Abi 7500 real time rt pcr system

The multiplex real-time PCR assays were carried out in a 96-well format using the One Step PrimeScript™ RT-PCR Kit (Takara, Dalian, China). Briefly, 5 µL of extracted viral RNA was transferred into a capillary containing 20 µL master mix containing a final concentration of 200 nM of each primer and 100 nM of each probe. RT-PCR was performed at the optimized condition, including 3 steps. Reverse transcription was performed at 42 °C for 30 min followed by denaturation at 95 °C for 10 s. Amplification was achieved by 40 cycles of 95 °C for 8 s and 60 °C for 34 s. All reactions were carried out on an ABI 7500 real-time RT-PCR system (Applied Biosystems, Foster City, CA, USA).
The results were analyzed using ABI 7500 software (Applied Biosystems). For each amplification plot, the Ct value, representing the PCR cycles at which the reporter dye fluorescence was detectable above the fluorescence threshold, was calculated automatically. The use of two fluorophores allowed the differentiation of the virus targets.

Rat hearts were removed and the left atrial tissue was isolated. Total RNA and miRNA were extracted using RNAeasy Mini Kit (Qiagen, Netherlands) and miRcute miRNA isolation kit (TIANGEN Biotech, China) following the manufacturer's instruction, respectively. Quantitative detection of total RNA and miRNA was performed by using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). Further detection of miR-27b-3p and Wnt3a levels and RNA reverse transcription to cDNA using miRcute Plus miRNA First-Strand cDNA Synthesis Kit (TIANGEN Biotech, China) and RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA) were performed according to their respective protocols. The primers of miR-27b-3p, Wnt3a, U6, and β-actin were designed and synthesized by TaKaRa (Kyoto, Japan) (Table 1). The RT-PCR assay for miR-27b-3p and Wnt3a was performed with the ABI 7500 Real-Time (RT) PCR System (Applied Biosystems, USA) with miRcute Plus miRNA qPCR Detection Kit (SYBR Green) (TIANGEN, China) and Fast SYBR Green Master Mix Kit (Applied Biosystems, USA) by the manufacturer's instructions, respectively. Fold changes in the miR-27b-3p and Wnt3a level were calculated using 2^-△△Ct methods and U6 or β-actin used as an internal control, respectively.