generally speaking, the real-time qPCR MasterMix is 2× concentrated solution, and only need to add templates and primers. 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.