DNA extraction procedures meaningfully influence qPCR-based mtDNA copy number determination

Abstract

Quantitative real time PCR (qPCR) is commonly used to determine cell mitochondrial DNA (mtDNA) copy number. This technique involves obtaining the ratio of an unknown variable (number of copies of an mtDNA gene) to a known parameter (number of copies of a nuclear DNA gene) within a genomic DNA sample. We considered the possibility that mtDNA:nuclear DNA (nDNA) ratio determinations could vary depending on the method of genomic DNA extraction used, and that these differences could substantively impact mtDNA copy number determination via qPCR. To test this we measured mtDNA:nDNA ratios in genomic DNA samples prepared using organic solvent (phenol-chloroform-isoamyl alcohol) extraction and two different silica-based column methods, and found mtDNA:nDNA ratio estimates were not uniform. We further evaluated whether different genomic DNA preparation methods could influence outcomes of experiments that use mtDNA:nDNA ratios as endpoints, and found the method of genomic DNA extraction can indeed alter experimental outcomes. We conclude genomic DNA sample preparation can meaningfully influence mtDNA copy number determination by qPCR.

Figure 1

mtDNA: nDNA ratios and reproducibility vary with different extraction techniques. (A) Ratios obtained when KIT-1, KIT-2, and PCIAA were used to prepare genomic DNA from frozen liver on two different days, with n=4 samples per day for each method. KIT-1 showed significant day-to-day variation, and the PCIAA technique had the least amount of intra-sample and day-to-day variation. (B) When all 8 samples for each method are used to obtain a mean mtDNA: nDNA ratio, the ratios are not uniformly comparable.

Figure 2

DNA loss as a function of the purification kit used and the type of DNA purified. (A) DNA is progressively lost with serial passage through KIT-1 columns. (B) mtDNA is preferentially lost when genomic DNA is serially passed through the KIT-1 column. (C) Direct assessment of mtDNA and nDNA recovery by the three methods.

Figure 3

mtDNA: nDNA ratios from myostatin knock-out and wild type mice. (A) KIT-1 produced lower ratios than KIT-2 and PCIAA extraction. PCIAA extraction produced the least amount of intra-sample variation. (B) PCIAA extraction but not column extractions found the mtDNA: nDNA ratio was increased in myostatin knock-out mice as compared to wild type mice. wt = wild type; ko = myostatin knock-out mice.

Figure 4

Genomic DNA extraction methods influence mtDNA: nDNA ratios when 3T3-L1 cells are differentiated. pre-diff=pre-differentiation procedure; post-dif=post-differentiation procedure.