Method for quantitative cDNA analysis in single-cell

Abstract

It is an object to provide a method of suitably analyzing the amount of gene expression of a single-cell. A method of detecting a nucleic acid comprising a step of sampling a single-cell from a sample containing at least a single-cell, a cell lysis step of lysing cell membrane of the sampled single-cell and extracting nucleic acids from the cell, a DNase treatment step of degrading DNA of the extracted nucleic acids with DNase, a step of hybridizing mRNA of the total RNA contained in the single-cell with oligo (dT) fixed onto a carrier, a step of performing reverse transcription of the mRNA hybridized with the oligo (dT) to fix cDNA derived from the single-cell onto the carrier, thereby preparing a single-cell derived cDNA library fixed onto a carrier, and a step of amplifying cDNA fixed onto the carrier and simultaneously detecting an amplification amount of the cDNA.

Description

technical field [0001] The present invention relates to a method for quantitatively analyzing gene expression of a single cell-derived cDNA-immobilized vector library obtained by immobilizing single-cell-derived cDNA onto a carrier as an analysis sample. The present invention also relates to a method for quantitatively analyzing gene expression of a cDNA-immobilized carrier library obtained by immobilizing cDNA derived from a small number of cells onto a carrier as an analysis sample. Background technique [0002] Typical gene expression analysis methods in the past include the microarray method and the real-time PCR method. Most of these conventional methods use a tissue sheet composed of a plurality of cells or a plurality of cultured cells (~10 6 ) as an analytical sample. In actual cells, gene expression levels vary over time, so even in the same tissue, gene expression in each cell differs in time depending on the location. Therefore, in analysis using a tissue or a p...

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Problems solved by technology

However, because the cDNA solution synthesized using this reagent contains residual reagents, which inhibits the PCR amplification reaction, it is difficult to perform real-time PCR analysis using all the cDNA of a single cell.

Therefore, in order to reduce the inhibition of PCR amplification caused by residual reagents, cDNA from a single cell must be used as an analysis sample again, resulting in a significant decrease in the detection sensitivity of real-time PCR analysis.

Also, with analysis, cDNA from a single cell is consumed, so the types of genes that can be analyzed may be limited

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