Reverse transcription reaction

NF-κB, caspase-3, KIM-1, and TGF-β expression was studied using RT-PCR. For this assay, drug-treated HK-2 was used to isolate total RNA using an RNase commercial kit obtained from Qiagen and processed as per the manufacturer’s instruction. NanoDrop 1000™ obtained from Thermo Scientific was used for the determination of quality as well as quantity of RNA. The obtained 1 μg of RNA was used for the cDNA preparation using reverse transcription reaction obtained from Bio-Rad. The sequence of a primer used in this RT-PCR is shown in Table 1. The efficacy of the primer was evaluated by the dilution and the gradient temperature technique (50–55). The PCR process was conducted using 20 μl of the prepared sample having one μl cDNA, 500 ng/μl final concentration of primer, and EvaGreen supermix qPCR obtained from Biorad. The 2^-ΔΔCt method was used for the estimation of fold change, and the experiment was conducted in triplicate (Heeba & Mahmoud, 2016 (link)).

First-strand synthesis was generated by mixing 5 µL of RNA with 500 nM of reverse primer in a 20 µL reverse transcription reaction (Bio-Rad, 1708897) containing 4 µL of reaction supermix, 2 µL of GSP enhancer solution, and 1 µL of reverse transcriptase. The mixture was incubated at 42 °C for 30 or 60 min, followed by a deactivation of the reverse transcriptase at 85 °C for 5 min. To generate ample product for gel electrophoresis analyses, the resultant cDNA was diluted 100-fold, and 1 µL was used as the template in a PCR amplification containing 0.5 µL of Q5 High-Fidelity DNA Polymerase (NEB, M0491S), 1x Q5 polymerase reaction buffer (NEB, B9072S), 0.5 uM of forward and reverse primer, 2.5 mM each of dATP (NEB, N0440S), dCTP (NEB, N0441S), dGTP (NEB, N0442S), dTTP (NEB, N0443S) in a 50 µL total reaction volume. The amplification conditions were 98 °C for 30 s and then 25 cycles of: 98 °C for 10 s, 55 °C for 20 s, 72 °C for 10 s with a final 72 °C extension step for 2 min. The products were assayed by gel electrophoresis and their concentrations were measured by Fragment Analyzer HS NGS Fragment Kit (Agilent Technologies Inc., DNF-474-0500).