Measurement of gene expression in archival paraffin-embedded tissues: development and performance of a 92-gene reverse transcriptase-polymerase chain reaction assay.
Throughout the last decade many laboratories have shown that mRNA levels in formalin-fixed and paraffin-embedded (FPE) tissue specimens can be quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) techniques despite the extensive RNA fragmentation that occurs in tissues so preserved. We have developed RT-PCR methods that are sensitive, precise, and that have multianalyte capability for potential wide use in clinical research and diagnostic assays. Here it is shown that the extent of fragmentation of extracted FPE tissue RNA significantly increases with archive storage time. Probe and primer sets for RT-PCR assays based on amplicons that are both short and homogeneous in length enable effective reference gene-based data normalization for cross comparison of specimens that differ substantially in age. A 48-gene assay used to compare gene expression profiles from the same breast cancer tissue that had been either frozen or FPE showed very similar profiles after reference gene-based normalization. A 92-gene assay, using RNA extracted from three 10- micro m FPE sections of archival breast cancer specimens (dating from 1985 to 2001) yielded analyzable data for these genes in all 62 tested specimens. The results were substantially concordant when estrogen receptor, progesterone receptor, and HER2 receptor status determined by RT-PCR was compared with immunohistochemistry assays for these receptors. Furthermore, the results highlight the advantages of RT-PCR over immunohistochemistry with respect to quantitation and dynamic range. These findings support the development of RT-PCR analysis of FPE tissue RNA as a platform for multianalyte clinical diagnostic tests.
Cronin
Maureen
M
Genomic Health Incorporated, Redwood City, California 94063, USA.
Pho
Mylan
M
Dutta
Debjani
D
Stephans
James C
JC
Shak
Steven
S
Kiefer
Michael C
MC
Esteban
Jose M
JM
Baker
Joffre B
JB
D016477:Artifacts; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods; D006801:Humans; D007150:Immunohistochemistry; D016612:Paraffin Embedding; D012334:RNA, Neoplasm / Q000032:analysis*; D015203:Reproducibility of Results; D012189:Retrospective Studies; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D013997:Time Factors
D012334:RNA, Neoplasm