Clontech human mitochondrial dna mtdna monitoring primer set

ARPE-19 cells were seeded in 6-well plates to confluence and serum starved for 2 days before adding TGFβ2 for up to 3 days. Genomic DNA (gDNA) was extracted using the NucleoSpin DNA RapidLyse kit (Macherey Nagel, Düren, Germany) and DNA concentration and purity was measured using the NanoDrop Spectrophotometer ND-1000 (ThermoFisher Scientific). Purity of the extracted DNA was based on the OD 260/280 nm absorbance ratio and were between 1.8 and 2.2. Each reaction was run in duplicate and consisted of 10 ng of gDNA, 5 μL of PowerUp SYBR Green Master Mix (ThermoFisher Scientific), 1 μL of the primer mix, all diluted in water to a final volume of 10 μL. The two primers specific for determination of nuclear DNA were SLCO2B1 and SERPINA1 and the two primers for detection of mtDNA were ND1 -NADH dehydrogenase subunit 1- and ND5, all provided in the Clontech Human Mitochondrial DNA (mtDNA) Monitoring Primer Set (Takara Bio Inc, Kusatsu, Shiga, Japan). Reactions were run on a 384-well plate using a LightCycler 480 (Roche). qPCR was performed as per the thermal cycles mentioned above. Based on the Ct values obtained for each of the four target genes, the copy number of mtDNA was defined as the average of the ratio of mtDNA to nuclear DNA using the two pairs of genes mt-ND5 with SERPINA1 and ND1 with SLCO2B1.

Genomic DNA (gDNA) was isolated using the Universal Genomic DNA Kit (CWBio #CW2298S). DNA concentration and purity was measured using the NanoDrop Spectrophotometer ND-1000. 10 ng of gDNA was used for the qPCR reactions and performed as previously described (Shu et al., 2021 (link)) using the Clontech Human Mitochondrial DNA (mtDNA) Monitoring Primer Set (Takara Bio Inc., Kusatsu, Shiga, Japan). mtDNA copy number was calculated as the average of the ratio of mtDNA to nuclear DNA using the two pairs of genes mt-ND5 with SERPINA1 and ND1 with SLCO2B1.