Sensimix sybr no rox one step kit

RNA was isolated and purified using a microRNA NucleoSpin^® Kit (Macherey & Nagel, Düren, Germany). The concentration and purity of the isolated RNA was measured and controlled using a NanoDrop spectrophotometer (Thermo Scientific, Peqlab Biotechnologie GmbH). The primers for qRT-PCR were designed using Primer3 Input software (v. 0.4.0, Whitehead Institute for Biomedical Research, Cambridge, MA, USA, Table S2) and purchased from Eurofins MWG (Ebersberg, Germany). One-step qRT-PCR was performed using a SensiMix™ SYBR No-ROX One-Step Kit (Bioline, Luckenwalde, Germany), including SYBR Green detection on a RotorGene 6000 cycler (Corbett Life Science, Sydney, Australia). The relative mRNA levels were calculated with an external standard curve using the average expression levels of housekeeping genes (Rn18S or β-actin).

Total RNA from the liver was isolated using the miRNA NucleoSpin® Kit (Macherey & Nagel, Düren, Germany) and quantified as described previously [32 (link), 55 (link)]. Total RNA from epididymal white adipose tissue (VAT) was isolated by optimized guanidine isothiocyanate/phenol-chloroform extraction using peqGOLD TriFast^TM (VWR International, Leuven, Belgium). The primers for the qRT-PCR were designed using Primer3 Input software version 4.1.0 (https://primer3.ut.ee/) and purchased from Eurofins MWG (Ebersberg, Gemany) (Table 1). The qRT-PCR was performed using the SensiMix™ SYBR No-ROX one step kit (Bioline, Luckenwalde, Germany) on a Rotorgene 6000 cycler (Corbett Life Science, Sydney, Australia). Target gene mRNA expression was calculated using an external standard curve and related to the housekeeper Rn18S (for liver) or the mean of Rn18S and Gt2b (for VAT).

Total RNA was isolated and purified from the treated cells using QIAzol lysis reagent (QIAGEN Sciences, USA) and RNA clean and Concentrator-25 (Inqaba Biotech, SA). Double stranded cDNA was synthesized from 3 μg of the total RNA using Superscript Reverse Transcriptase III (Invitrogen, USA). Real time quantitative PCR was done in triplicate using Rotor gene-3000 quantitative real-time PCR machine using Sensi Mix SYBR No-ROX One-Step Kit (Bioline, UK). The primers used were mouse Glut4 gene (Forward primer- 5’ GCA GCG AGT GAC TGG AAC A 3′; Reverse primer- 5’CCA GCC ACG TTG CAT TGT AG 3′), Nrf-1 gene (Forward primer- 5′ AAA CAC AAA CTC AGG CCA CC 3′; Reverse primer-5’ CCA TCA GCC ACA GCA GAG CA 3′) and Mef2a gene (Forward primer-5′ GTG TAC TCA GCA ATG CCG AC 3′; and Reverse primer-5’ AAC CCT GAG ATA ACT GCC CTC 3′). The amplification occurred in a 3-step cycle: denaturation at 95 °C for 5 s, annealing at 60 °C for 10 s, and extension at 72 °C for 15 s. Relative mRNA expression was normalised to mouse Actin reference gene (Forward primer- 5′ GAG ACC TTC AAC ACC CCA GCC 3′; Reverse primer- 5′ GGA GAG CAT AGC CCT CGT AG 3′) and calculated according to relative standard method.