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Quantitative analysis of microRNAs in tissue microarrays by in situ hybridization
Jason A. Hanna1, Hallie Wimberly1, Salil Kumar1, Frank Slack2, Seema Agarwal1, and David L. Rimm1
1Department of Pathology, Yale University Medical School, New Haven, CT
2Department of Molecular, Cellular and Developmental Biology, Yale University, Haven, CT
BioTechniques, Vol. 52, No. 4, April 2012, pp. 235–245
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Supplementary Material

MicroRNAs (miRNAs) have emerged as key regulators in the pathogenesis of cancers where they can act as either oncogenes or tumor suppressors. Most miRNA measurement methods require total RNA extracts which lack critical spatial information and present challenges for standardization. We have developed and validated a method for the quantitative analysis of miRNA expression by in situ hybridization (ISH) allowing for the direct assessment of tumor epithelial expression of miRNAs. This co-localization based approach (called qISH) utilizes DAPI and cytokeratin immunofluorescence to establish subcellular compartments in the tumor epithelia, then multiplexed with the miRNA ISH, allows for quantitative measurement of miRNA expression within these compartments. We use this approach to assess miR-21, miR-92a, miR-34a, and miR-221 expression in 473 breast cancer specimens on tissue microarrays. We found that miR-221 levels are prognostic in breast cancer illustrating the high-throughput method and confirming that miRNAs can be valuable biomarkers in cancer. Furthermore, in applying this method we found that the inverse relationship between miRNAs and proposed target proteins is difficult to discern in large population cohorts. Our method demonstrates an approach for large cohort, tissue microarray-based assessment of miRNA expression.

MicroRNAs (miRNAs) are short non-coding RNA molecules of 18–25 nucleotides that bind to target mRNAs and inhibit translation or promote degradation to ultimately down-regulate target protein expression (1). Each miRNA can potentially regulate hundreds of mRNAs, and it has been estimated that 30%–60% of all mRNAs are regulated by miRNAs playing roles in regulation of virtually every cellular process (2, 3). Improper miRNA regulation has been attributed to cancer (4) and scores of papers on miRNAs as prognostic and predictive biomarkers have been recently published. Since their discovery, the detection of miRNAs has been difficult, with most methods requiring total RNA extracts which lack critical spatial information. In situ hybridization (ISH), however, allows for the direct assessment of expression levels in tissue and, importantly, the evaluation of expression in malignant cells as well as stromal cells and invading lymphocytes.

Recent advances in ISH have enabled detection of miRNAs in formalin-fixed, paraffin embedded tissue (FFPE) using locked nucleic acid (LNA) modified probes and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) fixation, and numerous studies have been published utilizing these techniques (5-9). LNA probes utilize a modified ribose backbone where the 2'oxygen and 4'carbon atom are linked with a methylene bridge locking the sugar in the N-type conformation (10). This modification leads to greater thermodynamic stability and higher melting temperatures (Tm) of the probe and miRNA duplex allowing greater hybridization specificity. In addition, EDC reacts with the 5′phosphate of the miRNA, condensing it with amino groups in the protein matrix of the tissue. Without this EDC step, up to 50% of the miR-124 was released into the hybridization buffer suggesting the importance of EDC fixation especially for miRNAs expressed at low levels (11).

Here we describe an ISH assay to quantitatively measure miRNA expression in FFPE tissue microarrays (TMAs). In quantitatively measuring miRNA expression, we multiplex the ISH with cytokeratin immunofluorescence to apply the AQUA technology (15). This quantitative immunofluorescent (qIF) co-localization based approach employs DAPI and cytokeratin to establish subcellular compartments within tumor epithelia, allowing for the measurement of miRNA expression in these subcellular compartments that result in a score directly proportional to the number of molecules per unit of area. The assay permits standardization of miRNA measurement and the potential for rapid large cohort assessment, as well as testing for reproducibility in large TMA populations.

Materials and methods

miRNA in situ hybridization

FFPE tissue microarrays are first deparaffinized in xylene, rehydrated with an ethanol gradient, treated with 20 µg/mL Proteinase K (Roche Diagnostics, Indianapolis, IN, USA) for 10 min at 37°C, fixed with 4% formaldehyde (Thermo Scientific, Rockford, IL, USA) for 10 min, rinsed twice in 0.13M 1-Methylimidazole and refixed with 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, Thermo Scientific) for 1 h as described (11). Then endogenous peroxidases are blocked with 1% H2O2 for 30 min and slides are prehybridized at the hybridization temperature of 50°C for 30 min in hybridization buffer containing 50% formamide (American Bioanalytical), 5X SSC (American Bioanalytical, Natick, MA, USA), 50µg/mL Heparin (Sigma-Aldrich, St. Louis, MO, USA), 0.1% Tween 20 (Sigma), 500µg/mL yeast tRNA (Invitrogen, Carlsbad, CA, USA) adjusted to pH 6. Slides are hybridized for 1 h with 200 nM Double Digoxigenin (DIG) LNA modified probes (Exiqon, Copenhagen, Denmark) for miR-221 (Sequence: 5′-GAAACCCAGCAGA-CAATGTAGCT-3′), miR-21 (Sequence: 5′-TCAACATCAGT-CTGATAAGCTA-3′), miR-34a (Sequence: 5′-ACAACCAGCTAA-GACACTGCCA-3′), miR-205 (Sequence: 5′-CAGACTCCGGTGGAATGAAGGA-3′) and scrambled probe (Sequence: 5′-GTGTAACACGTCT-ATACGCCCA-3′) or 200 nM 5′DIG labeled miR-92a probe (Sequence: 5′-ACAGGCCGGGA-CAAGTGCAATA-3′) For the U6 Probe (Sequence: 5′-CACGAATTTGCGT-GTCATCCTT-3′), 25 nM 5′Dig labeled probe was used. Slides are then stringently washed in 2X SSC (once at hybridization temperature then twice at room temperature for 5 min each), blocked with 2% BSA (Sigma) for 30 min and incubated with Anti-Digoxigenin-POD, Fab fragments from sheep (Roche Diagnostics) diluted 1:100 and rabbit anti-cytokeratin (Dako Corp, Carpinteria, CA, USA) diluted 1:100 in block (2% BSA in PBS) for 1 h at room temperature. Following two washes with 0.1% Tween PBS (PBS-T) and one wash in PBS for 5 min each, the miRNA signal is detected with the TSA Plus Cyanine 5 system (Perkin Elmer, Norwalk, CT, USA), the slides are washed again with PBS-T and PBS as above, and cytokeratin is detected with Alexa 546-conjugated goat anti-rabbit secondary antibody (Molecular Probes) diluted 1:100 in block for one hour, and the slides are mounted with Prolong mounting medium containing 4’,6-Diamidino-2-phenylindole (DAPI, Molecular Probes, Eugene, OR, USA). Serial sections of small control index slides consisting of 43 breast cancer specimens were ran alongside each run to assess reproducibility and negative control scrambled probe and positive control U6 probe were also used for each run. miR-221 qISH was performed on two builds (redundant cores from different areas of same tumor specimens) of our breast cancer TMA cohort and the AQUA scores from the two cores were averaged for analysis. Any specimens with less than 0.17 mm2 tumors were excluded from analysis. miR-221 blocking oligo experiments were conducted as above. The miR-221 blocking oligo, consisting of the same sequence as endogenous mature miR-221 (sequence: 5′-AGCTACATTGTCT-GCTGGGTTTC-3′) was pre-incubated at 1.5 fold excess (300nM) with the miR-221 probe for 1 h at the hybridization temperature prior to hybridization on the TMA.

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