mtDNA quantification was performed using a quantitative PCR (qPCR)-based method similar to previously described assays41 (link). Twenty to 30 worms were collected in 30 µl of lysis buffer (50 mM KCl, 10 mM Tris-HCl pH 8.3, 2.5 mM MgCl2, 0.45% NP-40, 0.45% Tween 20, 0.01% gelatin, with freshly added 200 µg/ml proteinase K) and frozen at −80 °C for 20 min prior to lysis at 65 °C for 80 min. Relative quantification was used for determining the fold changes in mtDNA between samples. 1 µl of lysate was used in each triplicate qPCR reaction. qPCR was performed using the Thermo Scientific SyBr Green Maxima Mix and the MyiQ2 Two-Color Real-Time PCR Detection System (Bio-Rad Laboratories). Primers that specifically amplify mtDNA are listed in Supplementary Data file 6 . Primers that amplify a non-coding region near the nuclear-encoded ges-1 gene were used as a control. mtDNA was harvested from synchronized worms at the L4 stage. All qPCR results have been repeated at least three times and performed in triplicates. A Student’s t-test was employed to determine the level of statistical significance.
Sybr green maxima mix
Manufactured by Bio-Rad
SyBr Green Maxima Mix is a ready-to-use qPCR master mix that contains SYBR Green I dye, optimized buffer, and a DNA polymerase for real-time quantitative PCR analysis. It is designed for sensitive and reproducible quantification of DNA targets.
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3 protocols using sybr green maxima mix
1
Quantification of Mitochondrial DNA in C. elegans
2
Quantifying Mitochondrial Gene Expression
3
Quantifying Mitochondrial Gene Expression
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Worms were synchronized and raised in liquid culture until the L4 stage when they were harvested and compacted into pellets on ice. Total RNA was extracted from a 30–50 µl worm pellet using RNA STAT (Tel-Test). For the analysis of mtDNA-encoded mRNAs, the RNA extracts were then treated with DNAse using the DNA-free kit (Ambion) to reduce mtDNA contamination. 1 µg of RNA was used for synthesizing cDNA with the iScript cDNA Synthesis Kit (Bio-Rad Laboratories). qPCR was performed using the Thermo-Scientific SyBr Green Maxima Mix and the MyiQ2 Two-Color Real-Time PCR Detection System (Bio-Rad Laboratories). Primer sequences are listed in Supplementary Table 2 . All qPCR results are presented as technical replicates, but each experiment has been repeated 3 or more times. A Student’s t-test was employed to determine the level of statistical significance.
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