About quantitative PCR fluorescence signal intensity

1. If there is a standard curve, calculate it according to the standard curve;

2. Generally, it is a relative quantity, which is calculated by the deltadeltact method;

3. Primer or probe degradation: its integrity can be detected by PAGE electrophoresis;

4. Insufficient amount of template: for samples of unknown concentration, the highest concentration of serial diluted samples should be started;

5. Looking at the CT value is the most intuitive data for the analysis of fluorescence quantitative PCR, the CT value generally loses its reference value at about 35, and the starting value of the amplification curve measured by the biodai-PCR fluorescence quantitative method in our laboratory is basically about 30.

There are many factors that affect fluorescence PCR, but there are a few things that should be noted from an operational and technical point of view:

RNA integrity.

Because once the RNA is degraded, the quantified results do not correctly reflect the amount of mRNA in the sample, and the results obtained are also wrong. Therefore, in the RNA extraction process, it is necessary to strictly follow the operation specifications to avoid contamination by RNA enzymes.

Genomic DNA contamination in RNA samples.

The extracted RNA sample inevitably has a small amount of genomic DNA mixed in with it. Genomic DNA has two effects on the experiment: first, it affects the precise quantification of RNA; The second is to affect the specificity of PCR amplification.

Optimization of PCR reaction system.

The PCR amplification process must ensure that the amplification product has only a specific target product, and for this reason, the PCR reaction system must be optimized first. At present, many companies have kits for real-time quantitative PCR reactions, which can facilitate the operation of PCR reactions.

Reverse transcription. The amount of cDNA obtained by reverse transcription must be exactly equal to the amount of the corresponding mRNA, and reverse transcription is a critical step for real-time quantitative PCR. There are 2 types of reverse transcriptases commonly used at present:

AMV-RT and MMLV-R. Primers commonly used for reverse transcription are random primers, oligo-dt, and specific primer curvature. In contrast, Oligo-DT and specific primers are better suited for real-time PCR.

5.When using real-time quantitative PCR to study gene expression, internal standard genes must be used for relative quantification by relative quantification. A reliable internal standard gene should be a housekeeper gene that is stably expressed in different cell types and under different test conditions.

Commonly used internal standard genes are -tublin, actin,

GAPDH, 18SRNA, EFLA, and Cyclophilin, among others. Although in most cases the expression of these housekeeping genes is very stable, however.

Recently, it has been reported that the expression of these housekeeping genes changes in some cases. That is, not all internal standard genes are suitable.

Any trial. When selecting an internal standard gene, a variety of factors should be carefully considered to select the appropriate internal standard gene or combination of internal standard genes.

Real-time PCR refers to the method of adding fluorophores to the PCR amplification reaction system, detecting the fluorescence signal of each cycle product in the amplification reaction in real time, and finally quantifying the unknown template through the standard curve. The purpose of the control is to monitor all aspects of the test, so we follow the test process to sort out and flush the control. The workflow for a routine PCR is:

Sample collection - transportation - sample code processing - nucleic acid extraction - reversal of the Zen judgment (RNA virus) - amplification - result reading.

When there is incident light irradiating a substance, its molecules will absorb the energy of the incident light and be excited to make its electrons jump from a low energy level to a high energy level, at this time, the structure of the molecule in the excited state is not stable, and it will lose energy through the transition and return to the ground state, this process will be accompanied by the production of fluorescence or phosphorescence. As shown in Fig.

All fluorescent substances have two characteristic spectra, i.e., excitation and emission spectra.

Normally, the wavelength of the emitted light of a substance is always greater than the wavelength of its excitation light, because there is a certain amount of energy loss in the process of receiving excitation from the substance. The difference between the wavelengths of the excited and emitted light is called the Stoker shift.

The physical or chemical process that occurs between a molecule of a fluorescent substance and a solvent or solute molecule that results in a decrease in fluorescence intensity.

There are many forms of fluorescence quenching, and the mechanism is also complex. There are several main types:

Fluorescence quantitative PCR technology is a way to determine the relative and absolute amount of specific PCR fragments in a sample, and is a way to determine the content of specific PCR fragments. For example, the content of pathogens in patient samples, the content of a specific mRNA in experimental samples, etc. To achieve a highly efficient reaction, primers for real-time PCR applications should be designed to generate short amplicons.

The commonly used methods are the fluorescent dye method of SYBR Green and the TaqMan probe method, and the characteristics and applications of the two methods are shown in the following table

Due to the differences in the principle of different fluorescence quantitative PCR instruments and the spectral range of detection light provided, it is important to select the luminescent and quencher groups of the probe according to the range of detectable fluorescence signal set by the instrument model used (the probe method can be connected to the number of our fluorescence quantitative PCR kit and the corresponding machine model).

Dual-labeled fluorescent probes consisting of the quencher Tamra, Eclipse, or BHQ series dyes are often used as hydrolysis probes, or as they are called"taqman"Probes for real-time PCR experiments. Due to the different spectroscopic properties of these quenching dyes (see figure below), the characteristics of their use as quenching groups are also different, which are explained below

This is because the amount of DNA replication does not reach the level detected by the instrument, but the actual PCR cycle is real, which is reflected on the instrument curve as the baseline, and will not be detected by the instrument until the quantitative change reaches a qualitative change, and it becomes an exponential growth period.

Generally, the first 15 cycles of fluorescent PCR are used as the fluorescence background signal (baseline, baseline), that is, the fluorescence background value of the sample and the fluorescence value of the negative control, and the amplified fluorescence signal is masked by the fluorescence background signal.

The fluorescence threshold is an artificially set value on the fluorescence amplification curve, which can be set anywhere in the exponential amplification phase of the fluorescence signal, and the default setting of the fluorescence domain value is 10 times the standard deviation of the fluorescence signal for 3 15 cycles (automatically set by the machine).

When doing fluorescence quantitative PCR experiments, the manual setting is often used, and the principle of manual setting should be greater than the fluorescence background value of the sample and the maximum fluorescence value of the negative control, and at the same time, the initial stage of entering the exponential phase should be selected as much as possible, and the real signal is that the fluorescence signal exceeds the domain value.