Acoustic microstreaming increases the efficiency of reverse transcription reactions comprising single-cell quantities of RNA

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 similar to 9 and similar to 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/mu L, a single-cell equivalent) but had lesser effects at higher RNA concentrations (similar to 1 ng/mu L). This was supported by imaging experiments showing that micromixing improved mixing of a low concentration (20 pg/mu L) of fluorescence-labeled RNA but not a higher concentration (1 ng/mu L). We conclude that micromixing significantly increases RT yields obtainable from single-cell quantities of RNA.