Quantitative analysis of lymphokine mRNA expression by an automated, non-radioactive method.
Variable gene expression, thus giving rise to variable mRNA levels, constitutes a major mechanism for controlling cell development and cell function. In order to investigate these changed mRNA levels, a sensitive and quantitative assay is required. A quick and easy method is described to quantify specific mRNA's by a combination of the polymerase chain reaction (PCR) and an electro-chemiluminescenct (ECL) detection of the amplified products. Total cellular RNA is reverse transcribed and amplified with a biotinylated forward primer and a TBR (Tris (2,2'-bipyridine) ruthenium (II)) labelled 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. In the present study, cytokine mRNA expression in T lymphocytes was quantified. Cytokine mRNA was measured at the attomolar range in a dynamic range up to three orders of magnitude. The ECL detection is quantitative, rapid and accurate.
Vandevyver
C
C
Department of Biotechnology, Dr. L. Willems-Instituut, Diepenbeek, Belgium.
Raus
J
J
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D005069:Evaluation Studies as Topic; D015870:Gene Expression; D006801:Humans; D008163:Luminescent Measurements; D008222:Lymphokines / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D012333:RNA, Messenger / Q000235:genetics*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000276:immunology
D017931:DNA Primers; D008222:Lymphokines; D012333:RNA, Messenger