Mirna reverse transcription control mirtc

Real-time quantitative PCR (RT-qPCR) validation analysis was performed using the StepOnePlus^™ Real-Time PCR System (Applied Biosystems, Foster City, CA, United States) and the miScript PCR System (Qiagen) according to the manufacturer’s instructions. For miRNA abundance level detection, 250 ng of the total RNA were converted into complementary DNA (cDNA). The resulting cDNA was then diluted to have 0.5 ng/μL input material for miRNA detection. All RT-qPCR experiments were carried out using the Liquid Handling Robot QIAgility^™ (Qiagen) before performing RT-qPCR. All primer assays used in the current study were provided by Qiagen. Moreover, miRNA reverse transcription control (miRTC) (Qiagen) was performed to assess the performance of the reverse transcription reaction. The melting curve analysis was used to control the specificity of RT-qPCR products. Specificity of amplicons was further confirmed by agarose gel electrophoresis.

The StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA) and the miScript PCR System (Qiagen) were used to perform RT-qPCR validation of miRNA expression. All steps were performed according to the manufacturer’s instructions. The same samples used for microarray analysis were also used for RT-qPCR validation. Five miRNAs (miR-1231, miR-3127, miR-3198, miR-135b3p, miR-519e-5p) were chosen for RT-qPCR based on the significance of their abundance (highest fold-change) and biological significance in cardiovascular diseases^{9 (link),44 (link)}. Of the total RNA, 250 ng were converted into complementary DNA (cDNA). The resulting cDNA was diluted to 0.5 ng/μL, which was used for miRNA expression analysis. All PCR experiments were carried with the Liquid Handling Robot QIAgility (Qiagen). Primer assays were obtained from Qiagen. A miRNA reverse transcription control (miRTC) (Qiagen) was performed to assess the performance of the reverse transcription reaction. The melting curve analysis was used to determine the specificity of RT-qPCR products.