Both PCR and qPCR (Quantitative Real-time PCR) are commonly used molecular biology experimental techniques. Ordinary PCR can perform qualitative or semi-quantitative analysis through agarose gel electrophoresis of the PCR products. While qPCR, through real-time detection of fluorescence signal changes in each cycle of PCR amplification reaction, can perform quantitative analysis of the starting template by using the relationship between Ct value and standard curve. It has characteristics of high sensitivity and specificity, and is widely used in diagnosis and detection.
qPCR Detection Methods
qPCR is a mature method for mRNA/DNA quantification and SNP screening. Fluorescent dyes or specific fluorescently labeled nucleic acid probes can be used to monitor the reaction process in real time. There are two commonly used methods: the dye method and probe method.
The characteristics of the dye method are ease of use and low cost. It generally uses DNA dye SYBR Green I. In the qPCR experiment, the dye can bind to the double strands. In the free state, SYBR Green I almost has no fluorescence signal. However, after binding to DNA, it emits a fluorescence signal under the illumination of the excitation light source. Its signal strength represents the number of DNA molecules in the duplex. Therefore, the more PCR products, the more SYBR Green I binding, the stronger the fluorescence signal. In addition, the dye method also needs to do a melting curve to analyze the Tm value of the PCR product.
The TaqMan probe method is often used in qPCR experiments. A TaqMan probe is a linear oligonucleotide with a fluorophore group at the 5′ end and a quenched group at the 3′ end. When the qPCR probe is complete, the fluorescence is absorbed by the quenched group, and no fluorescence is emitted. During the process of PCR amplification, the 5′-3′ exonuclease activity of the Taq enzyme will cut the fluorophore group at the 5′ end of the qPCR probe so that the fluorophore signal can be detected after the separation of the fluorophore and quenched groups. The probe method has more specificity than the dye method, it can also be used for multi-system reactions, and it can predict and optimize reaction conditions in advance.
Common Considerations for qPCR Experiments
1. General rules for qPCR probe design
1) The length of the amplified fragment should not be too long. Generally less than 300bp in length.
2) The qPCR probe should not be complementary to any primer, and its length should be as short as possible on the premise of ensuring specificity. Generally not more than 30bp in length.
3) The Tm value of the qPCR probe is at least 5 degrees higher than that of the primer.
4) If the qPCR probe is used to detect polymorphic sites, the polymorphic sites should be as close to the middle of the probe as possible.
5) The 5 ‘end of the qPCR probe should not be base G, which can quench the fluorophore group.
2. The abnormal amplification curve
The normal amplification curve is generally S-shaped, and the Ct value is best between 20 and 30. Abnormal amplification curves include large Ct value, no plateauing, plateauing decline, etc.
1) Large Ct value (e.g., Ct value > 30)
· If the amount of template is low or the gene expression abundance is low, it is recommended to increase the amount of template and observe whether the Ct value can be reduced by a corresponding fold.
· Inappropriate qPCR reaction conditions or improper primer design leads to low amplification efficiency. It is recommended to confirm amplification efficiency through a standard curve.
· The amplified product is too long. It is recommended to use the three-step procedure to amplify or optimize the primers. The length of the amplified product should not exceed 300bp.
· There may be inhibitors in the system that affect enzyme activity. So, it is suggested to dilute the template gradient or prepare the template with higher purity.
2) The amplification curve could not reach a plateau
Due to low gene abundance and low number of cycles, it is suggested to increase the number of cycles or select products suitable for low gene abundance.
3) The amplifying curve decreased during the plateau
It may be that the baseline range is not set properly, which is usually caused by a high number of templates. It is recommended to reduce the baseline endpoint value (Ct value -4 is generally recommended).
4) The amplification curve is chaotic and irregular
The ROX concentration may not match the instrument. It is recommended to adjust the ROX concentration.
5) There was a dissolution curve, but no amplification curve
It may be that the amplification program was set incorrectly, and the fluorescence signal was not collected. It is suggested to conduct a new experiment and increase the fluorescence signal collection in the extension stage of the amplification program.
6) The melting curve is not a single peak
· Nonspecific amplification. It is suggested to design new primers, and the annealing temperature of primers can be optimized by gradient PCR.
· Primer dimers, and the primers are non-specific primers. It is recommended to optimize the amplification conditions, set the gradient Tm, and explore the optimal Tm value. Or, appropriately reduce the primer concentration.
· Genomic contamination in templates. Re-prepare the cDNA template.
3. The linear relation of the standard curve is poor
1) Sampling error. Increase the dilution ratio of the template and improve the sample volume.
2) Degradation of standard products. Reprepare the standard and repeat the experiment.
3) The template concentration is too high. Increase the dilution ratio of templates.
4. Repetition Sets
In general, repeat amplification 3 times for each gene in each specimen, and obtain data (Ct value) for the average value before subsequent calculation and analysis.
qPCR Probe & Oligo Synthesis by Synbio Technologies
Synbio Technologies is certified with ISO 9001 & ISO 13485 quality management system certifications with an established, independent laboratory and anti-false positive production processes. We can synthesize various types of qPCR probes, such as TaqMan probes, MGB probes, RPA probes, molecular beacon probes, double quenching probes, and COVID-19 probes. High quality qPCR probe synthesis guarantees the good performance of your qPCR experiment.
· Custom Oligo Synthesis
· Diagnostic Probes & Oligos
· Modified Oligos