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Chapter 41 : Molecular Analysis of Cytokines and Cytokine Receptors
During the last decades, analysis of cytokines and cytokine receptor research have gathered much interest. Numerous methods have been developed for the detection and quantification of cytokines and cytokine receptors, which may be detected at either the protein or the mRNA level. This chapter focuses on improvements of the recently developed real-time quantitative PCR that has been used in our laboratory for a couple of years in comparison to other available techniques. The methods described allow quantification of minute amounts of mRNA in small samples due to the exponential amplification of the target sequence and the generation of fluorescent molecules that are detected during the amplification reaction. The performance of amplification reactions is affected to a large extent by the concentrations of magnesium ions and oligonucleotide primers. Usually, specificity of primer annealing increases with decreasing magnesium ion concentration. With real-time PCR, specificity in terms of amplification of only the correct sequence is not the major goal, since hybridization and cleavage of the internal probe guarantee specificity of target quantification. A major advantage of the real-time PCR is its application as a high-throughput analytical system in routine diagnostics and research, as, in contrast to other conventional PCR-based techniques, no post-PCR processing of the reaction products is required.
Schematic representation of the quantification reaction. Fluorescence signals are generated during the PCR as, upon elongation of one primer, the internal probe is first displaced from the template strand and then cleaved by the Taq DNA polymerase. Separation of reporter and quencher dye leads to detectable amounts of reporter fluorescence. F, sense primer; R, antisense primer; P, probe.
Increase in reporter dye fluorescence during amplification reactions. Fluorescence intensities are measured in cycles 5 (dashed line) and 40 (solid line) in the range of 500 to 660 nm. While the TAMRA signal at 580 nm is hardly changed, the FAM signal at 535 nm strongly increases due to separation of reporter and quencher dyes by probe cleavage.
Multicomponent view. The changes in FAM, (squares), ROX (triangles), and TAMRA (circles) fluorescence intensities during PCR are shown. ROX serves as a passive reference dye and remains unchanged during PCR. The fluorescence of FAM, as the reporter dye, increases during PCR due to separation from the quencher upon probe cleavage. The TAMRA fluorescence decreases during PCR due to reduced energy transfer from the reporter dye after probe cleavage.
Nucleotide sequence of human IL-2 cDNA (HSIL2R). Sequence characteristics must be taken into account when oligonucleotide probes are chosen for quantification. Here, the cDNA sequence for human IL-2 is shown with exon boundaries double underlined and oligonucleotide binding sites underlined. The names of the oligonucleotides are indicated at the starting nucleotides. Note that the IL-2 probe spans an exon boundary.
Specificity of oligonucleotide primers for cDNA. Means of results in three amplification plots for no-template controls (solid squares), cDNA (open squares), genomic DNA (solid triangles), and coamplified cDNA and genomic DNA (open triangles) are shown. The amplification of NTC and genomic DNA does not generate a fluorescence signal. The amplification of cDNA and cDNA plus genomic DNA results in identical amplification plots. Delta Rn, normalized reporter fluorescence (see text).
Amplification of IL-2 plasmid standards with two different oligonucleotide combinations. IL-2 plasmid standard molecules (10 to 106) were amplified with oligonucleotides IL2S and IL2AS (A) or IL2S2 and IL2AS2 (B) within one experiment. The oligonucleotide binding sites are shown in Fig. 4 . With both primer combinations, the same probe (IL-2 probe) was used. Note that usage of oligonucleotides IL2S2 and IL2AS2 results in steeper amplification plots and higher fluorescence intensities. NTC, no-template controls; delta Rn, normalized reporter fluorescence.
IL-2 standard curves generated with two different oligonucleotide combinations. IL-2 plasmid standard molecules (10 to 106) were amplified with oligonucleotides IL2S and IL2AS (A) or IL2S2 and IL2AS2 (B) within one experiment. The oligonucleotide binding sites are shown in Fig. 4 . With both primer combinations, the same probe (IL-2 probe) was used. Note that usage of oligonucleotides IL2S2 and IL2AS2 results in earlier detection of significant fluorescence, i.e., higher sensitivity. Coeff, coefficient.
Increase in FAM (A) and TET (B) fluorescence during PCR amplification of different TNF-α haplotypes. Three different haplotypes of the human TNF-α promotor (DNA AG, DNA AA, and DNA GG) were amplified in the presence of two different internal probes. One is labeled with TET and specific for the wild type (TNFα-308G), the other is labeled with FAM and specific for the mutant promotor (TNFα-308A). If homozygous DNA TNFα-308A is present, the FAM fluorescence gives the higher intensity. If homozygous DNA TNFα-308G is present, the FAM fluorescence gives the lower intensity. The opposite holds true for the TET fluorescence. Heterozygous DNA in both cases gives intermediate intensities. Delta Rn, normalized reporter fluorescence (see text).
Identification of TNF-α haplotypes by allelic discrimination PCR. After PCR, the FAM and TET fluorescence intensities were determined for each sample. Plotting FAM fluorescence against TET fluorescence revealed different populations representing three haplotypes. High TET and low FAM intensities identify wild-type alleles; high FAM and low TET intensities identify mutant alleles. Heterozygous DNA gives intermediate results. NTC, no-template controls.
Comparison of methods for quantification of cytokine and cytokine receptor mRNA expression a