Sybr green master kit

Total RNA was extracted by using TRIzol (Invitrogen) and cDNA was synthesized by using cDNA kit (Toyobo, Japan). PCR analysis was performed by using SYBR^®-Green Master kit (Toyobo) (details in Supplementary Materials).

Total RNA was isolated from myocardial homogenate and cultured H9C2 cardiomyocytes using the TRIzol reagent (Thermo Fisher scientific, Shanghai, China) according to the manufacture’s instructions and stored at −80°C. Quantitative specific RNA expression was performed in one step with SYBR Green Master kit (TOYOBO). All reactions were performed in triplicate in ABI PRISM^® 7500 Sequence Detection System (Thermo Fisher scientific, Shanghai, China). After initial denaturation step at 95°C for 5 min, temperature cycling was initiated for 40 cycles of amplification (denaturation at 94°C for 30 s, annealing at 62°C for 30 s, and elongation at 72°C for 32s). The value obtained for the target gene expression were normalized to 18s rRNA and analyzed by the relative gene expression 2^−ΔΔCT method. The following primers were used to assess gene expression of STIM1 and 18s rRNA:

STIM1: Forward 5′-TGC CAA GGC TAG CTG TAA CAA-3′,

Reverse 5′-TCA GGT GAT TGT GGC GAGTC-3′;

Orai1: Forward 5′-AGG TGA TGA GCC TCA ACGAG-3′,

Reverse 5′-TAA CCC TGG CGG GTA GTCAT-3′;

TRPC1: Forward 5′-AGA TGT GCT TGG GAG AAA TGCT-3′,

Reverse 5′-CGT TCC ATA AGT TTC TGA CAA CCG-3′;

18s rRNA: Forward 5′-CCT GGA TAC CGC AGC TAGGA-3′,

Reverse 5′-GCG GCG CAA TAC GAA TGC CCC-3′.