Single-cell qPCR Assay with Massively Parallel Microfluidic System.

Abstract

The single-cell transcriptome is the set of messenger RNA molecules expressed in one cell. It is extremely variable and changes according to external, physical and biochemical conditions. Due to sensitivity shortages, most of genetic studies use bulk samples, providing only the average gene expression. Single-cell technologies have provided a powerful approach to a more detailed understanding of the heterogenic populations and minority cells. However, since it is still a quite novel technique, standardized protocol has to be established. Single-cell qPCR, although partly limited by the number of genes, is relatively simple to analyze. Therefore, its use is accessible without the necessity to recourse to complex bioinformatics analyses. The main steps for single-cell qPCR, as illustrated in this protocol, are composed by single-cell isolation, cell lysate, cDNA reverse-transcription synthesis, amplification for cDNA library generation, and finally, quantitative polymerase chain reaction.