Rt qpcr primers

RT-qPCR primers that were specific for MDA5, ALV-J Env, Gag, Pol, Mx1, and β-actin were designed using Premier Primer 5.0 software. RT-qPCR primers that were specific for genes in the MDA5 signaling pathway, including interferon-β promoter stimulator 1 (IPS-1), interferon regulatory factor-3 (IRF-3), interferon-β (IFNβ), double-stranded RNA-dependent protein kinase (PKR), 2′, 5′-oligoadenylate synthetase (OAS), myxovirus (influenza virus) resistance 1, interferon-inducible protein p78 (mouse) (Mx1), and major histocompatibility complex class I (MHC class I), are referenced in previous publications (Supplementary Table 1; Lee C. C. et al., 2014 (link)). PCR primers for the full-length MDA5 coding sequence clone and the 3′ UTR of the MDA5 clone were also designed using the Premier Primer 5.0 software (Supplementary Table 2). All the above primers were synthesized by Sangon Biotech Co., Ltd. (Guangzhou, China). A bulge-loop™ Reverse Transcription primer and RT-qPCR primers that were specific for gga-miR-34b-5p were designed and synthesized by RiboBio (Guangzhou, China).

Total RNAs from B. dorsalis testis and other body tissues were isolated using Triazole reagent (Invitrogen, Waltham, MA, USA). For orb2, cDNA was synthesized using the Prime Script RT-PCR Kit (TaKaRa, Japan) and quantitative real time (RT- PCR) PCR was performed using the SYBR Premix Ex Taq (TaKara). B-Actin was used as an internal control for normalization. Primers for qRT-PCR are given in Table S1 for miR-125-3p, miR-276b-3p, and U6 snRNA, cDNA was synthesized using Stem loop PCR primers. SYBR Green Master Mix (miScript SYBR Green PCR Kit, Qiagen, Hilden, Germany) was used to perform real-time PCR. All quantitative real-time PCR was done on an Applied Biosystems, Carlsbad, CA, USA. The stem loop primers, RT-qPCR primers for miRNA-125-3p, miR-276b-3p, and U6 snRNA were prepared from Ribobio (Guangzhou, China). The 2^−△△Ct method was used to normalize the expression of orb2 and miRNAs.