Prime script rt detection kit

RNA levels were measured by qRT-PCR. Briefly, RNAs of primary cardiomyocytes were extracted using RNAiso reagent (Takara Biotechnology). Subsequently, total RNA was reverse transcribed to complementary DNA (cDNA) according to the protocol of PrimeScript^TM RT detection kit (for mRNA) or PrimeScript^TM miRNA RT detection kit (for miRNA) (Takara Biotechnology). The RT primer of miRNA-143: 5′ GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACTGAGCT 3′; RT primer of U6: 5′ CGCTTCACGAATTTGCGTGTCAT 3′. qPCR Primer sets are indicated in Table 1. All primers were synthesized by Sangon Biotech (Shanghai, China). qPCR reactions were performed using the SYBR Premix Ex Taq^TM II kit as described by the protocol. The same thermocycling profile was used for all genes: 95 °C for 10 minutes, followed by 40 cycles of 95 °C for 15 seconds, 58 °C for 30 seconds, and 72 °C for 20 seconds.Table 1

List of primers.

Gene	Forward 5′-3′	Rerverse 5′-3′
ANP	ATACAGTGCGGTGTCCAACA	CGAGAGCACCTCCATCTCTC
BNP	GCTTTGGGCAGAAGATAGA	CAAGTTTGTGCTGGAAGATAA
β-MHC	TGCTGGCACCGTGGACTA	GCTTGAGGGAGGACTTCTGG
GAPDH	GCCAGCCTCGTCTCATAGACA	AGAGAAGGCAGCCCTGGTAAC
U6	CTCGCTTCGGCAGCACA	AACGCTTCACGAATTTGCGT
mir143	GGGTGAGATGAAGCACTGT	CAGTGCGTGTCGTGGAGT

Total RNA was isolated from mouse using RNAiso reagent (Takara Biotechnology). Prime-ScriptTM RT detection kit (Takara Biotechnology) were performed for real-time (RT)-PCR assays using ABI Prism 7500 sequence detection system. Relative levels of the sample mRNA expression were calculated and expressed as 2-DDCt. The primers used for qRT-PCR are shown: 5′-CCATGGCAGACGATGATCCC-3′ and 5′-GTATGGAAGTGATTGTCCAT-3′.