Abi 7500 real time pcr system

IL1R1 and IL1R2 genetic polymorphisms rs3917225, rs2234650 (also known as Pst-1 1970C/T), rs4141134, and rs2071008 were selected as the target SNPs, as referenced from earlier studies [13 (link), 14 (link)]. Genomic DNA was extracted from the peripheral blood using standard methods. Genotyping was performed using the TaqMan assay (ABI 7500 Real-Time PCR System, Applied Biosystems, Foster City, CA), and reactions were performed in 96-well microplates in ABI 9700 thermal cyclers (Applied Biosystems). Fluorescence was measured using the ABI 7500 Real-Time PCR System and analyzed using SDS software version 1.2.3 (Applied Biosystems). All SNPs were typed in each participant.

Reverse transcription was performed using a reverse-transcription system (Promega) according to the manufacturer’s protocols. Quantitative RT-PCR was performed using KAPA SYBR Fast qPCR kit (TAKARA, Code: DRR036A) and signals were detected with ABI 7500 Real-Time PCR System (Applied BioSystems). GAPDH was used as endogenous control. The RT-PCR was carried out in triplicate. All the primers used are listed in S1 Table.
Realtime quantification of microRNAs was carried out by stem-loop RT-PCR as described before [22 (link)]. Briefly, reverse transcription was performed using a reverse-transcription kit (TAKARA, code: DRRO47A) according to the manufacturer’s protocols. Quantitative RT-PCR was performed using KAPA SYBR Fast qPCR kit (TAKARA, Code: DRR420A) and signals were detected with ABI 7500 Real-Time PCR System (Applied BioSystems). RnU6 was used as endogenous control. The RT-PCR was carried out in triplicate. All the primers used are listed in S2 Table.

The RNA samples used for qRT-PCR analysis were the same as those for mRNA and miRNA sequence experiments. RNA was extracted as described under sRNA library preparation. First-strand cDNA synthesis was carried out using the Mir-X^TM miRNA first-strand cDNA synthesis kit (TaKaRa), and qRT-PCR of miRNAs was performed using the Mir-X^TM miRNA qRT-PCR SYBR kit (TaKaRa) and miRNA-specific primers in an ABI 7500 Real-Time PCR system (Applied Biosystems, Foster, USA). Small nuclear RNA U6 was used as an internal control. All samples included three technical repetitions. The primer sequences are shown in Supplementary Table S11.
Predicted target genes were validated by qRT-PCR using specific primers designed with Primer Premier 5.0. The first strand cDNA was synthesized from the RNAs using a PrimeScript^TM RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). qRT-PCR was performed in an ABI 7500 Real-Time PCR system (Applied Biosystems, Foster, USA) using the SYBR Premix Ex Taq^TM II Kit (TaKaRa, Dalian, China) according to the manufacturer’s instructions. The relative gene expression for qRT-PCR data was calculated by the 2^−ΔΔCt method. All of the analyzed target genes were tested with three replicates. The primer sequences are shown in Supplementary Table S12.

A total of four differently m6A methylated genes (EDAR, KRT2, FGF5 and TCHH) were selected for qRT-PCR, which was performed with SYBR Premix ExTaq (Takara, Dalian, China) on ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The cDNA synthesis of coding genes was performed using Quantscript RT Kit Quant cDNA (Tiangen Biotech, Beijing, China) with approximately 500 ng of total RNA as the template; SYBR Premix ExTaq (Tiangen, Beijing, China) was used for real-time PCR, which was available for an ABI7500 Real-Time PCR System (Applied Biosystems). The Tm values of each reaction were list in Table S1. The β-actin gene was used as a reference control. Each plate was repeated three times in independent runs for all references. Gene expression was evaluated by the 2^−∆∆Ct method [20 (link)].

Striatal tissues of rats were homogenized and total ribonucleic acid (RNA) was extracted by Trizol reagent (Invitrogen, USA). cDNA was generated from total RNA samples using the RevertAid First Strand cDNA Synthesis kit (Takara, Japan). Q-PCR was performed using the ABI 7500 Real-Time PCR System (Life Technologies, USA) according to the supplier’s instructions. After amplification, melting curve analysis and length verification by gel electrophoresis were carried out to confirm the specificity of PCR products. As a negative control, template RNA was replaced with PCR-grade water. Calculations of threshold cycle and difference were analyzed with ABI 7500 Real-Time PCR System (Life Technologies). Results were expressed as relative expression corrected to the housekeeping gene β-GAPDH. The detector used in real-time PCR reaction is SYBR Green.
The primer sequences used in this study were as follows:
5-GAGAATGAGGTTGCTTTGGAA-3 (forward) and 5-AGACGCTGGTAAGGAGTTGG-3 (reverse) for preproenkephalin B (PPEB) mRNA;
5-GACTCCAGGCGGAAACGGAT-3 (forward) and 5-TCGTAAGGGATCTTGCAGCC-3 (reverse) for FosB mRNA.

Total RNA was isolated from the roots using Tranzol (Transgene), in accordance with the manufacturer's protocol. The RNA purity was checked using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific). Only samples with absorbance ratios of OD_260/280 ≥ 1.8 and OD_260/230 ≥ 1.8 were selected for further use. The cDNA was prepared using a PrimeScript RT Reagent Kit (RR047A; Takara Bio). The relative expression levels were determined by performing a quantitative reverse transcription PCR (qRT-PCR) analysis using an ABI 7500 Real-Time PCR System (Thermo Fisher Scientific) or a ViiA 7 Real-Time PCR System (Life Technologies). The PCR thermocycling protocol was as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. After the amplification cycles, dissociation analyses were performed to confirm the validity of the target (Guo et al., 2018 (link); Jiao et al., 2019 (link); Li et al., 2019c (link)) Three biological replicates and three qRT-PCR technical replicates were performed for each sample. EF1α (AT5G60390) was used as the reference gene for normalization. The primer sequences used for the qRT-PCR analyses are listed in Table S1.