Quantification of cytokine mRNA expression by RT-PCR and electrochemiluminescence.

Abstract

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 (11) (TBR)-Iabeled 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.T h e quantitative measurement of specific mRNA species is of major importance to approach many fundamental questions in biology. Therefore, various strategies have been developed for quantitation of cDNA by PCR-based methods, (1-4) m o s t of them utilizing the principle of competitive PCR (C-PCR) in which a synthetic cDNA fragment is coamplified with the target cDNA segment.We were interested in comparing the expression of cytokine genes in numerous samples of antigen-specific T-cell clones. C-PCR is not suitable for this purpose because quantitative analysis of C-PCR reactions is tedious, with a large number of tubes per sample and because of separation of the two products by electrophoretic techniques or high performance liquid chromatography (HPLC), time-consuming Southern blot analysis, the use of radioactive isotopes, and tedious digitalization of the results. Therefore, we looked for a quantitative PCR method that is simple and fast and one in which the results could be digitalized easily--to provide numeric data--so that many samples can be processed at the same time.Recently, DiCesare and co-workers (s~ described an electrochemiluminescent (ECL)-based detection method for the quantitation of PCR-amplified DNA. This method involves the labeling of the amplified product by an oligonucleotide primer or probe coupled to Tris (2,2'-bipyridine) ruthenium (II) (TBR) chelate. TBR emits light at 620 nm in an electrochemical cell under appropriate redox conditions, allowing for direct and specific quantitation of DNA sequences.Here, we describe a protocol for directly quantitating PCR product formation by electrochemiluminescence and how these data can be used to determine the starting template concentration. In the present test, a single amplification is sufficient; therefore, several samples can be analyzed at the same time. No separation is required to quantify the amplified products. Although we used this method to quantify cytokine mRNA expression in long-term antigen-specific T-cell clones, it can be applied to any mRNA of interest. MATERIALS AND METHODS Preparation of mRNA Cytokine StandardsThe PCR standard RNAs (st-RNAs) were obtained from two nonhomologous, cloned synthetic DNA constructs...

Electrochemiluminescence from Tunicate, Tunichrome–Metal Complexes and Other Biological Samples

“…Electrochemiluminescence (ECL) has been described for several different types of molecular complexes including purely organic, organic-metal chelates and metallic halogen 'clusters' (1-3). ·Author In recent years, ECL has been employed in a number of very sensitive immunoassays and nucleic acid-based assays (4)(5)(6)(7)(8)(9)(10). The body of literature on ECL involves primarily synthetic ECL coordination complexes such as ruthenium (II) trisbipyridine (Ru(bpy)~+), Cr, Cu, Eu, Ir, Os, Pd, Pt, Re and other metal chelates (1- 3,11,12).…”

The Inherent Quantitative Capacity of the Reverse Transcription-Polymerase Chain Reaction

“…Furthermore, direct comparison has led some investigators to conclude that the use of competitor molecules does not significantly increase the quantitative accuracy or precision of PCR (23,24). Finally, it has been suggested that noncompetitive PCR yields quantitative results only during the exponential phase of the reaction (2,3,6,17,25,26). In the present study, we demonstrate that noncompetitive PCR run to the plateau phase provides a reliable measure of template input over an approximate 30-to 100-fold range above the lower limit of detection.…”

“…Second, the studies of Wang et al, 1989 (2) and Gilliland et al, 1990 (3) are widely cited as evidence that competitive PCR methods are essential for accurate quantitation by PCR. However, several noncompetitive PCR studies have demonstrated that product yields provide an accurate measure of template input when PCR product yield is measured in the exponential phase of amplification (17)(18)(19)(20)(21)(22). Furthermore, direct comparison has led some investigators to conclude that the use of competitor molecules does not significantly increase the quantitative accuracy or precision of PCR (23,24).…”

Isolation and Quantification of Episomal Expression Vectors in Human T Cells

“…The quantification procedure was described previously in detail (2,(5)(6)(7)(8)(9). Briefly, 35 µL of 1× PCR buffer (10 mM Tris-HCl, pH 8.4, 10 mM KCl and 0.1% gelatin; Perkin-Elmer), and 15 µL (2 mg/mL) of streptavidin-coated …”