Formalin-fixed paraffin-embedded (FFPE) tissues are routinely stored by most pathology departments and are a widely available resource for discovery of clinically useful biomarkers. We describe our method for optimizing quantitative reverse transcription PCR (RT-PCR) for expression analysis using frozen and archival tissue. Commonly used reference genes were evaluated for stability of expression in normal kidney and clear cell renal cell carcinoma (RCC). Optimal reference genes for calculating normalization factors for RT-PCR were ACTB, RPL13A, GUS, RPLP0, HPRT1, and SDHA when using FFPE RCC. The optimal reference genes when using frozen RCC were ACTB, RPL13A, and GUS, confirming that use of multiple reference genes improves accuracy when intact RNA from frozen renal tumors are used. Expression of 16 markers previously reported to have prognostic significance in RCC was determined in 23 matching frozen and FFPE renal tumors, representing a range of tumor grades and stages; correlation coefficient for expression measured in frozen and FFPE tumors was 0.921 (P<0.001). All markers predicted survival when frozen tumors were used and 14 of the 16 markers predicted survival when FFPE tumors were used as the source of RNA. An optimized RT-PCR assay can accurately measure expression of most prognostic tumor markers.
The prognosis associated with renal cell carcinoma (RCC) can vary widely. Small, incidentally diagnosed renal tumors can be observed safely for many years in patients who are poor surgical candidates (1). Larger, high-grade tumors carry risk of recurrence, which may be as high as 50%, following re-section of a clinically localized tumor (2). Accurate determination of prognosis using molecular markers may be useful for patient counseling and selecting treatment. However, there are no molecular markers of prognosis in routine clinical use for patients with RCC. Although microarray-based expression profiling studies have identified a large number of potentially prognostic markers, a barrier to development of molecular test for clinic use has been the lack of readily available clinical samples for validation of candidate markers.
Frozen tumor libraries are costly to establish and maintain. However, formalin-fixed paraffin-embedded (FFPE) tissues are routinely archived and stored by pathology departments. Therefore, FFPE tumor samples are widely available and can be annotated with clinical information. We describe steps for optimizing and validating a quantitative reverse transcription (RT-PCR) assay for expression profiling of FFPE RCC. We evaluate commonly used reference genes for stability of expression in RCC and determine the optimal number of reference genes necessary to calculate normalization factors when using frozen or archival tissue. We validate the assay by comparing expression values measured in frozen and archival tissue. Finally, we validate individual prognostic markers for use with archival tissue. These validated markers can be used in large-scale clinical studies, which are required before a marker can be recommended for clinical use. This approach can be applied across tumor types to develop assays for testing prognostic markers using archive tissue.Materials and Methods Patient Selection and Overall Study Design
To identify reference genes with stable expression in renal tissue, 20 pairs of clear cell RCC and adjacent normal kidney were used. An equal number of low-grade (Fuhrman grade 1 and 2) and high-grade (Fuhrman grade 3 and 4) tumors were represented. Matching pairs of frozen and FFPE, clear cell renal tumors from 23 patients were utilized to validate reference genes identified for use with FFPE renal tumors. All tissue samples were procured between 2002 and 2006 and were retrieved from the Department of Pathology following approval by the Institutional Review Board (EDR 53605). Commonly used reference genes (GUS, TFRC, TBP, RPLP0, ACTB, HPRT1, SDHA, B2M, and RPL13) (3,4) were evaluated for stable expression in renal tumors.RNA Extraction
For extraction of RNA from FFPE tissue, four 10-µm sections were cut from archival block. Excess paraffin was trimmed using a scalpel cleaned with RNaseZAP (Ambion, Austin, TX, USA), and the sections were placed in 1.5-mL RNase-free Eppendorf tubes. Sections were treated twice with 1 mL xylene for 30 min at 55°C while rocking. The sections were washed twice with 100% ethanol. RNA was extracted from the paraffin samples using the MasterPure kit (Epicentre Biotechnologies, Madison, WI, USA).
For frozen tissue, 0.5 g snap-frozen tissue was homogenized in TRIzol and placed in a 1.5-mL RNase-free Eppendorf tube. RNA was then extracted using the TRIzol protocol (Invitrogen, Carlsbad, CA, USA). RNA from both FFPE and frozen tissue was then treated with DNase I for 30 min. The samples were checked for residual genomic DNA by TaqMan RT-PCR for ACTB. If there was measurable DNA after 35 PCR cycles, the samples were treated with DNase I for an additional 30 min, and the assay for residual DNA was repeated.
The final RNA concentration (A260:0.025) and purity (A260:A280 ratio) was measured using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). RNA quality was assessed for 28S and 18S ribosomal degradation using a 2100 Bioanalyzer and an RNA 6000 Nano LabChip kit (both from Agilent Technologies, Santa Clara, CA, USA).Reverse Transcription and TaqMan PCR
Reverse transcription was performed in an Applied Biosystems 2720 thermal cycler using the cDNA Archive kit (Applied Biosystems, Foster City, CA, USA). The reaction was performed according to manufacturer's recommendation, using random primers and 1 µg total RNA. When utilized, gene-specific primers were included at 100 nmol/L for each gene. Gene-specific primers were designed using PerlPrimer v1.1.6 (perlprimer. sourceforge.net). All TaqMan primers and probes were custom designed using Primer Express, version 2.0 (Applied Biosystems), and appropriate mRNA consensus sequence from the NCBI Entrez nucleotide database. PCR product sizes were limited to 100 bases in length. TaqMan probes were labeled with 5′-FAM as a reporter and 3′-TAM as a quencher. All oligonucleotides and probes were supplied by Integrated DNA Technologies (Coralville, IA, USA).