Revertra ace qpcr rt kit

Total RNA was isolated from the intestinal samples using RNAiso Plus (Takara Bio Inc., Japan) according to the manufacturer’s instructions. After homogenization of the tissue with RNAiso in a bead beater (Taco Prep Bead Beater, Taichung City, Taiwan), the homogenate was extracted with chloroform, and then RNA was precipitated with isopropanol. The RNA concentration was measured using a nanodrop spectrophotometer (DeNovix DS-11FX, Wilmington, DE, USA). cDNA was synthesized from purified RNA using the TOYOBO ReverTra Ace qPCR RT kit (TOYOBO, Osaka, Japan) according to the manufacturer’s protocol. A quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed in triplicate with SYBR Premix Ex Taq II (Bioneer Corp., Daejeon, Korea) using the MyGo Pro PCR cycler (Diagnostic Technology, Belrose, Australia). The relative expression levels of mRNA from the target genes were compared with that of the endogenous control β-actin. Primer sequences used to measure cytokine (Table 1) expression were created by using Primer-BLAST software from the National Center for Biotechnology Information ( http://www.ncbi.nlm.nih.gov/). The sequences of the specific primers used to measure the relative expression of tight junction proteins (TJPs), Reg3 alpha (Reg3a), and Reg3 gamma (Reg3g) were obtained from previous studies [14 (link),15 (link)].

qPCR was performed using a LightCycler 1.0 system (Roche). Total RNA was extracted from the head of 10 male adults of the indicated genotype using an RNeasy Mini Kit (Qiagen, 74104). To quantify Camta transcript expression levels, RNA was converted into cDNA using a ReverTra Ace qPCR RT kit (TOYOBO, FSQ-101). cDNA was mixed with SYBR Premix Ex Taq II (TAKARA, RR820S) and 5 pmol of both forward (5’-AGCCGACAGTTTTCCATCAC-3’) and reverse (5’-CACTCGCCATGCTTATCAAA-3’) primers. RpL32 (rp49) was amplified as an internal control using the primer pair 5’-AGATCGTGAAGAAGCGCACCAAG-3’ (forward) and 5’-CACCAGGAACTTCTTGAATCCGG-3’ (reverse). qPCR was conducted at 95 °C for 30 sec (initial denaturation), followed by 40 cycles of denaturation at 95 °C for 5 sec, annealing at 55 °C for 30 sec and elongation at 72 °C for 30 sec. Data processing was performed using LightCycler Software Ver. 3.5 (Roche).

WT and SMS DKO tMEFs were infected with JEV for 1 h on ice. After washing unbound JEV, cells were incubated for 15 min at 37 °C. Then, cells were treated with 0.05% (w/v) trypsin for 5 min at 37 °C to remove JEV bound to cell surface. After inactivation of trypsin with DMEM containing 10% FBS, cells were washed and harvested. Total RNAs were extracted using an RNA purification kit (RNeasy, Qiagen, Hilden, Germany), and 1 μg of total RNA was converted to complementary DNA (cDNA) using ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan). Quantitative real-time PCR was performed using 12 K Flex Real-Time PCR System (ThermoFisher Scientific, Rockford, IL, USA), according to the standard TaqMan PCR kit protocol. The following primer and probe sets were used for JEV RNA quantification: forward primer 5′CTCAGCCTCTAAACGCCTATCC-3′, reverse primer 5′-GTCTCAGGTCCATCTACGACAAATG-3′, and probe 5′-AGAGAGCATTCTTTTTGCCCCGGAATTG-3′. The mixture of the primers and the probe for β-actin as an internal reference control was purchased from Thermo Fisher Scientific (TaqMan Gene Expression assay, Mm00607939_s1, Vic, Primer Limited). JEV RNA levels were normalized by β-actin mRNA.

Total RNA was extracted from the middle part of ileum using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. The ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan) was then used to make cDNA. On an Applied Biosystems StepOne Real-Time PCR System (Thermo, Waltham, MA, USA), all reactions were performed in triplicate. β-Actin was utilized as an endogenous control. The primer sequences used for real-time PCR analysis are as follows: β-Actin (forward, 5’-CTCTGTGTGGATTGGTGGCT-3’, reverse, 5’-CGCAGCTCAGTAACAGTCCG-3’), IL-4 (forward, 5’-TGTAGAGGTGTCAGCGGTCT-3’, reverse, 5’-TCAGTGTTGTGAGCGTGGAC-3’), IL-10 (forward, 5’-TAACTGCACCCACTTCCCAG-3’, reverse, 5’-TGGCAACCCAAGTAACCCTTAAA-3’).

RT-qPCR analysis was performed as previously reported [14 (link)]. Briefly, total RNA was isolated from the samples using the ReliaPrep RNA Cell Miniprep System (Promega). The purity and concentration of RNA were determined using a NanoDrop Lite spectrophotometer (Thermo Fisher Scientific). One hundred nanograms of total RNA were reverse-transcribed to cDNA using the ReverTra Ace qPCR RT Kit (TOYOBO, Japan). qRT-PCR was then performed using the PikoReal 96 Real-Time PCR system (Thermo Fisher Scientific) with THUNDERBIRD^® SYBR qPCR Mix (TOYOBO). The expression values were normalized to U36B4 expression and are expressed as the mean ± standard deviation (SD) of triplicate measurements. The primer sequences are listed in Table 1.

Total RNA from tissue was isolated with Trizol (Invitrogen) and then was reverse transcribed using ReverTraAce qPCR RT kit (Toyobo). qPCR was performed on the CFX96 Touch Real-Time PCR Detection System (Bio-Red) using SYBR Green reagent (Roche). Primer sequences are listed in Table S1.