Quantification of cytokine mRNA expression by RT-PCR and electrochemiluminescence.
Variable gene expression constitutes a major mechanism for controlling cell development and cell function. To investigate these changed mRNA levels, a sensitive and quantitative assay is required. We describe a quick and easy method to quantify specific mRNAs by a combination of PCR and an electrochemiluminescent (ECL) detection of the amplified products. Total cellular RNA is reverse-transcribed and amplified with a biotinylated forward primer and a Tris (2,2'-bipyridine) ruthenium (II) (TBR)-labeled reverse primer. The amplification product is captured on streptavidin-coated paramagnetic beads and quantified by ECL detection using the QPCR system 5000. The results can be converted to quantitative values with an external standard curve. This method permits accurate and reliable quantitation of cytokine mRNA expression.
Vandevyver
C
C
Department of Biotechnology, Dr. L. Willems-Instituut, Diepenbeek, Belgium. Caroline.Vandevyver@ping.be
Motmans
K
K
Raus
J
J
D002999:Clone Cells; D016207:Cytokines / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D008163:Luminescent Measurements; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D015203:Reproducibility of Results; D013601:T-Lymphocytes / Q000378:metabolism
D016207:Cytokines; D012333:RNA, Messenger