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, 181 (1), 58-66

The Optimization of TaqMan Real-Time RT-PCR Assay for Transcriptional Profiling of GABA-A Receptor Subunit Plasticity

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The Optimization of TaqMan Real-Time RT-PCR Assay for Transcriptional Profiling of GABA-A Receptor Subunit Plasticity

Omkaram Gangisetty et al. J Neurosci Methods.

Abstract

The GABA-A receptor plays a critical role in inhibitory neurotransmission in the brain. Quantitation of GABA-A receptor subunits in various brain regions is essential to understand their role in plasticity and brain disorders. However, conventional RNA assays are tedious and less sensitive for use in studies of subunit plasticity. Here we describe optimization of a sensitive assay of GABA-A receptor subunit gene expression by TaqMan real-time PCR. For each subunit gene, a set of primers and TaqMan fluorogenic probe were designed to specifically amplify the target template. The TaqMan methodology was optimized for quantification of mouse GABA-A receptor subunits (alpha(1-6), beta(1-3), gamma(2), and delta) and GAPDH. The TaqMan reaction detected very low levels of gene expression ( approximately 100 template copies of cDNA). A standard curve for GAPDH and one of the target genes, constructed using the cDNA, revealed slopes around -3.4 (r(2)=0.990), reflecting similar optimum PCR efficiencies. The methodology was utilized for quantification of the GABA-A receptor alpha(4)-subunit, which is known to upregulate following withdrawal from chronic progesterone or neurosteroids. Our results show that the alpha(4)-subunit expression increased threefold in the hippocampus following neurosteroid withdrawal in mice. The TaqMan PCR assay allows sensitive, high-throughput transcriptional profiling of complete GABA-A receptor subunit family, and thus provides specific tool for studies of GABA-A receptor subunit plasticity in neurological and psychiatric animal models.

Figures

Fig. 1
Fig. 1. Diagrammatic illustration of the TaqMan chemistry
The TaqMan PCR reaction exploits the 5′-nuclease activity of Taq DNA polymerase to cleave a TaqMan probe during PCR. The TaqMan probe is an oligonucleotide that contains a reporter dye (FAM, 6-carboxytetramethylrhodamine) at the 5′-end of the probe and a quencher dye (TAMRA, 6-carboxyfluorescein) at the 3′-end of the probe. During PCR, the probe specifically anneals to the target sequence between the forward and reverse primer sites. When the probe is intact, the quencher dye suppresses the fluorescence emission of the reporter dye primarily due to Forster-type energy transfer. During the PCR reaction, Taq DNA polymerase extends the primer through the polymerase activity, as it approaches the probe it displaces the probe and cleaves it through the 5′ to 3′ exonuclease activity. This separates the reporter dye and the quencher dye from the probe, which results in increased fluorescence of the reporter. Accumulation of PCR products is detected in “real-time” directly by monitoring the increase in fluorescence of the reporter dye with an automated PCR system.
Fig. 2
Fig. 2. Gel electrophoresis of PCR products of GABA-A receptor subunits and GAPDH
The target subunits and GAPDH gene expression from mouse whole brain cDNA were analyzed by RT-PCR using specific primers. A single band was detected near the expected size for each target subunit and GAPDH. No band was observed in the PCR products of negative control samples without template DNA. In the panel (M) indicates 100 bp molecular weight marker, (−) indicates PCR reaction with no cDNA template, (+) indicates PCR reaction with template cDNA.
Fig. 3
Fig. 3. Amplification plots of the GABA-A receptor α2-subunit in the TaqMan real-time PCR assay
TaqMan assay for the GABA-A receptor α2 subunit was performed using purified PCR product generated with specific primers and the template sample was serially diluted 10-fold from 109 to 102 copy numbers. The fluorescence emission measured Rn (reporter normalized fluorescence) was plotted against cycle number. In the amplification plots, fluorescence intensity increased as the PCR cycles increased. Negative control sample (no template DNA sample) showed no elevation of fluorescence. The legends (log 2 to 9) refer to the copy number of the template DNA used. TaqMan assay for GAPDH was performed similarly using purified PCR product serially diluted 10 fold from 108 to 102 copy numbers (data not shown). The threshold cycle (CT), the point at which the fluorescence exceeds a threshold limit is used as key parameter for calculating input cDNA for relative quantification of the target subunit expression.
Fig. 4
Fig. 4. Optimization of primer and TaqMan probe concentrations in the TaqMan assay
Primer concentration-dependent amplification plots of GAPDH (A) and the GABA-A receptor α1-subunit (B). Forward and reverse primers were tested at four different concentrations (100 – 600 nM) with a fixed probe concentration (300 nM) using whole brain cDNA for GAPDH and α1-subunit. The primer pair yielding the best amplification (400 nM) was chosen to determine the optimum probe concentration. TaqMan probe was tested at four different concentrations (50 – 300 nM) for GAPDH and α1-subunit and the CT values derived from the amplification plots are listed in Table 2. For most target subunits, 300 nM of probe yielded the best amplification with lowest CT values. The curves represent the mean of triplicate measurements.
Fig. 5
Fig. 5. Standard curves for GAPDH and selected GABA-A receptor subunits
Standard curves for GABA-A receptor α1 (A), α2 (B), α4 (C), and α6 subunit (D). Standard curves for each subunit and GAPDH were constructed using log cDNA copy number against CT values. The CT values and sample concentrations show a reverse linear correlation with nearly identical slopes of around (−3.4), indicating equivalent PCR efficiencies of target and GAPDH control. The correlation coefficient for standard curves ranged from 0.990 to 0.998, suggesting the accuracy of quantification protocol. Each point represents the mean of triplicate measurements for each subunit.
Fig. 6
Fig. 6. Standard curves for GAPDH and selected GABA-A receptor subunits
Standard curves for GABA-A receptor γ2 (A), δ (B), β2 (C), and β3 subunit (D). Standard curves for each subunit and GAPDH were constructed using log cDNA copy number against CT values. The CT values and sample concentrations show a reverse linear correlation with nearly identical slopes of around (−3.4), indicating equivalent PCR efficiencies of target and GAPDH control. The correlation coefficient for standard curves ranged from 0.990 to 0.998. Each point represents the mean of triplicate measurements for each subunit.
Fig. 7
Fig. 7. Changes in the GABA-A receptor α4-subunit expression in the hippocampus following neurosteroid withdrawal in mice
The upper panel (A) shows the absolute expression of α4 mRNA in terms of copy numbers, while the relative expression was shown in the lower panel (B) as the percent change in the ratio of α4/GAPDH copy numbers in each animal. The α4-subunit was up-regulated (~3-folds) in the hippocampus of mice chronically treated first with progesterone (25 mg/kg, sc, twice daily for 7 days) and then with finasteride (50 mg/kg, ip on day 7) which produces withdrawal of the neurosteroid allopregnanolone by inhibiting its synthesis from progesterone. Hippocampus was isolated 24 h after finasteride injection, total RNA was extracted and cDNA prepared from vehicle and neurosteroid withdrawal groups for α4-subunit analysis. The data was normalized in every assay using the internal GAPDH gene, which was not significantly altered following neurosteroid withdrawal. Data represent mean ± SEM (n = 8 mice per group). *p<0.01 versus control group.

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