Iscript adv cdna synthesis kit

To understand the exercise effect on sepsis, a set of 16 dysregulated genes were selected based on our rat sepsis-CLP model we previously identified with RNA-sequencing (RNA-seq) analysis [25 (link)]. Tissue RNA isolation, cDNA synthesis and real-time RT-PCR (qRT-PCR) analysis were performed as described previously by our lab [25 (link)]. In brief, liver total RNA was extracted using an RNA isolation kit (RNeasy plus mini kit, Qiagen) and the concentrations were estimated with NanoDrop (ThermoFisher Scientific). For cDNA synthesis, two micrograms of RNA was reverse-transcribed using the iScript-Adv cDNA Synthesis Kit (BioRad, USA) and 100 ng of cDNA was used for qPCR analysis in a final volume of 20 μl containing, iTaq universal SYBR^® Green supermix (BioRad, USA) and target specific qPCR gene expression primers (Primer sequence are listed in Additional file 1: Table S1). Amplification was performed using CFX-Connect Real-time qPCR system (BioRad, USA). mRNA expression changes were calculated using the 2 − ΔΔCT method [26 (link)] and expressed as fold-change compared to controls. β-actin was used as a normalization control.

Total RNA was extracted from the cell lines with Qiagen RNeasy Mini kit (Qiagen, Hilden, Germany) or SingleShot Cell Lysis Kit (Bio-Rad, Hercules, CA) and reverse transcribed by Superscript III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) or iScript Adv cDNA synthesis kit (Bio-Rad, Hercules, CA). All procedures were performed according to the manufacturer’s instructions. Quantitative real-time PCR was performed with Taqman Gene Expression Master Mix (Applied Biosystems) and probes (Table. S1) on StepOnePlus Real-Time PCR system (Applied Biosystems). The expression levels of each transcript were determined in triplicate and normalized to the level of GAPDH. Data analysis was performed with the comparative threshold cycle method (also known as the 2^−ΔΔCt method).

Total RNA was isolated from H9c2 cells in each experimental condition using a commercial total RNA isolation kit by following the manufacturer’s protocol (RNeasy plus mini kit, Qiagen) and the extracted RNA was quantified using a spectrophotometer- NanoDrop (Thermo Fisher Scientific, Inc). Total RNA (2–5 μg) was reverse transcribed to cDNA using the iScript-Adv cDNA Synthesis Kit according to the manufacturer’s recommendation (BioRad, United States). The cDNA (50–100 ng) was used for real-time PCR analysis in a final volume of 20 μl containing, iTaq universal SYBR^® Green supermix (BioRad, United States) and specific gene primers (Supplementary Table 1). qPCR was performed using the CFX Connect Real-time PCR system (BioRad, United States). Fold changes in expression were calculated using the 2- ΔΔCt method (27 (link)). Each reaction was run in duplicate or triplicate and Hprt1, or α-Tubulin was used as a normalization control.