Patent Description:
Cultured cells of a mammal or the like are widely used in the fields of medicine, research, and the like, and cells may deteriorate during the culture and storage. In order to confirm that cells are not deteriorated during the culture and storage, it is widely performed to control and ensure the quality of cells by gene expression analysis using real-time PCR (Patent Literature <NUM>).

In a real-time PCR assay for use in such a gene expression analysis, relative quantification using a calibration curve method or a comparative Ct method has been widely used. In a calibration curve method, serially diluted RNA extracted from a sample (cell) that highly expresses both of internal standard genes and target genes, or serially diluted cDNA synthesized from the RNA, is used as a standard. In a case of relative quantification, it is required to compare a reference calibrator sample with a target sample for each gene.

In performing research on a specific cell over a long period of time, in order to repeatedly perform relative quantification of gene expression as accurately as possible throughout the entire period of time, it is desirable to prepare a large amount of standards or calibrator samples in the initial stage of the research, and to use stored aliquots from the same cDNA/RNA pool in each gene expression analysis. However, it is difficult to obtain a sufficient amount of standards or calibrators to be required throughout the life cycle over a long period from development to market launch, and to stably store and maintain the obtained standards or calibrator samples. This is thus not realistic.

Further, in the regenerative medicine area and the like, the need for supplying a large amount of cells having a specific property over a long period of time has been increased, and gene expression analysis is used to control the quality of cells (Patent Literature <NUM>). It is difficult to supply a large amount of standards or calibrators to be required for the gene expression analysis for use in quality control of these cells, and a better method has been demanded.

In addition, in a case where cells/tissues of which the quality should be ensured are maintained in a large amount, it is assumed that these cells/tissues are required to be controlled in physically separated facilities, and when the quality control of cells is performed individually in these remote facilities, stable transfer of standards or calibrators may also become a problem. K Kruczek (<NUM>-<NUM>-<NUM>)discloses a method for absolute quantification with real-time PCR wherein a dilution series of plasmid DNA containing cDNA sequence for a particular mRNA was prepared. ALIAKBAR HADDAD-MASHADRIZEH ET AL (<NUM>-<NUM>-<NUM>) use a method for the absolute quantification of cDNA using real-time PCR. KARSTEN SCHROBBACK ET AL: (<NUM>-<NUM>-<NUM>) uses a method for the absolute quantification of cDNA using real-time PCR. RAGINA ET AL (<NUM>-<NUM>-<NUM>) relates to the use of absolute quantification using real-time PCR in the framework of regenerative medicine. <CIT> relates to the use of absolute quantification using real-time PCR in the framework of regenerative medicine. For absolute quantification of the HSVtk gene in JUNG JUYEON ET AL (<NUM>-<NUM>-<NUM>), a standard curve was generated, using a serial dilution of pTK629 plasmid DNA ranging from <NUM> to <NUM> copy. In <CIT>, absolute quantification of each gene was obtained using a standard curve of serial diluted genomic DNA and normalized to housekeeping genes β-ACTIN and Tata Box Binding protein. XIAO LING KUAI ET AL (<NUM>-<NUM>-<NUM>)relates to the use of absolute quantification using real-time PCR in the framework of regenerative medicine.

As for a cell/tissue to be used in research over a long period of time, in order to repeatedly perform relative quantification of gene expression of the cell/tissue as accurately as possible throughout the entire period of research, a large amount of standard or calibrator samples are required, and it is difficult to supply such a large amount of standards.

In particular, in the regenerative medicine area, the quality control to maintain constantly the property of a large amount of cells/tissues to be used over a long period of time is important, and in order to check that there is no change in the gene expression pattern of a cell, it is required to perform the gene expression analysis at fixed time intervals over a long period of time. A technique that enables the repeated gene expression analysis to be more stably performed over such a long period of time has been required.

The present invention stably provides uniform standards for the repeated gene expression analysis over a long period of time by employing an absolutely quantified standard including a synthetic DNA plasmid or the like. Further, the present invention enables the repeated gene expression analysis to be stably performed with high reproducibility by using such a standard.

By using the method according to the present invention, the quality of a cell having a specific property can be ensured over a long period of time, and thus the method is extremely useful in the field where cells having a specific property are required to be supplied in a large amount over a long period of time, for example, in the regenerative medicine area.

In addition, the method according to the present invention enables the efficacy or safety of a regenerative medicine product to be evaluated with high accuracy. Further, by using a synthetic DNA plasmid as the absolutely quantified standard, the repeated gene expression analysis over a long period of time can be performed stably while the standard can be prepared in each facility when performing the quality control of cells individually in a remote facility.

In addition, by employing an absolutely quantified standard, a sample for comparison is not necessarily required, and thus the evaluation can be performed with a standardized expression level obtained by dividing the copy number of a target gene in a target sample by the copy number of an internal standard gene, each of which has been obtained using a calibration curve.

In one aspect, the present invention is a method for analyzing gene expression in a cell or a tissue containing a cell, including using an absolutely quantified standard, as disclosed in claim <NUM>.

Further, in another aspect, the present invention is the method for analyzing gene expression described above, in which the method uses real-time PCR.

In addition, in another aspect, the present invention is the method for analyzing gene expression described above, in which the absolutely quantified standard being a synthetic DNA is used.

Further, in another aspect, the present invention is the method for analyzing gene expression described above, in which the absolutely quantified standard being a DNA plasmid is used.

In addition, in another aspect, not part of the invention, is a method for controlling quality of a cell or a tissue containing a cell, by using the method for analyzing gene expression described above.

Further, in another aspect, not part of the invention, is a method for controlling quality of a cell or a tissue containing a cell, in which the cell is a mammal cell.

In addition, in another aspect, not part of the invention, is a method for controlling quality of a cell or a tissue containing a cell, in which the cell is a somatic stem cell.

Further, in another aspect, not part of the invention, is a method for controlling quality of a cell or a tissue containing a cell, in which the cell or the tissue is used in the regenerative medicine area.

In addition, in another aspect, the present invention is a method for evaluating efficacy or safety of a regenerative medicine product containing a cell, by using the method for analyzing gene expression described in claims <NUM>-<NUM>.

Further, in another aspect, the present invention is the use of one or multiple absolutely quantified standards in the above method for analyzing gene expression as disclosed in claim <NUM> or in the above method for evaluating efficacy or safety of a regenerative medicine product as disclosed in claim <NUM>.

In the present invention, the expression "gene expression analysis" includes creating a population of what are derived from a cell or tissue sample and analyzing the population to determine which gene is expressed in the sample. Typically, by examining the presence of mRNA derived from each gene in the sample, the expression of the gene is evaluated.

The expression "quantitative gene expression analysis" means analysis to determine the relative or absolute value of the expression level of a gene. For example, the "quantitative gene expression analysis" includes analysis to determine the expression level of a gene based on the mRNA level in a sample.

The expression "PCR" or "polymerase chain reaction" means a technique for replicating a selected specific small fragment of DNA in vitro, even in the presence of excessive non-specific DNA. When primers are added to the selected DNA, the primers initiate duplication of the selected DNA by using nucleotides, a polymerase, and the like. By cycling the temperature, the selected DNA repeats denaturation and duplication. In some cases, a linear amplification method may be used as a substitute for PCR.

The expression "real-time PCR" means various PCR applications in which amplification in PCR is measured during the reaction rather than after completion of the reaction. In gene expression analysis using real-time PCR, the abundance of mRNA in a sample is calculated, and the gene expression level is determined. At this time, RNA itself may be used as a template for PCR, or a cDNA that has reverse transcribed from RNA may be used as a template for PCR. In a case where RNA is used as the template, a RT-PCR reaction solution in which a reverse transcriptase is added to a PCR reaction solution is prepared, and a cDNA is synthesized with the use of the RNA as the template by a reverse transcription reaction before being applied to temperature cycling of PCR reaction, and then a target sequence can be amplified with the use of the cDNA as the template by the subsequent PCR reaction (<NUM>-step RT-PCR). Further, the reverse transcription reaction and the PCR reaction can be separately performed (<NUM>-step RT-PCR). As the real-time PCR method, it is not particularly limited, and for example, a method of using a template-dependent nucleic acid polymerase probe, for example, a probe such as a hydrolysis probe using <NUM>'-exonuclease activity of Taq DNA polymerase, a method of using an intercalator such as SYBR Green, or the like can be used.

The expression "target gene" means a gene to be subjected to measurement of the abundance of the corresponding mRNA in real-time PCR.

The expression "internal standard gene" means a gene of which the expression level is measured to correct the expression level between experiments in real-time PCR. Typically, as the internal standard gene, a gene that is expressed in a certain amount in common in many tissues and cells, constantly expressed at all times, and essential for the maintenance and growth of cells(housekeeping gene), is used.

The expression "standard" means a sample used for obtaining a calibration curve showing the relationship between the Ct value (the number of cycles when the amplification curve reaches a constant signal intensity) and the copy number in the gene sample in real-time PCR, and is constituted of a dilution series. Typically, the standard contains RNA extracted from a sample (cell) that highly expresses both of an internal standard gene and a target gene, or a cDNA synthesized from the RNA by reverse transcription, and also contains newly synthesized RNA or DNA.

The expression "calibration sample" means a sample arbitrarily selected as a reference when the relative abundance of a target gene in multiple samples is measured.

The expression "expression profile" means a measurement result of the abundance ratio of multiple cell components. For example, the expression profile means the abundance of multiple RNAs or proteins, or of a combination of multiple RNAs and proteins. The expression profile may be a measurement result of, for example, the transcription state or the translation state.

The expression "quality control" includes maintaining a cell and a tissue having constant properties by confirming that the cell and tissue maintain the constant properties, by removing the cells and tissues that have lost the properties, and the like. Whether or not the constant properties are maintained is typically determined based on the uniformity of the expression profiles of genes of the cell and tissue.

The expression "cell" means a smallest structural unit of an organism that is constituted of one or more nuclei, cytoplasm, and various organelles, and the whole parts are surrounded by a semipermeable cell membrane or a cell wall, and can independently function, or means a unicellular organism. The cell may be a prokaryote, a eukaryote, or an archaeon. As the cell, a mammalian cell, in particular, a human cell is preferred. The cell may be a natural cell, or may be a cell modified to achieve the desired property, for example, by genetic manipulation or by successive cultivation.

The absolutely quantified standard of the present invention means a standard in which the content (the number of molecules) of RNA or DNA or an analog thereof, serving as a template for PCR reaction and being contained in each sample of a dilution series of a standard to be used in real-time PCR, has been determined in advance.

As the RNA or DNA or an analog thereof, ones in various forms such as single-stranded form, double-stranded form, straight chain form, and circular form can be used, and preferably, a circular plasmid DNA can be used. Further, the RNA or DNA or an analog thereof may contain a non-natural base pair.

The RNA or DNA to be used for the standard can have a sequence of a target gene and/or a sequence of an internal standard gene. A person skilled in the art can appropriately select such a sequence based on a known method. For example, a person skilled in the art can synthesize RNA or DNA by experiment, or by using a primer and the like determined with the use of sequence data and the like of a publicly available nucleotide sequence database (NCBI, GenBank, and the like), and amplify the RNA or DNA having a desired sequence by PCR, or can newly synthesize RNA or DNA.

In addition, as the method for determining the content of RNA or DNA, it is not particularly limited, and a known method can be used, for example, a method for measuring the absorbance of a specific absorption wavelength of a nucleic acid by using a spectrophotometer, a method for measuring the fluorescence emitted when bound to a specific target molecule (for example, Qubit fluorometer (Invitrogen)), a method in which nucleic acid subjected to agarose gel electrophoresis is stained, and the concentration of the stained nucleic acid is measured, a method of microchip-type electrophoresis (for example, Experion DNA analysis kit manufactured by Bio-Rad Laboratories, Inc. ), or the like can be used. In particular, microchip-type electrophoresis is preferably used.

The quantification of the standard by such a known method can be performed repeatedly with high accuracy, and for example, even in a case where a newly absolutely quantified standard is synthesized and used, comparison with the result of the analysis performed by using a standard that has been previously used can be performed.

When the expression level of a target gene is calculated by using the absolutely quantified standard of the present invention, the expression level (copy number) of target gene in the sample can be calculated by applying the Ct value obtained from a sample of which the content is unknown, to a calibration curve created by using the standard.

Any known method can be used to create a calibration curve and for example, a calibration curve can be created by using an amplification curve, where the amplification curve showing the relationship between the number of cycles and the amount of amplified DNA is obtained from the result of real-time PCR of a dilution series of a standard.

Further, by obtaining the expression level of an internal standard gene of each sample of which the expression level of a target gene has been obtained in the similar way, thereby standardizing the expression level of the target gene in each sample with the expression level of the standard gene, it is possible to obtain the standardized expression level of the target gene in which the variation between the respective samples has been corrected.

In addition, in order to examine the difference in the expression level of each target gene between various cells, it is possible to calculate the relative expression level to the comparison cell by dividing the standardized expression level of the cell to be analyzed by the value of a comparison cell.

When a cell or a tissue is cultured and stored over a long period of time, the expression levels of multiple genes constituting a gene expression profile that characterizes each cell or tissue are obtained at fixed time intervals by the above method, and by confirming that there is no or slight change over time, it can be confirmed and ensured that the cell or tissue has not been deteriorated.

Here, the type of and the number of multiple genes constituting a gene expression profile that characterizes each cell or tissue can be arbitrarily selected by a person skilled in the art based on known information.

As such a cell, any cell can be selected. Preferably, the cell is a mammal cell, in particular, a human cell. Further, the cell is preferably a somatic stem cell, or is in addition, preferably a cell that is used in the regenerative medicine area.

In such a quality control, by using a synthetic DNA as the absolutely quantified standard, the quality of the cell can be controlled by a unified reference even when the cell or tissue is controlled in a different facility.

In a regenerative medicine product containing a cell, in order to exert a desired therapeutic effect, a cell is necessary to maintain a constant property. By using a method for analyzing gene expression with the use of the absolutely quantified standard of the present invention, by confirming that there is no or slight change over time of an expression profile of the cell contained in a regenerative medicine product, evaluation for determining whether or not the cell maintains a constant property is performed, that is, the efficacy of a regenerative medicine product containing the cell can be evaluated. In such cases, for example, in a case where evaluation is performed on the basis of the amount of change in the relative or absolute expression level of a specific gene (group), the standard of the amount change can be set to, but not limited to, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or the like.

In addition, the application of a deteriorated cell causes a high risk of losing the safety of a regenerative medicine product. By using a method for analyzing gene expression with the use of the absolutely quantified standard of the present invention, the deteriorated cell is detected, and the safety of a regenerative medicine product can be evaluated.

A set of absolutely quantified standards selected for quality control of a specific cell or tissue is provided as a set of standards or as a single standard, for use in quality control of the cell or tissue.

When the set of absolutely quantified standards is provided as a set of standards or as a single standard, an additive agent that is usually used, such as a preservative can be used in combination, and the set can be provided in any form of a liquid, a dry powder, or the like.

The present invention will be described in more detail in the following Examples.

For each somatic stem cell isolated from a tissue that had been collected from each of donors A to F, a sample was prepared from each of the cells having different culture conditions (conditions <NUM> to <NUM>) and different passage numbers (P3, P4, and P7).

For the prepared DNA samples, the expressions of genes A to C were analyzed by real-time PCR according to the following procedures. Further, a commercially available fibroblast (comparison cell P3) derived from the same tissue was cultured, and similarly analyzed as the comparison cell.

RNA was extracted from a cell culture flask of <NUM>% confluent by the following procedures. A medium in the flask was removed, and the flask was washed with DPBS(-) twice, and then <NUM>µL of Buffer RLT (QIAGEN) was added to the washed flask, cells were peeled off by using a cell scraper, and a cell lysate was recovered in a <NUM>-mL tube. From this cell lysate, RNA was extracted by using RNeasy Mini Kit (QIAGEN) (genomic DNA was removed by using RNase-Free DNase Set (QIAGEN)), and finally the RNA was eluted with <NUM>µL of RNase free water.

By using an RNA solution, reverse transcription was performed with the use of QuantiTect Reverse Transcriptase kit (QIAGEN) to prepare a cDNA. In the reverse transcription reaction, a reaction volume of <NUM>µL and <NUM>µg of total RNA were used.

A plasmid in which a target sequence of each of genes containing an internal standard gene had been cloned was linearized and purified, and quantified by using a microchip-type electrophoresis device (Experion DNA analysis kit, Bio-Rad), and the concentration (ng/µL) obtained based on the molecular weight was converted to the copy number.

A primer-probe mix (in-house design) of the cryopreserved TaqMan Gene Expression Assay (target genes A to C) and the internal standard gene were thawed on ice, and a PCR reaction master mix was prepared for each gene on ice by using TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific). The master mix was aliquoted into respective wells of PCR tubes by <NUM>µL each, and the reverse transcription reaction solution prepared and diluted in advance (Nkx2. <NUM>: undiluted solution of reverse transcription reaction solution, GATA4, Tbx5, Mef2C: <NUM>-fold dilution, ACTB: <NUM>-fold dilution; n = <NUM> each), or a standard solution (<NUM><NUM> to <NUM><NUM> copies/µL; n = <NUM> each) was added into the respective wells by <NUM>µL each. The prepared PCR tubes were set in ABI7500 (Thermo Fisher Scientific), and were subjected to real-time PCR analysis.

In accordance with a conventional method, the copy numbers of each target gene and the internal standard gene in the reaction solution were calculated based on the calibration curve created for each gene. The standardized expression level of each target gene was calculated through the standardization where the obtained copy number of each target gene was divided by the copy number of the internal standard gene.

For the somatic stem cell sample derived from each donor, gene expression analysis was performed for each target gene by a calibration curve method, and the standardized expression level of each target gene was calculated.

In a similar manner, as a comparison, also for a fibroblast (comparison cell P3) derived from the same tissue as that of the above-described somatic stem cell, gene expression analysis was performed for each target gene by a calibration curve method and the standardized expression level of each target gene was calculated.

For each target gene, the relative expression level to the comparison cell was calculated by standardization where the standardized expression level in the somatic stem cell sample was divided by the standardized expression level in the comparison cell.

The test was considered valid when the coefficient of determination R2 of each calibration curve is <NUM> or more.

The standardized expression levels are shown in <FIG> by a heat map (the lower expression level is shown in darker and the higher expression level is shown in brighter). Gene expression profiles that are different from each other depending on each donor and the sample preparation condition were obtained.

The relative expression levels to a target gene in a comparison cell are shown in <FIG> by a heat map. With respect to each target gene, the difference in the expression profile depending on each donor and the sample preparation condition is clearly shown.

In the method for analyzing gene expression with the use of the absolutely quantified standard of the present invention, the state of each sample cell can be evaluated with the standardized expression level not necessarily with obtaining the relative expression level with the comparison cell.

Claim 1:
A method for analyzing gene expression in a cell or a tissue containing a cell, comprising:
- a step of preparing a first sample from the cell comprising mRNA expressed from a gene to be analyzed;
- a step of preparing a dilution series of absolutely quantified standards for the gene to be analyzed;
- preparing a second sample from the cell comprising mRNA expressed from an internal standard gene;
- a step of preparing a dilution series of absolutely quantified standards for the internal standard gene;
- a step of obtaining a calibration curve for the dilution series of absolutely quantified standards for the internal standard gene, and a calibration curve for the dilution series of absolutely quantified standards for the gene to be analyzed,
∘ wherein each calibration curve is based on an amplification curve obtained from a real-time PCR of the corresponding dilution series of the absolutely qualified standard, wherein the calibration curve shows a relationship between the number of cycles when the amplification curve reaches a constant rate of increase of signal intensity (Ct value) and the amount of amplified DNA;
- a step of calculating a copy number of the gene to be analyzed from the corresponding calibration curve based on the Ct value of the mRNA comprised in the first sample, determined by amplifying the mRNA from the first sample;
- a step of calculating a copy number of the internal standard gene from the corresponding calibration curve based on the Ct value of the mRNA comprised in the second sample, determined by amplifying the mRNA from the second sample; and
- a step of dividing the copy number of the gene to be analyzed by the copy number of the internal standard gene.