Profiling miRNA Expression in FFPE Tissues

RNA (miRNA) was extracted from formalin-fixed paraffin embedded tissues. We assessed slides and tumor blocks that were prepared over the duration of the study prior to the time of miRNA isolation to determine their suitability. The study pathologist (WS) reviewed slides to delineate tumor, normal, and polyp tissue. Cells were dissected from 1–4 sequential sections on aniline blue stained slides using an H&E slide for reference. Total RNA containing miRNA was extracted, isolated, and purified using the RecoverAll Total Nucleic Acid isolation kit (Ambion), RNA yields were determined using a NanoDrop spectrophotometer. 100 ng total RNA was labeled with cy3 and hybridized to Agilent Human miRNA Microarray V19.0 and were scanned on an Agilent SureScan microarray scanner model G2600D. The Agilent Human microarray was generated using known miRNA sequence information compiled in the Sanger miRBASE database v19.0. The microarray contains probes for 2006 unique human miRNAs, with one to four unique probes for each of the known miRNAs. The miRNA array contains 60,000 unique human sequences and averages 30 replicates per probe sequence. Data were extracted from the scanned image using Agilent Feature Extract software v.11.5.1.1. Data were required to pass stringent QC parameters established by Agilent that included tests for excessive background fluorescence, excessive variation among probe sequence replicates on the array, and measures of the total gene signal on the array to assess low signal. If samples failed to meet quality standards for any of these parameters, the sample was re-labeled, hybridized to arrays, and scanned. If a sample failed QC assessment a second time the sample was deemed to be of poor quality and the individual was excluded from down-stream analysis. Of the 1171 initial cases, 30 were excluded at this stage. A quantile normalization across arrays was done using preprocessCore¹¹ (www.bioconductor.org) to minimize differences that could be attributed to the array, amount of RNA, location on array, or other factors that could erroneously influence expression.
We refer to miRNAs using standard nomenclature used in the miRBase database^{12 (link)}. Briefly, the first three letters signifies the organism, followed by a unique number. The number is followed by a dash and number (i.e., 1) if more than one loci codes for the miRNA. A lettered suffix denotes closely related miRNAs. If two miRNAs are coded by the same precursor product then the minor product is assigned the suffix (*). If predominant/minor product status is not known then the suffix 5p and 3p are used to denote 5′ and 3′ arm respectively. Many of the miRNAs being replicated in this study were reported prior to current nomenclature. For instance, let-7 may be reported in the literature as being associated with tumor stage, however Let-7 has since been further delineated to several closely related mature sequences and genomic loci, for example let-7a-3p, let-7a-5p, and let-7b-3p. A complete list of the 121 miRNAs previously reported with stage and/or survivals that are being evaluated in this replication is included in Supplemental Table 1.