Revertaid h minus cdna synthesis kit

High quality intact RNA samples were processed for cDNA synthesis. First, 1 μg of each sample was DNase I (Fermentas, Thermo Fisher) treated and the DNA-free RNA was reverse transcribed according to the instructions in the Revert Aid H Minus cDNA Synthesis Kit (Thermo Fisher Scientific). Oligo-(dT)₁₈ primers were used for cDNA synthesis. cDNA reaction mixtures were diluted (1.5–5 ng template per reaction) and used in the qPCR reactions together with 250 nM of each primer and the Maxima SYBR Green qPCR Master Mixes (Thermo Fisher Scientific). Reactions were set up in 20 μl volume and run following the manufacturer’s instructions with the inclusion of melt curve analysis in the end of the run. qPCRs were performed in a StepOnePlus thermal cycler (Applied Biosystems, Carlsbad, CA, United States). All primers (Sigma-Aldrich) used in the qPCRs are listed in Supplementary Table S1. The gene encoding tryptophanyl-tRNA-synthetase (TtRNA) (GL50803_3032) was used as an endogenous control in the qPCR reactions (Einarsson et al., 2016b (link)). The fold change in gene expression was calculated using the ΔΔCt method. Significant changes in RNA levels between treatments and controls were assessed using one-way analysis of variance (ANOVA) at α < 0.05 followed by the Dunnett’s multiple comparison test at P < 0.05.

High-quality and DNaseI treated RNA samples were reverse transcribed to cDNA using the Revert Aid H Minus cDNA Synthesis Kit (Thermo Fisher) with oligo-(dT)18 primers, according to the manufacturer’s instructions. qPCR was performed using 2 ng cDNA template per reaction, 0.3μM of each primer and the SsoAdvanced Universal SYBR Green Supermix (2x) (Bio-Rad Laboratories). Reactions were set up in 10 μl volumes and run on a Bio-Rad CFX 384 instrument according to the manufacturer’s protocol. qPCR primers used in this study are listed in S5 Table. For all qPCRs, GAPDH was used as the housekeeping gene. The fold change values in gene expression were calculated using the 2^−ΔΔCT method [92 (link)]. Statistical significance in RNA levels was determined using Welchs’ t-test with Holm-type corrections for multiple testing.

RNA was isolated from the cell lysates and PAXgene tubes using the RNeasy Micro or Mini kit (QIAGEN Benelux BV) or the PAXgene RNA isolation kit (PreAnalytiX), respectively, according to the manufacturers’ protocols. In both procedures, a DNase (QIAGEN Benelux BV) step was included to remove any genomic DNA. RNA quantity and purity were determined using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Either 50 ng (cell fractions) or 250 ng (PAXgene whole blood) of RNA was used for complementary DNA (cDNA) synthesis, which was performed using the RevertAid H Minus cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA), according to the manufacturer’s protocol. Two CD19-enriched samples were excluded because of low RNA yield.

From each donor, blood was collected into a PAXgene tube (PreAnalytiX GmbH) at baseline and after 4 weeks and 13 weeks of treatment. The PAXgene tubes were stored at −20°C until further processing. After overnight thawing at room temperature, total RNA was isolated using the PAXgene Blood RNA kit (PreAnalytiX GmbH) according to the manufacturer's instructions. Total RNA concentration was measured using the Nanodrop spectrophotometer (ThermoFisher Scientific Inc.). From each sample, 250 ng RNA was reverse-transcribed into cDNA using a Revertaid H-minus cDNA synthesis kit (ThermoFisher Scientific Inc.).

Reverse transcription and quantitative real-time-PCR were performed as described previously [34 (link),35 (link)]. Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and reverse transcribed using the RevertAid H Minus cDNA Synthesis Kit (ThermoFisher Scientific, Darmstadt, Germany). Reactions where RNA or reverse transcriptase had been omitted were used as negative controls. PCR products were obtained using gene-specific primers reported previously [36 (link)] and SYBR Green (Applied Biosystems, Darmstadt, Germany) in a 7000 ABI Prism Instrument (Applied Biosystems, Darmstadt, Germany). The levels of the target genes were normalized to beta actin (Actb) housekeeping mRNA levels. The primer sequences were the following: Esr1 forward 5’-CAGGACCAGCCCGATTCC-3’ and reverse 5’-TTAGGGTACATGGGTGAGAGTTTG-3’; Esr2 forward 5’-CGCTCGGCATGGACAAC-3’ and reverse 5’-CCCATGCGGTGGAGAGTAAT-3’; Actb forward 5’-TGCCCCTCGTGCTGTTTT-3’ and reverse 5’-TCTGTCCCATGCCAACCAT-3’.

Freshly dissected aortic tissues were snap‐frozen in liquid nitrogen. Frozen tissues were pulverized and RNA was isolated using Qiagen RNeasy Mini kit following the manufacturer's instructions. For reverse transcription, 1 microgram of RNA was used for conversion into cDNA using the RevertAid H minus cDNA Synthesis Kit (ThermoFisher Scientific). Real‐time PCR was performed on the Applied Biosystems 7900HT real‐time PCR machine as described previously.18 Gapdh was used as endogenous control. Refer to Data S1 for qPCR primers.

To quantify mRNA-levels real-time qPCR was performed using Maxima-SYBR-Green-qPCR-Master-Mix (Thermo Scientific, Waltham, MA, USA) and GAPDH as reference-gene. Total-RNA was isolated from 5 × 10⁶ cells using TRI-Reagent (Sigma-Aldrich). RNA (1 μg) was transcribed to cDNA using RevertAid-H-Minus-cDNA-Synthesis Kit (Thermo Scientific). Oligonucleotides for BIM, NOXA, BCLXL, BIRC5, SESN3 and GAPDH are listed in Supplementary Table S3. qRT–PCR-reactions were performed in triplicates in a Bio-Rad-iCycler-instrument and repeated three times. After normalization to GAPDH, regulation was calculated between treated and untreated cells.