A complete RNA isolation was performed using the Cell Total RNA Isolation kit (ForeGene). Using RT Easy™ II (Fore Gene), according to the manufacturer's protocol, cDNA synthesis was performed. Following which, mRNA expression and miRNA expression levels were measured by RT-qPCR using the Bio-Rad CFX96 Touch system (Bio-Rad Laboratories, Inc.) and iTaq™ Universal SYBR GREEN Supermix (Bio-Rad Laboratories, Inc.). The RT-qPCR thermocycling conditions were: 50°C for 2 min, at 95°C for 30 sec, and then 40 cycles of 15 sec at 95°C and 30 sec at 60°C. Two reference genes U6 and GAPDH were used to quantify miRNA and mRNA, respectively. To calculate mRNA expression, the 2−ΔΔCq (25 (link)) method was employed. The sequences of primers are listed in Table I .
Rt easy 2
Manufactured by Foregene
Sourced in China
The RT Easy™ II is a compact and efficient laboratory instrument designed for performing reverse transcription (RT) reactions. It enables the conversion of RNA into complementary DNA (cDNA) for downstream molecular biology applications such as qPCR and gene expression analysis. The device provides precise temperature control and automated cycling to ensure consistent and reliable results.
Automatically generated - may contain errors
3 protocols using rt easy 2
1
Comprehensive RNA Isolation and Expression Analysis
2
Quantitative PCR Analysis of VSMC mRNA
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The relative mRNA levels of these genes were determined using real-time quantitative PCR. Briefly, total RNA was isolated from VSMCs and aortic tissue using a Total RNA Isolation Kit (RE-03111) according to the manufacturer’s protocol. RNA purity was determined based on a ratio of the absorbance at 260 to 280 nm of 1.9–2.1. cDNA was synthesised using RT Easy™ II (FORE GENE; RT-01022). Quantitative reverse transcription‒polymerase chain reaction (PCR) was performed using Real Time PCR Easy™-SYBR Green I (FORE GENE, QP-01012). The relative mRNA expression level was calculated using the delta-delta-CT method, with β-actin as the reference gene.
3
Total RNA Extraction and qPCR Analysis
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Total RNA were extracted using Trizol reagent and reverse transcribed using RT Easy™ II (Foregene, Chengdu, China) according to the protocol of the manufacturer. Real-time qPCR was performed using the SYBR Green PCR Kit (Qiagen, Hilden, Germany), and specific primers were used (Supplementary Table S1 ). Samples were run in triplicate, and gene expression levels were analyzed and normalized with GAPDH using the comparative threshold method (2‐△△Ct).
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