Vii 7 real time pcr platform

RNA was extracted using miRVana microRNA isolation kit (Ambion) and PCR with reverse transcription (RT–PCR) was performed using multiplexed TaqMan primers (Applied Biosystems). The miR profiles were generated with a 384-well microfluidic card based TaqMan human microRNA panel (Applied Biosystems) amplified on a 7900 HT Fast Real Time PCR system (Applied Biosystems). HUVECs were treated with either a 20 Gy dose of radiation or 10 μM cisplatin or 200 μM hydrogen peroxide or 10ng ml⁻¹ of TNF-α for 6 h. At the end of the treatment, RNA was extracted using miRVana microRNA isolation kit (Ambion). 1,000 ng of RNA was reverse transcribed without pre-amplification using the Taqman Megaplex primer pools A and B and real-time PCR was performed on the microfluidic cards for the human microRNA panels A & B per manufacturer's recommendation (Applied Biosystems). Data was normalized to internal control small RNA RNU48 or U6 small RNAs. mRNAs were normalized to either β-actin or GAPDH. Individual RT–PCRs were performed using predesigned TaqMan Assays for mature miRs, primary miRs or mRNAs (Applied Biosystems) on a SmartCycler (Cepheid) or Vii-7 real-time PCR platform (Applied Biosystems) according to manufacturer's instructions.

RNA was extracted using the miRVana microRNA isolation kit (Ambion/Life Technologies). Affymetrix microRNA array v4.0 profiles were generated by the OHSU Genome Profiling Core facility. Individual RT-PCRs were performed using predesigned TaqMan Assays for mature miRs, primary miRs or mRNAs (Applied Biosystems) on a Vii-7 real time PCR platform (Applied Biosystems) according to manufacturer’s instructions. Data was normalized to internal control small RNA RNU48 or U6 small RNAs. mRNAs were normalized to either β-actin or GAPDH.
Nanostring microRNA profiling was performed per manufacturer’s instructions and data was analyzed using the N-solver software. Raw data was normalized to housekeeping genes.