2.8. Reverse Transcription Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR)

The FRDA fibroblasts were plated at a density of 1 × 10⁵ cells per well in a 6-well plate and incubated for 24 h at 37 ± 2 °C in a 5% CO₂-humidified incubator. The medium was replaced with a fresh medium containing adenosine ranging from 0 to 600 µM or 5 µM idebenone and incubated for 2 h, followed by 12.50 mM BSO [38] for 24 h. Fibroblasts in DMEM without any treatment or pre-treated with 5 μM idebenone [36] served as the negative and positive controls, respectively. Fibroblasts were subjected to total RNA extraction using TRIzol^® reagent according to the manufacturer’s protocol. The concentration of total RNA was determined using the NanoDrop™ 2000/2000c Spectrophotometers (Thermo Fisher Scientific, Waltham, MA, USA). Approximately 300 ng of total RNA was converted to cDNA using RevertAid First Strand cDNA Synthesis Kit according to the manufacturer’s protocol and Veriti^® 96-Well Thermal Cycler (Applied Biosystems Inc., Foster City, CA, USA). The RT-qPCR was performed using 15 ng/µL cDNA template in a pooled solution containing qPCRBIO SyGreen Mix Separate-ROX, 10 pmol/µL forward and reverse oligonucleotide primers, and OmniPur^® water. The primer sequences used in this study were synthesized by Genewiz (South Plainfield, NJ, USA), as shown in Table 1. Amplification was performed for 40 cycles in the StepOne Plus™ Real-Time PCR System (Applied Biosystems Inc., Foster City, CA, USA) [39,40,41]. The expression of the gene of interest was normalized to the reference gene actin beta (ACTB). Fold change of gene expression was determined using the comparative threshold cycle method (ΔΔCt) [42,43].

RT-qPCR primers.

ACTB, actin beta; NFE2L2, NFE2-like bZIP transcription factor 2; NRF1, nuclear respiratory factor 1; PPARGC1A PPARG coactivator 1 alpha; TFAM, transcription factor A, mitochondrial.