M mlv cdna synthesis kit

Total RNA was prepared as described previously [18 (link)]. cDNA was synthesized from total RNA by reverse transcriptase (RT) using an M-MLV cDNA synthesis kit (Enzynomics, Daejeon, Kr). STE12 mRNA levels were measured by quantitative real-time PCR (qRT-PCR) using SYBR green I (GenetBio, Daejeon, Kr). All values were normalized to the level of ACT1 (actin) mRNA. STE12-specific primers and ACT1-specific primers (Table 2) were used to amplify 100-bp fragments of each coding sequence.

Total RNA from S. pseudintermedius ATCC 49051 was isolated as described above, and cDNA was synthesized using the M-MLV cDNA synthesis kit (Enzynomics, Daejeon, Korea). qPCR was performed using an ABI Step One Plus Real-Time System (Applied Biosystems, Waltham, MA, USA). TOPreal™ q-PCR 2X PreMix (SYBR Green with high ROX; Enzynomics) was used. Total RNA was also isolated from the dissected left ears of mice in in vivo experiments. The primers used to validate qPCR and the expression of cytokine genes are listed in Supplementary Table S1. The expression level was standardized against constitutively expressed 16S rRNA and glyceraldehyde 3-phosphate dehydrogenase expression levels and quantified using the 2^-△△Ct method. qPCR was performed in three independent experiments.

The total RNA was isolated from the intestines using TRIzol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. First-strand cDNA synthesis from the total RNA template was performed using M-MLV cDNA Synthesis Kit (Enzynomics, Daejeon, Korea). The resulting cDNA was subjected to real-time PCR using qPCR 2 × PreMIX SYBR (Enzynomics, Daejeon, Korea) and a QuantStidio3 (Applied Biosystems, TermoFisher Scientific, MA, USA). The expression level of genes was normalized with the housekeeping gene GAPDH. Use the following primers: murine GAPDH, forward, 5′-AGG TCG GTG TGA ACG GAT TTG-3′, reverse, 5′-AGG TTT GAT TCA GGC AGA TGT T-3′; murine IL-6, forward, 5′-TAG TCC TTC CTA CCC CAA TTT CC-3′, reverse, 5′-TTG GTC CTT AGC CAC TCC TTC-3′; murine IL-13, forward, 5′-CCT GGC TCT TGC TTG CCT T-3′, reverse, 5′-GGT CTT GTG TGA TGT TGC TCA-3′; murine IL-10, forward, 5′-ATT TGA ATT CCC TGG GTG AGA AG-3′, reverse, 5′-CAC AGG GGA GAA ATC GAT GAC A-3′; murine ZO-1, forward, 5′-TTT TTG ACA GGG GGA GTG G-3′, reverse, 5′-TGC TGC AGA GGT CAA AGT TCA AG-3′; human IL-8, forward, 5′-TTT TGC CAA GGA GTG CTA AAG A-3′, reverse, R: 5′-AAC CCT CTG CAC CCA GTT TTC-3′.

Mouse brains (P12) were sectioned using Brain Matrix, and random punches from the slices were processed with Qiagen RNeasy Plus Mini Kit to obtain RNA. cDNAs were synthesized from the obtained RNAs using Enzynomics M-MLV cDNA synthesis kit. Following primers were used to detected wild-type (979 bp) and Δ9 (912 bp) alleles. Forward: CTACGGGCTA TTCCAGCCTC CCTC, Reverse: GTTGATATCA CTGGCTGAGCGCTG.

To determine mRNA levels, RNAs were extracted from the whole cortex of WT and Ank2-cKO mice at P7, P14, P21, and P28 using TRIzol™ Reagent (Ambion, 15596026). cDNAs were synthesized using M-MLV cDNA synthesis kit (Enzynomics, EZ006M), and real-time qPCR (RT-qPCR) was performed using THUNDERBIRD SYBR qPCR Mix (Toyobo, QPS-201) and CFX96 Real-Time System. For RT-qPCR, the primers in the Key Resources Table were used. The Gapdh gene was used as a control gene.

Total RNA was extracted using a Hybrid-R RNA extraction kit (GeneAll Biotechnology, Seoul, South Korea). cDNA was synthesized by M-MLV cDNA Synthesis kit (Enzynomics, Daejeon, South Korea) according to the supplier’s instructions. Quantitative real-time PCR was performed using TOPrealTM qPCR 2X PreMIX (Enzynomics) on a CFX Connect Real-Time PCR Detection system (Bio-Rad Laboratories, Hercules, CA, USA). Primers used were 5’-AGGGCATCATCAATTTCGAG-3’ (sense) and 5’-TGCCTCTCTTCATCCTTTGG-3’ (antisense) for human SOD1 (NCBI gene ID: 6647); 5’-GTGTGATGGTCCTTCCAACC-3’ (sense) and 5’-CTGACATCCTCTGGCTCACA-3’ (antisense) for human Prdx6 (NCBI gene ID: 9588); 5’-CAGTCTCAAGTATGTCCGT-3’ (sense) and 5’-AGGCTCAATGTTGATGGT-3’ (antisense) for human Gpx2 (NCBI gene ID: 2877); 5’-TTGGCTACCTTGCAGTTCGT-3’ (sense) and 5’-ATGTGAACCATCGCAGGCA-3’ (antisense) for human CISD2 (NCBI gene ID: 493856); 5’-CATGTACGTTGCTATCCAGGC-3’ (sense) and 5’-CTCCTTAATGTCACGCACGAT-3’ (antisense) for human β-actin (NCBI gene ID: 60). Ratio of target gene fold-change was normalized to human β-actin expression using comparative CT (2-ΔΔCt) method.