Quantification of Cardiac Gene Expression

Total RNA was prepared from mouse hearts using TRIzol (Invitrogen)^{43 (link)}. cDNA was generated using 300 ng total RNA and SuperScript III Reverse Transcriptase (ThermoFisher). Using Maxima SYBR Green qPCR master mix (Fermentas), real-time RT-PCR was performed under the following conditions: 94 °C for 10 min; 40 cycles of 94 °C for 15 s, 58 °C for 30 s, 72 °C for 30 s; and a final elongation at 72 °C for 15 min. Relative mRNA expression was determined by the ΔΔ-Ct method normalized to the ribosomal RNA (18S) level. The following oligonucleotide primers were used: NPPA, sense 5’-ATGGGCTCCTTCTCCATCAC-3’ and antisense 5′-ATCTTCGGTACCGGAAGCTG-3′; NPPB, sense 5′-AAGTCCTAGCCAGTCTCCAGA-3′ and antisense 5′-GAGCTGTCTCTGGGCCATTTC-3′; MYH7, sense 5′-GCCAACACCAACCTGTCCAAGTTC-3′ and antisense 5′- TGCAAAGGCTCCAGGTCTGAGGGC-3′; Rcan1.4, sense 5′-TCCAGCTTGGGCTTGACTGAG-3′ and antisense 5′- ACTGGAAGGTGGTGTCCTTGT-3′; 18 S rRNA, sense 5′-CGCGGTTCTATTTTGTTGGT-3′ and antisense 5′-AGTCGGCATCGTTTATGGTC-3′.