Extraction and Molecular Screening of Decade-Old mRNA from Archived Breast Cancer Tissues
David E Nowak, Leonardo P Roquero, Dhanajay A Chitale. Henry Ford Hospital, Detroit, MI
Background: Since the explosion of molecular techniques in pathology over recent years, a common goal has been to develop retrospective studies where patient outcomes are known. Abundant formalin-fixed paraffin embedded (FFPE) material is available at any institution generally archived for decades. Extraction of amplifiable mRNA from old blocks has been a challenge and has been sporadically reported. Our aim was to test mRNA quality extracted from archived FFPE blocks from breast cancer patients, where the inherent fatty nature of the tissue impedes optimal fixation.
Design: Tumor FFPE tissues from breast cancer cases were retrieved between years 2000-2001. 34 cases were randomly selected for this pilot project based on morphological similarity and availability of tumor blocks with >75% tumor content. Total RNA was isolated (Recover-All Nucleic Acid Isolation Kit, Ambion), reverse transcribed (RT First Strand kit, Qiagen), and analyzed by real-time PCR on a Roche Lightcycler 480. Total RNA quantity was assessed on Nanodrop machine. Samples with more than 1ug of RNA yield were considered adequate for validation test with HPRT1 gene as primary screening housekeeping gene. Then the samples were run on Human Breast Cancer Signaling Array (Qiagen, PAH-131) that contained 84 key genes commonly involved in the dysregulation of signal transduction in breast cancer and 5 house keeping genes (Beta2 Microglobulin, GAPDH, HPRT1, ACTB, RPL13A). Positive calls were set at an arbitrary cycle threshold (CT) of 40 cycles.
Results: Total RNA extractions yielded 65 ng to 18.75 ug RNA in 34 samples. 26/34 cases (77%) yielded more than 1ug of RNA and all showed successful HPRT1 gene amplification. 19/26 cases were run on the cancer signaling array. All cases had the housekeeping genes consistently amplified with CT of 26.6-38.7. Eighty-four breast cancer associated genes showed CT value ranging from 12.8-39.8 with most arrays yielding at least 65% positive calls.
Conclusions: We have developed a protocol for the extraction and gene expression screening of decade old mRNA. While the cycle thresholds determined during the screen indicate either low relative expression or simply low recovery, the efficacy as a screening tool is readily apparent. Moreover, the process can go from paraffin block to usable screening mRNA expression data in a matter of a few hours. Applying this type of technology to additional breast data sets and other neoplasms will undoubtably increase the feasibility of using molecular techniques to retrieve valuable retrospective information currently locked away in every institution's FFPE storage archives.
Monday, March 19, 2012 1:00 PM
Poster Session II # 296, Monday Afternoon