2. Generally, it is a relative quantity, so it is calculated by Delta △△CT method.
3. Examples are as follows: the CT value of gene A in the control group is 20, and the CT value of internal reference (such as β actin) is 15. In the experimental group, the gene A CT value was 18, and the internal reference CT value was 14.
4. Calculate the sample size first: δ CT =15-14 =1. The power of 1 of 2 is 2. In other words, the sample size of the experimental group is twice that of the control group. Gene A:δCT = 20- 18 = 2. The second power of 2 is 4. In other words, the amount of A gene in the experimental group is four times that in the control group. However, since the amount of sample added is twice, the four bits are 2=2, and the final relative amount is twice.
5. Several points for 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 power of 2 is only a theoretical value, and the actual amplification efficiency is lower than 2. It is best not to use Cybergreen.
Extended data:
PCR principle:
The basic principle of 1 and PCR technology is similar to the natural replication process of DNA, and its specificity depends on oligonucleotide primers complementary to both ends of the target sequence.
2.PCR consists of three basic reaction steps: denaturation-annealing-extension:
(1) denaturation of template DNA: After the template DNA is heated to about 93℃ for a certain period of time, the double-stranded DNA of the template DNA or the double-stranded DNA formed by PCR amplification is dissociated, so as to combine with primers and prepare for the next round of reaction;
(2) Annealing (renaturation) of template DNA and primer: after the template DNA is denatured into single strand by heating, the temperature is reduced to about 55℃, and the primer and the complementary sequence of the template DNA single strand are paired and combined;
③ Primer extension: Using DNA template-primer conjugate, at 72℃, using dNTP as reaction raw material and target sequence as template, according to the principle of base complementary pairing and semi-conservative replication, under the action of DNA polymerase (such as TaqDNA polymerase), a new semi-conservative replication chain complementary to the template DNA chain was synthesized. It takes 2 ~ 4 minutes to complete a cycle, and the target gene to be amplified can be amplified by a million times in 2 ~ 3 hours.
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