Proflex 2 flat block thermal cycler

Digital PCR and quantification of the absolute levels of serum miRNAs were performed using the Quant-Studio 3D Digital PCR System (Thermo Fisher Scientific). Data were analyzed using the QuantStudio 3D Analysis Suite Cloud Software (Thermo Fisher Scientific). The digital PCR mixture contained 5.0 μL of the RT product, 1.0 μL nuclease-free H₂O, 7.50 μL of the QuantStudio™ 3D Digital PCR Master Mix, and 0.75 μL of the TaqMan MiRNA Assay-1 (20X) for let-7d [18 (link)]. Samples were individually loaded onto the QuantStudio 3D digital PCR 20K chip kit v2 using the QuantStudio 3D digital PCR Chip Loader. Digital PCR was performed in a Proflex 2× flat block thermal cycler (Applied Biosystems) using standard conditions: 96°C for 10 min followed by 39 cycles of 60°C for 2 min, 98°C for 30 sec, and 60°C for 2 min. Chips were read on the QuantStudio 3D Digital PCR instrument and the number of FAM-positive and FAM-negative (empty) wells quantified [20 (link)].

Total DNA from control and KSS fibroblast cell lines were used as templates for digital PCR (dPCR) analysis using multiplex ND4 and ND1 primer probes on the QuantStudio 3D Digital PCR System and with supplied reagents (Applied Biosystems, Waltham, MA). In titration experiments, relative mtDNA inputs were normalized by the ND1 assay in qPCR as determined above. The dPCR was prepared according to the manufacturer’s protocol and loaded onto the individual QuantStudio v1 dPCR chips using the QuantStudio 3D Digital PCR Chip Loader. Chip PCR amplification was performed in a Proflex 2× flat block thermal cycler (Applied Biosystems, Waltham, MA) using standard conditions: 96 °C for 10 min; 39 cycles of 60 °C for 2 min and 98 °C for 30 sec; and 60 °C for 2 min. Chips were then read on the QuantStudio 3D instrument to obtain the number of wells positive for the VIC/HEX and FAM channels, the number of wells without DNA (ROX positive, VIC and FAM negative) and empty wells (ROX negative). Data analysis and chip quality were assessed using the QuantStudio 3d Analysis Suite. All chips were between 25–75% empty well ensuring suitability for quantitation. The number of ND4⁺ wells was divided by the total ND1⁺ wells to calculate the fraction of normal (WT) mtDNA content. The ND4⁻ content is one minus that value. All dilutions were performed in at least triplicate to determine error.