Quantitative polymerase chain reaction using an external control mRNA for determination of gene expression in a heterogeneous cell population

Gene expression can be evaluated quantitatively by conventional RT-PCR or N orthern blotting with the aid of a correction based on the expression of an internal control gene. However, this approach is not suitable for quantita ting gene expression in a group of heterogeneous cell subsets, because the internal control gene expression may vary among the subsets, Therefore, we developed a new method for quantitative PCR using rat poly(A)(+) RNA as an external control, We used this method to investigate cytokine gene expressi on in lymph node cells from mice during the induction of contact hypersensi tivity. Expression of the murine glyceraldehydephosphate dehydrogenase (GAP DH) gene, a candidate internal control, was not constant in cells from trin itrochlorobenzene- and vehicle-applied animals, suggesting that GAPDH gene expression changes in heterogeneous lymph node-cell subsets during inductio n of contact hypersensitivity. Therefore, we decided to use rat GAPDH mRNA as an external control. Cytokine gene expression was measured by quantitati ve PCR and was corrected based on external rat GAPDH cDNA, The reliability of this quantitative PCR was superior to that of the conventional method wi th an internal control.