JXB Advance Access originally published online on June 18, 2004
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Journal of Experimental Botany, Vol. 55, No. 402, pp. 1445-1454, July 2004
Journal of Experimental Botany, Vol. 55, No. 402, © Society for Experimental Biology 2004; all rights reserved
REVIEW ARTICLE |
Real-time PCR: what relevance to plant studies?
Institut de Biotechnologie des Plantes, UMR CNRS 8618, Université Paris-Sud, F-91405 Orsay cedex, France
* To whom correspondence should be addressed. Fax: +33 1 6915 3425. E-mail: charrier{at}ibp.u-psud.fr
Received 19 January 2004; Accepted 22 April 2004
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Abstract |
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 Top Abstract IntroductionRelevant features of real-time...Detection and quantification of...Quantification of specific...Limitations and future...ConclusionsReferences |
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The appearance of genetically modified organisms on the food market a few years ago, and the demand for more precise and reliable techniques to detect foreign (transgenic or pathogenic) DNA in edible plants, have been the driving force for the introduction of real-time PCR techniques in plant research. This was followed by numerous fundamental research applications aiming to study the expression profiles of endogenous genes and multigene families. Since then, the interest in this technique in the plant scientist community has increased exponentially. This review describes the technical features of quantitative real-time PCR that are especially relevant to plant research, and summarizes its present and future applications.
Key words: Expression, fluorochrome, genetically modified organism, Molecular Beacon, pathogen, ScorpionTM, SYBRgreen®, TaqMan®, technique, transgene
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Introduction |
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TopAbstractIntroductionRelevant features of real-time...Detection and quantification of...Quantification of specific...Limitations and future...ConclusionsReferences |
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Real-time PCR differs from classical PCR by the measurement of the amplified PCR product at each cycle throughout the PCR reaction. In practice, a video camera records the light emitted by a fluorochrome incorporated into the newly synthesized PCR product. Thus, real-time PCR allows the amplification to be followed in real-time during the exponential phase of the run, and thus allows the amount of starting material to be determined precisely. Contrary to end-point PCR techniques, the result is independent from the plateau corresponding to the saturation of the reaction, the latter leading to inaccurate quantification. Figure 1 shows the main principles of the real-time PCR process, while the details and requirements necessary to obtain reliable data have been reviewed by Freeman et al. (1999)
and Bustin (2000, 2002). Besides being an alternative to some well-established laboratory techniques, real-time PCR has a number of features which makes it the choice for several types of study. Compared with the other techniques presently available, it allows the detection of a given nucleic acid target in a rapid, specific and very sensitive way. In addition, it affords the absolute quantification of the initial target. To date, the reliability of real-time PCR has never been questioned.
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Relevant features of real-time PCR |
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TopAbstractIntroductionRelevant features of real-time...Detection and quantification of...Quantification of specific...Limitations and future...ConclusionsReferences |
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Rapidity
Compared with classical PCR, one of the main advantages of real-time PCR is its rapidity to provide reliable data. Typically, the time of a whole real-time PCR run ranges from 20 min to 2 h. Indeed, the time needed to shift temperature is a major limiting factor responsible for the duration of a classical PCR experiment. The LightCyclerTM PCR machine (Roche) uses capillaries instead of tubes, which are heated by light instead of a heating block. As a result, the time necessary to heat the PCR mixture is considerably reduced (from 15 s to 1–2 s). In addition, recording the amplification in real-time avoids collecting samples at different steps of the PCR experiment, making the process less tedious and time-consuming. Moreover, some machines accommodate 384 well plates and can process queuing plates over 24 h non-stop (Wurmbach et al., 2003
), which might be a determining advantage for high throughput studies, or if rapid sample processing is required (Schnerr et al., 2001).
Sensitivity
Real-time PCR provides a high sensitivity for the detection of DNA or RNA due to a combination of the amplification performed by the PCR step and the system of detection (Bustin, 2000). It is therefore a very convenient technique for studies with a limited amount of starting material (Bago et al., 2002), or for assessing the expression of a high number of genes from minute quantities of RNA. The detection is based on the measurement of the fluorescence emitted by probes incorporated into the newly formed PCR product, or alternatively released into the buffer during the amplification of the PCR product. Intercalating agents and fluorogenic probes are the two main types of molecules currently used to detect PCR amplification in real-time. The first intercalating agent used was ethidium bromide, but in 1997, Wittwer et al. (1997a, b) proposed replacing it with the SYBRgreen® molecule, because of its higher affinity for double-stranded DNA. As intercalating agents bind regardless of the nucleotide nature, they can be used for any type of sequence. This is an economical advantage for a laboratory testing a large number of genes. However, a disadvantage of SYBRgreen® is that it is equally incorporated into every amplicon, and should unspecific sequences be amplified, the signal measured would correspond to both non-specific and specific products, thereby compromising the accurate quantification of the latter (see section ‘Specificity’ for further developments on this issue).
In order to bypass this potential problem, intercalating agents can be replaced by labelled oligonucleotides or probes, which specifically bind to the target sequence. This technology relies on the use of probes labelled with two different fluorochromes, one of which, when excited, is able to transfer its energy to the other via Fluorescence Resonance Energy Transfer (FRET). This non-radiative energy transfer only occurs if the two molecules are in close proximity to each other (a few nanometres). Depending on the proximity of the second fluorochrome, the first one may either emit light or transfer its energy to the second, which in turn fluoresces. Thus, bringing the two fluorochromes in close proximity to each other results in the fluorescence quenching of the first one, and fluorescence emission of the second one.
As the fluorescence from the emitting fluorochrome increases proportionally with the amount of newly synthesized DNA, both effects can be recorded to follow the amplification of the target DNA. Hence, several strategies have been developed, all of which rely on placing them in the vicinity of each other (excitation) or conversely ensuring their separation (quenching) during the amplification phase. So far, FRET-mediated excitation has rarely been used in plant studies (Busch et al., 2002), and its use, which requires four oligonucleotides, should be limited to studies requiring a very high level of specificity. The application of quenching systems has been more common in plant studies, being initiated by the use of the TaqMan® probes, followed by the Molecular Beacons and ScorpionTM probes. As they require the design of a labelled oligonucleotide specific for each sequence, they are economically relevant only if many experiments are to be performed on the same target. Figure 2 illustrates the main strategies currently developed and used in plant studies.
Specificity
Surprisingly, in a study carried out on four pea thioredoxin h (TRXh) encoding genes, Montrichard et al. (2003) noticed that real-time PCR yielded weaker signals than expected from northern blot analyses. This observation was explained by a cross-hybridization of the probe to the RNA encoding another isoform during the northern blot procedure. Indeed, in contrast to techniques requiring the hybridization of nucleic acids several hundreds base pairs long, such as cDNA-based microarray and northern blotting, short oligonucleotide-mediated real-time PCR guarantees a high specificity in the detection of the target sequence. In fact, specificity is achieved by the use of two target sequence-specific oligonucleotides, and this can be enhanced by increasing the number of oligonucleotides nested within the initial amplification product. In this respect, FRET-mediated probes seem to ensure a higher specificity than SYBRgreen® (Shimada et al., 2003). In any case, specificity of the process can be checked after completion of the PCR run, by testing the nature of the amplified product with gel electrophoresis, melting curves, and sequencing data.
Quantification
Most importantly, the quantification range of real-time PCR is up to seven orders of magnitude as originally illustrated by Higuchi et al. (1993), Heid et al. (1996), and more recently in plants by Charrier et al. (2002), Filion et al. (2003), and Hernández et al. (2003a). This results from the capacity of this technique to calculate, for every sample within an extremely low to high concentrations range, the number of cycles necessary to reach the Ct (see Fig. 1 for definition). The absolute amount of the target is calculated from a calibration curve. Alternatively, a relative quantification can be deduced considering Ct differences between samples and standards as nicely illustrated by Bovy et al. (2002), and improved by Pfaffl (2001).
Basically, real-time quantitative PCR may be used for quantifying DNA or RNA abundance, leading to two major types of applications: foreign DNA (e.g. transgenes or contaminating micro-organisms) detection and quantification, and gene expression studies.
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Detection and quantification of foreign DNA |
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TopAbstractIntroductionRelevant features of real-time...Detection and quantification of...Quantification of specific...Limitations and future...ConclusionsReferences |
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Quantification of pathogenic or symbiotic micro-organisms associated with plants
Real-time PCR assays aiming at quantifying the level of plant infection by a pathogen have been increasing for the last few years, since the first report by Böhm et al. (1999)
. Most of them rely on the relative quantification of two specific plant and pathogen DNA sequences. They are faster, more specific and more sensitive when compared with traditional protocols based on symptom recording or on conidiophore or colony counting (Winton et al., 2003), and, most importantly, may be transposed to virtually every pathosystem. For those reasons, they are being widely used for the diagnosis of diseases in the field and for applied purposes. For instance, seed potatoes cannot be sold in the EU unless they are devoid of the potato brown rot agent Ralstonia solanacearum (Council directive 2002/56/EC). Classical detection methods require a labour-intensive culture and pathogenicity test on tomato seedlings. However, real-time PCR has been shown to enable the quantitative detection of R. solanacearum in a rapid and reliable manner, thus providing an improved alternative assay that could be implemented on a large scale (Weller et al., 2000).
Likewise, food contamination by mycotoxins is of great concern, since many have been found to be carcinogenic and they are not easily removed during food processing. However, since toxin abundance does not correlate with fungal contamination, but is linked to the toxinogenic properties of each strain, real-time PCR detection assays targeted at genes involved in toxinogenesis have been developed for trichotecene-producing Fusarium and aflatoxin-producing Aspergillus species (Mayer et al., 2003; Schnerr et al., 2001). Petit et al. (2003) recently implemented a refinement of this technique based on the quantification of the nor1 mRNA, which directly addresses aflatoxin biosynthesis in infected wheat. As many countries are becoming more and more concerned about food safety, the market for such applications is growing rapidly.
Real-time PCR application in fundamental studies is still lagging behind, and only a few real-time PCR-based pathogenicity assays have been reported in this field (van Wees et al., 2003; Hiriart et al., 2003). Most of the currently used resistance tests rely on visual assessment of the symptoms and spore or colony counting. However, Brouwer et al. (2003) recently implemented real-time PCR tests to quantify a number of pathogens on Arabidopsis and demonstrated that they are a very interesting alternative to classical tests. More details about pathogen detection assays can be found in Schaad and Frederick (2002), McCartney et al. (2003), and Schaad et al. (2003).
Contamination of processed food by foreign DNA
A requirement for methods capable of accurately quantifying food contaminants has emerged, due to the introduction of stringent food safety regulations. Since most of them enforce a tolerable level of contamination, accurate quantification is of crucial importance for agribusiness companies. Indeed, on the one hand, they have to comply with those maximal authorized levels, while on the other hand, the rejection of batches falsely labelled as contaminated would lead to unnecessary costs. In this context, real-time quantitative PCR is becoming the technique of choice for assessing food contamination or adulteration. Compared with ELISA, PCR assays are easier to develop since they do not require raising specific antibodies. They provide a higher sensitivity and are better suited for the detection of unwanted food ingredients in highly processed food, notably because DNA is more thermo-stable than proteins. For example, the absence of gluten in baby food can be controlled by an amplification test of cereal genes by real-time PCR (Sandberg et al., 2003). Likewise, real-time quantitative PCR has been shown to be an ideal tool for assessing common wheat (Triticum aestivum) adulteration in durum wheat pasta (Alary et al., 2002; Terzi et al., 2003), as Spanish, Italian, and French regulations enforce a 3% maximal level of common wheat contamination in pasta and semolina.
Another field of applications was born with the launching of genetically modified organisms on the market. Indeed, the European Community Council recently enacted a new regulation (EC Regulation no. 1829/2003) enforcing the compulsory labelling of food containing more than 0.9% GMOs. However, GMO detection is not trivial and current assays present a number of worrying limitations, which have been nicely reviewed by Ahmed (2002) and Auer (2003). Briefly, protocols aimed at detecting transgenic DNA contamination were developed mainly for transgenic soybean (Berdal and Holst-Jensen, 2001; Hird et al., 2003) and maize (Brodmann et al., 2002; Vaïtilingom et al., 1999), and more rarely for other species such as rapeseed (Zeitler et al., 2002). A major limitation of PCR-based detection assays is that a new set of oligonucleotides has to be designed for each transgenic line, except when they are targeted to common DNA regions such as the CaMV35S promoter (Hohne et al., 2002). Alternatively, event-specific protocols have been developed for unique lines, such as Starlink (Windels et al., 2003) and Bt11 (Ronning et al., 2003) transgenic maize. In order to make the detection specific for only one given and identifiable event, scientists have cloned the borders separating the transgene from the rest of the host genome, and used them as specific markers of the given event (Hernández et al., 2003b; Taverniers et al., 2001).
Genetics of transgenes
Transgenic plants are easily amenable to genetic analyses when the transgene is inserted as a single copy within the host genome. Ingham et al. (2001) showed that duplex real-time PCR can be used to determine transgene copy number in transformed plants. They found a strict correlation between their results and Southern blot analyses, except for two lines (out of 37) in which the discrepancy could be ascribed to multiple insertions at a single locus. Overall, they demonstrated that real-time PCR provided a fast and robust method for this application, which could easily be automated and applied to a large number of samples. In lines containing a single insertion, multiplexed PCR could even discriminate between homozygous and hemizygous plants. If previously genotyped lines are not available to calibrate the assay, German et al. (2003) proposed using hemizygous T0 plants as a standard. In this way, homozygous plants can be selected before they produce seeds, thereby avoiding laborious segregation analyses.
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Quantification of specific transcripts |
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TopAbstractIntroductionRelevant features of real-time...Detection and quantification of...Quantification of specific...Limitations and future...ConclusionsReferences |
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Integrated expression analyses
Integrative developmental biology requires the parallel analyses of genes involved in the same physiological process. The study of their expression profile may be useful in understanding the cellular function of the encoded proteins. Indeed, in yeast, groups of genes displaying the same expression profile are enriched in genes encoding proteins interacting physically (Ge et al., 2001
). However, comparisons between numerous gene expression profiles require a sensitive and reliable technique avoiding errors inherent in independent RNA preparations. Because of its sensitivity, real-time PCR is able to meet this technical requirement, as the expression of numerous genes may be tested on the same RNA preparation. Taking advantage of this, Shimada et al. (2003) used real-time PCR to investigate the expression of eight genes involved in brassinosteroid (BR) biosynthesis, degradation or signal transduction. They were able to correlate the expression of the BR synthesizing enzymes with the presence of BR in planta, thus providing a more complete view of BR metabolism at the whole plant level. Likewise, Anterola et al. (2002) surveyed the expression of genes involved in phenylpropanoid biosynthesis in pine, following phenylalanine feeding. Their study provided very interesting insights about the coordinate regulation of seven key genes (among them phenylalanine-ammonia-lyase and coumarate-4-hydroxylase) involved in lignin precursors metabolism.
Gene families
Multigenic families are a distinctive feature of plant genomes, as opposed to animals. For instance, 66% of A. thaliana genes belong to families, with half of them containing at least five members (The Arabidopsis Genome Initiative, 2000), and information available from ESTs programs on other plant species shows that this particularity is not restricted to Arabidopsis. Analysing all the members of a gene family is necessary in order to obtain an accurate view of its overall function. Often, studies describing the expression profile of multigene family members have been performed in A. thaliana because of the possibility of an exhaustive survey. However, other plants with a high number of publicly available ESTs, such as wheat, maize, tomato, rice, potato, and the moss Physcomitrella patens, could be used for similar analyses. However, the amplification of several highly similar sequences cannot be excluded in those organisms, and data interpretation should be performed very cautiously.
In this context, several requirements have led plant scientists to use real-time RT-PCR methods rather than northern blotting. First, the analysis of more than ten genes by northern blotting is a fairly tedious and repetitive work, as several identical blots have to be prepared (Dong et al., 2003). Instead, Yokoyama and Nishitani (2001) analysed the expression profile of the 33 members of the xyloglucan transglucosylase/hydrolase (XTH) gene family in five organs of A. thaliana and in response to four hormones by real-time RT-PCR.
Secondly, genes expressed at a very low level remain difficult to detect by northern blot techniques (Brown et al., 2003; Jakab et al., 2003). In A. thaliana, the ARIADNE gene family is composed of 16 genes that have been identified as putative E3-ligases, based on their homology with Drosophila and mouse genes involved in the ubiquitin-mediated protein degradation pathway. Their expression was studied by real-time RT-PCR (Mladek et al., 2003). The transcripts of seven genes were undetectable or close to the background level, while the remaining genes were expressed, but with different absolute mRNA levels that varied up to 25-fold. The absolute transcript level remained very low, with fewer than 50 copies per nanogram of total RNA for eight of the nine AtARI genes analysed. The sensitivity of real-time RT-PCR showed that, while the expression profile was quasi-constitutive for nearly all of the AtARI genes, two of the very low expressed genes displayed a specific expression pattern (Mladek et al., 2003).
Thirdly, closely related genes that are very similar at the sequence level may cross-hybridize during northern blot procedures (Montrichard et al., 2003), and therefore, it may be difficult to determine the RNA level of a specific member of a gene family. This problem is resolved by the high specificity of real-time RT-PCR guaranteed by the use of at least two specific oligonucleotides (Fig. 2). The GSK3/Shaggy kinase family is composed of ten genes in A. thaliana (AtSK). Their expression level is rather low (2-fold lower than actin; Charrier et al., 2002), and difficult to study by northern blotting, although possible, as reported by Dornelas et al. (1999). However, some of these genes display a very high level of nucleotide sequence identity (up to 98.2%; Charrier et al., 2002), and finding probes specific for each of the ten genes requires them to be designed in the 5' or 3' UTR regions. In order to overcome these difficulties, real-time RT-PCR was used to study the expression profile of the entire gene family, both in several organs of the plant and in response to a number of abiotic stresses. Relative analyses showed that, while most of the AtSK genes were expressed constitutively, two of them displayed either some organ specificity or responded to osmotic stress (Charrier et al., 2002).
Knowing the exhaustive expression pattern of a gene family opens up the additional investigation branch of molecular and functional evolution. Indeed, several studies have shown that the expression profile of gene family members was not strictly related to the position of the corresponding proteins within a phylogenetic tree (Mladek et al., 2003; Orsel et al., 2002; Panchuk et al., 2002; Tan et al., 2003; Yokoyama and Nishitani, 2001). This suggested that the regulation of gene expression had undergone different molecular evolutionary mechanisms compared with those influencing protein sequences.
To date, only a limited number of additional studies have described gene family expression profiles using real-time RT-PCR (Balbi and Lomax, 2003; Berger et al., 2002; Kürsteiner et al., 2003; Reintanz et al., 2002; Shimada et al., 2003; Teramoto et al., 2002). Alternative methods to address the transcriptional behaviour of members of large gene families are available, such as microarray hybridization, as illustrated for 142 members of the Arabidopsis cytochrome P450 gene family (Xu et al., 2001). However, due to their specific limitations, an exhaustive picture can only be obtained by the parallel use of those methods.
Confirmation of microarray experiments
One of the fastest expanding applications of real-time RT-PCR is the confirmation of data obtained from microarray studies. Indeed, the reliability of microarray experiments may sometimes be questioned. Since plants display a high number of multigene families, cross-hybridization between cDNA representatives of members of gene families on cDNA-based chips may lead to false interpretations. On the other hand, microarray experiments can analyse thousands of genes in one step, whereas real-time PCR is often limited to far fewer genes. Real-time PCR requires the design of specific oligonucleotides for each gene to be analysed, and because of the limited number of both fluorophores and light spectra detected by real-time PCR machines, this allows the detection of fewer than five genes per multiplex PCR run. However, a maximum of two genes are analysed routinely in the same tube. Therefore, a widely used strategy is to point out a handful of potentially interesting genes with microarray experiments and to confirm those candidates by real-time RT-PCR analysis (Klok et al., 2002). As an illustration, Rider et al. (2003) randomly picked 12 candidates among a pool of 185 genes previously identified by AffimetrixTM microarray experiments as being up-regulated in the seeds of the Arabidopsis pickel mutant compared with wild-type seeds. They confirmed the up-regulation for 10 of those genes by real-time RT-PCR experiments.
While some studies are purely confirmatory, others have used real-time RT-PCR to analyse the expression pattern of the candidate genes further, either to determine fine-tuned kinetics (Goda et al., 2002; Goto and Naito, 2002), or in conditions where the available material was sparse, thereby taking advantage of the technique's sensitivity. The hybridization of cDNA microarrays with RNA of Arabidopsis seedlings infected with the incompatible fungus Alternaria brassicicola led to the identification of functional groups of genes involved in systemic acquired resistance (Schenk et al., 2003). Then, 23 genes, each representative of one of these groups, were chosen to perform a time-course study in response to A. brassicicola infection in local and distal leaf tissues by real-time RT-PCR (Schenk et al., 2003).
Whereas numerous studies have obtained similar results by real-time PCR and microarray experiments, with a linear correlation of up to five orders of magnitude (Maguire et al., 2002), several other articles report that the n-fold variation measured by real-time PCR is generally lower (up to 10 times) than that measured using cDNA microarray, where cross-hybridization may occur for highly similar, yet distinct gene sequences (Schenk et al., 2003).
Even with the AffimetrixTM chips, which are oligonucleotide-based and therefore more specific than PCR-fragment-based microarray slides, discrepancies between microarray data and real-time PCR data have been noticed. Wang et al. (2003) showed that real-time PCR data could display higher induction ratios compared with microarrays, yet conserving a good correlation between the two techniques. However, Hammond et al. (2003) noticed that the magnitude and the kinetics of the response of several genes to phosphate starvation differed between the two techniques, including opposite responses for some of them. These conflicting results were explained by the fact that the two experiments were performed with two different biological samples and different oligonucleotides.
In all cases, real-time RT-PCR is considered to be the most reliable technique and discrepancies can most often be ascribed to the normalization or background subtraction methods used in microarray analysis. Hence, real-time RT-PCR has even been used as a reference to compare different methods of microarray analyses (Puthoff et al., 2003).
The application range of real-time quantitative PCR is much broader than what can possibly be exposed in the framework of this review. For example, in the domain of DNA quantification, Jones et al. (2001) nicely exploited real-time PCR to quantify the proportion of restricted DNA corresponding to a transgene, after methylation as a result of post-transcriptional gene silencing. While the rapidity and the reliability of real-time RT-PCR has allowed the description of the expression pattern of numerous individual genes in wild-type plants (Blanco-Portales et al., 2002; Choi et al., 2002; Dambrauskas et al., 2003; Holmberg et al., 2002; Nolan et al., 2003; Shen et al., 2003; Suzuki et al., 2002; Thomas et al., 2003), mutants (Chae et al., 2003) and transgenic plants (Busch et al., 2002; Chang et al., 2003; Itoh et al., 2001), as well as the description of their molecular phenotypes (Balbi and Lomax, 2003; Maruyama-Nakashita et al., 2003), the technique's sensitivity has also been used to calculate gene silencing efficiency (Busch et al., 2002; Lacomme et al., 2003), and to determine the frequency of alternative splicing (Halterman et al., 2003).
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Limitations and future developments |
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TopAbstractIntroductionRelevant features of real-time...Detection and quantification of...Quantification of specific...Limitations and future...ConclusionsReferences |
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Despite numerous advantages, real-time PCR has some limitations. Since it is performed on small DNA fragments, real-time quantitative PCR might fail to detect biologically relevant processes like alternative splicing or partial transcript degradation occurring during post-transcriptional gene silencing events. Performing northern blots in parallel with real-time PCR should help to overcome this difficulty, as suggested by Montrichard et al. (2003)
in their study of the NADPH/NADP-thioredoxin gene expression. Once specific splicing sites are known, appropriate intron-hybridizing primers can be designed to monitor the accumulation of a specific transcript, as nicely illustrated by Halterman et al. (2003).
For convenience, most real-time PCR analyses are currently performed at the organ level, but further studies may take advantage of the technique's unequalled sensitivity, and address gene expression at the cellular level. For instance, Philippar et al. (2003) were able to study the expression pattern of a potassium channel-encoding gene after manually dissecting epidermal, mesophyll, and vascular tissues from maize leaves. Nakazono et al. (2003) also nicely demonstrated that laser-capture micro-dissection allowed epidermal cells and vascular tissue to be dissected from maize coleoptiles, obtaining about 40 ng total RNA for each tissue, corresponding to 10 000 cells. However, complementary approaches using reporter genes or in situ RNA hybridizations are still useful for addressing gene expression profiles at the cellular level (Berger et al., 2002; Reintanz et al., 2002).
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Conclusions |
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TopAbstractIntroductionRelevant features of real-time...Detection and quantification of...Quantification of specific...Limitations and future...ConclusionsReferences |
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Real-time quantitative PCR was first developed to meet specific technical requirements, such as a high sensitivity and specificity, which were not easily achieved with other classical techniques. It is now becoming a routine tool, and it is believed that, thanks to its experienced reliability, its applications will proliferate in the forthcoming years. Thanks to its rapidity, it should even replace some widely used protocols, like Southern blotting for transgenic plant analysis. A comparison of real-time PCR with other laboratory techniques with regard to their most common applications is provided in Table 1. Most evidently, real-time PCR development is still limited by the high costs of the machine and reagents, but hopefully, future will make this technology economically more widely accessible.
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Acknowledgements |
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We are grateful to Y Henry, A Picaud and M Hodges (IBP, Université Paris-Sud, Orsay, France), F Vedele and M Orsel (INRA, Versailles, France), and FC Küpper (The Scottish Association for Marine Science, Oban, UK) for critical reading of the manuscript and helpful suggestions.
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Footnotes |
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Abbreviations: FRET, fluorescence resonance energy transfer; GMO, genetically modified organism; GMP, genetically modified plant; PCR, polymerase chain reaction.
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