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Page | 149 exceeds a detection threshold, the Ct (threshold cycle) correlates with the number of target sequences present. Higher the initial number of target sequences in the sample, faster the fluorescence intensity will increase during the PCR reaction.
The cycle in which fluorescence intensity can be detected is termed as quantitation cycle (Cq), and lower Cq values mean higher initial copies of target sequences. The threshold cycle (Ct) or the quantitation cycle (Cq) are determined by the standard curve dilution series, also known as “absolute” quantification. The standard curve approach is employed when the objective of the experiment is to measure the exact number of target sequences. The templates for the standard curve quantification can include genomic DNA, cDNA, total RNA, or plasmids containing the cloned gene of interest. The log values of the initial template copy number of each dilution are plotted against the Ct generated to prepare the standard curve, and the linear regression line is calculated. The Ct values of unknown samples can be compared to the standard curve for quantification of initial copy numbers. The standard curve dilution series need to consider the entire range of concentrations that will be measured in the unknown samples. The linearity of the regression line, denoted by R2 or Pearson Correlation Coefficient, should be close to 1. The standard templates (DNA or RNA) are to be quantified using the standard methods (UV spectrophotometry or nucleic acid binding dyes) in replicates, with no template controls (NTC).
The ‘relative quantification’ approach is employed to examine ‘gene expression’ by measuring the relative concentration of the gene of interest in unknown samples compared to a calibrator.
The calibrator, which is a baseline for the expression of a target gene serves as a benchmark to which other samples can be compared. The differences in Ct values as fold-changes between the unknown sample and the calibrator are expressed to state whether the gene of interest is up- or down-regulated. In addition to comparing the expression of target gene in control versus experimental sample, normalization using a reference gene whose expression is constant in both the control and experimental samples are desired. The actual amplification efficiencies of target and normalizer need to be established using the multiple standard curve approach to verify the reproducibility of efficiency measurements.
The qPCR chemistries include the non-specific DNA binding dyes such as SYBR Green I and EvaGreen. These non-binding dyes are easy to use, reasonably priced, and have relatively low fluorescence when free in solution. The fluorescence intensity increases by more than 1000- fold, and proportionately to the double DNA concentration. The inherent disadvantage of non- specific DNA binding dyes is their non-specificity. Hence, the primer dimer formation due to non-specific binding of primers needs to be avoided. The presence of non-specific signal can be detected by the melt-curve analysis. The amplified sequences can be characterized in the melting curve analysis using their apparent melting temperature (Tm), which is a function of product length and base composition. Following the PCR amplification, the amplified sequences can be slowly melted while fluorescence from the dye is monitored. The amplified
Page | 150 DNA melts with increasing temperature and the fluorescence density gets decreased. In the PCR amplification where the amplified sequences consist of molecules of homogeneous length and base composition, a single thermal transition is detected. Otherwise, multiple thermal transitions in the fluorescence intensity will be detected. The specific and non-specific amplicons based on the melting temperature (Tm) of the reaction end-products can be differentiated from the fluorescence versus temperature curve (also known as the dissociation curve).
The probe-based chemistries provide a higher level of detection specificity. The internal, fluorescent probe will not fluoresce, remain quenched in the absence of a specific target sequence. When the probe hybridizes to the target sequence, the fluorescence of reporter dye can be detected. TaqMan probes which are the third oligonucleotide in the PCR reaction and have the fluorescent reported dye (FAM) attached to the 5’ end and a quencher (TAMRA) or a dark quencher (Black Hole Quencher) at the 3’ end. The fluorescence from the reporter dye is quenched as long as both the reporter and quencher are in close proximity. TaqMan probes use the FRET (Fluorescence Resonance Energy Transfer) quenching mechanism. The probe designing is done to anneal to one strand of the target sequence, just slightly downstream of one of the primers. Taq polymerase which has 5’-3’ nuclease activity encounters the probe, displaces and degrades the 5’ end, and releases the reporter dye free into solution. The separation of reporter dye and quencher will help to detect the reporter dye. TaqMan chemistry (specialized probes such as MGB or LNA probes) is used in a multiplex reaction where a separate probe is designed for each target sequence (allele) and each probe labelled with different fluorophores (e.g., FAM and HEX).
The qPCRmethods with detection of deviations in the amplification efficiency of individual reactions are more precise than end-point determinations. The most challenging steps of qPCR analysis are the primer and probe designing, and the fluorescence detection chemistry. The techniques of qPCR and reverse transcription (RT)-qPCR have become very popular now and are used in biomedical and agricultural research, and biotechnological applications. To improve the quality, reproducibility, and interpretability of qPCR data, the ‘minimum information for the publication of quantitative real-time PCR experiments’ (MIQE) needs to be considered in all the applications, more so for genotyping and diagnosis (Taylor et al. 2010; Bustin and Nolan 2017). The applications of qPCR are growing rapidly, which indicates that qPCR will continue to remain one of the sought-after technique in medicine, molecular life sciences and agricultural sciences.
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