Peripheral venous blood samples were collected in ethylenediaminetetraacetic acid-containing vacutainer tubes. Plasma was recovered following centrifugation at 3000 revolutions per minute for 15 minutes, and stored in small aliquots at −20°C pending analysis. Genomic DNA was extracted by the salting-out method.29 AGT genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Briefly, PCR was performed in a final-volume 15 µl containing genomic DNA (50 ng), 0.2 μM of forward and reverse primers, 0.2 mM deoxynucleotide mix (Invitrogen, Carlsbad, CA, USA) and 1 U GoTaqDNA polymerase (Promega, Madison, WI, USA). For M235T, the following primers were used: forward, 5’-CAgggTgCTgTC CAC ACT ggA CCC C-3’, and reverse, 5’- CCg TTT gTgCAgggCCTggCT CTC T-3’. For T174M, the following primers were used: forward, 5’-gATgCg CAC AAggTC CTg-3’, and reverse, 5’, CAg ggTgCTgTC CAC ACT ggCTCg C-3’. The 165 base pair (bp) M235T and 303 bp T174M amplicons were digested with Thermus thermophilus strain 111 Tth111I at 65°C or Nocardia corallina (NcoI) at 37°C, respectively (New England Biolabs, Ipswich, MA, USA). Digested fragments were separated by electrophoresis on 3% agarose gel, and yielded 141 bp + 24 bp for M235T, and 211 bp + 92 bp for T174M, respectively.
Deoxynucleotide mix
Manufactured by Thermo Fisher Scientific
Sourced in United States
Deoxynucleotide mix is a solution containing the four essential deoxynucleotides (dATP, dCTP, dGTP, and dTTP) required for DNA synthesis and amplification. The mix is commonly used in various molecular biology applications, such as polymerase chain reaction (PCR), DNA sequencing, and other DNA-based experiments.
Automatically generated - may contain errors
2 protocols using deoxynucleotide mix
1
Genotyping of AGT gene variants
2
Reverse Transcription for cDNA Synthesis
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cDNA was synthesized using Transcriptor First Strand cDNA Synthesis Kit (Roche, Germany) by reverse transcription according to the manufacturer’s instructions. Reverse transcription was carried out in 20 μl solution that contained 0.5 μl 20 U/μl Transcriptor reverse transcriptase, 4μl Transcriptor RT Reaction Buffer (5x concentrated), 2 μl Deoxynucleotide Mix, 0.5 μl 40 U/μl protector RNase inhibitor, 2 μl 10 mmol/ml stem-loop RT primers (Invitrogen), 7 μl DEPC water (Invitrogen, USA), and 4 μl total RNA template. After being mixed gently, the reaction mixtures were incubated at 25°C for 10 min, 55°C for 30 min and then 85°C for 5 min. The final cDNA products were stored at −20°C until use. U6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as the reference genes. The sequence of all primers used in the present study is provided in Table 2
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