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

详细信息    查看全文

• 作者：Wen Guo ; Lan Jiang ; Shalender Bhasin ; Shaharyar M. Khan ; Russell H. Swerdlow
• 关键词：Mitochondrial DNA ; Phenol extraction ; Real-times PCR ; DNA isolation
• 刊名：Mitochondrion
• 出版年：2009
• 出版时间：July 2009
• 年：2009
• 卷：9
• 期：4
• 页码：261-265
• 全文大小：314 K

文摘

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.