Multisource rna miniprep kit

Total RNA was isolated from infected cells using a Multisource RNA miniprep kit (Axygen catalog no. AP-MN-MS-RNA-250) and reverse transcribed to cDNA using an iScript cDNA synthesis kit (Bio-Rad catalog no. 1708890). The viral burden was quantified with qRT-PCR using iTaq Universal SYBR green supermix (Bio-Rad catalog no. 1725121) on the Bio-Rad CFX-96 Touch real-time detection system. Primer sequences are shown in Table S1 in the supplemental material. The amount of virus was normalized to the human GAPDH gene (GenBank accession no. NC_000012.12).

Lung tissue total RNA was isolated using a Multisource RNA Miniprep kit (Axygen, USA). The total RNA was reverse transcribed into cDNA using PrimeScript™ RT Master Mix (Perfect Real Time) Kit (Takara, China). qPCR was performed using an ABI Prism 7500 instrument following the SYBR-Green reagent protocol (Takara, China). Data were analyzed as fold change by the 2^−ΔΔCt method. Primers used in this study are listed below: β-actin forward: 5′-gccaaccgtgaaaagatg-3′, β-actin reverse: 5′-tgccagtggtacgaccag-3′; Rras2 forward: 5′-gaccatggcttttgcttgct-3′, Rras2 reverse: 5′-tagcggggacattgaacgtg-3′; Ttll12 forward: 5′-gcatccagagagttcgcaga-3′, Ttll12 reverse: 5′-gggtctcgggtgtaacacag-3′; Nog forward: 5′-tgtacgcgtggaacgaccta-3′, Nog reverse: 5′-ggcttacacaccatgccctc-3′; Vangl2 forward: 5′-tgatccccgattgcttggtc-3′, Vangl2 reverse: 5′-ccagaccactcggctgttt-3′; Gli3 forward: 5′-atcagccctgctttgagctt-3′, Gli3 reverse: 5′-gatgggtctctgcgttggaa-3′; Trps1 forward: 5′-gagcagcagaggatctggag-3′, Trps1 reverse: 5′-cctagtctgctccccgtttg-3′; Tc1 forward: 5′- ctcttcgagtaagcccgtcc-3′, Tc1 reverse: 5′-gggctcgagttttctcctcc-3’.

Total RNA was isolated either from homogenized mosquitoes or infected cell supernatant using a Multisource RNA Miniprep Kit (AP-MN-MS-RNA-250, Axygen) according to the manufacturer’s protocol. Total RNA was reverse transcribed to cDNA using an iScript cDNA Synthesis Kit (1708890, Bio–Rad) in a mixture of 1 μl of iScript Mix and 4 μl of RNA. Reverse transcription was performed at 25°C for 5 min, 46°C for 60 min and 95°C for 1 min. Viral genomes were then quantified by qPCR using iTaq Universal SYBR Green Supermix (1725121, Bio–Rad) according to the manufacturer’s protocol using 1 μl of cDNA for each reaction. RT–qPCR was performed on a Bio–Rad CFX-96 Touch Real-Time Detection System. The RT–qPCR cycle was set to 95°C for 2 min, 40 cycles of 95°C for 10 sec and 60°C for 30 sec, detection of melting curve and finally at 25°C for 2 min. Primer sequences are shown in S1 Table.