title: 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.
authors.affiliation: Florey Neuroscience Institutes, The University of Melbourne, Parkville, Victoria, Australia.
authors.forename: Wah Chin
authors.initials: WC
authors.lastname: Boon
authors.forename: Karolina
authors.initials: K
authors.lastname: Petkovic-Duran
authors.forename: Kylie
authors.initials: K
authors.lastname: White
authors.forename: Elena
authors.initials: E
authors.lastname: Tucker
authors.forename: Anthony
authors.initials: A
authors.lastname: Albiston
authors.forename: Richard
authors.initials: R
authors.lastname: Manasseh
authors.forename: Malcolm Kenneth
authors.initials: MK
authors.lastname: Horne
authors.forename: Tim Douglas
authors.initials: TD
authors.lastname: Aumann
mesh_terms: D000162:Acoustics; D000818:Animals; D001921:Brain / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics*; D051379:Mice; D012313:RNA / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D048348:Reverse Transcription; D012680:Sensitivity and Specificity
chemical_list: D018076:DNA, Complementary; D012313:RNA