2, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA
3, Department of Pathology, St. Mary's Hospital, McGill University, Montreal, Quebec, Canada
4, Center for Medical Genomics, Emory University School of Medicine, Atlanta, GA
5, Indiana University Cancer Center, Indiana Cancer Pavilion, Indianapolis, IN
6, Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA, USA
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Formalin-fixed paraffin-embedded (FFPE) tissue samples make up a vast archive of pathologically well-characterized clinical samples from randomized trials and are an immense virtually untapped resource that can be used for conducting biomarker investigations. Even though the degradation of RNA that occurs due to the formalin fixation process results in RNA species with an average size of ∼200 nt (1), it is feasible to extract and purify RNA from such FFPE tissue and to perform real-time reverse transcription-polymerase chain reaction (RT-PCR)-based gene expression profiling. Some studies have addressed improvements to the process of isolating high quality FFPE RNA suitable for RT-PCR or high-throughput genome-wide gene expression profiling (2,3,4).
The DASL (cDNA-mediated annealing, selection, extension and ligation) assay is based upon massively multiplex RT-PCR applied in a microarray format that allows for the determination of expression of up to 512 genes (502 genes in the cancer panel used in this study), using RNA isolated from 96 FFPE tumor tissue samples in a high-throughput format (5,6). The DASL assay has been used recently to identify a 16-gene set that correlates with prostate cancer relapse (7).
Here we have compared four FFPE RNA preparation methodologies resulting in an optimized protocol for performance in the DASL assay. Although Illumina Inc. (San Diego, CA, USA) recommends using the High Pure RNA kit (Roche, Mannheim, Germany) based on earlier studies (5,6), several new kits (from Ambion, Austin, TX, USA; Qiagen, Valencia, CA, USA; and SuperArray, Frederick, MD, USA) have not been previously tested for utility in downstream DASL assays. The four commercially available kits for RNA extraction from FFPE tissues that were used in this study are: Ambion's RecoverAll kit, Roche's High Pure kit, Qiagen's RNeasy FFPE kit, and SuperArray's ArrayGrade FFPE RNA Isolation kit. In our experiments, FFPE blocks from six different breast cancer patients who were estrogen receptor positive (ER + ) and epidermal growth factor receptor 2-positive (HER2 + ) (as determined by immunohistochemistry [IHC]), and were sectioned to enable direct comparison of the same tissues with six individual samples. All samples were then run in the DASL assay as two technical replicates. Here we report our findings on performance of these FFPE RNA extraction methods as well as the finalized optimal protocol in the DASL assay.
Total cellular RNA was extracted with overall yields ranging from 0 to 9 µg with concentrations from 0 to over 300 ng/µL. For each kit, longer digestion with Proteinase K yielded more RNA ((Figure 1)A), consistent with previous studies (2). Often limited information is available on how the tissue was processed prior to fixation and embedding, but processing likely strongly influences the quantity and quality of nucleic acids recovered from the sample.
Figure 1.
Most kits tested in this study produced RNA with an A260/A280 ratio close to 2 ((Figure 1)B). The most variability in the A260/A280 ratio was seen when using the SuperArray ArrayGrade FFPE RNA Isolation kit. When the A260/A280 ratio decreases, there is a tendency for the RPL13a TaqMan cycle threshold (CT) values to increase and the DASL replicate reproducibility to decrease. Agilent 2100 bioanalysis (Asilent Technologies, Santa Clara, CA, USA) determined that the median RNA size was approximately 100–200 bp ((Figure 1)E), with profiles typical for FFPE RNA (8), but the RNA integrity number (RIN) values were not predictive of the utility of RNA samples.
