Prostate lobes were dissected in RNase-free PBS, and placed immediately in lysis buffer from the mirVana miRNA Isolation Kit (Ambion AM1560) or RNeasy Plus Mini Kit (Qiagen 74134). Cells were homogenized in lysis buffer using a pestle, and RNA extracted following respective kit instructions. Colon and bladder samples were snap-frozen in liquid nitrogen, homogenized using a mortar and pestle, and RNA extracted using the RNeasy Plus Mini Kit (Qiagen). For quantitative RT-PCR of mRNA transcripts, first-strand cDNA was made using SuperScript III First-Strand Synthesis SuperMix (Invitrogen 18080–400). Quantitative RT-PCR was performed using iQ SYBR Green Supermix (Bio-Rad 170–8880) and the Bio-Rad iCycler. All assay values were normalized to the HPRT1 internal control.
For quantitative RT-PCR of mature miRNA, cDNA was made using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) and primers from respective TaqMan MicroRNA Assays according to manufacturer’s instructions. Quantitative RT-PCR was performed using the TaqMan Universal PCR Master Mix (Applied Biosystems 4324018), TaqMan MicroRNA Assays for miR-26a (Applied Biosystems, Assay 000405) or miR-26b (Applied Biosystems, Assay 000407) or Sno202 (Applied Biosystems, Assay 001232) and the Applied Biosystems 7300 Real-Time PCR System. All assay values were normalized to the Sno202 internal control.
For quantitative RT-PCR of mature miRNA, cDNA was made using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) and primers from respective TaqMan MicroRNA Assays according to manufacturer’s instructions. Quantitative RT-PCR was performed using the TaqMan Universal PCR Master Mix (Applied Biosystems 4324018), TaqMan MicroRNA Assays for miR-26a (Applied Biosystems, Assay 000405) or miR-26b (Applied Biosystems, Assay 000407) or Sno202 (Applied Biosystems, Assay 001232) and the Applied Biosystems 7300 Real-Time PCR System. All assay values were normalized to the Sno202 internal control.