How are the results of real time PCR analyzed?

The **Effect of different analysis modes on the results of real-time PCR results. Methods A total of 77 serum HBV DNA samples of hepatitis B patients in outpatient and ward patients were detected by LINE-Gene FQD-33A quantitative PCR detection system, and the results were analyzed by the maximum second derivative method and sample fitting method, respectively. Results The overall correlation between the two methods was good (r=0 978).

However, when the HBV DNA copy number content < 10 5, the correlation between the two modes was poor (R=0 706), and the value obtained by the maximum second derivative method was lower than that obtained by the sample fitting method (T=2 61, P<0 05), and false negative results were prone to occur. Conclusion Although there are many steps in the sample fitting method, the results are more reliable, so it is recommended to use the sample fitting method when analyzing the results. Moreover, when the HBV DNA copy number in the sample is <10 5, the result can only be analyzed using the sample fitting method.

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Based on the set standard value, it is automatically calculated.

Probably a problem with my own computer.

The principle of real-time quantitative PCR usually refers to the principle of real-time quantitative PCR, that is, the fluorophore is added to the PCR reaction system, and the PCR amplification is detected in real time by using the change of fluorescence signal.

Changes in the amount of amplified product per cycle in the reaction, by cycling thresholds and standard curves.

The analysis was performed to quantify the number of starting template copies in the specimens.

Real-time PCR is a real-time detection of the PCR process through fluorescence signals during PCR amplification. Because there is a relationship between the CT value of the template and the starting copy number of the template during the exponential period of PCR amplification, it has become the basis for quantification.

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Quantitative PCR has absolute quantification and relative quantification, and absolute quantification is to first use the concentration gradient of the plasmid of known concentration (make a standard curve, and then calculate its expression by doing the CT value of real-time PCR; Relative quantification is the comparison of housekeeping genes (...)

This in the quantitative should be the parameter value of the measurement in it, and it can be judged by this parameter.

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The amount of cDNA is determined to be 16 srnarRNA is based on the quality of RNA extraction.

For some quantitative information, you should check the professional explanation of this aspect.

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But what exactly is the amount? Some quantities based on some of his measurements and stuff, of course.

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

2. Generally, it is a relative quantity, and it is calculated by the delta delta ct method.

3. For example, the CT value of gene A in the control group is 20, and the CT value of the internal control (such as Actin) is 15. The CT value of gene A in the experimental group was 18, and the CT value of internal control was 14.

4. First calculate the amount of sample added: the power of delta ct=15-14= is 2. In other words, the amount of sample added in the experimental group was twice that of the control group.

Gene A: Delta ct=20-18= to the 2nd power is 4. In other words, the amount of gene A in the experimental group was 4 times that of the control group.

However, since the amount of sample is doubled, 2=2 is used at 4 places, and the final relative amount is 2x.

5. A few points of attention: the specificity of amplification must be determined, only the CT value of the same target can be subtracted (the amplification efficiency may be different), a certain power of 2 is only a theoretical value, the actual amplification efficiency is lower than 2, it is best not to use syber green.

The full name of PCR is: polymerase chain reaction, as the saying goes, it is to artificially create an environment for double-stranded gene fragments to replicate and amplify.

Gene of interest: This is the gene fragment you want to amplify.

Denaturation: It is to unwind the double strands in a hot environment, generally 94 degrees Celsius.

Primer: The initiation of guided replication is a single-stranded fragment of RNA that guides the start of replication.

Polymerase: Taq enzyme is a heat-resistant enzyme found in volcanic craters, under the action of which four deoxynucleotides (DNTPs) are used as raw materials to synthesize double strands.

Extension: Generally speaking, it is the process of forming a new double chain between the original rotten template chain and the newly synthesized single chain, and the length of extension time and temperature will affect the hunger and destitude quality of the new double chain.

The general process of PCR.

1. Template DNA

2. (First cycle): template DNA denaturation and primer renaturation.

3. (First Cycle): Primer extension.

4. (Second cycle): newly synthesized double-stranded denaturation and primer refolding.

5. (Second Cycle): Primer extension.

And so on until the end.

After that, you can soak an agarose electrophoresis to see the amplification, and you can also measure the OD value and calculate the concentration.

Now there is also Real Time PCR, real-time quantitative PCR, which can measure the concentration of reactants at equal intervals and obtain relatively accurate data, which is relatively simple.

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The results are the same. It can explain the amplification efficiency and dissolution temperature of the whole process of special DNA replication in vitro.

Differences: 1. The composition of the two systems is different.

Compared with ordinary PCR instruments, the fluorescence quantitative PCR instrument has more fluorescence signal acquisition system and computer analysis and processing system.

2. The principles of the two are different.

Real-time PCR monitors the fluorescence excited by the fluorescent dye bound to the DNA in real time, and the amount of PCR final product is determined by detecting the amount of dye inserted into the DNA.

3. The response requirements of the two are different.

Fluorescence quantitative PCR has high requirements for amplified fragments, generally 100-300bp, and ordinary PCR can amplify fragments with long spots.

4. The application of the two is different.

Fluorescence quantitative PCR is mainly used to quantitatively analyze and determine the level of gene transcription, ordinary PCR instrument is to do qualitative analysis and amplification of gene fragments, quantitative PCR instrument can do the work of ordinary PCR instrument, not vice versa.

5. The measurement cost of the two is different.

Quantitative PCR machines** are more expensive, and ordinary gene cyclers (PCRs) can only qualitatively analyze the presence or absence of target fragments. But it's a lot lower, and the running costs are also much lower.

Real-time PCR technology effectively solves the limitations of traditional quantitative detection of endpoints, realizes the detection of the intensity of the fluorescence signal once in each cycle, and records it in the computer software, and obtains the quantitative results according to the standard curve through the calculation of the CT value of each sample.