Cfx manager real time pcr detection system software version 3

RNA was isolated from FFPE sections using PureLink FFPE Total RNA Isolation kit (Invitrogen, cat. K156002), in accordance with the manufacturer’s instructions. RNA samples were quantified spectrophotometrically and loaded onto 1.5% agarose gel to visualize the degree of RNA integrity. The reverse transcription was carried out with Thermo Scientific RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, cat. K1622) in accordance with the manufacturer’s instructions. Gene expression was evaluated with specific primers for PKM2, SHMT2, HIF-1α, and IL-6 (all from Qiagen) by a CFX Connect™ Real-Time PCR Detection System (BioRad Laboratories) using a SYBR-Green fluorophore based real-time reaction (Brilliant SYBR Green QPCR Master Mix, Thermo Fisher Scientific). Gene expression analysis was performed using CFX Manager™ Real Time PCR Detection System Software, Version 3.1 (BioRad).

Total RNA was extracted from cells using TRIzol reagents (Immunological Science) in accordance with the manufacturer’s instructions. RNA was quantified spectrophotometrically, and its quality was assessed by 1% agarose gel electrophoresis and staining with ethidium bromide. The reverse transcription was carried out with Super Script II R-Transcriptase (FS-RT-3022, Fisher Molecular Biology). Gene expression was evaluated with specific primers for SOD2, BIRC-5, c-MYC, MMP-2 and CRP (RT²-qPCR Primer Assays: Human CRP (NM_000567). Cat.no PPH02632A, Human BIRC5 (NM_001012270) Cat.no PPH00271E, Human MMP2 (NM_001127891) Cat.no PPH00151B and Human SOD2 (NM_000636) Cat.no PPH01716B (all from Qiagen S.r.l., Milano, Italy) were evaluated using the CFX Connect Real-Time PCR Detection System (BioRad Laboratories, Segrate, Italy) with a SYBR green fluorophore-based real-time reaction (Brilliant SYBR Green QPCR Master Mix, Thermo Fisher Scientific). Expression data were analyzed using CFX Manager Real Time PCR Detection System Software, version 3.1 (BioRad).

Total RNA was extracted from treated cells using TRIzol reagent (Invitrogen, cat. 15596026) in accordance with the manufacturer’s instructions. RNA was quantified spectrophotometrically and its quality was assessed by 1.5% agarose gel electrophoresis and staining with ethidium bromide. The reverse transcription was carried out by Thermo Scientific RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Life Technologies, Monza, Italy, cat. K1622) in accordance with the manufacturer’s instructions. Gene expression was evaluated with specific primers for HIF-1α, β-actin, PKM2, SHMT2 and PSA (all from Qiagen) using CFX Connect™ Real-Time PCR Detection System (BioRad Laboratories) with a SYBR-Green fluorophore based real-time reaction (Brilliant SYBR Green QPCR Master Mix, Thermo Fisher Scientific). Gene expression analysis was performed using CFX Manager™ Real Time PCR Detection System Software, Version 3.1 (BioRad).