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 Northern blotting with the aid of a correction based on the expression of an internal control gene. However, this approach is not suitable for quantitating 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 expression in lymph node cells from mice during the induction of contact hypersensitivity. Expression of the murine glyceraldehydephosphate dehydrogenase (GAPDH) gene, a candidate internal control, was not constant in cells from trinitrochlorobenzene- and vehicle-applied animals, suggesting that GAPDH gene expression changes in heterogeneous lymph node-cell subsets during induction of contact hypersensitivity. Therefore, we decided to use rat GAPDH mRNA as an external control. Cytokine gene expression was measured by quantitative PCR and was corrected based on external rat GAPDH cDNA. The reliability of this quantitative PCR was superior to that of the conventional method with an internal control.
Shiseido Skin Biology Research Laboratories, Yokohama, Japan. shibata_michio@po.shiseido.co.jp
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M
Shibata
T
T
Hariya
M
M
Hatao
T
T
Ashikaga
H
H
Ichikawa
D000818:Animals; D002478:Cells, Cultured; D016207:Cytokines / Q000235:genetics*; D018076:DNA, Complementary / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D005260:Female; D015870:Gene Expression; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics*; D008198:Lymph Nodes / Q000502:physiology*; D051379:Mice; D008809:Mice, Inbred C3H; D010853:Picryl Chloride / Q000494:pharmacology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D015203:Reproducibility of Results
D016207:Cytokines; D018076:DNA, Complementary; D012333:RNA, Messenger; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010853:Picryl Chloride