A slope of less than —3. A slope of greater than —3.
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This can occur when values are measured in the nonlinear phase of the reaction or it can indicate the presence of inhibitors in the reaction. The efficiency of a real-time PCR assay can be calculated by analyzing a template dilution series, plotting the C T values against the log template amount, and determining the slope of the resulting standard curve.
Fluorescence is measured during each cycle, which greatly increases the dynamic range of the reaction, since the amount of fluorescence is proportional to the amount of PCR product. PCR products can be detected using either fluorescent dyes that bind to double-stranded DNA or fluorescently labeled sequence-specific probes. The excitation and emission maxima of SYBR Green I are at nm and nm, respectively, allowing use of the dye with any real-time cycler.
Detection takes place in the extension step of real-time PCR. Signal intensity increases with increasing cycle number due to the accumulation of PCR product. Use of SYBR Green enables analysis of many different targets without having to synthesize target-specific labeled probes. However, nonspecific PCR products and primer—dimers will also contribute to the fluorescent signal.
Fluorescently labeled probes provide a highly sensitive method of detection, as only the desired PCR product is detected. However, PCR specificity is also important when using sequence-specific probes. Amplification artifacts such as nonspecific PCR products and primer—dimers may also be produced, which can result in reduced yields of the desired PCR product. Competition between the specific product and reaction artifacts for reaction components can compromise assay sensitivity and efficiency. The following probe chemistries are frequently used.
TaqMan probes : sequence-specific oligonucleotide probes carrying a fluorophore and a quencher moiety. The fluorophore is attached at the 5' end of the probe and the quencher moiety is located at the 3' end. This results in detectable fluorescence that is proportional to the amount of accumulated PCR product.
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When the 2 probes bind, their fluorophores come into close proximity, allowing energy transfer from a donor fluorophore to an acceptor fluorophore. Therefore, fluorescence is detected during the annealing phase of PCR and is proportional to the amount of PCR product. As the FRET system uses 2 primers and 2 probes, good design of the primers and probes is critical for successful results. Dyes used for fluorogenic probes in real-time PCR : For real-time PCR with sequence-specific probes, various fluorescent dyes are available, each with its own excitation and emission maxima see table Dyes commonly used for quantitative, real-time PCR.
The wide variety of dyes makes multiplex, real-time PCR possible detection of 2 or more amplicons in the same reaction , provided the dyes are compatible with the excitation and detection capabilities of the real-time cycler used, and the emission spectra of the chosen dyes are sufficiently distinct from one another. Therefore, when carrying out multiplex PCR, it is best practice to use dyes with the widest channel separation possible to avoid any potential signal crosstalk.
Other probes : Many probe suppliers have developed their own proprietary dyes. For further information, please refer to the web pages of the respective suppliers. Target nucleic acids can be quantified using either absolute quantification or relative quantification.
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Absolute quantification determines the absolute amount of target expressed as copy number or concentration , whereas relative quantification determines, as the first step of analysis, the ratio between the amount of target and the amount of a control e. Subsequently, this normalized value can then be used to compare, for example, differential gene expression in different samples.
Use of external standards enables the level of a gene to be given as an absolute copy number. For gene expression analysis, the most accurate standards are RNA molecules of known copy number or concentration. Depending on the sequence and structure of the target and the efficiency of reverse transcription, only a proportion of the target RNA in the RNA sample will be reverse transcribed. The use of RNA standards takes into account the variable efficiency of reverse transcription. The amount of unknown target should fall within the range tested.
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Amplification of the standard dilution series and of the target sequence is carried out in separate wells. The C T values of the standard samples are determined. Then, the C T value of the unknown sample is compared with the standard curve to determine the amount of target in the unknown sample.
It is important to select an appropriate standard for the type of nucleic acid to be quantified. The copy number or concentration of the nucleic acids used as standards must be known. In addition, standards should have the following features:. RNA standards can be created by cloning part or all of the transcript of interest into a standard cloning vector.
Ensure that in vitro transcription of the insert leads to generation of the sense transcript. Furthermore, ensure that the RNA used as a standard does not contain any degradation products or aberrant transcripts by checking that it migrates as a single band in gel or capillary electrophoresis.
After determination of RNA concentration by spectrophotometry, the copy number of standard RNA molecules can be calculated using the following formula:. Advantages of this method are that large amounts of standard can be produced, its identity can be verified by sequencing, and the DNA can easily be quantified by spectrophotometry. Plasmid standards should be linearized upstream or downstream of the target sequence, rather than using supercoiled plasmid for amplification.
This is because the amplification efficiency of a linearized plasmid often differs from that of the supercoiled conformation and more closely simulates the amplification efficiency of genomic DNA or cDNA. After spectrophotometric determination of plasmid DNA concentration, the copy number of standard DNA molecules can be calculated using the following formula:.
We recommend including at least 20 bp upstream and downstream of the primer binding sites of the amplicons. The copy number of the target present in the genomic DNA can be directly calculated if the genome size of the organism is known. For example, the genome size haploid of Mus musculus is 2. In relative quantification, the ratio between the amounts of a target gene and a control gene e. This ratio is then compared between different samples.
In gene expression analysis, housekeeping or maintenance genes are usually chosen as an endogenous reference. The target and reference gene are amplified from the same sample, either separately or in the same reaction duplex, real-time PCR. The normalized value is determined for each sample and can be used, for example, to compare differential expression of a gene in different tissues or to compare gene expression between siRNA-transfected cells and untransfected cells.
However, the expression level of the endogenous reference gene must not vary under different experimental conditions or in different states of the tissue e. When gene expression levels are compared between samples, the expression level of the target is referred to as being, for example, fold higher in stimulated cells than in unstimulated cells.
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The quantification procedure differs depending on whether the target and the endogenous reference gene are amplified with comparable or different efficiencies. The amplification efficiency of 2 genes target A and target B can be compared by preparing a dilution series for both genes from a reference RNA or cDNA sample. Each dilution series is then amplified in real-time one-step or two-step RT-PCR and the CT values obtained are used to construct standard curves for target A and target B.
The amplification efficiency E for each target can be calculated according to the following equation:. To compare the amplification efficiencies of the 2 target sequences, the C T values of target A are subtracted from the C T values of target B. The difference in C T values is then plotted against the logarithm of the template amount see figure Efficiency comparison. Amplification efficiencies of the target gene and the endogenous reference gene are usually different since efficiency of primer annealing, GC-content of the sequences to be amplified, and PCR product size usually vary between the 2 genes.
In this case, a standard curve needs to be prepared for the target gene as well as for the endogenous reference gene, for example, using total RNA prepared from a reference cell line calibrator or reference sample. Due to differences in PCR efficiency, the resulting standard curves will not be parallel and the differences in C T values of the target and the reference will not be constant when the template amounts are varied see figure Different PCR efficiencies.
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If the amplification efficiencies of the target gene and the endogenous reference gene are comparable, one standard curve for the reference gene is sufficient. The differences in C T values of the target and the reference will be constant when the amounts of template are varied see figure Same PCR efficiencies. The amounts of target and reference in an unknown sample are calculated by comparing the C T values with the standard curve for the reference gene.