Total RNA was isolated from biopsy using the RNA Trizol reagent (TaKaRa, Japan). cDNA synthesis was performed using HiScript QRT SuperMix + gDNA wiper (Vazyme Biotech, China). Real-time quantitative PCR was done on the ABI 7500 Real-Time PCR Detection System, using ChamQ SYBR qPCR Master Mix (Vazyme Biotech, China). Primer sequences were as follows: TSLP forward, 5′-CCCAGGCTATTCGGAAACTCAG-3′, and reverse, 5′-CGCCACAATCCTTGTAATTGTG-3′ (these primers do not distinguish between the two TSLP isoforms); long TSLP forward, 5′-CACCGTCTCTTGTAGCAATCG-3′, and reverse, 5′-TAGCCTGGGCACCAGATAGC-3′; short TSLP forward, 5′-CCGCCTATGAGCAGCCAC-3′, and reverse, 5′-CCTGAGTAGCATTTATCTGAG-3′. Typically, 40 cycles of 20 s at 95 °C and 20 s at 60 °C, followed by the thermal dissociation protocol for Fast SYBR green detection. PCR reactions were normalized by expression analysis of GAPDH with the following primers: GAPDH forward, 5′-GCACCGTCAAGGCTGAGAAC-3′ and reverse, 5′-ATGGTGGTGAAGACGCCAGT-3′. The amount of each mRNA was normalized to the amount of GAPDH in the same sample. Relative increases in mRNA expression were calculated using the 2−ΔΔCT method.
Real time pcr detection system
Manufactured by Vazyme
Sourced in China
The Real-Time PCR Detection System is a laboratory equipment designed to perform real-time polymerase chain reaction (PCR) analysis. It is used to amplify and detect specific DNA or RNA sequences in a sample, allowing for accurate quantification and analysis of genetic material.
Automatically generated - may contain errors
Lab products found in correlation
Chamq sybr qpcr master mix, by Vazyme (2 mentions)
Rna trizol reagent, by Takara Bio (1 mentions)
Hiscript q rt supermix, by Vazyme (1 mentions)
Rna isolater, by Vazyme (1 mentions)
Du800, by Beckman Coulter (1 mentions)
Hiscript 2 q rt supermix for qpcr gdna wiper, by Vazyme (1 mentions)
Trizol reagent, by Vazyme (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Nanodrop nd 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Sybr green master mix, by Vazyme (1 mentions)
4 protocols using real time pcr detection system
1
Quantification of TSLP Isoforms by qPCR
2
Quantifying Hippocampal Gene Expression
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Total RNA was extracted from the hippocampus tissues homogenized in RNA isolater (Vazyme Biotech Co.,Ltd, Nanjing, China). Using a spectrophotometer (DU800, Beckman Coulter Inc., Brea, CA, USA), the total RNA concentration was confirmed at 260 nm and 280 nm. Then, 1 μg of purified RNA was reverse-transcripted for RT-PCR to generate cDNA with HiScript II Q RT SuperMix for qPCR (+g DNA wiper) (Vazyme Biotech Co.,Ltd, Nanjing, China). qPCR was performed using the ChamQ SYBR qPCR Master Mix (Vazyme Biotech Co.,Ltd, Nanjing, China) and determined on a real-time PCR detection system (Roche, Switzerland). The relative mRNA expression level was determined with the 2-ΔΔCt method with β-actin as the internal reference control. Primer sequences were shown in Supplementary Table 1
3
Piezo1 Gene Expression Quantification
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Total cellular RNA was extracted with TRIzol reagent (Vazyme Biotech, China), reverse-transcribed into cDNA by reverse transcriptase and oligo (dT) primers (Vazyme Biotech, China), and then amplified in a Real-time PCR Detection System with SYBR Green master mix and specific primers. Gene expression was normalized to Gapdh using the 2−ΔΔCT method. The results were from three independent experiments performed in triplicate. Primers were purchased from Sangon Biotech, China. The primer sequences used were listed: Piezo1-F: 5′-CCCTGTTACGCTTCAATGCT-3′, Piezo1-R: 5′-GCTACCGTTTTGTCCCAGAA-3′; Gapdh-F:5′-AGGTCGGTGTGAACGGATTTG-3′, Gapdh-R 5′-TGTAGACCATGTAGTTGAGGT-3′.
4
Real-Time PCR for CCN1 Expression
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Total RNA from tissues was extracted by using TRIzol reagent (TakaraBIO, Dalian, China). RNA purity and concentration were determined by a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA was reverse transcribed using a Prime ScriptTM RT Reagent Kit (Takara Bio, Dalian, China) according to the manufacturer’s protocol. The reactions were performed in a Bio-Rad Real-Time PCR Detection System using SYBR® Green Master Mix (Vazyme Biotech, Nanjing, China). The primers used for the real-time PCR reaction were as follows: human CCN1: forward, 5′-CTGAGTGTATGCCATTCGG-3′, reverse, 5′-ACTGCTGTATCCCAATAAG-3′; mouse CCN1: forward, 5′-GTGCCGCCTGGTGAAAGAGA-3′, reverse, 5′-GCTGCATTTCTTGCCCTTTTT
TAG-3′; human β-actin: forward, 5′-GGACTTCGAGCAAGAGATGG-3′, reverse, 5′-AGGAAGGAAGGCTGGAAGAG-3′; mouse β-actin: forward, 5′-CTAAGGCC
AACCGTGAAAAG-3′, reverse, 5′-GGTACGACCAGAGGCATACA-3′. After a pre-denaturation step at 95 °C for 5 min, 40 cycles of PCR were performed as follows: 10 s denaturation at 95 °C and 30 s annealing at 60 °C. The fold amplification for each gene was calculated by using the comparative 2−ΔΔCt method.
TAG-3′; human β-actin: forward, 5′-GGACTTCGAGCAAGAGATGG-3′, reverse, 5′-AGGAAGGAAGGCTGGAAGAG-3′; mouse β-actin: forward, 5′-CTAAGGCC
AACCGTGAAAAG-3′, reverse, 5′-GGTACGACCAGAGGCATACA-3′. After a pre-denaturation step at 95 °C for 5 min, 40 cycles of PCR were performed as follows: 10 s denaturation at 95 °C and 30 s annealing at 60 °C. The fold amplification for each gene was calculated by using the comparative 2−ΔΔCt method.
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