Soadvanced universal sybr green supermix

Real-time PCR reactions were performed using soAdvanced Universal SYBR Green Supermix (Bio-Rad) with cDNAs synthesized from iScript Advanced cDNA synthesis kit (Bio-Rad). 20 µl reaction mix with 2 µl cDNA (~ 1–10 ng) were monitored on StepOnePlus Thermal Cycler (Applied Biosystems) in “fast mode”. Cycling conditions: 95^oC, 30’, 40 or 45 cycles of 95oC, 15’ and 60^oC, 30’ with plate reading, and a final melt curve stage using default conditions. Primers used are listed in Table S2.

A total of twelve candidate genes belonging to different categories from RNA-seq were used for gene expression analysis via qPCR. First-strand cDNA was synthesized from total RNA using a Transcript First Strand cDNA Synthesis Kit (Roche, Upper Bavaria Germany). qPCR was performed using the SoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules CA, USA). The qPCR reactions were performed in a final volume of 10 μL containing 5 μL of 2 × MasterMix, 0.5 μL of 100 μM of each primer, 1 μL of ddH₂0 and 3 μL of cDNA. The reactions occurred at 95 °C for 30 sec, followed by 40 cycles of 95 °C for 10 sec, 58 °C for 30 sec, and melting curve analysis from 65 °C to 95 °C at 0.5 °C increments. Primers for qPCR were designed based on our predicted gene sequences (Supplementary Table S11). Primers for 18 S were used for internal controls, and all of the qPCR primers were tested with RT-PCR before use. Fold changes were determined by the 2^−ΔΔCt method based on three technical replicates per sample. All qPCR reactions were repeated at least three times.

Total RNA was isolated from the frozen cardiac tissue of all control and diabetic rats using TRIZOL reagent (Ambion; life technologies). RNA (1 μg) was reverse‐transcribed to single‐stranded cDNA using iScript cDNA synthesis kit (Bio‐Rad) as per the manufacturer's instructions. Real‐time qPCR was performed with CFX96 Real‐Time PCR Detection System (Bio‐Rad) using SoAdvancedTM Universal SYBR® Green Supermix (Bio‐Rad) and performed on CFX96 RT‐PCR Detection System (Bio‐Rad) in triplicate. Three independent technical replicates were run for each cardiac sample. The specificity of each primer set was monitored by analysing the dissociation curve. GAPDH was used as a housekeeping gene. Expression of the following genes was analysed: ANF, Epac1/2, SERCA2a, PLB and TnI. Table S1 shows the sequence of forward and reverse primers that were used. The relative expression level of each gene was determined using the comparative cycle threshold (Ct) method (2^−∆∆Ct) normalized to control GAPDH gene.