Mirneasy mini kit reagent

Molecular markers associated with morphological alterations were detected by qRT-PCR. Total RNA was extracted from the leukocyte samples with the miRNeasy mini kit reagent (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA quality and amount were determined using a Nanodrop TM Lite Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and by gel electrophoresis. For the analysis, 500 ng of total RNA was retrotranscribed using the iScriptTM cDNA Synthesis Kit (BioRad, Hercules, CA, USA) following the manufacturer’s instructions. For gene expression analysis, 5 ng of cDNA was processed in duplicate in a Rotor-Gene Q real-time machine (Qiagen, Hilden, Germany) using the SsoAdvanced Universal SYBR^® Green SuperMix (BioRad, Hercules, CA, USA). The PCR conditions were as follows: 30 s of initial denaturation (95–98°) and then 40 cycles at 95 °C for 5 s, 60 °C for 20 s. To assess product specificity, a melting curve analysis from 65 °C to 95 °C was performed. The gene transcript values were normalized using GAPDH as a reference gene. The relative quantification of the samples was performed by the Rotor-Gene AssayManager^® v1.0 (Qiagen, Hilden, Germany). The complete list of the primer sequences is shown in Table 2.

Total RNA was extracted from tissue samples and NRCM with the miRNeasy mini kit reagent (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The cDNA was synthesized from 1 μg RNA using the QuantiTect Reverse Transcription Kit (Qiagen) as directed by the manufacturer.
For gene expression analyses, 10 ng of cDNA from cardiac tissue or NRCM were processed in triplicate in a Rotor-Gene Q real-time machine (Qiagen) using the Quantifast SYBR Green Mix (Qiagen). PCR conditions were as follows: 5 min of initial denaturation and then 40 cycles of 95 °C for 10 s, 58 °C for 20 s, 72 °C for 10 s. To assess product specificity, a melting curve analysis from 65 °C to 95 °C with a heating rate of 0.1 °C/s with a continuous fluorescence acquisition was constructed. Gene transcript values were normalized with those obtained from the amplification of Hprt, Hmbs, and Gapdh. The relative quantification of samples was performed by Rotor Gene Q-Series Software and expressed as mean ± standard error of the mean (SEM). The complete list of primer sequences is shown in Table S2.

Total RNA was extracted from tissue samples and NRCM with the miRNeasy mini kit reagent (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA quality and amount were determined using the Agilent Bioanalyzer 2100 and the RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA, USA) The cDNA was synthesized from 1 μg RNA using the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany) or miScript II RT kit (Qiagen, Hilden, Germany) as indicated by the manufacturer.
For gene and miRNA expression analyses, 10 ng of cDNA were processed in triplicate in a Rotor-Gene Q real-time machine (Qiagen, Hilden, Germany) using the Quantifast SYBR Green Mix (Qiagen, Hilden, Germany). PCR conditions were as follows: 5 min of initial denaturation and then 40 cycles of 95 °C for 10 s, 58 °C for 20 s, and 72 °C for 10 s. To assess product specificity, a melting curve analysis from 65 °C to 95 °C with a heating rate of 0.1 °C/s with a continuous fluorescence acquisition was constructed. Gene transcript values were normalized using Hprt and Hmbs reference genes. miRNA transcript values were normalized using U6 and U1 reference miRNA. The relative quantification of samples was performed by Rotor Gene Q-Series Software (Qiagen, Hilden, Germany) and expressed as mean ± standard error of the mean (SEM). The complete list of primer sequences is shown in Table S2.