Improved normalization of real-time reverse transcriptase polymerase chain reaction data using an external RNA control.

No golden standard exists for normalization of real-time reverse transcriptase polymerase chain reaction (RT PCR) data and procedures used are often not validated. Numerous studies have indicated that current approaches are inadequate. Here, we report the development of an external RNA control approach. It is the first to add external RNA to tissue on a per unit weight basis, and we demonstrate its accuracy, suitability, and necessity in experiments involving severe physiological challenges. We utilized the approach to examine the expression of the internal RNA control genes (reference genes) beta-actin, cyclophilin A, and glyceraldehyde 3-phospate dehydrogenase in brain and heart of normoxic and anoxic crucian carp (Carassius carassius). The internal RNA control genes differed significantly in expression in experimental groups, especially in heart. We also demonstrate that the external RNA control approach provides a more accurate normalization of target genes. For example, it revealed a 2.5-fold increase in the expression of the stress-response gene HSC70, which was not detected using beta-actin or geNorm. Further, we demonstrate and discuss the need for using the optimized and standardized external RNA control protocol reported. Collectively, our data suggest that the normalization of real-time RT PCR data is considerably improved by adding an external RNA control to the samples.