ANALYSIS OF HOUSEKEEPING GENE STABILIZATION IN ISOLATED MITOCHONDRIA

Objective: The mitochondria isolation method is used to investigate mitochondrial processes. RT-qPCR measurement of gene transcription levels is frequently used in these studies. In this method, it is important that the housekeeping genes used in the normalisation of the results are stable. The study aimed to determine which of two housekeeping genes is more stable in isolated mitochondria. Material and Method: Mitochondria were isolated from FHC cells. Membrane integrity and functionality were measured by mitotracker and JC-1 stainings and ROS ratio by flow cytometry. Housekeeping genes GAPDH and β-actin were used in RT-qPCR, and gene stability was calculated using ΔCq method and percentage coefficient of variance. For gene validation, CAT and SOD1 transcriptions were calculated by the 2-ΔΔCq method. These genes were selected because oxidative stress status has been detected. Result and Discussion: Isolated mitochondria membrane integrity was preserved and the membrane potential ratio was found 91%. ROS ratio was 1.6% and there was no oxidative stress that would affect the antioxidant enzymes levels. ΔCq values were 4.54±0.06 for β-actin and 0.69±0.16 for GAPDH, with 1.33% and 23.95% percentage coefficient of variance respectively. CAT transcription levels were 25.7±2.6 and 1.8±0.33, while SOD1 levels were 70.1±12.7 and 4.8±0.6 correspondingly. It was concluded that β-actin may be more stable than GAPDH. The potential impact of housekeeping gene selection on outcomes should be considered.