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

Abstract：

: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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Stanley KK,Szewczuk E

doi

10.1093/nar/gni182

keywords:

subject

Has Abstract

pub_date

2005-11-24 00:00:00

pages

e180

issue

20

eissn

0305-1048

issn

1362-4962

pii

33/20/e180

journal_volume

33

pub_type

杂志文章

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