7500 system sequence detection software

Buccal epithelial cells from the subjects were obtained using 3 mL of 3% glucose mouthwash for 2 min. DNA was extracted with sequential phenol/chloroform/isoamyl alcohol (25:24:1) solution and precipitated with salt ethanol solution [35 (link)]. The analysis of genetic polymorphisms was performed using real-time polymerase chain reaction (qPCR). The −1026(A>C) (rs2779249) and +2087(A>G) (rs2297518) SNPs in the NOS2 gene were genotyped using predesigned TaqMan SNP Genotyping Assays (Applied Biosystems, Foster City, CA, USA). DNA amplification was carried out in a 13 µL volume containing 20 ng genomic DNA, 6.25 µL of 2× Master Mix (Applied Biosystems), 0.63 µL 20× TaqMan Assay Mix (Applied Biosystems), and 4.63 µL of RNA/DNAase-free water (Applied Biosystems). The amplifications in 96-well plates were made in a 7500 Real Time PCR System (Applied Biosystems, Foster City, CA, USA). Subsequently, end-point fluorescence was determined using 7500 System Sequence Detection Software (Applied Biosystems, Foster City, CA, USA).

The total RNA of homogenized heart tissues was extracted with TRIZOL Reagent (Invitrogen, Carlsbad, USA), and then reversely transcribed into cDNA with a Reverse Transcription Kit (catalog RR047A, Takara, Tokyo) according to the manufacturer’s instructions. Primers were designed by Primer Premier 5.0. The following specific primers were used: CVB3: Sense: 5′-CGGTACCTTTGTGCGCCTG T-3′; Anti-sense: 5′-CAGGCCGCCAACGCAGCC-3′. The housekeeping gene β-actin: Sense: 5′-AATTCCATCATGAAGTGTGA-3′; Anti-sense: 5′-ACTCCTGCTTGCTGATCCAC-3′. RT-PCR was performed with an initial denaturation step for 3 min at 94 °C, a three-step cycling procedure (denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 60 s) for 35 cycles. The gene expressions were normalized to the level of β-actin transcripts and quantified by the CT method using the 7500 System Sequence Detection software (Applied Biosystems, Waltham, MA, USA). All reactions were performed in duplicate for each sample.

Total RNA including the small RNA fraction of homogenized heart tissues was extracted with TRIZOL Reagent® (Invitrogen, USA), and then reverse transcripted into cDNA with an Reverse Transcription kit (Ferma, CA) according to the manufacturer’s instructions. Primer for IL-21 and the housekeeping gene GAPDH are designed by Primer Premier 5.0. Real time-polymerase chain reaction (RT-PCR) was performed using an ABI 7500 Sequence Detection System (Applied Biosystems, Foster City, CA) using SYBR green. The amplification steps consisted of denaturation at 95°C, followed by 40 cycles of denaturation at 95°C for 15 s and then annealing at 60°C for 1 min. The relative gene expressions were normalized to the level of GAPDH transcripts and quantified by the ^∆∆CT method using 7500 System Sequence Detection software (Applied Biosystems). All reactions were performed in at least triplicate for each sample.

The cDNA was analysed using real‐time quantitative PCR (qPCR). For optimal quantification, primers were designed using Primer Express software (Applied Biosystems, Foster City, CA, USA). The qPCR reactions were performed using the ABI PRISM 7500 system and SYBR™ Premix Ex Taq II (Takara Bio Inc., Shiga, Japan). All samples were run in triplicate as technical replicates. The following amplification procedure was employed. Data were analysed using the 7500 System Sequence Detection software (Applied Biosystems). All samples had the same starting quantities of all candidate reference genes, based on the standard curves generated for those genes. All procedures and data analyses followed MIQE guidelines.34 The specific primer sequences targeting genes for stemness, differentiation, EMT, Shh signalling and the neural crest are listed in Table 2.

Genomic DNA was extracted from 200 μl of whole blood collected in ethylenediaminetetraacetate-coated tubes using a NucleoSpin^® Blood QuickPure kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. To evaluate the quantity and quality of extracted DNA, the optical density was measured using a NanoDrop™ 1000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). The DNA concentration was adjusted to 10 ng/μl before assays were performed.
Genotyping was performed using the TaqMan™ Drug Metabolism Genotyping Assay (Applied Biosystems, Foster City, CA, USA) for the following SNPs: PON1 Q192R (rs662; c.575A>G rs662 or p.Gln192Arg), CYP2C19*2 (rs4244285; c.681G>A or p.Pro227Pro), CYP2C19*3 (rs4986893; c.636G>A or p.Trp212Ter), and CYP2C19*17 (rs12248560; c.-806C>T). The total reaction volume (25 μl) consisted of 2 × TaqMan Universal Polymerase Chain Reaction Master Mix II (Applied Biosystems), 20 × primer and probe mix (TaqMan Drug Metabolism Genotyping Assay Mix), and 20 ng of template DNA according to the manufacturer’s instructions. Polymerase chain reaction was performed using the 7500 Real-Time PCR System (Applied Biosystems). Allele discrimination was manually and automatically performed using the 7500 System Sequence Detection software (Applied Biosystems).

Total RNA from CA1 tissue was isolated by using the RNAspin mini kit (GE Healthcare). The cDNA was generated from total RNA with Superscript III reverse transcriptase (Invitrogen). Real-time PCR analysis was performed by using the ABI PRISM 7500 real-time PCR system with Power SYBR Green PCR Master Mix according to the instruction manual (Applied Biosystems, Foster City, CA). The forward and reverse primer sequences used for Ndfip1 are shown in Table 1. The primer sequences used for HRPT are also shown in Table 1. The cycle threshold (Ct) value and data were analyzed by using the 7500 system Sequence Detection Software (Applied Biosystems). Quantitative analysis of Ndfip1 gene expression was normalized to that of HPRT gene expression.

Total RNA was extracted from PBMCs using a E.Z.N.A.^® Total RNA Kit I (OMEGA, USA), then the cDNAs were synthesized using a PrimeScript™ RT reagent Kit (TaKaRa, RR037S, Dalian, China), following the instructions provided by the manufacturer. Based on mRNA sequences of RORγt in Genbank, the primers were designed with primer premier 5.0 software and synthesized by Sangon Biotech Co.,Ltd (Shanghai, China). The following primers were used: RORγt, 5′-CCTGGGCTCCTCGCCTGACC-3′ (forward primer) and 5′-TCTCTCTGCCCTCAGCCTTGCC-3′ (reverse primer) with the amplicon size of 169 bp, β-actin, 5′-CACGAAACTACCTTCAACTCC-3′ (forward primer) and 5′-CATACTCCTGCTTGCTGATC-3′ (reverse primer) with the amplicon size of 262 bp. The qRT-PCR was performed with a 7500 Real Time PCR system (Applied Biosystems, USA) using FastStart Universal SYBR Green Master (ROX) (Roche, Swiss) in a 20 µl reaction volume containing 200 nM primers and 5 ng cDNA. Thermal cycling was initiated with a 10-min denaturation at 95 °C, followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. The relative RORγt mRNA expressions were normalized to the level of β-actin (the housekeeping gene) transcripts and quantified by the 2^−∆∆Ct method using 7500 System Sequence Detection software (Applied Biosystems).