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Simultaneous quantification of mtDNA copy number and transcripts by Selfie-dPCR

AuthorsPodlesniy, Petar ; Trullas, Ramón
Issue Date23-Mar-2017
CitationCOST Acction CA15214 Workshop (2017)
EuroCellNet Workshop (2017)
AbstractTo understand the mechanisms that regulate mitochondrial DNA (mtDNA) transcription and replication it is necessary to have a precise measurement of the number of transcripts per miDNA strand. Taking into account that mtDNA ¡is present in a number of copies that varies depending on cell type and conditions, it is expressed in a polycistronic transcript and both light and heavy chains express their own transcript in a differentially regulated way, this is a highly challenging task. The number of mtDNA copies is not constant and there is no transcript that can be used as a reference standard. The presence of just two mtDNA encoded transcripts that are regulated independently prevents their use as reference transcripts. Importantly also distinction between the expression of the transcripts of the light and heavy chain is usually not achieved in the standard PCR workflow. Here we present a novel method named Selfie-PCR that allows the precise simultaneous measurement of both mtDNA and mitDNA transcripts in the same sample. The number of transcripts per encoding mtDNA can be assessed in a locus specific and strand specific manner. Selfie-PCR permits the quantification of transcription initiation events in both strands and the assessment of miDNA transcription progress. As this method uses the mtDNA present in the sample as the own reference standard and does not rely on any external reference it can be used in cells and tissues from different origins with different mtDNA abundance or metabolic state. The Selfie-PCR method is applicable in either quantitative or digital PCR approaches, although the latter has the advantage that it allows the absolute measurement of miDNA targets. The Selfie-PCR method permits for the first time the monitoring of miDNA transcription and replication in the same sample and can be used in a wide range of biological systems.
DescriptionTrabajo presentado en el COST Acction CA15214 Workshop: An integrative action for multidisciplinary studies on cellular structural networks / EuroCellNet Workshop, celebrado en Praga los días 23 y 24 de marzo de 2017
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