Sybr green fast qpcr mix

The total RNA of myocardium was extracted by Trizol separation reagent according to the reagent instructions. RNA sample was subjected to reverse transcription with the following procedure: 37°C 15 min, 85°C 5 s, and 4°C forever. The cDNAs were amplified with SYBR Green Fast qPCR mix (Takara, Otsu, Shiga, Japan) using ABI StepOne PCR System (ABI, Carlsbad, CA, USA). 18S was serviced as the house-keep gene. The average cycle threshold (CT) values from triplicate experiments were normalized to 18S, and the group of WT + NS was served as control samples. Relative mRNA expressions were calculated as the fold of control samples. The structures of all primers used are listed in Table 1.

Total RNA was extracted from A549 cells using Trizol reagent (Invitrogen Corporation, CA, USA) followed by reverse transcription using a PrimeScript™ RT MasterMix Kit (Takara, Shiga, Japan). Real-time PCR was performed with the above cDNA using SYBR Green Fast qPCR mix (Takara) with an iCyler iQ Real-time PCR Detection System (Bio-Rad Laboratories Inc., USA). 18S was used as a housekeeping gene. Experimental cycle threshold values were normalized to 18S, and relative mRNA expression was calculated versus a control sample (the primers used are listed in Table 1).

Total RNA from the frozen heart tissues or primary cardiomyocytes was extracted using the TRIzol Reagent (Invitrogen Life Technologies, USA). Subsequently, the RNA was reverse transcribed to cDNA using a PrimeScript™ RT Master Mix Kit (Takara). Quantitative real-time PCR was performed using a SYBR Green Fast qPCR mix (Takara) with the ABI 7500 Real-Time PCR System (ABI, Carlsbad, CA, USA). The expression levels of the target genes were normalized to that of GAPDH. The primer sequences used in this procedure are listed in Supplementary Table S1 and S2.

Total RNA was extracted from myocardial tissue using Trizol reagent (Takara, Kyoto, Japan), and then the purified RNA was reversely transcribed into cDNA using PrimeScript™ RT Master Mix Kit (Takara, Kyoto, Japan). SYBR Green Fast qPCR mix (Takara, Kyoto, Japan) and ABI 7500 real-time PCR system (ABI, Carlsbad, CA, USA) were used to perform qRT-PCR. All primers used were as follows: atrial natriuretic peptide (ANP)-F 5′-GAGAAGATGCCGGTAGAAGA-3′, ANP-R 5′-GAGAAGATGCCGGTAGAAGA-3′, brain natriuretic peptide (BNP)-F, 5′-CTGCTGGAGCTGATAAGAGA-3′, BNP-R 5′-TGCC CAAAGCAGCTTGAGAT-3′; CaMKIIδ A-F 5′-CGAGAAATTTTTCAGCAGCC-3′, CaMKIIδ A-R 5′-ACAGTAGTTTGGGGCTCCAG-3′; CaMKIIδ B-F 5′-CGAGAAATTTTTCAGCAGCC-3′, CaMKIIδ B-R 5′-GCTCTCAGTTGACTCCATCATC-3′; CaMKIIδ C-F 5′-CGAGAAATTTTTCAGCAGCC-3, CaMKIIδ C-R 5′-CTCAGTTGACTCCTTTACCCC-3′; 18S-F 5′-AGTCCCTGCCCTTTGTACACA-3′, 18S-R 5′-CGATCCGAGGGCCTCACTA-3′. 18S was used as a housekeeping gene. Each experiment was performed in triplicate. The relative expressions of target genes were calculated with the 2^−∆∆Ct method.

The total RNA of the myocardium was extracted from the myocardium using a Trizol separation reagent. The cDNA was then synthesized by the Prime Script™ RT Master Mix Kit (Takara, Kyoto, Japan) before quantitative real-time PCR was performed using the SYBR Green Fast qPCR mix (Takara) with the ABI StepOne PCR System (ABI, Carlsbad, CA, USA). All primers used were the following: atrial natriuretic peptide (ANP)—F 5′-GAGAAGATGCCGGTAGAAGA-3′; ANP—R 5′-AAGCACTGCCGTCTCTCAGA-3′; brain natriuretic peptide (BNP)—F 5′-CTGCTGGAGCTGATAAGAGA-3′; BNP—R 5′-TGCCCAAAGCAGCTTGAGAT-3′; CaMKII δA—F 5′-CGAGAAATTTTTCAGCAGCC-3′; CaMKII δA—R 5′-ACAGTAGTTTGGGGCTCCAG-3′; CaMKII δB—F 5′-CGAGAAATTTTTCAGCAGCC-3′; CaMKII δB—R 5′-GCTCTCAGTTGACTCCATCATC-3′; CaMKII δC—F 5′-CGAGAAATTTTTCAGCAGCC-3′; CaMKII δC—R 5′-CTCAGTTGACTCCTTTACCCC-3′; 18S—F 5′-AGTCCCTGCCCTTTGTACACA-3′, 18S—R 5′-CGATCCGAGGGCCTCACTA-3′.
We normalized the experimental period threshold to 18S, a housekeeping gene, and calculated relative mRNA expression relative to control samples.

Total RNA was extracted from myocardial tissue using Trizol reagent (Takara, Kyoto, Japan), and then the purified RNA was reversely transcribed into cDNA using PrimeScript™ RT Master Mix Kit (Takara). SYBR Green Fast qPCR mix (Takara) and ABI 7500 real-time PCR system (ABI, Carlsbad, CA, United States) were used to perform Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). 18S was used as a housekeeping gene. Each experiment was performed in triplicate. The relative expressions of target genes were calculated using the 2^–ΔΔCt method.