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

Abstract
To understand the mechanisms that regulate DNA gene tran- scription and replication it is necessary to have a precise measurement of the number of transcripts per gene. However, in the case of mitochondrial DNA (mtDNA), precise measurement of mtDNA transcription requires to take 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 dif- ferentially regulated way. The presence of two mtDNA encoded transcripts that are regulated independently prevents their use as reference transcripts. In addition, distinction between the expres- sion 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 simulta- neous measurement of both genomic and mtDNA transcripts in the same sample. The number of transcripts per encoding gene 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 gene transcription progress. As this method uses genes present in the sample as the own reference stan- dards and does not rely on any external reference it can be used in cells and tissues from different origins with different gene copy number or metabolic state.
Acknowledgement: Supported by SAF2014-56644-R and CIBERNED PI2016/06-3 grants.
http://dx.doi.org/10.1016/j.bdq.2017.02.042