gene PBEF1 in a test sample of brain tissue cells obtained from the said human subject and in a control sample of known normal brain tissue cells wherein higher level of expression of the gene in the test sample as compared to the normal sample, indicates poorer survival of the human subject from which the test sample has been obtained. It also relates to a kit to distinguish a human subject with glioblastoma of poorer survival from that of better survival.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a method of diagnosing the presence of a astrocytoma, malignant astrocytoma, glioblastoma and to identify the type of glioblastoma in a human subject. Diffuse infiltrating astrocytomas include the following entities: 1. Diffuse astrocytoma (DA; WHO Gr. II), 2. Anaplastic astrocytoma (AA; WHO Gr. III) and 3. Glioblastoma (GBM; WHO Gr. IV). AA and GBM constitute malignant astrocytomas and are the most common intrinsic CNS neoplasms. The development of GBM has been described to occur through at least two genetic pathways resulting in the formation of primary and secondary GBMs. Primary GBM represents the most frequently presenting variant occurring de novo without an evidence of a less malignant precursor. Patients under this category are commonly of older age and have a high rate of epidermal growth factor receptor (EGFR) gene amplification, p16 INK4A deletion, mutations in PTEN gene and MDM2 amplification. In contrast, secondary GBM occurs after a preceding diagnosis of lower grade astrocytomas. Mutations in p53 gene, RB alterations and PDGFR amplification and over-expression are thought to be more common in the development of secondary GBM. In spite of these genetic differences, it remains uncertain whether these subtypes differ significantly with respect to prognosis.
The inventive method involves collecting or otherwise obtaining a sample of a bodily substance derived from the human subject, which sample contains human nucleic acid or protein originating from the subject, and quantitatively or semi-quantitatively determining therein the level of expression of single or combination of genes selected from the groups comprising of:

    1. SYT1, RAB26, DIRAS2 and RAB13. A characteristic expression pattern of the said genes is diagnostic for the presence of astrocytoma.
    2. IGFBP7. A characteristic expression pattern of the said gene is diagnostic for the malignant astrocytoma (AA, GBM).
    3. COL6A1, DCN, PLAT, LGALS3, FABP7, LOX, LAMB1, IGFBP3, GADD45A, FSTL1, RHOC, B2M and PBEF1. A characteristic expression pattern of the said genes is diagnostic for the presence of glioblastoma.
    4. AEBP1 and SOD2. A characteristic expression pattern of the said genes is diagnostic for the type of glioblastoma.
    5. PBEF1. A characteristic expression of said gene to determine the prognosis of glioblastoma


This includes detection by means of measuring of proteins or specific nucleic acids, such as RNA or cDNA.
The sequences used in the present invention are provided here as under. However, they are not arranged according to the SEQ ID but according to the genes to which they correspond [the sequences arranged as per the SEQ ID as well as the key to the Sequences are provided in the later pages in the patentIn 3.5 format].
1. Synaptotagmin I (SYT1)
Accession number: NM—001135806
Sequence:







ACCACCAAGAATAAAATAGTTGTTTGTCCCCTACAGTAGAACAAGTTT


 


GCCCATTCATCCTTGTGATAGATATGCATGCAAAACCAAAATGAAATC


 


AAATCCCCACAGATGGCTCGTAAGTCAAAAACACTGTTTAATTCTTTC


 


ACTGCATCCCTTTGGGAAGCCTGGCCCTTGAAAAACAGAATAATTCTG


 


AAAGAAAGAAAACAAAGAAAAACATACTCCAGAATTCCTAATAGAACA


 


CTTCACCTGAACCTAAAATGGTGAGCGAGAGTCACCATGAGGCCCTGG


 


CAGCCCCGCCTGTCACCACTGTCGCGACTGTTCTGCCAAGCAACGCCA


 


CAGAGCCAGCCAGTCCTGGAGAAGGAAAGGAAGATGCATTTTCTAAGC


 


TGAAGGAGAAGTTTATGAATGAGTTGCATAAAATTCCATTGCCACCGT


 


GGGCCTTAATTGCAATAGCCATAGTCGCAGTCCTTTTAGTCCTGACCT


 


GCTGCTTTTGTATCTGTAAGAAATGTTTGTTCAAAAAGAAAAACAAGA


 


AGAAGGGAAAGGAAAAAGGAGGGAAGAATGCCATTAACATGAAAGATG


 


TAAAAGACTTAGGGAAGACGATGAAAGATCAGGCCCTCAAGGATGATG


 


ATGCTGAAACTGGATTGACAGATGGAGAAGAAAAAGAAGAACCCAAAG


 


AAGAGGAGAAACTGGGAAAACTTCAGTATTCACTGGATTATGATTTCC


 


AAAATAACCAGCTGCTGGTAGGGATCATTCAGGCTGCCGAACTGCCCG


 


CCTTGGACATGGGGGGCACATCTGATCCTTACGTGAAAGTGTTTCTGC


 


TACCTGATAAGAAGAAGAAATTTGAGACAAAAGTCCACCGAAAAACCC


 


TTAATCCTGTCTTCAATGAGCAATTTACTTTCAAGGTACCATACTCGG


 


AATTGGGTGGCAAAACCCTAGTGATGGCTGTATATGATTTTGATCGTT


 


TCTCTAAGCATGACATCATT


 


GGAGAATTTAAAGTCCCTATGAACACAGTGGATTTTGGCCATGTAACT


 


GAGGAATGGCGTGACCTGCAAAGTGCTGAGAAGGAAGAGCAAGAGAAA


 


TTGGGTGATATCTGCTTCTCCCTTCGCTACGTACCTACTGCTGGTAAG


 


CTGACTGTTGTCATTCTGGAGGCAAAGAACCTGAAGAAGATGGATGTG


 


GGTGGCTTATCCGATCCTTATGTGAAGATTCATCTGATGCAGAATGGT


 


AAGAGGCTGAAGAAGAAAAAGACAACAATTAAAAAGAACACACTTAAC


 


CCCTACTACAATGAGTCATTCAGCTTTGAAGTACCTTTTGAACAAATC


 


CAGAAAGTGCAGGTGGTGGTAACTGTTTTGGACTATGACAAGATTGGC


 


AAGAACGATGCCATCGGCAAAGTCTTTGTGGGCTACAACAGCACCGGC


 


GCGGAGCTGCGACACTGGTCAGACATGCTGGCCAACCCCAGGCGACCT


 


ATTGCCCAGTGGCACACCCTGCAGGTAGAGGAGGAAGTTGATGCCATG


 


CTGGCCGTCAAGAAGTAAAGGAAAGAAGAAGCCTTTCTGCATTTGCCC


 


ATATAGTGCTCTTTAGCCAGTATCTGTAAATACCTCAGTAATATGGGT


 


CCTTTCATTTTTCCAGCCATGCATTCCTAACACAATTCAGTGGTACTT


 


GGAATCCTGTTTTAATTTGCACAAATTTAAATGTAGAGAGCCCCTAAG


 


TCCTTCATCATACCACTGCCCTCCAAATCTACTCTTCTTTTAAGCAAT


 


ATGATGTGTAGATAGAGCATGAATGAAATTATTTATTGTATCACACTG


 


TTGTATATACCAGTATGCTAAAGATTTATTTCTAGTTTGTGTATTTGT


 


ATGTTGTAAGCGTTTCCTAATCTGTGTATATCTAGATGTTTTTAATAA


 


GATGTTCTATTTTAAACTATGTAAATTGACTGAGATATAGGAGAGCTG


 


ATAATATATTATACGGTAAATATAGTATCGTCTGCATTCCAGCAAAAA


 


TATCAACTCGTAAGGCACTAGTACAGTTAAACTGACATCTTAAAGGAC


 


AACTTAAACCTGAGCTTTCTATTGAATCATTTGAGTACCAAGATAAAC


 


TTACACCACATACTTGGTGGGTGAATCCAATTTTGTAGAATTCCTACA


 


CAGGCAAAATAGCATGATCTGAGCAGCAGCATCCAGGCTGACCTCAAG


 


GAAGCATAGCCACAAAACAGAATAGCACCTGTCTGTACATATTTACAA


 


AGCTAAAATAATGGCTTCACTCTTATATTTGAGGAAGCAACTGAACAG


 


GAGTCAATGATTTCATATTACTGCATATAGAATAACAACAAGGTGTTC


 


CGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGCACATTTGTTTG


 


GGGATGGGGGAGAAGAAGCTAAGGGGAGAAGTCAACATTTATGAAATA


 


TTGCCTGACTATTTAAAAAGAAAAAAGTAGCTCTCCATTATCACCTTT


 


ATACAAAATGTACATCCTGTGAATTCTGTTCCAGATTTCACACCTACA


 


ATAATTCCAAAAGGTTTGCACATTAGAGTTTGTAACAAAATATTTTAT


 


TATATAAAACCAGGTTAGAAGGAATGCAGGATATTTTTAACACAACAA


 


TCTGTGCTTATTACACAAAATTACTTTGTGGTAAACAGACAGTATTGT


 


AATCCCATCAAAAGATGAAAGAAAAACAAAAACAAAAACCAACAACAA


 


TTAGCCATAGTTCTGAATGCACTTCAATTAAGCCAAAACAGACAGCTA


 


GTGATCTTTTTATATGCTCTTTTTACTTAAGTTTTAATTTGTCCTTTA


 


AAAAAAGGTGAAACAAACCAAGAACAAGTTCTAGAAAACTGAAGCAAC


 


CTCTTATGTATACTAGATGCTTGATTTAGGAGGAGTTTTTAAACGTTT


 


TCAATGTTATTATGTAGTAAATGACACTATTATGAAGCTACTAGTCAT


 


TCCATAAGAGTCTTAAAGGACTGCTCTGTGTAACACTGTGACTGCCGT


 


GTGTGCTTAGACCCGTAGTTTCCTCAGTGGATAGCACTCAATTTATTC


 


CGTAGTGATATTGTAACAATACTGCCATTCCCTTCTACTGCACTGCCC


 


AAGGTGTGTGTAGCACAAACAGTTCTCATTACAAAGGACCAATTCAGA


 


ACTGAAAAGCTATGCATAGGACAAGGAAGATACATAGAATGGGGTGGA


 


ACACAGCATTTTGTCAAGCACTGTGCAATATTCCATATTTTTCCCCAC


 


TATGGTAGACAACCATTTCGTGGAAGGGCAGCCTATTATCCCACACTG


 


CATCTAGCCTTTTGTCCCATTCACTTCTGTGATCCATTTTAATTTCCA


 


GGCCACAAGACAGTAGTGATGCTCTGAAATGAAAGTTTGTCTTCACAA


 


ATATCAAAACAAAATGGAGGAAAACTAAGCATTGGCCTCATGTTCAGT


 


CTTCAGGATATCACACCACGTCTTTTCAAAAACTAAAGAGAATTCAAA


 


AAGGGCTGATGGTAGGCTTTGAACATGGGGTTGGCTGTTTCCCAGTAA


 


AACTGGAATTCCTGTCGTTACTGTTTCCTTATCAAAGAAGGGGCAAGC


 


TCTTTTGCCTTTTAGGCCAGACATAGCAAACGCTTTATAATTGGCATA


 


GACATAAAGGATAAAAGGAAAATAACCGTCTGCCGATGGTCCGTACTT


 


CTTAAAAAACATAGGTAATAGAAAATATACACAAGTCAGAATGTGAAA


 


TTAAATAATGGTTTGAACAGAAAATTCAAACAAGACTCTTTCCAATTT


 


AAAGGGCCAAACCCTACCAAAGAGAGGGAGTTGACTGGCTTTTAAAAA


 


GTATTTAAATACCACAAATGACATTTAATTTCACTGTATTCAGCTTTA


 


AGTTGTTCACAATGAAACCACACTTTCAAACAAGCAGGTTCAAGCTGC


 


TGAATAGACATTATTTCTTGCATTAAAATACCACTAATGCATTCTCTT


 


GCAACACTGCCAGACATGGGATTGTCACCATAGAATTAGTTGGTACTA


 


TGCCATCTTTCACTCTTTCACAAGTCAGTGATGGAACCTGCTTTATGA


 


CCAAGATTCATCCTCAAATAAGCCACATGTACCCTTCTGACAAAGCTG


 


TGTAAAGTATTAGAATCTGATGCTCTAGAAAGATCCTAGTTGCCTTTG


 


TGTATATTTACTGCCTGCTTGAGTGTTTCTATGTGTGGGTTTTCCCTG


 


TATCTTGTAGAAATGTTGGGGTGTTTTCCTCTGCCATATGGCTCGTGG


 


CCTGCGAGCCAACTATTTCAGCTGTATTTTACCTTCATTTTTGATGAG


 


GTGATTTAAATTTTGTTTCACTTTGTGTAGTGAATTCCACAGTAGTTT


 


TCTGATTGTTGTTAAAAATGACTTAACATATTACACAGATATTCAATA


 


AAAATGTTTTATTTCCTGTTGAAAAAAAAAAAAAAAAAA






Primers used:










Forward primer sequence:



GGTTGGCTGTTTCCCAGTAAAAC



 



Reverse primer sequence:



TTTTAAGAAGTACGGACCATCGG






Amplicon length: 171

Amplicon sequence:








GGTTGGCTGTTTCCCAGTAAAACTGGAATTCCTGTCGTTACTGTTTCC


 


TTATCAAAGAAGGGGCAAGCTCTTTTGCCTTTTAGGCCAGACATAGCA


 


AACGCTTTATAATTGGCATAGACATAAAGGATAAAAGGAAAATAACCG


 


TCTGCCGATGGTCCGTACTTCTTAAAA






2. RAB26, Member RAS Oncogene Family (RAB26)

Accession number: NM—014353.

Sequence:








GCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCAGGGGAAGGGTTCGG


 


GTCCGGGTCGGGCTCGGCGGGCGCGGGGTGCGGGACGGCCCAGGGCAC


 


GGCGGCTGCAGCGGGAGCACACTGAGCGCCCGCCCGCCATGTCCAGGA


 


AGAAGACCCCCAAGAGCAAAGGGGCCAGCACCCCCGCTGCCTCCACGC


 


TGCCCACCGCCAACGGGGCCCGACCGGCGCGCTCCGGGACTGCGCTTT


 


CCGGCCCCGACGCGCCGCCCAACGGGCCCTTGCAGCCCGGCCGGCCCT


 


CGCTTGGCGGCGGTGTCGACTTCTACGACGTCGCCTTCAAGGTCATGC


 


TGGTGGGGGACTCGGGTGTGGGGAAGACCTGTCTGCTGGTGCGATTCA


 


AGGATGGTGCTTTCCTGGCGGGGACCTTCATCTCCACCGTAGGCATTG


 


ACTTCCGGAACAAAGTTCTGGACGTGGATGGTGTGAAGGTGAAGCTGC


 


AGATGTGGGACACAGCTGGTCAGGAGCGGTTCCGdAGTGTTACCCATG


 


CCTACTACCGGGATGCTCATGCTCTGCTGCTGCTCTACGATGTCACCA


 


ACAAGGCCTCCTTTGACAACATCCAGGCCTGGCTGACCGAGATCCACG


 


AGTACGCCCAGCACGACGTGGCGCTCATGCTGCTGGGGAACAAGGTGG


 


ACTCTGCCCATGAGCGTGTGGTGAAGAGGGAGGACGGGGAGAAGCTGG


 


CCAAGGAGTATGGACTGCCCTTCATGGAGACCAGCGCCAAGACGGGCC


 


TCAACGTGGACTTGGCCTTCACAGCCATAGCAAAGGAGTTGAAGCAGC


 


GCTCCATGAAGGCTCCCAGCGAGCCGCGCTTCCGGCTGCATGATTACG


 


TTAAGAGGGAGGGTCGAGGGGCCTCCTGCTGCCGCCCTTGAACCTGGC


 


TGAGCTCAGTCCTCTGGAGGAAGCCGCCCAGTCCCTAGAAGGCTGGAC


 


AGAGGGTCTCCAGGCCCTTCTGACTTTGTTGCCCAGTGGCCAACGCCC


 


GAGTGTCTGTTTTCAGGAGCCCCAGGTCAAGCCTTGTCCCTTCCTCCT


 


CCCAGCAACAGTCCCAACAAGCAGGCTTCTGAGAGCCCGTGGCCGCAC


 


ACTGGCCGCCACGGAAAAGCAGTCTTCTGCACGGGACGGGGAGCGGCA


 


AGTGGACAGACTTTGCCACGGTGCTCTGCTGCCCCCTCCTGGGCACGT


 


CCAGGTGAGGGAGGGCTGGGGCTGGCACCACGCACAGTGCCTAACCCT


 


AGAAAAGCCATGTCTTCAGCCGCACATGCTCAGGCAGCTAAGGGAGGA


 


CGCCTGCCCACGCCTGGGACAGAAGGCTTCACTGCTAATCACATCGTG


 


CATCTGTGTGTCCTGGGAGCTGCCTGCTCCCGGCCCACCCTCTAGGAG


 


GCTCTGGCTCAAACAGCAATAGGGTCTTCCTCACTGACCTTGGAGGAT


 


GCCTGTGGCCTTGTGATAAAATGTGGGAAATCACAGAAAACACCAGAA


 


ACAACAACTGCCAGCCCGGCCTGGCCACAGGTGAGGTCTGTGATTTCC


 


GAGCACGCTCCACCTTGCACTCAACTTGGCCTTTTGATTGCACAAGCC


 


TTTGTTTTCAGTCCTAGTGAATAAAGTTGTGTTTTCTGGAAAAAAAAA


 


AAAAAAAAA






Primers used:










Forward primer sequence:



GTCTGCTGGTGCGATTCAAG



 



Reverse primer sequence:



GCATGGGTAACACTGCGGA






Amplicon length: 163

Amplicon sequence:








GTCTGCTGGTGCGATTCAAGGATGGTGCTTTCCTGGCGGGGACCTTCA


 


TCTCCACCGTAGGCATTGACTTCCGGAACAAAGTTCTGGACGTGGATG


 


GTGTGAAGGTGAAGCTGCAGATGTGGGACACAGCTGGTCAGGAGCGGT


 


TCCGCAGTGTTACCCATGC






3. DIRAS Family, GTP-Binding RAS-Like 2(DIRAS2)

Accession number: NM—017594

Sequence








ACACACCCTGCGCTGCCCTGTCCTGCGCGAGTGGAGCTCTGAAGAAGC


 


TCTGAGCGGAGTTGTGTTCTTCCCCAGGTGCGTCCTGGCTGAGAGTTG


 


GAGCTCTCCAGCAACATGCCTGAGCAGAGTAACGATTACCGGGTGGCC


 


GTGTTTGGGGCTGGCGGTGTTGGCAAGAGCTCCCTGGTGTTGAGGTTT


 


GTGAAAGGCACATTCCGGGAGAGCTACATCCCGACGGTGGAAGACACC


 


TACCGGCAAGTGATCAGCTGTGACAAGAGCATATGCACATTGCAGATC


 


ACCGACACGACGGGGAGCCACCAGTTCCCGGCCATGCAGCGGCTGTCC


 


ATCTCCAAAGGGCACGCCTTCATCCTGGTGTACTCCATTACCAGCCGA


 


CAGTCCTTGGAGGAGCTCAAGCCCATCTACGAACAAATCTGCGAGATC


 


AAAGGGGACGTGGAGAGCATCCCCATCATGCTGGTGGGGAACAAGTGT


 


GATGAGAGCCCCAGCCGCGAGGTGCAGAGCAGCGAGGCGGAGGCCTTG


 


GCCCGCACATGGAAGTGTGCCTTCATGGAGACCTCAGCCAAGCTCAAC


 


CATAACGTGAAGGAGCTTTTCCAGGAGCTGCTCAACCTGGAGAAGCGC


 


AGGACCGTGAGTCTCCAGATCGACGGGAAAAAGAGCAAGCAGCAGAAA


 


AGGAAAGAGAAGCTCAAAGGCAAGTGCGTGATCATGTGAAGGCCCTTC


 


CTGCGGGAGGAGCAGCTGTGTGTCCCCGGCACCTCACTCCCCCAAAAT


 


GACACCCACCGTCGTCAGGGTAGCATGTATAATGCCCACGTGTTAAAC


 


ATTGCATTTAATCGAGATGCGTCCTATTGTCCTTAAGAGGGCGTTTCA


 


CACCACCAACAGTAAGCCACCCACTCTGGAGTCACAGAATCTGCCAGG


 


CGGTTCAAGTGAAAACCAACACACTCAGCATCCCTGGGAACTGAGAGG


 


TGCCAGCAATTGCTGAAGGTGGCGATGAACACCCGAAGGTGGGAGGGA


 


GGACTGGTACCCACAAAGCAACATGTACCGAGAGGACTAAATGTCATC


 


TACGTGCATGTGAGAGCGTGTTAACCTAGAGTTACCTGCACCAACCCC


 


AGACAGAAGCCAATCACATCTTTGGGGGAGGGGAGGGGCAGGAAGAGG


 


TGAGAAGATCAGATGGTCCAAAGTGGACCACACTTGGTCCATTTTACA


 


CTTTTTTAAAGGGGATTAAAAAACACAGCCTCTCCCCCAAAGGGTGTC


 


CGTTCTTAATTCCCACCTGGCCTGTTAGGAGCCTTGCTACCCTGAGGG


 


GATGTGTTCACCTTACCTAGACCTAGTTAGGAAGTATCATTTTAAGCT


 


ATTAGAGTATTTATCTTCATGTGCAGGGATAAGTGCACTAACAGTGTG


 


CTGCTCTGTCGGAAGTTCTTCAGTTTTTAAGTGAGGATATCGTGACAG


 


TATTAAAACATCGCAATAATGTTCCTGTGTGTTATACATCGAGGGTTT


 


TAGAAATGTGATTTTCTTCTTTTGACCTGTGAGGAGTATAACTTCTTT


 


CAGCCCTCAGATTTTAAATACAAGCAAATAAACTCACTATTTTTAGAC


 


GTTTTTTTCCTCCAAGGTGGTTTTCTTCTCTTAAATAACTCGATCTGT


 


ACCCAGCTGGGTAGCAGCCAGCAAAGGCCATCAGACAACCAGAAGCAC


 


ATCCATTTTTGTAGTGTCACAAACATGTATATGCCACACTTTGCACCT


 


TAATGAAATACTTTGAAACAGAAGTTATTCACTGTGTTTTTGATGATC


 


TATCTGTATTGGAAATATGTTCCTGGAAAATGCATTTAAATAATAGTA


 


AATTCTCTTGCATGTTCCATTATACGTGTCTTCTAAGAGCTGTTCAAT


 


ACAGTATTCACTCTAGAAACAATTATCTTTTTCTCTTAATGATTTTGT


 


GTGCATCTTTAATCTTTCAAGCCAAATTACAGCTATTTCAGGTTTCCT


 


GTGTTAGCTTGGGGATAGGATGGTGGCTGGAGACAGGCAGGCTTCTCT


 


GCCCTGGGAAGAGCCCACTCAGCTTAATTGCTCTGCCATCGTAGAGCC


 


TGGTTGGACTTGGCTTCCTGAAAACTCCCACTGATAGTGCCTGTTAGA


 


TCTCCTGTTTGTTTCAGTTGGCAGAACATTTACTGGCCCCAACTGTGG


 


CATCATCCTCTCAGCAGTCTTCCTGTCACCCGCCTGGCAGGCAGAAGG


 


AGCTGCAGTCCTACGTGGGCCTGCCTGGGGGGGTGGGGGCTGCATGGC


 


TGTTGGGTGGCAGTGTCAGCACAGGGAGGGCTTAAGTTGGGGATGTTT


 


GACCAGGCCACCTCCTGCAACTGCTGTTTCTCCTGTCCCTCCTATGCA


 


GGGCTTGCAGCAGCAGCAGTGTGGCCATCTCCATCCCCCAAAGCACAC


 


TTGCTCTCTCAATATGTCCTAGTTTTCTTCAGCCTTTTCTGGTTCAGT


 


TCCCTTGTCCTGATCTCATCCTCTCTGGTCTCCCAATAACTCACCCTT


 


GGGATGTGTTTAGAGCGTGGGAGGTGCCTTTGAGAACTGCTTGACTCC


 


ATGATCTCCTAGAACAAAACCGCCCTGACTTTACAGGGGGAACACTCA


 


TGCTGAGCTGAGAAAGCAGAGAAGTGGCGTGGGAGCCAGCTGGGGGTG


 


AAGAGCATTTGGGCCAGTCCCGTGGCCCCCTTCAGATTCCTCAAGCAG


 


GATTGTTCTGTTCTAAAAAGCTGTTGCACAGCATTCGCAATGAGATCT


 


TTAGTTGGCGGATTTTCTGGAACATTTGTTTTTCAACTTGTCCCGACA


 


TTTTTTTTCTGTTTCTATTCTGAGAGAGAGATGATCAAGTTTTAATTT


 


GGGTATAGGTTAAATGGAAGAAGAAACAGAACTTCATGGCCAAAGTAG


 


ACCTATAGATTTTGATTGGGTTCTTTGTTAACAGTAGAATGCGATCTT


 


TGCCACTGACTGTAGTATTAATAAGGTTTTAATGTGAGATATTCCTGC


 


AAACCATCCCATTTCTACTGATTGTAAGTCAGAATTTCTTTTATCCCT


 


TTCAAATCAGTTTCTACATGTTTAAGTGTTCAGGGCTTCATCAGCATG


 


AGAAGTTTGTAATTACTGAAAGTCTGATTTCATTCAGGACACATTTTT


 


TCCTTCATATTTTTTCTGTGAATTTATAGGCTAGGAAGGCTATTGAAG


 


CCTCAATTATGGGTCTTCATTTTGAGATCGTTTTCTATGAGCTGAACT


 


GGAGATATCAATGGTTATCTCAAAATCGTCTTTTAGGAGATCCCCAAT


 


TGACTCAGAGTTTGAGGAGTTAGTATCACAGAATTAGATTTTTTTAAA


 


GCATTTGTACGTTTCCATTCCCAAATATGTAGCTGTGGTTCTTGAAAA


 


CACATCCTACATTGCATATGGGCATAGCAGTTTTTGACCCAGGCAGAA


 


TAAGTTAATATTTAATTAAATATTGCTTTGAAGATGGCGCTCTGGGCA


 


TGAGCATGGGGCTCCATGACTTCCCTTCTATCCCCATGAGCCCCTCCT


 


CCATCCAGCGACAAGCCATGGGCATGCATACAATGCAGCAAGACCAAC


 


ACAAGAGCAATATTGAATTGTTCATTCTATCTAAAATTACATGTATAT


 


AAAATATATAATTTATCTTCCTGCATTTTTGAAGTATAAAGTCATAAA


 


TTGTACATATCTGTAAGCTAGTATATTTGTTTCACTGTTTGTAATATT


 


TAAGAAATGCTCATTCTTTGTAGAACAAAAATGTATTAAATATTTTAA


 


AAATTGCTCTGTGATACTTAATTTTTTTCCCCAAAATTTGTAATGTGT


 


TGCTTCTACATAAGTTCTCTGGAAATATCTACAACTAATAGGACACAT


 


GTAAATCCTTGAAGACACATCCTGGAATTCATACCCCACAAGGACAGT


 


GTGTATACAAAGTATTTGCAGAGCATGACTTTTATATGTGTGGGATAT


 


CAATGTGTATATTTATATTTAAAGTGTATTTATTGTTACAAGTCTATT


 


CTCTATTATATTTTATTTACTCTGCGGTTATAAAAATCACCCTTGCAT


 


ACAAGTTTCTAGTTGCCAGTGATGTTCTGGAAATAATGGGAGATATTA


 


CAATAAAGCTACAGTTATGACACCCTG






Primers used:










Forward primer sequence:



CTGGTGTTGAGGTTTGTGAAAGG



 



Reverse primer sequence:



CCGTCGTGTCGGTGATCTG






Amplicon length: 124

Amplicon sequence:








CTGGTGTTGAGGTTTGTGAAAGGCACATTCCGGGAGAGCTACATCCCG


 


ACGGTGGAAGACACCTACCGGCAAGTGATCAGCTGTGACAAGAGCATA


 


TGCACATTGCAGATCACCGACACGACGG






4. RAB13, Member RAS Oncogene Family (RAB13)

Accession number: NM—002870

Sequence:








CTGGGCTCCGTGCCGCTCTGTTTGCCAACCGTCCAGTCCCGCCTACCA


 


GTGCCGGGCGCTCCCCACCCCTCCCCCGGCTCCCCCGGTGTCCGCCAT


 


GGCCAAAGCCTACGACCACCTCTTCAAGTTGCTGCTGATCGGGGACTC


 


GGGGGTGGGCAAGACTTGTCTGATCATTCGCTTTGCAGAGGACAACTT


 


CAACAACACTTACATCTCCACCATCGGAATTGATTTCAAGATCCGCAC


 


TGTGGATATAGAGGGGAAGAAGATCAAACTACAAGTCTGGGACACGGC


 


TGGCCAAGAGCGGTTCAAGACAATAACTACTGCCTACTACCGTGGAGC


 


CATGGGCATTATCCTAGTATACGACATCACGGATGAGAAATCTTTCGA


 


GAATATTCAGAACTGGATGAAAAGCATCAAGGAGAATGCCTCGGCTGG


 


GGTGGAGCGCCTCTTGCTGGGGAACAAATGTGACATGGAGGCCAAGAG


 


GAAGGTGCAGAAGGAGCAGGCCGATAAGTTGGCTCGAGAGCATGGAAT


 


CCGATTTTTCGAAACTAGTGCTAAATCCAGTATGAATGTGGATGAGGC


 


TTTTAGTTCCCTGGCCCGGGACATCTTGCTCAAGTCAGGAGGCCGGAG


 


ATCAGGAAACGGCAACAAGCCTCCCAGTACTGACCTGAAAACTTGTGA


 


CAAGAAGAACACCAACAAGTGCTCCCTGGGCTGAGGACCCTTTCTTGC


 


CTCCCCACCCCGGAAGCTGAACCTGAGGGAGACAACGGCAGAGGGAGT


 


GAGCAGGGGAGAAATAGCAGAGGGGCTTGGAGGGTCACATAGGTAGAT


 


GGTAAAGAGAATGAGGAGAAAAAGGAGAAAAGGGAAAAGCAGAAAGGA


 


AAAAAAGGAAGAGAGAGGAAGGGAGAAGGGAGAGGAATGAATTGAGGA


 


AGTGAAAGAAGGCAAGGAGGTAGGAAGAGAGGGAGGAGGAAAGGAAGG


 


AGAGATGCCTCAGGCTTCAGACCTTACCTGGGTTTTCAGGGCAAACAT


 


AAATGTAAATACACTGATTTATTCTGTTACTAGATCAGGTTTTAGGGT


 


CCTGCAAAAGGCTAGCTCGGCACTACACTAGGGAATTTGCTCCTGTTC


 


TGTCACTTGTCATGGTCTTTCTTGGTATTAAAGGCCACCATTTGCACA


 


AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA


 


AAAAAAAAAAA






Primers used:










Forward primer sequence:



ATAACTACTGCCTACTACCGTGG



 



Reverse primer sequence:



CCATGTCACATTTGTTCCCCAG






Amplicon length: 160

Amplicon sequence:








ATAACTACTGCCTACTACCGTGGAGCCATGGGCATTATCCTAGTATAC


 


GACATCACGGATGAGAAATCTTTCGAGAATATTCAGAACTGGATGAAA


 


AGCATCAAGGAGAATGCCTCGGCTGGGGTGGAGCGCCTCTTGCTGGGG


 


AACAAATGTGACATGG






5. Insulin-Like Growth Factor Binding Protein 7 (IGFBP7)

Accession number: NM—001553

Sequence:








GCCGCTGCCACCGCACCCCGCCATGGAGCGGCCGTCGCTGCGCGCCCT


 


GCTCCTCGGCGCCGCTGGGCTGCTGCTCCTGCTCCTGCCCCTCTCCTC


 


TTCCTCCTCTTCGGACACCTGCGGCCCCTGCGAGCCGGCCTCCTGCCC


 


GCCCCTGCCCCCGCTGGGCTGCCTGCTGGGCGAGACCCGCGACGCGTG


 


CGGCTGCTGCCCTATGTGCGCCCGCGGCGAGGGCGAGCCGTGCGGGGG


 


TGGCGGCGCCGGCAGGGGGTACTGCGCGCCGGGCATGGAGTGCGTGAA


 


GAGCCGCAAGAGGCGGAAGGGTAAAGCCGGGGCAGCAGCCGGCGGTCC


 


GGGTGTAAGCGGCGTGTGCGTGTGCAAGAGCCGCTACCCGGTGTGCGG


 


CAGCGACGGCACCACCTACCCGAGCGGCTGCCAGCTGCGCGCCGCCAG


 


CCAGAGGGCCGAGAGCCGCGGGGAGAAGGCCATCACCCAGGTCAGCAA


 


GGGCACCTGCGAGCAAGGTCCTTCCATAGTGACGCCCCCCAAGGACAT


 


CTGGAATGTCACTGGTGCCCAGGTGTACTTGAGCTGTGAGGTCATCGG


 


AATCCCGACACCTGTCCTCATCTGGAACAAGGTAAAAAGGGGTCACTA


 


TGGAGTTCAAAGGACAGAACTCCTGCCTGGTGACCGGGACAACCTGGC


 


CATTCAGACCCGGGGTGGCCCAGAAAAGCATGAAGTAACTGGCTGGGT


 


GCTGGTATCTCCTCTAAGTAAGGAAGATGCTGGAGAATATGAGTGCCA


 


TGCATCCAATTCCCAAGGACAGGCTTCAGCATCAGCAAAAATTACAGT


 


GGTTGATGCCTTACATGAAATACCAGTGAAAAAAGGTGAAGGTGCCGA


 


GCTATAAACCTCCAGAATATTATTAGTCTGCATGGTTAAAAGTAGTCA


 


TGGATAACTACATTACCTGTTCTTGCCTAATAAGTTTCTTTTAATCCA


 


ATCCACTAACACTTTAGTTATATTCACTGGTTTTACACAGAGAAATAC


 


AAAATAAAGATCACACATCAAGACTATCTACAAAAATTTATTATATAT


 


TTACAGAAGAAAAGCATGCATATCATTAAACAAATAAAATACTTTTTA


 


TCACAAAAAAAAAAAAAAAA






Primers used:










Forward primer sequence:



GGTCCTTCCATAGTGACGCC



 



Reverse primer sequence:



TCTGAATGGCCAGGTTGTCC






Amplicon length: 184

Amplicon sequence:








GGTCCTTCCATAGTGACGCCCCCCAAGGACATCTGGAATGTCACTGGT


 


GCCCAGGTGTACTTGAGCTGTGAGGTCATCGGAATCCCGACACCTGTC


 


CTCATCTGGAACAAGGTAAAAAGGGGTCACTATGGAGTTCAAAGGACA


 


GAACTCCTGCCTGGTGACCGGGACAACCTGGCCATTCAGA






6. Collagen, Type VI, Alpha 1 (COL6A1)

Accession number: NM—001848

Sequence:








GCTCTCACTCTGGCTGGGAGCAGAAGGCAGCCTCGGTCTCTGGGCGGC


 


GGCGGCGGCCCACTCTGCCCTGGCCGCGCTGTGTGGTGACCGCAGGCC


 


CCAGACATGAGGGCGGCCCGTGCTCTGCTGCCCCTGCTGCTGCAGGCC


 


TGCTGGACAGCCGCGCAGGATGAGCCGGAGACCCCGAGGGCCGTGGCC


 


TTCCAGGACTGCCCCGTGGACCTGTTCTTTGTGCTGGACACCTCTGAG


 


AGCGTGGCCCTGAGGCTGAAGCCCTACGGGGCCCTCGTGGACAAAGTC


 


AAGTCCTTCACCAAGCGCTTCATCGACAACCTGAGGGACAGGTACTAC


 


CGCTGTGACCGAAACCTGGTGTGGAACGCAGGCGCGCTGCACTACAGT


 


GACGAGGTGGAGATCATCCAAGGCCTCACGCGCATGCCTGGCGGCCGC


 


GACGCACTCAAAAGCAGCGTGGACGCGGTCAAGTACTTTGGGAAGGGC


 


ACCTACACCGACTGCGCTATCAAGAAGGGGCTGGAGCAGCTCCTCGTG


 


GGGGGCTCCCACCTGAAGGAGAATAAGTACCTGATTGTGGTGACCGAC


 


GGGCACCCCCTGGAGGGCTACAAGGAACCCTGTGGGGGGCTGGAGGAT


 


GCTGTGAACGAGGCCAAGCACCTGGGCGTCAAAGTCTTCTCGGTGGCC


 


ATCACACCCGACCACCTGGAGCCGCGTCTGAGCATCATCGCCACGGAC


 


CACACGTACCGGCGCAACTTCACGGCGGCTGACTGGGGCCAGAGCCGC


 


GACGCAGAGGAGGCCATCAGCCAGACCATCGACACCATCGTGGACATG


 


ATCAAAAATAACGTGGAGCAAGTGTGCTGCTCCTTCGAATGCCAGCCT


 


GCAAGAGGACCTCCGGGGCTCCGGGGCGACCCCGGCTTTGAGGGAGAA


 


CGAGGCAAGCCGGGGCTCCCAGGAGAGAAGGGAGAAGCCGGAGATCCT


 


GGAAGACCCGGGGACCTCGGACCTGTTGGGTACCAGGGAATGAAGGGA


 


GAAAAAGGGAGCCGTGGGGAGAAGGGCTCCAGGGGACCCAAGGGCTAC


 


AAGGGAGAGAAGGGCAAGCGTGGCATCGACGGGGTGGACGGCGTGAAG


 


GGGGAGATGGGGTACCCAGGCCTGCCAGGCTGCAAGGGCTCGCCCGGG


 


TTTGACGGCATTCAAGGACCCCCTGGCCCCAAGGGAGACCCCGGTGCC


 


TTTGGACTGAAAGGAGAAAAGGGCGAGCCTGGAGCTGACGGGGAGGCG


 


GGGAGACCAGGGAGCTCGGGACCATCTGGAGACGAGGGCCAGCCGGGA


 


GAGCCTGGGCCCCCCGGAGAGAAAGGAGAGGCGGGCGACGAGGGGAAC


 


CCAGGACCTGACGGTGCCCCCGGGGAGCGGGGTGGCCCTGGAGAGAGA


 


GGACCACGGGGGACCCCAGGCACGCGGGGACCAAGAGGAGACCCTGGT


 


GAAGCTGGCCCGCAGGGTGATCAGGGAAGAGAAGGCCCCGTTGGTGTC


 


CCTGGAGACCCGGGCGAGGCTGGCCCTATCGGACCTAAAGGCTACCGA


 


GGCGATGAGGGTCCCCCAGGGTCCGAGGGTGCCAGAGGAGCCCCAGGA


 


CCTGCCGGACCCCCTGGAGACCCGGGGCTGATGGGTGAAAGGGGAGAA


 


GACGGCCCCGCTGGAAATGGCACCGAGGGCTTCCCCGGCTTCCCCGGG


 


TATCCGGGCAACAGGGGCGCTCCCGGGATAAACGGCACGAAGGGCTAC


 


CCCGGCCTCAAGGGGGACGAGGGAGAAGCCGGGGACCCCGGAGACGAT


 


AACAACGACATTGCACCCCGAGGAGTCAAAGGAGCAAAGGGGTACCGG


 


GGTCCCGAGGGCCCCCAGGGACCCCCAGGACACCAAGGACCGCCTGGG


 


CCGGACGAATGCGAGATTTTGGACATCATCATGAAAATGTGCTCTTGC


 


TGTGAATGCAAGTGCGGCCCCATCGACCTCCTGTTCGTGCTGGACAGC


 


TCAGAGAGCATTGGCCTGCAGAACTTCGAGATTGCCAAGGACTTCGTC


 


GTCAAGGTCATCGACCGGCTGAGCCGGGACGAGCTGGTCAAGTTCGAG


 


CCAGGGCAGTCGTACGCGGGTGTGGTGCAGTACAGCCACAGCCAGATG


 


CAGGAGCACGTGAGCCTGCGCAGCCCCAGCATCCGGAACGTGCAGGAG


 


CTCAAGGAAGCCATCAAGAGCCTGCAGTGGATGGCGGGCGGCACCTTC


 


ACGGGGGAGGCCCTGCAGTACACGCGGGACCAGCTGCTGCCGCCCAGC


 


CCGAACAACCGCATCGCCCTGGTCATCACTGACGGGCGCTCAGACACT


 


CAGAGGGACACCACACCGCTCAACGTGCTCTGCAGCCCCGGCATCCAG


 


GTGGTCTCCGTGGGCATCAAAGACGTGTTTGACTTCATCCCAGGCTCA


 


GACCAGCTCAATGTCATTTCTTGCCAAGGCCTGGCACCATCCCAGGGC


 


CGGCCCGGCCTCTCGCTGGTCAAGGAGAACTATGCAGAGCTGCTGGAG


 


GATGCCTTCCTGAAGAATGTCACCGCCCAGATCTGCATAGACAAGAAG


 


TGTCCAGATTACACCTGCCCCATCACGTTCTCCTCCCCGGCTGACATC


 


ACCATCCTGCTGGACGGCTCCGCCAGCGTGGGCAGCCACAACTTTGAC


 


ACCACCAAGCGCTTCGCCAAGCGCCTGGCCGAGCGCTTCCTCACAGCG


 


GGCAGGACGGACCCCGCCCACGACGTGCGGGTGGCGGTGGTGCAGTAC


 


AGCGGCACGGGCCAGCAGCGCCCAGAGCGGGCGTCGCTGCAGTTCCTG


 


CAGAACTACACGGCCCTGGCCAGTGCCGTCGATGCCATGGACTTTATC


 


AACGACGCCACCGACGTCAACGATGCCCTGGGCTATGTGACCCGCTTC


 


TACCGCGAGGCCTCGTCCGGCGCTGCCAAGAAGAGGCTGCTGCTCTTC


 


TCAGATGGCAACTCGCAGGGCGCCACGCCCGCTGCCATCGAGAAGGCC


 


GTGCAGGAAGCCCAGCGGGCAGGCATCGAGATCTTCGTGGTGGTCGTG


 


GGCCGCCAGGTGAATGAGCCCCACATCCGCGTCCTGGTCACCGGCAAG


 


ACGGCCGAGTACGACGTGGCCTACGGCGAGAGCCACCTGTTCCGTGTC


 


CCCAGCTACCAGGCCCTGCTCCGCGGTGTCTTCCACCAGACAGTCTCC


 


AGGAAGGTGGCGCTGGGCTAGCCCACCCTGCACGCCGGCACCAAACCC


 


TGTCCTCCCACCCCTCCCCACTCATCACTAAACAGAGTAAAATGTGAT


 


GCGAATTTTCCCGACCAACCTGATTCGCTAGATTTTTTTTAAGGAAAA


 


GCTTGGAAAGCCAGGACACAACGCTGCTGCCTGCTTTGTGCAGGGTCC


 


TCCGGGGCTCAGCCCTGAGTTGGCATCACCTGCGCAGGGCCCTCTGGG


 


GCTCAGCCCTGAGCTAGTGTCACCTGCACAGGGCCCTCTGAGGCTCAG


 


CCCTGAGCTGGCGTCACCTGTGCAGGGCCCTCTGGGGCTCAGCCCTGA


 


GCTGGCCTCACCTGGGTTCCCCACCCCGGGCTCTCCTGCCCTGCCCTC


 


CTGCCCGCCCTCCCTCCTGCCTGCGCAGCTCCTTCCCTAGGCACCTCT


 


GTGCTGCATCCCACCAGCCTGAGCAAGACGCCCTCTCGGGGCCTGTGC


 


CGCACTAGCCTCCCTCTCCTCTGTCCCCATAGCTGGTTTTTCCCACCA


 


ATCCTCACCTAACAGTTACTTTACAATTAAACTCAAAGCAAGCTCTTC


 


TCCTCAGCTTGGGGCAGCCATTGGCCTCTGTCTCGTTTTGGGAAACCA


 


AGGTCAGGAGGCCGTTGCAGACATAAATCTCGGCGACTCGGCCCCGTC


 


TCCTGAGGGTCCTGCTGGTGACCGGCCTGGACCTTGGCCCTACAGCCC


 


TGGAGGCCGCTGCTGACCAGCACTGACCCCGACCTCAGAGAGTACTCG


 


CAGGGGCGCTGGCTGCACTCAAGACCCTCGAGATTAACGGTGCTAACC


 


CCGTCTGCTCCTCCCTCCCGCAGAGACTGGGGCCTGGACTGGACATGA


 


GAGCCCCTTGGTGCCACAGAGGGCTGTGTCTTACTAGAAACAACGCAA


 


ACCTCTCCTTCCTCAGAATAGTGATGTGTTCGACGTTTTATCAAAGGC


 


CCCCTTTCTATGTTCATGTTAGTTTTGCTCCTTCTGTGTTTTTTTCTG


 


AACCATATCCATGTTGCTGACTTTTCCAAATAAAGGTTTTCACTCCT


 


CTAAAAAAAAAAAAAAAAAAAAA






Primers used:










Forward primer sequence:



ACAGTGACGAGGTGGAGATCA



 



Reverse primer sequence:



GATAGCGCAGTCGGTGTAGG






Amplicon length: 122

Amplicon sequence:








ACAGTGACGAGGTGGAGATCATCCAAGGCCTCACGCGCATGCCTGGCG


 


GCCGCGACGCACTCAAAAGCAGCGTGGACGCGGTCAAGTACTTTGGGA


 


AGGGCACCTACACCGACTGCGCTATC






7. Decorin (DCN)

Accession number: NM 001920
Sequence:







>gi|47419925|ref|NM_001920.3| Homo sapiens


decorin (DCN), transcript variant A1, mRNA


GAATCTACAATAAGACAAATTTCAAATCAAGTTGCTCCACTATACTGC


 


ATAAGCAGTTTAGAATCTTAAGCAGATGCAAAAAGAATAAAGCAAATG


 


GGAGGAAAAAAAAGGCCGATAAAGTTTCTGGCTACAATACAAGAGACA


 


TATCATTACCATATGATCTAATGTGGGTGTCAGCCGGATTGTGTTCAT


 


TGAGGGAAACCTTATTTTTTAACTGTGCTATGGAGTAGAAGCAGGAGG


 


TTTTCAACCTAGTCACAGAGCAGCACCTACCCCCTCCTCCTTTCCACA


 


CCTGCAAACTCTTTTACTTGGGCTGAATATTTAGTGTAATTACATCTC


 


AGCTTTGAGGGCTCCTGTGGCAAATTCCCGGATTAAAAGGTTCCCTGG


 


TTGTGAAAATACATGAGATAAATCATGAAGGCCACTATCATCCTCCTT


 


CTGCTTGCACAAGTTTCCTGGGCTGGACCGTTTCAACAGAGAGGCTTA


 


TTTGACTTTATGCTAGAAGATGAGGCTTCTGGGATAGGCCCAGAAGTT


 


CCTGATGACCGCGACTTCGAGCCCTCCCTAGGCCCAGTGTGCCCCTTC


 


CGCTGTCAATGCCATCTTCGAGTGGTCCAGTGTTCTGATTTGGGTCTG


 


GACAAAGTGCCAAAGGATCTTCCCCCTGACACAACTCTGCTAGACCTG


 


CAAAACAACAAAATAACCGAAATCAAAGATGGAGACTTTAAGAACCTG


 


AAGAACCTTCACGCATTGATTCTTGTCAACAATAAAATTAGCAAAGTT


 


AGTCCTGGAGCATTTACACCTTTGGTGAAGTTGGAACGACTTTATCTG


 


TCCAAGAATCAGCTGAAGGAATTGCCAGAAAAAATGCCCAAAACTCTT


 


CAGGAGCTGCGTGCCCATGAGAATGAGATCACCAAAGTGCGAAAAGTT


 


ACTTTCAATGGACTGAACCAGATGATTGTCATAGAACTGGGCACCAAT


 


CCGCTGAAGAGCTCAGGAATTGAAAATGGGGCTTTCCAGGGAATGAAG


 


AAGCTCTCCTACATCCGCATTGCTGATACCAATATCACCAGCATTCCT


 


CAAGGTCTTCCTCCTTCCCTTACGGAATTACATCTTGATGGCAACAAA


 


ATCAGCAGAGTTGATGCAGCTAGCCTGAAAGGACTGAATAATTTGGCT


 


AAGTTGGGATTGAGTTTCAACAGCATCTCTGCTGTTGACAATGGCTCT


 


CTGGCCAACACGCCTCATCTGAGGGAGCTTCACTTGGACAACAACAAG


 


CTTACCAGAGTACCTGGTGGGCTGGCAGAGCATAAGTACATCCAGGTT


 


GTCTACCTTCATAACAACAATATCTCTGTAGTTGGATCAAGTGACTTC


 


TGCCCACCTGGACACAACACCAAAAAGGCTTCTTATTCGGGTGTGAGT


 


CTTTTCAGCAACCCGGTCCAGTACTGGGAGATACAGCCATCCACCTTC


 


AGATGTGTCTACGTGCGCTCTGCCATTCAACTCGGAAACTATAAGTAA


 


TTCTCAAGAAAGCCCTCATTTTTATAACCTGGCAAAATCTTGTTAATG


 


TCATTGCTAAAAAATAAATAAAAGCTAGATACTGGAAACCTAACTGCA


 


ATGTGGATGTTTTACCCACATGACTTATTATGCATAAAGCCAAATTTC


 


CAGTTTAAGTAATTGCCTACAATAAAAAGAAATTTTGCCTGCCATTTT


 


CAGAATCATCTTTTGAAGCTTTCTGTTGATGTTAACTGAGCTACTAGA


 


GATATTCTTATTTCACTAAATGTAAAATTTGGAGTAAATATATATGTC


 


AATATTTAGTAAAGCTTTTCTTTTTTAATTTCCAGGAAAAAATAAAAA


 


GAGTATGAGTCTTCTGTAATTCATTGAGCAGTTAGCTCATTTGAGATA


 


AAGTCAAATGCCAAACACTAGCTCTGTATTAATCCCCATCATTACTGG


 


TAAAGCCTCATTTGAATGTGTGAATTCAATACAGGCTATGTAAAATTT


 


TTACTAATGTCATTATTTTGAAAAAATAAATTTAAAAATACATTCAAA


 


ATTACTATTGTATACAAGCTTAATTGTTAATATTCCCTAAACACAATT


 


TTATGAAGGGAGAAGACATTGGTTTGTTGACAATAACAGTACATCTTT


 


TCAAGTTCTCAGCTATTTCTTCTACCTCTCCCTATCTTACATTTGAGT


 


ATGGTAACTTATGTCATCTATGTTGAATGTAAGCTTATAAAGCACAAA


 


GCATACATTTCCTGACTGGTCTAGAGAACTGATGTTTCAATTTACCCC


 


TCTGCTAAATAAATATTAAAACTATCATGTGAAAAAAAAAAAAAAAA


 


AA






Primers used:









Forward primer sequence:



AGTTGGAACGACTTTATCTGTCC



 



Reverse primer sequence:



GTGCCCAGTTCTATGACAATCA






Amplicon length: 160
Amplicon sequence:







AGTTGGAACGACTTTATCTGTCCAAGAATCAGCTGAAGGAATTGCCAG


 


AAAAAATGCCCAAAACTCTTCAGGAGCTGCGTGCCCATGAGAATGAGA


 


TCACCAAAGTGCGAAAAGTTACTTTCAATGGACTGAACCAGATGATTG


 


TCATAGAACTGGGCAC






8. Plasminogen Activator, Tissue (PLAT)

Accession number: N—000930
Sequence:







ATGGCCCTGTCCACTGAGCATCCTCCCGCCACACAGAAACCCGCCCAG


 


CCGGGGCCACCGACCCCACCCCCTGCCTGGAAACTTAAAGGAGGCCGG


 


AGCTGTGGGGAGCTCAGAGCTGAGATCCTACAGGAGTCCAGGGCTGGA


 


GAGAAAACCTCTGCGAGGAAAGGGAAGGAGCAAGCCGTGAATTTAAGG


 


GACGCTGTGAAGCAATCATGGATGCAATGAAGAGAGGGCTCTGCTGTG


 


TGCTGCTGCTGTGTGGAGCAGTCTTCGTTTCGCCCAGCCAGGAAATCC


 


ATGCCCGATTCAGAAGAGGAGCCAGATCTTACCAAGTGATCTGCAGAG


 


ATGAAAAAACGCAGATGATATACCAGCAACATCAGTCATGGCTGCGCC


 


CTGTGCTCAGAAGCAACCGGGTGGAATATTGCTGGTGCAACAGTGGCA


 


GGGCACAGTGCCACTCAGTGCCTGTCAAAAGTTGCAGCGAGCCAAGGT


 


GTTTCAACGGGGGCACCTGCCAGCAGGCCCTGTACTTCTCAGATTTCG


 


TGTGCCAGTGCCCCGAAGGATTTGCTGGGAAGTGCTGTGAAATAGATA


 


CCAGGGCCACGTGCTACGAGGACCAGGGCATCAGCTACAGGGGCACGT


 


GGAGCACAGCGGAGAGTGGCGCCGAGTGCACCAACTGGAACAGCAGCG


 


CGTTGGCCCAGAAGCCCTACAGCGGGCGGAGGCCAGACGCCATCAGGC


 


TGGGCCTGGGGAACCACAACTACTGCAGAAACCCAGATCGAGACTCAA


 


AGCCCTGGTGCTACGTCTTTAAGGCGGGGAAGTACAGCTCAGAGTTCT


 


GCAGCACCCCTGCCTGCTCTGAGGGAAACAGTGACTGCTACTTTGGGA


 


ATGGGTCAGCCTACCGTGGCACGCACAGCCTCACCGAGTCGGGTGCCT


 


CCTGCCTCCCGTGGAATTCCATGATCCTGATAGGCAAGGTTTACACAG


 


CACAGAACCCCAGTGCCCAGGCACTGGGCCTGGGCAAACATAATTACT


 


GCCGGAATCCTGATGGGGATGCCAAGCCCTGGTGCCACGTGCTGAAGA


 


ACCGCAGGCTGACGTGGGAGTACTGTGATGTGCCCTCCTGCTCCACCT


 


GCGGCCTGAGACAGTACAGCCAGCCTCAGTTTCGCATCAAAGGAGGGC


 


TCTTCGCCGACATCGCCTCCCACCCCTGGCAGGCTGCCATCTTTGCCA


 


AGCACAGGAGGTCGCCCGGAGAGCGGTTCCTGTGCGGGGGCATACTCA


 


TCAGCTCCTGCTGGATTCTCTCTGCCGCCCACTGCTTCCAGGAGAGGT


 


TTCCGCCCCACCACCTGACGGTGATCTTGGGCAGAACATACCGGGTGG


 


TCCCTGGCGAGGAGGAGCAGAAATTTGAAGTCGAAAAATACATTGTCC


 


ATAAGGAATTCGATGATGACACTTACGACAATGACATTGCGCTGCTGC


 


AGCTGAAATCGGATTCGTCCCGCTGTGCCCAGGAGAGCAGCGTGGTCC


 


GCACTGTGTGCCTTCCCCCGGCGGACCTGCAGCTGCCGGACTGGACGG


 


AGTGTGAGCTCTCCGGCTACGGCAAGCATGAGGCCTTGTCTCCTTTCT


 


ATTCGGAGCGGCTGAAGGAGGCTCATGTCAGACTGTACCCATCCAGCC


 


GCTGCACATCACAACATTTACTTAACAGAACAGTCACCGACAACATGC


 


TGTGTGCTGGAGACACTCGGAGCGGCGGGCCCCAGGCAAACTTGCACG


 


ACGCCTGCCAGGGCGATTCGGGAGGCCCCCTGGTGTGTCTGAACGATG


 


GCCGCATGACTTTGGTGGGCATCATCAGCTGGGGCCTGGGCTGTGGAC


 


AGAAGGATGTCCCGGGTGTGTACACCAAGGTTACCAACTACCTAGACT


 


GGATTCGTGACAACATGCGACCGTGACCAGGAACACCCGACTCCTCAA


 


AAGCAAATGAGATCCCGCCTCTTCTTCTTCAGAAGACACTGCAAAGGC


 


GCAGTGCTTCTCTACAGACTTCTCCAGACCCACCACACCGCAGAAGCG


 


GGACGAGACCCTACAGGAGAGGGAAGAGTGCATTTTCCCAGATACTTC


 


CCATTTTGGAAGTTTTCAGGACTTGGTCTGATTTCAGGATACTCTGTC


 


AGATGGGAAGACATGAATGCACACTAGCCTCTCCAGGAATGCCTCCTC


 


CCTGGGCAGAAAGTGGCCATGCCACCCTGTTTTCAGCTAAAGCCCAAC


 


CTCCTGACCTGTCACCGTGAGCAGCTTTGGAAACAGGACCACAAAAAT


 


GAAAGCATGTCTCAATAGTAAAAGATAACAAGATCTTTCAGGAAAGAC


 


GGATTGCATTAGAAATAGACAGTATATTTATAGTCACAAGAGCCCAGC


 


AGGGCCTCAAAGTTGGGGCAGGCTGGCTGGCCCGTCATGTTCCTCAAA


 


AGCACCCTTGACGTCAAGTCTCCTTCCCCTTTCCCCACTCCCTGGCTC


 


TCAGAAGGTATTCCTTTTGTGTACAGTGTGTAAAGTGTAAATCCTTTT


 


TCTTTATAAACTTTAGAGTAGCATGAGAGAATTGTATCATTTGAACAA


 


CTAGGCTTCAGCATATTTATAGCAATCCATGTTAGTTTTTACTTTCTG


 


TTGCCACAACCCTGTTTTATACTGTACTTAATAAATTCAGATATATTT


 


TTCACAGTTTTTCCAAAATCAGAGTGGAATGGTTTTGTTATAGATGCT


 


GTATCCCACTCTTTATTCATGTTCACATTTTAAAATCATTTGGAATTC


 


TGCTTCACTCGCTTAACATATACACAACACCTGTAACATACAAGGCAA


 


TGGGCTAGGTGCTCCAGACCGGGAAAAGGAGGGACAGGAATGCTTGGT


 


CTGATGGGCTAATATGGCATTTAGAGAAGTACCAAGGTACAGTGGAGC


 


CGGTCACAAAAGGGCAGACTTGTAGTAGAATTCAGTTGCAAGAGGGAT


 


TGGGGAATCTTAAGGAAAAAATAGAATCTTAAGGAAAAAATAACTGGG


 


TGAGACGTGGACTGTGGACAGGTGTGGAAAAGGCACTCTCCATGGAGG


 


TATGAATATGTAGAGGGCCAAGAGAGGGGAGTACAGGGAGAAATGAGT


 


TGAGCTTGTCTGAAGTGAACTTCAGGAAGAGGAACATAGGCTGGAATT


 


TAGATTATGGGGGCTCTGAACACCAAACTGAGTTTGGACTTAATTGAC


 


TTCTG






Primers used:










Forward primer sequence:



ACTGCCGGAATCCTGATGG



 



Reverse primer sequence:



TGTGCTTGGCAAAGATGGC






Amplicon length: 201

Amplicon sequence:








ACTGCCGGAATCCTGATGGGGATGCCAAGCCCTGGTGCCACGTGCTGA


 


AGAACCGCAGGCTGACGTGGGAGTACTGTGATGTGCCCTCCTGCTCCA


 


CCTGCGGCCTGAGACAGTACAGCCAGCCTCAGTTTCGCATCAAAGGAG


 


GGCTCTTCGCCGACATCGCCTCCCACCCCTGGCAGGCTGCCATCTTTG


 


CCAAGCACA






9. Lectin, Galactoside-Binding, Soluble, 3 (LGALS3)

Accession number: NM—002306.

Sequence:








GGAGAGGACTGGCTGGGCAGGGGCGCCGCCCCGCCTCGGGAGAGGCGG


 


GCCGGGCGGGGCTGGGAGTATTTGAGGCTCGGAGCCACCGCCCCGCCG


 


GCGCCCGCAGCACCTCCTCGCCAGCAGCCGTCCGGAGCCAGCCAACGA


 


GCGGAAAATGGCAGACAATTTTTCGCTCCATGATGCGTTATCTGGGTC


 


TGGAAACCCAAACCCTCAAGGATGGCCTGGCGCATGGGGGAACCAGCC


 


TGCTGGGGCAGGGGGCTACCCAGGGGCTTCCTATCCTGGGGCCTACCC


 


CGGGCAGGCACCCCCAGGGGCTTATCCTGGACAGGCACCTCCAGGCGC


 


CTACCCTGGAGCACCTGGAGCTTATCCCGGAGCACCTGCACCTGGAGT


 


CTACCCAGGGCCACCCAGCGGCCCTGGGGCCTACCCATCTTCTGGACA


 


GCCAAGTGCCACCGGAGCCTACCCTGCCACTGGCCCCTATGGCGCCCC


 


TGCTGGGCCACTGATTGTGCCTTATAACCTGCCTTTGCCTGGGGGAGT


 


GGTGCCTCGCATGCTGATAACAATTCTGGGCACGGTGAAGCCCAATGC


 


AAACAGAATTGCTTTAGATTTCCAAAGAGGGAATGATGTTGCCTTCCA


 


CTTTAACCCACGCTTCAATGAGAACAACAGGAGAGTCATTGTTTGCAA


 


TACAAAGCTGGATAATAACTGGGGAAGGGAAGAAAGACAGTCGGTTTT


 


CCCATTTGAAAGTGGGAAACCATTCAAAATACAAGTACTGGTTGAACC


 


TGACCACTTCAAGGTTGCAGTGAATGATGCTCACTTGTTGCAGTACAA


 


TCATCGGGTTAAAAAACTCAATGAAATCAGCAAACTGGGAATTTCTGG


 


TGACATAGACCTCACCAGTGCTTCATATACCATGATATAATCTGAAAG


 


GGGCAGATTAAAAAAAAAAAAAGAATCTAAACCTTACATGTGTAAAGG


 


TTTCATGTTCACTGTGAGTGAAAATTTTTACATTCATCAATATCCCTC


 


TTGTAAGTCATCTACTTAATAAATATTACAGTGAATTACCTGTCTCAA


 


TATGTCAAAAAAAAAAAAAAAAAA






Primers used:










Forward primer sequence:



TGCTGATAACAATTCTGGGCAC



 



Reverse primer sequence:



TGAAGCGTGGGTTAAAGTGGA






Amplicon length: 102

Amplicon sequence:








TGCTGATAACAATTCTGGGCACGGTGAAGCCCAATGCAAACAGAATTG


 


CTTTAGATTTCCAAAGAGGGAATGATGTTGCCTTCCACTTTAACCCAC


 


GCTTCA






10. Fatty Acid Binding Protein 7, Brain (FABP7)

Accession number: NM—001446.

Sequence:








GAGGATTGGGAGGAACTCGACCTACTCCGCTAACCCAGTGGCCTGAGC


 


CAATCACAAAGAGGATTGGAGCCTCACTCGAGCGCTCCTTCCCTTCTC


 


CTCTCTCTGTGACAGCCTCTTGGAAAGAGGGACACTGGAGGGGTGTGT


 


TTGCAATTTAAATCACTGGATTTTTGCCCACCCTCTTTCCAAATAAGA


 


AGGCAGGAGCTGCTTGCTGAGGTGTAAAGGGTCTTCTGAGCTGCAGTG


 


GCAATTAGACCAGAAGATCCCCGCTCCTGTCTCTAAAGAGGGGAAAGG


 


GCAAGGATGGTGGAGGCTTTCTGTGCTACCTGGAAGCTGACCAACAGT


 


CAGAACTTTGATGAGTACATGAAGGCTCTAGGCGTGGGCTTTGCCACT


 


AGGCAGGTGGGAAATGTGACCAAACCAACGGTAATTATCAGTCAAGAA


 


GGAGACAAAGTGGTCATCAGGACTCTCAGCACATTCAAGAACACGGAG


 


ATTAGTTTCCAGCTGGGAGAAGAGTTTGATGAAACCACTGCAGATGAT


 


AGAAACTGTAAGTCTGTTGTTAGCCTGGATGGAGACAAACTTGTTCAC


 


ATACAGAAATGGGATGGCAAAGAAACAAATTTTGTAAGAGAAATTAAG


 


GATGGCAAAATGGTTATGACCCTTACTTTTGGTGATGTGGTTGCTGTT


 


CGCCACTATGAGAAGGCATAAAAATGTTCCTGGTCGGGGCTTGGAAGA


 


GCTCTTCAGTTTTTCTGTTTCCTCAAGTCTCAGTGCTATCCTATTACA


 


ACATGGCTGATCATTAATTAGAAGGTTATCCTTGGTGTGGAGGTGGAA


 


AATGGTGATTTAAAAACTTGTTACTCCAAGCAACTTGCCCAATTTTAA


 


TCTGAAAATTTATCATGTTTTATAATTTGAATTAAAGTTTTGTCCCCC


 


CCCCCCTTTTTTTTATAAACAAGTGAATACATTTTATAATTTCTTTTG


 


GAATGTAAATCAAATTTGAATAAAAATCTTACACGTGAAAAAAAA






Primers used:










Forward primer sequence:



CTCTCAGCACATTCAAGAACACG



 



Reverse primer sequence:



GCGAACAGCAACCACATCAC






Amplicon length: 221

Amplicon sequence








CTCTCAGCACATTCAAGAACACGGAGATTAGTTTCCAGCTGGGAGAAG


 


AGTTTGATGAAACCACTGCAGATGATAGAAACTGTAAGTCTGTTGTTA


 


GCCTGGATGGAGACAAACTTGTTCACATACAGAAATGGGATGGCAAAG


 


AAACAAATTTTGTAAGAGAAATTAAGGATGGCAAAATGGTTATGACCC


 


TTACTTTTGGTGATGTGGTTGCTGTTCGC






11. Lysyl Oxidase (LOX)

Accession number: NM—002317

Sequence:








ATTACGTGAACAAATAGCTGAGGGGCGGCCGGGCCAGAACGGCTTGTG


 


TAACTTTGCAAACGTGCCAGAAAGTTTAAAATCTCTCCTCCTTCCTTC


 


ACTCCAGACACTGCCCGCTCTCCGGGACTGCCGCGCCGCTCCCCGTTG


 


CCTTCCAGGACTGAGAAAGGGGAAAGGGAAGGGTGCCACGTCCGAGCA


 


GCCGCCTTGACTGGGGAAGGGTCTGAATCCCACCCTTGGCATTGCTTG


 


GTGGAGACTGAGATACCCGTGCTCCGCTCGCCTCCTTGGTTGAAGATT


 


TCTCCTTCCCTCACGTGATTTGAGCCCCGTTTTTATTTTCTGTGAGCC


 


ACGTCCTCCTCGAGCGGGGTCAATCTGGCAAAAGGAGTGATGCGCTTC


 


GCCTGGACCGTGCTCCTGCTCGGGCCTTTGCAGCTCTGCGCGCTAGTG


 


CACTGCGCCCCTCCCGCCGCCGGCCAACAGCAGCCCCCGCGCGAGCCG


 


CCGGCGGCTCCGGGCGCCTGGCGCCAGCAGATCCAATGGGAGAACAAC


 


GGGCAGGTGTTCAGCTTGCTGAGCCTGGGCTCACAGTACCAGCCTCAG


 


CGCCGCCGGGACCCGGGCGCCGCCGTCCCTGGTGCAGCCAACGCCTCC


 


GCCCAGCAGCCCCGCACTCCGATCCTGCTGATCCGCGACAACCGCACC


 


GCCGCGGCGCGAACGCGGACGGCCGGCTCATCTGGAGTCACCGCTGGC


 


CGCCCCAGGCCCACCGCCCGTCACTGGTTCCAAGCTGGCTACTCGACA


 


TCTAGAGCCCGCGAAGCTGGCGCCTCGCGCGCGGAGAACCAGACAGCG


 


CCGGGAGAAGTTCCTGCGCTCAGTAACCTGCGGCCGCCCAGCCGCGTG


 


GACGGCATGGTGGGCGACGACCCTTACAACCCCTACAAGTACTCTGAC


 


GACAACCCTTATTACAACTACTACGATACTTATGAAAGGCCCAGACCT


 


GGGGGCAGGTACCGGCCCGGATACGGCACTGGCTACTTCCAGTACGGT


 


CTCCCAGACCTGGTGGCCGACCCCTACTACATCCAGGCGTCCACGTAC


 


GTGCAGAAGATGTCCATGTACAACCTGAGATGCGCGGCGGAGGAAAAC


 


TGTCTGGCCAGTACAGCATACAGGGCAGATGTCAGAGATTATGATCAC


 


AGGGTGCTGCTCAGATTTCCCCAAAGAGTGAAAAACCAAGGGACATCA


 


GATTTCTTACCCAGCCGACCAAGATATTCCTGGGAATGGCACAGTTGT


 


CATCAACATTACCACAGTATGGATGAGTTTAGCCACTATGACCTGCTT


 


GATGCCAACACCCAGAGGAGAGTGGCTGAAGGCCACAAAGCAAGTTTC


 


TGTCTTGAAGACACATCCTGTGACTATGGCTACCACAGGCGATTTGCA


 


TGTACTGCACACACACAGGGATTGAGTCCTGGCTGTTATGATACCTAT


 


GGTGCAGACATAGACTGCCAGTGGATTGATATTACAGATGTAAAACCT


 


GGAAACTATATCCTAAAGGTCAGTGTAAACCCCAGCTACCTGGTTCCT


 


GAATCTGACTATACCAACAATGTTGTGCGCTGTGACATTCGCTACACA


 


GGACATCATGCGTATGCCTCAGGCTGCACAATTTCACCGTATTAGAAG


 


GCAAAGCAAAACTCCCAATGGATAAATCAGTGCCTGGTGTTCTGAAGT


 


GGGAAAAAATAGACTAACTTCAGTAGGATTTATGTATTTTGAAAAAGA


 


GAACAGAAAACAACAAAAGAATTTTTGTTTGGACTGTTTTCAATAACA


 


AAGCACATAACTGGATTTTGAACGCTTAAGTCATCATTACTTGGGAAA


 


TTTTTAATGTTTATTATTTACATCACTTTGTGAATTAACACAGTGTTT


 


CAATTCTGTAATTACATATTTGACTCTTTCAAAGAAATCCAAATTTCT


 


CATGTTCCTTTTGAAATTGTAGTGCAAAATGGTCAGTATTATCTAAAT


 


GAATGAGCCAAAATGACTTTGAACTGAAACTTTTCTAAAGTGCTGGAA


 


CTTTAGTGAAACATAATAATAATGGGTTTATATATGTCATAGCATAGA


 


TGAATTTAGAAACAATGCTCCTACTGTTTAAATACATATGGACACATC


 


TGGTGCTGAGAAAGAAACAAACACATTACCATTGGTGTCAAGAAATAT


 


TACTATATAGCAGAGAAATGGCAATACATGTACTCAGATAGTTACATC


 


CCTATATAAAAAGTATGTTTACATTTAAAAAATTAGTAGATAACTTCC


 


TTTCTTTCAAGTGCACAATTTCATTTTGACTTGAGTCAACTTTTGTTT


 


TGGAACAAATTAAGTAAGGGAGCTGCCCAATCCTGTCTGATATTTCTT


 


GAGGCTGCCCTCTATCATTTTATCTTTCCCATGGGCAGAGATGTTGTA


 


AGTGGGATTCTTAATATCACCATTCTTGGGACTGGTATACATAAGGCA


 


GCCGTGAAACTGGAAAGTCATTTTGATGACTGATGTGATACATCCAGA


 


GGTAAAATGCATTTAAACATATTAAAGTATTTGCCAAAGATACAATTT


 


TCTTGCTGACATAAAAATCACACAAACAAGTCCCCCCCAAACCACAAC


 


TGTCTCTCAAATAGCTTAAAAAAATTGAAAAACATTTTAGGATTTTTC


 


AAGTTTTCTAGATTTTAAAAAGATGTTCAGCTATTAGAGGAATGTTAA


 


AAATTTTATATTATCTAGAACACAGGAACATCATCCTGGGTTATTCAG


 


GAATCAGTCACACATGTGTGTGTGTCTGAGATATAGTCTAAATTAGCA


 


AAGCACATAGTATTACATACTTGAGGGGTTGGTGAACAAAGGAAAAAT


 


ATACTTTCTGCAAAACCAAGGACTGTGCTGCGTAATGAGACAGCTGTG


 


ATTTCATTTGAAACTGTGAAACCATGTGCCATAATAGAATTTTGAGAA


 


TTTTGCTTTTACCTAAATTCAAGAAAATGAAATTACACTTTTAAGTTA


 


GTGGTGCTTAAGCATAATTTTTCCTATATTAACCAGTATTAAAATCTC


 


AAGTAAGATTTTCCAGTGCCAGAACATGTTAGGTGGAATTTTAAAAGT


 


GCCTCGGCATCCTGTATTACATGTCATAGAATTGTAAAGTCAACATCA


 


ATTACTAGTAATCATTCTGCACTCACTGGGTGCATAGCATGGTTAGAG


 


GGGCTAGAGATGGACAGTCATCAACTGGCGGATATAGCGGTACATATG


 


ATCCTTAGCCACCAGGGCACAAGCTTACCAGTAGACAATACAGACAGA


 


GCTTTTGTTGAGCTGTAACTGAGCTATGGAATAGCTTCTTTGATGTAC


 


CTCTTTGCCTTAAATTGCTTTTTAGTTCTAAGATTGTAGAATGATCCT


 


TTCAAATTGTAATCTTTTCTAACAGAGATATTTTAATATACTTGCTTT


 


CTTAAAAAACAAAAAAACTACTGTCAGTATTAATACTGAGCCAGACTG


 


GCATCTACAGATTTCAGATCTATCATTTTATTGATTCTTAAGCTTGTA


 


TTAAAAACTAGGCAATATCATCATGGATACATAGGAGAAGACACATTT


 


ACAATCATTCATTGGGCCTTTTATCTGTCTATCCATCCATCATCATTT


 


GAAGGCCTAATATATGCCAAGTACTCACATGGTATGCATTGAGACATA


 


AAAAAGACTGTCTATAACCTCAATAAGTATTAAAAATCCCATTATTAC


 


CCATAAGGTTCATCTTATTTCATTTTTAGGGAATAAAATTACATGTCT


 


ATGAAATTTCAATTTTAAGCACTATTGTTTTTCATGACCATAATTTAT


 


TTTTAAAAATAAATTAAAGGTTAATTATATGCATGTATGTATTTCTAA


 


TAATTAAAAATGTGTTCAATCCCTGAAATGTCTGCCTTTTAAATATAA


 


CACCTACTATTTGGTTAAAAAAAAAAAAAAAAAAAAA






Primers used:










Forward primer sequence:



CAGGGTGCTGCTCAGATTTCC



 



Reverse primer sequence:



GGTAATGTTGATGACAACTGTGC






Amplicon length: 110

Amplicon sequence:








CAGGGTGCTGCTCAGATTTCCCCAAAGAGTGAAAAACCAAGGGACATCAG


 


ATTTCTTACCCAGCCGACCAAGATATTCCTGGGAATGGCACAGTTGTCAT


 


CAACATTACC






12. Laminin, Beta 1 (LAMB1)

Accession number: NM—002291

Sequence:








GGGACCTGGAAGCGCCCCAGCCCCGCAGCGATCGCAGATTCGGCTTTCAA


 


ACAAAAGAGGCGCCCCGGGGGGTGGGACCGGGACCTCACCCGGTCCTCGC


 


AGAGTTGCGGCCGCCCGCCCCTTCAGCCCCGGCTCTCCGTATGCGCATGA


 


GCAGAGGCGCCTCCCTCTGTTCCTCCCAAGGCTAAACTTTCTAATTCCCT


 


TCTTTGGGCTCGGGGGCTCCCGGAGCAGGGCGAGAGCTCGCGTCGCCGGA


 


AAGGAAGACGGGAAGAAAGGGCAGGCGGCTCGGCGGGCGTCTTCTCCACT


 


CCTCTGCCGCGTCCCCGTGGCTGCAGGGAGCCGGCATGGGGCTTCTCCAG


 


TTGCTAGCTTTCAGTTTCTTAGCCCTGTGCAGAGCCCGAGTGCGCGCTCA


 


GGAACCCGAGTTCAGCTACGGCTGCGCAGAAGGCAGCTGCTATCCCGCCA


 


CGGGCGACCTTCTCATCGGCCGAGCACAGAAGCTTTCGGTGACCTCGACG


 


TGCGGGCTGCACAAGCCCGAACCCTACTGTATCGTCAGCCACTTGCAGGA


 


GGACAAAAAATGCTTCATATGCAATTCCCAAGATCCTTATCATGAGACCC


 


TGAATCCTGACAGCCATCTCATTGAAAATGTGGTCACTACATTTGCTCCA


 


AACCGCCTTAAGATTTGGTGGCAATCTGAAAATGGTGTGGAAAATGTAAC


 


TATCCAACTGGATTTGGAAGCAGAATTCCATTTTACTCATCTCATAATGA


 


CTTTCAAGACATTCCGTCCAGCTGCTATGCTGATAGAACGATCGTCCGAC


 


TTTGGGAAAACCTGGGGTGTGTATAGATACTTCGCCTATGACTGTGAGGC


 


CTCGTTTCCAGGCATTTCAACTGGCCCCATGAAAAAAGTCGATGACATAA


 


TTTGTGATTCTCGATATTCTGACATTGAACCCTCAACTGAAGGAGAGGTG


 


ATATTTCGTGCTTTAGATCCTGCTTTCAAAATAGAAGATCCTTATAGCCC


 


AAGGATACAGAATTTATTAAAAATTACCAACTTGAGAATCAAGTTTGTGA


 


AACTGCATACTTTGGGAGATAACCTTCTGGATTCCAGGATGGAAATCAGA


 


GAAAAGTATTATTATGCAGTTTATGATATGGTGGTTCGAGGAAATTGCTT


 


CTGCTATGGTCATGCCAGCGAATGTGCCCCTGTGGATGGATTCAATGAAG


 


AAGTGGAAGGAATGGTTCACGGACACTGCATGTGCAGGCATAACACCAAG


 


GGCTTAAACTGTGAACTCTGCATGGATTTCTACCATGATTTACCTTGGAG


 


ACCTGCTGAAGGCCGAAACAGCAACGCCTGTAAAAAATGTAACTGCAATG


 


AACATTCCATCTCTTGTCACTTTGACATGGCTGTTTACCTGGCCACGGGG


 


AACGTCAGCGGAGGCGTGTGTGATGACTGTCAGCACAACACCATGGGGCG


 


CAACTGTGAGCAGTGCAAGCCGTTTTACTACCAGCACCCAGAGAGGGACA


 


TCCGAGATCCTAATTTCTGTGAACGATGTACGTGTGACCCAGCTGGCTCT


 


CAAAATGAGGGAATTTGTGACAGCTATACTGATTTTTCTACTGGTCTCAT


 


TGCTGGCCAGTGTCGGTGTAAATTAAATGTGGAAGGAGAACATTGTGATG


 


TTTGCAAAGAAGGCTTCTATGATTTAAGCAGTGAAGATCCATTTGGTTGT


 


AAATCTTGTGCTTGCAATCCTCTGGGAACAATTCCTGGAGGGAATCCTTG


 


TGATTCCGAGACAGGTCACTGCTACTGCAAGCGTCTGGTGACAGGACAGC


 


ATTGTGACCAGTGCCTGCCAGAGCACTGGGGCTTAAGCAATGATTTGGAT


 


GGATGTCGACCATGTGACTGTGACCTTGGGGGAGCCTTAAACAACAGTTG


 


CTTTGCGGAGTCAGGCCAGTGCTCATGCCGGCCTCACATGATTGGACGTC


 


AGTGCAACGAAGTGGAACCTGGTTACTACTTTGCCACCCTGGATCACTAC


 


CTCTATGAAGCGGAGGAAGCCAACTTGGGGCCTGGGGTTAGCATAGTGGA


 


GCGGCAATATATCCAGGACCGGATTCCCTCCTGGACTGGAGCCGGCTTCG


 


TCCGAGTGCCTGAAGGGGCTTATTTGGAGTTTTTCATTGACAACATACCA


 


TATTCCATGGAGTACGACATCCTAATTCGCTACGAGCCACAGCTACCCGA


 


CCACTGGGAAAAAGCTGTCATCACAGTGCAGCGACCTGGAAGGATTCCAA


 


CCAGCAGCCGATGTGGTAATACCATCCCCGATGATGACAACCAGGTGGTG


 


TCATTATCACCAGGCTCAAGATATGTCGTCCTTCCTCGGCCGGTGTGCTT


 


TGAGAAGGGAACAAACTACACGGTGAGGTTGGAGCTGCCTCAGTACACCT


 


CCTCTGATAGCGACGTGGAGAGCCCCTACACGCTGATCGATTCTCTTGTT


 


CTCATGCCATACTGTAAATCACTGGACATCTTCACCGTGGGAGGTTCAGG


 


AGATGGGGTGGTCACCAACAGTGCCTGGGAAACCTTTCAGAGATACCGAT


 


GTCTAGAGAACAGCAGAAGCGTTGTGAAAACACCGATGACAGATGTTTGC


 


AGAAACATCATCTTTAGCATTTCTGCCCTGTTACACCAGACAGGCCTGGC


 


TTGTGAATGCGACCCTCAGGGTTCGTTAAGTTCCGTGTGTGATCCCAACG


 


GAGGCCAGTGCCAGTGCCGGCCCAACGTGGTTGGAAGAACCTGCAACAGA


 


TGTGCACCTGGAACTTTTGGCTTTGGCCCCAGTGGATGCAAACCTTGTGA


 


GTGCCATCTGCAAGGATCTGTCAATGCCTTCTGCAATCCCGTCACTGGCC


 


AGTGCCACTGTTTCCAGGGAGTGTATGCTCGGCAGTGTGATCGGTGCTTA


 


CCTGGGCACTGGGGCTTTCCAAGTTGCCAGCCCTGCCAGTGCAATGGCCA


 


CGCCGATGACTGCGACCCAGTGACTGGGGAGTGCTTGAACTGCCAGGACT


 


ACACCATGGGTCATAACTGTGAAAGGTGCTTGGCTGGTTACTATGGCGAC


 


CCCATCATTGGGTCAGGAGATCACTGCCGCCCTTGCCCTTGCCCAGATGG


 


TCCCGACAGTGGACGCCAGTTTGCCAGGAGCTGCTACCAAGATCCTGTTA


 


CTTTACAGCTTGCCTGTGTTTGTGATCCTGGATACATTGGTTCCAGATGT


 


GACGACTGTGCCTCAGGATACTTTGGCAATCCATCAGAAGTTGGGGGGTC


 


GTGTCAGCCTTGCCAGTGTCACAACAACATTGACACGACAGACCCAGAAG


 


CCTGTGACAAGGAGACTGGGAGGTGTCTCAAGTGCCTGTACCACACGGAA


 


GGGGAACACTGTCAGTTCTGCCGGTTTGGATACTATGGTGATGCCCTCCA


 


GCAGGACTGTCGAAAGTGTGTCTGTAATTACCTGGGCACCGTGCAAGAGC


 


ACTGTAACGGCTCTGACTGCCAGTGCGACAAAGCCACTGGTCAGTGCTTG


 


TGTCTTCCTAATGTGATCGGGCAGAACTGTGACCGCTGTGCGCCCAATAC


 


CTGGCAGCTGGCCAGTGGCACTGGCTGTGACCCATGCAACTGCAATGCTG


 


CTCATTCCTTCGGGCCATCTTGCAATGAGTTCACGGGGCAGTGCCAGTGC


 


ATGCCTGGGTTTGGAGGCCGCACCTGCAGCGAGTGCCAGGAACTCTTCTG


 


GGGAGACCCCGACGTGGAGTGCCGAGCCTGTGACTGTGACCCCAGGGGCA


 


TTGAGACGCCACAGTGTGACCAGTCCACGGGCCAGTGTGTCTGCGTTGAG


 


GGTGTTGAGGGTCCACGCTGTGACAAGTGCACGCGAGGGTACTCGGGGGT


 


CTTCCCTGACTGCACACCCTGCCACCAGTGCTTTGCTCTCTGGGATGTGA


 


TCATTGCCGAGCTGACCAACAGGACACACAGATTCCTGGAGAAAGCCAAG


 


GCCTTGAAGATCAGTGGTGTGATCGGGCCTTACCGTGAGACTGTGGACTC


 


GGTGGAGAGGAAAGTCAGCGAGATAAAAGACATCCTGGCGCAGAGCCCCG


 


CAGCAGAGCCACTGAAAAACATTGGGAATCTCTTTGAGGAAGCAGAGAAA


 


CTGATTAAAGATGTTACAGAAATGATGGCTCAAGTAGAAGTGAAATTATC


 


TGACACAACTTCCCAAAGCAACAGCACAGCCAAAGAACTGGATTCTCTAC


 


AGACAGAAGCCGAAAGCCTAGACAACACTGTGAAAGAACTTGCTGAACAA


 


CTGGAATTTATCAAAAACTCAGATATTCGGGGTGCCTTGGATAGCATTAC


 


CAAGTATTTCCAGATGTCTCTTGAGGCAGAGGAGAGGGTGAATGCCTCCA


 


CCACAGAACCCAACAGCACTGTGGAGCAGTCAGCCCTCATGAGAGACAGA


 


GTAGAAGACGTGATGATGGAGCGGAAATCCCAGTTCAAGGAAAAACAAGA


 


GGAGCAGGCTCGCCTCCTTGATGAACTGGCAGGCAAGCTACAAAGCCTAG


 


ACCTTTCAGCCGCTGCCGAAATGACCTGTGGAACACCCCCAGGGGCCTCC


 


TGTTCCGAGACTGAATGTGGCGGGCCAAACTGCAGAACTGACGAAGGAGA


 


GAGGAAGTGTGGGGGGCCTGGCTGTGGTGGTCTGGTTACTGTTGCACACA


 


ACGCCTGGCAGAAAGCCATGGACTTGGACCAAGATGTCCTGAGTGCCCTG


 


GCTGAAGTGGAACAGCTCTCCAAGATGGTCTCTGAAGCAAAACTGAGGGC


 


AGATGAGGCAAAACAAAGTGCTGAAGACATTCTGTTGAAGACAAATGCTA


 


CCAAAGAAAAAATGGACAAGAGCAATGAGGAGCTGAGAAATCTAATCAAG


 


CAAATCAGAAACTTTTTGACCCAGGATAGTGCTGATTTGGACAGCATTGA


 


AGCAGTTGCTAATGAAGTATTGAAAATGGAGATGCCTAGCACCCCACAGC


 


AGTTACAGAACTTGACAGAAGATATACGTGAACGAGTTGAAAGCCTTTCT


 


CAAGTAGAGGTTATTCTTCAGCATAGTGCTGCTGACATTGCCAGAGCTGA


 


GATGTTGTTAGAAGAAGCTAAAAGAGCAAGCAAAAGTGCAACAGATGTTA


 


AAGTCACTGCAGATATGGTAAAGGAAGCTCTGGAAGAAGCAGAAAAGGCC


 


CAGGTCGCAGCAGAGAAGGCAATTAAACAAGCAGATGAAGACATTCAAGG


 


AACCCAGAACCTGTTAACTTCGATTGAGTCTGAAACAGCAGCTTCTGAGG


 


AAACCTTGTTCAACGCGTCCCAGCGCATCAGCGAGTTAGAGAGGAATGTG


 


GAAGAACTTAAGCGGAAAGCTGCCCAAAACTCCGGGGAGGCAGAATATAT


 


TGAAAAAGTAGTATATACTGTGAAGCAAAGTGCAGAAGATGTTAAGAAGA


 


CTTTAGATGGTGAACTTGATGAAAAGTATAAAAAAGTAGAAAATTTAATT


 


GCCAAAAAAACTGAAGAGTCAGCTGATGCCAGAAGGAAAGCCGAAATGCT


 


ACAAAATGAAGCAAAAACTCTTTTAGCTCAAGCAAATAGCAAGCTGCAAC


 


TGCTCAAAGATTTAGAAAGAAAATATGAAGACAATCAAAGATACTTAGAA


 


GATAAAGCTCAAGAATTAGCAAGACTGGAAGGAGAAGTCCGTTCACTCCT


 


AAAGGATATAAGCCAGAAAGTTGCTGTGTATAGCACATGCTTGTAACAGA


 


GGAGAATAAAAAATGGCTGAGGTGAACAAGGTAAAACAACTACATTTTAA


 


AAACTGACTTAATGCTCTTCAAAATAAAACATCACCTATTTAATGTTTTT


 


AATCACATTTTGTATGGAGTTAAATAAAGTACAGTGCTTTTGTATAAAAA


 


AAAAAAAAAAAAAAAA






Primers used:









Forward primer sequence:
ACAAGCCCGAACCCTACTGTA


 


Reverse primer sequence:
GACCACATTTTCAATGAGATGGC






Amplicon length: 125

Amplicon sequence:








ACAAGCCCGAACCCTACTGTATCGTCAGCCACTTGCAGGAGGACAAAAAA


 


TGCTTCATATGCAATTCCCAAGATCCTTATCATGAGACCCTGAATCCTGA


 


CAGCCATCTCATTGAAAATG






13. Insulin-Like Growth Factor Binding Protein 3 (IGFBP3)

Accession number: NM—000598.

Sequence:








AGATGCGAGCACTGCGGCTGGGCGCTGAGGATCAGCCGCTTCCTGCCTGG


 


ATTCCACAGCTTCGCGCCGTGTACTGTCGCCCCATCCCTGCGCGCCCAGC


 


CTGCCAAGCAGCGTGCCCCGGTTGCAGGCGTCATGCAGCGGGCGCGACCC


 


ACGCTCTGGGCCGCTGCGCTGACTCTGCTGGTGCTGCTCCGCGGGCCGCC


 


GGTGGCGCGGGCTGGCGCGAGCTCGGCGGGCTTGGGTCCCGTGGTGCGCT


 


GCGAGCCGTGCGACGCGCGTGCACTGGCCCAGTGCGCGCCTCCGCCCGCC


 


GTGTGCGCGGAGCTGGTGCGCGAGCCGGGCTGCGGCTGCTGCCTGACGTG


 


CGCACTGAGCGAGGGCCAGCCGTGCGGCATCTACACCGAGCGCTGTGGCT


 


CCGGCCTTCGCTGCCAGCCGTCGCCCGACGAGGCGCGACCGCTGCAGGCG


 


CTGCTGGACGGCCGCGGGCTCTGCGTCAACGCTAGTGCCGTCAGCCGCCT


 


GCGCGCCTACCTGCTGCCAGCGCCGCCAGCTCCAGGAAATGCTAGTGAGT


 


CGGAGGAAGACCGCAGCGCCGGCAGTGTGGAGAGCCCGTCCGTCTCCAGC


 


ACGCACCGGGTGTCTGATCCCAAGTTCCACCCCCTCCATTCAAAGATAAT


 


CATCATCAAGAAAGGGCATGCTAAAGACAGCCAGCGCTACAAAGTTGACT


 


ACGAGTCTCAGAGCACAGATACCCAGAACTTCTCCItCGAGTCCAAGCGG


 


GAGACAGAATATGGTCCCTGCCGTAGAGAAATGGAAGACACACTGAATCA


 


CCTGAAGTTCCTCAATGTGCTGAGTCCCAGGGGTGTACACATTCCCAACT


 


GTGACAAGAAGGGATTTTATAAGAAAAAGCAGTGTCGCCCTTCCAAAGGC


 


AGGAAGCGGGGCTTCTGCTGGTGTGTGGATAAGTATGGGCAGCCTCTCCC


 


AGGCTACACCACCAAGGGGAAGGAGGACGTGCACTGCTACAGCATGCAGA


 


GCAAGTAGACGCCTGCCGCAAGGTTAATGTGGAGCTCAAATATGCCTTAT


 


TTTGCACAAAAGACTGCCAAGGACATGACCAGCAGCTGGCTACAGCCTCG


 


ATTTATATTTCTGTTTGTGGTGAACTGATTTTTTTTAAACCAAAGTTTAG


 


AAAGAGGTTTTTGAAATGCCTATGGTTTCTTTGAATGGTAAACTTGAGCA


 


TCTTTTCACTTTCCAGTAGTCAGCAAAGAGCAGTTTGAATTTTCTTGTCG


 


CTTCCTATCAAAATATTCAGAGACTCGAGCACAGCACCCAGACTTCATGC


 


GCCCGTGGAATGCTCACCACATGTTGGTCGAAGCGGCCGACCACTGACTT


 


TGTGACTTAGGCGGCTGTGTTGCCTATGTAGAGAACACGCTTCACCCCCA


 


CTCCCCGTACAGTGCGCACAGGCTTTATCGAGAATAGGAAAACCTTTAAA


 


CCCCGGTCATCCGGACATCCCAACGCATGCTCCTGGAGCTCACAGCCTTC


 


TGTGGTGTCATTTCTGAAACAAGGGCGTGGATCCCTCAACCAAGAAGAAT


 


GTTTATGTCTTCAAGTGACCTGTACTGCTTGGGGACTATTGGAGAAAATA


 


AGGTGGAGTCCTACTTGTTTAAAAAATATGTATCTAAGAATGTTCTAGGG


 


CACTCTGGGAACCTATAAAGGCAGGTATTTCGGGCCCTCCTCTTCAGGAA


 


TCTTCCTGAAGACATGGCCCAGTCGAAGGCCCAGGATGGCTTTTGCTGCG


 


GCCCCGTGGGGTAGGAGGGACAGAGAGACAGGGAGAGTCAGCCTCCACAT


 


TCAGAGGCATCACAAGTAATGGCACAATTCTTCGGATGACTGCAGAAAAT


 


AGTGTTTTGTAGTTCAACAACTCAAGACGAAGCTTATTTCTGAGGATAAG


 


CTCTTTAAAGGCAAAGCTTTATTTTCATCTCTCATCTTTTGTCCTCCTTA


 


GCACAATGTAAAAAAGAATAGTAATATCAGAACAGGAAGGAGGAATGGCT


 


TGCTGGGGAGCCCATCCAGGACACTGGGAGCACATAGAGATTCACCCATG


 


TTTGTTGAACTTAGAGTCATTCTCATGCTTTTCTTTATAATTCACACATA


 


TATGCAGAGAAGATATGTTCTTGTTAACATTGTATACAACATAGCCCCAA


 


ATATAGTAAGATCTATACTAGATAATCCTAGATGAAATGTTAGAGATGCT


 


ATATGATACAACTGTGGCCATGACTGAGGAAAGGAGCTCACGCCCAGAGA


 


CTGGGCTGCTCTCCCGGAGGCCAAACCCAAGAAGGTCTGGCAAAGTCAGG


 


CTCAGGGAGACTCTGCCCTGCTGCAGACCTCGGTGTGGACACACGCTGCA


 


TAGAGCTCTCCTTGAAAACAGAGGGGTCTCAAGACATTCTGCCTACCTAT


 


TAGCTTTTCTTTATTTTTTTAACTTTTTGGGGGGAAAAGTATTTTTGAGA


 


AGTTTGTCTTGCAATGTATTTATAAATAGTAAATAAAGTTTTTACCATTA


 


AAAAAATATCTTTCCCTTTGTTATTGACCATCTCTGGGCTTTGTATCACT


 


AATTATTTTATTTTATTATATAATAATTATTTTATTATAATAAAATCCTG


 


AAAGGGGAAAATAAAAAAAA






Primers used:









Forward primer sequence:
AGAGCACAGATACCCAGAACT


 


Reverse primer sequence:
TGAGGAACTTCAGGTGATTCAGT






Amplicon length: 105

Amplicon sequence:








AGAGCACAGATACCCAGAACTTCTCCTCCGAGTCCAAGCGGGAGACAGAA


 


TATGGTCCCTGCCGTAGAGAAATGGAAGACACACTGAATCACCTGAAGTT


 


CCTCA






14. Growth Arrest and DNA-Damage-Inducible, Alpha (GADD45A)

Accession number: NM—001924

Sequence:








CAGTGGCTGGTAGGCAGTGGCTGGGAGGCAGCGGCCCAATTAGTGTCGTG


 


CGGCCCGTGGCGAGGCGAGGTCCGGGGAGCGAGCGAGCAAGCAAGGCGGG


 


AGGGGTGGCCGGAGCTGCGGCGGCTGGCACAGGAGGAGGAGCCCGGGCGG


 


GCGAGGGGCGGCCGGAGAGCGCCAGGGCCTGAGCTGCCGGAGCGGCGCCT


 


GTGAGTGAGTGCAGAAAGCAGGCGCCCGCGCGCTAGCCGTGGCAGGAGCA


 


GCCCGCACGCCGCGCTCTCTCCCTGGGCGACCTGCAGTTTGCAATATGAC


 


TTTGGAGGAATTCTCGGCTGGAGAGCAGAAGACCGAAAGGATGGATAAGG


 


TGGGGGATGCCCTGGAGGAAGTGCTCAGCAAAGCCCTGAGTCAGCGCACG


 


ATCACTGTCGGGGTGTACGAAGCGGCCAAGCTGCTCAACGTCGACCCCGA


 


TAACGTGGTGTTGTGCCTGCTGGCGGCGGACGAGGACGACGACAGAGATG


 


TGGCTCTGCAGATCCACTTCACCCTGATCCAGGCGTTTTGCTGCGAGAAC


 


GACATCAACATCCTGCGCGTCAGCAACCCGGGCCGGCTGGCGGAGCTCCT


 


GCTCTTGGAGACCGACGCTGGCCCCGCGGCGAGCGAGGGCGCCGAGCAGC


 


CCCCGGACCTGCACTGCGTGCTGGTGACGAATCCACATTCATCTCAATGG


 


AAGGATCCTGCCTTAAGTCAACTTATTTGTTTTTGCCGGGAAAGTCGCTA


 


CATGGATCAATGGGTTCCAGTGATTAATCTCCCTGAACGGTGATGGCATC


 


TGAATGAAAATAACTGAACCAAATTGCACTGAAGTTTTTGAAATACCTTT


 


GTAGTTACTCAAGCAGTTACTCCCTACACTGATGCAAGGATTACAGAAAC


 


TGATGCCAAGGGGCTGAGTGAGTTCAACTACATGTTCTGGGGGCCCGGAG


 


ATAGATGACTTTGCAGATGGAAAGAGGTGAAAATGAAGAAGGAAGCTGTG


 


TTGAAACAGAAAAATAAGTCAAAAGGAACAAAAATTACAAAGAACCATGC


 


AGGAAGGAAAACTATGTATTAATTTAGAATGGTTGAGTTACATTAAAATA


 


AACCAAATATGTTAAAGTTTAAGTGTGCAGCCATAGTTTGGGTATTTTTG


 


GTTTATATGCCCTCAAGTAAAAGAAAAGCCGAAAGGGTTAATCATATTTG


 


AAAACCATATTTTATTGTATTTTGATGAGATATTAAATTCTCAAAGTTTT


 


ATTATAAATTCTACTAAGTTATTTTATGACATGAAAAGTTATTTATGCTA


 


TAAATTTTTTGAAACACAATACCTACAATAAACTGGTATGAATAATTGCA


 


TCATT






Primers used:









Forward primer sequence:
GAGAGCAGAAGACCGAAAGGA


 


Reverse primer sequence:
CACAACACCACGTTATCGGG






Amplicon length: 145

Amplicon sequence:








GAGAGCAGAAGACCGAAAGGATGGATAAGGTGGGGGATGCCCTGGAGGAA


 


GTGCTCAGCAAAGCCCTGAGTCAGCGCACGATCACTGTCGGGGTGTACGA


 


AGCGGCCAAGCTGCTCAACGTCGACCCCGATAACGTGGTGTTGTG






15. Follistatin-Like 1 (FSTL1)

Accession number: BX647421

Sequence:








ATTTCCTCCGAGGCTGGCGATCGGCGGAGCTCCCACCTCCGCTTACAGCT


 


CGCTGCCGCCGTCCTGCCCCGCGCCCCCAGGAGACCTGGACCAGACCACG


 


ATGTGGAAACGCTGGCTCGCGCTCGCGCTCGCGCTGGTGGCGGTCGCCTG


 


GGTCCGCGCCGAGGAAGAGCTAAGGAGCAAATCCAAGATCTGTGCCAATG


 


TGTTTTGTGGAGCCGGCCGGGAATGTGCAGTCACAGAGAAAGGGGAACCC


 


ACCTGTCTCTGCATTGAGCAATGCAAACCTCACAAGAGGCCTGTGTGTGG


 


CAGTAATGGCAAGACCTACCTCAACCACTGTGAACTGCATCGAGATGCCT


 


GCCTCACTGGATCCAAAATCCAGGTTGATTACGATGGACACTGCAAAGAG


 


AAGAAATCCGTAAGTCCATCTGCCAGCCCAGTTGTTTGCTATCAGTCCAA


 


CCGTGATGAGCTCCGACGTCGCATCATCCAGTGGCTGGAAGCTGAGATCA


 


TTCCAGATGGCTGGTTCTCTAAAGGCAGCAACTACAGTGAAATCCTAGAC


 


AAGTATTTTAAGAACTTTGATAATGGTGATTCTCGCCTGGACTCCAGTGA


 


ATTCCTGAAGTTTGTGGAACAGAATGAAACTGCCATCAATATTACAACGT


 


ATCCAGACCAGGAGAACAACGAGTTGCTTAGGGGACTCTGTGTTGATGCT


 


CTCATTGAACTGTCTGATGAAAATGCTGATTGGAAACTCAGCTTCCAAGA


 


GTTTCTCAAGTGCCTCAACCCATCTTTCAACCCTCCTGAGAAGAAGTGTG


 


CCCTGGAGGATGAAACGTATGCAGATGGAGCTGAGACCGAGGTGGACTGT


 


AACCGCTGTGTCTGTGCCTGTGGAAATTGGGTCTGTACAGCCATGACCTG


 


TGACGGAAAGAATCAGAAGGGGGCCCAGACCCAGACAGAGGAGGAGATGA


 


CCAGATATGTCCAGGAGCTCCAAAAGCATCAGGAAACAGCTGAAAAGACC


 


AAGAGAGTGAGCACCAAAGAGATCTAATGAGGAGGCACAGACCAGTGTCT


 


GGATCCCAGCATCTTCTCCACTTCAGCGCTGAGTTCAGTATACACAAGTG


 


TCTGCTACAGTCGCCAAATCACCAGTATTTGCTTATATAGCAATGAGTTT


 


TATTTTGTTTATTTGTTTTGCAATAAAGGATATGAAGGTGGCTGGCTAGG


 


AAGGGAAGGGCCACAGCCTTCATTTCTAGGAGTGCTTTAAGAGAAACTGT


 


AAATGGTGCTCTGGGGCTGGAGGCTAGTAAGGAAACTGCATCACGATTGA


 


AAGAGGAACAGACCCAAATCTGAACCTCTTTTGAGTTTACTGCATCTGTC


 


AGCAGGCTGCAGGGAGTGCACACGATGCCAGAGAGAACTTAGCAGGGTGT


 


CCCCGGAGGAGAGGTTTGGGAAGCTCCACGGAGAGGAACGCTCTCTGCTT


 


CCAGCCTCTTTCCATTGCCGTCAGCATGACAGACCTCCAGCATCCACGCA


 


TCTCTTGGTCCCAATAACTGCCTCTAGATACATAGCCATACTGCTAGTTA


 


ACCCAGTGTCCCTCAGACTTGGATGGAGTTTCTGGGAGGGTACACCCAAA


 


TGATGCAGATACTTGTATACTTTGAGCCCCTTAGCGACCTAACCAAATTT


 


TAAAAATACTTTTTACCAAAGGTGCTATTTCTCTGTAAAACACTTTTTTT


 


TGGCAGGTTGACTTTATTCTTCAATTATTATCATTATATTATTGTTTTTT


 


AATATTTTATTTTCTTGACTAGGTATTAAGCTTTTGTAATTATTTTTCAG


 


TAGTCCCACCACTTCATAGGTGGAAGGAGTTTGGGGTTCTTCCTGGTGCA


 


GGGGCTGAAATAACCCAGATGCCCCCACCCTGCCACATACTAGATGCAGC


 


CCATAGTTGGCCCCCCTAGCTTCCAGCAGTCCACTATCTGCCAGAGGAGC


 


AAGGGTGCCTTAGACCGAAGCCAGGGGAAGAAGCATCTTCATAAAAAACT


 


TTCAAGATCCAAACATTAATTTGTTTTTATTTATTCTGAGAAGTTGAGGC


 


AAATCAGTATTCCCAAGGATGGCGACAAGGGCAGCCAAGCAGGGCTTAGG


 


ATATCCCAGCCTACCAATATGCTCATTCGACTAACTAGGAGGGTGAGTTG


 


GCCCTGTCTCTTCTTTTTTCTGGACCTCAGTTTCCTCAGTGAGCTGGTAA


 


GAATGCACTAACCTTTTGATTTGATAAGTTATAAATTCTGTGGTTCTGAT


 


CATTGGTCCAGAGGGGAGATAGGTTCCTGTGATTTTTCCTTCTTCTCTAT


 


AGAATAAATGAAATCTTGTTACTAGAACAAGAAATGTCAGATGGCCAAAA


 


ACAAGATGACCAGATTTGATCTCAGCCTGATGACCCTACAGGTCGTGCTA


 


TGATATGGAGTCCTCATGGGTAAAGCAGGAAGAGAGTGGGAAAGAGAACC


 


ACCCCACTCTGTCTTCATATTTGCATTTCATGTTTAACCTCCGGCTGGAA


 


ATAGAAAGCATTCCCTTAGAGATGAGGATAAAAGAAAGTTTCAGATTCAA


 


CAGGGGGAAGAAAATGGAGATTTAATCCTAAAACTGTGACTTGGGGAGGT


 


CAGTCATTTACAGTTAGTCCTGTGTCTTTCGACTTCTGTGATTATTAACC


 


CCACTCACTACCCTGTTTCAGATGCATTTGGAATACCAAAGATTAAATCC


 


TTGACATAAGATCTCATTTGCAGAAAGCAGATTAAAGACCATCAGAAGGA


 


AATTATTTAGGTTGTAATGCACAGGCAACTGTGAGAAACTGTTGTGCCAA


 


AAATAGAATTCCTTCTAGTTTTTCTTGTTCTCATTTGAAAGGAGAAAATT


 


CCACTTTGTTTAGCATTTCAAGCTTTTATGTATCCATCCCATCTAAAAAC


 


TCTTCAAACTCCACTTGTTCAGTCTGAAATGCAGCTCCCTGTCCAAGTGC


 


CTTGGAGAACTCACAGCAGCACGCCTTAATCAAAGGTTTTACCAGCCCTT


 


GGACACTATGGGAGGAGGGCAAGAGTACACCAATTTGTTAAAAGCAAGAA


 


ACCACAGTGTCTCTTCACTAGTCATTTAGAACATGGTTATCATCCAAGAC


 


TACTCTACCCTGCAACATTGAACTCCCAAGAGCAAATCCACATTCCTCTT


 


GAGTTCTGCAGCTTCTGTGTAAATAGGGCAGCTGTCGTCTATGCCGTAGA


 


ATCACATGATCTGAGGACCATTCATGGAAGCTGCTAAATAGCCTAGTCTG


 


GGGAGTCTTCCATAAAGTTTTGCATGGAGCAAACAAACAGGATTAAACTA


 


GGTTTGGTTCCTTCAGCCCTCTAAAAGCATAGGGCTTAGCCTGCAGGCTT


 


CCTTGGGCTTTCTCTGTGTGTGTAGTTTTGTAAACACTATAGCATCTGTT


 


AAGATCCAGTGTCCATGGAAACATTCCCACATGCCGTGACTCTGGACTAT


 


ATCAGTTTTTGGAAAGCAGGGTTCCTCTGCCTGCTAACAAGCCCACGTGG


 


ACCAGTCTGAATGTCTTTCCTTTACACCTATGTTTTTAAGTAGTCAAACT


 


TCAAGAAACAATCTAAACAAGTTTCTGTTGCATATGTGTTTGTGAACTTG


 


TATTTGTATTTAGTAGGCTTCTATATTGCATTTAACTTGTTTTTGTAACT


 


CCTGATTCTTCCTTTTCGGATACTATTGATGAATAAAGAAATTAAAGTGA


 


AAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAAAAAAA






Primers used:









Forward primer sequence:
CAACCACTGTGAACTGCATCG


 


Reverse primer sequence:
CCTTTAGAGAACCAGCCATCTG






Amplicon length: 204

Amplicon sequence:








CAACCACTGTGAACTGCATCGAGATGCCTGCCTCACTGGATCCAAAATCC


 


AGGTTGATTACGATGGACACTGCAAAGAGAAGAAATCCGTAAGTCCATCT


 


GCCAGCCCAGTTGTTTGCTATCAGTCCAACCGTGATGAGCTCCGACGTCG


 


CATCATCCAGTGGCTGGAAGCTGAGATCATTCCAGATGGCTGGTTCTCTA


 


AAGG






16. Ras Homolog Gene Family, Member C(RHOC)

Accession number: AK094474

Sequence:








GGAGCCTGTAGCCTTTATTCATGCCCCCCTGACCAAATGCAGTGAGAGAC


 


AAGGCCCCTGCCGAAAACAACTCCAGGGGCCTGGGACTCTGGGTCCCCTA


 


CTGCAGACACTTTCCTGTGAGCCAGAAGTGTATAAAGTGCTGGTGTGTGA


 


CCATCCTTTGGGGAAGGTCAAAGGGGGCAAGATCCCCAGGGGCCCTGAGG


 


AAGGGCAGGGCATAGGCGTGGCTCCCAGAGCGCTGGGAGGGAGGGCCCGT


 


GCCACCACCTCGGGGCTAGAAAACAATGCAGTCCTGGGCAGGAGGGAACT


 


GAAAATGGGAGCCTTCAGCATGGAGCCCTCAGGAGGCTGGGGTTGTAGGG


 


GGATAATTTCTGTACCCCTGTGAAGGGAGGGGGCATGTAGGAAAGGCCTT


 


GGGGATCTCAGAGAATGGGACAGCCCCTCCGACGCTTGTTCTTGCGGACC


 


TGGAGGCCAGCCCGAGTGGCCATCTCAAACACCTCCCGCACTCCCTCCTT


 


GGTCTTGGCTGAGCACTCAAGGTAGCCAAAGGCACTGATCCGGTTCGCCA


 


TGTCCCGGCCTTCCTCAGACCGAACGGGCTCCTGCTTCATCTTGGCCAGC


 


TCTCTCCTGGTGTGCTCGTCTTGCCTCAGGTCCTTCTTATTCCCCACCAG


 


GATGATGGGCACGTTGGGGCAGAAGTGCTTCACCTCTGGGGTCCACTTCT


 


CAGGAATGTTTTCCAGGCTGTCAGGGCTGTCGATGGAGAAGCACATGAGG


 


ATGACATCAGTGTCCGGGTAGGAGAGAGGCCGCAGTCGATCATAGTCTTC


 


CTGCCCTGCTGTGTCCCACAGAGCCAGCTCCACCTGCTTGCCGTCCACCT


 


CAATGTCCGCAATATAGTTCTCAAAGACAGTAGGGACGTAGACCTCCGGA


 


AACTGATCCTTGCTGAAGACGATGAGGAGGCAGGTCTTCCCACAGGCACC


 


ATCCCCAACGATCACCAGCTTCTTTCGGATTGCAGCCATGGTGGGGCTCC


 


AGCCGGCTGAAGTTCCCAGGCTGCAGGAAGAGAGGGCGGGCTCTGGAGCT


 


GAGATGAAGTCAAGGCTGTTGGGAAGGGGGAGGGGGCTAGAGTCTGGGCT


 


GGGAGGAGCCCCAAAAGAAGAGACAAATGAGGGCCAGTCCCAGCACCAAC


 


CAGGCAGGGAGCAGTTAAGAAAGCGACGGTAACCTGATCTCAGCCTCAAA


 


CCTAGCTTTTTCTCTCAGTCCCACATCCTGTCAAACTGGGCTGACTGAAC


 


GCCTCTACTCCCCACACCCCACCACCACCTCACACTGCCCTTTAGGAAGC


 


GAATACTCCAGCCCCAGGCCTCTTCCCTTCAACATAGATCCTGAGTGGCC


 


CTTCCCTTGCCTCCAGACACATTCACAAAACTGTTGGTTTTGTGGACATG


 


AGTCAGAGAATTTACAGGAGTTCAAAGTACACAGCCACACTCTTCCCACC


 


ACAAAACGGACTCTCTCTGATTCCCCAGAAGACAAGCAAGAAGGCATTCA


 


CCCTGTCGGCAGATCGCCTCCAGAAATGGAAACCATCCTCCAAAAAGAGG


 


GTTCCTTGGGAATTCTATCCCGGTGACTGACGCTGGGATTTCTTCCAACT


 


CCTCCACCCACTCCATTAGTTCACCTTGCCCTGTTTTGTAAAGATGGGCT


 


GGGGTAGCCCCAACCTGGGGTGGGCAGTGTTGATGGAGGGCAATCACTAC


 


TGGGGTGAAAGCCAGTCACTTAGGCATGAGTATGCCACTGCTGTCCCCCC


 


AGCAGGGTAATTCAGACGGCACCAGAGTGGTGGGAGGCAGAGGACAGAAA


 


CCCGGGGTTGAGGCATGCGTTAAGGGACCTGGAGCCTCCAGCCCAATTAG


 


AAGACTTTCCCTCCAGGCTATGATTGGGCCAGAACAGCAGGCAGCCCAGG


 


CCAGGACACTAGGCCCAAGGCCAAGATGGCATGGACAACTCCCTGTGAGA


 


GGCAGCCCCAGAGGGACTGTCCCACTGACCCCTTAAGAGGGGCAACTGAG


 


CCCCACACAGGGCCTGGAATGGAGCCTGGAACTTCTGGGGCCTTCCCCAA


 


GACAAGACAGTGTGGATACATCAGACCTCTCTCCAATCGCTCTCTTGAAT


 


TCCCAGATGATCCAGAGCGGCCGGTTGACTTTGCCGGCCCACCCTACACC


 


TTCCGCTCCGCCGCCTCCAGCTGCGCGGCCGGTGCCGGAGGCTCAGACT






Primers used:









Forward primer sequence:
CCTGCCTCCTCATCGTCTTC


 


Reverse primer sequence:
AGCACATGAGGATGACATCAGTG






Amplicon length: 195

Amplicon sequence:








CCTGCCTCCTCATCGTCTTCAGCAAGGATCAGTTTCCGGAGGTCTACG


 


TCCCTACTGTCTTTGAGAACTATATTGCGGACATTGAGGTGGACGGCA


 


AGCAGGTGGAGCTGGCTCTGTGGGACACAGCAGGGCAGGAAGACTATG


 


ATCGACTGCGGCCTCTCTCCTACCCGGACACTGATGTCATCCTCATGT


 


GCT






17. AE Binding Protein 1(AEBP1)

Accession number: NM—001129

Sequence:








CGGCTATCCGCGCGGGAGTGCGCCACGCGGGGCCGGAGCGCCTATTAG


 


CCGCCAGGACCTCGGAGCGCCCCGACCACCCCTGAGCCCCTCTGGCTT


 


CGGAGCCCCCCAGCACCCCTTCCCGGGTCCCCTCGCCCACCCTAATCC


 


ACTCTCCCTCCCTTTCCCGGATTCCCTCGCTCACCCCATCCTCTCTCC


 


CGCCCCTTCCTGGATTCCCTCACCCGTCTCGATCCCCTCTCCGCCCTT


 


TCCCAGAGACCCAGAGCCCCTGACCCCCCGCGCCCTCCCCGGAGCCCC


 


CCGCGCGTGCCGCGGCCATGGCGGCCGTGCGCGGGGCGCCCCTGCTCA


 


GCTGCCTCCTGGCGTTGCTGGCCCTGTGCCCTGGAGGGCGCCCGCAGA


 


CGGTGCTGACCGACGACGAGATCGAGGAGTTCCTCGAGGGCTTCCTGT


 


CAGAGCTAGAACCTGAGCCCCGGGAGGACGACGTGGAGGCCCCGCCGC


 


CTCCCGAGCCCACCCCGCGGGTCCGAAAAGCCCAGGCGGGGGGCAAGC


 


CAGGGAAGCGGCCAGGGACGGCCGCAGAAGTGCCTCCGGAAAAGACCA


 


AAGACAAAGGGAAGAAAGGCAAGAAAGACAAAGGCCCCAAGGTGCCCA


 


AGGAGTCCTTGGAGGGGTCCCCCAGGCCGCCCAAGAAGGGGAAGGAGA


 


AGCCACCCAAGGCCACCAAGAAGCCCAAGGAGAAGCCACCTAAGGCCA


 


CCAAGAAGCCCAAGGAGAAGCCACCCAAGGCCACCAAGAAGCCCAAAG


 


AGAAGCCACCCAAGGCCACCAAGAAGCCCCCGTCAGGGAAGAGGCCCC


 


CCATTCTGGCTCCCTCAGAAACCCTGGAGTGGCCACTGCCCCCACCCC


 


CCAGCCCTGGCCCCGAGGAGCTACCCCAGGAGGGAGGGGCGCCCCTCT


 


CAAATAACTGGCAGAATCCAGGAGAGGAGACCCATGTGGAGGCACGGG


 


AGCACCAGCCTGAGCCGGAGGAGGAGACCGAGCAACCCACACTGGACT


 


ACAATGACCAGATCGAGAGGGAGGACTATGAGGACTTTGAGTACATTC


 


GGCGCCAGAAGCAACCCAGGCCACCCCCAAGCAGAAGGAGGAGGCCCG


 


AGCGGGTCTGGCCAGAGCCCCCTGAGGAGAAGGCCCCGGCCCCAGCCC


 


CGGAGGAGAGGATTGAGCCTCCTGTGAAGCCTCTGCTGCCCCCGCTGC


 


CCCCTGACTATGGTGATGGTTACGTGATCCCCAACTACGATGACATGG


 


ACTATTACTTTGGGCCTCCTCCGCCCCAGAAGCCCGATGCTGAGCGCC


 


AGACGGACGAAGAGAAGGAGGAGCTGAAGAAACCCAAAAAGGAGGACA


 


GCAGCCCCAAGGAGGAGACCGACAAGTGGGCAGTGGAGAAGGGCAAGG


 


ACCACAAAGAGCCCCGAAAGGGCGAGGAGTTGGAGGAGGAGTGGACGC


 


CTACGGAGAAAGTCAAGTGTCCCCCCATTGGGATGGAGTCACACCGTA


 


TTGAGGACAACCAGATCCGAGCCTCCTCCATGCTGCGCCACGGCCTGG


 


GGGCACAGCGCGGCCGGCTCAACATGCAGACCGGTGCCACTGAGGACG


 


ACTACTATGATGGTGCGTGGTGTGCCGAGGACGATGCCAGGACCCAGT


 


GGATAGAGGTGGACACCAGGAGGACTACCCGGTTCACAGGCGTCATCA


 


CCCAGGGCAGAGACTCCAGCATCCATGACGATTTTGTGACCACCTTCT


 


TCGTGGGCTTCAGCAATGACAGCCAGACATGGGTGATGTACACCAACG


 


GCTATGAGGAAATGACCTTTCATGGGAACGTGGACAAGGACACACCCG


 


TGCTGAGTGAGCTCCCAGAGCCGGTGGTGGCTCGTTTCATCCGCATCT


 


ACCCACTCACCTGGAATGGCAGCCTGTGCATGCGCCTGGAGGTGCTGG


 


GGTGCTCTGTGGCCCCTGTCTACAGCTACTACGCACAGAATGAGGTGG


 


TGGCCACCGATGACCTGGATTTCCGGCACCACAGCTACAAGGACATGC


 


GCCAGCTCATGAAGGTGGTGAACGAGGAGTGCCCCACCATCACCCGCA


 


CTTACAGCCTGGGCAAGAGCTCACGAGGCCTCAAGATCTATGCCATGG


 


AGATCTCAGACAACCCTGGGGAGCATGAACTGGGGGAGCCCGAGTTCC


 


GCTACACTGCTGGGATCCATGGCAACGAGGTGCTGGGCCGAGAGCTGT


 


TGCTGCTGCTCATGCAGTACCTGTGCCGAGAGTACCGCGATGGGAACC


 


CACGTGTGCGCAGCCTGGTGCAGGACACACGCATCCACCTGGTGCCCT


 


CACTGAACCCTGATGGCTACGAGGTGGCAGCGCAGATGGGCTCAGAGT


 


TTGGGAACTGGGCGCTGGGACTGTGGACTGAGGAGGGCTTTGACATCT


 


TTGAAGATTTCCCGGATCTCAACTCTGTGCTCTGGGGAGCTGAGGAGA


 


GGAAATGGGTCCCCTACCGGGTCCCCAACAATAACTTGCCCATCCCTG


 


AACGCTACCTTTCGCCAGATGCCACGGTATCCACGGAGGTCCGGGCCA


 


TCATTGCCTGGATGGAGAAGAACCCCTTCGTGCTGGGAGCAAATCTGA


 


ACGGCGGCGAGCGGCTAGTATCCTACCCCTACGATATGGCCCGCACGC


 


CTACCCAGGAGCAGCTGCTGGCCGCAGCCATGGCAGCAGCCCGGGGGG


 


AGGATGAGGACGAGGTCTCCGAGGCCCAGGAGACTCCAGACCACGCCA


 


TCTTCCGGTGGCTTGCCATCTCCTTCGCCTCCGCACACCTCACCTTGA


 


CCGAGCCCTACCGCGGAGGCTGCCAAGCCCAGGACTACACCGGCGGCA


 


TGGGCATCGTCAACGGGGCCAAGTGGAACCCCCGGACCGGGACTATCA


 


ATGACTTCAGTTACCTGCATACCAACTGCCTGGAGCTCTCCTTCTACC


 


TGGGCTGTGACAAGTTCCCTCATGAGAGTGAGCTGCCCCGCGAGTGGG


 


AGAACAACAAGGAGGCGCTGCTCACCTTCATGGAGCAGGTGCACCGCG


 


GCATTAAGGGGGTGGTGACGGACGAGCAAGGCATCCCCATTGCCAACG


 


CCACCATCTCTGTGAGTGGCATTAATCACGGCGTGAAGACAGCCAGTG


 


GTGGTGATTACTGGCGAATCTTGAACCCGGGTGAGTACCGCGTGACAG


 


CCCACGCGGAGGGCTACACCCCGAGCGCCAAGACCTGCAATGTTGACT


 


ATGACATCGGGGCCACTCAGTGCAACTTCATCCTGGCTCGCTCCAACT


 


GGAAGCGCATCCGGGAGATCATGGCCATGAACGGGAACCGGCCTATCC


 


CACACATAGACCCATCGCGCCCTATGACCCCCCAACAGCGACGCCTGC


 


AGCAGCGACGCCTACAACACCGCCTGCGGCTTCGGGCACAGATGCGGC


 


TGCGGCGCCTCAACGCCACCACCACCCTAGGCCCCCACACTGTGCCTC


 


CCACGCTGCCCCCTGCCCCTGCCACCACCCTGAGCACTACCATAGAGC


 


CCTGGGGCCTCATACCGCCAACCACCGCTGGCTGGGAGGAGTCGGAGA


 


CTGAGACCTACACAGAGGTGGTGACAGAGTTTGGGACCGAGGTGGAGC


 


CCGAGTTTGGGACCAAGGTGGAGCCCGAGTTTGAGACCCAGTTGGAGC


 


CTGAGTTTGAGACCCAGCTGGAACCCGAGTTTGAGGAAGAGGAGGAGG


 


AGGAGAAAGAGGAGGAGATAGCCACTGGCCAGGCATTCCCCTTCACAA


 


CAGTAGAGACCTACACAGTGAACTTTGGGGACTTCTGAGATCAGCGTC


 


CTACCAAGACCCCAGCCCAACTCAAGCTACAGCAGCAGCACTTCCCAA


 


GCCTGCTGACCACAGTCACATCACCCATCAGCACATGGAAGGCCCCTG


 


GTATGGACACTGAAAGGAAGGGCTGGTCCTGCCCCTTTGAGGGGGTGC


 


AAACATGACTGGGACCTAAGAGCCAGAGGCTGTGTAGAGGCTCCTGCT


 


CCACCTGCCAGTCTCGTAAGAGATGGGGTTGCTGCAGTGTTGGAGTAG


 


GGGCAGAGGGAGGGAGCCAAGGTCACTCCAATAAAACAAGCTCATGGC


 


AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA






Primers used:










Forward primer sequence:



AAAGGGCGAGGAGTTGGAG



 



Reverse primer sequence:



GAGGCTCGGATCTGGTTGT Amplicon length: 105






Amplicon sequence:










AAAGGGC GAGGAGTTGG AGGAGGAGTG GACGCCTACG



 



GAGAAAGTCA AGTGTCCCCC CATTGGGATG GAGTCACACC



 



GTATTGAGGA CAACCAGATC CGAGCCTC






18. Beta 2 Microglobulin (B2M)

Accession number: NM—004048

Sequence:








AATATAAGTGGAGGCGTCGCGCTGGCGGGCATTCCTGAAGCTGACAGC


 


ATTCGGGCCGAGATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGCGCTA


 


CTCTCTCTTTCTGGCCTGGAGGCTATCCAGCGTACTCCAAAGATTCAG


 


GTTTACTCACGTCATCCAGCAGAGAATGGAAAGTCAAATTTCCTGAAT


 


TGCTATGTGTCTGGGTTTCATCCATCCGACATTGAAGTTGACTTACTG


 


AAGAATGGAGAGAGAATTGAAAAAGTGGAGCATTCAGACTTGTCTTTC


 


AGCAAGGACTGGTCTTTCTATCTCTTGTACTACACTGAATTCACCCCC


 


ACTGAAAAAGATGAGTATGCCTGCCGTGTGAACCATGTGACTTTGTCA


 


CAGCCCAAGATAGTTAAGTGGGATCGAGACATGTAAGCAGCATCATGG


 


AGGTTTGAAGATGCCGCATTTGGATTGGATGAATTCCAAATTCTGCTT


 


GCTTGCTTTTTAATATTGATATGCTTATACACTTACACTTTATGCACA


 


AAATGTAGGGTTATAATAATGTTAACATGGACATGATCTTCTTTATAA


 


TTCTACTTTGAGTGCTGTCTCCATGTTTGATGTATCTGAGCAGGTTGC


 


TCCACAGGTAGCTCTAGGAGGGCTGGCAACTTAGAGGTGGGGAGCAGA


 


GAATTCTCTTATCCAACATCAACATCTTGGTCAGATTTGAACTCTTCA


 


ATCTCTTGCACTCAAAGCTTGTTAAGATAGTTAAGCGTGCATAAGTTA


 


ACTTCCAATTTACATACTCTGCTTAGAATTTGGGGGAAAATTTAGAAA


 


TATAATTGACAGGATTATTGGAAATTTGTTATAATGAATGAAACATTT


 


TGTCATATAAGATTCATATTTACTTCTTATACATTTGATAAAGTAAGG


 


CATGGTTGTGGTTAATCTGGTTTATTTTTGTTCCACAAGTTAAATAAA


 


TCATAAAACTTGATGTGTTATCTCTTA






Primers used:










Forward primer sequence:



AGGCTATCCAGCGTACTCCAA



 



Reverse primer sequence:



AATGCGGCATCTTCAAACC






Amplicon length: 337

Amplicon sequence:








AGGCTATCCAGCGTACTCCAAAGATTCAGGTTTACTCACGTCATCCAG


 


CAGAGAATGGAAAGTCAAATTTCCTGAATTGCTATGTGTCTGGGTTTC


 


ATCCATCCGACATTGAAGTTGACTTACTGAAGAATGGAGAGAGAATTG


 


AAAAAGTGGAGCATTCAGACTTGTCTTTCAGCAAGGACTGGTCTTTCT


 


ATCTCTTGTACTACACTGAATTCACCCCCACTGAAAAAGATGAGTATG


 


CCTGCCGTGTGAACCATGTGACTTTGTCACAGCCCAAGATAGTTAAGT


 


GGGATCGAGACATGTAAGCAGCATCATGGAGGTTTGAAGATGCCGCA


 


TT






19. Superoxide Dismutase 2, Mitochondrial (SOD2)

Accession number: NM—001024465.

Sequence:








GCGGTGCCCTTGCGGCGCAGCTGGGGTCGCGGCCCTGCTCCCCGCGCT


 


TTCTTAAGGCCCGCGGGCGGCGCAGGAGCGGCACTCGTGGCTGTGGTG


 


GCTTCGGCAGCGGCTTCAGCAGATCGGCGGCATCAGCGGTAGCACCAG


 


CACTAGCAGCATGTTGAGCCGGGCAGTGTGCGGCACCAGCAGGCAGCT


 


GGCTCCGGTTTTGGGGTATCTGGGCTCCAGGCAGAAGCACAGCCTCCC


 


CGACCTGCCCTACGACTACGGCGCCCTGGAACCTCACATCAACGCGCA


 


GATCATGCAGCTGCACCACAGCAAGCACCACGCGGCCTACGTGAACAA


 


CCTGAACGTCACCGAGGAGAAGTACCAGGAGGCGTTGGCCAAGGGAGA


 


TGTTACAGCCCAGATAGCTCTTCAGCCTGCACTGAAGTTCAATGGTGG


 


TGGTCATATCAATCATAGCATTTTCTGGACAAACCTCAGCCCTAACGG


 


TGGTGGAGAACCCAAAGGGGAGTTGCTGGAAGCCATCAAACGTGACTT


 


TGGTTCCTTTGACAAGTTTAAGGAGAAGCTGACGGCTGCATCTGTTGG


 


TGTCCAAGGCTCAGGTTGGGGTTGGCTTGGTTTCAATAAGGAACGGGG


 


ACACTTACAAATTGCTGCTTGTCCAAATCAGGATCCACTGCAAGGAAC


 


AACAGGCCTTATTCCACTGCTGGGGATTGATGTGTGGGAGCACGCTTA


 


CTACCTTCAGTATAAAAATGTCAGGCCTGATTATCTAAAAGCTATTTG


 


GAATGTAATCAACTGGGAGAATGTAACTGAAAGATACATGGCTTGCAA


 


AAAGTAAACCACGATCGTTATGCTGATCATACCCTAATGATCCCAGCA


 


AGATAATGTCCTGTCTTCTAAGATGTGCATCAAGCCTGGTACATACTG


 


AAAACCCTATAAGGTCCTGGATAATTTTTGTTTGATTATTCATTGAAG


 


AAACATTTATTTTCCAATTGTGTGAAGTTTTTGACTGTTAATAAAAGA


 


ATCTGTCAACCATCAAAAAAAAAAAAA






Primers used:










Forward primer sequence:



AACCTCAGCCCTAACGGTG



 



Reverse primer sequence:



AGCAGCAATTTGTAAGTGTCCC






Amplicon length: 180

Amplicon sequence:








AACCTCAGCCCTAACGGTGGTGGAGAACCCAAAGGGGAGTTGCTGGAA


 


GCCATCAAACGTGACTTTGGTTCCTTTGACAAGTTTAAGGAGAAGCTG


 


ACGGCTGCATCTGTTGGTGTCCAAGGCTCAGGTTGGGGTTGGCTTGGT


 


TTCAATAAGGAACGGGGACACTTACAAATTGCTCT






20. Nicotinamide Phosphoribosyltransferase (PBEF1)

Accession number: NM—005746

Sequence:








GCTGCCGCGCCCCGCCCTTTCTCGGCCCCCGGAGGGTGACGGGGTGAA


 


GGCGGGGGAACCGAGGTGGGGAGTCCGCCAGAGCTCCCAGACTGCGAG


 


CACGCGAGCCGCCGCAGCCGTCACCCGCGCCGCGTCACGGCTCCCGGG


 


CCCGCCCTCCTCTGACCCCTCCCCTCTCTCCGTTTCCCCCTCTCCCCC


 


TCCTCCGCCGACCGAGCAGTGACTTAAGCAACGGAGCGCGGTGAAGCT


 


CATTTTTCTCCTTCCTCGCAGCCGCGCCAGGGAGCTCGCGGCGCGCGG


 


CCCCTGTCCTCCGGCCCGAGATGAATCCTGCGGCAGAAGCCGAGTTCA


 


ACATCCTCCTGGCCACCGACTCCTACAAGGTTACTCACTATAAACAAT


 


ATCCACCCAACACAAGCAAAGTTTATTCCTACTTTGAATGCCGTGAAA


 


AGAAGACAGAAAACTCCAAATTAAGGAAGGTGAAATATGAGGAAACAG


 


TATTTTATGGGTTGCAGTACATTCTTAATAAGTACTTAAAAGGTAAAG


 


TAGTAACCAAAGAGAAAATCCAGGAAGCCAAAGATGTCTACAAAGAAC


 


ATTTCCAAGATGATGTCTTTAATGAAAAGGGATGGAACTACATTCTTG


 


AGAAGTATGATGGGCATCTTCCAATAGAAATAAAAGCTGTTCCTGAGG


 


GCTTTGTCATTCCCAGAGGAAATGTTCTCTTCACGGTGGAAAACACAG


 


ATCCAGAGTGTTACTGGCTTACAAATTGGATTGAGACTATTCTTGTTC


 


AGTCCTGGTATCCAATCACAGTGGCCACAAATTCTAGAGAGCAGAAGA


 


AAATATTGGCCAAATATTTGTTAGAAACTTCTGGTAACTTAGATGGTC


 


TGGAATACAAGTTACATGATTTTGGCTACAGAGGAGTCTCTTCCCAAG


 


AGACTGCTGGCATAGGAGCATCTGCTCACTTGGTTAACTTCAAAGGAA


 


CAGATACAGTAGCAGGACTTGCTCTAATTAAAAAATATTATGGAACGA


 


AAGATCCTGTTCCAGGCTATTCTGTTCCAGCAGCAGAACACAGTACCA


 


TAACAGCTTGGGGGAAAGACCATGAAAAAGATGCTTTTGAACATATTG


 


TAACACAGTTTTCATCAGTGCCTGTATCTGTGGTCAGCGATAGCTATG


 


ACATTTATAATGCGTGTGAGAAAATATGGGGTGAAGATCTAAGACATT


 


TAATAGTATCAAGAAGTACACAGGCACCACTAATAATCAGACCTGATT


 


CTGGAAACCCTCTTGACACTGTGTTAAAGGTTTTGGAGATTTTAGGTA


 


AGAAGTTTCCTGTTACTGAGAACTCAAAGGGTTACAAGTTGCTGCCAC


 


CTTATCTTAGAGTTATTCAAGGGGATGGAGTAGATATTAATACCTTAC


 


AAGAGATTGTAGAAGGCATGAAACAAAAAATGTGGAGTATTGAAAATA


 


TTGCCTTCGGTTCTGGTGGAGGTTTGCTACAGAAGTTGACAAGAGATC


 


TCTTGAATTGTTCCTTCAAGTGTAGCTATGTTGTAACTAATGGCCTTG


 


GGATTAACGTCTTCAAGGACCCAGTTGCTGATCCCAACAAAAGGTCCA


 


AAAAGGGCCGATTATCTTTACATAGGACGCCAGCAGGGAATTTTGTTA


 


CACTGGAGGAAGGAAAAGGAGACCTTGAGGAATATGGTCAGGATCTTC


 


TCCATACTGTCTTCAAGAATGGCAAGGTGACAAAAAGCTATTCATTTG


 


ATGAAATAAGAAAAAATGCACAGCTGAATATTGAACTGGAAGCAGCAC


 


ATCATTAGGCTTTATGACTGGGTGTGTGTTGTGTGTATGTAATACATA


 


ATGTTTATTGTACAGATGTGTGGGGTTTGTGTTTTATGATACATTACA


 


GCCAAATTATTTGTTGGTTTATGGACATACTGCCCTTTCATTTTTTTT


 


CTTTTCCAGTGTTTAGGTGATCTCAAATTAGGAAATGCATTTAACCAT


 


GTAAAAGATGAGTGCTAAAGTAAGCTTTTTAGGGCCCTTTGCCAATAG


 


GTAGTCATTCAATCTGGTATTGATCTTTTCACAAATAACAGAACTGAG


 


AAACTTTTATATATAACTGATGATCACATAAAACAGATTTGCATAAAA


 


TTACCATGATTGCTTTATGTTTATATTTAACTTGTATTTTTGTACAAA


 


CAAGATTGTGTAAGATATATTTGAAGTTTCAGTGATTTAACAGTCTTT


 


CCAACTTTTCATGATTTTTATGAGCACAGACTTTCAAGAAAATACTTG


 


AAAATAAATTACATTGCCTTTTGTCCATTAATCAGCAAATAAAACATG


 


GCCTTAACAAAGTTGTTTGTGTTATTGTACAATTTGAAAATTATGTCG


 


GGACATACCCTATAGAATTACTAACCTTACTGCCCCTTGTAGAATATG


 


TATTAATCATTCTACATTAAAGAAAATAATGGTTCTTACTGGAATGTC


 


TAGGCACTGTACAGTTATTATATATCTTGGTTGTTGTATTGTACCAGT


 


GAAATGCCAAATTTGAAAGGCCTGTACTGCAATTTTATATGTCAGAGA


 


TTGCCTGTGGCTCTAATATGCACCTCAAGATTTTAAGGAGATAATGTT


 


TTTAGAGAGAATTTCTGCTTCCACTATAGAATATATACATAAATGTAA


 


AATACTTACAAAAGTGGAAGTAGTGTATTTTAAAGTAATTACACTTCT


 


GAATTTATTTTTCATATTCTATAGTTGGTATGACTTAAATGAATTACT


 


GGAGTGGGTAGTGAGTGTACTTAAATGTTTCAATTCTGTTATATTTTT


 


TATTAAGTTTTTAAAAAATTAAATTGGATATTAAATTGTATGGACATC


 


ATTTATTAATTTTAAACTGAATGCCCTCAATAAGTAATACTGAAGCAC


 


ATTCTTAAATGAAGATAAATTATCTCCAATGAAAAGCATGACATGTGT


 


TTCAATAGAAGAATCTTAAGTTGGCTAAATTCAAAGTGCTTGACATCA


 


AAATGTTCTAGAGTGATTAGCTACTAGATTCTGAATCATACATCACAT


 


CTGACTAGAGACCAGTTTCTTTCGAATGATTCTTTTATGTATGTAGAT


 


CTGTTCTTCTGAGGCAGCGGTTGGCCAACTATAGCCCAAAGGCCAAAT


 


TTGGACTTCTTTTTATAAATGCAGATTGTCTATGGCTGCTTTCCCACT


 


ACTCCAGCCTAAGGTAAACAGCTGCAATAGAAGCCAAATGAGAATCGC


 


AAAGCCCAAAATGTTTATTAACCTGCCCTTTACACAAAATTACACAAA


 


AAGTTTCCTGATCTCTGTTCTAAGAAAAGGAGTGTGCCTTGCATTTAA


 


AAGGAAATGTTGGTTTCTAGGGAAGGGAGGAGGCTAAATAATTGATAC


 


GGAATTTTCCTCTTTTGTCTTCTTTTTTCTCACTTAAGAATCCGATAC


 


TGGAAGACTGATTTAGAAAAGTTTTTAACATGACATTAAATGTGAAAT


 


TTTAAAAATTGAAAAGCCATAAATCATCTGTTTTAAATAGTTACATGA


 


GAAAATGATCACTAGAATAACCTAATTAGAAGTGTTATCTTCATTAAA


 


TGTTTTTTGTAAGTGGTATTAGAAAGAATATGTTTTTCAGATGGTTCT


 


TTAAACATGTAGTGAGAACAATAAGCATTATTCACTTTTAGTAAGTCT


 


TCTGTAATCCATGATATAAAATAATTTTAAAATGATTTTTTAATGTAT


 


TTGAGTAAAGATGAGTAGTATTAAGAAAAACACACATTTCTTCACAAA


 


ATGTGCTAAGGGGCGTGTAAAGAATCAAAAGAAACTATTACCAATAAT


 


AGTTTTGATAATCACCCATAATTTTGTGTTTAAACATTGAAATTATAG


 


TACAGACAGTATTCTCTGTGTTCTGTGAATTTCAGCAGCTTCAGAATA


 


GAGTTTAATTTAGAAATTTGCAGTGAAAAAAGCTATCTCTTTGTTCAC


 


AACCATAAATCAGGAGATGGAGATTAATTCTATTGGCTCTTAGTCACT


 


TGGAACTGATTAATTCTGACTTTCTGTCACTAAGCACTTGGTATTTGG


 


CCATCTCCATTCTGAGCACCAAACGGTTAACACGAATGTCCACTAGAA


 


CTCTGCTGTGTGTCACCCTTAAATCAGTCTAAATCTTCCAGACAAAAG


 


CAAATGGCATTTATGGATTTAAGTCATTAGATTTTCAACTGACATTAA


 


TTAATCCCTCTTGATTGATTATATCATCAAGTATTTATATCTTAAATA


 


GGAGGTAGGATTTCTGTGTTAAGACTCTTATTTGTACCCTATAATTAA


 


AGTAAAATGTTTTTTATGAGTATCCCTTGTTTTCCCTTCTTAAATTGT


 


TATCAAACAATTTTTATAATGAAATCTATCTTGGAAAATTAGAAAGAA


 


AAATGGCAAGGTATTTATTGTTCTGTTTGCCATAATTTAGAACTCACA


 


CTTAAGTATTTTGTAGTTTTACATTCCTTTTTAACCCATTCAGTGGAG


 


AATGTCAGCTTTTCTCCCAAGTTGTATGTTAAGTCTATTCTAATATGT


 


ACTCAACATCAAGTTATAAACATGTAATAAACATGGAAATAAAGTTTA


 


GCTCTATTAGTGAAGTGTTAAAAAAAAAAAAAA






Primers used:










Forward primer sequence:



ATTGCCTTCGGTTCTGGTGG



 



Reverse primer sequence:



CGGCCCTTTTTGGACCTTTTG Amplicon length: 155






Amplicon sequence:








ATTGCCTTCGGTTCTGGTGGAGGTTTGCTACAGAAGTTGACAAGAGAT


 


CTCTTGAATTGTTCCTTCAAGTGTAGCTATGTTGTAACTAATGGCCTT


 


GGGATTAACGTCTTCAAGGACCCAGTTGCTGATCCCAACAAAAGGTCC


 


AAAAAGGGCCG






The list of sequences given above are represented by SEQ ID No. 1 to 80, used in the present invention, which are being provided herein in PatentIn 3.5 format. The sequence listings are also being provided in hard copy as well as in electronic format on a CD_ROM. The complementary DNA sequences [cDNA] provided in the specification are complementary to the respective gene sequences and the said cDNA sequences are represented by SEQ ID Nos 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77 and 79. It may be noted that the cDNA sequences are equivalent to mRNA sequences except that the base Thymine [T] in cDNA is replaced by the base Uracil [U] in mRNA. Further, the probe sequences for the genes useful in the detection of astrocytoma, it's grades and glioblastoma prognosis are represented by SEQ ID No. 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78 and 80.
A ready reference for the said sequences used in the present invention is being provided here as under:
1. SEQ ID No. 1 represents forward primer of SYT1 gene
2. SEQ ID No. 2 represents reverse primer of SYT1 gene
3. SEQ ID No. 3 represents forward primer of RAB26 gene
4. SEQ ID No. 4 represents reverse primer of RAB26 gene
5. SEQ ID No. 5 represents forward primer of DIRAS2 gene
6. SEQ ID No. 6 represents reverse primer of DIRAS2 gene
7. SEQ ID No. 7 represents forward primer of RAB13 gene
8. SEQ ID No. 8 represents reverse primer of RAB13 gene
9. SEQ ID No.9 represents forward primer of IGFBP7 gene
10. SEQ ID No 10. represents reverse primer of IGFBP7 gene
11. SEQ ID No. 11 represents forward primer of COL6A1 gene
12. SEQ ID No. 12 represents reverse primer of COL6A1 gene
13. SEQ ID No. 13 represents forward primer of DCN gene
14. SEQ ID No. 14 represents reverse primer of DCN gene
15. SEQ ID No 15. represents forward primer of PLAT gene
16. SEQ ID No 16. represents reverse primer of PALT gene
17. SEQ ID No. 17 represents forward primer of LGALS3 gene
18. SEQ ID No. 18 represents reverse primer of LGALS3 gene
19. SEQ ID No. 19 represents forward primer of FABP7 gene
20. SEQ ID No. 20 represents reverse primer of FABP7 gene
21. SEQ ID No. 21 represents forward primer of LOX gene
22. SEQ ID No. 22 represents reverse primer of LOX gene
23. SEQ ID No. 23 represents forward primer of LAMB1 gene
24. SEQ ID No. 24 represents reverse primer of LAMB1 gene
25. SEQ ID No. 25 represents forward primer of IGFBP3 gene
26. SEQ ID No. 26 represents reverse primer of IGFBP3 gene
27. SEQ ID No. 27 represents forward primer of GADD45A gene
28. SEQ ID No. 28 represents reverse primer of GADD45A gene
29. SEQ ID No. 29 represents forward primer of FSTL1 gene
30. SEQ ID No. 30 represents reverse primer of FSTL1 gene
31. SEQ ID No. 31 represents forward primer of RHOC gene
32. SEQ ID No.32 represents reverse primer of RHOC gene
33. SEQ ID No. 33 represents forward primer of AEBP1 gene
34. SEQ ID No. 34 represents reverse primer of AEBP1 gene
35. SEQ ID No. 35 represents forward primer of B2M gene
36. SEQ ID No. 36 represents reverse primer of B2M gene
37. SEQ ID No. 37 represents forward primer of SOD2 gene
38. SEQ ID No.38 represents reverse primer of SOD2 gene
39. SEQ ID No.39 represents forward primer of PBEF1 gene
40. SEQ ID No. 40 represents reverse primer of PBEF1 gene
41. SEQ ID No. 41 represents cDNA of SYT1 gene
42. SEQ ID No. 42 represents an amplicon or nucleic acid probe for SYT1 gene
43. SEQ ID No.43 represents cDNA of RAB26 gene
44. SEQ ID No. 44 represents an amplicon or nucleic acid probe for RAB26 gene
45. SEQ ID No. 45 represents cDNA of DIRAS2 gene
46. SEQ ID No. 46 represents an amplicon or nucleic acid probe for DIRAS2 gene
47. SEQ ID No. 47 represents cDNA of RAB13 gene
48. SEQ ID No. 48 represents an amplicon or nucleic acid probe for RAB13 gene
49. SEQ ID No.49 represents cDNA of IGFBP7 gene
50. SEQ ID No 50 represents an amplicon or nucleic acid probe for IGFBP7 gene
51. SEQ ID No. 51 represents cDNA of COL6A1 gene
52. SEQ ID No. 52 represents an amplicon or nucleic acid probe for COL6A1 gene
53. SEQ ID No. 53 represents cDNA of DCN gene
54. SEQ ID No. 54 represents an amplicon or nucleic acid probe for DCN gene
55. SEQ ID No 55. represents cDNA of PLAT gene
56. SEQ ID No 56. represents an amplicon or nucleic acid probe for PALT gene
57. SEQ ID No. 57 represents cDNA of LGALS3 gene
58. SEQ ID No. 58 represents an amplicon or nucleic acid probe for LGALS3 gene
59. SEQ ID No. 59 represents cDNA of FABP7 gene
60. SEQ ID No. 60 represents an amplicon or nucleic acid probe for FABP7 gene
61. SEQ ID No. 61 represents cDNA of LOX gene
62. SEQ ID No. 62 represents an amplicon or nucleic acid probe for LOX gene.
63. SEQ ID No. 63 represents cDNA of LAMB 1 gene
64. SEQ ID No. 64 represents an amplicon or nucleic acid probe for LAMB 1 gene
65. SEQ ID No. 65 represents cDNA of IGFBP3 gene
66. SEQ ID No. 66 represents an amplicon or nucleic acid probe for IGFBP3 gene
67. SEQ ID No. 67 represents cDNA of GADD45A gene
68. SEQ ID No. 68 represents an amplicon or nucleic acid probe for GADD45A gene
69. SEQ ID No. 69 represents cDNA of FSTL1 gene
70. SEQ ID No. 70 represents an amplicon or nucleic acid probe for FSTL1 gene
71. SEQ ID No. 71 represents cDNA of RHOC gene
72. SEQ ID No.72 represents an amplicon or nucleic acid probe for RHOC gene
73. SEQ ID No. 73 represents cDNA of AEBP1 gene
74. SEQ ID No. 74 represents an amplicon or nucleic acid probe for AEBP1 gene
75. SEQ ID No. 75 represents cDNA of B2M gene
76. SEQ ID No. 76 represents an amplicon or nucleic acid probe for B2M gene
77. SEQ ID No. 77 represents cDNA of SOD2 gene
78. SEQ ID No.78 represents an amplicon or nucleic acid probe for SOD2 gene
79. SEQ ID No.79 represents cDNA of PBEF1 gene
80. SEQ ID No. 80 represents an amplicon or nucleic acid probe for PBEF1 gene
The sample is preferably collected directly from the human subject's body. Preferred and convenient substances for sampling include blood, lymph or plasma, cerebrospinal fluid, other biopsy sample of cellular material from brain tissue. Cellular material includes any sample containing human cells, including samples of tissue, expressed tissue fluids (e.g., lymph or plasma) or tissue wash and the like. Tissue samples that can be collected include, but are not limited to, cell-containing material from the brain. This includes normal brain tissue, tumor tissue, tumor-adjacent tissue, and/or blood plasma from a site within the brain.
In accordance with the inventive methods, the tissue sample preferably contains cells that express a plurality of protein species and mRNA species, which proteins and/or mRNA species are detectably distinct from one another. “Obtaining” and “collecting” the sample are used interchangeably herein and encompass sampling, resecting, removing from in situ, aspirating, receiving, gathering, and/or transporting the tissue sample or a concentrate, sediment, precipitate, supernatant, filtrate, aspirate, or other fraction of any of these. For example, conventional biopsy methods are useful for obtaining the tissue sample. These include percutaneous biopsy, laparoscopic biopsy, surgical resection, tissue scrapes and swabs, sampling via stents, catheters, endoscopes, needles, surgical resection, and other known means. For example, to obtain a sample from inside the skull of the human subject; typically, Magnetic Resonance Imaging (MRI)-guided stereotactic techniques are employed, but other methods can be used.
The sample is alternatively derived from cultured human cells, cell-free extracts, or other specimens indirectly derived from a subject's body, as well as from substances taken directly from a subject's body. Samples may be stored before detection methods are applied (for example nucleic acid amplification and/or analysis, or immunochemical detection) by well known storage means that will preserve nucleic acids or proteins in a detectable and/or analyzable condition, such as quick freezing, or a controlled freezing regime, in the presence of a cryoprotectant, for example, dimethyl sulfoxide (DMSO), trehalose, glycerol, or propanediol-sucrose. Samples may also be pooled before or after storage for purposes of amplifying the nucleic acids specific for the said genes for analysis and detection, or for purposes of detecting the respective proteins.
The sample is used immediately or optionally pre-treated by refrigerated or frozen storage overnight, by dilution, by phenol-chloroform extraction, or by other like means, to remove factors that may inhibit various amplification reactions. The level of expression in the sample for the said proteins or their messenger ribonucleic acid (mRNA) is then detected quantitatively or semi-quantitatively.
Polynucleotides specific for the said genes, including mRNA species, are determined by base sequence similarity or homology to known nucleotide sequences. Base sequence homology is determined by conducting a base sequence similarity search of a genomics data base, such as the GenBank database of the National Center for Biotechnology Information (NCBI; www.ncbi.nlm.nih.gov/BLAST/), using a computerized algorithm, such as PowerBLAST, QBLAST, PSI-BLAST, PHI-BLAST, gapped or ungapped BLAST, or the “Align” program through the Baylor College of Medicine server (www.hgsc.bcm.tmc.edu/seq_clata). (Altchul, et al., 1997; Zhang and Madden, 1997; Madden et al., 1996; Altschul et al., 1990).
Preferably, polynucleotide sequences specific to the said genes, including an mRNA sequence, is at least 5 to 30 contiguous nucleotides long, more preferably at least 6 to 15 contiguous nucleotides long, and most preferably at least 7 to 10 contiguous nucleotides long. mRNA specific to any of the said genes can be, but is not necessarily, an mRNA species containing a nucleotide sequence that encodes a functional version of the said genes or fragments thereof. Also included among mRNAs specific to the said genes are splice variants.
Quantitative detection of levels of mRNAs specific to the said genes or their proteins, or of other proteins or mRNA species of interest in accordance with the present invention is done by any known method that provides a quantitative or semi-quantitative determination of expression. A quantitative method can be absolute or relative. An absolute quantitation provides an absolute value for the amount or level of expression in comparison to a standard, which amount or level is typically a mole, mass, or activity value normalized in terms of a specified mass of protein, mass of nucleic acid, number or mass of cells, body weight, or the like. Additionally, the quantitative or absolute value is optionally normalized in terms of a specified time period, i.e., expression level as a rate. A relative detection method provides a unitless relative value for the amount or level of expression, for example, in terms of a ratio of expression in a given sample relative to a control, such as normal tissue or the expression of a selected “housekeeping” gene. The skilled artisan is aware of other examples of quantitative and semi-quantitative detection methods.
In accordance with the inventive methods, the expression level of the proteins encoded by the said genes is optionally detected by immunochemical means, such as, but not limited to, enzyme-linked immunosorbent assay (ELISA), immunofluorescent assay (IFA), immunoelectrophoresis, immunochromatographic assay or immunohistochemical staining, employing polyclonal or monoclonal antibodies or antibody fragments against the said gene products. Antibodies or antibody fragments that target the said proteins are available commercially or can be produced by conventional means.
Similarly, the expression levels of other proteins of interest, in accordance with the inventive methods, can be detected by conventional immunochemical means as described above. Most preferably, quantitative or semi-quantitative detection of the expression level of mRNA species is accomplished by any of numerous methods of nucleic acid amplification (e.g., amplification of specific nucleic acid segments) in the form of RNA or cDNA, which RNA or cDNA amplification product is ultimately measured after amplification. The final amplification product of RNA or cDNA is measured by any conventional means, such as, but not limited to, densitometry, fluorescence detection, or any other suitable biochemical or physical assay system. Before amplification, it is preferable to extract or separate mRNA from genomic DNA in the sample and to amplify nucleic acids remaining in that fraction of the sample separated from the DNA, to avoid false positives that are caused by amplification of contaminating genomic DNA in the original specimen.
Histopathological means of classifying malignant tumors into grades are known for various kinds of malignant tumor, including astrocytomas. (Daumas-Duport et al., 1988).
The present inventive method can be used to diagnose astrocytoma, wherein a higher level of expression of RAB13 or a lower level of expression of any or a combination of genes from the group consisting of, but not limited to SYT1, RAB26 and DIRAS2 is indicative of the presence of astrocytoma (See FIG. 1).
The present inventive method can be used to diagnose malignant astrocytoma (AA, GBM), wherein a higher level of expression of IGFBP7 is indicative of the presence of malignant astrocytoma. (See FIG. 2)
The present inventive method can be used to diagnose the presence of glioblastoma, since GBMs generally over-express any or a combination of genes from the group consisting of, but not limited to COL6A1, DCN, PLAT, LGALS3, FABP7, LOX, LAMB1, IGFBP3, GADD45A, FSTL1, RHOC, B2M and PBEF1. (See FIG. 3). Primary and secondary GBMs are frequently indistinguishable with conventional histopathological methods, but using the inventive method, these types are readily distinguished, since primary GBMs generally over express any or a combination of genes from the group consisting of, but not limited to AEBP1 and SOD2. (See FIG. 4).
In a preferred embodiment the above identified genes are used in combination with known markers such as
EGFR, p53 and Ki-67 for determining the presence of astrocytoma.
Further, present invention method can also be used for the prognosis of glioblastoma in human subjects where in higher expression of PBEF1 indicates the poorer survival of the human subject from which the test sample has been obtained (FIG. 11)
The foregoing descriptions of the methods of the present invention are only illustrative and by no means exhaustive. When these features of the present invention are employed, diagnostic and treatment decisions can be more appropriately optimized for the individual astrocytoma and glioblastoma patient, and the prospects for his or her survival can be enhanced.
Transcriptome Analysis of Astrocytomas
We analyzed the expression profile of 18981 human genes using 19k cDNA microarrays for twenty five samples of diffusely infiltrating astrocytoma comprising four diffuse astrocytoma (DA; Gr II), five AA (Gr. III) and sixteen GBM (Gr IV; six secondary and ten primary). 14,929 genes for which the data was available for more than 50% of the samples within each grade were only considered for subsequent analysis. The data obtained from image analysis was filtered, normalized and log 2 transformed before being used for further studies. To identify the significantly differentially regulated genes between normal and astrocytoma samples as well as between different grades of astrocytoma, the data were subjected to Significance Analysis of Microarrays using the one-class and two-class option as required.
SAM Analysis Identifies Grade Specific Genes
Examination of 14,929 genes data set derived from 25 astrocytoma samples through SAM analysis found 385 to be up regulated and 911 genes to be down regulated in all groups of astrocytoma in comparison to normal brain sample. DAs (Gr. II) are diffusely infiltrating low-grade astrocytoma which progress over to malignant grade III anaplastic astrocytoma and grade IV GBM over period of 5-10 yrs (4, 5, 12). AAs (Gr. III) are less malignant than GBMs with AA patients having a mean survival of 2-3 years in comparison to GBM patients whose mean survival is less than a year (3, 5, 13, 14). To identify the genes which are differentially regulated between GBM and lower-grade astrocytoma (LGA) i.e., DA and AA, we subjected the data obtained from 9 of the later group (4 DA and 5 AA) and 16 GBM (10 primary and 6 secondary) through SAM analysis. We found 41 genes up regulated in LGA (DA & AA) as against GBM and 37 genes up regulated in GBM as against LGA. Since secondary GBM progresses from lower grades, i.e.; DA or AA, it is likely that these tumors share an expression profile with DA or AA as against primary GBM. Thus to obtain the expression profile specific to progressive astrocytoma (DA, AA, Secondary GBM) as against primary GBM (de novo GBM), we compared the data obtained from 15 samples belonging to progressive astrocytoma (4 DA, 5 AA and 6 secondary GBM) and 10 samples belonging to primary GBM through SAM analysis. We found 20 genes up regulated in progressive astrocytoma as against primary GBM and 45 genes up regulated in primary GBM as against progressive astrocytoma.
Real-Time RT-Quantitative PCR Validation of SAM Identified Genes
In order to identify genes whose differential expression characterize astrocytomas, malignant astrocytomas (AA, GBM) or primary glioblastomas, we critically analyzed the data obtained from SAM analysis and some of the interesting genes were selected for further validation. Expression pattern of the selected was confirmed by real-time RT-quantitative PCR analysis on independent set of 100 samples of different grades of astrocytomas and glioblastoma. Sample set includes 5 DAs, 31 AAs, 20 secondary GBMs, 35 primary GBMs and 9 normal brain samples. Statistical significance of expression pattern of a gene was examined by Mann-Whitney test. P value of equal to or less than 0.05 was considered statistically significant.
Identification of Down- or Up-Regulated, Genes in Astrocytoma
Using SAM analysis and subsequent RT-qPCR analysis, we have identified the genes SYT1, RAB26, DIRAS2 to be down-regulated in all grades of astrocytoma where as RAB13 is up-regulated in all grades of astrocytoma.
Synaptotagmin I (SYT1) was found to be down regulated by more than 4 fold in DAs(5/5; 100%), AAs (25/31; 80%), secondary GBMs (17/20; 85%) and primary GBMs (31/35; 88.5%) but not in normal brain samples (0/9; 0%). P value found to be statistically significant between Normal and all grades of astrocytoma viz., Normal vs. DA (0.001), Normal vs. AA(<0.001), Normal vs. secondary GBM (<0.001), Normal vs. primary GBM (<0.001) (FIG. 1. A).
RAB26, member RAS oncogene family (RAB26) was found to be down regulated by more than 4 fold in DAs (5/5; 100%), AAs (21/30; 70%), secondary GBMs (19/20; 95%) and primary GBMs (31/35; 88.5%) but not in normal brain samples (0/9; 0%). P value found to be statistically significant between Normal and all grades of astrocytoma viz., Normal vs. DA (0.001), Normal vs. AA (<0.001), Normal vs. secondary GBM (<0.001), Normal vs. primary GBM (<0.001) (FIG. 1. B).
DIRAS family, GTP-binding RAS-like 2 (DIRAS2) was found to be down regulated by more than 4 fold in DAs (4/5; 80%), AAs (19/31; 61.2%), secondary GBMs (20/20; 100%) and primary GBMs (29/35; 82.8%) but not in normal brain samples (0/9; 0%). P value found to be statistically significant between Normal and all grades of astrocytoma viz., Normal vs. DA (0.001), Normal vs. AA (<0.001), Normal vs. secondary GBM (<0.001), Normal vs. primary GBM (<0.001) (FIG. 1. C).
RAB13, member RAS oncogene family (RAB13) was found to be down regulated by more than 4 fold in majority of DAs (4/5; 80%), AAs(24/30; 80%), secondary GBMs (17/20; 85%) and primary GBMs (34/35; 97.1%) but not in normal brain samples (1/9; 11.1%). P value found to be statistically significant between Normal and all grades of astrocytoma viz., Normal vs. DA (0.059), Normal vs. AA (<0.001), Normal vs. secondary GBM (<0.001), Normal vs. primary GBM (<0.001) (FIG. 1. D).
Identification of Up-Regulated Gene(s) in Malignant Astrocytoma (AA &GBM)
Insulin-like growth factor binding protein 7 (IGFBP7) was found to be up-regulated mainly in malignant astrocytomas (AA, GBM). Over-expression in malignant astrocytomas in comparison to controls is found to be statistically significant—Normal vs. AA (P=0.002), Normal vs. secondary GBM (P=0.01), Normal vs. primary GBM (P<0.001)—but not in benign astrocytomas (Normal vs. DA (P=0.297). IGFBP7 is found to be up-regulated by more than 4 fold majority of AAs (25/31; 80%), secondary GBMs (14/20; 70%) and primary GBMs (32/35; 91%) but not in controls (2/9; 22%), and DAs (3/5; 60%) (FIG. 2).
In addition, protein expression pattern of IGFBP7 was analyzed by immunohistochemistry. In the normal brain, neurons and glial cells were negative for IGFBP-7. There was a faint to moderate staining of parenchymal and leptomeningeal vessels {data not shown}. Only 1/9 of DAs and 40% (4/10) cases of AA showed positive staining of tumor cell cytoplasm. Among GBMs, 80% (12/15) cases of secondary GBM and 100% (19/19) of primary GBMs showed positive staining for IGFBP-7 in the tumor cells (FIG. 5).
Identification of GBM Specific Genes
Using SAM analysis and subsequent RT-qPCR analysis, the genes COL6A1, DCN, PLAT, LGALS3, FABP7, LOX, LAMB1, IGFBP3, GADD45A, FSTL1, RHOC, B2M were found to be over-expressed mainly in GBMs. Their over-expression in GBMs in comparison to controls as well as lower-grade samples (LGA), i.e., DA and AA was found to be statistically significant.
Collagen, type VI, alpha 1 (COL6A1) is found to be up-regulated by more than 3 fold in majority of GBMs (31/55; 54.5%) but in fewer no. of controls (1/9; 11%) and LGAs (11/36; 30.5%). P value obtained from Mann-Whitney U test for the comparison of GBM vs. Normal is <0.001 and for the comparison of GBM vs. LGA is 0.009 (FIG. 3. A).
Decorin (DCN) is found to be up-regulated by more than 4 fold in majority of GBMs (43/54; 79.6%) but in fewer no. of controls (4/9; 44%) and in LGAs (14/36; 38.9%). P value obtained from Mann-Whitney U test for the comparison of GBM vs. Normal is 0.003 and for the comparison of GBM vs. LGA is <0.001 (FIG. 3. B).
Plasminogen activator, tissue (PLAT) is found to be up-regulated by more than 4 fold in approximately half the no. of GBMs (28/54; 51.8%) but in none of the controls (0/9; 0%) and in very few LGAs (4/36; 11.1%). P value obtained from Mann-Whitney U test for the comparison of GBM vs. Normal is 0.001 and for the comparison of GBM vs. LGA is <0.001 (FIG. 3. C).
Lectin, galactoside-binding, soluble, 3 (LGALS3) is found to be up-regulated by more than 4 fold in majority of GBMs (37/55; 67.2%) but in fewer no. of controls (1/9; 11%) and LGAs (6/36; 16.7%). P value obtained from Mann-Whitney U test for the comparison of GBM vs. Normal is 0.001 and for the comparison of GBM vs. LGA is <0.001 (FIG. 3. D).
Fatty acid binding protein 7, brain (FABP7) is found to be up-regulated by more than 4 fold in majority of GBMs (35/55; 63.6%) but in fewer no. of controls (1/9; 11%) and in none of LGAs (0/36; 0%). P value obtained from Mann-Whitney U test for the comparison of GBM vs. Normal is 0.04 and for the comparison of GBM vs. LGA is <0.001 (FIG. 3. E).
Lysyl oxidase (LOX) is found to be up-regulated by more than 4 fold in majority of GBMs (34/55; 61.8%) but in fewer no. of controls (2/9; 22%) and LGAs (10/36; 27.8%). P value obtained from Mann-Whitney U test for the comparison of GBM vs. Normal is 0.03 and for the comparison of GBM vs. LGA is <0.001 (FIG. 3. F).
Laminin, beta 1 (LAMB1) is found to be up-regulated by more than 2 fold in majority of GBMs (46/55; 83.6%) but in fewer no. of controls (1/9; 11%) and LGAs (5/36; 13.9%). P value obtained from Mann-Whitney U test for the comparison of GBM vs. Normal is <0.001 and for the comparison of GBM vs. LGA is <0.001 (FIG. 3. G).
Insulin-like growth factor binding protein 3 (IGFBP3) is found to be up-regulated by more than 4 fold in majority of GBMs (34/55; 61.8%) but in fewer no. of controls (0/9; 0%) and LGAs (8/36; 22.2%). P value obtained from Mann-Whitney U test for the comparison of GBM vs. Normal is <0.001 and for the comparison of GBM vs. LGA is <0.001 (FIG. 3.11). In addition to this, protein expression pattern of IGFBP-3 was analyzed by immunohistochemistry. In the normal brain, IGFBP-3 showed a faint cytoplasmic labeling (1+) of the neurons. The astrocytes, oligodendrocytes and blood vessels were not labeled {data not shown}. Among astrocytomas, 33.33% (3/9) of DAs and 80.0% (8/10) of AAs showed positive staining. Among GBMs, 86.66% (13/15) of secondary GBMs and 100% (19/19) of primary GBMs showed positive staining for IGFBP-3. (FIG. 6)
Growth arrest and DNA-damage-inducible, alpha (GADD45A) is found to be up-regulated by more than 4 fold in majority of GBMs (32/55; 58.2%) but in fewer no. of controls (0/9; 0%) and LGAs (8/36; 22.2%). P value obtained from Mann-Whitney U test for the comparison of GBM vs. Normal is <0.001 and for the comparison of GBM vs. LGA is <0.001 (FIG. 3. I). We also analyzed the expression pattern of GADD45A by immunohistochemical staining (FIG. 7). Among primary and secondary GBM samples tested, 58.0% (7/12) and 50.0% (3/6) of them respectively showed strong positive cytoplasmic staining for GADD45A. The percentage positively for GADD45A staining in AA (Gr. and DA (Gr. II) was found to be 14.0% (1/7) and 00% (0/3) respectively. The average percent positive tumor cells were found to be higher among primary GBM samples (30.0%) followed by 25.0% and 20.0% among secondary GBM and AA respectively. Analysis of five normal brain samples showed glial cells to be negative for GADD45A staining.
Follistatin-like 1 (FSTL1) is found to be up-regulated by more than 4 fold in majority of GBMs (39/55; 70.9%) but in fewer no. of controls (0/9; 0%) and LGAs (4/36; 11.1%). P value obtained from Mann-Whitney U test for the comparison of GBM vs. Normal is <0.001 and for the comparison of GBM vs. LGA is <0.001 (FIG. 3.1). Immunohistochemical analysis revealed that 85.0% (18/21) and 86.0% (9/15) of primary and secondary GBM respectively are positive for FSTL1 protein staining (FIG. 8). As expected, reduced positively was found among AA (30.0%; 3/10) and DA (0.0%; 0/10). Staining of normal brain revealed glial cells to be negative for FSTL1 staining. The average percent positive tumor cells was found to be higher among primary GBM and secondary GBM (31.0 and 24% respectively) as against only 13.0% among AA cases suggesting the fact FSTL1 is expressed in very high levels among GBMs.
Ras homolog gene family, member C(RHOC) is found to be up-regulated by more than 2 fold in majority of GBMs (37/55; 67.3%) but in fewer no. of controls (1/9; 11.1%) and LGAs (16/36; 44.4%). P value obtained from Mann-Whitney U test for the comparison of GBM vs. Normal is <0.001 and for the comparison of GBM vs. LGA is 0.021 (FIG. 3. K).
Beta-2-microglobulin (B2M) is found to be up-regulated by more than 4 fold in majority of GBMs (34/55; 61.8%) but in fewer no. of controls (1/9; 11.1%) and LGAs (17/36; 47.2%). P value obtained from Mann-Whitney U test for the comparison of GBM vs. Normal is 0.004 and for the comparison of GBM vs. LGA is 0.022 (FIG. 3.L). Immuno-histochemical staining for the expression of B2M confirmed the mRNA expression pattern (FIG. 9). While most GBM samples tested positive for a strong cytoplasmic staining (82.3%; 14/17), only 28.57% of anaplastic astrocytoma (2/7) and none of diffuse astrocytoma samples (0/3) showed positive staining.
PBEF1 transcript levels were up regulated more than three fold (Log 2 ratio=1.585) in majority of GBMs (63.60%; 35/55 with a mean log 2 ratio of 2.48) in comparison to AAs (9.60%; 3/31 with a mean log 2 ratio of 0.44), DAs (0.00%; 0/5 with a mean log 2 ratio of 0.50) and normal brain samples (0.00%; 0/9) with a P value <0.001 (FIG. 3 M). Statistical significance was assessed by Mann-Whitney test using GraphPad PRISM software. The staining pattern of PBEF1 was generally found to be granular and diffuse cytoplasmic. We found a vast majority of GBMs (72.22%; 26/36) found to be positive for PBEF1 staining (FIG. 10 E, F). As expected, reduced percentage of samples among AAs (40.00%; 4/10) and DAs (20.00%; 2/10) showed PBEF1 positive staining (FIG. 10 B, C). Further, the average percent positive tumor cells were+found to be high among GBMs (37.50%) as against 22.00% and 8.50% among AAs and DAs respectively suggesting the fact that PBEF1 is expressed in very high levels among GBMs. The staining of normal brain revealed that the glial cells are negative for PBEF1 staining (FIG. 10 A).
Identification of Primary GBM Specific Genes
Using SAM analysis and subsequent RT-qPCR analysis, we have also found two genes, namely, AEBP1 and SOD2 whose over-expression is characteristic to primary GBM.
AE binding protein 1 (AEBP1) is found to be up-regulated by more than 2 fold in majority of primary GBMs (29/35; 82.8%) but in fewer no. of controls (1/9; 11.1%), DAs (2/5; 40%), AAs (9/31; 29%) and secondary GBMs (9/20; 45%). The difference in transcript levels between primary GBM and all other groups is statistically significant: primary GBM vs. controls (P=0.002); primary GBM vs. DAs (P=0.043); primary GBM vs. AAs (P<0.001) and primary GBM vs. secondary GBMs (P=0.027) (FIG. 4. A).
Superoxide dismutase 2, mitochondrial (SOD2) is found to be up-regulated by more than 4 fold in majority of primary GBMs (25/35; 71.4%) but in fewer no. of controls (0/9; 0%), DAs (0/5; 0%), AAs (5/31; 16.1%) and secondary GBMs (4/20; 20%). The difference in transcript levels between primary GBM and all other groups is statistically significant: primary GBM vs. controls (P=0.001); primary GBM vs. DAs (P=0.012); primary GBM vs. AAs (P<0.001) and primary GBM vs. secondary GBMs (P=0.001) (FIG. 4. B).
Survival Value of PBEF1
To analyze the survival value of PBEF1, we subjected a different set of 51 retrospective GBM cases where follow up was available, for expression of PBEF1. In GBM patients, while the prognostic significance of clinical variables in predicting survival have been clearly defined, altered protein expression of the well known genetic alteration found in these tumors, like over-expression of p53 and EGFR expression, have individually failed to give a clear cut prognostic significance, with confounding results in different studies. Therefore, for the purpose of multivariate analysis, we immunostained the sections to study the expression of p53 and EGFR in order to analyze the significance of their co-expression with PBEF1 with respect to patient survival.
Correlating the expression of PBEF1 with survival among GBM patients, in univariate analysis, the median survival of the group which was positive for PBEF1 was lesser than that of the group negative for PBEF1, albeit with lack of statistical significance (12 months vs. 16 months respectively, p=0.16; FIG. 11 A). In multivariate analysis, while PBEF-1 expression by itself did not correlate with survival, its co-expression with p53 showed a trend towards poorer survival. The median survival of the group positive for both the markers was 8 months (red line), as compared to 14 months (green line) of the group negative for both or either of them (P=0.08; FIG. 11 B). We also noted that radiotherapy, KPS at presentation and p53 expression were significant independent predictors of survival at all steps in the multivariate analysis (data not shown). The data put together suggest that PBEF1 is a potential prognostic marker amongst GBM patients particularly in combination with aberrant p53 expression.
Accordingly the present invention comprises the determination of level of expression of single or combination of genes selected from the group comprising of SYT1, RAB26, DIRAS2 and RAB13 in a test sample of brain tissue cells obtained from said human subject and in a control sample of known normal brain tissue cells, wherein a higher level of expression of RAB13 in the test sample or a lower level of expression of SYT1, RAB26, DIRAS2 in the test sample, as compared to the control sample, indicates the presence of astrocytoma in the human subject from which the test sample is obtained.
In an embodiment of the present invention, the expression level of said genes is determined by checking the level of RNA transcripts of the said genes by employing an oligonucleotide in nucleic acid-based detection methods such as in situ hybridization, RT-PCR analysis etc. or optionally the expression level of said genes is determined by checking the level of respective proteins of said genes by employing an antibody in protein-based detection methods such as immunohistochemistry, western blot analysis and ELISA etc.
Accordingly the present invention, the present invention also provides a kit for characterizing astrocytoma in a human subject, wherein the said kit comprising:

    a) reagent capable of specifically detecting the presence or absence of a single or combination of, said genes such as SYT1, RAB26, DIRAS2 and RAB13.
    b) instructions for using said kit for detecting the presence of astrocytoma in said human subject.


In another embodiment kit for detecting the presence of astrocytoma in a human subject, wherein the said kit comprises:

    
    
        [a] reagents capable of specifically detecting the level of expression of single or combination of genes selected from the group comprising of SYT1, RAB26, DIRAS2 and RAB13;
        [b] instructions for using said kit for characterizing astrocytoma in said human subject. The reagents used in such kits preferably comprise of an amplicon or nucleic acid probe represented by SEQ ID No. 42, 44, 46 and 48.
    
    


In another embodiment, the present invention also provides a kit for identifying the malignant astrocytoma (AA, GBM) in a human subject, wherein the said kit comprising:

    
    
        a) a reagent capable of specifically detecting the level of expression of a the gene IGFBP7.
        b) instructions for using said kit for identifying the malignant astrocytoma (AA, GBM) in said human subject. The reagents used in such kits preferably comprise of an amplicon or nucleic acid probe represented by SEQ ID No. 50.
    
    


The present invention also provides a kit for detecting the presence of malignant astrocytoma in a human subject, wherein the said kit comprises:

    a) reagent comprises an antibody that specifically binds to proteins encoded by the said genes.
    b) instructions for using said kit for characterizing astrocytoma in said human subject


In another embodiment, the present invention also provides a kit for detecting the presence of glioblastoma (GBM) in a human subject, wherein the said kit comprising:

    a) reagent capable of specifically detecting the level of expression of a single or combination of said genes such as COL6A1, DCN, PLAT, LGALS3, FABP7, LOX, LAMB1, IGFBP3, GADD45A, FSTL1, RHOC, B2M and PBEF1
    b) instructions for using said kit for detecting the presence of glioblastoma in said human subject. The reagents used in such kits preferably comprise of an amplicon or nucleic acid probe represented by SEQ ID No. 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 76 and 80.


The present invention also provides a kit for detecting the presence of glioblastoma in a human subject, wherein the said kit comprises:

    a) reagent comprises an antibody that specifically binds to proteins encoded by the said genes.
    b) instructions for using said kit for detecting the presence of glioblastoma in said human subject


In yet other embodiment, the present invention provides a kit for identifying the type of glioblastoma in a human subject, wherein the said kit comprising:

    a) reagent capable of specifically detecting the level of expression of a single or the combination of said, genes such as AEBP1, SOD2.
    b) instructions for using said kit for identifying the type of glioblastoma in said human subject. The reagents used in such kits preferably comprise of an amplicon or nucleic acid probe represented by SEQ ID No. 74 and 78.


The present invention also provides a kit for identifying the type of glioblastoma in a human subject, wherein the said kit comprises:

    a) the reagent comprises an antibody that specifically binds to proteins encoded by the said genes.
    b) instructions for using said kit for identifying the type of glioblastoma in said human subject


The present invention also provides a kit to determine the prognosis of glioblastoma in a human subject, wherein the said kit comprising:

    a) reagent capable of specifically detecting the presence or absence of a PBEF1.
    b) instructions for using said kit to determine the prognosis of glioblastoma in said human subject.


The present invention also provides a kit for determining the prognosis of glioblastoma in a human subject, wherein the said kit comprises:

    
    
        [a] reagent capable of specifically detecting the level of expression of the gene PBEF1;
        [b] instructions for using said kit for determining the prognosis of glioblastoma in said human subject. The reagents used in such kits preferably comprise an amplicon or nucleic acid probe represented by SEQ ID No. 80.
    
    





BRIEF DESCRIPTION OF FIGURES
FIG. 1. Scatter plots of the genes that are differentially regulated in astrocytoma in comparison to normal brain tissue.
Log 2-transformed gene expression ratios obtained from real-time quantitative PCR analysis are plotted for SYT1 i.e FIG. 1A, RAB26 i.e FIG. 1B, DIRAS2 i.e FIG. 1C and RAB13 i.e FIG. 1D. Each dot represents a data derived from one sample. Horizontal bar represents the median log 2 ratio of the corresponding group.
FIG. 2. Scatter plots of the genes that are up-regulated in malignant astrocytoma (AA, GBM) in comparison to normal brain tissue.
Log 2-transformed gene expression ratios obtained from real-time quantitative PCR analysis are plotted for IGFBP7. Each dot represents a data derived from one sample. Horizontal bar represents the median log 2 ratio of the corresponding group.
FIG. 3. Scatter plots of glioblastoma specific genes.
Log 2-transformed gene expression ratios obtained from real-time quantitative PCR analysis are plotted for COL6A1 i.e. FIG. 3A, DCN i.e. FIG. 3B, PLAT i.e FIG. 3C, LGALS3 i.e 3D, FABP7 i.e FIG. 3E, LOX i.e FIG. 3F, LAMB1 i.e FIG. 3G, IGFBP3 i.e FIG. 3H, GADD45A i.e FIG. 3I, FSTL1 i.e FIG. 3J, RHOC i.e FIG. 3K, B2M i.e FIG. 3L and PBEF1 i.e FIG. 3M. Each dot represents a data derived from one sample. Horizontal bar represents the median log 2 ratio of the corresponding group.
FIG. 4. Scatter plots of primary glioblastoma specific genes.
Log 2-transformed gene expression ratios obtained from real-time quantitative PCR analysis are plotted for AEBP1 i.e FIG. 4A and SOD2 i.e FIG. 4B. Each dot represents a data derived from one sample. Horizontal bar represents the median log 2 ratio of the corresponding group.
FIG. 5(Q-T). Immunohistochemical validation of IGFBP7.
Representative micrographs showing IGFBP-7 staining in diffusely infiltrating astrocytomas (DA, AA, s/p GBM). This isoform shows staining in malignant astrocytomas (AA, GBM). IGFBP-7 staining is shown in panels Q, R, S and T. A few tumor cells are labeled in DA (Q). Gradual increase in the number of labeled cells is seen in AA (R), secondary GBM (S), primary GBM (T).
FIG. 6(E-H). Immunohistochemical validation of IGFBP3.
Representative micrographs showing IGFBP-3 staining in diffusely infiltrating astrocytomas (DA, AA, s/p GBM). This isoform shows maximal staining pattern in GBMs. IGFBP-3 staining is shown in panels E, F, G and H. A moderate number of tumor cells show cytoplasmic staining in DA (E) and AA (F) while secondary GBM (G) and primary GBM (H) show strong (3+) cytoplasmic staining of several tumor cells.
FIG. 7(A-E). Immunohistochemical validation of GADD45A.
Immuno-histochemical validation of GADD45A overexpression in glioblastoma. Sections from normal brain—negative for staining (A), DA—negative for staining (B), AA—negative for staining (C), secondary GBMs—positive for staining (D); Primary GBM—positive for staining (E) were stained for GADD45A.
FIG. 8(A-E). Immunohistochemical validation of FSTL1.
Immunohistochemical validation of FSTL1 overexpression in glioblastoma. Sections from normal brain—negative for staining (A), DA—negative for staining (B), AA—negative for staining (C), secondary—positive for staining GBMs (D); Primary GBMs—positive for staining (E) were stained for FSTL1.
FIG. 9(A-E). Immunohistochemical validation of B2M.
Immunohistochemical validation of B2M overexpression in glioblastoma. Sections from normal brain-whitematter (A), DA (B) and AA (C) are negatively stained for B2M while Secondary GBM (D) and Primary GBM (E, F) are positively stained. Note that few parenchymal venules in normal brain (A) and in DA (B) are faintly labeled.
FIG. 10. Immunohistochemical validation of PBEF1.
Sections from normal brain—negative for staining (A), DA—negative for staining (B), AA—negative for staining (C), AA—positive for staining (D), GBMs—positive for staining (E and F) were stained for PBEF1
FIG. 11. PBEF1 expression and survival of patients with GBM.
Kaplan-Meier survival estimates for 51 GBM patients are calculated for p53, EGFR1 and PBEF1 staining. (A) Survival curves for the groups positive and negative for PBEF1 in univariate analysis. The cases which are positive for PBEF1 (red line) had a poorer survival than the cases which were negative (green line) (P=0.16), (B) Survival curves for the groups positive and negative for co-expression of p53 and PBEF1, in multivariate analysis. The group positive for both the markers (red line) had a poor survival as compared to the group negative for both or either of them (green line).



EXAMPLES
The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.
Example 1
Tissue Collection
Astrocytoma tissue samples were collected from patients, who underwent surgery at Sri Satya Sai Institute of Higher Medical Sciences and Manipal Hospital, Bangalore, India at the time of surgical resection. Controls comprised non-tumrous brain tissue samples (temporal lobe) collected from patients who underwent surgery for intractable epilepsy. A total of thirty-seven astrocytoma samples of different grades were used in this study. Tissues were bisected and one half was snap-frozen in liquid nitrogen and stored at −80° C. until RNA isolation. The other half was fixed in formalin and processed for paraffin sections and these were used to identify the histopathological grade and the type of astrocytoma.
Example 2
RNA Isolation
Total RNA was extracted from the frozen tissue by a combination of the TRIzol method (Invitrogen, USA) and RNeasy Midi kit (Qiagen) according to the manufacturer's instructions. The RNA samples were quantified by measuring the absorbance using a spectrophotometer and visualized on a MOPS-Formaldehyde gel for quantity and quality assurance.
Example 3
Quantitative RT-PCR
The relative quantitation of expression levels of selected genes was carried out using a two-step strategy: in the first step, cDNA was generated from RNA derived from different tissue samples using the High-capacity cDNA archive kit (ABI PRISM); subsequently real-time quantitative PCR was carried out with the cDNA as template using the following gene-specific primer sets and DyNAmo HS SYBR Green qPCR kit (Finnzymes, Finland): All the primers used were designed using the Primer Express software Version 2.0.0 from Applied Biosystems.
The list of primer pairs is given below.













Sl. NO:
Gene
Forward Primer (5′ to 3′)
Reverse Primer (5′ to 3′)


















 1
SYT1
GGTTGGCTGTTTCCCAGTAAAAC
TTTTAAGAAGTACGGACCATCGG


 


 2
RAB26
GTCTGCTGGTGCGATTCAAG
GCATGGGTAACACTGCGGA


 


 3
DIRAS2
CTGGTGTTGAGGTTTGTGAAAGG
CCGTCGTGTCGGTGATCTG


 


 4
RAB13
ATAACTACTGCCTACTACCGTGG
CCATGTCACATTTGTTCCCCAG


 


 5
IGFBP7
GGTCCTTCCATAGTGACGCC
TCTGAATGGCCAGGTTGTCC


 


 6
COL6A1
ACAGTGACGAGGTGGAGATCA
GATAGCGCAGTCGGTGTAGG


 


 7
DCN
AGTTGGAACGACTTTATCTGTCC
GTGCCCAGTTCTATGACAATCA


 


 8
PLAT
ACTGCCGGAATCCTGATGG
TGTGCTTGGCAAAGATGGC


 


 9
LGALS3
TGCTGATAACAATTCTGGGCAC
TGAAGCGTGGGTTAAAGTGGA


 


10
FABP7
CTCTCAGCACATTCAAGAACACG
GCGAACAGCAACCACATCAC


 


11
LOX
CAGGGTGCTGCTCAGATTTCC
GGTAATGTTGATGACAACTGTGC


 


12
LAMB1
ACAAGCCCGAACCCTACTGTA
GACCACATTTTCAATGAGATGGC


 


13
IGFBP3
AGAGCACAGATACCCAGAACT
TGAGGAACTTCAGGTGATTCAGT


 


14
GADD45A
GAGAGCAGAAGACCGAAAGGA
CACAACACCACGTTATCGGG


 


15
FSTL1
CAACCACTGTGAACTGCATCG
CCTTTAGAGAACCAGCCATCTG


 


16
RHOC
CCTGCCTCCTCATCGTCTTC
AGCACATGAGGATGACATCAGTG


 


17
AEBP1
AAAGGGCGAGGAGTTGGAG
GAGGCTCGGATCTGGTTGT


 


18
B2M
AGGCTATCCAGCGTACTCCAA
AATGCGGCATCTTCAAACC


 


19
SOD2
AACCTCAGCCCTAACGGTG
AGCAGCAATTTGTAAGTGTCCC


 


20
PBEF1
ATTGCCTTCGGTTCTGGTGG
CGGCCCTTTTTGGACCTTTTG









Real-time quantitative PCR was carried out in ABI PRISM 7900 (Applied Biosystems) sequence detection system with the cDNA as template using gene specific primer sets and Dynamo kit containing SYBR green dye (Finnzyme, Finland). All measurements were made in triplicate. The genes RPL35A (ribosomal protein L35a), AGPAT1 (1-acylglycerol-3-phosphate O-acyltransferase 1), ATP5G1 (ATP synthase, H+ transporting, mitochondrial F0 complex, subunit C1 (subunit 9)) and GARS (glycyl-tRNA synthetase) were used as internal controls as their expression levels were found to be unaltered in the array experiments. Normal brain tissue samples from nine different epilepsy patients were used as reference. For each sample, fold change in gene expression is calculated over its mean expression in normal brain sample. Statistical significance was tested by Mann-Whitney test using GraphPad PRISM® software. For each gene, depending on its expression, a suitable threshold fold change was used to calculate the percentage of regulated samples.
Example 4
Histopathology and Immunohistochemistry (IHC)
Histological sections of normal brain and tumor tissues were examined by light microscopy using hematoxylin and eosin (H&E) preparation. Tumor sections of diffusely infiltrating astrocytomas were graded using the WHO grading scheme (11). Paraffin sections (4 μm) from the tumor tissue and controls were collected on silane coated slides for IHC. The primary antibodies used are as follows: 1. IGFBP7 (H-102, SC-3095, diluted to 1:25); 2. IGFBP3 (H-98, SC-9028, diluted to 1:50); 3. GADD45A (Santa cruz, clone C-20, rabbit polyclonal-1:50 dilution); 4. F STL1 (Rabbit polyclonal antibody against purified GST-FSTL1, 1:100 dilution); 5. B2M (Santa cruz, mouse monoclonal, 1:50 dilution); 6. PBEF1 (Rabbit polyclonal antibody against purified GST-PBEF1; 1:1000 dilution). Microwave antigen retrieval was done at 400 watt for 18 minutes in 10 mM citrate buffer, pH 6.0. The antibodies used mainly for the purpose of sub-classifying GBM cases were p53 (monoclonal: DO-7, Biogenix-USA, diluted to 1:200) and EGFR (monoclonal: E-30, Biogenix-USA, diluted to 1:50). For p53, antigen retrieval was performed by heat treatment of the deparaffinized sections in a microwave oven for 25-35 minutes at 700 W in citrate buffer (10 mM, Ph 6.0). For EGFR staining, the sections were pretreated with Tris-EDTA pH9.0 at 600 W for 30 minutes. All sections were further treated with methanol and 5% hydrogen peroxide to block endogenous peroxidase followed by washes with PBS buffer (pH 7.6). Skimmed milk powder (5%) was used to block background staining for 45 minutes. The sections were incubated with the primary antibody overnight at 4° C. This was followed by incubation with supersensitive non-biotin HRP detection system (QD440-XAK, Biogenex). “3,3′-Diaminobenzidine” (Sigma) was used as the chromogenic substrate.
Brain tumor samples previously characterized for over-expression of p53 and EGFR were used as positive controls. p53 and EGFR immunoreactivity was considered positive when more than 20% of tumor cells stained positively (nuclear and membrane cytoplasmic labeling respectively). GBMs were classified as primary and secondary, taking into consideration the clinical profile of patients, expression of EGFR and p53 (5). The mean age of patients with primary GBM was 50.6 years and mean duration of symptoms was 2.7 months. Uniform staining for EGFR was evident in all cases and five revealed additionally p53 expression. Among secondary GBMs, the mean age of the patients was 33.8 years and mean duration of symptoms was 5.3 months. p53 immunoreactivity was uniformly evident in all cases and two revealed additionally EGFR over-expression.
ADVANTAGES
The advantages of the present invention are:

    
    
        1. It provides a useful method for diagnosing the presence of astrocytoma.
        2. It provides a useful method for diagnosing the presence of malignant astrocytoma (AA or GBM).
        3. It provides a useful method for identifying the type of Glioblastoma—namely primary and secondary.
        4. It provides a useful method to determine the prognosis of glioblastoma.
        5. The method is useful both before and after clinical symptoms have appeared.
        6. The method can also be applied to monitor the effectiveness of anti-cancer treatments.

## Claims

1. A method of identifying and grading glioma in a human subject comprising determining the level of expression of a combination of genes including
SYT1, RAB26, DIRAS2, RAB13, IGFBP7, COL6A1, DCN, PLAT, LGALS3, FABP7, LOX, LAMB1, IGFBP3, GADD45A, FSTL1, RHOC, B2M, PBEF1, AEBP1,and SOD2 in a test sample of brain tissue cells obtained from the human subject,
wherein the level of expression of said combination of genes is determined by using the primer pairs:








SEQ ID NO: 1 as a Forward Primer of SYT1 gene:


5′ GGTTGGCTGTTTCCCAGTAAAAC 3′;


 


SEQ ID NO: 2 as a Reverse Primer of SYT1 gene:


5′ TTTTAAGAAGTACGGACCATCGG 3′;


 


SEQ ID NO: 3 as a Forward Primer of RAB26 gene:


5′ GTCTGCTGGTGCGATTCAAG 3′;


 


SEQ ID NO: 4 as a Reverse Primer of RAB26 gene:


5′ GCATGGGTAACACTGCGGA 3′;


 


SEQ ID NO: 5 as a Forward Primer of DIRAS2 gene:


5′ CTGGTGTTGAGGTTTGTGAAAGG 3′


 


SEQ ID NO: 6 as a Reverse Primer of DIRAS2 gene:


5′ CCGTCGTGTCGGTGATCTG 3′;


 


SEQ ID NO: 7 as a Forward Primer of RAB13 gene:


5′ ATAACTACTGCCTACTACCGTGG 3′;


 


SEQ ID NO: 8 as a Reverse Primer of RAB13 gene:


5′ CCATGTCACATTTGTTCCCCAG 3′;


 


SEQ ID NO: 9 as a Forward Primer of IGFBP7 gene:


5′ GGTCCTTCCATAGTGACGCC 3′;


 


SEQ ID No 10 as a Reverse Primer of IGFBP7 gene:


5′ TCTGAATGGCCAGGTTGTCC 3′;


 


SEQ ID NO: 11 as a Forward Primer of COL6A1 gene:


5′ ACAGTGACGAGGTGGAGATCA 3′;


 


SEQ ID NO: 12 as a Reverse Primer of COL6A1 gene:


5′ GATAGCGCAGTCGGTGTAGG 3′;


 


SEQ ID NO: 13 as a Forward Primer of DCN gene:


5′ AGTTGGAACGACTTTATCTGTCC 3′;


 


SEQ ID NO: 14 as a Reverse Primer of DCN gene:


5′ GTGCCCAGTTCTATGACAATCA 3′;


 


SEQ ID No 15 as a Forward Primer of PLAT gene:


5′ ACTGCCGGAATCCTGATGG 3′;


 


SEQ ID No 16. as a Reverse Primer of PALT gene:


5′ TGTGCTTGGCAAAGATGGC 3′;


 


SEQ ID NO: 17 as a Forward Primer of LGALS3 gene:


5′ TGCTGATAACAATTCTGGGCAC 3′;


 


SEQ ID NO: 18 as a Reverse Primer of LGALS3 gene:


5′ TGAAGCGTGGGTTAAAGTGGA 3′;


 


SEQ ID NO: 19 as a Forward Primer of FABP7 gene:


5′ CTCTCAGCACATTCAAGAACACG 3′;


 


SEQ ID NO: 20 as a Reverse Primer of FABP7 gene:


5′ GCGAACAGCAACCACATCAC 3′;


 


SEQ ID NO: 21 as a Forward Primer of LOX gene:


5′ CAGGGTGCTGCTCAGATTTCC 3′;


 


SEQ ID NO: 22 as a Reverse Primer of LOX gene:


5′ GGTAATGTTGATGACAACTGTGC 3′;


 


SEQ ID NO: 23 as a Forward Primer of LAMB1 gene:


5′ ACAAGCCCGAACCCTACTGTA 3′;


 


SEQ ID NO: 24 as a Reverse Primer of LAMB1 gene:


5′ GACCACATTTTCAATGAGATGGC 3′;


 


SEQ ID NO: 25 as a Forward Primer of IGFBP3 gene:


5′ AGAGCACAGATACCCAGAACT 3′;


 


SEQ ID NO: 26 as a Reverse Primer of IGFBP3 gene:


5′ TGAGGAACTTCAGGTGATTCAGT 3′;


 


SEQ ID NO: 27 as a Forward Primer of GADD45A gene:


5′ GAGAGCAGAAGACCGAAAGGA 3′;


 


SEQ ID NO: 28 as a Reverse Primer of GADD45A gene:


5′ CACAACACCACGTTATCGGG 3′;


 


SEQ ID NO: 29 as a Forward'rimer of FSTL1 gene:


5′ CAACCACTGTGAACTGCATCG 3′;


 


SEQ ID NO: 30 as a Reverse Primer of FSTL1 gene:


5′ CCTTTAGAGAACCAGCCATCTG 3′;


 


SEQ ID NO: 31 as a Forward Primer of RHOC gene:


5′ CCTGCCTCCTCATCGTCTTC 3′;


 


SEQ ID NO: 32 as a Reverse Primer of RHOC gene:


5′ AGCACATGAGGATGACATCAGTG 3′;


 


SEQ ID NO: 33 as a Forward Primer of AEBP1 gene:


5′ AAAGGGCGAGGAGTTGGAG 3′;


 


SEQ ID NO: 34 as a Reverse Primer of AEBP1 gene:


5′ GAGGCTCGGATCTGGTTGT 3′;


 


SEQ ID NO: 35 as a Forward Primer of B2M gene:


5′ AGGCTATCCAGCGTACTCCAA 3′;


 


SEQ ID NO: 36 as a Reverse Primer of B2M gene:


5′ AATGCGGCATCTTCAAACC 3′;


 


SEQ ID NO: 37 as a Forward Primer of S002 gene:


5′ AACCTCAGCCCTAACGGTG 3′;


 


SEQ ID NO: 38 as a Reverse Primer of S002 gene:


5′ AGCAGCAATTTGTAAGTGTCCC 3′;


 


SEQ ID NO: 39 as a Forward Primer of PBEF1 gene:


5′ ATTGCCTTCGGTTCTGGTGG 3′; and


 


SEQ ID NO: 40 as a Reverse Primer of PBEF1 gene:


5′ CGGCCCTTTTTGGACCTTTTG 3′.

2. The method of claim 1, wherein a higher level of expression of RAB13 in the test sample or a lower level of expression of SYT1, RAB26, and/or DIRAS2 in the test sample as compared to a control sample indicates the presence of astrocytoma in the human subject and a higher level of expression of IGFBP7 in the test sample as compared to a control sample indicates the presence of malignant astrocytoma in the human subject.

3. The method of claim 2, wherein a higher level of expression of COL6A1, DCN, PLAT, LGALS3, FABP7, LOX, LAMB1, IGFBP3, GADD45A, FSTL1, RHOC, B2M, and/or PBEF1 in the test sample as compared to a control sample indicates the presence of glioblastoma in the human subject and a higher level of expression of AEBP1 and/or SOD2 in the test sample as compared to a control sample indicates the presence of primary glioblastoma in the human subject and a higher level of expression of PBEF1 in the test sample, as compared to a control sample indicates a poor prognosis for survival of the human subject.

4. The method of claim 1, wherein determining the level of gene expression comprises RT-PCR analysis.

5. The method of claim 1, further comprising detecting the presence or levels of EGFR, p53, and/or Ki-67 proteins.