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

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Title: Acoustic microstreaming increases the efficiency of reverse transcription reactions comprising single-cell quantities of RNA
Author(s): Boon, Wah Chin; Petkovic-Duran, Karolina; White, Kylie; Tucker, Elena; Albiston, Anthony; Manasseh, Richard; Horne, Malcolm Kenneth; Aumann, Tim Douglas
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.
Publication type: Journal article
Research centre: Swinburne University of Technology. Faculty of Engineering and Industrial Sciences
Source: BioTechniques, Vol. 50, no. 2 (Feb 2011), pp. 116-119
Publication year: 2011
FOR Code(s): 06 Biological Sciences; 10 Technology
Keyword(s): cDNA; Micromixing; MRNA; qPCR; RT-PCR
Publisher: Informa BioSciences
ISSN: 0736-6205
Publisher URL: http://dx.doi.org/10.2144/000113587
Additional information: The authors acknowledge support from the National Health and Medical Research Council of Australia (project grant no. 6288480) and the Scobie and Clare MacKinnon Trust.
Peer reviewed