1. DNA preparation
The most important step to ensure successful PCR is the preparation of high-quality DNA. The integrity and purity of the DNA template are essential. Quantitative PCR involves multiple rounds of enzymatic reactions and is, therefore, more sensitive to impurities such as proteins, phenol/chloroform, salts, EDTA, and other chemical solvents. Contaminants can also interfere with fluorescence detection. The ratio of absorbance values at 260 nM and 280 nM gives an estimate of the purity of the DNA. Pure DNA has an A260 / A280 ratio of 1.8-2.0. Lower proportions indicate the presence of contaminants such as proteins.
Very few copies of the target nucleic acid (equivalent to approximately 100 pg of gDNA or cDNA) are needed to initiate qPCR. To minimize contamination with reaction inhibitors, the amount of starting template should be kept to the minimum required for accurate quantitation. When the starting material is RNA, primer design and DNase I treatment will reduce the signals that can be generated from gDNA contamination.
3. Primer design
Whether using a dsDNA-binding dye or probe-based detection chemistry, the design of high-quality primers is one of the most crucial pre-experimental steps in qPCR. PCR-specific primers must be designed with the help of primer design software to eliminate complications introduced with primer dimers and secondary structures. Lower primer concentrations decrease the accumulation of primer dimer formation and the formation of nonspecific products, which is critical in the use of SYBR Green I dye in quantitative PCR.
Standard PCR / qPCR master mixes contain dATP, dCTP, dGTP, and dTTP. However, there are some mixes available that replace dTTP with dUTP. The products of previous reactions run with dUTP will contain uracil instead of thymine. These are then susceptible to cleavage by uracil-DNA-glycosylase (UNG). Therefore, pre-incubation of subsequent reactions with UNG avoids carryover of contamination between reactions. To be effective, all reactions in the laboratory must use dUTP.
5. Magnesium concentration
Magnesium chloride (MgCl2) is required for the activity of reverse transcriptase, Taq DNA polymerase, and 5 ‘to 3’ exonuclease of Taq DNA. Optimal Mg2 + concentrations for reactions containing DLP are usually between 3 and 6 mM. Lower magnesium chloride concentrations generally result in the formation of fewer nonspecific products. Some ReadyMix solutions are provided at a 2X concentration of 7 mM magnesium chloride (3.5 mM final concentration). In some cases, a vial of a 25 mM magnesium chloride solution is provided to further optimize the final magnesium chloride concentration if necessary. Sometimes a reaction mixture that does not contain MgCl2 may be necessary to be able to use a low concentration, e.g. Eg when using Scorpion probe detection.
6. Reverse transcriptase
A reverse transcriptase enzyme that provides high yields of cDNA, while preserving activity at high temperature, is critical to the success of RT-qPCR. Performance at high temperatures helps ensure that regions of RNA with a significant secondary structure are destabilized and accessible for hybridization and subsequent amplification. When performing one-step RT-qPCR, high-temperature performance allows the use of gene-specific primers with high melting temperatures (Tm), increasing the specificity of the reaction. When performing two-step protocols, it is important to ensure that the enzyme results in a linear and proportional yield of cDNA from RNA. Minimizing pipetting can reduce variability. Some ReadyMixes contain primers and other reagents necessary to perform RT, for example, ReadyScript cDNA Synthesis Mix (RDRT).
7. Taq DNA polymerase
As with the selection of the most appropriate reverse transcriptase for RT, the selection of the appropriate enzyme is vital. A fundamental problem with natural Taq DNA polymerase is that the enzyme has residual activity at low temperatures. The binding of nonspecific primers leads to the formation of nonspecific products as a result of this residual polymerase activity. Taq DNA polymerases blocked by antibodies or chemically blocked (“hot start”) help rectify this situation by preventing enzymatic activity until the high-temperature denaturation step begins. See the PCR Mix Selection Guide define the best hot-start polymerase for your application.
A positive control is always useful to ensure that all components of the kit are working properly. A negative / no template control is not required to determine whether there is contamination. A signal in the no template control demonstrates the presence of DNA contamination or primer dimer formation.
Reaction buffers or master mixes typically contain dNTPs, a Taq DNA polymerase, MgCl2, and stabilizers. SYBR Green I, ROX ™, fluorescein, and inert charge dyes may also be included, depending on detection chemistry, instrument, and reaction requirements. Stabilizers and PCR buffer components are generally the property of the manufacturer. If purchased separately, maximum flexibility is possible, as each ingredient can be individually optimized in the reaction. However, on the contrary, while buying the ingredients together as a master mix reduces flexibility, increases batch consistency and convenience while reducing the number of pipetting steps and therefore the chances of error and pollution.
10. Data analysis
Follow the recommendations of the real-time instrument used to perform quantitative SYBR Green PCR. The following may help new instrument users. Generally, the number of cycles is plotted against fluorescence. Threshold cycles (CT) or crossover points are used to determine the amount of templates in each sample. The threshold cycle or crossover point is the first cycle that shows a detectable increase in fluorescence due to the formation of PCR products. The cycles before the crossover point are the baseline cycles. The baseline cycles do not show a detectable increase in fluorescence due to the PCR products. The threshold used to determine when the first detectable increase in fluorescence occurs can also be adjusted manually. The threshold should always be done on a logarithmic amplification plot. In a logarithmic amplification plot, the threshold should be set in the log-linear range and not in the plateau phase.
11. Melt curves
Performing a melting curve analysis at the end of the run will help to analyze only the PCR product of interest. Follow the real-time instrument manufacturer’s instructions for melting curve analysis. Successive runs with the same primers can be modified to eliminate the contribution of primer dimer formation to the product signal by collecting data in an additional cycle step, the temperature of which must be between the already determined dimer and temperatures. product melt (TM).