First stand complementary dna cdna synthesis kit

Total RNA from the cell samples was reverse-transcribed using a First-Stand complementary DNA (cDNA) synthesis kit according to the manufacturer instructions (Promega, Madison, WI, USA). For reverse transcription (RT), 3 µg of the total RNA was mixed with 1 µL of Oligo (dT)₁₅ (0.5 µg/reaction) and nuclease-free water and heated in a 70 °C heat block for five minutes. After pre-incubation, the reverse transcription reaction mix containing: 4 µL GoScript 5× reaction buffer (Promega, Madison, WI, USA), 3µL MgCl₂ (final concentration 1.5–5.0 mM), 1 µL dNTPs (10 mM), 1 µL Recombinant RNases Ribonuclease Inhibitor (20 U/µL), and 1 µL GoScript Reverse Transcriptase was prepared.

Total RNA from the tissue samples was reverse-transcribed using a First-Stand complementary DNA (cDNA) synthesis kit according to the manufacturer instructions (Promega, Madison, WI, USA). For the reverse transcription (RT), 3 μg of the total RNA was mixed with 1 μL of Oligo(dT)₁₈ primer (0.5 μg/µL) and nuclease-free water and heated in a 70 °C heat block for five minutes. After preincubation, the reverse transcription reaction mix containing 4 μL GoScript^TM 5× reaction buffer, 3 μL 25 mM MgCl₂, 1 μL deoxyribonucleotide triphosphates (dNTPs, 10 mM), 1 μL Recombinant RNases Ribonuclease Inhibitor (20 U/μL) and 1 μL GoScript^TM Reverse Transcriptase (160 U/μL), was added to the mixture with RNA in the total volume of 20 µL. The mixture was first incubated for 5 min at 25 °C, then for 60 min at 42 °C, and for the final 15 min at 70 °C. The solution of cDNA was stored at −20 °C.