Absolute measurement of gene transcripts with Selfie-digital PCR

Abstract

Absolute measurement of the number of RNA transcripts per gene is necessary to compare gene transcription among different tissues or experimental conditions and to assess transcription of genes that have a variable copy number per cell such as mitochondrial DNA. Here, we present a method called Selfie-digital PCR that measures the absolute amount of an RNA transcript produced by its own coding DNA at a particular moment. Overcoming the limitations of previous approaches, Selfie-digital PCR allows for the quantification of nuclear and mitochondrial gene transcription in a strand-specific manner that is comparable among tissues and cell types that differ in gene copy number or metabolic state. Using Selfie-digital PCR, we found that, with the exception of the liver, different organs exhibit marked variations in mitochondrial DNA copy number but similar transcription of mitochondrial DNA heavy and light chains, thus suggesting a preferential role of mitochondrial DNA abundance over its transcription in organ function. Moreover, the strand-specific analysis of mitochondrial transcription afforded by Selfie-digital PCR showed that transcription of the heavy strand was significantly higher than that of the light strand in all the tissues studied.