Abstract * Introduction * Real time * Fluorescent Markers * Analysis of the melting curve * Threshold and threshold cycle values

(cycle threshold values) * Optimization of real-time PCR * Strategies for mRNA quantification in real-time PCR * Reference Genes * Applications of real-time PCR * Future prospects for the real-time PCR * Conclusion * References Summary The identification of gene expression and specific sequences of DNA or RNA is a crucial factor when working with molecular biology techniques. To get this information some tests have been developed, the PCR being one of the most used. However, this technique has some limitations such as the difficulty of quantifying the PCR product and the risk of lead contamination in the laboratory. To address these shortcomings, real-time PCR has been developed. This technique provides a large amount of data with high sensitivity and specificity using modern platforms that prevent pollution can be caused by nucleic acid in the laboratory. On the other hand, some considerations such as choosing the strategy of quantification and fluorescent markers, and the interpretation of the data collected must be taken into account when using real-time PCR. This article reviews the basics of real-time PCR and the management of technical data from this laboratory. Keywords: real-time PCR | quantification Genetics | Molecular biology | reference genes | cDNA | reverse transcriptase (Real Time PCR: the new age of genetic information) Abstract

Identification of gene expression and specific sequences of DNA or RNA is a crucial fact-When working with molecular biology techniques. In order to Obtain this information Some Have Been Developed tests, PCR Being one of the Most used ones. However, this technique has Some Limitations as the Difficulty to QUANTIFY the PCR product and the Risk to carry out laboratory contamination. In order to face These real time PCR Disadvantages Has Been Developed. This technique eat out with an elevated output data with a high sensibility and specificity using modern laboratory contamination Platforms That Avoid That Could Be Caused by nucleic acids. Nevertheless, Several Considerations, like the choice of the strategy of Quantification and fluorescent markers as well as data interpretation, Must Be taken Into account when to working with real time PCR. This review goes Through the basic concepts on the real time PCR technique and handling of data Obtained. Keywords: real time PCR | genetic Quantification | Molecular biology | reference genes | complementary DNA | reverse transcriptase Job Type: literature review article Description of resources: text, images, 1.46 MB approx. 1 INTRODUCTION In all living cells regulate their activities through the activation or deactivation of the expression of their genes. Gene expression is generally proportional to the number of copies of messenger RNA (mRNA) of a particular gene. This fact is crucial when it comes to identifying the presence of specific cellular products and the mRNA is translated on ribosomes to form proteins. Therefore it is possible to obtain data on the production of biological agents if the expression of genes in a cell is known (McPherson et al., 2008). To address this premise, a technique known as northern blotting has been widely used. In this method purified RNA is separated by agarose gel electrophoresis and then identified with specific DNA probes (Smith & Old, 1993). Although this technique is still used to investigate the cellular gene expression, the main disadvantage is the requirement of relatively large amounts of RNA, preventing their use when limited amounts of mRNA (McGuire et al., 2004). To overcome this limitation has been developed the technique of real-time PCR, also called realtime quantitative PCR. This technique is based on polymerase chain reaction (PCR) and used to amplify and simultaneously quantify molecules of DNA or complementary DNA (cDNA) specific and thus have access to reliable and accurate data on gene expression in cells study (Heid et al., 1996).> The real-time PCR amplicons can generate very small (60 bp) making it ideal for the detection of quantitative changes in gene expression during the course of experimental or pathological cellular changes as well as for quantification of mRNA levels tissue samples with partially degraded RNA (Bustin, 2002).

An important feature of real-time PCR is its wide dynamic range. This implies that a wide range of ratios in the studied genes and normalizing genes can be analyzed with similar sensitivity and specificity (Dorak, 2008). In this test, the PCR product is measured at the end of each cycle. The data can be analyzed using computer software and the number of copies of mRNA or gene expression relative number of samples can be calculated (Heid et al., 1996). 2 Reverse transcription in real-time PCR A limitation of real-time PCR is to be used as target sequence DNA, since DNA polymerase can not amplify RNA in a similar manner. This problem can be overcome by using the enzyme reverse transcriptase to generate cDNA from an RNA template (Valasek & Repa, 2005). There are several commonly used reverse transcriptases, including reverse transcriptase have avian virus and mioblastosis the reverse transcriptase of murine leukemia virus, the first being the most robust of them. It is also possible to use mixtures of reverse transcriptase, these may lead to improved efficiency of reverse transcription enzymes individual component (Bustin, 2005b). For this reason, the real-time PCR with reverse transcriptase (RT-PCR in real time) has become the method of choice for rapid and quantitative examination of the expression of specific genes, which is not possible with previous methods ( Walker, 2002) Finally, it is important to note that since the real-time PCR and reverse transcription was used in combination, the final signal obtained from RT-PCR in real time depends on the efficiency of the reverse transcriptase reaction (Bustin et al. , 2005). 3 highlighters Several methods have been developed to quantify the PCR product. However, PCR has been used primarily as a qualitative method, since to carry out the quantification in the products of these reactions take much time and methods to realize it is relatively difficult to implement. It should also be noted that in recent cycles of PCR reaction, the amount of product is unrelated to the initial amount of DNA or cDNA in the samples analyzed (Lee et al., 2004; Valasek & Repa, 2005 .) Conversely and in contrast to previous methods which require additional components, one of the most important benefits of real-time PCR is that the entire process is performed in the thermocycler. This feature helps reduce the risk of subsequent contamination in the laboratory and can increase the performance of real-time test (Valasek & Repa, 2005). To obtain these results, this system includes two components: the thermal cycler integrated optical elements (Figure 1) and fluorescent markers that provide information on amplification along the cycles of PCR (Heid et al., 1996). Figure 1

Optical components in the thermal cycler that allows the identification of fluorescent markers. There are two main types of fluorescent markers for real-time PCR: general and specific. Techniques using specific fluorescent markers used nucleic acid probes that bind to specific amplicons (PCR product). Methods using fluorescent probes have the advantage of being very precise and to avoid possible artifacts or nonspecific sequences present in the PCR product. However, this approach requires specific sequences designed for use as probes and is therefore more laborious and costly (Lee et al., 2004). The generic detection is based on the use of dyes that bind to any double stranded sequences of DNA in a PCR reaction (Figure 2). Once the dye binds to nucleic acid formed in the reaction, and emits a fluorescent signal is processed in real time (Walker, 2002). Therefore, an increase of PCR product leads to an increase in fluorescence detected in each PCR cycle, this allows the concentration of DNA or cDNA can be quantified (Ririe et al., 1997). Several of these markers have been described but are most commonly used dyes SYBR (R) Green and SYBR (R) Gold (Lee et al., 2004). These markers are popular for use in real-time PCR because not only cheaper but also are distributed by suppliers of items such as cocktails ready to use and require no additional experimental design (Ririe et al., 1997; Giglio et al., 2003). The main limitation of these markers is that by joining the total nucleic acid in the PCR reaction, emits a light signal, both for specific products and for those who are not (primer-dimers). To cope with this situation is an analysis of the results in the melting curve (Melt Curve). This analysis allows non-specific products can be discriminated from the specific amplicon (Ririe et al., 1997). Figure 2 Generic fluorescent markers in real-time PCR emit a light signal to all double-stranded sequences. The light signal can be measured later. 4 Analysis of the melting curve The data obtained with different mixtures of SYBR (R) Green (trade cocktails or mixtures in the laboratory) may vary. But there is always that the amplicons detected with SYBR (R) Green I are significantly larger and have a high GC content (Giglio et al., 2003). Consequently, it is possible to identify specific PCR products by analyzing the melting temperature expressed in a curve whose shape is related to GC content, size of amplicons and their sequence (Ririe et al. 1997). The analysis of the melting curve can be carried out in most available platforms for real-time PCR, usually at the end of the amplification reaction. The measurement of temperaturedependent fluorescence is performed while the temperature in the thermocycler increases of about 50 ^0 C to 95 ^0 C, the fluorescence detected dependent on the presence of doublestranded sequences of DNA or cDNA (Giglio et al ., 2003, Lee et al., 2004).

When the double strands of DNA or cDNA was separated from the effects of temperature, fluorescence decreases because the dye is no longer attached to the PCR product. Most instruments provide an analysis of these data taking into account the point where it appears the first negative differential fluorescence signal with respect to temperature and melting temperature (Figure 3). This point appears as one or more peaks that represent the temperatures at which the highest levels of change in fluorescence occurs, corresponding to a particular product such PCR (Lee et al., 2004). To discriminate specific PCR products is necessary to know that these are dissociated at a temperature higher than artifacts like primer dimers (Ririe et al., 1997). Figure 3 Analysis of the melting curve. The peaks of the curves show that the specific products of realtime PCR have a higher melting temperature than that of nonspecific products. It is worth noting that the method of analysis of the melting curve is similar to that of agarose gel electrophoresis (Giglio et al., 2003). Both methods can detect the presence of non-specific products, but are also prone to errors. These cases occur when the molecular weights of PCR products in question are similar. Additionally, the accuracy of this data also depends on the hardware platforms used (Lee et al., 2004). 5 Threshold and threshold cycle values (cycle threshold values) As described, the results of real-time PCR is based on the detection and quantification of fluorescent markers along the PCR reaction. This lets you know the amount of fluorescence emitted during the exponential phase of the reaction, where a significant increase in PCR product correlates with the initial amount of DNA or cDNA under study (Walker, 2002). To obtain these results, the cycle threshold values (Ct for its acronym in English) should be obtained. Previously, an appropriate threshold should be set by the operator at a point significantly above baseline (Figure 4). Ct values are determined by identifying the cycle in which the emission of fluorescent intensity rises above background noise in the exponential phase of PCR reaction (Figure 5). In other words, the Ct value is represented by the cycle in which the production of fluorescence crosses the threshold (Bustin, 2005a). It is important to consider that a Ct value greater than 40 cycles indicates no amplification and therefore should not be included in the calculations. There are now software that can determine Ct values by a mathematical analysis of the growth curve and may have better reproducibility in testing real-time PCR (Dorak, 2008) Figure 4 Threshold (threshold) set prior to the determination of Ct values.

Figure 5 The cycle numbers at which the curve crosses the fluorescence threshold corresponding to the Ct values to be used in subsequent calculations. 6 Optimised real-time PCR To obtain reliable results in testing with real-time PCR reaction must be optimized in the laboratory. Optimization is to make the normal test variations do not cause significant effects on the Ct values and have a minimal impact on the amount of fluorescence observed. The most important criteria for optimization are specificity, sensitivity, efficiency and reproducibility of real-time PCR (Edwards, 2004). The factors to be optimized are the master mixes of reagents, the concentration of the primers, concentration of fluorescent markers and the concentration of the sample. The best reagent concentrations and PCR conditions are those in which there is optimum results in terms of the melting curve and the efficiency of amplification reactions conducted with positive controls or calibrators (Edwards, 2004). It is also useful to agarose gel electrophoresis when optimizing real-time PCR. The results of this analysis provide data to relate the length of the product with the peaks of the analysis of the melting curve and the possible presence of artifacts as the first dimmers (Dussault & Pouliot, 2006). 7 Strategies for quantification of mRNA in real-time PCR Two strategies are commonly used to carry out the quantification of gene expression with realtime PCR. These strategies are: quantifying absolute and relative quantification of real-time PCR. 7.1 Absolute quantification The technique of absolute quantification relates the PCR signal obtained with the real-time fixed number of copies of a standard sequence using a calibration curve. Calibration curves are highly reproducible and allow the generation of specific and sensitive. However, the model of external calibration curves must be rigorously validated with absolute accuracy for the quantification of gene expression in real-time PCR depends exclusively on the accuracy of the materials used (Pfaffl, 2008). In addition, the design of standard sequences, production and accurate determination of their concentrations and their long-term stability and storage are complex and may have some problems (Pfaffl, 2004). These considerations make an absolute quantification procedure laborious, expensive and can not always take place in all laboratories. 7.2 Relative quantification

The relative quantification does not require standards with concentrations determined. This technique is used to obtain the magnitude of the physiological changes in gene expression levels of a gene in the study compared with one or more reference gene (Pfaffl, 2004). We must take into account that the expression of reference genes should be constant in the studied cells. Therefore, the sequences used for relative quantification genes are usually housekeeping (Ambion, 2008). The calculations in relative quantification of gene expression based on the comparison of Ct values using the efficiency of the PCR reaction as a correction factor. However, there is a model that does not require the efficiency of the reaction to access a correction factor. This model assumes optimum efficiency and identity (corresponding to 100%) in the efficiency of reaction in real-time PCR gene in both the study and the reference gene (Livak & Schmittgen, 2001). Method 2 This is the delta-delta Ct is only applicable for a quick estimate of the relative proportion of gene expression study (Pfaffl, 2001). Method 2-delta delta Ct is the proportion obtained from the relationship between Ct values of the sample and control Ct values as shown in the following equation: Another model for relative quantification has been published by Pfaffl (2001). In this model the different PCR efficiencies for both genes under study and for reference genes are taken into account as shown in the following equation: In this equation the ratio of the gene under study is expressed in a sample against a control compared with a reference gene. Etarget represents the efficiency of real-time PCR amplicon under study, ESRD represents the efficiency of real-time PCR reference gene; CPtarget is the Ct deviation of control unless the sample studied gene, and is CPref Ct deviation of control unless the sample of reference gene. As mentioned, this model is also necessary to know the efficiency of PCR for each gene studied. The efficiencies of real-time PCR was calculated from the slopes of the standard curve obtained after serial dilutions with the reactions of real-time PCR (Pfaffl, 2004) according to the formula:

E = 10 [-1/slope] -1. It is noteworthy that the efficiency of real-time PCR is the ability of the reaction of doubling the number of copies of DNA or cDNA chains in each cycle (Bustin & Nolan, 2004a) It was observed that the relative quantification of mRNA has some limitations. First, you can introduce a significant statistical bias when there are large differences in gene expression levels in the study and the normalizing gene which can lead to a wrong biological interpretation and secondly, it is difficult to find suitable reference genes ( Bustin et al., 2005). For these reasons it is advisable to use more than one reference gene in order to have reliable data in research (Bustin & Nolan, 2004b). 8 reference genes

Some of the most commonly used reference genes include -actin, Glyceraldehyde 3-phosphate dehydrogenase, hypoxanthine guanine phosphoribosyl-transferase and 18S ribosomal RNA (Huggett et al., 2005). The correct choice of reference genes for normalization of real-time PCR is essential to reflect reliable data on the biological processes of the proteins under study (Robinson et al., 2007). It has also been shown that the use of a single gene as reference gene is susceptible to error in interpreting the results of real-time PCR (Lee et al., 2002). Consequently, to normalize the expression of genes when working with real-time PCR is necessary to use more than one reference gene, especially when you can not find a single reference gene with optimal characteristics for relative quantification (Huggett et al., 2005, Robinson et al., 2007) Currently there appropriate software for the analysis of suitability are reference genes for each experiment is performed and can be found freely in internet. An example is BestKeeper with which you can access the status of up to 10 reference genes and can be downloaded from http://www.gene-quantification.info/. 9 Applications of real-time PCR. The real-time PCR has wide applications in scientific research and diagnostic tool. It is worth noting that there have been great achievements in the study of viruses that affect humans and animals (Bustin, 2005a) and in the investigation of pathogenic bacteria and fungi, for this test offers high sensitivity and specificity in less time and at lower cost. It is also widely used to identify genetic mutations or polymorphisms using specific probes and results in the analysis of changes in the melting curve (Valasek & Repa, 2005). Also real-time PCR was used to access data relevant to the biodynamic of disease organisms (Clementi et al., 1993). However, one of the major application fields of this technique is the investigation of changes in gene expression in cells through the quantification of its mRNA, allowing you to associate these changes to cell physiological states, the presence of drugs, infectious agents, etc ( Bustin et al., 2005). An additional advantage of this technique is that to implement it is sufficient to have a small amount of sample. This makes it ideal when used in conjunction with laser microsurgery to take biopsies of tissue or specific cells and study, for example, the behavior of cancer cells (Edwards et al., 2004, Johnson et al., 2005). The real-time PCR is also being used in other fields such as forensics and biosecurity. In these cases the investigations were based on the identification with high sensitivity and specificity of pathogens or saprophytes widespread in the environment. The characterization of small quantities of genetic material from such agencies makes it possible to know its source and identify its dynamics (Johnson et al., 2005). Another area that uses real-time PCR is food security. Here, the identification of genetically modified organisms (GMOs) in food or food additives is a need can be met using this technology. Under the revised principles above can also be accessed on the quantification of

GMOs using reference genes to normalize the cDNA amount entered in the trials (Bustin, 2005a). 10 Future prospects for the real-time PCR One use that emerges from the technology used in real-time PCR is a molecular diagnostic testing in the same place where you take the sample to be analyzed and not in distant laboratories. This not only would this type of cheaper technologies, but the data collected will be easier to process and are available to researchers immediately. These views, together with the development of portable hardware platforms will give way to the beginning of the era of individualized genetic testing (Walker, 2002). The availability and ease of use to future projects with real-time PCR also make it possible to use these platforms in educational institutions to teach in a practical and clear the characteristics and implications of molecular biology (Valasek & Repa, 2005) . 11 Conclusion Currently, research based on molecular biology is opening a vast spectrum of possibilities for a better understanding of biological phenomena that surround us. The implications of this new knowledge covering such diverse fields as medicine, environmental conservation and industry. In this context the evidence and real-time PCR is a promising aid for the development of applications that allow for molecular biology research by obtaining a large flow of accurate and reliable data on the organisms studied. But further research and development will be needed to make this event accessible to a wide audience and the results interpreted in a direct and reliable. REFERENCES Biosystems Ambion. 2008. Use of Internal and External Standards or Reference RNAs for Accurate Quantitation of RNA Levels. Bustin, SA 2002. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. Journal of Molecular Endocrinology 29: 23-39. Bustin, SA 2005a. Real-Time PCR. In: Encyclopedia of Diagnostic Genomics and Proteomics J. Fuchs & M. Podda, editors. Marcel Dekker.New York, p. 1131-1135. Bustin, SA 2005b. Real-Time Reverse Transcription PCR. In: Encyclopedia of Diagnostic Genomics and Proteomics J. Fuchs & M. Podda, editors. Marcel Dekker.New York, p. 11311135. Bustin, SA, Benes, V., Nolan, T. & Pfaffl, M.W. 2005. Quantitative real-time RT-PCR perspective. Journal of Molecular Endocrinology 34: 597-601. Bustin, SA & Nolan, T. 2004a. Analysis of mRNA Expression by Real-Time PCR. In: RealTime PCR: An Essential Guide. KJEdwards et al., Editors. Bioscience.Wymondham Horizon, p. 125-184.

Bustin, SA & Nolan, T. 2004b. Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. Journal of Biomolecular Techniques 15: 155-166. Clementi, M., Menzo, S., Bagnarelli, P., Manzin, A., Valenza, A. & Varaldo, E. 1993. Quantitative PCR and RT-PCR in virology. Genome Research 2: 191-196. Dorak, M.T. 2008. Real-Time PCR. http://dorakmt.tripod.com/genetics/realtime.html Dussault, A.A. & Pouliot, M. 2006. Rapid and simple comparison of messenger RNA Levels using real-time PCR. Biological Procedures Online 8: 1-10. Edwards, K.J. 2004. Performing Real-Time PCR. In: Real-Time PCR: An Essential Guide. KJEdwards et al., Editors. Bioscience.Wymondham Horizon, p. 71-84. Edwards, K. J., Logan, Julie, & Saunders, Nick, 2004. Real-Time PCR: An Essential Guide. Horizon Bioscience, Wymondham. Giglio, S., Monis, P.T. & Saint, c.p. 2003. Demonstration of Preferential binding of SYBR Green I to specific DNA fragments in real-time multiplex PCR. Nucleic Acids Research 31: E136. Heid, C.A., Stevens, J., Livak, K.J. & Williams, P.M. 1996. Real time quantitative PCR. Genome Research 6: 986-994. Huggett, J., Dheda, K., Bustin, SA & ZUML, A. 2005. Real-time RT-PCR normalization; Strategies and considerations. Genes and Immunity 6: 279-284. Johnson, M., Petrauskene, O., Brzoska.P & Melancon.C. 2005. Using Real-Time PCR for Pathogen Detection. Genetic Engineering News 25. Lee, M.A., Squirrell, D.J., Leslie, D.L. & Brown, T. 2004. Fluorescent Homogeneous Chemistri. In: Real-Time PCR: An Essential Guide. KJEdwards et al., Editors. Bioscience.Wymondham Horizon, p. 31-70. Lee, P. S., Sladek, R., Greenwood, C.M. & Hudson, T.J. 2002. Control genes and variability: Absence of ubiquitous reference transcripts in diverse mammalian expression studies. Genome Research 12: 292-297. Livak, K.J. & Schmittgen, T.D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C (T)) Method. Methods 25: 402-408. McGuire, K., Anju, M., Russell, GC, Springbett, A., Craigmile, SC, Nichani, AK, Malhotra, DV & Glass, e.g. 2004. Quantitative analysis of pro-inflammatory cytokine mRNA expression in Theileria annulata-infected cell lines derived from resistant and susceptible cattle. Veterinary Immunology and immunopathology. 99: 87-98.

McPherson, M. J., Hames, B. D., & Taylor, G. R, 2008. PCR Practical aproach. First Editon edn. Oxford University Press Oxford. Pfaffl, M.W. 2001. A New Mathematical model for relative Quantification in real-time RT-PCR. Nucleic Acids Research 29: E45. Pfaffl, M.W. 2004. Quantification Strategies in Real-Time PCR. In: AZ of Quantitative PCR SABustin, editor. Line.La Jolla International University, p. 87-120. Pfaffl, M.W. 2008. Gene-Quantification. http://www.gene-quantification.de/ Ririe, K.M. Rasmussen R.P. & Wittwer, C.T. 1997. Product Differentiation by analysis of DNA melting curves During The polymerase chain reaction. Analytical Biochemistry 245: 154-160. Robinson, T.L., Sutherland, I.A. & Sutherland, J. 2007. Validation of candidate bovine reference genes for use with real-time PCR. Veterinary Immunology and immunopathology. 115: 160-165. Smith, D.P. & Old, R.W. 1993. Structure, Function and Analysis. In: R. Rapley & Molecular Diagnostics MRWalker, editors. Publication.Victoria Blackwell Scientific, p. 3-25. Valasek, M.A. & Repa, J.J. 2005. The power of real-time PCR. Advances in Physiology Education 29: 151-159. Walker, N.J. 2002. Tech.Sight. A Technique Whose Time Has Come. Science 296: 557-559.