Quantification of cytokine gene expression by competitive PCR using a colorimetric assay.
Competitive reverse transcription-polymerase chain reaction (RT-PCR) is a new technique allowing quantification of cytokine gene expression from either experimental or clinical samples. In this assay, a time-consuming step is the quantification of amplified products. To improve this step, we set up a colorimetric assay in which the amplified product from either the cDNA or the competitor can be reliably quantified. Using this approach, which can be completely automatized, up to 320 PCR products can be quantified each day. In this report, we describe the quantification of IL-10 mRNA molecules as compared to that of beta-actin mRNA molecules. The sensitivity of the quantification was 7.7 x 10(7) molecules for the amplified beta-actin cDNA and the amplified IL-10 cDNA, corresponding to approximately 9.6 pg amplified beta-actin cDNA and 11 pg amplified IL-10 cDNA, respectively. The intra-assay variation coefficient was < 12%. This technique can be readily extended to all cytokines, and it thus allows routine monitoring of cytokine gene expression, either from experimental samples or from clinical trials.
Zou
W
W
INSERM U131, Institut Paris-Sud sur les Cytokines, Clamart, France.
Durand-Gasselin
I
I
Dulioust
A
A
Maillot
M C
MC
Galanaud
P
P
Emilie
D
D
D000199:Actins / Q000235:genetics; D001483:Base Sequence; D001667:Binding, Competitive; D003124:Colorimetry / Q000379:methods; D016207:Cytokines / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005069:Evaluation Studies as Topic; D005784:Gene Amplification; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D016753:Interleukin-10 / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D012680:Sensitivity and Specificity
D000199:Actins; D016207:Cytokines; D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D016753:Interleukin-10