Primerscript rt reagents kit

Total RNA was isolated and subjected to reverse transcription and qPCR analysis. β-actin served as internal control. The primer pairs used in this study are: 5′-atccaggctgtgctgtcc-3′ as forward primer and 5′-gaggatcttcatgaggtagtcg-3′ as reverse primer for β-actin; 5′-gaacgctccctctgcttgc-3′ as forward primer and 5′-aaactcaacctgaaaacgttaccttc-3′ as reverse primer for ORF7. The ΔΔCt method [16 ] for relative quantification of gene expression was used to determine viral RNA levels. For analyzing the inhibitory effect on RNA levels, SYBR Green real-time PCR was performed. Briefly, reverse transcription was carried out using Takara PrimerScript RT reagents kit in a volume of 10 μl at 37 °C for 15 min. One microliter of reverse transcription reaction mixture was used for qPCR by using gene-specific primers and master mix from SYBR Primer Ex Taq II kit (Takara). All reactions were performed in a 25 μl reaction volume. The reaction was carried out at 95 °C for 30 s, followed by 35 cycles at 95 °C for 30 s and 60 °C for 5 s. Relative amount of PRRSV RNA were normalized to β-actin mRNA. Amplification and detection of samples were performed with the CFX96 Touch Real-Time PCR Detection System (Bio-Rad).

As we previously described [5 (link)], total RNA was isolated from Marc-145 cells or PAMs approximately 60 h post PRRSV infection and AS-ON transfection using the RNAiso Plus RNA isolation kit (Takara Dalian, China), and subjected to reverse transcription (Takara PrimerScript RT reagents kit) and qPCR analysis (SYBR Primer Ex Taq II kit). β-actin served as an internal control. The primer pairs used in this study are listed in Table 1. The ΔΔCt method [27 (link)] for relative quantification of gene expression was applied to determine viral RNA levels using SYBR Green real-time PCR. The relative amount of PRRSV RNA was normalized to β-actin mRNA. Amplification and detection of samples were performed with the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA).

As we previously described^{32 (link)}, total RNA was isolated from Marc-145 cells at different time points post transfection and PRRSV infection using the RNAiso Plus RNA isolation kit (Takara Dalian, China, Cat. #9108/9109), and subjected to reverse transcription (Takara PrimerScript RT reagents kit, Cat. #RR037A) and qPCR analysis (Clontech, SYBR Primer Ex Taq II kit, Cat. # RR820Q). The reverse transcription was performed in 10 µl reaction incubated at 37 °C for 15 mins and then at 85 °C for 5 s, containing 2 µl of 5× Prime Script Buffer, 0.5 µl of Prime Script RT Enzyme Mix 1, 0.5 µl of Oligo dT primer, 0.5 µl of Random 6 mers, 3 µl of RNA (480 ng) and 3.5 µl of water. The qPCR reaction was carried out in 20 µl reaction, containing 10 µl of SYBR Premix Ex Taq^TM II (2×), 0.4 µM forward primer, 0.4 µM reverse primer and 1.5 µl of cDNA template (480 ng). The primer pairs used in this study are listed in Table 1. Amplification and detection of samples were performed with the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA). The ΔΔCt method^{33 (link)} (ABI PRISM 7700 Sequence Detection System. 1999. User Bulletin # 2.) for relative quantification of gene expression was applied to determine viral RNA (ORF7) and cellular RACK1 and IFNα mRNA levels using SYBR Green real-time PCR. GAPDH served as an internal control.