Cfx connect st system

Total mRNA from cell cultures was extracted using Bio-Tri RNA lysis buffer (Bio-Lab, Jerusalem, Israel), followed by DNase I treatment (Thermo Scientific, IL, USA), and then reverse transcribed using the Iscript cDNA kit (Bio-Rad, CA, USA). Real-time PCR was performed using the iTaq Universal SYBR Green Supermix (Bio-Rad, CA, USA) and the CFX connect ST system (Bio-Rad, CA, USA). Primers are listed in Table 1.

Total mRNA from liver and muscle was extracted with Bio-Tri RNA lysis buffer (Bio-Lab, Israel) followed by DNase I treatment (Thermo Scientific, IL, USA) and reverse transcribed using the Qscript cDNA Synthesis Kit (Quanta Biosciences, MA). Real-time PCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad, CA) and the CFX connect ST system (Bio-Rad, CA), and gene expression quantification was done using the 2∧(-ΔΔCT) method. The primers were as follows: Col1α1 (forward-5′-TTCTCCTGGCAAAGACGGACTCAA-3′, reverse-5′-GGAAGCTGAAGTCATAACCGCCA-3′), Tgfβ1 (forward-5′-GCGGACTACTATGCTAAAGAGG-3′, reverse-5′-GTAGAGTTCCACATGTTGCTCC-3′), Glut4 (forward-5′-ATTTGGGGCCCTAGGTTGTT-3′, reverse-5′-ATACAGCAGCCCTTGGGTTT-3′), Cxcl10 (forward-5′-GGATGGCTGTCCTAGCTCTG-3′, reverse-5′-TGAGCTAGGGAGGACAAGGA-3′), Stat6 (forward-5′-AGCCCAGAAACAAAGCCTCTT-3′, reverse-5′-TTCGAGCATTAACACCCCACT-3′), Klf4 (forward-5′-AGAAGTGTGACAGGGCCTTTT-3′, reverse-5′-TCGTGGGAAGACAGTGTGAAA-3′), Acc (forward-5′-GGACCACTGCATGGAATGTTAA-3′, reverse-5′-TGAGTGACTGCCGAAACATCTC-3′). QuantiTect Primer (Qiagen) was used to evaluate the expression of Scd1 (QT00291151), and Cd36 (QT0105825). Normalization was performed against Ubc (forward-5′-GCCCAGTGTTACCACCAAGA-3′, reverse-5′-CCCATCACACCCAAGAACA-3′) and Gapdh (forward-5′-AGGTCGGTGTGAACGGATTTG-3′, reverse-5′-TGTAGACCATGTAGTTGAGGTCA-3′).

Total mRNA from in vivo studies (kidney homogenate) and in vitro studies (HK-2 or LLCPK-1 cells) was extracted using Bio-Tri RNA lysis buffer (Bio-Lab, Israel) followed by DNase I treatment (Thermo Fisher Scientific, Waltham, MA, USA). RNA purity was validated spectrophotometrically by nanodrop 2000 (Thermo Fischer Scientific, Waltham, MA, USA), and reverse transcribed using the iScript cDNA kit (Bio-Rad, Hercules, CA, USA). Real-time PCR analysis of 6 biological replicates in duplicates was performed using iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) and the CFX connect ST system (Bio-Rad, Hercules, CA, USA). The primers, used to assess the expression of various genes, were validated by blast sequencing, checked for specificity using the melting curve and Cq of the no template control (NTC) as well as optimized by defining the best annealing temperature, are listed in Table 1.

For total mRNA isolation, tissue samples or hepatocytes were washed in 1× PBS and harvested using Bio-Tri RNA lysis buffer (Bio-Lab, Israel). Extracted RNA was treated with DNase I (Thermo Scientific, IL), and reverse transcribed using the Iscript cDNA kit (Bio-Rad Laboratories, CA). Quantitative PCR reactions for Lepr-s, Ddit3, or Ppp1r15a were performed using iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories, CA), and the CFX connect ST system (Bio-Rad Laboratories, CA). Relative quantity (RQ) values of all tested genes were normalized to Ubc. Primers are listed in Supplementary file 1.