C100 touch thermal cycler cfx96 real time system

Atria from seven mice were dissected and immediately placed into All protect tissue solution (Qiagen, Germany). Atria were then stored at −80°C until RNA extraction. Total RNA was extracted from atria and purified using the QIAzol reagent (Qiagen) and the Qiagen RNeasy Kit (Qiagen). RNA quantity was then measured by spectrophotometry using the NanoDrop (Thermofisher, USA). 100 ng of RNA was reversed transcribed using cDNA Reverse Transcription kit (BIO-RAD). Primers were synthesized and obtained from BIO-RAD. RT-qPCR was performed in a 10 μL reaction volume composed by cDNA, SYBR Green mix (BIO-RAD, USA), ddH2O and primers on the BIO-RAD C100 Touch Thermal Cycler/CFX96 Real-time System. Expression levels for RAPGEF3 (EPAC1) and RAPGEF4 (EPAC2) were normalized using CFX Manager software (BIO-RAD) and housekeeping genes, GAPDH and HPRT1.

Samples (ventricular myocardium and PF) from three pig hearts were dissected out and immediately placed into RNA later Buffer. Biopsies were then stored at 4 °C until RNA extraction. Total RNA was extracted from tissues using QIAzol reagent (QIAGEN). RNA was purified and DNase treated using the QIAGEN RNeasy Kit (QIAGEN). RNA quantity was assessed by spectrophotometry (NanoDrop/Thermofisher).
Sequences for primers were obtained from Ensembl Genome Browser. Primers were designed using Primer designing tool (NCBI) and synthesized at Sigma Aldrich/Merck. 400 ng of RNA was reversed transcribed using a cDNA Reverse Transcription kit (BIO-RAD) according to the manufacturer’s protocol. RT-qPCR was performed in a 10-μL reaction volume (1 μL cDNA, 5 μL of SYBR Green mix (BIO-RAD), a volume with a concentration of 10 µm upstream and downstream primers respectively, and added ddH2O to 10 μL on the BIO-RAD C100 Touch Thermal Cycler/CFX96 Real-time System. Expression levels for Col1a1 (collagen type I) and Col3a1 (collagen type III) were normalized using a normalization factor calculated by CFX Manager software (BIO-RAD) and based on RT-qPCR results for two selected housekeeping genes, HPRT1 and GUSB.