Viia7 rt pcr instrument

Total mRNA was isolated from whole spleen using Qiagen RNeasy minispin columns and using the manufacturer’s protocol. In brief, tissues were homogenized and treated with RLT lysis buffer to obtain lysates for RNA isolation. Isolated RNA was reverse transcribed using a cloned avian myeloblastosis virus reverse transcriptase kit (Invitrogen, Grand Island, NY) to make cDNA. qRT-PCR reactions were performed on ViiA7 RT-PCR instrument (Applied Biosystems, USA) using TaqMan PreAmp Master Mix (ThermoFisher Scientific, USA. Custom TaqMan Array cards (pre-loaded 384-well microfluidic cards) were purchased from ThermoFisher Scientific (list of primers in Table A in S1 File). All reactions were performed according to the manufacturer’s protocols. Gene expression analyses were performed by ΔΔCT method [29 (link)] using generated using DataAssist3.1 with 18s and HPRT as endogenous controls as previously described [30 (link)]. WNV infected samples were compared to mock-infected samples. PCR-amplification of each cDNA sample was done in triplicates.

RNA from platelets was extracted using the TRIzol reagent according to the standard manufacturer’s recommendations. The SuperMix for qPCR (Vazyme, China) was used to obtain complementary DNA. RT-PCR was performed with qPCR Master Mix (Vazyme, China) and analyzed using the ViiA7 RT-PCR instrument (Applied Biosystems, United States). The reference gene GAPDH was used for the normalization. The primers are listed in Table 2.