generally speaking, the real-time qPCR MasterMix is a 2× concentrated solution, and only templates and primers need to be added. Due to the high sensitivity of real-time qPCR, at least three parallel holes should be made for each sample, so as to prevent the statistical analysis from being carried out in the later data analysis due to the large difference of Ct or the large SD. Generally speaking, the final concentration of lead in the reaction system is 1-4 mm; The template is generally 1ng-5 for total RNA, and it should be adjusted according to the expression abundance of the target gene if the cDNA is usually 1ul or 1 times dilution of 1ul. Of course, these are all empirical values, and in the process of operation, it is necessary to optimize according to the differences of MasterMix, template and primer used to achieve an optimal reaction system. In the process of preparing the reaction system, the following points should be noted:

1. MasterMix should not be frozen and thawed repeatedly. If it is used frequently, it is best to dissolve it and put it at 4 degrees.

2. Mix more to reduce the error of sample addition. It is best to operate on ice.

3. each tube or hole should be replaced with a new gun head! Don't use the same gun head to add samples continuously!

4. After all the ingredients are added, centrifuge to remove bubbles.

5. Each sample has at least 3 parallel holes.

reference dye (passive dye) is commonly used as ROXTM (now it is a registered trademark of ABI! ) or other dyes, as long as it does not affect the fluorescence value of the detected PCR products. The function of reference dye is to standardize the non-PCR oscillation in fluorescence quantitative reaction, correct the loading error or the error between holes, and provide a stable baseline. Nowadays, many companies have formulated ROXTM in MasterMix or Premix. If the reaction curve is good or the reaction system has been optimized, ROXTM dye can not be added for correction.

generally speaking, the reaction procedure of real-time qPCR does not need three steps of denaturation, annealing and extension, as in conventional PCR. Because the length of the product is between 8 and 15 BP, only denaturation and annealing are needed. For dye methods such as SYBR@Green, it is best to add a dissolution program after the PCR amplification program is finished to form a dissolution curve and judge the specific amplification of PCR products. The dissolution procedures and instruments all have default settings, or are slightly different, but they all collect fluorescence signals when the product is dissolved.

3. Instrument settings

All instruments are basically the same in operation. The setup includes plate setup and program setup. Let's take ABI StepOne as an example and take a detailed look at the reaction settings: < P > A. First, the choice of experimental purpose: quantitative or other. We named it "BioTeke" and conducted a "quantitative" experiment.

B. Selection of experimental methods: We chose SYBR Green method for comparing Ct, Fast program, and used cDNA as a template.

C. setting of target genes: there are several target genes and their names.

D. sample setting: including which is the experimental group and which is the control group. And the setting of negative control and biological repetition.

e. setting of control group and reference gene: this is to prepare for later quantification

F. setting of reaction program: setting of PCR reaction program should be based on different companies' MasterMix. For example, BioTeke can activate DNA polymerase at 95℃ for 2 minutes (ABI takes 1 minutes). The cyclic reaction is 4 cycles at 95℃ for 15 seconds and 6℃ for 15 seconds. The dissolution curve program can use the default settings of the instrument. Or the procedure suggested in the instrument manual.

g. Setting of reaction system:

These five steps A-G are simply set, and you can save, modify the reaction program or react immediately.

it should be noted that the ABI instrument needs to add ROX reference dye, and the default is ROX. Some companies put ROX or other dyes in MasteMix; Also have a plenty of separate. The choice of this step should be made according to the MasterMix of different companies. There is no reference dye in BioTeke's MasterMix, so choose "none".

after setting, you can put the configured PCR tube into the instrument and click RUN!

v. analysis of Real-time qPCR data

1. Common parameters of real-time qpcr

Baseline

Usually 3-15 cycles of fluorescence signal

Baseline

threshold)

Automatic setting is 3-15.

thresholds can be set separately for different genes in the same reaction, but the same threshold must be used for the same gene amplification.

Ct value: linear with the logarithm of initial concentration.

Ct:15-35 is usually taken for quantitative analysis. Too big or too small will lead to inaccurate quantification.

Rn(Normalized reporter) is the ratio of the fluorescence emission intensity of the fluorescent reporter group to that of the reference dye.

△ Rn: △ rn is the standardized result obtained by subtracting the baseline from RN (△Rn=Rn- baseline).

2. key factors affecting Ct value

template concentration

template concentration is the most important factor determining Ct. Control in a suitable range, so that the Ct is between 15 and 35.

influence of composition of reaction solution

the fluorescence emission of any molecule is influenced by environmental factors-such as pH value and salt concentration of the solution.

the efficiency of PCR reaction

the efficiency of PCR reaction will also affect the Ct value. Continuous gradient dilution amplification under the condition of low PCR amplification efficiency may produce standard curves with different slopes compared with the condition of high PCR amplification efficiency. The efficiency of PCR depends on the experiment, the performance of reaction mixture and the quality of sample. Generally speaking, the reaction efficiency is acceptable between 9-11%.

3. How to evaluate the effect of real-time quantitative PCR reaction

PCR amplification efficiency: In order to correctly evaluate the PCR amplification efficiency, it is necessary to do at least three parallel repetitions and at least five orders of magnitude multiples (5logs) of continuous gradient dilution of template concentration.

Frequently asked questions

1. No Ct value appears

The steps of detecting fluorescence signals are wrong: SG method generally adopts 72℃ extension to collect signals, while Taqman method generally collects signals at the end of annealing or extension.

degradation of primer or probe: its integrity can be detected by PAGE electrophoresis.

insufficient template quantity: for samples with unknown concentration, we should start with the highest concentration of serial diluted samples.

template degradation: avoid the introduction of impurities and repeated freezing and thawing in sample preparation.

2. The CT value appears too late (CT >; 38)

Low amplification efficiency: the reaction conditions are not optimized enough. Design better primers or probes; The reaction is carried out by a three-step method; Appropriately reducing the annealing temperature; Increase the concentration of magnesium ions, etc.

degradation of various reaction components in p>PCR or insufficient sample loading.

the p>PCR product is too long: generally, the product length is 8-15bp.

3. The linearity of the standard curve is not good

There is an error in the sample addition: the standard is not graded.

degradation of standards: avoid repeated freezing and thawing of standards, or re-prepare and dilute standards.

poor primers or probes: redesign better primers and probes.

There are inhibitors in the template, or the template concentration is too high

4. Negative control has signals

The primer design is not optimized enough: the appearance of primer dimer and hairpin structure should be avoided.

poor primer concentration: appropriately reduce the primer concentration, and pay attention to the concentration ratio of upstream and downstream primers.

magnesium ion concentration is too high: reduce the magnesium ion concentration appropriately, or choose a more suitable mix kit.

the template is polluted by genome: avoid the introduction of genomic DNA during RNA extraction, or avoid non-specific amplification through primer design.

5. The dissolution curve has more than one main peak

The primer design is not optimized enough: the appearance of primer dimer and hairpin structure should be avoided.

poor primer concentration: appropriately reduce the primer concentration, and pay attention to the concentration ratio of upstream and downstream primers.

magnesium ion concentration is too high: reduce the magnesium ion concentration appropriately, or choose a more suitable mix kit.

the template is polluted by genome: avoid the introduction of genomic DNA during RNA extraction, or avoid non-specific amplification through primer design.

6. Low amplification efficiency

Some components in the reaction reagent, especially fluorescent dyes, are degraded.

the reaction conditions are not optimized enough: the annealing temperature can be reduced appropriately or the three-step amplification method can be changed.

there are inhibitors of PCR reaction in the reaction system: generally, they are introduced when the template is added, so the template should be diluted moderately first, and then added into the reaction system to reduce the influence of inhibitors.

7. the same reagent produces different curves on different instruments. how to judge?

judging criteria: amplification efficiency, sensitivity and specificity

if the amplification efficiency is 9%-11%, it is all specific amplification, and the data can be used for analysis.

8. Abnormal amplification curve? Like an "S" curve?

the reference dye is not set correctly (when the reference dye is not added to the MasterMix, select NONE)

the concentration of the template is too high or the fluorescent dye is degraded

the problems of fluorescent quantitative PCR are summarized

1. What is the on-off sequence of the quantitative PCR instrument?

operating according to the correct switching sequence is helpful to prolong the service life of the instrument and reduce the frequency of instrument failure.

boot sequence: turn on the computer first, then turn on the host of the quantitative PCR instrument after the computer is fully started, and then turn on the quantitative PCR collection software after the green light on the host panel, and conduct experiments.

shutdown sequence: after confirming that the experiment has ended, first turn off the signal collection software, then turn off the power supply of the quantitative PCR instrument host, and finally turn off the computer.

2. What kinds of reaction tubes and covers are suitable for quantitative PCR experiments? What should I pay attention to?

the following consumables can be used in quantitative PCR experiment: 96-hole optical reaction plate with optical film, .2 ml optical octal reaction tube with optical film, and .2 ml optical octal reaction tube with flat cover. See the following table for the specific usage method and article number of quantitative PCR consumables produced by ABI company:

3. Why do you need to defragment the computer regularly? How to organize?

when running the real-time quantitative PCR instrument and analyzing the experimental results with software, the computer will delete and create several files, and the free space of the computer hard disk will be divided into more and more small pieces. When the file on the hard disk drive is stored in decomposed fragments, the program needs longer time to access the file, because the file fragments must be searched many times to access different fragments. The defragmentation utility combines multiple fragments of a file and stores them in the same location on the hard disk, so as to remove the file fragments and optimize the system performance.

The defragmentation method is as follows:

On the Windows desktop, select start, My computer.

in the (My Computer) window, right-click the hard disk drive and select (property).

in the (properties) dialog box, select the Tools tab and click Defragment now.

click defragmentation.

when the defragmentation dialog box is displayed, click (ok).

in the local disk properties dialog box, click (ok).

repeat the above steps for the remaining drives in the computer.

4. when do I update the windows service pack?

do not perform this operation. Do not update the operating system of the computer that controls the quantitative PCR instrument unless the representative of American Applied Biological Systems Company informs you to update the operating system. The new version of Microsoft Windows operating system may conflict with SDS software, and the instrument may not run normally. If you want to install a service pack to update the operating system, you should check the release notes provided with the SDS software to avoid compatibility problems.

5. what data should be backed up?

you should back up your experimental data regularly, and the frequency of backup is recommended to be once a week, and burn it on CD. At the same time, you should also back up all kinds of pure fluorescence spectrum correction files, background files and installation verification experimental data of quantitative PCR instrument. The directory where these files are located is C:/Applied Biosystems/SDS Document. The following figure is a sample of the calibration file.

6. what kind of laboratory environment can ensure the normal operation of instruments and equipment?

A good laboratory environment helps to prolong the service life of the instrument and reduce the frequency of instrument failure. The following aspects are recommended:

power supply: it is recommended to equip with appropriate UPS or voltage regulator.

ventilation: the ventilation of the instrument should be unobstructed.

temperature: it is recommended that the laboratory be equipped with air conditioning, and the temperature should be controlled between 1-3 C ..

humidity: 2-8%; For humid provinces, it is recommended that laboratories be equipped with dehumidifiers.

space: easy to operate and safe.

7. how to judge whether the sample heating block of quantitative pcr instrument is polluted? How to eliminate pollution?

One way is to run the background correction reaction plate. When one or more reaction holes continuously show abnormally high signals, it indicates that the holes may be contaminated by fluorescent pollutants.

Another method is to correct the ROI without putting anything on the sample block. When the signal of a hole is obviously higher than other holes, it indicates that the hole is polluted.

The steps to remove the contamination of the sample heating block are as follows:

Use a pipette to suck a small amount of ethanol and drop it into each contaminated reaction well.

blow several times.

suck the waste liquid into the waste liquid cup.

repeat the above steps: ethanol for three times and deionized water for three times.

make sure that the residual liquid in the reaction hole is completely evaporated.

8. what is background correction? How often do you perform background correction?

the background correction program measures the blank fluorescence intensity of the reaction tube and water used in the quantitative PCR instrument. During the calibration procedure, the quantitative PCR instrument runs for 1 minutes.