Cfx384 c1000 touch thermal cycler

Total RNA from cell lines and mice tissues was isolated using Trizol reagent (Invitrogen, Carlsbad, CA USA). RNA was treated with DNaseI using the DNaseI Kit (Ambion, Ambion Inc., Austin, TX, USA). cDNA was synthesized from 0.5–1 μg total RNA using the Quanta Biosciences qScript ™ (Quanta Biosciences Inc., Gaithersburg, MD, USA) cDNA Synthesis Kit (95047-100) for mRNA analysis and the qScript™ microRNA cDNA Synthesis Kit (95107-100) for miRNA analysis. Quantiative PCR (qPCR) of miRNAs and mRNAs in RNA isolated from cultured cell or mice livers was performed using the CFX384, C1000 touch thermal cycler (Bio-Rad, Hercoles, CA, USA) and a SYBR Green PCR Kit: Quanta Cat. #84018 and #84071, respectively. All experiments were performed in triplicate. qRT-PCR of RNA isolated from human samples was performed using the following specific TaqMan (TAqMan, Invitrogen, CA, USA) predesigned probes: Hs00399294_g1 (H19),Hs04187499_m1 (FADD), and hsa-miR-675-002005 (miR-675).

Total RNA isolation was performed by using the miRNeasy Mini Kit (Qiagen) according to manufacturer instructions. The kit enables the purification of total RNA, including small RNAs and is based on silica-spin columns for optimal RNA binding. RNA concentration and purity were assessed by NanoDrop™ Spectrophotometer (260/280 ratio = 1.8/2.0; 260/230 ratio = 1.8/2.0). cDNA was synthesized using iScript cDNA Synthesis Kit (Biorad) for mRNA and miRCURY LNA RT Kit for microRNAs (Qiagen) following manufacturer instructions. Real-time quantitative PCR (RT-qPCR) was performed using SYBR Green master mix (Primer Design) for the mRNA analysis; the miRCURY LNA miRNA PCR Assay kit (Qiagen) was used for microRNAs amplification. Primers were designed and checked for specificity using BLAST Primer Design Tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast) and purchased from Sigma-Aldrich; microRNA-specific primer mix were obtained from Qiagen. Results were analyzed upon a CFX384 C1000 Touch Thermal Cycler (Biorad), using the 2(-Delta Ct) analysis method. Each biological sample analyzed represents the average value of 3 technical replicates. Primer sequences are listed in Supplementary Table 3.

The cDNA was diluted 1/5 with dH₂O and 2.5 μL was used for qPCR analysis (10 μL total volume; Bio-Rad, iTaq Universal SYBR Green Supermix; Art. No.: 172-5124) in a Bio-Rad CFX384 C1000 Touch Thermal cycler. The qPCRs were done in technical duplicates. Only primer pairs with an efficiency between 90 and 110% were considered for qPCR analysis. The genes of interest were compared with the housekeeping genes actA (AN6542) and benA (AN1182) and the relative fold change in expression was calculated according to the 2^-∆∆Ct method (Livak and Schmittgen 2001 (link)). The qPCR program was run as follows: 95 °C (3 min)—40 repetitions of (95 °C (10 s)–60 °C or 64 °C (10 s)–72 °C (30 s))–95 °C (10 s)–melting curve (65 °C–95 °C at 0.5 °C intervals and 5-s hold at each interval). All qPCR primer pairs were run at an annealing temperature of 60 °C apart from primers for mdpG which were run at 64 °C. A complete list of qPCR primers can be found in Supplementary Table S1.