Abstract C32: Non-uniform recovery of microRNAs from formalin-fixed paraffin-embedded tissue
Authors: Hou Zizheng , Barrack Evelyn R. 
Published: May 2013

Abstract
Background and Objective: The availability of formalin-fixed paraffin-embedded (FFPE) tissue provides an enormous resource for the discovery of molecular markers. Several reports indicate that microRNA (also referred to as miRNA or miR, a class of small noncoding RNAs 19-24 nucleotides in length that negatively regulate translation of protein-coding mRNAs) is preserved and readily extracted from FFPE tissue. Our goal in this study was to identify a method for optimal recovery of miRNA from FFPE prostate tissue.
Experimental Approach: We compared five commercially available kits that are designed to isolate miRNA from FFPE tissue: SABiosciences RT2 FFPE RNA Extraction Kit (kit 1), Applied Biosystems RecoverAll Total Nucleic Acid Isolation Kit (kit 2), Roche High Pure miRNA Isolation Kit (kit 3), Qiagen miRNeasy FFPE Kit (kit 4), and MO BIO UltraClean FFPE RNA Isolation Kit (kit 5). All kits were used to process serial sections (10 μm thickness) from the same FFPE tissue block. We assessed purity and yield of total RNA based on NanoDrop analysis of absorbance at 230, 260, and 280 nm. Equal amounts of total RNA were converted to cDNA using reverse transcriptase (RT) and miR-specific primers, and real-time PCR using TaqMan miR probes was used to quantitate 378 miRs and 3 small nucleolar RNA species (TaqMan Low Density Array).
Results: The total RNA yield/section was 5.3, 1.6, 3.0, 2.5, and 3.2 μg from kits 1-5, respectively (average of two independent extractions of serial sections from the same FFPE tissue block). Nevertheless, as expected, analysis of the same amount of RNA from each kit showed that all samples had the same level (based on CT) of the small nucleolar RNA RNU48; thus, the recovery of RNU48 per unit amount of total RNA was the same for all kits. Unexpectedly, however, only a limited number of miRs were present at the same level (same CT) in all kit preparations; thus, per unit amount of total RNA, the recovery of only a limited number of miRs was independent of the method used to isolate RNA. For a substantial proportion of miRs, recovery was dependent on the kit used, and recovery differed among miRs. To quantitate differences in recovery, we normalized transcript levels to RNU48 and calculated the ratio of the level of each miR in different kits. Ratios of miRx(kit a)/miRx(kit b) = 1.0 vs. miRy(kit a)/miRy(kit b) = 2.0 would mean that the recovery of miRs x and y is not uniform. Comparing kit 4 vs. kit 1, 10% of miRs had a kit 4/kit 1 ratio &lt;0.75, 17% had a ratio ∼1, 28% had a ratio between 1 and 2, 33% had a ratio between 2 and 4, and 12% had a ratio &gt;4; these data indicate non-uniform recovery of different miRs. This non-uniform recovery of miRs was independent of relative abundance, affecting both high abundance and low abundance transcripts. Notably, the observation of non-uniform recovery of different miRs required the analysis of different kit preparations.
Conclusions: Different miRs are not uniformly recovered from total RNA of FFPE tissue. This suggests that a miR profile in an RNA extract may not accurately represent the tissue in vivo, and that the profile observed may depend on the RNA extraction method used. We suggest that analysis of miRs in tumor relative to nontumor tissue of the same patient will provide a way to normalize for differences in miR recovery, and thus provide a reliable approach to identify miRs as markers of disease. The basis for non-uniform recovery of miRs from FFPE tissue is not known, nor whether this effect is unique to FFPE tissue. [Supported by a grant from the DOD Prostate Cancer Research Program.]
Citation Format: Zizheng Hou, Evelyn R. Barrack. Non-uniform recovery of microRNAs from formalin-fixed paraffin-embedded tissue [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C32.