The use of TaqMan RT-PCR assays for semiquantitative analysis of gene expression in CNS tissues and disease models
Introduction
Tissue localisation of mRNA expression and changes in gene expression induced in various CNS animal models have traditionally been studied using Northern blot analysis, competitive PCR, in situ hybridisation or RNAse protection assays. For example, brain-derived neurotrophic factor (BDNF) mRNA expression has been shown to be upregulated after KCl-induced cortical spreading depression (CSD) in rats by in situ hybridisation (Matsushima et al., 1998) and by Northern blotting (Kawahara et al., 1997). In addition, in the rat permanent middle cerebral artery occlusion (pMCAO) model of cerebral ischaemia, endogenous interleukin 1 receptor antagonist (IL-1ra) mRNA has been shown to be upregulated using RT-PCR (Wang et al., 1997). Although these techniques have proven successful and reliable, they can require large amounts of RNA, can be difficult to optimise and quantify, and may require time consuming downstream processing of samples such as gel electrophoresis. Furthermore, they are not generally amenable to rapid analysis of multiple gene targets or large sample numbers.
TaqMan RT-PCR is a recently developed technique which enables the measurement of an accumulating PCR product in real time by utilising a dual-labelled TaqMan fluorogenic probe (Heid et al., 1996, Lang et al., 1997, Lie and Petropoulos, 1998). The fluorogenic 5′-nuclease PCR assay utilises the endogenous 5′–3′ nuclease activity of Taq polymerase to digest the TaqMan probe which hybridises to the target sequence during PCR. When the probe is intact the reporter fluorophore emission is suppressed by the quencher fluorophore. During PCR the probe anneals to the DNA template and the 5′–3′-nuclease activity of Taq polymerase releases the reporter from the vicinity of the quencher dye resulting in increased reporter fluorescence (Fig. 1). The remaining probe dissociates from the target sequence allowing the polymerase reaction to continue. The intensity of fluorescence is directly related to the amount of input target DNA. The fluorescent signal is captured using an ABI Prism 7700 sequence detection system (PE Applied Biosystems) which allows for the rapid screening of many transcripts from a single pool of tissue samples in 96-well format.
Due to a lack of reports describing the use of TaqMan technology in animal models of CNS diseases and CNS localisation experiments, the aim of this study was to validate the TaqMan technology for various neuroscience applications, by investigating the localisation of genes with known expression profiles, and also by studying genes previously shown to be upregulated in animal models. Our results show TaqMan data are consistent with other mRNA expression techniques such as in situ hybridisation and Northern blotting. Critical parameters important for the correct evaluation of gene expression by TaqMan analysis are also discussed.
Section snippets
Tissue acquisition and RNA extraction
Human polyA+ mRNA samples extracted from CNS and peripheral tissues were obtained from Clontech. These were pooled samples from multiple non-diseased individuals, consisting of both males and females aged 10–78. For animal studies, CNS and peripheral tissues were obtained from Sprague–Dawley rats. Permanent middle cerebral artery occlusion (pMCAO) of the left middle cerebral artery was carried out by inserting an intraluminal filament at the origin from the Circle of Willis in
Determination of PCR sensitivity and efficiency
The sensitivity of PCR using different primer/probe sets was established from the threshold cycle (Ct) values obtained using known quantities of genomic DNA. For the majority of primer/probe sets a Ct of 21–26 was obtained with 65 000 copies of human genomic DNA or 25 000 copies of rat genomic DNA (Table 1). The exception to this was the rat GAPDH primer/probe set which was particularly sensitive (Ct=16).
To determine the PCR efficiency and whether the relationship between Ct and log starting
Discussion
In this study, we have demonstrated the successful utilisation of TaqMan RT-PCR technology for the mapping of mRNA distribution in both human and rat tissues, as well as for the identification of genes upregulated in CNS animals models. We have clearly demonstrated distribution profiles and changes in gene expression in animal models similar to those described previously using other techniques such as in situ hybridisation, Northern blotting, RT-PCR or receptor autoradiography. For example, the
References (18)
- et al.
Ischemia-induced changes in α-tubulin and β-actin mRNA in the gerbil brain and effects of bifemaline hydrochloride
Brain Res.
(1993) - et al.
A polymerase chain reaction assay for the detection and quantification of cytokine gene expression in small numbers of cells
J. Immunol. Methods
(1992) - et al.
Dopamine receptors and brain function
Neuropharmacology
(1996) - et al.
Cortical spreading depression induces long-term alterations of BDNF levels in cortex and hippocampus distinct from lesion effects: implications for ischaemic tolerance
Neurosci. Res.
(1997) - et al.
Quantification of cytokine mRNA expression by RT-PCR in samples of previously frozen blood
J. Immunol. Methods
(1997) - et al.
A rapid method for semiquantitative analysis of the human Vβ-repertoire using TaqMan PCR
J. Immunol. Methods
(1997) - et al.
Rapid feline immunodeficiency virus provirus quantitation by polymerase chain reaction using the TaqMan fluorogenic real-time detection system
J. Virol. Methods
(1999) - et al.
Advances in quantitative PCR technology: 5′ nuclease assays
Curr. Opin. Biotechnol.
(1998) Allelic discrimination using fluorogenic probes and the 5′-nuclease assay
Gen. Anal. Biomol. Eng.
(1999)
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2013, Neurobiology of DiseaseCitation Excerpt :Six μl of the striatal RNA or 14 μl STN RNA, respectively, were converted into cDNA using the iScript cDNA synthesis kit including a blend of oligo (dT) and random hexamers (BioRad). Primer sequences were designed with NCBI primer3 software, except for Dat, Gapdh, Gad67, Hprt1, Ppia, and Th, which were adapted from the literature (Hommel et al., 2003; Medhurst et al., 2000; Schiff et al., 2009; Zhang et al., 2010), and spanned exon–intron boundaries (Table 2). The qRT-PCR was performed in 96-well plates using the StepOnePlus instrument (Applied Biosystems) as described previously (Ratzka et al., 2011).