Cfx96 c1000 qpcr machine

Quantitative PCR reactions using TaqMan chemistry (PrimeTime Std qPCR Assay, IDT) were performed on a Bio-Rad CFX96/C1000 qPCR machine. We used the comparative ΔΔCt method to determine the relative quantity of mtDNA (33 (link)). The levels of different mtDNA species between nontransduced and transduced samples were determined by quantifying the levels of total mtDNA/genomic DNA (Nd1/Actb) or (D-loop/Actb). 5 to 10 ng/sample of total DNA, 2x IDT PrimeTime Gene Expression Master Mix (IDT# 1055770), the primers (final conc. 250 nM), and probes (final conc. 125 nM) below were mixed to a volume of 10 μl using the following cycling conditions (95 °C 3 min; [95 °C 5 s; 60 °C 30 s; 40 cycles]) and default imaging parameters. Primer/Probe Pairs.

qPCR was performed using PrimeTime qPCR Assay (Integrated DNA Technologies) and the primers/probes indicated in Supplemental Table 9. PCR was performed on a CFX96/C1000 qPCR machine (Bio-Rad). Cycling conditions were as follows: one cycle of 95 ºC for 3 min, 39 cycles of 95 ºC for 15 s, 60 ºC for 1 min. Comparative Ct method was used to determine relative reads. Total mtDNA levels were determined by comparing mtDNA MT-ND1 or MT-COX1 to nDNA ACTIN.

Quantitative PCRs using SYBR/ROX chemistry (172–5,264; Bio‐Rad, SsoAdvanced SYBR Green) on a Bio‐Rad CFX96/C1000 qPCR machine were performed, and the ΔΔC_T values were calculated using manufacturers’ software (Bacman et al, 2013). We determined the levels of mtDNA with the following primers: ND1 (F‐CGA TTC CGC TAC GAC CAAC T; R‐AGG TTT GAG GGG GAA TGC TG) and the nuclear DNA‐encoded β‐actin (ACTB) primers were used (F‐GCG CAA GTA CTC TGT GTG GA; R‐GCG CAA GTA CTC TGT GTG GA, located in exon 6) to normalize the results (Bacman et al, 2013). Alternatively, and to confirm the SYBR results, the qPCR was also performed with PrimeTime qPCR probes (IDT, Integrated DNA Technologies) according to the manufacturers’ protocol (IDT) for both mtDNA (COXI) and nuclear DNA (ACTB). The probe sequence for ACTB is proprietary (ref. Hs.PT.56a.41086380.g from IDT), but the primers sequence used were F‐ GCC ATG TAC GTT GCT ATC CA and R‐ GTC ACC GGA GTC CAT CAC). For COXI, the primers/probe sequences were as follows: F‐TTC TGA CTC TTA CCT CCC TCT C; R‐TGG GAG TAG TTC CCT GCT AA; probe:/56‐FAM/TC CTA CTC C/ZEN/T GCT CGC ATC TGC TA/3IABkFQ/. The results were expressed as percentage of the untransfected cells (%UNT).

To determine the total levels of mtDNA present in samples, we performed qPCR using TaqMan reagents (PrimeTime Std qPCR Assay, Integrated DNA Technologies)^{20 (link)}. Samples were run on a BioRad CFX96/C1000 qPCR machine. Comparative cycle threshold (Ct) method was used to determine relative reads and total mtDNA levels were determined by comparing mtDNA (ND1 and ND5) to genomic DNA (18S). The following primer/probe sets were used^{20 (link)}: