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. 2021 Nov 16;2(4):100952. doi: 10.1016/j.xpro.2021.100952

Figure 7.

Figure 7

Quantification of mtDNA enrichment after CS-MAP

Two different types of quantification (semi-quantitative and quantitative PCR (qPCR)) were performed on the same samples for comparison.

(A) Semi-quantitative PCR showing the enrichment of mitochondrial DNA (mtDNA) over nuclear DNA (nDNA) after CS-MAP. To evaluate mtDNA enrichment, the primers AA113 and CD15 (Ahier et al., 2018) were used with Phusion DNA Polymerase (Biolabs, M0530) under the following cycle conditions: 98°C for 30 s, followed by 34 cycles of 98°C for 10 s, 65°C for 15 s, and 72°C for 1 min. To evaluate nDNA levels, the primers ges-1F1 and ges-1R1 (Ahier et al., 2018) were used with the aforementioned polymerase under the following cycle conditions: 98°C for 30 s, followed by 34 cycles of 98°C for 10 s, 55°C for 15 s, and 72°C for 1 min. 1 μL of the input diluted at 10 ng·μL-1 and 1 μL of the CS-MAP sample were used per reaction. nDNA has occasionally been detected in the CS-MAP sample. GeneRuler DNA Ladder Mix (SM0331, Thermo Scientific) was used for this gel.

(B and C) The samples used in A) were analyzed by qPCR using a Rotor-Gene Q real-time PCR (QIAGEN) machine. To quantify mtDNA levels, the primers AA178 and AA179 were used, while the primers SZ35 and SZ36 (Ahier et al., 2021) were used to quantify nDNA. qPCR was performed using the following cycle conditions: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 58°C for 40 s using the Sensifast SYBR No-ROX Kit (BIO-98005, Bioline). 1 μL of the input diluted at 10 ng·μL-1 and 1 μL of the undiluted CS-MAP sample were used per technical triplicate. The efficiency of enrichment can vary due to experimental variability and/or cell-type used. We usually observe a 20- to 50-fold enrichment with some rare cases where nuclear DNA was not detected within 40 qPCR cycles. (B) Table showing the obtained Ct values and the calculation steps of the applied comparative Ct method. ΔCt corresponds to nDNA average Ct value - mtDNA average Ct value and relative enrichment (RE) corresponds to 2ΔCt. (C) Quantification of mtDNA enrichment relative to nDNA. An 18.4-fold enrichment was observed in this experiment. IP: ImmunoPurified mitochondria; Input: Corresponding input 1.