Real-time quantitative PCR experiment

Summary

Real-time quantitative PCR (qPCR) was first introduced by Applied Biosystems in 1996. Conventional PCR methods allow for exponential amplification of specific DNA fragments and qualitative analysis of the amplification products by gel electrophoresis or quantitative analysis by optical density scanning after radionuclide labeling. Both qualitative and quantitative analyses are tests of the end products of the PCR reaction. However, in many cases it is more important to determine the amount of starting template before PCR signal amplification. Since this technique not only realizes the leap from qualitative to quantitative PCR, but also has the characteristics of stronger specificity, more accurate and reliable results, and a higher degree of automation compared with conventional PCR.

Principle

The basic principle of real-time quantitative PCR is to rely on fluorescent markers and automated instrumentation that reads fluorescence intensity at each cycle and monitors PCR products in real time as the reaction progresses, thus enabling more accurate quantitative and qualitative analysis of template samples. The basis of real-time quantitative PCR is the quantitative relationship between the amount of template DNA at the start of the reaction and the amount of amplification product in the exponential phase of the cycle, which can be reflected by real-time monitoring and calculation of the fluorescence signal. Early in the PCR reaction, the level of fluorescence produced is not clearly distinguishable from the background, and then the fluorescence production enters an exponential, linear, and ultimately a plateau phase, so that the amount of PCR product can be detected at a point in the exponential phase of the PCR reaction, and from this the initial amount of template can be deduced.

Appliance

Real-time quantitative PCR technology is mainly used in the following aspects: 1. real-time quantitative PCR technology used in tumor diagnosis and research 2. real-time quantitative PCR technology used in gene mutation and its polymorphism 3. real-time quantitative PCR technology used in the detection of pathogens

Operation method

Real-time quantitative PCR experiment

Materials and Instruments

Reagents:
Primers, fluorescent probes/fluorescent dyes
Instrument:
Real-time quantitative PCR instrument

Move

The basic process of real-time quantitative PCR technique can be divided into the following steps:

(i) Design of primers and probes

Check the comparison sequence, choose the position of the probe first, and then design the primers so that they are as close to the probe as possible.
The principles of probe design are:
1. as short as possible, not more than 30bp;
2. Tm value should be between 68 - 70 ℃;
3. Avoid the 5'-end of the guanosine (G) to avoid pining inactivation;
4. Select the chain of cytidylic acid (C) more than guanosine (G) as a probe, the content of G is more than the content of C will reduce the efficiency of the reaction.

(ii) PCR reaction and data recording

Extract total RNA from cells or tissues, reverse transcription PCR. PCR reaction needs to be carried out in the special reaction plate or tube of the real-time quantitative PCR instrument. After adding all the reaction reagents, put the reaction plate into the instrument and complete the reaction and data recording according to the instrument operation instructions.

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Generally speaking, the fluorescence amplification curve can be divided into three phases: the fluorescence background signal phase, the fluorescence signal exponential amplification phase and the plateau phase, and its shape is a smooth "S" curve.

1. Absolute quantification by the standard curve method uses a series of standards of known concentration to produce a standard curve. The standards can be purified plasmid DNA, in vitro transcribed RNA, or in vitro synthesized single-stranded DNA.

2. Relative quantification by the standard curve method is its own relative standard curve. The relative dilution of the standard is sufficient. The amount of target sequence in the sample throughout the experiment is derived from its own standard curve and must be divided by the amount of the reference.

3. Relative Quantification of Ct Comparison Ct is the number of cycles the thermal cycler goes through to detect a fluorescent signal that reaches a set threshold. Assuming a 1-fold increase in the amount of product per cycle, the Ct value obtained during the exponential phase of the PCR reaction reflects the amount of starting template, and a difference of one cycle corresponds to a 2-fold difference in the amount of starting template. This method eliminates the need for a standard curve, but assumes that the target gene and the internal reference gene have similar amplification efficiencies.

Caveat

Common Problems and SolutionsCommon problems in the real-time quantitative PCR process are:(a) Factors affecting specificityThe Ct value appears too late or no Ct signal appears due to the degradation of primers or probes, or improper design of primers or probes. Optimization is to design better primers or probes; optimize primer concentration and annealing temperature. Degradation of primers or probes can be avoided by checking their integrity by electrophoresis before performing real-time quantitative PCR experiments.Non-specific amplification occurs when the template has genomic contamination. Optimization is to avoid DNA contamination during RNA extraction or to avoid non-specific amplification by primer design.(ii) Factors affecting reproducibilityThe initial copy number of the target gene is low, resulting in poor reproducibility of the results. Samples with higher initial concentration should be used or the dilution of samples should be reduced. If the amount of the target gene in the sample to be tested is near the detection limit of the reaction system, then it is better to use duplicate wells to ensure the reliability of the results. If the researcher is conducting the first experiment, then a range of dilution concentrations of the template should be chosen to conduct the experiment to select the most appropriate template concentration, in general, so that Ct is located in 15 - 30 cycles is more appropriate.(iii) Poor linearity of the standard curveThe reasons may be1. Inadmissible addition of samples, so that the standard is not gradient;2. Degradation of the standard;3. The template concentration is too high. The ideal standard should be highly homologous to the sample, although the difference between the standard and the sample always exists. When making the standard curve, at least 5 dilutions of the standard should be chosen to cover the full range of possible concentrations of the amount of the target gene in the sample to be tested.(iv) Factors affecting the sensitivity levelDue to the use of fluorescent material as a quantitative tool, the sensitivity of real-time quantitative PCR can usually reach 100 copies/ml, and the linear range of logarithmic analysis is very wide, 0 - 1011 copies/ml.There are many factors affecting the sensitivity of real-time quantitative PCR, and in addition to the usual PCR reaction factors such as the reaction system and Taq enzyme activity, the following factors should be noted:The specific product competes with the primer dimer for the fluorescent dye SYBR Green, thus reducing the sensitivity of real-time PCR. The TaqMan probe can be used instead of SYBR Green.Use a hot-start method to enhance specificity. Adding a reaction component only when the reaction system reaches the primer annealing temperature is effective in minimizing the formation of primer dimers, since the formation of primer dimers begins as soon as the reagents are mixed.It is important to optimize the primer design as much as possible. Making the GC content of the two primers approximately the same, using purified primers for experiments, etc. can help prevent the formation of primer dimers.Mg2 +The concentration of Mg2+.Mg2+ is a key factor affecting Taq enzyme activity.Mg2+ is a key factor affecting the activity of the Taq enzyme.Mg2+ is a key factor in Taq enzyme activity, as a low concentration of Mg2+ will not optimize Taq enzyme activity, while a high concentration will increase the formation of primer dimers. In general, for PCR reactions using DNA or cDNA as templates, a concentration of 2 - 5 mmol/L MgCl2+ should be selected.2For RT-PCR with mRNA as the template, choose 4-8 mmol/L MgCl22For mRNA template RT-PCR, choose 4 - 8 mmol/L MgCl2.

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