Real Time Pcr Essay Example for Free

Real Time Pcr EssayPROBE-BASED detection SYSTEMS14 Hybridization dig intos ( as well as called FRET probes)16 MELTING frizzle ANALYSIS16 Multiplex real time PCR18 APPLICATIONS OF substantive judgment of conviction PCR18 GENE EXPRESSION ANALYSIS18 SNP GENOTYPING19 human immunodeficiency virus DETECTION19 CYSTIC FIBROSIS (CF) DETECTION20 THE ADVANTAGES OF received- cartridge holder PCR20 THE DISADVANTAGES21 REFRENCES21 REAL TIME PCR TRADITIONAL PCR The polymerase chemical chain reaction (PCR) is peerless of the near powerful technologies in molecular biology. Using PCR, detail whiles within a deoxyribonucleic sexually transmitted disease or complementary deoxyribonucleic acid template can be copied, or amplified, umteen thousand- to a millionfold.In traditional (end shoot) PCR, spying and quantitation of the amplified sequence are performed at the end of the reaction after the last PCR cycle, and involve post-PCR analysis such as gel electrophoresis and image analysi s. REAL-TIME QUANTITATIVE PCR (qPCR) In real-time quantitative PCR (qPCR), the arrive of PCR product is measu red ink at each cycle. This ability to monitoring device the reaction during its exponential cast enables users to determine the initial amount of target with great precision. WHATS WRONG WITH AGAROSE GELS? * Poor precision. * Low sensitivity. Short dynamic break away lt 2 logs. * Low re issue. * Non-automated. * Size-based dissimilarity only * Ethidium bromide staining is non very(prenominal) quantitative REAL TIME PCR VS PCR . BASIC PRINCIPLE Quantitative PCRis carried come to the fore in athermic cyclerwith the content to illuminate each sample with a beam of light of a specified wavelength and detect the fluorescence emitted by the unrestrainedfluorochrome. The thermal cycler is also able to rapidly heat and chill samples thereby taking advantage of the physicochemical properties of thenucleic acidsand deoxyribonucleic acid polymerase.The PCR process genera lly consists of a series of temperature changes that are repeated 25 40 times, these cycles normally consist of three coifs the first, at around 95C, allows the separation of the nucleic acids double chain the second, at a temperature of around 50-60C, allows the alignment of the primers with the DNA templatethe third at betwixt 68 72C, facilitates thepolymerizationcarried out by the DNA polymerase In real-time PCR, * the amount of DNA is measured after each cycle by the use of light markers that are incorporated into the PCR product. The increase in fluorescent signal is directly proportional to the number of PCR product molecules (amplicons) generated in the exponential phase of the reaction. * Fluorescent reporters use include double-stranded DNA (dsDNA)- binding tints, or color molecules attached to PCR primers or probes that are incorporated into the product during amplification. * The change in fluorescence everyplace the course of the reaction is measured by an instr ument that combines thermal pass with scanning capability.By plotting fluorescence against the cycle number, the real-time PCR instrument gene place an amplification plot that re frames the accumulation of product everywhere the duration of the entire PCR reaction ( betoken 1). Figure 1Amplification plots are created when the fluorescent signal from each sample is plan against cycle number therefore, amplification plots represent the accumulation of product over the duration of the real-time PCR experiment. The samples existence amplified in this example are a dilution series of the template. TYPES OF PCRQuantitative PCR Qualitative qPCR A limited or non- peculiar(prenominal) detection chemical science allows the quantification ofthe amplified product. In qualitative qPCR, the goal is to detect the presence or absence of a certain sequence. The amount detected at a certain point of the slope is directly related to theinitial amount of target in the sample For virus sub-typ ing and bacterial species identification. Can also be employ for allelic discrimination between wild type and mutant, between diametrical SNPs or between different splicing forms. common pplications of quantitative PCR are gene looking analysis, pathogen detection/quantification and microribonucleic acid quantification Different fluorophores can be apply for the 2 alleles, and the ratio of the fluorophores signals correlates to the related amount of one form compared to the other one. Quantitative PCR software uses the exponential phase of PCR for quantification. Specific detection rules such as Double-Dye probe systems are more often use for theseApplications Overview of real-time PCR real-time PCR is a variation of the example PCR technique used to quantify DNA or ribonucleic acid in a sample.Using sequence-specific primers, the relative number of copies of a particular DNA or ribonucleic acid sequence can be determined.. Quantification of amplified product is obtained using fluorescent probes or fluorescent DNA binding dyes and real time PCR instruments that measure fluorescence while execute temperature changes needed for the PCR cycles. qPCR STEPS There are three major steps that make up a qPCR reaction. Reactions are generally run for 40 cycles. 1. DenaturationThe temperature should be appropriate to the polymerase chosen (usually 95C).The denaturation time can be increased if template GC content is high. 2. AnnealingUse appropriate temperatures based on the calculated releaseing temperature (Tm) of the primers (5C below the Tm of the primer). 3. ExtensionAt 7072C, the activity of the DNA polymerase is optimal, and primer extension occurs at rates of up to 100 bases per second. When an amplicon in qPCR is small, this step is often combined with the annealing step using 60C as the temperature. BASICS OF REAL TIME PCR Baseline The baseline phase contains all the amplification that is below the level of detection of the real time instrument.T hreshold where the threshold and the amplification plot baffle defines CT. Can be set manually/automatically CT (cycle threshold) the cycle number where the fluorescence passes the threshold Rn (Rn-baseline) NTC no template control Rn is plotted against cycle numbers to produce the amplification curves and gives the CT value. ONE-STEP OR TWO-STEP REACTION qRT-PCR can be one step or two step. 1. cardinal-step qRT-PCR Two-step qRT-PCR starts with the reverse transcription of either total ribonucleic acid or poly(A)+ RNA into complementary DNA using a reverse transcriptase (RT).This first-strand cDNA subtraction reaction can be primed using random hexamers, oligo(dT), or gene-specific primers (GSPs). To give an equal representation of all targets in real-time PCR applications and to countermand the 3? prejudice of oligo(dT), it is usually recommended that random hexamers or a mixture of oligo(dT) and random hexamers are used. The temperature used for cDNA synthesis depends on the RT enzyme chosen. Following the first-strand synthesis reaction, the cDNA is transferred to a separate tube for the qPCR reaction. In general, only 10% of the first strand reaction is used for each qPCR. . virtuoso-step qRT-PCR One-step qRT-PCR combines the first-strand cDNA synthesis reaction and qPCR reaction in the comparable tube, simplifying reaction setup and reducing the possibility of contamination. Gene-specifi c primers (GSP) are required. This is because using oligo(dT) or random primers result generate nonspecific products in the one-step procedure and subdue the amount of product of interest. Overview of qPCR and qRT-PCR components This voice provides an overview of the major reaction components and parameters snarled in real-time PCR experiments. * DNA polymerase One of the important factors affecting PCR specificity is the fact that Taq DNA polymerase has residual activity at low temperatures. Primers can anneal nonspecifically to DNA, allowing the polymera se to synthesize nonspecific product. The hassle of nonspecific products resulting from mispriming can be minimized by using a hot-start enzyme. Using a hot-start enzyme ensures that no active Taq is present during reaction setup and the initial DNA denaturation step. * Template Anywhere from 10 to 1,000 copies of template nucleic acid should be used for each real-time PCR reaction. This is equivalent to approximately 100 pg to 1 ? of genomic DNA, or cDNA, generated from 1 pg to 100 ng of total RNA. Excess template may increase the amount of contaminants and narrow efficiency. If the template is RNA, care should be taken to reduce the chance of genomic DNA contamination. One option is to treat the template with DNaseI. Ultrapure, intact RNA is essential for full-length, high-quality cDNA synthesis and accurate mRNA quantification. RNA should be devoid of any RNase contamination, and aseptic conditions should be maintained. * Reverse transcriptase The reverse transcriptase (RT) is as critical to the success of qRT-PCR as the DNA polymerase.It is alpha to choose an RT that not only provides high yields of full-length cDNA but also has good activity at high temperatures. High-temperature performance is also very important for tackling RNA with secondary structure or when working with gene-specific primers (GSPs). * dNTPs It is recommended that both the dNTPs and the Taq DNA polymerase be purchased from the same vendor, as it is not uncommon to see shifts of one full threshold cycle (Ct) in experiments that employ these items from separate vendors. * Magnesium concentration In qPCR, magnesium chloride or magnesium sulfate is typically used at a fi nal concentration of 3 mM.This concentration works well for intimately targets however, the optimal magnesium concentration may vary between 3 and 6 mM. * UNG The Uracil-N-Glycosylase is an enzyme that hydrolyses all single-stranded and double-stranded DNA containing dUTPs. Consequently, if all PCR amplifications are performed in the presence of a dNTPs/dUTPs blend, by carrying a UNG step before every run it is possible to get rid of any previous PCR product. * ROX Some thermocyclers require MasterMix containing ROX dye for normalization. This is the case for the ABI and Eppendorf machines, and optional on the Stratagene machines.If you work with such machines, it is easier to work with the ROX dye already incorporated in the MasterMix rather than adding it manually. It guarantees a high level of reproducibility and homogeneity of your balks. * Fluorescein For iCycler iQ, My iQ and iQ5 machines (BioRad thermocyclers), the normalization method for SYBR Green chit uses Fluorescein to create a virtual background. As in the case for the ROX, it is better and easier to use a MasterMix that contains pre-diluted Fluorescein, guaranteeing higher reproducibility and homogeneity of your assays. REAL TIME PCR SYSTEMSystem Features Four interchangeable block formats Optional Automation Accessory amp B arcode Scanner Argon ion laser/CCD camera swooning to Use Software, Multiple Applications Set up Wizards QC Filtering/Flag System Flexible data reports amp exporting SOFTWARES FOR DATA ANALYSIS AND PRIMER DESIGNING 1 ) Light Cycler Relative Quantification Software The first commercially available software was the Light Cycler Relative Quantification Software (2001). 2 ) REST In 2002, the relative expression software tool (REST ) was established as a new tool. 3 ) Q-GeneRecently a second software tool, Q-Gene, was developed, which is able to perform a statistical test of the real-time data. Q-Gene manages and expedites the planning, performance and evaluation of quantitative real-time PCR experiments. 4) OligoPerfect A primer design software program such as OligoPerfect, available on the Web at www. invitrogen. com/oligoperfect, can automatically evaluate a target sequence and design primers for it based on the criteria STEPS OF REAL TIME PCR Real-time reaction mix (final conce ntrations) 1x 2 x AmpliTaq Gold 0. 5 ? M 5 primer 0. 5 ? M 3 primer 0. 2 ? M probe 0. 4 ? Rox reference dye 20 ? l Final Volume (including sample and dH20) STANDARD REAL-TIME PCR PROTOCOL checkout DESIGN This persona describes the stages of real-time PCR assay design and implementation. We will identify sources of variability, the role they play in data accuracy, and guidelines for optimization in the following areas 1Target amplicon and primer design 2. Nucleic acid purification 3. Reverse transcription 4. Controls and normalization 5. Standard curve evaluation of efficiency, sensitivity, and reproducibility Good primer (pair) properties One way to minimize efficiency bias is to amplify relatively short targets.Amplifying a 100 bp region is more likely to result in complete synthesis in a disposed(p) cycle than, say, amplifying a 1,200 bp target. For this reason, real-time PCR target lengths are generally in the range of 60 bp to 200 bp. In addition, shorter amplicons act as a buff er against variations in template integrity. Primers designed to amplify larger regions are less likely to anneal with the same fragment in a moderately degraded nucleic acid sample. PURIFICATION Phenol-based organic extraction is a very effective method for purifying RNA from a wide variety of kiosk and tissue types.During sample lysis, phenol and guanidine isothiocyanate disrupt cells and dissolve cell components. while maintaining the integrity of the nucleic acids by protecting them from RNases. Chloroform is added and the mixture is separated by centrifugation, which separates the solution into an aqueous phase and an organic phase. RNA remains exclusively in the aqueous phase in the presence of guanidine isothiocyanate, while DNA and protein are goaded into the organic phase and interphase. The RNA is then recovered from the aqueous phase by precipitation with isopropyl alcohol. REVERSE TRANSCRIPTION CONSIDERATIONSMost reverse transcriptases employed in qRT-PCR are deri ved from avian myeloblastosis virus (AMV) or Moloney murine leukemia virus (M-MLV). An ideal reverse transcriptase will exhibit the following attributes * Ther most(prenominal)ability thermostable RTs function at the higher end of (or above) this range and allow for successful reverse transcription of GC-rich regions. * RNase H activity RNase H activity can drastically reduce the yield and ratio of full-length cDNA, which translates to poor sensitivity. Several RTs, most notably SuperScript II and III, have been engineered for reduced RNase H activity. NORMALIZATION AND QUANTIFICATIONWhen analyzing and comparing results of Real-Time qPCR assays many researchers are confronted with several uncontrolled variables, which can lead to misinterpretation of the results. Those uncontrolled variables can be the amount of starting material, enzymatic efficiencies, and differences between tissues, individuals or experimental conditions. In order to make a good comparison, normalization can be used as a correction method, for these variables. The most commonly k instantern and used ways of normalization are * normalization to the original number of cells, * normalization to the total RNA mass, normalization to one or more housekeeping genes, * normalization to an internal or external calibrator. Normalization to number of cells can actually only be done for cell destination and blood samples. The two majors methods of normalization are the absolute quantification and the relative quantification . Absolute quantification Absolute quantification requires a standard curve of known copy numbers. The amplicon being studied can be cloned, or a synthetic oligonucleotide (RNA or DNA) can be used. The standard must be amplified using the same primers as the gene of interest and must amplify with the same efficiency.The standards must also be quantified accurately. This can be carried out by reading the absorbance at A260, although this does not distinguish between DNA and RNA, or by using a fluorescent ribonucleic acid stain such as RiboGreen. Relative quantification Relative quantification is the most widely used technique. Gene expression levels are calculated by the ratio between the amount of target gene and an endogenous reference gene, which is present in all samples. The reference gene has to be chosen so that its expression does not change under the experimental conditions or between different tissue.There are simple and more complex methods for relative quantification, depending on the PCR efficiency, and the number of reference genes used. STANDARD CURVE TO ASSESS EFFICIENCY, SENSITIVITY, AND REPRODUCIBILITY The final stage before assay employment is validating that all the experimental design parameters result in a highly efficient, sensitive, and reproducible experiment. * Reaction efficiency One hundred percent efficiency corresponds to a perfect doubling of template at every cycle, but the acceptable range is 90110% for assay validation.This efficiency range corresponds to standard curve slopes of 3. 6 to 3. 1. The graph in Figure shows the measurement bias resulting solely from differences in reaction efficiency.. A standard curve is generated by plotting a dilution series of template against the Ct for each dilution. To some, sensitivity is measured by how early a target Ct appears in the amplification plot. However, the true gauge of sensitivity of an assay is whether a given low amount of template fits to the standard curve while maintaining a preferred efficiency. The most dilute sample that fits determines reaction sensitivity.The standard curve also includes an R2 value, which is a measure of replicate reproducibility. Standard curves may be repeated over time to assess whether the consistency, and therefore the data accuracy for the samples. Real-Time PCR Fluorescence Detection Systems Several different fluorescence detection technologies can be used for realtime PCR, and each has specific assay design require ments. All are based on the generation of a fluorescent signal that is proportional to the amount of PCR product formed. The three main fluorescence detection systems are * DNA-binding agents (e. g. SYBR Green and SYBR GreenER technologies * Fluorescent primers (e. g. , LUX Fluorogenic Primers and Amplifluor qPCR primers) * Fluorescent probes (e. g. , TaqMan probes, Scorpions, Molecular Beacons) The detection method plays a critical role in the success of real-time PCR. DNA-Binding Dyes The most common system for detection of amplified DNA is the use of intercalating dyes that fluoresce when butt on to dsDNA. SYBR Green I and SYBR GreenER technologies use this type of detection method. The fluorescence of DNA-binding dyes importantly increases when bound to double-stranded DNA (dsDNA).The intensity of the fluorescent signal depends on the amount of dsDNA that is present. As dsDNA accumulates, the dye generates a signal that is proportional to the DNA concentration and can be detect ed using real-time PCR instruments. SYBR Green I advantages Low cost assay Easy design and set up SYBR Green I disadvantages Non specific system Not adapted to multiplex Non suitable for qualitative qPCR Primer-Based Detection Systems Primer-based fluorescence detection technologies can provide highly sensitive and specific detection of DNA and RNA.In these systems, the fluorophores is attached to a target-specific PCR primer that increases in fluorescence when incorporated into the PCR product during amplification. * Amplifluor Real-Time PCR Primers Amplifluor real-time PCR primers are designed with both a fluorophore and quencher on the same primer. The primer adopts a hairpin configuration that brings the fluorophore in exclude proximity to the quencher. The fluorescent signal increases when the primer is unfolded and the fluorophore and quencher are de-coupled during incorporation into an amplification product.Figure Ampliflour primer PROBE-BASED DETECTION SYSTEMS Probe-ba sed systems provide highly sensitive and specifi c detection of DNA and RNA. However, dual-labeling and complex design specifi cations make them expensive and more diffi cult to use than primer-based systems or DNAbinding dyes. TaqMan probes = Double-Dye probes TaqMan probes, also called Double-Dye Oligonucleotides, Double-Dye Probes, or Dual Labelled probes, are the most widely used type of probes. A fluorophore is attached to the 5 end of the probe and a quencher to the 3 end.The fluorophores is excited by the machine and passes its energy, via FRET (Fluorescence Resonance Energy Transfer) to the quencher. TaqMan probes can be used for both quantification and mutation detection, and most designs appear to work well. TaqMan ASSAY DENATURATION ANNEALING OF PRIMERS AND PROBE POLYMERIZATION AND PROBE CLEAVAGE Molecular Beacons In addition to two sequence-specific primers, molecular beacon light assays employ a sequence-specific, fluorescently labeled oligonucleotide probe called a mol ecular beacon, which is a dye-labeled oligonucleotide (2540 nt) that forms a hairpin structure with a stem and a loop .A fluorescent reporter is attached to the 5 end of the molecular beacon and a quencher is attached to the 3 end. The loop is designed to hybridize specifically to a 1530 nucleotide section of the target sequence Figure Moleculer Beacon They are highly specific, can be used for multiplexing, and if the target sequence does not match the beacon sequence exactly, crosswalk and fluorescence will not occur a desirable quality for allelic discrimination experiments. Hybridization probes (also called FRET probes) Roche has developed hybridization probes for use with their LightCycler.Two probes are designed to bind adjacent to one another on the amplicon. One has a 3 label of FAM, whilst the other has a 5 LC dye, LC red 640 or 705. When the probes are not bound to the target sequence, the fluorescent signal from the reporter dye is not detected. However, when the probes h ybridize to the target sequence during the PCR annealing step, the close proximity of the two fluorophores allows energy transfer from the donor to the acceptor dye, resulting in a fluorescent signal that is detected. FRET probe principle and light cyclerMELTING CURVE ANALYSIS Melting curve analysis can only be performed with real-time PCR detection technologies in which the fluorophore remains associated with the amplicon. Amplifications that have used SYBR Green I or SYBR GreenER dye primers can be subjected to melting curve analysis. Dual-labeled probe detection systems such as TaqMan probes are not compatible because they produce an irreversible change in signal by cleaving and releasing the fluorophore into solution during the PCR however, the increased specificity of this method makes this less of a concern.The level of fluorescence of both SYBR Green I and SYBR GreenER dyes significantly increases upon binding to dsDNA. By monitoring the dsDNA as it melts, a decrease in fluor escence will be seen as soon as the DNA becomes single-stranded and the dye dissociates from the DNA. Figure Melting curve analysis can detect the presence of nonspecifc products, as shown by the additional peaks to the left of the peak for the amplified product in the melt curve. How to perform melting curve analysisTo perform melting curve analysis, the real-time PCR instrument can be programmed to include a melting visibility immediately following the thermocycling protocol. After amplification is complete, the instrument will reheat your amplified products to give complete melting curve data. Most real-time PCR instrument platforms now incorporate this feature into their analysis packages. In general, the program steps will be 1. Rapid heating of the amplified sample to 94C to denature the DNA. 2. modify the sample to 60C. 3.Slowly heating (by increasing the temperature 0. 2C/second) the sample while plotting fluorescence signal vs. temperature. (As the temperature increases an d the dsDNA strands melt, the fluorescence signal will decrease. ) Figure Example of a melting curve thermal profile setup on an Applied Biosystems instrument (rapid heating to 94C to denature the DNA, followed by cooling to 60C. ) Multiplex real-time PCR In multiplex real-time PCR, more than one set of gene-specific primers is used to amplify separate genes from the template DNA or RNA in a single tube.Typically, multiplex reactions are used to amplify a gene of interest and a housekeeping gene (e. g. , -actin or GAPDH), which is used as a normalize for the reaction. Because more than one PCR product will be quantified in the same tube, different fluorescent reporter dyes are used to label the separate primers or probes for each gene. More Samples Analyzed per Plate. Target and normalizer in same reaction and Less sample consumed. APPLICATIONS OF REAL TIME PCR GENE EXPRESSION ANALYSIS A sample gene expression analysis using a multiplex TaqMan assay is presented in the following sec tions.In this example, were interested in the relative expression of three genes in the polyamine biosynthesis pathway, ornithine decarboxylase (ODC), ODC antizyme (OAZ), and antizyme inhibitor (AZI), in two different samples, sample A and sample B. 1. RNA was isolated from sample A and sample B. 2. RNA was reverse transcribed into cDNA. 3. The amount of the target genes (ODC, OAZ, and AZI) and the reference gene (b-actin) was determined in each of the cDNA samples using a multiplex qPCR assay. 4. Data were analyzed and the relative expression of each of the target genes in the two samples was calculated.EXAMPLE BRCA1 is a gene involved in tumor suppression. BRCA1 controls the expression of other genes. In order to monitor level of expression of BRCA1, real-time PCR is used. SNP GENOTYPING In order to perform SNP genotyping, two specific probes labeled with different dyes are used, the first for the wild type allele and the second for the mutant allele. If the assay results in the g eneration of only the first fluorescent color, then the individual is homozygous wild type at that locus. If the assay results in the generation of only the second fluorescent color, then the individual is homozygous mutant.And finally, if both fluorescent colors are produced, then the individual is heterozygous. At the end of the reaction, hydrolysis probes are digested. The quality of a hydrolysis probe is given by the hybridization efficiency, the quenching of the intact probe and the cleavage activity of Taq polymerase. HIV DETECTION Nowadays HIV is strikingly spreading out whole the world. so in order to diminish its distribution , it is necessary to detect it as soon as possible amp for this purpose, Real time PCR is recommended by scientist. In this method , pol gen of the virus, is amplified in thermocycler. 6 patient have been studied. infection in these patients was confirmed by ELISA amp western blot. * Sampling amp RNA extracting from patients. * Cloning of target segmen t by using Xba I amp Hind III. And 180 bp primers. * Standard virus mRNA was extracted. * Quantitative analysis of HIV virus by SYBR-green Real Time RT-PCR. CYSTIC FIBROSIS (CF) DETECTION Cystic fibrosis (CF) is the most common inherited disease among Caucasian populations with an incidence of 1 in 2500 births. A3 base pair (bp) deletion, designated DF508, accounts for nearly 70% of CF cases and causes severe manifestations of the disease.It results in the absence of phenylalanine at position 508 of the cystic fibrosis transmembrane conductance regulator (CFTR) protein and this error prevents normal touch and translocation of the polypeptide chain to apical membranes of epithelial cells. This deletion can be detected by molecular beacons in real time PCR. FigureExamples of specific molecular beacon fluorescence increase during real-time PCR in samples containing single lymphoblasts homozygous normal for CF (green), heterozygous DF508 (blue), or homozygous DF508 (red). A) Fluorescen t signal from the molecular beacon detecting the normal allele. (B) Fluorescent signal from the molecular beacon detecting the DF508 allele. Dashed lines indicate the threshold of 200 units (10 SD above baseline readings) used for determining CT values. THE ADVANTAGES OF REAL-TIME PCR * The ability to monitor the progress of the PCR reaction as it occurs in real time * The ability to precisely measure the amount of amplicon at each cycle * An increased dynamic range of detection * The combination of amplification and detection in a single tube, which eliminates post-PCR manipulations.Rapid cycling times (1 hour) * High sample throughput (200 samples/day) * Low contamination risk (sealed reactions) * Very sensitive (3pg or 1 genome eq of DNA) * Broad dynamic range (10 1010 copies) * Reproducible (CV lt 2. 0 %) * Allows for quantitation of results * Software driven operation * No more expensive than in house PCR ($15/test) THE DISADVANTAGES * Current technology has limited capacity f or multiplexing. Simultaneous detection of 2 targets is the limit. * Development of protocols needs high level of technical skill and/or support. Requires RampD capacity and with child(p)) * High capital equipment costs ($ 50,000 -160,000). REFRENCES * http//www. icmb. utexas. edu/core/DNA/qPCR/QiagenRT-PCR. pdf www. icmb. utexas. edu * http//books. google. com. pk/books? id=-v-U-mXWg-gCampprintsec=frontcoverampdq=real+time+pcramphl=enampsa=Xampei=Bph1UezKIceDhQeUh4CwCAampved=0CDAQ6AEwAQv=onepageampq=real%20time%20pcrampf=false books. google. com. pk * PCR/Real-Time PCR Protocols www. protocol-online. org Real-Time Pcr An Essential Guide Google Books books. google. com * * http//www. gene-quantification. e/bio-rad-CFX96-bulletin-5589. pdf www. gene-quantification. de * https//www. google. com. pk/output=searchampsclient=psy-abampq=fret+rt-qpcrampoq=fret+in+rtampgs_l=serp. 1. 1. 0i22i30l2. 1583. 4622. 1. 10196. 6. 6. 0. 0. 0. 0. 551. 2584. 3-3j1j2. 6. 0 0. 0 1c. 1. 9. serp. 97Wjtm9 UCU4amppsj=1ampbav=on. 2,or. r_cp. r_qf. ampfp=f6d28cf5fd703914ampbiw=1366ampbih=600 www. google. com. pk * BioTechniques Real-time PCR for mRNA quantitation www. biotechniques. com * http//env1. gist. ac. kr/joint_unugist/file/g_class11_real_time_pcr_vt. pdf env1. gist. ac. kr