Typical uses of real-time PCR include pathogen detection, gene expression analysis, single nucleotide polymorphism SNP analysis, analysis of chromosome aberrations, and most recently also protein detection by real-time immuno PCR. Wittwer Clinical Chemistry , 55 4 : Vandesompele, M. Kubista and M. Chapter 5 - Data Analysis Software M. Pfaffl, J. Vandesompele and M.
The Road from Qualitative to Quantitative Assay. What is next?
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Bustin, page - Cambridge University Press. Among the many factors that determine the sensitivity, accuracy, and reliability of a real-time quantitative reverse transcription polymerase chain reaction qRT—PCR 1 assay, template quality is one of the most important determinants of reproducibility and biological relevance .
This is a well-recognized problem , and there are numerous reports that describe the significant reduction in the sensitivity and kinetics of qPCR assays caused by inhibitory components frequently found in biological samples , , , ,  and . The inhibiting agents may be reagents used during nucleic acid extraction or copurified components from the biological sample such as bile salts, urea, haeme, heparin, and immunoglobulin G. At best, inhibitors can generate inaccurate quantitative results; at worst, a high degree of inhibition will create false-negative results.
The most common procedure used to account for any differences in PCR efficiencies between samples is to amplify a reference gene in parallel with the reporter gene and to relate their expression levels. However, this approach assumes that the two assays are inhibited to the same degree. The problem is even more pronounced in absolute quantification, where an external calibration curve is used to calculate the number of transcripts in the test samples, an approach that is commonly adopted for quantification of pathogens.
Some, or all, of the biological samples may contain inhibitors that are not present in the nucleic acid samples used to construct the calibration curve, leading to an underestimation of the mRNA levels in the test samples . The increasing interest in extracting nucleic acids from formalin-fixed paraffin-embedded FFPE archival material undoubtedly will lead to an exacerbation of this problem. Obviously, such inhibitors are likely to distort any comparative quantitative data. PCR technology is based on a simple principle; an enzymatic reaction that increases the initial amount of nucleic acids.
This method makes it possible to detect specific mRNA transcripts in any biological sample. Problems arise when optimization of the experimental work flow becomes necessary because of high technical variations. Therefore the source of experimental variances can often be found in the pre-PCR analytical steps.
Usually this is neglected and optimization is done for PCR reaction only. In this chapter — RT-PCR optimization strategies - the whole workflow of RT-PCR experiment will be discussed, because the identification of the source of variability is only possible following error accumulation in every single step. Reliable data can be created when the technical variance caused by the experimental steps is kept as low as possible.
In this chapter many recommendations to decrease the technical variance can be found. Working with low-quality RNA may strongly compromise the experimental results of downstream applications which are often labour-intensive, time-consuming, and highly expensive. To verify RNA quality nowadays commercially available automated capillary-electrophoresis systems are available which are on the way to become the standard in RNA quality assessment.
Advantage and disadvantages of RNA quantity and quality assessment are shown in performed applications of various tissues and cell cultures. Go the RNA Integrity sub-domain. Quantitative real-time PCR for cancer detection: the lymphoma case. Expert Rev Mol Diagn.
Introduction for Genetics and Oncology Volume Rapid Cycle Real-Time PCR Methods and Applications
Advances in the biologic sciences and technology are providing molecular targets for diagnosis and treatment of cancer. Lymphoma is a group of cancers with diverse clinical courses. Gene profiling opens new possibilities to classify the disease into subtypes and guide a differentiated treatment. Real-time PCR is characterized by high sensitivity, excellent precision and large dynamic range, and has become the method of choice for quantitative gene expression measurements. For accurate gene expression profiling by real-time PCR, several parameters must be considered and carefully validated.
These include the use of reference genes and compensation for PCR inhibition in data normalization. Quantification by real-time PCR may be performed as either absolute measurements using an external standard, or as relative measurements, comparing the expression of a reporter gene with that of a presumed constantly expressed reference gene.
Sometimes it is possible to compare expression of reporter genes only, which improves the accuracy of prediction. The amount of biologic material required for real-time PCR analysis is much lower than that required for analysis by traditional methods due to the very high sensitivity of PCR. Fine-needle aspirates and even single cells contain enough material for accurate real-time PCR analysis. Cells in all organisms regulate gene expression by turnover of gene transcripts single stranded RNA : The amount of an expressed gene in a cell can be measured by the number of copies of an RNA transcript of that gene present in a sample.
In order to robustly detect and quantify gene expression from small amounts of RNA, amplification of the gene transcript is necessary. In order to amplify small amounts of DNA, the same methodology is used as in conventional PCR using a DNA template, at least one pair of specific primers , deoxyribonucleotides , a suitable buffer solution and a thermo-stable DNA polymerase. A substance marked with a fluorophore is added to this mixture in a thermal cycler that contains sensors for measuring the fluorescence of the fluorophore after it has been excited at the required wavelength allowing the generation rate to be measured for one or more specific products.
This allows the rate of generation of the amplified product to be measured at each PCR cycle. The data thus generated can be analysed by computer software to calculate relative gene expression or mRNA copy number in several samples. Quantitative PCR can also be applied to the detection and quantification of DNA in samples to determine the presence and abundance of a particular DNA sequence in these samples.
Evaluation of digital real-time PCR assay as a molecular diagnostic tool for single-cell analysis
Northern blotting is often used to estimate the expression level of a gene by visualizing the abundance of its mRNA transcript in a sample. In this method, purified RNA is separated by agarose gel electrophoresis , transferred to a solid matrix such as a nylon membrane , and probed with a specific DNA or RNA probe that is complementary to the gene of interest. Although this technique is still used to assess gene expression, it requires relatively large amounts of RNA and provides only qualitative or semi quantitative information of mRNA levels.
For this reason a number of standardization systems often called normalization methods have been developed. Some have been developed for quantifying total gene expression, but the most common are aimed at quantifying the specific gene being studied in relation to another gene called a normalizing gene, which is selected for its almost constant level of expression. These genes are often selected from housekeeping genes as their functions related to basic cellular survival normally imply constitutive gene expression.
The most commonly used normalizing genes are those that code for the following molecules: tubulin , glyceraldehydephosphate dehydrogenase , albumin , cyclophilin , and ribosomal RNAs. Real-time PCR is carried out in a thermal cycler with the capacity to illuminate each sample with a beam of light of at least one specified wavelength and detect the fluorescence emitted by the excited fluorophore.
The thermal cycler is also able to rapidly heat and chill samples, thereby taking advantage of the physicochemical properties of the nucleic acids and DNA polymerase. The PCR process generally consists of a series of temperature changes that are repeated 25 — 50 times. Due to the small size of the fragments the last step is usually omitted in this type of PCR as the enzyme is able to increase their number during the change between the alignment stage and the denaturing stage.
Real-time PCR technique can be classified by the chemistry used to detect the PCR product, specific or non-specific fluorochromes.
An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity measured at each cycle. This can potentially interfere with, or prevent, accurate monitoring of the intended target sequence.
Gene Quantification & real-time PCR / kinetic RT-PCR
Then the reaction is run in a real-time PCR instrument , and after each cycle, the intensity of fluorescence is measured with a detector; the dye only fluoresces when bound to the dsDNA i. This method has the advantage of only needing a pair of primers to carry out the amplification, which keeps costs down; multiple target sequences can be monitored in a tube by using different types of dyes.
Fluorescent reporter probes detect only the DNA containing the sequence complementary to the probe; therefore, use of the reporter probe significantly increases specificity, and enables performing the technique even in the presence of other dsDNA. Using different-coloured labels, fluorescent probes can be used in multiplex assays for monitoring several target sequences in the same tube.
The specificity of fluorescent reporter probes also prevents interference of measurements caused by primer dimers , which are undesirable potential by-products in PCR. However, fluorescent reporter probes do not prevent the inhibitory effect of the primer dimers, which may depress accumulation of the desired products in the reaction. The method relies on a DNA-based probe with a fluorescent reporter at one end and a quencher of fluorescence at the opposite end of the probe. The close proximity of the reporter to the quencher prevents detection of its fluorescence; breakdown of the probe by the 5' to 3' exonuclease activity of the Taq polymerase breaks the reporter-quencher proximity and thus allows unquenched emission of fluorescence, which can be detected after excitation with a laser.
An increase in the product targeted by the reporter probe at each PCR cycle therefore causes a proportional increase in fluorescence due to the breakdown of the probe and release of the reporter. Real-time PCR permits the identification of specific, amplified DNA fragments using analysis of their melting temperature also called T m value, from m elting t emperature.
The DNA melting temperature is specific to the amplified fragment. The results of this technique are obtained by comparing the dissociation curves of the analysed DNA samples. Unlike conventional PCR, this method avoids the previous use of electrophoresis techniques to demonstrate the results of all the samples.
This is because, despite being a kinetic technique, quantitative PCR is usually evaluated at a distinct end point. Unlike end point PCR conventional PCR , real time PCR allows monitoring of the desired product at any point in the amplification process by measuring fluorescence in real time frame, measurement is made of its level over a given threshold. A commonly employed method of DNA quantification by real-time PCR relies on plotting fluorescence against the number of cycles on a logarithmic scale.
A threshold for detection of DNA-based fluorescence is set times of the standard deviation of the signal noise above background. The number of cycles at which the fluorescence exceeds the threshold is called the threshold cycle C t or, according to the MIQE guidelines, quantification cycle C q. During the exponential amplification phase, the quantity of the target DNA template amplicon doubles every cycle.
However, the efficiency of amplification is often variable among primers and templates. Therefore, the efficiency of a primer-template combination is assessed in a titration experiment with serial dilutions of DNA template to create a standard curve of the change in C q with each dilution.