Lightcycler 480 sybr green 1 master

Relative quantitation of the 22 paired mRNAs was performed using quantitative real-time polymerase chain reaction (qRT-PCR) analysis (LightCycler 480 SYBR Green I Master, Thermo Fisher) to determine whether the two genes had a predictive role in our Guilin dataset (HBV-related HCC patients with tumor and para-tumor tissues). Amplification reactions were performed in a volume of 20 μl under the following conditions: 95 °C for 5 min, followed by 45 cycles of 95 °C for 30 s, 55 °C for 20 s, and 77 °C for 30 s, followed by 42 °C for 10 s. RBM15-specific primers were as follows: forward primer, (5′- ACCGCAGTCCAGAATTGAGC-3′); reverse primer, (5′- ACTTCAGCTTGGAGGAAGCAG-3′) (position: 1948–2206, length: 279 bp). HNRNPA2B1-specific primers were used as follows: forward primer, (5′- GCAGGAAGTTCAGAGTTCTAGG-3′); reverse primer, (5′- AGTTACTTCCTGGTCCTGGTC-3′) (position: 748–828, length: 101 bp). GAPDH-specific primers were used as follows: forward primer, (5′- GTCTTCACCACCATGGAGAAG-3′); reverse primer, (5′- CATGAGTCCTTCCACGATACC-3′) (position: 323–527, length: 225 bp). Data analysis was calculated using the ΔΔCT method.

Gene expression was determined by quantitative RT-PCR. Total RNA was extracted and purified from LV tissue and isolated cardiomyocytes with the RNeasy Fibrous Tissue Mini Kit (QIAGEN), following the manufacturer’s protocol. RNA (500 ng) was reverse transcribed into cDNA using the SuperScript III First-Strand Synthesis SuperMix kit (Invitrogen). cDNA was subjected to PCR amplification to detect ANP (Nppa), BNP (Nppb), α-SA (Acta1), collagen III (Col3a1), and Trpm4 gene expression, performed with the CFX384 Touch Real-Time PCR Detection System (Bio-Rad), PCR master mix LightCycler 480 SYBR Green I Master (Invitrogen). Samples were run in technical triplicate, and the mRNA expression levels were normalised to those of GAPDH to calculate relative gene expression using delta-delta Ct method. The mouse RT-PCR primers (Sigma-Aldrich) used are shown in (Key resources table).