Andrew D Medhurst and Menelas N Pangalos 1 Introduction

TaqMan reverse transcription-polymerase chain reaction (TaqMan RT-PCR) is a recently developed technique (1) that has been used to study gene expression in tissues of the central nervous system (CNS) including the striatum. For example, TaqMan has been used to profile mRNA distribution patterns across the brain for y-aminobutyric acid-B (GABAb) receptor subunits (2), 5-hydroxytryptamine4 (5-HT4) receptor splice variants (3), novel G-protein-coupled receptors (4), and ion channels including vanilloid receptors (5) and two pore potassium channels (6). More specifically, TaqMan RT-PCR studies have demonstrated a huge enrichment of dopamine D2 (7) and D3 (8) receptors in human striatal tissues compared to other brain regions. In addition, the technique has been utilized for the analysis of gene expression changes in animal models of CNS diseases including Parkinsons's disease (9), stroke (10), and migraine (7).

The TaqMan RT-PCR technique enables the measurement of an accumulating PCR product in real time by utilizing a dual-labeled TaqMan fluorogenic probe (1,11,12; Fig. 1A). It allows a more accurate comparison of mRNA expression levels between large numbers of samples, compared to other techniques such as Northern blotting, standard RT-PCR, and in situ hybridization. It is also useful for validating samples prior to microarray analysis.

The chemistry of TaqMan RT-PCR is based around the endogenous 5'-3' exonuclease activity of Thermus aquaticus (Taq) polymerase (1,11,12). During polymerization, the gene-specific TaqMan probe anneals to the specific DNA

From: Methods in Molecular Medicine, vol. 79: Drugs of Abuse: Neurological Reviews and Protocols Edited by: J. Q. Wang © Humana Press Inc., Totowa, NJ

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Fig. 1. Schematic representation of TaqMan PCR assay. (A) TaqMan probe with fluorescent reporter dye FAM (6-carboxyfluorescein) attached to the 5' end and a quencher dye TAMRA (6-carboxytetramethylrhodamine) attached to the 3' end. (B) During polymerization the TaqMan probe containing a reporter dye (R) and a quencher dye (Q) specifically anneals to one strand of the DNA template between forward and reverse PCR primers. During amplification, the fluorogenic probe is displaced by Taq polymerase and then cleaved (C), releasing the reporter dye. After cleavage, the shortened probe dissociates from the template, allowing polymerization of the strand to complete. Fluorescence is proportional to the amount of product accumulated.

Fig. 1. Schematic representation of TaqMan PCR assay. (A) TaqMan probe with fluorescent reporter dye FAM (6-carboxyfluorescein) attached to the 5' end and a quencher dye TAMRA (6-carboxytetramethylrhodamine) attached to the 3' end. (B) During polymerization the TaqMan probe containing a reporter dye (R) and a quencher dye (Q) specifically anneals to one strand of the DNA template between forward and reverse PCR primers. During amplification, the fluorogenic probe is displaced by Taq polymerase and then cleaved (C), releasing the reporter dye. After cleavage, the shortened probe dissociates from the template, allowing polymerization of the strand to complete. Fluorescence is proportional to the amount of product accumulated.

template between forward and reverse PCR primers (Fig. 1B). When the probe is intact the reporter fluorophore emission is suppressed by a quencher fluorophore. The 5'-3'-nuclease activity of Taq polymerase releases the reporter dye from the vicinity of the quencher dye, resulting in the generation of reporter fluorescence (Fig. 1C). The remaining probe fragment dissociates from the target sequence, allowing polymerization to continue. The intensity of fluorescence is proportional to the amount of DNA amplified, and this reaction occurs during every cycle of PCR. The fluorescent signal is captured using an ABI Prism 7700 sequence detection system (PE Applied Biosystems) which allows the rapid analysis of 96 PCR samples simultaneously, without any time-consuming downstream gel electrophoresis steps. Data are easily captured using Sequence Detector Software (Applied Biosystems) linked to the ABI Prism 7700, and can then be exported to Microsoft Excel worksheets for further analysis.

The standard techniques used to generate mRNA expression data in human and rat CNS tissues using TaqMan RT-PCR are described, including protocols we have used for RNA extraction, cDNA synthesis, primer and probe design and quality control, TaqMan PCR assays, and data analysis (7).

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