Real time PCR

Real-time PCR is also referred to as quantitative PCR (QT-PCR). With this technique, the formation of the reaction products can be monitored as the reaction proceeds. Usually a fluorescent marker is employed. The increase in products after each cycle can be recorded as an increase in fluorescence. Such an amplification plot provides a more complete picture of the PCR process than measuring the product accumulation at the end of a fixed number of cycles. Two different types of assays can be used for real-time PCR dsDNA binding dye assays and probe based assays. 

The binding dye is a small molecule that fluoresces upon binding to double-stranded DNA. As PCR amplification produces more and more dsDNA molecules, the fluorescence signal increases (Fig. 6.6). Dyes are classified into two categories 

As multiple dye molecules bind to one DNA molecule, the method is very sensitive especially during the first cycles. The dyes bind to any dsDNA present. This makes the method very versatile as the same dye may be used for any sequence amplification. On the other hand, the dyes do not distinguish between specific and non-specific dsDNA. Mismatches and primer-dimers also give fluorescence signals. 

With the probe based method, only specific products are detected. Multiple probes with different reporters can be used for simultaneous detection of a number of distinct sequences. 

Real-time PCR for DNA amplification and detection was facilitated using the microfluidic chip. 

FIGURE 9.7 The contact angle of PDMS after various surface treatments (a) 3 h after 02 plasma treatment (28° C), (b) 48 h after 02 plasma treatment (70° C), (c) 144 h after 02 plasma treatment (90° C) and (d) parylene-coated PDMS (92° C)[1000]. Reprinted with permission from the Institute of Physics Publishing. 

FIGURE 9.8 Temperature profile of the PCR chamber (center) fabricated on different substrates silicon (solid line), glass (broken line), PDMS with bottom heating (dotted line), and PDMS with both top and bottom heating (chain line). Top inset shows the temperature profile in the expanded scale (90-94°C) [1000]. Reprinted with permission from Institute of Physics Publishing. 

The use of an intercalating dye, SYBR Green I, for real-time PCR can only be performed in an all-glass device, rather than one of PDMS-glass, because the dye and DNA appeared to migrate into the PDMS (Sylard 184) polymer [447], 

Real-time PCR was also performed by on-chip thermal cycling for the detection of Hantavirus, HIV, orthopoxviruses (266-281 bp), Borrelia burgdorferi, human (3-actin (294 bp), and the human complement C6 gene (73 bp) [944]. In another report, real-time PCR was performed for DNA extracted from Bacillus subtilis in a plastic microfluidic cassette. The spore samples (lOVmL) were sampled, filtered, and sonicated in the presence of 6-pm glass beads for spore disruption to release the DNA [945], 

Estimation of the titer of pathogenic agents (different viruses or bacteria) in blood or body fluids to evaluate the success of the therapy  

Detection of minimal cancer disease and the concentration oftumor cells in blood or bone marrow to monitor the success of the tumor therapy  

Detection of single nudeotide polymorphisms (SNPs) in genomic sequences. 

Different methods have been developed to measure the fluorescence of the amplified PCR product. The most popular methods are the SYBR Green I method and the FRET method in addition to the cycling probe technology method. 

Detection of SYBR Green I Fluorescence The SYBR Green I is a simple, inexpensive and sensitive method. SYBR Green I is an intercalating dye that binds to the minor groove of double stranded DNA (dsDNA) molecules, regardless its sequence. After binding the dye to the dsDNA, the intensity of the fluorescence emission increases more than 300-fold, which provide excellent sensitivity to quantify the dsDNA 

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Lok and McMahon 2007), but the ideal outcome is undetectable HBV DNA (< 10-30lU/ml) in highly sensitive real-time PCR-based assays. In HBeAg-positive patients, loss of HBeAg followed by the emergence of anti-HBe antibodies ( e seroconversion) indicates a sustained response to therapy when it persists after treatment cessation. HBs seroconversion (loss of HBsAg and emergence of anti-HBs antibodies) is the most desirable endpoint, as it indicates a complete response with sustained remission from HBV disease. It is rarely achieved with cnrrent therapies. 

Adjunctive to flux control analysis, other components of metabolism that contribute to product accumulation are needed including (1) substrate/precursor pool sizes (metabolomics), (2) co-factor capacities (metabolomics), (3) gene expression profiles (transcriptomics and quanfifafive real-time PCR), (4) protein profiles (pro-... 

The three main categories of hybridization probes for real-time PCR are (1) cleavage based assays such as TaqMan, (2) displaceable probe assays such as Molecular Beacons and (3) probes which are incorporated directly into primers such as Scorpions. 

The PCR technique is very useful during all stages of the research and development of biotech crops. PCR analysis is used for gene discovery, event selection, screening, transformant identification, line selection and plant breeding. Quantitative real-time PCR is used to determine the number of transgene copies inserted in experimental... 

CCL5 i 1 Serum Synovium TB FLS SC (nonendothelium) OB, MNC Main CCR5 ligand Real-time PCR (mRNA), ELISA, IHC, IF 77, 130, 131, 133, 142, 143... 

CCL17 SF PBMCs MDDC CCR4 ligand. Th2 chemokine Real-time PCR, ELISA 139, 144... 

CCL18 SF Synovium PBMCs MDDC Expressed in DC. Can act as antagonist of CCL11 and CCL13 for binding to CCR3. ELISA, real-time PCR (mRNA), IHC 139, 145... 

IHC, IF, real-time PCR (mRNA), ELISA, RT-PCR, DNA microarray Real-time PCR (mRNA), IF, ELISA, RT-PCR Real-time PCR (mRNA), RT-PCR, Northern, ISH, IHC, IF, ELISA RT-PCR, IHC, ISH (mRNA)... 

CCL17 Synovium Real-time PCR Elevated levels compared with OA or normal controls. 139... 

Synovium Real-time PCR ELISA Elevated levels compared with tissue obtained from amputation for diabetes or other etiologies. ... 

Although PCR amplification begins at an exponential rate, it enters a stationary phase after approximately 30 cycles, and additional cycles do not increase the concentration of amplicons. For this reason it is not practical to use PCR for quantifying bacteria directly, and other methods, such as real-time PCR, are used for this purpose. 

Real-time PCR is a quantitative method for measuring amplicons as they are produced by measuring the increase in fluorescence of a dye added to the reaction mixture.12,104,105 Methods using fluorescent reporters, such as SYBR Green,104,106 TaqMan ,107,108 or molecular beacons,9 collect quantitative data at the time when DNA is in the exponential phase of amplification. 

The instrumentation for real-time PCR includes a thermal cycler with a computer, a spectrophotometer for fluorescence detection, and software for acquisition and analysis of data.105,109... 

Makino, S. I. Cheun, H. I. Application of the real-time PCR for the detection of airborne microbial pathogens in reference to the anthrax spores. J. Microbiol. Meth. 2003,53,141-147. 

Beilin, T. Pulz, M. Matussek, A. Hempen, H.-G. Gunzer, F. Rapid detection of enterohemorrhagic Escherichia coli by real-time PCR with fluorescent hybridization probes. J. Clin. Microbiol. 2001,39, 370-374. 

Wittwer, C. Hahn, M. Kaul, K. (Eds.). Rapid Cycle Real-Time PCR Methods and Applications. Springer-Verlag Berlin, 2004. 

Ambion, Inc. Real-time PCR goes prime time. TechNotes 2001,8,1-4. 

Classical PCR involves detection of a PCR product by electophoretic mobility on a gel, which is time consuming. Real-time PCR is distinct from classical PCR, in that electrophoresis is avoided and the PCR product is detected... 

Fig. 11.3 Effect of HU on ET-1 mRNA expression in the TrHBMEC (a) and EA-hy 926 (b) endothelial cells in culture. Quantitative real-time PCR was used to assess the level of ET-1 mRNA in at least four independent experiments in duplicate. Results are expressed in percentage of residual ET-1 mRNA expression for HU-treated cells as compared to the control (culture with or without cytokines). The TATA-binding protein mRNA was used as an internal control. The abbreviations are the same as in the legend for Figure 11.2.

Shi, R., and Chiang, V. L. (2005). Facile means for quantifying microRNA expression by real-time PCR. Biotechniques 39, 519-525. 

To quantify the amount of firefly and Renilla luciferase RNAs, total RNA is extracted from transfected neurons, reverse transcribed, and subjected to real-time PCR amplification using QuantiTech SYBR Green PCR mixture (Qiagen). 

Degradation of 5 -terminal sequences in luciferase mRNA containing 60-nt poly(A) tract and capped with the indicated anti reverse cap analogs. The mRNAs half-lives were determined by real time PCR with primers directed against the 5 -end of luciferase mRNA as described in the text. 

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