Exogenous reference RNA for normalization of real-time quantitative PCR

We have utilized an in vitro transcribed 3′ mRNA fragment of the plant gene ribulose bisphosphate carboxylase (RuBisCO) as an exogenous standard for normalization of quantitative PCR data. Both K562 cells and primary erythroid CD34⁺ progenitor cells were treated with sodium butyrate and changes in γ-globin mRNA levels were assayed using a previously published TaqMan® probe and primer set, while RuBisCO levels were assayed by a SYBR® Green detection assay. The data presented show that a correction to measured γ-globin induction was necessary with both cell types. The correction for the CD34⁺ progenitor cells was a striking 95% increase, while that for the K562 cells was 44%. The use of an exogenous reference such as in vitro transcribed mRNA for the RuBisCO plant gene provides a robust and sample-independent method for the normalization of quantitative PCR data in bacterial and animal cells.