Total RNA was isolated using an RNeasy Kit (QIAGEN) and quantified using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Wilmington, DE). cDNA was synthesized using a PrimeScript RT Reagent Kit (Roche) using 2.5 μg of total cellular RNA. The level of mRNA was determined by real-time PCR analysis using a Bio-Rad CFX96 instrument with a QuantiTect SYBR-Green PCR Master Mix (TaKaRa, Dalian, China) and the appropriate primers (Table 1 ). Complementary primers were added and the PCR reaction was performed for 40 cycles of 95°C for 1 min, 56°C for 30 s, and 72°C for 1 min. Relative changes in gene expression levels were determined using the 2−△△Ct method [17 (link)]. The cycle number at which the transcripts were detectable (Ct) was normalized to the cycle number at which the GAPDH gene was detected, referred to as ΔCt.
Cfx96 instrument
Manufactured by Takara Bio
Sourced in Japan
The CFX96 instrument is a real-time PCR detection system designed for gene expression analysis, genotyping, and basic research applications. It features 96-well sample capacity and uses fluorescence detection technology to monitor and quantify target DNA sequences during the amplification process.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy kit, by Qiagen (1 mentions)
Primescript rt reagent kit, by Roche (1 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Quantitect sybr green pcr master mix, by Takara Bio (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Sybr green real time pcr kit, by Takara Bio (1 mentions)
Primescript rt regent kit, by Takara Bio (1 mentions)
Sybr premix ex taq kit, by Takara Bio (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Primescript rt master mix, by Takara Bio (1 mentions)
3 protocols using cfx96 instrument
1
Quantitative Real-Time PCR Analysis
2
Hepatic Gene Expression Analysis by RT-qPCR
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The hepatic total RNA was extracted on ice with Trizol reagent (Takara, Japan) and then was reverse-transcribed into first-strand cDNA with the PrimeScript™ RT Regent kit (Takara). Real-Time Quantitative PCR (RT-qPCR) reactions were conducted in a BioRad CFX96 instrument using the SYBR Green Real-time PCR kit (Takara). The specific primes are listed in Table 1 and the procedure of PCR amplification was set as follows: 95°C for 2min, 40cycles of 95°C for 10s, 57°C for 10s, and 72°C for 20s, followed by a melting curve analysis. The gene β-actin was used as the house gene after its stability being verified, and relative expression of target genes involved in lipid metabolism and antioxidant response was calculated with the 2-ΔΔCT method (Livak and Schmittgen, 2001 (link)).
3
Quantitative Analysis of Gene Expression in HCC
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Total RNA was extracted from HCC tissues and cells using TRIzol Reagent (Invitrogen, USA) according to the manufacturer's instructions. In brief, 1 µg of total RNA was reverse transcribed to cDNA with PrimeScript RT Master Mix (TaKaRa, Japan). Then, qRT-PCR analysis was performed in triplicate on a Bio-Rad CFX96 instrument with a SYBR Premix Ex Taq Kit (TaKaRa, Japan). β-actin was used as the internal control, and the relative expression of each target gene was calculated by the 2 -ΔΔCt method. The primers used for PCR are listed in Supplemental Table 2.
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