Applied biosystems high capacity cdna reverse transcription kit

First strand cDNA was generated using the Applied Biosystems® High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), using 10 μl RNA (~500 ng) and random primers. The final composition of cDNA reaction mixtures was 2 μl of 10X buffer, 2 μl of 10X random primers, 0.8 μl of 100 mM dNTP mix, 1 μl RNAse inhibitor, and 1 μl reverse transcriptase in a final volume of 20 μl.

Total RNA wa isolated using RNeasy Mini Kit (Qiagen, #74104) and 1μg of total RNA were reverse transcribed to complementary DNA (cDNA) using Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, #4368814). Quantitative real-time RT-PCR (qRT-PCR) was performed with Applied Biosystems StepOnePlus RT-PCR System available in the Michigan MicroArray Core using the Power SYBR Green PCR Master Mix (Applied Biosystems, #4367659). A housekeeping gene (GAPDH) was used as an internal standard. The primers used in this study are described in Supplementary Table 1.

All total RNA extractions from tissue specimens as well as Hs 578T and BT-549 cells were performed using Ribozol RNA Extraction Reagent (Thomas Scientific). Following reverse transfections performed using Applied Biosystems™ High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, U.S.A.), all qPCR reaction systems were prepared using qScript One-Step RT-qPCR Kit (Quantabio, U.S.A.) to detect the expression of NRON and snaR with the endogenous control of 18S rRNA. The qPCR reactions were performed in triplicate manner and data were analyzed using the 2^−ΔΔC_T method.

Prior to total RNA-extraction cells were washed once in PBS (Sigma). Total RNA was extracted using the Omega BIO-TEK E.Z.N.A.® Total RNA Kit I (Omega BIO-TEK; Cat.# R6834-02) according to the manufacturer’s instructions. DNA digestion was performed on-column using the Qiagen RNase-Free DNase Set (Qiagen; Cat.# 79256). cDNA was generated from 1 µg RNA (assessed by Nanodrop2000 (Thermo Fisher Scientific) measurement) using the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems; Cat.#4368814). Real-time qPCRs were performed in 96-well plates using the PrecisionPLUS qPCR Mater Mix (Primer Design; Cat.#4368814) and the CFX96 Touch Real-Time PCR Detection System machine (BIO-RAD) and BIO-RAD FX96 Touch Real-Time PCR Detection System - CFX Maestro Software. The expression of transcripts was normalized to expression of Large Ribosomal Protein (RPLPO). Data analysis was performed using the Cycles threshold (ddCt) method and expressed as mRNA relative expression ddCt. The following TaqMan probes (Applied Biosystems) were used for qPCR analysis: RPLPO (Hs99999902_m1), SBNO2 (ISO1/ISO2) (Hs00922127_m1), SBNO2 (ISO1) (Hs00209130_m1), IL20 (Hs00218888_m1), IL23A (Hs00900828_g1), IL24 (Hs01114274_m1), CXCR2 (Hs00174304_m1), CXCR4 (Hs00976734_m1), KREMEN1 (Hs00230750_m1)

RNA was extracted using TRIzol Reagent (Invitrogen, #15596026) and reverse-transcribed by Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, #4368814) as previously described^{74 (link)}. Selected gene expression differences were analyzed via real-time quantitative polymerase chain (qPCR) using SsoAdvanced SYBR Green Supermix (BIO-RAD, #172527) via CFX Connect (BIO-RAD, Hercules, CA, USA). Quantified mRNA expression was normalized to housekeeping gene Rpl7 (ribosomal protein L7) (or ribosomal 5 S RNA for RNA immunoprecipitation studies), and expression was presented relative to control levels using the ∆∆CT method of analysis. The following primers were used: mouse Ythdf2 5′-TGGTTCTGTGCATCAAAAGGA-3′ and 5’-CACCTCCAGTAGACCAAGCA-3′; mouse Murf1 5’-GCTGGTGGAAAACATCATTGACAT-3’ and 5’-CATCGGGTGGCTGCCTTT-3’; mouse Mafbx 5’-CTTTCAACAGACTGGACTTCTCGA-3’ and 5’-CAGCTCCAACAGCCTTACTACGT-3’; mouse Rpl7 5’-TGGAACCATGGAGGCTGT-3’ and 5’-CACAGCGGGAACCTTTTTC-3’; mouse 5 S 5’-CTACGGCCATACCACCCTG-3’ and 5’-CCTACAGCACCCGGTATTCC-3’; mouse Asb2 5’-GCAGAGAACACCTGGATTGCCT-3’ and 5’-TTGGCGTCTGCGTTGTATCGCA-3’; rat Asb2 5’-GCACTTCAGCGCTCTACTTC-3’ and 5’-ATGTAGGCGTCGATGTTTGC-3’; mouse Smad3 5’-GCTTTGAGGCTGTCTACCAGCT-3’ and 5’-GTGAGGACCTTGACAAGCCACT-3’.

Total RNA was extracted from hearts and cultured cardiomyocytes using a RNeasy Plus Mini Kit (Qiagen) following the manufactures’ instructions. 0.5 – 2 μg of RNA was reverse transcribed to cDNA using Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (cat. No 4368813, ThermoFisher). Quantitative real-time PCR was performed with SSoAdvanced Universal Probe Supermix (Bio-rad) and sequence specific Taqman probes (ThermoFisher) as indicated in Table 1. Samples were run in duplicates on a Quantstudio 3 (Applied Biosystems, ThermoFisher) Real Time detection instrument. Relative gene expression was normalized to expression levels of housekeeping genes β2-microglobulin (for in vitro studies) or 18S rRNA (for in vivo studies). Results were evaluated using the 2^−ΔΔCt method and expressed as mean ± standard error of the mean (SEM).