The total RNA was extracted with Trizol solution according to the instructions for use (Promega, Beijing, China), and the cDNA was synthesized using the first strand cDNA synthesis kit. qRT-PCR was performed using SYBR Green qPCR SuperMix. The reaction conditions were as follows: 50°C for 2 min, 95°C for 2 min, 95°C for 15 s, 60°C for 32 s, 40 cycles. The melting curves were analyzed at 60–95°C. Each sample was detected for three times by qRT-PCR (Biorad IQ5). The relative mRNA expression was normalized to the corresponding β-actin expression and analyzed by 2−△△Ct method. The nucleotide sequences of primer pairs used to quantify gene expression were as follows:PUMA-F:5′-GGGTGTCTGCGTGAGTCC-3′, PUMA-R:5′-CCAGGCAAGCG ACAGA TAC A-3′, β-actin-F:5′-CTCTGTGTGGATCGGTGGCT-3′, β-actin-R:5′-TGTAAAACGCAGCTCAGTAA CAG-3'.
Trizol solution
Manufactured by Promega
Sourced in United States
Trizol solution is a monophasic solution of phenol, guanidine isothiocyanate, and other components used for the isolation of total RNA from various biological samples, including cells, tissues, and microorganisms. It facilitates the separation of RNA from DNA, proteins, and other cellular components during the RNA extraction process.
Automatically generated - may contain errors
2 protocols using trizol solution
1
Quantitative RT-PCR Analysis of PUMA Expression
2
RT-qPCR Gene Expression Analysis
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We extracted total RNA according to the instruction of TRIzol solution (Promega, Madison, Wisconsin, WI, USA). Then, we utilized a PrimeScript RT reagent Kit (Takara, Dalian, China) to reverse-transcribed RNA to cDNA. Using specific primers, the gene expressions were qualified by RT-qPCR based on a commercial kit, SYBR® Premix DimerEraser Kit (Takara, Dalian, China), β-actin as control. PCR program was conducted on a Real-Time PCR System (ABI7500, Applied Biosystems, Waltham, MA, USA) and the detailed process went as follows: 1 min at 95 °C, 20 s at 95 °C and 10 s at 56 °C and 15 s at 72 °C for 35 cycles, then held at 4 °C. Finally, the calculation of gene expression was achieved by using the 2−ΔΔCt method.
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