Tripure total rna extraction kit

Human ES A673 cells were seeded in 6-well plate (5 × 10⁵ cells/well) and transfected with 15 nM nCAR/miR-34a-5p, control RNA, or vehicle for 48 h. Cells were harvested with TRIpure Total RNA Extraction kit (EP013; ELK Biotechnology, Wuhan, China). Reverse transcription was performed using M-MLV Reverse Transcriptase (EQ002; ELK Biotechnology, Wuhan, China) and random hexamers (for U6) or stem-loop primer 5′-CTC AAC TGG TGT CGT GGA GTC GGC AAT TCA GTT GAG ACA ACC AG-3′ (for miR-34a-5p). Then qPCR analyses were performed with QuFast SYBR Green PCR Master Mix (EQ001; ELK Biotechnology, Wuhan, China) and the following gene specific primers (44 (link)), forward 5′-AGG CAG TGT CTT AGC TGG TTG T-3′, reverse 5′-CTC AAC TGG TGT CGT GGA GTC-3′ for miR-34a-5p, and forward 5′-CTC GCT TCG GCA GCA CAT-3′, reverse 5′-AAC GCT TCA CGA ATT TGC GT-3′ for U6, on a StepOne™ Real-Time PCR system (Life technologies, Carlsbad, CA, USA). Relative mature miR-34a-5p levels were calculated by using the comparative threshold cycle (Ct) method with the formula 2^−ΔΔCt.

Total RNA was extracted from the cardiomyocytes using TRIpure Total RNA Extraction kit (Cat: EP013) form ELK Biotechnology Co. Ltd., China according to the manufacturer’s instructions. Reverse transcription to make cDNA was conducted using M-MLV Reverse Transcriptase kit (Cat: EQ002) also from ELK Biotechnology Co. Ltd., China. Quantitative real-time PCR was performed using StepOne™ Real-Time PCR system (Life Technologies, CA, USA) using QuFast SYBR Green PCR Master Mix (Cat: EQ001, ELK Biotechnology Co.Ltd., China. A 10-μl total reaction volume was used, and the reaction were as follows: 95 °C for 1 min; 40 cycles of 95 for 15 s, 58 °C for 20 s and 72 °C for 45 s, melting curve 60 °C → 95 °C, with 1 °C temperature increase every 20 s. Beta-actin was used as the endogenous control for data normalization and the double delta method used to calculate gene expression. Ct values used for calculations were averages of three independent repeats. All primer information is provided in Additional file 1: Table S1.