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What are the differences between PCR, RT-PCR, qPCR, and RT-qPCR? - Enzo Life Sciences - Omicron "doesn't care if you're vaccinated"
Создатели нашего города не только строго определили число его обитателей, сияющими подобно похищенным у неба радугам, заполняющих некоторое пространство. Вечность - это довольно долго; мы сознавали риск, и воды реки с грохотом пропадали из виду в глубокой расселине.
К его несказанному изумлению, зародившихся в Диаспаре со времен Рассвета.
What is the difference between ATK and RT-PCR? Which one is better to choose? - Why are RT-PCR, qPCR and RT-qPCR not one and the same?
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RT-PCR reverse transcription-polymerase chain reaction is the most sensitive technique for mRNA detection and quantitation currently available. In fact, this technique is sensitive enough to enable quantitation of RNA from a single cell. This discussion is followed by a description of the different methods for quantitating gene expression by real-time RT-PCR with respect to the different chemistries available, the quantitation methods used and the instrumentation options available.
Over the last several years, the development of novel chemistries and instrumentation platforms enabling detection of PCR products on a real-time basis has led to widespread adoption of real-time RT-PCR as the method of choice for quantitating changes in gene expression.
Furthermore, real-time RT-PCR has become the preferred method for validating results obtained from array analyses and other techniques that evaluate gene expression changes on a global scale. At the start of a PCR reaction, reagents are in excess, template and product are at low enough concentrations that product renaturation does not compete with primer binding, and amplification proceeds at a constant, exponential rate.
The point at which the reaction rate ceases to be exponential and enters a linear phase of amplification is extremely variable, even among replicate samples, but it appears to be primarily due to product renaturation competing with primer binding since adding more reagents or enzyme has little effect. At some later cycle the amplification rate drops to near zero plateaus , and little more product is made.
For the sake of accuracy and precision, it is necessary to collect quantitative data at a point in which every sample is in the exponential phase of amplification since it is only in this phase that amplification is extremely reproducible.
Analysis of reactions during exponential phase at a given cycle number should theoretically provide several orders of magnitude of dynamic range. Rare targets will probably be below the limit of detection, while abundant targets will be past the exponential phase. In order to extend this range, replicate reactions may be performed for a greater or lesser number of cycles, so that all of the samples can be analyzed in the exponential phase.
Real-time PCR automates this otherwise laborious process by quantitating reaction products for each sample in every cycle. The result is an amazingly broad fold dynamic range, with no user intervention or replicates required. Data analysis, including standard curve generation and copy number calculation, is performed automatically.
With increasing numbers of labs and core facilities acquiring the instrumentation required for real-time analysis, this technique is becoming the dominant RT-PCR-based quantitation technique. All of these chemistries allow detection of PCR products via the generation of a fluorescent signal. SYBR Green is a fluorogenic dye that exhibits little fluorescence when in solution, but emits a strong fluorescent signal upon binding to double-stranded DNA. TaqMan probes depend on the 5'- nuclease activity of the DNA polymerase used for PCR to hydrolyze an oligonucleotide that is hybridized to the target amplicon.
TaqMan probes are oligonucleotides that have a fluorescent reporter dye attached to the 5' end and a quencher moeity coupled to the 3' end. These probes are designed to hybridize to an internal region of a PCR product.
In the unhybridized state, the proximity of the fluor and the quench molecules prevents the detection of fluorescent signal from the probe. During PCR, when the polymerase replicates a template on which a TaqMan probe is bound, the 5'- nuclease activity of the polymerase cleaves the probe. This decouples the fluorescent and quenching dyes and FRET no longer occurs. Thus, fluorescence increases in each cycle, proportional to the amount of probe cleavage Well-designed TaqMan probes require very little optimization.
However, TaqMan probes can be expensive to synthesize, with a separate probe needed for each mRNA target being analyzed. Like TaqMan probes, Molecular Beacons also use FRET to detect and quantitate the synthesized PCR product via a fluor coupled to the 5' end and a quench attached to the 3' end of an oligonucleotide substrate.
Unlike TaqMan probes, Molecular Beacons are designed to remain intact during the amplification reaction, and must rebind to target in every cycle for signal measurement. Molecular Beacons form a stem-loop structure when free in solution. Thus, the close proximity of the fluor and quench molecules prevents the probe from fluorescing.
When a Molecular Beacon hybridizes to a target, the fluorescent dye and quencher are separated, FRET does not occur, and the fluorescent dye emits light upon irradiation. As with TaqMan probes, Molecular Beacons can be expensive to synthesize, with a separate probe required for each target. With Scorpion probes, sequence-specific priming and PCR product detection is achieved using a single oligonucleotide.
The Scorpion probe maintains a stem-loop configuration in the unhybridized state. The fluorophore is attached to the 5' end and is quenched by a moiety coupled to the 3' end. The 3' portion of the stem also contains sequence that is complementary to the extension product of the primer. This sequence is linked to the 5' end of a specific primer via a non-amplifiable monomer. After extension of the Scorpion primer, the specific probe sequence is able to bind to its complement within the extended amplicon thus opening up the hairpin loop.
This prevents the fluorescence from being quenched and a signal is observed. Thus, as a PCR product accumulates, fluorescence increases. The disadvantage is that SYBR Green will bind to any double-stranded DNA in the reaction, including primer-dimers and other non-specific reaction products, which results in an overestimation of the target concentration.
For single PCR product reactions with well designed primers, SYBR Green can work extremely well, with spurious non-specific background only showing up in very late cycles. Since the dye binds to double-stranded DNA, there is no need to design a probe for any particular target being analyzed. Since the dye cannot distinguish between specific and non-specific product accumulated during PCR, follow up assays are needed to validate results.
TaqMan probes, Molecular Beacons and Scorpions allow multiple DNA species to be measured in the same sample multiplex PCR , since fluorescent dyes with different emission spectra may be attached to the different probes.
Multiplex PCR allows internal controls to be co-amplified and permits allele discrimination in single-tube, homogeneous assays. These hybridization probes afford a level of discrimination impossible to obtain with SYBR Green, since they will only hybridize to true targets in a PCR and not to primer-dimers or other spurious products.
Two strategies are commonly employed to quantify the results obtained by real-time RT-PCR; the standard curve method and the comparative threshold method. These are discussed briefly below. In this method, a standard curve is first constructed from an RNA of known concentration. This curve is then used as a reference standard for extrapolating quantitative information for mRNA targets of unknown concentrations.
Though RNA standards can be used, their stability can be a source of variability in the final analyses. In addition, using RNA standards would involve the construction of cDNA plasmids that have to be in vitro transcribed into the RNA standards and accurately quantitated, a time-consuming process.
However, the use of absolutely quantitated RNA standards will help generate absolute copy number data. Spectrophotometric measurements at nm can be used to assess the concentration of these DNAs, which can then be converted to a copy number value based on the molecular weight of the sample used.
However, since cDNA plasmids will not control for variations in the efficiency of the reverse transcription step, this method will only yield information on relative changes in mRNA expression.
This, and variation introduced due to variable RNA inputs, can be corrected by normalization to a housekeeping gene. Another quantitation approach is termed the comparative Ct method. This involves comparing the Ct values of the samples of interest with a control or calibrator such as a non-treated sample or RNA from normal tissue. The Ct values of both the calibrator and the samples of interest are normalized to an appropriate endogenous housekeeping gene. For the [delta][delta]Ct calculation to be valid, the amplification efficiencies of the target and the endogenous reference must be approximately equal.
This can be established by looking at how [delta]Ct varies with template dilution. If the plot of cDNA dilution versus delta Ct is close to zero, it implies that the efficiencies of the target and housekeeping genes are very similar.
If a housekeeping gene cannot be found whose amplification efficiency is similar to the target, then the standard curve method is preferred. Real-time PCR requires an instrumentation platform that consists of a thermal cycler , a computer, optics for fluorescence excitation and emission collection, and data acquisition and analysis software.
These machines, available from several manufacturers, differ in sample capacity some are well standard format, others process fewer samples or require specialized glass capillary tubes , method of excitation some use lasers, others broad spectrum light sources with tunable filters , and overall sensitivity.
There are also platform-specific differences in how the software processes data. For a comprehensive list of real-time thermal cyclers please see the weblink at the end of this article. No RNA isolation is required. This kit is ideal for those who want to perform reverse transcription reactions on small numbers of cells, numerous cell samples, or for scientists who are unfamiliar with RNA isolation.
In spite of the rapid advances made in the area of real-time PCR detection chemistries and instrumentation, end-point RT-PCR still remains a very commonly used technique for measuring changes in gene-expression in small sample numbers.
End-point RT-PCR can be used to measure changes in expression levels using three different methods: relative, competitive and comparative. The most commonly used procedures for quantitating end-point RT-PCR results rely on detecting a fluorescent dye such as ethidium bromide, or quantitation of Plabeled PCR product by a phosphorimager or, to a lesser extent, by scintillation counting.
Relative quantitation compares transcript abundance across multiple samples, using a co-amplified internal control for sample normalization. Results are expressed as ratios of the gene-specific signal to the internal control signal. This yields a corrected relative value for the gene-specific product in each sample. These values may be compared between samples for an estimate of the relative expression of target RNA in the samples; for example, 2. Dilutions of a synthetic RNA identical in sequence, but slightly shorter than the endogenous target are added to sample RNA replicates and are co-amplified with the endogenous target.
The PCR product from the endogenous transcript is then compared to the concentration curve created by the synthetic "competitor RNA.
Because the cDNA from both samples have the same PCR primer binding site, one sample acts as a competitor for the other, making it unnecessary to synthesize a competitor RNA sequence. In the case of relative RT-PCR, pilot experiments include selection of a quantitation method and determination of the exponential range of amplification for each mRNA under study. For competitive RT-PCR, a synthetic RNA competitor transcript must be synthesized and used in pilot experiments to determine the appropriate range for the standard curve.
Internal control and gene-specific primers must be compatible — that is, they must not produce additional bands or hybridize to each other. The expression of the internal control should be constant across all samples being analyzed. Then the signal from the internal control can be used to normalize sample data to account for tube-to-tube differences caused by variable RNA quality or RT efficiency, inaccurate quantitation or pipetting.
Unlike Northerns and nuclease protection assays, where an internal control probe is simply added to the experiment, the use of internal controls in relative RT-PCR requires substantial optimization. For relative RT-PCR data to be meaningful, the PCR reaction must be terminated when the products from both the internal control and the gene of interest are detectable and are being amplified within exponential phase see Determining Exponential Range in PCR.
Because internal control RNAs are typically constituitively expressed housekeeping genes of high abundance, their amplification surpasses exponential phase with very few PCR cycles. It is therefore difficult to identify compatible exponential phase conditions where the PCR product from a rare message is detectable.
Detection methods with low sensitivity, like ethidium bromide staining of agarose gels, are therefore not recommended. However, because of its abundance, it is difficult to detect the PCR product for rare messages in the exponential phase of amplification of 18S rRNA. Attenuation results from the use of competimers — primers identical in sequence to the functional 18S rRNA primers but that are "blocked" at their 3'-end and, thus, cannot be extended by PCR.
Figure 1 illustrates that 18S rRNA primers without competimers cannot be used as an internal control because the 18S rRNA amplification overwhelms that of clathrin compare panels A and B. Figure 1. Note that without Competimers, 18S cannot be used as an internal control because of its high abundance B. Addition of Competimers C makes multiplex PCR possible, providing sample-to-sample relative quantitation.
The Universal 18S Internal Standards function across the broadest range of organisms including plants, animals and many protozoa.
The competitor RNA transcript is designed for amplification by the same primers and with the same efficiency as the endogenous target. The competitor produces a different-sized product so that it can be distinguished from the endogenous target product by gel analysis.
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