Quantitative Estimation of Multiple miRNAs and mRNAs from a Single Cell

Abstract

INTRODUCTIONAlthough all cells of an individual contain the same genetic material, each cell is unique with respect to its gene expression profile. Recent studies have demonstrated that small regulatory noncoding RNAs, microRNAs (miRNAs), negatively regulate gene expression by targeting mature messenger RNAs (mRNAs). Understanding the abundance of miRNAs and target gene transcripts from a single cell has therefore gained importance in research and clinical fields. In several circumstances, multiple gene transcript analysis of single cells from heterogenous populations provides an important tool in assessing variability in gene expression within a population. Transcript analysis at the single-cell level is also necessary when the starting material is limited, such as in the case of fetal cells present in the amniotic fluid. Here, we provide a protocol for simultaneous detection of miRNAs and multiple gene transcripts from a single cell using TaqMan-based real-time polymerase chain reaction (PCR). The protocol details isolation of RNA from single cells, cDNA synthesis, and transcript detection via TaqMan real-time PCR. This method can be used for efficacious detection of at least 10 different gene transcripts (single-plex) or five different gene transcripts and five miRNAs from a single cell, or, using gene specific primer-probes for duplex PCR, up to 20 different gene transcripts. The entire procedure from isolation of RNA to acquisition of real-time PCR data takes 8 h.