Mmu mir 155

Total RNA was obtained from cell cultures or tissues using miRNeasy Mini RNA Extraction Kit (Qiagen). A total of 10 ng total RNA were reverse transcribed using a reverse transcriptase and stem-loop primers as indicated by the manufacturer (TaqMan MicroRNA Reverse Transcription Kit, Applied Biosystems). One and a half microliters of the reaction was used as a template for the qPCR amplification reaction (TaqMan Universal Master Mix, No AmpErase UNG, Applied Biosystems) in a thermocycler (ViiA 7 Real-Time PCR system, Applied Biosystems). Quantitative miRNA RT-PCR expression data were normalized to small nucleolar RNA U6 expression (RNU6B). Stem-loop primers and qPCR probes were purchased from TaqMan MicroRNA assay (Applied Biosystems): RNU6B (AB ID: 001093), mmu-miR-155 (AB ID: 002571), mmu-miR-802 (AB ID: 002029).

Total RNA was extracted from frozen scraped colon mucosa according to the manufacturer's instructions for use of the TRIzol reagent (Invitrogen, Carlsbad, CA). RNA integrity was assessed using the Agilent 2100 Bioanalyzer RNA 600 nano assay (Agilent Technologies, Santa Clara, CA). MicroRNA microarray profiling was conducted as previously described [32] (link). Five mg of total RNA was labeled and hybridized to the microRNA microarray (Ohio State microRNA microarray version 4.0, Columbus, OH). Microarray data was deposited into Gene Expression Omnibus (accession number GSE56025).
Quantitative RT-PCR was used to validate specific microRNAs. The microRNAs that were validated included: hsa-miR-16; mmu-let-7f; mmu-miR-351; has-miR-150; has-miR-425; hsa-miR-196a; hsa-miR-138; and mmu-miR-155 (Applied Biosystems, Foster City, CA). Taqman MicroRNA assays (Applied Biosystems) were used according to the manufacturer's instructions in a 7500 real-time RT-PCR system (Applied Biosystems). Mouse specific small nuclear (sn)/small nucleolar (sno), snoRNA 202, snoRNA 234, and snoRNA 142 endogenous controls were used as the normalization controls (Applied Biosystems). All assays were performed in triplicate.