Hairpin it micrornas quantitation pcr kit

For mRNA quantification, PrimeScript™ RT Master Mix (Takara, Japan) was used to synthesize first-strand cDNA. qRT-PCR was performed with the SYBR Premix Ex Taq™ (Takara), using gene-specific primers for IRF3, RBMX, and PIN1, β-actin was used as an internal standard (Table S3), The 2^−ΔΔCT method was adopted to analyze the expression of the different genes. All the expression data were subjected to a one-way ANOVA, and statistical significance was assumed at P < 0.05.
For miRNA quantification, the Hairpin-it™ MicroRNAs Quantitation PCR Kit (GenePharma, China) was used to quantify mature miRNAs according to the manufacturer’s instructions. Total RNA was isolated from kidney organs of CyHV-2 infected fish, and 1 μg of total RNAs were used for cDNA synthesis. PCR amplification was performed in a 20 μL reaction containing 2 μL of cDNA, 10 μL of Real-time PCR Master Mix (FAM), 10 μM of miRNA specific Primer, and 10 μM of miRNA specific probe, 1U DNA polymerase. Synthetic miRNA (GenePharma, China) was used as the standard. Data was normalized to total RNA and used to determine the relative miRNA copy number per 1 μg of total RNA. Each reaction was performed in triplicate and the data were calculated as the mean ± SD as described above. All reactions were performed in triplicate on the CFX96 Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA).

Total RNA was extracted from cells using a kit from Qiagen. The extracted RNA (1 μg) was subjected to reversely transcribed using the cDNA using the PrimeScript RT Reagent (TAKARA), and the qPCR was carried out using the SYBR Premix Ex Taq™ (Takara). For the miRNA, qPCR was carried out using Hairpin‐it™ MicroRNAs Quantitation PCR Kit (GenePharma). GAPDH was used as an internal control for lncRNA or mRNA, and U6 was used as the internal control for miRNA. The relative level of RNA was calculated using the 2^−ΔΔCt method. The primer sequences were shown in Additional Table S1.