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Nucleic Acids Research 2005 33(20):e180; doi:10.1093/nar/gni182
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Published online 24 November 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oxfordjournals.org

Methods Online

Multiplexed tandem PCR: gene profiling from small amounts of RNA using SYBR Green detection

Keith K. Stanley1,2,* and Elektra Szewczuk1

1Corbett Research Pty Ltd 14 Hilly Street, Mortlake NSW 2137, Sydney, Australia 2University of New South Wales Centre for Immunology Building, Cnr West and Boundary Streets, Darlinghurst, NSW 2010, Australia

*To whom correspondence should be addressed. Tel: +612 8382 2833; Fax: +612 8382 2391; Email: keith.stanley{at}corbettresearch.com

Received October 10, 2005. Revised November 3, 2005. Accepted November 3, 2005.

Multiplexed tandem PCR (MT-PCR) is a process for highly multiplexed gene expression profiling. In the first step, multiple primer pairs are added to the RNA to be analysed together with reverse transcriptase and Taq DNA polymerase. Following reverse transcription, the multiplexed amplicons are simultaneously amplified for a small number of cycles so as to avoid competition between amplicons. The reaction product is then diluted and analysed in multiple individual PCRs using primers nested inside the primers used for the multiplexed amplification. As the second PCR uses a template enriched in the amplicons of interest, the conditions can be optimized to significantly reduce ‘primer dimer’ formation allowing SYBR Green chemistry to be used for quantification. MT-PCR can be configured for as little as 10 pg RNA (equivalent to a single mammalian cell) and works well with RNA extracted from archival formalin-fixed paraffin-embedded sections. We illustrate MT-PCR with gene expression profiles of breast cancer cell lines.


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