Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using 5′ nuclease quantitative reverse transcription-polymerase chain reaction

Tony E. Godfrey, Sun Hun Kim, Marielena Chavira, David W. Ruff, Robert S. Warren, Joe Gray, Ronald H. Jensen

Research output: Contribution to journalArticle

255 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)84-91
Number of pages8
JournalJournal of Molecular Diagnostics
Volume2
Issue number2
StatePublished - May 2000
Externally publishedYes

Fingerprint

Paraffin
Formaldehyde
Reverse Transcription
Polymerase Chain Reaction
Messenger RNA
Gene Expression
Aptitude
Operating Rooms
Uncertainty
Real-Time Polymerase Chain Reaction
Decision Making
Complementary DNA
RNA
Pathology
Research

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using 5′ nuclease quantitative reverse transcription-polymerase chain reaction. / Godfrey, Tony E.; Kim, Sun Hun; Chavira, Marielena; Ruff, David W.; Warren, Robert S.; Gray, Joe; Jensen, Ronald H.

In: Journal of Molecular Diagnostics, Vol. 2, No. 2, 05.2000, p. 84-91.

Research output: Contribution to journalArticle

Godfrey, Tony E. ; Kim, Sun Hun ; Chavira, Marielena ; Ruff, David W. ; Warren, Robert S. ; Gray, Joe ; Jensen, Ronald H. / Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using 5′ nuclease quantitative reverse transcription-polymerase chain reaction. In: Journal of Molecular Diagnostics. 2000 ; Vol. 2, No. 2. pp. 84-91.
@article{2dd6d021fbf7432e97195a88366071c5,
title = "Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using 5′ nuclease quantitative reverse transcription-polymerase chain reaction",
abstract = "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.",
author = "Godfrey, {Tony E.} and Kim, {Sun Hun} and Marielena Chavira and Ruff, {David W.} and Warren, {Robert S.} and Joe Gray and Jensen, {Ronald H.}",
year = "2000",
month = "5",
language = "English (US)",
volume = "2",
pages = "84--91",
journal = "Journal of Molecular Diagnostics",
issn = "1525-1578",
publisher = "Association of Molecular Pathology",
number = "2",

}

TY - JOUR

T1 - Quantitative mRNA expression analysis from formalin-fixed, paraffin-embedded tissues using 5′ nuclease quantitative reverse transcription-polymerase chain reaction

AU - Godfrey, Tony E.

AU - Kim, Sun Hun

AU - Chavira, Marielena

AU - Ruff, David W.

AU - Warren, Robert S.

AU - Gray, Joe

AU - Jensen, Ronald H.

PY - 2000/5

Y1 - 2000/5

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0034189805&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034189805&partnerID=8YFLogxK

M3 - Article

C2 - 11272893

AN - SCOPUS:0034189805

VL - 2

SP - 84

EP - 91

JO - Journal of Molecular Diagnostics

JF - Journal of Molecular Diagnostics

SN - 1525-1578

IS - 2

ER -