Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using 5' nuclease quantitative reverse transcription-polymerase chain reaction.
Analysis of gene expression and correlation with clinical parameters has the potential to become an important factor in therapeutic decision making. The ability to analyze gene expression in archived tissues, for which clinical followup is already available, will greatly facilitate research in this area. A major obstacle to this approach, however, has been the uncertainty about whether gene expression analyses from routinely archived tissues accurately reflect expression before fixation. In the present study we have optimized the RNA isolation and reverse transcription steps for quantitative reverse transcription-polymerase chain reaction (RT-PCR) on archival material. Using tissue taken directly from the operating room, mRNAs with half-lives from 10 minutes to >8 hours were isolated and reverse transcribed. Subsequent real-time quantitative PCR methodology (TaqMan) on these cDNAs gives a measurement of gene expression in the fixed tissues comparable to that in the fresh tissue. In addition, we simulated routine pathology handling and demonstrate that this method of mRNA quantitation is insensitive to pre-fixation times (time from excision to fixation) of up to 12 hours. Therefore, it should be feasible to analyze gene expression in archived tissues where tissue collection procedures are largely unknown.
Department of Laboratory Medicine, University of California San Francisco Cancer Center, USA. godfreyte@msx.upmc.edu
T E
TE
Godfrey
S H
SH
Kim
M
M
Chavira
D W
DW
Ruff
R S
RS
Warren
J W
JW
Gray
R H
RH
Jensen
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D008099:Liver / Q000378:metabolism; D008297:Male; D016612:Paraffin Embedding; D011467:Prostate / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014158:Transcription, Genetic
D017931:DNA Primers; D012333:RNA, Messenger