Quantstudio 3d analysis suite

Reverse transcription-quantitative (RT-q)PCR was performed to detect mRNA levels of miR-155, atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and B-myosin heavy chain (B-MHC). Primer3 Plus (http://www.primer3plus.com/cgi-bin/dev/primer3plus.cgi) was used to design the primers. According to the manufacturer's protocol, mouse myocardial tissues or isolated rat ventricular myocytes (NRVMs, 1×10⁶) were treated with TRIzol^® (cat. no. 15596026; Thermo Fisher Scientific, Inc.) to extract total RNA. According to the manufacturer's protocol, reverse transcription of RNA was then performed using a miRNA reverse transcription kit (cat. no. 4366597; Thermo Fisher Scientific, Inc.) and PrimeScript RT-PCR kit (cat. no. RR014A; Takara Bio, Inc.). The cDNA was used as a template and RT-qPCR was performed on QuantStudio 3D PCR system (Thermo Fisher Scientific, Inc.) with QuantStudio 3D AnalysisSuite (Thermo Fisher Scientific, Inc.). qPCR thermocycling conditions were as follows: Denaturation at 94°C for 5 min, 37 cycles of 95°C for 15 sec, 60°C for 30 sec and 72°C for 30 sec. All reactions were repeated three times. U6 and GAPDH were the internal controls. The relative expressions of miR-155, ANP, BNP and B-MHC were expressed as a function of cycle quantification (Cq) and analyzed by the 2^−ΔΔCq method (27 (link)). The primers are listed in Table I.