Where can you have a complete introduction to fluorescent PCR? Urgent!!!

Is it real-time quantitative PCR, I have the courseware to send to you to see?

Fluorescence PCR refers to the method of adding fluorophores to the PCR reaction system, using fluorescence signal accumulation to monitor the entire PCR process in real time, and finally quantifying the unknown template through the standard curve. It is also called real-time PCR.

Fluorescence-PCR technical principle:After mixing the TaqMan probe labeled with fluorescein and the template DNA, the thermal cycle of high-temperature denaturation, low-temperature renaturation, and temperature extension was completed, and the polymerase chain reaction law was observed, and the TaqMan probe that was complementary to the template DNA was cut off, and the fluorescein was free in the reaction system.

Fluorescence under specific light excitation, with the increase of the number of cycles, the amplified target gene fragment increases exponentially, by real-time detection of the corresponding fluorescence signal intensity with amplification, the Ct value is obtained, and at the same time, several standards with known template concentrations are used as a control, and the copy number of the target gene of the specimen to be tested can be obtained.

A method to measure the total amount of products after each polymerase chain reaction (PCR) in a DNA amplification reaction using fluorescent chemicals. A method for quantifying specific DNA sequences in the sample to be tested by internal or external control.

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.

Technical principle. After mixing the TaqMan probe labeled with fluorescein and the template DNA, the thermal cycle of high-temperature denaturation, low-temperature renaturation, and temperature-appropriate extension was completed, and the TaqMan probe with the polymerase chain defibrillation closure law was observed, and the TaqMan probe with the template DNA complementary pair was cut off, and the fluorescein was free in the reaction scatter hood system.

Fluorescence under specific light excitation, with the increase of the number of cycles, the amplified target gene fragment increases exponentially, by real-time detection of the corresponding fluorescence signal intensity with amplification, the Ct value is obtained, and at the same time, several standards with known template concentrations are used as a control, and the copy number of the target gene of the specimen to be tested can be obtained.

The instructions of the German DBI brand fluorometric reagents are very clear.

BioGHC EliteFast SYBR Master Mix contains DNTP MIX, Mg2+, SYBR Green I, and BioG hot-start polymerase, generally speaking, the final primer concentration in the reaction system can obtain a good amplification effect, and when the reaction results are not satisfactory, the primer concentration can be adjusted in the range. The qPCR reaction is highly sensitive, and the accuracy of the template amount has a great impact on the results, so it is recommended to add the template after moderate dilution, or use a 50 ul reaction system. The volume of the template should not exceed 10% of the reaction volume, avoid strong light exposure during operation, and the length of the amplification product should be in the range of 100bp-500bp, and the reaction conditions:

95 Heat for 6-10 min to activate the warm-start DNA polymerase, then 95 for 5-15 sec, 60 for 30-35 sec for 40 cycles. The reaction conditions can be adjusted according to the size of the target fragment, the TM value of the primers, etc.

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.

This is 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 primers. 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.

How should it be calculated using the 2-CT method?

Assuming that gene A is detected, the CT is (remember here that the larger the CT, the lower the relative content) of the common group test1:28

Experimental group 1 test2: 29

Experimental group 2 test3: 30

At this time, it is necessary to set up a control, assuming that test1 is the control group (ordinary group), of course, the ordinary group is the control.

Then, the relative content is:

Normal group test1:1

Experimental group 1 test2:2 -(28-29)=1 2=experimental group 2 test3:2 -(28-30)=1 4=The expression of gene a in experimental group 1 and experimental group 2 decreased by 2 times and 4 times, respectively!

So, it's out, the result.

For each group of different parts, is it possible to compare one of them as 1?

Such as the experimental group 1testis

brainliver

muscle

You can use any of them as 1 as a comparison, but you have to indicate it in the legend of the figure, and just make it clear!

If you compare the same part of the three groups, should you also treat one of the groups as 1?

Yes, you can, such as.

brain, normal group, test1

Experimental group 1 test2

Experimental group 2 test3

Take the ordinary group test1, as the control group, set it to 1, and the others are relative contents, which should also be noted in the legend of the figure to make it clear!

This one is simple. You set the relative amount of a part of the normal group to 1. Others, both within and between groups, use CT to calculate relative quantities.

Let's compare. The basic idea is to base one point and the others are relative. Don't set a 1 every time.