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Home List of Titles Acoustic microstreaming increases the efficiency of reverse transcription reactions comprising single-cell quantities of RNA
Please use this identifier to cite or link to this item: http://hdl.handle.net/1959.3/157100
- Acoustic microstreaming increases the efficiency of reverse transcription reactions comprising single-cell quantities of RNA
- Boon, Wah Chin; Petkovic-Duran, Karolina; White, Kylie; Tucker, Elena; Albiston, Anthony; Manasseh, Richard; Horne, Malcolm Kenneth; Aumann, Tim Douglas
- 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
- BioTechniques, Vol. 50, no. 2 (Feb 2011), pp. 116-119
- Publication year
- FOR Code(s)
- 06 Biological Sciences; 10 Technology
- cDNA; Micromixing; MRNA; qPCR; RT-PCR
- Informa BioSciences
- Publisher URL
- Copyright © 2011.
- 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