Mysticq microrna cdna synthesis mix

RNA was extracted from cells using the miRNeasy miRNA isolation mini kit (#217004, Qiagen). To quantify mRNA of interest, cDNA synthesis and quantitative PCR (qPCR) were conducted using the GoTaq 2-Step RT-qPCR dye-based detection system (#A6010, Promega) and a Roche 480 Lifecycler (Roche, Sussex, UK). Quantification of microRNAs was achieved using the MystiCq microRNA cDNA synthesis mix and MystiCq microRNA qPCR dye-based assay primer system (#MIRRT, Sigma). Primers are detailed in Supplementary Table 3. For RNAseq samples were prepared using the Truseq small RNA library (#RS-200-0012, Illumina, Essex, UK), Truseq Stranded RNA library (#20020597, Illumina) and sequenced using the NextSeq 500 High Output Run (150 cycles) for two biological replicates. Two biological replicates were sequenced and analysis conducted using Cutadtapt, SHRiMP, SAMtools for trimming, mapping to GRch38 build, generating the raw counts. Data available from the Gene Expression Omnibus GSE117744. FunRich software was used to conduct enrichment analysis with Fishers exact test to generate P values [45 (link)].

SCC25 and SCC090 cells (2 × 10⁵ cells collected at 24 h post-transfection) or 0.15 ml plasma was mixed with 1 ml Trizol reagent (Invitrogen, USA) to extract total RNAs. Following DNase I digestion, SensiFAST™ cDNA Synthesis Kit (Bioline, USA) and KAPA SYBR® FAST Universal Kit (Sigma-Aldrich, USA) were used to perform reverse transcription and prepare qPCR reaction mixtures. The expression of NCK1-AS1 was detected with 18S rRNA as the internal control. The miRNA Isolation Kit (RMI050, Geneaid, USA) was used to extract miRNA from SCC25 and SCC090 cells (2 × 10⁵ cells collected at 24 h post-transfection) or 0.15 ml plasma. MystiCq® microRNA cDNA Synthesis Mix (Sigma-Aldrich, USA) was used to synthesize cDNA and qPCR reactions were carried out using miScript SYBR Green PCR Kit (QIAGEN, Shanghai, China). The expression of miR-100 was determined with U6 as endogenous control. Primer sequences were 5′-AGTTCAGCCCCCACTGCTCT-3′ (forward) and 5′-TGGTTTGAGTTCCCATTTCTC-3′ (reverse) for NCK1-AS1; 5′-TACCACATCCAAGGAAGCA-3′ (forward) and 5′-TTTTTCGTCACTACCTCCCC-3′ (reverse) for 18S rRNA; 5′-ATATGGAACGCTTCACGAATTT-3′ (forward) and 5′-TCGCTTCGGCAGCACATATAC-3′ (reverse) for U6. The forward primer of miR-100 was 5′-AACCCGUAGAUCCGAACUUG-3′. Poly (T) was used as the reverse primer of miR-100. Each experiment included 3 replicates. All Ct values were calculated based on the 2^−ΔΔCT method.

miRNA was collected from EVs isolated from infected (MOI, 1:1) or control human monocytes (5 × 10⁶) using the exosomal RNA isolation kit according to the manufacturer’s instructions (Norgen Biotek). Polyadenylation and cDNA conversion were performed using MystiCq microRNA cDNA synthesis mix according to the manufacturer’s instructions (Sigma-Aldrich). For qPCR of hsa-miR-21-5p, hsa-miR-24-3p, hsa-miR-146b-5p, hsa-miR-155-5p, and hsa-miR-299-5p, the appropriate specific MystiCq microRNA qPCR assay forward primer (Sigma-Aldrich) was used. In each case, MystiCq microRNA qPCR universal reverse primers were used. Human MystiCq qPCR assay primer SNORD44 (Sigma-Aldrich) was used as a housekeeping control for comparative qPCR of human samples, whereas MystiCq qPCR assay primer SNORD85 (Sigma-Aldrich) was used as a housekeeping control for comparative qPCR of mouse cell samples. To detect mmu-miR-24-3p during in vivo infection, quantitative qPCR was performed by obtaining a standard curve for synthetic mmu-miR-24-3p. qPCR was performed in a StepOnePlus real-time PCR system with MystiCq microRNA SYBR green qPCR ReadyMix with ROX (Sigma-Aldrich) with the following parameters: initial denaturation at 95°C for 2 min; 40 cycles of PCR; denaturation at 95°C for 5 s; and annealing, extension, and read (Table 1).

For validation of mature miRNAs expression, 200 ng of total RNA was reverse transcribed in vitro to cDNA using the MystiCq microRNA cDNA Synthesis Mix (Sigma-Aldrich, Saint Louis, MO, USA) according to the manufacturer’s instructions. Relative expression was calculated using the Ct method with uninfected as the reference and small nuclear RNA U6 as endogenous controls.
JEV RNA was quantified as described earlier14 (link). Similarly, RNA was extracted from JEV infected mice brain and uninfected control brain. Notch1, Dll1, JAG1, Hes1 expression were examined with specific primer by qRT-PCR method. The RNA transcript levels were normalized to that of

RNA isolation from cells and cell culture supernatants was performed using TRIzol^® Reagent (Thermo Fisher Scientific) according to the manufacturer’s instruction with the exception that 30 µg GlycoBlue™ Blue Coprecipitant (Thermo Fisher Scientific) was added to the aqueous phase before RNA precipitation. Reverse transcription for mRNA expression analyses was performed using the Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Fisher Scientific). For miR analysis, cDNA was transcribed using the MystiCq™ microRNA cDNA Synthesis Mix (Sigma-Aldrich) according to the manufacturer’s instructions. The QuantStudio 3/5 Real-Time PCR System and PowerUP™ SYBR™ Green Master Mix (both from Thermo Fisher Scientific) were used to perform real-time quantitative PCR (qPCR). Primers for SNORD44 came from Sigma-Aldrich and all other primers from Biomers (Ulm, Germany). Primer sequences are presented in Table 1. Relative mRNA/miR expression was calculated using the QuantStudio qPCR data analysis software (Thermo Fisher Scientific) and the ∆∆Ct method and was normalized to the indicated respective control RNAs.

For the validation of methylated genes expression, we used primers for Socs1, Ep300, p16, Mgmt and Dapk (QuantiTect Primer Assay, Qiagen); for the evaluation of miR-466e-5p gene targets expression, we used primers for Zfp704, Cplx2 and Cnot7[41] (link), [42] (link); for the validation of miRNA microarrays, we used primers for miR-466e-5p, miR-21-3p and miR-185-3p (MystiCq microRNA qPCR Assay Primer, Sigma-Aldrich). Mouse liver RNA was reverse-transcribed using the RT2 first strand kit (SABiosciences, Frederick, MD, USA) for gene expression experiments and MystiCq microRNA cDNA Synthesis Mix (Sigma-Aldrich) for miRNA expression experiments. Then quantitative real-time PCR reactions were performed in triplicate with an Applied Biosystems 7300 Real-Time PCR system (Life Technologies), using the RT2 SYBR Green PCR Master Mix (SABiosciences) for gene expression experiments and the MystiCq microRNA SYBR Green qPCR ReadyMix for miRNA expression experiments, according to the manufacturers’ instructions.