Chamq sybr qpcr master mix kit

We took 10 mg of kidney tissue samples from rats in each group. We crushed the samples by adding nine times the volume of 0.85% normal saline, and then centrifuged them to obtain the supernatant. The total RNA was transcribed into cDNA using the PrimeScript™ RT Master Mix kits. The mRNA expressions of ACE, ACE 2, MAS, and β-actin (with the β-actin gene serving as an endogenous control for the assay) were quantified using a StepOne Plus real-time PCR system (Applied Biosystems, Waltham, MA, USA) following the instructions provided with the ChamQ SYBR qPCR Master Mix kit [29 (link)]. The RT-reaction was as follows: the first stage was 30 s at 95 °C, the second stage was 40 cycles of 10 s at 95 °C and 30 s at 60 °C, and the third stage was 15 s at 95 °C, 60 s at 60 °C and 15 s at 90 °C.

Total RNA was extracted from the samples mentioned above with the FastPure Cell/Tissue Total RNA Isolation Kit according to the kit instructions. Then, cDNA was synthetized using a PCR instrument (2720 Thermal Cycler PCR instrument, Applied Biosystems, USA) with the HiScript II Q RT SuperMix for qPCR (+gDNA wiper) Kit. Finally, the semi-quantitative analysis of viral replication was conducted using a real-time PCR instrument (Step One Plus™ Real Time PCR instrument, Applied Biosystems) with the ChamQ™ SYBR qPCR Master Mix Kit. The primers for DHAV-1 and β-actin were designed in our previous study. The primer sequences designed in our previous research [43 ] were as follows: DHAV-1 forward, 5′-GCCACCCTTCCTGAGTTTGT-3′; DHAV-1 reverse, 5′-TACCATTCCACTTCTCCTGCTT-3′; β-actin forward, 5′-CTTTCTTGGGTATGGAGTCCTG-3′; and β-actin reverse, 5′-TGATTTTCATCGTGCTGGGT-3′. The reaction parameters were as follows: 95 °C for 30 s, 95 °C for 5 s (40 cycles) and 60 °C for 30 s.