Applied biosystems high capacity cdna reverse transcription kit with rnase inhibitor

Total cellular RNA was isolated and prepared using RNeasy Mini Kit (Qiagen, Valencia, CA), following the manufacturer’s protocol. For quantitative real-time reverse transcriptase-PCR (qRT-PCR) experiment, cDNA was produced using the Applied Biosystems™ High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Thermo Fisher Scientific) and 50 ng of cDNA per sample was analyzed using an Applied Biosystems™ StepOne™ Real-Time PCR System together with gene-specific primers and TaqMan® Gene Expression Master Mix. Accompanying software was used for the acquisition of threshold cycle (Ct) values. Fold changes in expression quantities was calculated according to the 2^-ΔΔCT method.

The excised thoracic aortas were frozen in liquid nitrogen, pulverized in a mill, and then homogenized with a POLYTRON PT 2500 E (Kinematica AG, Luzern, Schweiz). Homogenized tissues were lysed with 900 μL of QIAzol Lysis Reagent (QIAGEN K.K., Tokyo, Japan). Genomic DNA contamination of the aqueous phase was reduced with gDNA Eliminator Solution (QIAGEN K.K.). After separating the phases with the addition of 180 μL of chloroform, RNA was isolated using an RNeasy Mini Kit (QIAGEN K.K.). An Applied Biosystems High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Thermo Fisher Scientific K.K., Tokyo, Japan) was used to synthesize cDNAs from 1 μg of total RNA. Quantitative PCR was performed using an Applied Biosystems StepOnePlus Real-Time PCR System with TaqMan™ Fast Universal PCR Master Mix (Thermo Fisher Scientific K.K.). Relative gene expression was calculated using the ΔΔCt method. Gene expression was normalized to GAPDH. The primers and probes used in the present study are listed in S1 Table (Thermo Fisher Scientific K.K.).

Total RNA was isolated using Invitrogen TRIzol Reagent (#15-596-018, Thermo Fisher Scientific). Reverse transcription was performed with random primers using Applied Biosystems High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (#43-749-66, Thermo Fisher Scientific). Quantitative Real-Time PCR was performed using PowerUp SYBR Green Master Mix (#A25777, Thermo Fisher Scientific) in a QuantStudio 5 Real-Time PCR System (Thermo Fisher Scientific). Relative mRNA expression was calculated using the ΔΔCT method.