A method for the quantitative analysis of human heat shock gene expression using a multiplex RT-PCR assay.
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 hsp90alpha, hsp90beta, 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.
Wang
S M
SM
Department of Biochemistry, Molecular Biology and Cell Biology, Rice Institute for Biomedical Research, Northwestern University, Evanston, IL 60208, USA.
Khandekar
J D
JD
Kaul
K L
KL
Winchester
D J
DJ
Morimoto
R I
RI
D002460:Cell Line; D017931:DNA Primers; D000091342:Endoplasmic Reticulum Chaperone BiP; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000235:genetics*; D006801:Humans; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
D017931:DNA Primers; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins