Evaluation and Validation of Total RNA Extraction Methods for MicroRNA Expression Analyses in Formalin-Fixed, Paraffin-Embedded Tissues Martina Doleshal,

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Evaluation and Validation of Total RNA Extraction Methods for MicroRNA Expression Analyses in Formalin-Fixed, Paraffin-Embedded Tissues Martina Doleshal, Amber A. Magotra, Bhavna Choudhury, Brian D. Cannon, Emmanuel Labourier, Anna E. Szafranska The Journal of Molecular Diagnostics Volume 10, Issue 3, Pages 203-211 (May 2008) DOI: 10.2353/jmoldx.2008.070153 Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 qRT-PCR analysis of RNA samples isolated with the RecoverAll or RNeasy kit. Total RNA was extracted in triplicate from five different FFPE tissue blocks using the indicated method. Each RNA sample was analyzed in duplicate using qRT-PCR assays specific for miR-24, -103, -191, 18S rRNA, or hGUSB mRNA. The Journal of Molecular Diagnostics 2008 10, 203-211DOI: (10.2353/jmoldx.2008.070153) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Comparison between FFPE and matching frozen samples. A: Average expression levels for miR-24, -103, and -191 in RNA samples isolated from FFPE and matched frozen breast (three specimens), cervix (three specimens), and gall bladder (two specimens) tissues. Each qRT-PCR reaction was performed in duplicate. B: Same as A for 18S rRNA and hGUSB mRNA. C: Average differential expression (ΔCt) between frozen and matching FFPE samples for miRNAs or mRNA and rRNA species. D: Average standard deviation of qRT-PCR signal (Ct) within the 13 FFPE tissues blocks described in Table 1 for miRNA or mRNA and rRNA species. The Journal of Molecular Diagnostics 2008 10, 203-211DOI: (10.2353/jmoldx.2008.070153) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Scale-up of the RecoverAll procedure. RNA isolation was performed with four, eight, 12, or 16 FFPE tissue slices (20-μm) from two prostate cancer FFPE blocks that differed in tissue cross-sectional area. For the block with a tissue area >150 mm2, twice the recommended amount of proteinase K was used. The Journal of Molecular Diagnostics 2008 10, 203-211DOI: (10.2353/jmoldx.2008.070153) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Armored RNA Quant as a process control. RNA isolation was performed using 16 20-μm slices from 15 pairs of matched FFPE lung tumor and NAT specimens with 1010 copies of ARQ spiked at the proteinase K digestion step. qRT-PCR were performed in duplicate using 2 μl of purified RNA for ARQ and 10 ng of purified RNA for miR-24, -191, -103, hGUSB mRNA, and 18S rRNA. Samples with abnormally high Ct for endogenous RNAs and ARQ or for endogenous RNAs only are indicated by a circle and a star, respectively. The Journal of Molecular Diagnostics 2008 10, 203-211DOI: (10.2353/jmoldx.2008.070153) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions