Mirna qpcr quantitation kit

Trizol reagent (Thermo Fisher Scientific) was used to extract total RNA from tissues and cells according to the instructions. The extracted total RNA was measured with NanoDorp. 5 μg of total RNA was used for reverse-transcription into cDNA using miRNA qPCR Quantitation Kit (GenePharma, Shanghai, China) and PrimeScript RT Reagent Kit (Takara, Japan). StepOnePlus™ System (Applied Biosystems, Foster City, CA, USA) was used for qPCR analysis using SYBR Green Master Mix (Takara, Japan). U6 snRNA and GAPDH genes were used as internal references for relative gene expression by the 2–∆∆Ct method. Sequences of all primers were shown in Supplementary Materials and Methods.

Total RNA was extracted from the femoral vein tissues of the rats in each group using RNA extraction kits (Invitrogen, Carlsbad, CA, USA). The primers (Table 1 and Supplementary Table 1) for miR-136-5p, IL-6, CRP, U6, and β-actin were synthesized by Takara Biotech-nology Ltd. (Dalian, Liaoning, China). Subsequently, the extracted total RNA was reverse transcribed into complementary DNA (cDNA) using the PrimeScript reverse transcription kits. RT-qPCR for miR-136-5p was carried out using miRNA qPCR Quantitation Kit (Shanghai GenePharma Co., Ltd., Shanghai, China) [40 (link)] and that for IL-6 and CRP was performed using SYBR Premix Ex Taq II kit (Takara, Tokyo, Japan), in accordance with the manufacturer’s instructions. U6 was used as the internal control for miR-136-5p and β-actin was used for IL-6 and CRP assays. The expression levels of miR-136-5p, IL-6, and CRP were calculated by applying the 2^-ΔΔCt method.

To synthesize cDNA, total RNA was reverse transcribed using a PrimeScript RT Reagent Kit (Takara, Japan) and a miRNA qPCR Quantitation Kit (GenePharma, Shanghai, China) according to the manufacturer’s instructions. Quantitative real-time RT-PCR was performed using a StepOnePlus™ System (Applied Biosystems, Foster City, CA, USA) with SYBR Green Master Mix (Takara, Japan). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 snRNA were used as endogenous controls for mRNA and miRNA, respectively. All samples were normalized to internal controls, and the fold changes were calculated using a relative quantification method (2^−ΔΔCt). Real-time PCR reactions were performed in triplicate. The primer sequences are listed in Additional file 1: Supplemental Information.