Following its invention 25 years ago, PCR has been adapted for numerous molecular biology applications. Gene expression analysis by reverse-transcription quantitative PCR (RT-qPCR) has been a key enabling technology of the post-genome era. Since the founding of BioTechniques, this journal has been a resource for the improvements in qPCR technology, experimental design, and data analysis. qPCR and, more specifically, real-time qPCR has become a routine and robust approach for measuring the expression of genes of interest, validating microarray experiments, and monitoring biomarkers. The use of real-time qPCR has nearly supplanted other approaches (e.g., Northern blotting, RNase protection assays). This review examines the current state of qPCR for gene expression analysis now that the method has reached a mature stage of development and implementation. Specifically, the different fluorescent reporter technologies of real-time qPCR are discussed as well as the selection of endogenous controls. The conceptual framework for data analysis methods is also presented to demystify these analysis techniques. The future of qPCR remains bright as the technology becomes more rapid, cost-effective, easier to use, and capable of higher throughput.

Reverse transcription PCR (RT-PCR) represents a sensitive and powerful tool for analyzing RNA. While it has tremendous potential for quantitative applications, a comprehensive knowledge of its technical aspects is required. Successful quantitative RT-PCR involves correction for experimental variations in individual RT and PCR efficiencies. This review addresses the mathematics of RT-PCR, choice of RNA standards (internal vs. external) and quantification strategies (competitive, noncompetitive and kinetic [real-time] amplification). Finally, the discussion turns to practical considerations in experimental design. It is hoped that this review will be appropriate for those undertaking these experiments for the first time or wishing to improve (or validate) a technique in what is frequently a confusing and contradictory field.

Tyrosine hydroxylase catalyzes the rate-limiting step in the biosynthesis of the catecholamines dopamine, norepinephrine, and epinephrine. Therefore, the regulation of tyrosine hydroxylase enzyme number and intrinsic enzyme activity represents the central means for controlling the synthesis of these important biogenic amines. An intricate scheme has evolved whereby tyrosine hydroxylase activity is modulated by nearly every documented form of regulation. Beginning with the genomic DNA, evidence exists for the transcriptional regulation of tyrosine hydroxylase mRNA levels, alternative RNA processing, and the regulation of RNA stability. There is also experimental support for the role of both translational control and enzyme stability in establishing steady-state levels of active tyrosine hydroxylase protein. Finally, mechanisms have been proposed for feedback inhibition of the enzyme by catecholamine products, allosteric modulation of enzyme activity, and phosphorylation-dependent activation of the enzyme by various different kinase systems. Given the growing literature suggesting that different tissues regulate tyrosine hydroxylase mRNA levels and activity in different ways, regulatory mechanisms provide not only redundancy but also diversity in the control of catecholamine biosynthesis.

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A variety of in vitro and in vivo studies demonstrate that dopamine is a toxic molecule that may contribute to neurodegenerative disorders such as Parkinson's disease and ischemia-induced striatal damage. While much attention has focused on the fact that the metabolism of dopamine produces reactive oxygen species (peroxide, superoxide, and hydroxyl radical), growing evidence suggests that the neurotransmitter itself may play a direct role in the neurodegenerative process. Oxidation of the dopamine molecule produces a reactive quinone moiety that is capable of covalently modifying and damaging cellular macromolecules. This quinone formation occurs spontaneously, can be accelerated by metal ions (manganese or iron), and also arises from selected enzyme-catalyzed reactions. Macromolecular damage, combined with increased oxidant stress, may trigger cellular responses that eventually lead to cell death. Reactive quinones have long been known to represent environmental toxicants and, within the context of dopamine metabolism, may also play a role in pathological processes associated with neurodegeneration. The present discussion will review the oxidative metabolism of dopamine and describe experimental evidence suggesting that dopamine quinone may contribute to the cytotoxic and genotoxic potential of this essential neurotransmitter.

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Functional deletion mutants of the trans-acting factor TFIIIA, truncated at both ends of the molecule, have been expressed by in vitro transcription of a cDNA clone and subsequent cell-free translation of the synthetic mRNAs. A region of TFIIIA 19 amino acids or less, near the carboxyl terminus, is critical for maximal transcription and lies outside the DNA-binding domain. The elongated protein can be aligned over the internal control region (ICR) of the Xenopus 5S RNA gene with its carboxyl terminus oriented toward the 5' end of the gene and its amino terminus oriented toward the 3' end of the gene. The nine "zinc fingers" and the linkers that separate them comprise 80% of the protein mass and correspond to the DNA-binding domain of TFIIIA. The zinc fingers near the amino terminus of the protein contribute more to the overall binding energy of the protein to the ICR than do the zinc fingers near the carboxyl end. The most striking feature of TFIIIA is its modular structure. This is demonstrated by the fact that each zinc finger binds to just one of three short nucleotide sequences within the ICR.TFIIIA is a positive, trans-acting factor required for transcription of 5S RNA genes in Xenopus laevis. It was first purified from Xenopus ovaries and shown to be a protein of about 38,500 daltons (12). One molecule of TFIIIA binds to the internal control region (ICR) of a 5S RNA gene (31,42) nucleating the formation of a transcription complex (6) that involves at least two other factors (22,41). This complex is required for RNA polymerase III to initiate transcription accurately at the start site of the 5S RNA gene.Analysis of TFIIIA proteolytic fragments suggests that the protein is composed of a transcription and a DNA-binding domain (42). The carboxyl terminus of TFIIIA (43) is required for the full transcriptional activity of the factor but not for the sequence-specific binding of the protein to the ICR (42). These data suggested that the binding domain of the protein can be aligned over the ICR with its carboxyl terminus toward the 5' end and its amino terminus toward the 3' end of the ICR. This picture of an asymmetric, elongated protein molecule is reinforced by its physical properties in solution (3). Deletion mutants of the ICR show clearly that binding of the protein is not uniform over the ICR since binding to the 3' end of the ICR is required for binding at the 5' end but not the reverse (39). Protection of G residues from methylation by TFIIIA is strongest at the 3' end of the ICR and weakest at the 5' end (13).The gene for TFIIIA has been isolated from cDNA libraries prepared from Xenopus ovaries (16,43). The protein sequence predicted from the cDNA sequence is composed predominantly of nine imperfect, tandemly repeated regions, each approximately 30 amino acids in length (10, 30). The conserved sequence includes pairs of cysteines and histidines, amino acids known to bind metal ions in proteins. It had been shown previously that TFIIIA contains zinc (19). Each adjacent repeated polypeptide and its associ...

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