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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Neurosci Methods. Author manuscript; available in PMC 2010 June 30.
Published in final edited form as:
PMCID: PMC2696555
NIHMSID: NIHMS113896

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

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 (α1–6, β1–3, γ2, and δ) and GAPDH. The TaqMan reaction detected very low levels of gene expression (~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 (r2=0.990), reflecting similar optimum PCR efficiencies. The methodology was utilized for quantification of the GABA-A receptor α4 subunit, which is known to upregulate following withdrawal from chronic progesterone or neurosteroids. Our results show that the α4-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.

Keywords: Real-time PCR, TaqMan, GABAA receptor, Neurosteroid withdrawal, GAPDH

1. Introduction

GABA (γ-aminobutyric acid) is the major inhibitory neurotransmitter in the brain where it acts at GABA-A and GABA-B receptors. The GABA-A receptor is a transmembrane-gated ion channel that mediates both phasic inhibitory synaptic transmission and tonic perisynaptic inhibition, and thereby plays a critical role in the pathophysiology of neuropsychiatric conditions including anxiety and epilepsy. The GABA-A receptor has binding sites for GABA, benzodiazepines and neurosteroids (Hosie et al., 2007), and therefore, it is a major target receptor for several clinically used anxiolytic, antiepileptic and anesthetic agents. The receptor exists as a pentamer of 4 transmembrane subunits that form an intrinsic chloride channel. Seven different classes of subunits with multiple variants have been reported in mammals (α1–6, β1–3, γ1–3, ρ1–3, δ, ε, θ) (Korpi et al., 2002; Whiting, 2003; Sieghart, 2006). Many GABA-A receptor subtypes contain α, β and γ-subunits with the likely stoichiometry 2α,2β,1γ (Korpi et al., 2002; Fritschy and Brunig, 2003). Functional properties of GABA-A receptor depend on its subunit composition, which are differentially expressed both temporally and spatially throughout the brain (Sigel et al., 1990; Pirker et al., 2000; Wohlfarth et al., 2002). Most GABA-A receptors contain a single type of α- and β-subunit variant. The α1β2γ2 hetero-oligomer constitutes the largest population of GABA-A receptors in the brain, followed by the α2β3γ2 and α3β3γ2 isoforms. Receptors that contain the α4-, α5-, α6-, or the β1-, γ1-, γ3-, δ-, ε- and θ-subunits are less abundant but play important functions. The α6- and δ-subunits in cerebellar granule cells, or the α4- and δ-subunits, typically present at extracellular/perisynaptic sites in dentate granule cells and thalamic neurons, mediate tonic current in response to ambient levels of GABA (Mody and Pearce, 2004; Farrant and Nusser, 2005; Walker and Semyanov, 2008). Mutations in certain GABA-A receptor subunits cause epilepsy (DeLorey et al., 1998; Baulac et al., 2001; Macdonald and Kang, 2008) and also alter drug sensitivity (Rudolph et al., 2001; Lambert et al., 2003). A variety of neuroendocrine conditions are associated with profound alterations in GABA-A receptor subunit expression (Smith et al., 1998; Fellosa et al., 1998; Maguire et al., 2005).

Analysis of subunit expression is important to understand the pharmacology and functional significance of GABA-A receptor subunits in various regions in the brain. However, studies utilizing traditional RNA assay techniques, such as the RNAase protection assay, the in-situ hybridization and the competitive PCR, are tedious, require large quantities of RNA, and often difficult to analyze multiple genes in large sample size (Wisden et al., 1992; Follesa et al., 1998; Liu et al., 2002). The reverse transcription followed by PCR (RT- PCR) is a standard technique routinely used for detecting the gene expression, but its sensitivity is very limited and not amenable for rapid analysis.

Therefore, a more sensitive and reliable technique is required for accurate quantification of GABA-A receptor subunit expression in brain tissue samples. Real-time PCR is a versatile technique for rapid analysis of multiple samples. The use of fluorescent dyes such as SYBR green allows quantitation of the starting amount of nucleic acid by measuring the fluorescence intensity with PCR instrumentation. A non-regulated reference gene such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is used as endogenous control for relative analysis of gene expression. The “TaqMan” real-time PCR, which measures PCR-product accumulation during the exponential phase of the PCR reaction using a dual-labelled fluorogenic probe (referred as “TaqMan probe”) has been developed and used extensively (Holland et al., 1991; Gibson et al., 1996; Heid et al., 1996; Lie and Petropoulos, 1998). The TaqMan assay is based on the 5′–3′ exonuclease activity of Taq DNA polymerase to cleave a dual-labeled probe, which is designed to hybridize to a target sequence during amplification (Fig. 1). Disintegration of the probe during PCR releases reporter fluorescence and the intensity of the fluorescence signal measured during the exponential phase of the PCR reaction is proportional to the amount of input target DNA. Unlike the intercalating SYBR green that binds to all double-stranded DNA products, the TaqMan probe avoids detection of non-specific amplification products because of its stringent design to bind to the target gene sequence. The TaqMan assay has been widely used for reliable and sensitive analysis of gene expression of glutamate receptor subunits (Medhurst et al., 2000; Pfaffl, 2001; Horii et al., 2002; Langmann et al., 2003). Studies using the TaqMan technology for GABA-A receptor subunits have appeared, but are restricted to a few of the subunits (Floyd et al., 2004; Linneman et al., 2006; Byrnes et al., 2007). However, there are few studies that describe systematic optimization of TaqMan real-time PCR protocol for GABA-A receptor subunit superfamily.

Fig. 1
Diagrammatic illustration of the TaqMan chemistry

In this study, we describe the optimization of TaqMan real-time RT-PCR assay for quantification of GABA-A receptor subunit family using the GAPDH as reference gene. The assay was utilized to determine changes in expression of the hippocampal GABA-A receptor α4 subunit gene, which is known to increase following withdrawal from chronic progesterone and neurosteroids. Our results show that the α4-subunit was upregulated threefold following neurosteroid withdrawal in mice.

2. Materials and Methods

2.1. Animals

Female adult mice (25–30 g) of C57BL/6J strain were used in the study. Total RNA extraction was performed from whole brain and hippocampus in untreated adult mice or mice with neurosteroid withdrawal treatment. To induce neurosteroid withdrawal, mice were treated with progesterone (25 mg/kg, s.c., twice daily for 7 days) followed by single injection of finasteride (50 mg/kg, i.p.) on seventh day. Twenty-four hours after finasteride injection, mice were anaesthetized using isoflurane and the hippocampus was rapidly dissected for RNA isolation. Chronic treatment with progesterone is associated with high circulating levels of neurosteroids such as allopregnanolone because progesterone is converted in the brain into allopregnanolone (Reddy et al., 2004). Administration of finasteride, a 5α-reductase inhibitor that blocks conversion of progesterone into allopregnanolone, induces a state of neurosteroid withdrawal. This treatment group was referred to as neurosteroid withdrawal group. The control group received vehicle (15% β-cyclodextrin solution) for 7 days and the brain tissue was collected for RNA isolation. All animal procedures were approved by the Institutional Animal Care and Use Committee.

2.2. Extraction of total RNA

Total RNA was extracted from whole brain and hippocampus using a Trizol reagent from Invitrogen (Carlsbad, CA) as per the manufacturer’s instructions. The quality of RNA samples was ascertained by measuring optical density (OD, 260/280) absorption ratio of 1.7 (range 1.62 – 2.1). The integrity of RNA was verified by the detection of 18S and 28S bands after agarose-formaldehyde gel electrophoresis. Total RNA samples were stored at −80 °C. To remove residual DNA contamination, the RNA samples were incubated with RNAase-free DNAse I at 37 °C for 20 minutes, and then the DNAase I was inactivated at 65 °C for 10min (Ambion, Austin, TX). The purified total RNA was used to generate cDNA.

2.3. Preparation of cDNA

Total RNA from each sample was used to synthesize cDNA using a Superscript II first-strand cDNA synthesis kit (Invitrogen Inc., Carlsbad, CA) with oligo (dT) primers, according to the manufacturer’s protocol. Briefly, 2μg of DNAse-treated total RNA was used as starting material, to which we added 1 μl of oligo (dT), 1 μl of 10 mM dNTPs, 2μl of 10× first strand buffer, 4 μl 25 mM MgCl2, 2 μl of 0.1 M DTT, and 1 μl of RNAase out. The reagents, RNA, oligo (dT), and dNTPs were mixed first, then heated at 65 °C for 5 min and then chilled on ice until the other components were added. The samples were incubated at 42 °C for 2 min. Then 1 μl of Superscript II (40 U/μl) was added, and the samples were incubated at 42 °C for 50 min. The reaction was inactivated at 70 °C for 15 min. The samples were kept on ice, centrifuged briefly, added 1 μl of RNAse H and incubated at 37 °C for 20 min. Parallel reactions for each RNA sample were run in the absence of Superscript II (no RT control) to assess any genomic DNA contamination.

2.4. Design of primers and probes

The PCR primers and TaqMan probe specific for each GABA-A receptor subunit gene and GAPDH were designed using Beacon Designer software (Bio-Rad Inc., Hercules, CA). Like the Primer Express software (Applied Biosystems Inc., Foster City, CA), this program selects probe and primer sets with optimized melting temperatures, secondary structure, base composition, and amplicon lengths. The specificity of the primers and probes for each subunit was confirmed by a homology search. Highly purified salt-free primers were obtained commercially (IDT Inc., Coralville, IA) and TaqMan probes were procured from Applied Biosystems (Foster City, CA). The TaqMan probe was designed with FAM (6-carboxyfluorescein) as the reporter dye on the 5′ end and TAMRA (6-carboxytetramethylrhodamine) as the quencher dye on the 3′ end (Fig. 1). The parameters for primers and probes were optimized as per the guidelines of the TaqMan assay (Applied Biosystems).

2.5. TaqMan real-time PCR

TaqMan real-time quantitative PCR amplification reactions were carried out in an AB 7500 fast real-time system (Applied Biosystems). Real-time PCR was carried out with TaqMan Universal PCR Master Mix (Applied Biosystems), which contained AmpliTaq Gold DNA Polymerase, AmpErase, UNG, dNTPs with dUTP, and optimized buffer components. AmpErase UNG treatment was used to prevent the possible reamplification of carry-over PCR products. Briefly, a 25-μl reaction mixture consists of 12.5-μl TaqMan Universal PCR Master mix, 400 nM primers (a range of 100 – 600 nM were used in optimization studies), 300 nM TaqMan probe (a range of 50–300 nM were used in optimization studies) for each of the target genes. A master mix was prepared, vortexed and aliquoted (24μl) into the wells of a 0.2-mL optical-grade 96-well PCR plate (Applied Biosystems). Then, 1μl of cDNA template was added to a final volume of 25μl. All reactions were carried out in triplicate with no template control as well as no-RT sample. The thermal cycle conditions were: 50 °C for 2 min (AMPerase activation), 95 °C for 10 min (Taq activation), 95 °C for 15 sec for denaturation, and 60 °C for 1 min for annealing and extension. The AB 7500 system measures a fluorescent accumulation of PCR product by continuous monitoring. The program was set to monitor the complete amplification for 50 cycles. All TaqMan PCR data were captured using the SDS software (Applied Biosystems). For every sample, an amplification plot was generated showing the increase in the reporter dye fluorescence (Rn) with each cycle of PCR. Quantification of input target amount was analyzed by cycle threshold (CT) value, the point at which the sample PCR amplification plot crosses the threshold.

2.6. Optimizing primer and probe concentrations

For each target gene, the optimum primer and probe concentration was determined with a fixed template level (104 copy numbers). A combination of four concentrations (100 – 600 nM) of both forward and reverse primers with a constant probe concentration was determined. The combination showing the highest fluorescence was tested thereafter at four different TaqMan probe concentrations (50 – 300 nM). The combination of primer and TaqMan probe concentration that yielded optimal assay performance was chosen for further experiments.

2.7. Generation of calibration curves

A calibration curve approach was used to establish the results obtained by the TaqMan PCR assay. For this purpose, we prepared mouse whole brain cDNA using purified total RNA. PCR products were generated for each target subunit with specific primers using mouse whole brain cDNA as template. The specificity of PCR products generated for each subunit was confirmed by agarose-gel electrophoresis and ethidium bromide staining. The single bands were excised from the gel and eluted using the gel extraction kit (Quiagen, Valencia, CA). PCR products were sequence verified by sequence service center (MWG, High Point, NC). Each target subunit PCR product was quantified using spectrophotometer. Copy number (C, copies/μl) for each subunit template was calculated using the following equation: C = PA/GMX, where P is the PCR product concentration (ng/μl), A is Avogadro’s constant (6 × 1023), G is the amplicon size (bp), M is the multiplying constant (1 × 109) and X is the average molecular weight of the 1 bp in the amplicon (650 Daltons). Copy numbers for each target subunit were serially diluted from 1010 to 101 copies and were used as template for TaqMan assay. A calibration curve was plotted for each target subunit and GAPDH using known copy number verses CT values.

2.8. Data analysis

Linear regression and correlation analysis of real-time PCR amplification data was performed using SDS software (Applied Biosystems). Standard curves for each subunit gene were plotted showing CT value versus log of initial copy number of cDNA. The slope of the standard curve was calculated to describe the efficiency of PCR (if the PCR amplification is exponential, resulting in a doubling of product in every cycle, the slope will be −3.3 as 3.3 cycles are required to generate a 10-fold increase in product). The GABA-A receptor subunit expression was analyzed based on the relative quantification approach as outlined in AB 7500 User Bulletin (Applied Biosystems). The relative quantification provides accurate change in expression of the target gene in the experimental sample relative to a control calibrator sample. The initial target input amount for each target subunit and GAPDH were calculated using standard curve method. The quantity calculated for target subunit was normalized with GAPDH as the endogenous control. The normalized value of control (vehicle group) was taken as the calibrator to calculate the relative expression levels of target genes in the experimental group. The target gene expression in the samples was expressed as percent change from control. The unpaired t-test was used to compare the effect of neurosteroid withdrawal on α4 subunit expression in mice. The criterion for statistical significance was p< 0.05.

3. Results

3.1. Specificity of TaqMan probe and primers

The TaqMan probe and primer pair designed for GABA-A receptor subunit genes are listed in Table 1. Specificity of primers and probes were verified by homology search. To confirm the specificity of primers for each target subunit, a traditional RT-PCR followed by agarose gel electrophoresis was performed using mouse whole brain cDNA as a template. As shown in Fig. 2, a single PCR product with the desired length was observed for each target subunit gene and the reference GAPDH. No band was observed in the negative control samples without RT (data not shown). To ascertain the possible contamination or non-specific products in the PCR reaction, all primer pairs were also tested with no template cDNA as negative control that showed no band (Fig. 2). Moreover, nucleotide sequence analysis of the RT-PCR products revealed an identical sequence to each target GABA-A receptor subunit amplicons as listed in Table 1. Therefore, the probe and primers pairs were highly specific to target genes and do not produce non-specific amplicons.

Fig. 2
Gel electrophoresis of PCR products of GABA-A receptor subunits and GAPDH
TABLE 1
Sequences of primers and TaqMan probes for mouse GABA-A receptor subunits.

3.2. Amplification plots and sensitivity of TaqMan reactions

Amplification plots were derived for GABA-A receptor subunits (α1–6, β1–3, γ2, and δ) and GAPDH using increased template copy numbers ranging from 102 to 109. A representative amplification plot of the α2-subunit was shown in Fig. 3. The plot shows that fluorescence (Rn) increases with increased copy number of template cDNA in the samples. The samples with highest target subunit template were detected earlier in the PCR cycles as shown by the leftward shift of the amplification plot. Amplification plots show that the TaqMan assay was highly sensitive to detect very low levels of gene expression (~100 copies of the template cDNA). The amplification plots provided two valuable parameters – threshold (a level of delta Rn that was automatically set to be above the baseline) and cycle threshold, also known as CT value (the fractional cycle number at which the fluorescence passes the threshold). Quantification of input target amount was analyzed by CT value, which in turn predicts the quantity of input target cDNA. The CT value was utilized for construction of standard curves and data analysis in the TaqMan assay.

Fig. 3
Amplification plots of the GABA-A receptor α2-subunit in the TaqMan real-time PCR assay

3.3. Optimization of primers and probe concentrations

We optimized the probe and primer concentrations by performing a series of experiments with varying primer-probe combinations. Figure 4 shows the amplification plots for all primer concentration combinations for the GABA-A receptor α1 subunit and GAPDH. The CT values derived from these plots were listed in Table 2. The combination of 400 nM forward and reverse primer combination showed a plot with both the highest florescence and lowest CT value. At this primer concentration, the probe combination of 300 nM yielded a plot with both highest florescence and lowest CT value (Table 2). Further experiments were carried out using a combination of 400 nM primers and 300 nM probe concentration.

Fig. 4
Optimization of primer and TaqMan probe concentrations in the TaqMan assay
TABLE 2
CT values of GAPDH and the GABA-A receptor α1-subunit derived from amplification plots with various primer and probe combinations.

3.4. Selection of endogenous reference gene

To quantify levels of target mRNA expression, the GAPDH was used as an endogenous reference gene for relative analysis of gene expression. A specific set of primers and probe was designed for mouse GAPDH gene and the levels of GAPDH mRNA in samples from control and experimental groups were determined by TaqMan real-time PCR. Data from extensive series of experiments shows that CT values were similar in different groups (CT values ranged 17– 18), indicating that GAPDH expression was found to be unaffected by treatments.

3.5. Standard curves of GABA-A receptor subunit genes

To determine the PCR efficiency and linearity of template amplification, standard curves were constructed for each of the GABA-A receptor subunit genes and GAPDH using mouse whole brain cDNA as template. As illustrated in methods, known copy numbers calculated from the purified PCR products ranging from 102 to 108 copies of the templates were used. The TaqMan assay was performed in triplicate for 50 cycles and CT values obtained were plotted against the log cDNA copy numbers. Standard curves derived for α1, α2, α4, and α6 subunit and GAPDH were shown in Fig. 5. Standard curves for other GABA-A receptor subunits (β2, β3, γ2, and δ) were shown in Fig. 6. A reverse linear relationship was detected over four orders of magnitude for each of the target subunits and GAPDH. PCR amplification efficiency was assessed for each primer and probe set from the slope of the standard curve. In most cases, the slope of the standard curve was close to –3.4, indicating maximal PCR amplification efficiency. The CT values and cDNA template concentrations show a reverse linear correlation with nearly identical slopes. The correlation coefficients were in the range of 0.990 to 0.998, indicating the accuracy of the amplification efficiency.

Fig. 5
Standard curves for GAPDH and selected GABA-A receptor subunits
Fig. 6
Standard curves for GAPDH and selected GABA-A receptor subunits

3.6. Effect of neurosteroid withdrawal on α4 subunit expression

The optimized protocol for TaqMan assay was utilized to determine changes in the expression of the hippocampal GABA-A receptor α4 subunit gene, which is known to significantly upregulate in animals undergoing withdrawal from chronic progesterone or neurosteroid treatment. Mice were treated chronically with progesterone to induce high levels of neurosteroids and then withdrawn by finasteride injection to inhibit biosynthesis of the neurosteroid allopregnanolone. As shown in Fig. 7B, the α4-subunit expression was increased significantly (~3-fold) in the hippocampus following neurosteroid withdrawal in mice. The absolute expression, represented as the mRNA copy number, was found to be similar and consistent with the pattern of change noted in relative expression using GAPDH as an internal control (Fig. 7A). There was no change in expression levels of GAPDH following neurosteroid withdrawal (control: 205,937 ± 9,868 copy numbers; withdrawal: 210,062 ± 29,339 copy numbers). These results are consistent with the previous studies demonstrating that progesterone or neurosteroid withdrawal increases the α4 subunit expression in the hippocampus (Smith et al., 1998; Gulinello et al. 2001).

Fig. 7
Changes in the GABA-A receptor α4-subunit expression in the hippocampus following neurosteroid withdrawal in mice

4. Discussion

The present study shows successful optimization of TaqMan real-time RT-PCR assay for analysis of GABA-A receptor subunit expression in brain tissues. The key findings of this study are: (i) The TaqMan technology is optimized for quantification of mouse GABA-A receptor α1–6, β1–3, γ2 and δ subunits; (ii) The primers and probes used in the assay selectively amplify the specific target gene as verified by the electrophoresis of PCR products and sequencing of the products; (iii) The TaqMan assay is highly sensitive and can detect very low levels of gene expression (~10 to 100 copies of template mRNA), and thus requires very small amount of mRNA (low ng levels) compared to traditional RNA assays; (iv) The TaqMan assay is superior in specificity and accurately measures target subunit expression using the GAPDH as internal control; and (v) The TaqMan assay is effectively utilized by confirming the neurosteroid withdrawal-induced upregulation of the α4-subunit expression.

The real-time PCR is a versatile tool that can be optimized for enhanced sensitivity, high throughput analysis, and minimal post-PCR manipulations. Two types of fluorescent regents (SYBR green dye and TaqMan probe) allow for real-time monitoring of the PCR. The SYBR green method is widely used to measure the fluorescence of amplified products in the real-time quantitative PCR assay (Gutala and Reddy, 2004). The SYBR green assay is a less-expensive alternative to TaqMan assay. However, SYBR green chemistry is less specific and can detect non-specific amplification products because of its intercalating mechanism. Since SYBR green nonspecifically detects all double-stranded DNA, it is not possible to distinguish between the specific amplified product and the primer-dimers formed during the reaction. Unlike SYBR green technology, the TaqMan assay is highly specific because the probe is designed for highly specific hybridization with target cDNA sequences. The TaqMan PCR is more suitable for transcriptional profiling of GABA-A receptor subunit plasticity because of several closely related subunits.

In the present study, we optimized primers and probes for mouse GABA-A receptor subunit family (Table 1). The sequences are retrieved from NCBI web site. Primers and probes are designed for each subunit gene using optimum parameters according to the guidelines described for real-time PCR. In the design of TaqMan probes, we considered several important factors such as Tm, the presence at 3′ end of the primer of A’s or T’s, and shorter amplicon size. Primers for each subunit are chosen to select from two different exons. This type of intron flanking primer design distinguishes the amplicons by their size from genomic DNA contamination. The γ2 subunit has two splice variants such as γ2L, γ2S which differ by 24bp. Primers and probes for γ2 subunit are designed from a common sequence for both splice variants, so that in these one would recognize expression of both the splice variants. Primers for all subunit genes are validated by performing regular RT PCR using mouse whole brain cDNA as a template. The specificity of primers is confirmed by a single band with correct size amplicon detected on agarose gel-electrophoresis. These PCR products are gel purified and further confirmed by DNA sequencing. Thus, the TaqMan PCR offers additional sensitivity because of the dual-labeled probe, which is specific for each target subunit. This is a highly desirable feature when analyzing a complete superfamily of GABA-A receptor subunits that have multiple closely related homologous variants.

In real-time PCR assays, a quantitative relationship exists between amount of input target template and amount of PCR product at any given cycle number. Therefore, amplification of the target gene at high efficiency over dynamic input ranges is important for accurate quantitation of gene expression. In the present study, a standard curve for GAPDH and each target gene is constructed (102 – 109 copy numbers of template cDNA). Purified PCR products generated using the specific primers of each target subunit are used as a template to generate standard curves. The standard curve demonstrated excellent correlation coefficient ranging from 0.990 to 0.998 indicating accurate amplification of each target subunit gene. The slopes for GAPDH and target genes are around −3.4, reflecting similar optimum PCR efficiencies. The template concentrations are prepared with suitable dilution so that the input levels can be detected from the standard curves. It is possible that the amplification may not be equally scaled at certain dilutions, especially lower dilutions (higher copy numbers). However, such exceptions do not appear to affect the amplification of target subunits because standard curves are highly consistent with ideal amplification of target subunits with slopes around −3.4 (r2=0.99). Amplification plots can be further optimized using alternative primers and probe for maximum efficiency and specificity.

The present study utilized the relative expression approach for quantification of target gene expression. Generally two quantification strategies can be utilized in real-time PCR: an absolute and a relative quantification. In absolute quantification, the absolute mRNA copy number is determined by comparison to appropriate external calibration curve (Pfall, 2001; Pfaffl and Hageleit, 2001). The relative expression is based on the expression levels of a target gene versus a reference gene and is adequate for most purposes to investigate physiological changes in gene expression levels. In this study, GAPDH is chosen as a reference gene. Reliable endogenous controls are critical for achieving accurate results in real-time PCR. Ideally, the endogenous control should not be altered in the disease or under treatment conditions, and should be expressed roughly the same level as the RNA under study (Thellin et al., 1999; Bustin, 2000). GAPDH is the most widely used reference gene (Uno et al., 2002; Horii et al., 2002). In the present study, the expression of GAPDH is not altered under the neurosteroid withdrawal conditions. Alternative reference genes such as β-actin, β2-microglobulin or 18S RNA or other unregulated housekeeping genes can be considered if the GAPDH is likely to change under the experimental conditions (Gutala and Reddy, 2004; Floyd et al., 2004). There are several advantages of using more than one housekeeping gene for the purpose of data normalization such as avoiding false positive changes and accurate detection or expression of fold change in target gene expression.

The TaqMan assay for GABA-A receptor subunit expression can be used to accurately measure changes in subunit expression under various physiological or pharmacological conditions. Progesterone exposure and withdrawal has been shown to distinctly affect GABA-A receptor subunit plasticity (Follesa et al., 1998; Reddy, 2004). The most striking finding following withdrawal from progesterone or the neurosteroid allopregnanolone is the marked up regulation of the α4-subunit in the hippocampus (Smith et al., 1998; Gulinello et al., 2001). It has been suggested that the increased expression of the α4- subunit might decrease the net GABA-A receptor-mediated inhibition and promote excitability leading to increased seizure exacerbation (Reddy, 2004). Most of those subunit plasticity studies utilized conventional RNA assays (Wisden et al., 1992; Follesa et al., 1998; Liu et al., 2002) or Western blots (Sun et al., 2004; Maguire et al., 2005). Despite some reports using real-time PCR (Linneman et al., 2006; Byrnes et al., 2007), a detailed protocol describing TaqMan methodology for GABA-A receptor subunits is lacking. In the present study, the TaqMan assay is used to determine the effect of neurosteroid withdrawal on GABA-A receptor α4- subunit expression in the hippocampus in mice. We found that α4- subunit is upregulated significantly following neurosteroid withdrawal. These results are highly consistent with previous reports on α4-subunit expression (Smith et al., 1998; Gulinello et al. 2001).

The TaqMan RT-PCR has several advantages over conventional RT-PCR methods. The TaqMan PCR is a “closed-tube” homogenous system avoiding time-consuming and hazardous post-PCR processing and decreasing the potential risk of contamination. The TaqMan PCR allows precise and reproducible quantification because it is based on CT values rather than end-point detection, where the PCR components are rate limiting. The amount of cDNA template required (1 to 25 ng) is much lower than the amount needed for the conventional RNA assays such as Northern blot analysis (μg). This method is very useful for analysis of GABA-A receptor subunits in single cells or in samples from single RNA pool with low copy number of the subunit genes. Other advantages include the availability of automated real-time PCR systems that rapidly monitor a large number of samples in a 96- or 384-well format that permits a high-throughput screening of large sample size.

In conclusion, these results suggest that the real-time TaqMan RT-PCR assay is a precise, reliable, and sensitive method for quantification of GABA-A receptor subunits in brain tissues. This TaqMan assay allows rapid, high-throughput profiling of complete GABA-A receptor superfamily, and thus provides a powerful tool for investigations of GABA-A receptor subunit plasticity in animal models of neurological and psychiatric conditions.

Acknowledgments

This work was supported by the U.S. National Institutes of Health (NIH) grant R01 NS051398 (to DSR).

Footnotes

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