Isolation of High Quality miRNA from Decalcified, Formalin-Fixed, Paraffin-Embedded Bone Marrow Biopsy Samples
JA Schumacher, ME Salama, P Szankasi, AK Ho, TW Kelley. ARUP Institute for Clinical & Experimental Pathology, Salt Lake City, UT; University of Utah, Salt Lake City, UT
Background: Decalcified, formalin fixed, paraffin embedded (D-FFPE) bone marrow biopsy samples present a challenge for molecular testing due to nucleic acid degradation. However, the integrity of micro RNA (miRNA) in D-FFPE tissues has not been well characterized. We evaluated the ability to obtain high quality miRNA from D-FFPE bone marrow biopsies that could be quantified by an array-based real time PCR technique.
Design: Thirty three D-FFPE bone marrow biopsy samples from the hematopathology archives (2006-present) were de-identified. Total RNA was extracted using either Qiagen miRNeasy FFPE or Ambion Recoverall™ kits. Absorbance measurements at 280, 260, and 230nm were performed to assess RNA concentration and purity. Following the manufacturer's protocol, total RNA (60-1090 ng) was reverse transcribed and subjected to quantitative RT-PCR to measure 92 miRNAs using the miFinder RT2 miRNA PCR array from SA Biosciences. Relative expression values were calculated using the ΔΔCt calculation with data normalized to the average of the geometric mean of 4-housekeeping miRNAs.
Results: Four cases were excluded from analysis due to low RNA yields (<9ng/µL). The remaining 29 cases yielded a mean RNA concentration of 79.4ng/µL (range 12.6-217.9ng/µL), and mean 260/280 ratio of 1.87 (range of 1.7-2.02). The miRNeasy extraction kit yielded the highest quality RNA with a mean 260/230 ratio of 1.64 (range 0.91-2.15; n=23). The Recoverall™ extraction yielded RNA with a mean 260/230 ratio of 0.47 (range 0.2-0.77; n=6). Those cases with average crossing points for 4-housekeeping miRNAs ≤27 cycles were designated as passing QC for the purposes of this analysis. This maximized the number of evaluated miRNAs with crossing points <30 cycles, within the linear amplification range of the assay. Of 29 cases analyzed, 15 met our QC criterion. 2/5 cases (40%) with RNA isolated using Recoverall™ passed QC and 13/24 cases (54%) with RNA isolated using miRNeasy passed QC. As expected, we found that cases where >100ng total RNA was reverse transcribed had higher QC success rates (14/24) (58%) than those using <100ng total RNA (1/5) (20%). QC failures did not correlate with the age of the tissue block. There was a 51.7% QC pass rate for the samples using our QC criterion of ≤27 cycles for the average of the geometric mean of 4-housekeeping miRNAs.
Conclusions: Array-quality miRNA can be obtained from D-FFPE bone marrow biopsy samples.
Category: Pan-genomic/Pan-proteomic Approaches to Diseases
Monday, March 22, 2010 1:00 PM
Poster Session II # 237, Monday Afternoon