Molecular biology related experimental steps and precautions (3) qPCR articles
——Fluorescence quantitative PCR articles—— Quantitative Real-time PCR is the addition of a fluorescently labeled probe or a corresponding fluorescent dye to a conventional PCR. Each time one cycle, a fluorescence intensity signal is collected. As the PCR reaction proceeds, the PCR reaction product continuously Accumulated, the fluorescence signal intensity also increases in proportion. In general, the fluorescence amplification curve can be divided into three phases: a fluorescent background signal phase, a fluorescent signal index amplification phase, and a plateau phase. In the fluorescence signal index amplification phase, there is a linear relationship between the logarithm of the amount of PCR product and the amount of starting template, and quantitative analysis can be selected at this stage. (1) Fluorescence threshold and CT value The fluorescence threshold is a value artificially set on the fluorescence amplification curve. It can be set at any position in the fluorescence signal exponential amplification stage, but the default (default) setting of the general fluorescence domain value is 3-15. 10 times the standard deviation of the circulating fluorescent signal. The CT value refers to the number of cycles experienced by the fluorescent signal in each reaction tube reaching the set domain value during PCR amplification. Relationship between CT values ​​and starting template: Studies have shown that there is a linear relationship between the Ct value of each template and the logarithm of the starting copy number of the template. The more the starting copy number, the smaller the Ct value, the formula is as follows: Ct= -1/lg(1+Ex)*lgX0+lgN/lg(1+Ex) n is the number of cycles of the amplification reaction, X0 is the initial template amount, Ex is the amplification efficiency, and N is the amount of the amplification product when the fluorescence amplification signal reaches the threshold intensity. A standard curve can be made using a standard of known starting copy number, where the abscissa represents the logarithm of the starting copy number and the ordinate represents the Ct value. Therefore, as long as the Ct value of the unknown sample is obtained, the initial copy number of the sample can be calculated from the standard curve. (2) Fluorescent probes and fluorescent dyes Fluorescence quantitative detection can be divided into fluorescent probes and fluorescent dyes depending on the label used. SYBR Green I is the most commonly used DNA binding dye for real-time PCR and non-specifically binds to double-stranded DNA. In the free state, SYBR Green I emits weak fluorescence, but after binding to double-stranded DNA, its fluorescence is greatly enhanced. SYBR Green I is used for qPCR without probes, and the designed program is versatile and relatively inexpensive. Fluorescent dyes can be used to indicate the nature of the melting point of double-stranded DNA, and the presence or absence of PCR bands, primer dimers, and the like can be identified by melting point curve analysis. However, SYBR Green I has no template specificity and therefore causes false positives that affect the accuracy of quantification. The TaqMan probe is an oligonucleotide probe designed to sequence with the upstream and downstream primers of the target sequence with higher specificity. (3) qPCR considerations 1. RNA used for qPCR reactions should avoid DNA contamination, and care should be taken during RNA extraction and reversal. 2. Protect from light during operation and operate on ice. 3. The cDNA template avoids repeated freezing and thawing, otherwise it will cause degradation of the template. 4. Insufficient amount of template or degradation will result in CT values ​​appearing too late or not appearing. Samples of unknown concentration should be taken from the highest concentration of serially diluted samples. 5. When adding the template, it should be diluted moderately and then added to the reaction system to improve the amplification efficiency. However, if the template concentration is too low, the reproducibility is worse, and the dilution factor of the sample should be reduced. 6. When the primer is designed, the amplified fragment should not be too long, generally 80-300 bp; the annealing temperature should be high, generally above 60 °C. Also pay special attention to avoid the presence of primer dimers and non-specific amplification. 7. Excessive concentration of primers in the system or primer dimers will cause more than one main peak in the dissolution curve. 8. Set up a negative control to prevent contamination of water and mix. 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