Single cell molecular analyses

We then determined the molecular basis of observed mitochondrial CI defects at the single cell level. Serial cryo-sections (15μm) were placed on PEN membrane slides (Zeiss). CI deficient, intermediate, and normal myofibres were captured by laser microdissection into individual 0.2ml tubes containing 15μL single-cell lysis buffer (0.5M Tris-HCl, 0.5% Tween 20, 1% Proteinase K, pH 8.5) using a PALM system (Zeiss), and incubated at 55°C for 3 hours followed by heat inactivation for 10 minutes at 95°C.

Quantitative real-time PCR (qPCR) was performed on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) using a multiplex assay targeting the mtDNA-encoded genes MT-ND1 (forward primer L3485-3504; reverse primer H3532-3553; probe L3506-3529) and MT-ND4 (forward primer L12087-12109; reverse primer H12140-12170; probe L12111-12138), as described previously [21]. MT-ND1 is located in the minor arc of the mtDNA genome and MT-ND4 in the major arc. Targets in MT-ND1 and MT-ND4 are commonly used to screen for large-scale mtDNA deletions by qPCR as the majority of large-scale mtDNA deletions affect the major arc and thus remove MT-ND4 but retain MT-ND1. 2μL of DNA lysate from individual myofibres were amplified in triplicate. Mastermix comprised: iTaq (Bio-Rad); 75nM of each primer; 200nM of each probe. Amplification conditions were: 3 minutes at 95°C, then 39 cycles of 10 seconds at 95°C followed by 1 minute at 62°C. DNA extract from whole blood of a healthy individual was also included on each qPCR run as a control sample as this contains negligible levels of mtDNA deletions.

We screened for mtDNA deletions in individual myofibres by comparing threshold cycle (C_t) values of MT-ND1 to MT-ND4 relative to those C_t values for the control sample. That is, δδC_t = [C_t(MT-ND1)_sample - C_t(MT-ND4)_sample] – [C_t(MT-ND1)_control - C_t(MT-ND4)_control]. We screened for mtDNA depletion (reduction in overall cellular mtDNA content) in individual CI-deficient myofibres by considering the calculated starting quantity (SQ) of mtDNA relative to the 5^th centile of SQ in CI-normal myofibres from the same individuals.