High capacity cdna reverse transcription kit

Kidneys were collected 24 h after CLP and snap frozen in liquid nitrogen. Total RNA was purified from tissue samples using Trizol reagent (Thermo Fisher Scientific, Waltham, Massachusetts). cDNA was created using a High Capacity cDNA Reverse Transcription Kit and qRT‐PCR was performed using a Sensimix II kit (Bioline, Taunton, Massachusetts) and TaqMan gene expression assays for kidney injury marker 1 (KIM‐1, Havcr1), neutrophil gelatinase‐associated lipocalin (NGAL, Lcn2), interleukin 6 (Il6), IL‐1β (Il1b), tumor necrosis factor α (Tnfa), fatty acid translocase (cluster of differentiation 36, Cd36), fatty acid transport proteins 1 and 2 (Slc27a1, Slc27a2), medium chain acyl‐CoA dehydrogenase (Acadm), very long‐chain acyl‐CoA dehydrogenase (Acadvl), acyl‐coenzyme A oxidase 1 (Acox1), acetyl‐CoA carboxylase, carnitine palmitoyltransferase 1a and 2 (Cpt1a and Cpt2) and peroxisome proliferator‐activated receptor α (PPARα) (Thermo Fisher Scientific, Waltham, Massachusetts). Relative gene expression was calculated using the ΔΔ‐Ct method normalizing the results to WT sham condition. On preliminary review of the gene expression data, it became apparent that the PPARα primer/probe set had been contaminated and this target was thus eliminated from final analysis.

Total RNA was isolated (Bioline, London, UK) and 700 ng reverse transcribed using High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor. Quantitative PCR was performed using a 7900HT Fast Real-Time PCR System with thermal cycles of 50 °C (2 min) and 95 °C (10 min) followed by 40 cycles of 95 °C (15 s) and 60 °C (1 min). For DNMT detection the reaction mix consisted of 300 nM of both forward and reverse primers (Sigma, Poole, UK), 125 nM FAM-labelled probe specific to DNMT 1, 3a and 3b [34 (link)], 2X TaqMan^® mastermix, 0.5 µL β-2-Microglobulin (β2M) reference control with VIC-reporter dye, and 35 ng cDNA. Inventoried TaqMan^® FAM-labelled probes were used to measure expression of DAPK1 (Hs00234480_m1), TET1 (Hs00286756_m1) and PUMA (Hs00248075_m1). β-2-Microglobulin (Hs00984230_m1) with a VIC-reporter dye was used as a reference control gene. Relative change in gene expression was calculated using the 2^−ΔΔCt method.