Purelink rna isolation kit

LNCaP95 cells (180,000 cells/well) were plated in 6-well plates. The cells were treated with vehicle, ENZA, and EPI-7170 for one hour prior to treating with, or without 1 nM R1881 for 48 h. Total RNA was isolated using pure link RNA isolation kit (ThermoFisher Scientific) and reverse transcribed with high capacity RNA to cDNA kit (ThermoFisher Scientific). Quantitative real-time PCR was performed using n = 3 independent experiments with technical triplicates for each sample. Expression levels were normalized to levels of RPL13A transcript. Primers have been described [11 (link)].

RT-qPCR analysis of gene expression was performed as previously described [18] (link). Briefly, total adipocyte and liver RNA was extracted using TRIzol method. Total RNA from eWAT and BAT was isolated using PureLink RNA isolation kit (ThermoFisher). For RT-qPCR, 2 μg of total RNA was reverse-transcribed using MMLV-RT followed by qPCR using SYBR Green (Life Technologies). Relative mRNA expression was normalized to the levels of ribosomal protein 36B4. Total eWAT lysates were prepared by tissue homogenization in a lysis buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 5 mM NaF, 25 mM β-glycerolphosphate, 1 mM sodium orthovanadate, 10% glycerol, 1% tritonX-100, 1 mM dithiothreitol, and freshly added protease inhibitors. Immunoblotting was performed using specific antibodies against adipsin (Santa Cruz Biotech, sc-50419).

In order to determine the mRNA expression levels of specific markers at each stage of differentiation, livers were sacrificed at the end of each stage and approximately 2/3 of the livers were used for RNA extraction (n = 3 per stage). The liver tissues were flash frozen in liquid nitrogen and ground using a mortar and pestle. The resulting tissue was then used for RNA isolation using a PureLink RNA isolation kit (Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s instructions. The resulting RNA was used for cDNA synthesis using an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA), following the manufacturer’s instructions. The cDNA was then used in qRT-PCR analysis using a ViiA7 Real time PCR system (Thermo Fisher Scientific) and a power SYBR Green PCR master mix kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. The list of primers used is provided in Supplementary Table S1. All expression levels were normalized to GAPDH expression. Results for pluripotency and endoderm marker expression were represented relative to undifferentiated iPSCs cultured on geltrex-coated well plates. Results for early and mature hepatic marker expression were represented relative to cells differentiated on geltrex-coated well plates unless stated otherwise.

Total cellular RNA was extracted using the Pure Link RNA isolation kit (ID:12183018A, Thermo Fisher Scientific) quantified at 260/280 nm and tested by 1% agarose gel electrophoresis to check the integrity of the samples. Aliquots corresponding to 1 µg of total RNA were reverse transcribed by using the SuperScriptVilo cDNA synthesis kit (ID: 11754-(50), Thermo Fisher Scientific) following the manufacturer’s protocol. The cDNAs (corresponding to 50 ng of the original RNA) were subjected to real-time PCR with the QuantiTect SYBR Green PCR Kit (ID: 204143, Qiagen, Valencia, CA, USA) following the manufacturer’s instructions. The mRNA levels of STAT3-regulated genes, including IL-6, Bcl-2, Cyclin D1 and Survivin, were analyzed by quantitative real-time PCR. SDHA was used as the internal control.