To estimate the expression level of each gene by RT-PCR, total RNA was extracted from cells lysed with TRI reagent (Molecular Research Center), and equal amounts of total RNA from each sample were converted to cDNA by reverse transcription using random nine-mer primer with SuperScript III Reverse Transcriptase (RT) (Life Technologies) as previously described [12, 13] . Each cDNA was added to a PCR mixture for ampli cation (Taq PCR Kit, Takara). The PCR primers used in this study were as follows: CREBRF sense primer, 5'-AAGTCAAGATCAACCCTGTG-3', CREBRF antisense primer, 5'-TTGGTTGGCTGTTCTCTCAT - 3'; G3PDH sense primer, 5'-ACCACAGTCCATGCCATCAC-3', G3PDH antisense primer, 5'-TCCACCACCCTGTTGCTGTA-3'. The typical reaction cycling conditions were as follows: 30 sec at 96°C, 30 sec at 58°C and 30 sec at 72°C. The results represent 21 or 28 cycles of ampli cation. The products were separated by electrophoresis on 2.0% agarose gels and visualized using ethidium bromide. The expression level of each mRNA was analyzed using the ImageJ software (National Institutes of Health), and the relative amount of CREBRF mRNA was calculated based on the G3PDH value obtained from the identical cDNA. The CREBRF mRNA expression of each cDNA was normalized to the values obtained from the untreated control cells [13] .
Taq pcr kit
Manufactured by Takara Bio
The Taq PCR Kit is a reagent kit designed for performing polymerase chain reaction (PCR) experiments. It contains a thermostable DNA polymerase enzyme, Taq, along with necessary buffers and reagents required for DNA amplification.
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2 protocols using taq pcr kit
1
Quantifying CREBRF Gene Expression by RT-PCR
2
Quantifying CREBRF Gene Expression by RT-PCR
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To estimate the expression level of each gene by RT-PCR, total RNA was extracted from cells lysed with TRI reagent (Molecular Research Center), and equal amounts of total RNA from each sample were converted to cDNA by reverse transcription using random nine-mer primer with SuperScript III Reverse Transcriptase (RT) (Life Technologies) as previously described [12, 13] . Each cDNA was added to a PCR mixture for ampli cation (Taq PCR Kit, Takara). The PCR primers used in this study were as follows: CREBRF sense primer, 5'-AAGTCAAGATCAACCCTGTG-3', CREBRF antisense primer, 5'-TTGGTTGGCTGTTCTCTCAT - 3'; G3PDH sense primer, 5'-ACCACAGTCCATGCCATCAC-3', G3PDH antisense primer, 5'-TCCACCACCCTGTTGCTGTA-3'. The typical reaction cycling conditions were as follows: 30 sec at 96°C, 30 sec at 58°C and 30 sec at 72°C. The results represent 21 or 28 cycles of ampli cation. The products were separated by electrophoresis on 2.0% agarose gels and visualized using ethidium bromide. The expression level of each mRNA was analyzed using the ImageJ software (National Institutes of Health), and the relative amount of CREBRF mRNA was calculated based on the G3PDH value obtained from the identical cDNA. The CREBRF mRNA expression of each cDNA was normalized to the values obtained from the untreated control cells [13] .
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