Real time pcr stepone plus

Total small RNA was extracted with miRVana miRNA isolation kit (Thermo Fisher), and quality and quantity determined by NanoDrop spectrophotometer. miRNA was assessed by real-time PCR using TaqMan probe (Thermo Fisher) and primer sets using Applied Biosystems Real Time PCR StepOne Plus. Briefly, total small RNA (10 ng) was reverse-transcribed using Taqman MicroRNA Reverse Transcription Kit and amplified using TaqMan 2x Universal PCR Master Mix, No AmpErase UNG (Thermo Fisher). To determine VEGFA, ACTB and EP300 mRNAs, one-step reverse transcription coupled with real-time PCR was performed with 2x VeriQuest Probe One-Step qRT-PCR Master Mix (Affymetrix) using total RNA (0.5 μg) extracted with Trizol in an Applied Biosystems Real Time PCR StepOne Plus machine. RT-qPCR probes for the Taqman Gene Expression Assay, i.e. VEGFA (Hs00900055_m1), ACTB (Hs99999903_m1) and EP300 (Hs00914223_m1) were from Thermo Fisher (Cat. #4331182): The primers for semi-quantitative RT-PCR were: RT_ACTB-f: 5΄-ATGGATGATGATATCGCCGCG-3΄; RT_ACTB-r: 5΄-CTAGAAGCATTTGCGGTGGAC-3΄; RT_VEGFA-f: 5΄-ACAGAACGATCGATACAGAA-3΄; RT_VEGFA-r: 5΄-AAAGATCATGCCAGAGTCTC-3΄.