A method for the quantitative analysis of human heat shock gene expression using a multiplex RT-PCR assay

San Ming Wang, Janardan D. Khandekar, Karen L. Kaul, David J. Winchester, Richard I. Morimoto*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

33 Scopus citations

Abstract

A quantitative multiplex RT-PCR assay is described to measure the levels of messenger RNAs for eight human genes encoding the heat shock proteins (HSP) and molecular chaperones hsp90α, hsp90β, hsp70, hsc70, mtHsp75, Grp78 (BiP), hsp60 and hsp27. The basis of this assay is reverse transcription of total RNA isolated from human cells followed by amplification with PCR. By the careful selection of pairs of oligonucleotide primers corresponding to unique regions of each heat shock gene, selectivity can be attained such that messenger RNAs of multiple heat shock genes can be analyzed simultaneously in a single reaction. This method provides both the absolute and relative levels of each heat shock message by including in the reaction, reference control RNAs corresponding to in vitro transcripts of heat shock gene plasmids carrying small internal deletions.

Original languageEnglish (US)
Pages (from-to)153-161
Number of pages9
JournalCell Stress and Chaperones
Volume4
Issue number3
DOIs
StatePublished - Sep 1999

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology

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