Qscript supermix

RNA was isolated using RNeasy mini kit (Qiagen). Digested with DNaseI (Sigma-Aldrich) for 15 min at RT to remove DNA contamination, and subsequently, DNaseI was inactivated by heating at 70 °C for 10 min. cDNA was synthesized using Qscript supermix (Quanta Biosciences). Real-time PCR was performed using SSO fast SYBR green supermix (Bio-Rad) in a 7500 real-time PCR system (Applied Biosystems) using 7500 software v2.0.1. Melting curve analysis was performed to ensure the amplification of a single product. Primers used were the following: TCF-1-F-5′-AGGCCAAGAAGCCAACCATCAAGA and TCF-1-R-5′-ACTCTGCAATGACCTTGGCTCTCA; TCF-3-F-5′-TGCAGTGAGCGTGAAATCACCAGT and TCF-3-R-5′-AATGGCTGCACTTTCCTTCAGGGT; TCF-4-F-5′-TCGGCAGAGAGGGATTTAGCTGATGT and TCF-4-R-5′-CTTTCCCGGGATTTGTCTCGGAAACT; LEF1-F-5′-AAGCATCCAGATGGAGGCCTCTACAA and LEF1-R-5′-TGATGTTCTCGGGATGGGTGGAGAAA; β-catenin-F-5′-TCTTGCCCTTTGTCCCGCAAATCA and β-catenin-R-5′-TCCACAAATTGCTGCGTCCCA; EAAT2-F-5′-CCAAGCTTGGATCACTGCCCTGG and EAAT2-R-5′-CCAGCCCCAAAAGAGTCACCCACAA; GS-F-5′-TTGAGAAACTAAGCAAGCGGCACC and GS-R-5′-ATCCAGTTAGACGTCGGGCATTGT; and GAPDH-F-5′-CTTCAACGACCACTTTGT and GAPDH-R-5′-TGGTCCAGGGGTCTTACT. Fold change in messenger RNA (mRNA) expression was calculated by relative quantification using the comparative C_T method with GAPDH as the endogenous control.

RNA was isolated using RNeasy Lipid Tissue Mini Kit with on-column DNase treatment (Qiagen) and then reverse transcribed into cDNA using qScript Supermix (Quanta Biosciences). Relative gene expression was then determined by quantitative PCR (qPCR; ABI QuantStudio3 System) using the standard curve method after amplification of cDNA with gene-specific primer-probe sets using PerfeCTa Fastmix II Fastmix (Quantabio). Taqman Gene Expression Assays were Mm01299053_m1 for Crebrf and Mm02342430_g1 for reference gene Ppia (ThermoFisher). qPCR was conducted in accordance with MIQE guidelines [26 (link)].

Viral RNA was extracted from approximately 10 mg of spleen using the NucleoMag^® Vet kit (MACHEREY-NAGEL, Germany) on a KingFisher^™ Flex Purification System, according to the manufacturer’s instructions, and eluted with 100 μL of the supplied elution buffer. Complementary DNA (cDNA) was made using 2 μL qScript^™ Supermix (Quanta Biosciences, USA) and 8 μL RNA in a 10 μL reaction, according to the manufacturer’s instructions. Spleen samples (n = 191) were tested in duplicate on a Mic qPCR instrument (Bio Molecular Systems), using 1 μL template and PowerUp^™ SYBR^® Green Master Mix (Applied Biosystems, USA), with the primer concentration and cycling conditions as described previously [13 (link)]. Primers targeted the RNA dependent RNA polymerase gene within ORF1b [13 (link)]. Samples were considered positive if the amplification curve crossed the automatically defined threshold and the melting peak was between 85 °C and 86.5 °C. Samples were considered equivocal if only one of the duplicates was positive with the Cq value >33 or if both duplicates showed the correct melting peak, but the Cq was >37. Samples with equivocal results were retested in duplicate with 2 μL and 5 μL template, using a conventional PCR targeting a 321 bp conserved region in ORF1b [14 (link)].