Evaluating miRNA Expression Integrity in FFPE Samples with qRT-PCR by Using Patient-matched Formalin-fixed and Fresh-frozen Tissues from Renal Cell Carcinoma (RCC) Patients
Van Le, B.A.1, David Juan, M.A.1, Derek Beaton, M.A.2, Benjamin Keenan, M.A.1, Yasmin Akbari, B.A.1, Joon Hee Lee, B.A.1, Tanmay Parekh, B.A.1, Travis Antes, PhD3, Louis Liou, MD/PhD1.
1Boston University, Boston, MA, USA, 2University of Massachusetts, Dartmouth, MA, USA, 3System Biosciences, Mountain View, CA, USA.
BACKGROUND:Recent discoveries have shown that miRNAs regulate many important cellular processes_controlling many oncogenes and tumor suppressors. Profiling miRNA expressions will provide new methods for diagnosing, finding therapeutic targets, and identifying prognostic factors for cancers. However, the majority of the published studies were performed with fresh-frozen tissues which are usually prospectively banked and are scarce. Using formalin-fixed, paraffin-embedded (FFPE) samples are preferable because they are more readily available since hospitals have a massive retrospective collection of tissues fixed in the manner for clinical purposes. The challenge in using FFPE blocks for molecular profiling is volatility of the nucleic acids. They are modified and degraded during the fixation period and with long-term storage at ambient temperature and oxygen level, their original molecular signature can be affected. Therefore, it is important to validate that formalin does not alter significantly the miRNA signature in clear cell RCC for future data in FFPE tissue to have any significant meaning.
METHODS: IRB approval was obtained from our institution. Kidney tissues taken at the time of surgeries for each patient were made into FFPE blocks and flash frozen. RNA from FFPE blocks and fresh-frozen tissues of six patients were extracted using RecoverAll Total Nucleic Acid Isolation Kit (Ambion, Texas) and mirVana™ miRNA Isolation Kit (Ambion, California), respectively. All samples were one to three years old. Extracted total RNA was then quantified with NanoDrop ND-1000 Spectrophotometer and reverse transcribed to cDNA using Quantimir (Systems Bioscience, California). Real-time qRT-PCR was performed on 60 miRNAs of interest in triplicate using the Oncomir kit (Systems Bioscience, California).
RESULTS: To evaluate the integrity of miRNA expression patterns in FFPE blocks, miRNA expression patterns in FFPE tissues were compared to the corresponding fresh-frozen tissues. Data analysis showed that there was a high correlation (R2 = 0.96, p < 0.01) between formalin-fixed and fresh-frozen miRNAs. (See Figure 1)
CONCLUSIONS:Analysis showed that miRNA expression patterns are mostly unaffected by formalin fixation and long-term storage. This suggests that miRNA expressions in archived FFPE blocks are robust enough and will be an invaluable resource to perform retrospective studies to find prognostic factors in kidney cancer by using real-time qRT-PCR.