Sybr green master mixture

Myocardial total RNA was extracted with TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Complementary DNA was synthesized using the PrimeScript RT Master Mix kit (Takara Bio, Inc.) and then used for qPCR with a SYBR-Green Master Mixture (Takara Bio, Inc.) on the ABI StepOnePlus RT-PCR system (Applied Biosystems, Thermo Fisher Scientific, Inc.). The reverse transcription conditions were as follows: 37°C for 15 min and 85°C for 5 sec. The qPCR cycling conditions were as follows: 95°C for 10 min and 40 cycles of 95°C for 15 sec and 60°C for 15 sec. qPCR was performed with 3 replicates of each sample. The relative expression quantity was analyzed by normalizing to the GAPDH level and calculated with the 2^−ΔΔCq method (38 (link)). The primers were as follows: 5′-AGG TCG GTG TGA ACG GAT TTG-3′ and 5′-AGG TCG GTG TGA ACG GAT TTG-3′ for GAPDH, 5′-GCT TCC AGG CCA TAT TGG AG-3′ and 5′-GGG GGC ATG ACC TCA TCT T-3′ for natriuretic peptide type A (ANP), 5′-GAG GTC ACT CCT ATC CTC TGG-3′ and 5′-GCC ATT TCC TCC GAC TTT TCT C-3′ for natriuretic peptide type B (BNP), 5′-ACT GTC AAC ACT AAG AGG GTC A-3′ and 5′-TTG GAT GAT TTG ATC TTC CAG GG-3′ for myosin heavy polypeptide 7 (β-MHC), 5′-GCT CCT CTT AGG GGC CAC T-3′ and 5′-CCA CGT CTC ACC ATT GGG G-3′ for collagen I, 5′-ACG TAG ATG AAT TGG GAT GCA G-3′ and 5′-GGG TTG GGG CAG TCT AGT G-3′ for collagen III.

Delphinidin (>98% pure), a VEGFR2 inhibitor was obtained from Extrasynthase (Lyon, France). Dulbecco’s Modified Eagle medium (DMEM) and fetal bovine serum (FBS) were from Thermo Fisher Scientific Inc (Waltham, MA, USA); TRIzol Reagent and Lipofectamine 2000 were from Invitrogen (Carlsbad, CA, USA); M-MLV Reverse Transcriptase was from Promega (Madison, WI, USA); SYBR Green Master Mixture was from Takara (Otsu, Japan). ECL-PLUS/Kit was from GE Healthcare (Piscataway, NJ, USA). Cell apoptosis kit [Propidium Iodide (PI), RNase A, Annexin V-FITC] was from KeyGEN biology (Nanjing, China).