Acoustic Microstreaming Increases the Efficiency of Reverse Transcription Reactions Comprising Single-Cell Quantities of RNA

Abstract

Correlating gene expression with behavior at the single-cell level is difficult, largely because the small amount of available mRNA (<1 pg) degrades before it can be reverse transcribed into a more stable cDNA copy. This study tested the capacity for a novel acoustic microstreaming method ("micromixing"), which stirs fluid at microliter scales, to improve cDNA yields from reverse transcription (RT) reactions comprising single-cell quantities of RNA. Micromixing significantly decreased the number of qPCR cycles to detect cDNA representing mRNA for hypoxanthine phosphoribosyl-transferase (Hprt) and nuclear receptor-related 1 (Nurr1) by ~9 and ~15 cycles, respectively. The improvement was equivalent to performing RT with 10- to 100-fold more cDNA in the absence of micromixing. Micromixing enabled reliable detection of the otherwise undetectable, low-abundance transcript, Nurr1. It was most effective when RNA concentrations were low (0.1-1 pg/µL, a "single-cell equivalent") but had lesser effects at higher RNA concentrations (~1 ng/µL). This was supported by imaging experiments showing that micromixing improved mixing of a low concentration (20 pg/µL) of fluorescence-labeled RNA but not a higher concentration (1 ng/µL). We conclude that micromixing significantly increases RT yields obtainable from single-cell quantities of RNA.