Transcription level of messenger RNA per gene copy determined with dual-spike-in strategy.

To quantify the transcription level of a gene, we have conceived a novel concept, transcription level of messenger RNA (mRNA) per gene copy, which was determined with a dual-spike-in strategy. In this strategy, an exogenous DNA was added as the spike reference for target DNA in addition to the exogenous RNA as the reference for target RNA. After the mRNA-to-DNA ratio of a target gene was estimated by real-time polymerase chain reaction (PCR), it was first normalized with the mRNA-to-DNA ratio of the exogenous reference. The normalized ratio was multiplied by the ratio of exogenous RNA to exogenous DNA to obtain the transcription level of mRNA per gene copy. This quantified transcription value allows one to compare the expression of a target gene in different tissues or the expression in a specified tissue under different conditions.