Real-time Quantitative RT-PCR Shows Variable, Assay-dependent Sensitivity to Formalin Fixation: Implications for Direct Comparison of Transcript Levels in Paraffin-embedded Tissues

Abstract

Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) is a versatile tool for precise quantification of gene expression. Formalin-fixed and paraffin-embedded (FFPE) tissue is well suited for qRT-PCR, if RNA extraction is optimized and small amplicon sizes are used. However, little is known whether individual assays may show variable sensitivity to fixation. This is of great importance, if a direct comparison of different transcripts is performed within the same sample, such as for mRNA splice variants. We established a cell culture model to test for and quantify differences in performance of individual qRT-PCR assays on FFPE as compared with fresh material, using TaqMan methodology. RNA was isolated from 7 different cell lines either directly or after preparation of a FFPE cell block. RNA from both sources was reverse transcribed and gene expression quantified using 13 different TaqMan assays. All assays allowed highly reproducible target quantification, using both fresh and FFPE-derived cDNA. The 13 assays showed an average Ct difference of 3.2 between fresh and FFPE cells, if identical amounts of cDNA were used as template. However, the Ct shifts varied from 1.8 to 5.1 for individual assays, indicating variable resistance to fixation. These Ct shift differences were statistically highly significant in 27/78 (35%) of all possible combinations of assays. Because the Ct shift remained constant for each assay, they could be used for calculation of correction factors which rendered FFPE-derived expression data highly comparable to those obtained from fresh material, and as a consequence among each other. Thus, a standardized assessment of qRT-PCR assay efficiencies in FFPE allows for precise intraindividual comparison of mRNA species, such as splice variants with different biologic functions, in archival tissues.

Quantitative RT-PCR assays for the determination of urokinase-type plasminogen activator and plasminogen activator inhibitor type 1 mRNA in primary tumor tissue of breast cancer patients: Comparison to antigen quantification by ELISA

“…For the preceding mRNA isolation, only small amounts of tumor tissue were required. The amplicon lengths of the QPCR assays for uPA and PAI-1 were minimized as far as possible to potentially enable quantification of partially fragmented mRNA extracted from formalin-fixed, paraffinembedded tissue (38). Recently, several QPCR assays for determination of uPA and/or PAI-1 mRNA expression have been introduced with rather large amplicon sizes (13)(14)(15)17,18,39,40).…”

The impact of cyclin D1 mRNA isoforms, morphology and p53 in mantle cell lymphoma: p53 alterations and blastoid morphology are strong predictors of a high proliferation index

“…The amount of short CyD1 isoform was calculated by the DDCt method. 25,27 Reactive lymphoid tissue was used as the control for background CyD1 levels, and small intestinal samples, which show higher physiological CyD1 expression, served as a control to assess the normal range of the CyD1 isoform ratio (CyD1 isoform ratio=2 -DDCt , DDCt=[Ctvalue CyD1 3'UTR(tumor)-Ct-value CyD1 total(tumor)]-[(mean Ct-value CyD1 3'UTR(controls) -(mean Ct-value CyD1 total (controls)]. A ratio (proximal 3'UTR/exon1-2) of 0.6 or lower was regarded as evidence of significant expression of the short D3'UTR-deficient CyD1 isoform.…”

Specific lentiviral shRNA-mediated knockdown of cyclin D1 in mantle cell lymphoma has minimal effects on cell survival and reveals a regulatory circuit with cyclin D2

“…The sequences for cyclin D2 were 5 0 -CGCAAGCATGCTCAGACCTT-3 0 , 5 0 -TG CGATCATCGACGGTGG-3 0 and 5 0 -FAM-TGCCACCGACTTT AAGTTTGCCATGT-TAMRA-3 0 ; the sequences for cyclin D3 and TATA box-binding protein (TBP) as the housekeeping gene have already been described. 17,18 Before the experiments started, the linear range of the assays was validated with a dilution curve using transcribed RNA from uninfected cell lines. Data were analyzed using the DC T method as previously described.…”