Real-time PCR instrument   Q: What is the difference between RT-PCR, QPCR, Real-time PCR, and real-time RT-PCR?

A: RT-PCR is reverse transcription PCR (RT-PCR), which is a widely used variant of polymerase chain reaction (PCR). In RT-PCR, an RNA strand is reverse transcribed into complementary DNA, which is then used as a template for DNA amplification by PCR.

Real-time-PCR and qPCR (Quantitative Rea-ltime-PCR) are the same thing, both are real-time quantitative PCR, which means that each cycle in the PCR process has a real-time record of data, so the number of starting templates can be measured. Precise analysis. Although Real-time PCR (real-time fluorescence quantitative PCR) and Reverse transcription PCR (reverse transcription PCR) seem to be abbreviated as RT-PCR, the international convention is that RT-PCR refers specifically to reverse transcription PCR, Real-time PCR is generally abbreviated as qPCR (quantitative real-time PCR). Real-time RT-PCR (RT-qPCR) is a reverse transcription PCR combined with fluorescence quantitative technology: first, cDNA (RT) is obtained from RNA reverse transcription, and then real-time PCR is used for quantitative analysis (qPCR) .

Q: Why should the fragment length of the amplified product of real-time PCR be controlled within the range of 80-300bp? A: The length of each gene sequence is different, some are several kb, some are hundreds of bp, but when we design primers, we only need to require the length of the product to be 80-300bp, too short or too long is not suitable for quantitative PCR detection . The product fragment is too short to be distinguished from the primer-dimer. The length of the primer-dimer is about 30-40bp, and it is difficult to distinguish whether it is a primer-dimer or a product when it is less than 80bp. The product fragment is too long, more than 300bp, which easily leads to low amplification efficiency and cannot effectively detect the amount of the gene. For example, when you count the number of people in a classroom, you only need to count how many mouths there are. The same is true when detecting genes. You only need to detect a certain sequence of a gene to represent it. The entire sequence will do. If you need to count both the number of mouths and the number of noses, ears, and glasses in order to count people, it is easy to make a mistake.

Q: What is the length of primer design? A: Generally speaking, the primer length is about 20-24bp. Of course, when designing primers, we must pay attention to the TM value of the primers, because this is related to the annealing temperature. After a lot of experiments, 60 ℃ is a good TM value. If the annealing temperature is too low, it is easy to cause non-specific amplification. If the annealing temperature is too high, the amplification efficiency will generally be low, the peak of the amplification curve will be late, and the CT value will be delayed.

Q: Will the amount of samples collected affect the experimental results?

A: No. Obviously, the more samples are collected, the more RNA is extracted, the more cDNA, and the more target fragments. For absolute quantification, to calculate the copy number of a target fragment, the amount of samples collected will definitely affect the experimental results. For example, detecting the hepatitis B virus HBV in the blood is to detect the HBV content in a certain amount (1ml) of the blood. For relative quantification commonly used in scientific research, the sample size has nothing to do with the experimental results, because relative quantification refers to the comparison between the target gene and the reference gene, you can think that they are upstream and downstream fragments existing in the same nucleic acid chain, if the sample size is large The internal reference gene and the target gene were both increased in equal proportions at the same time, which did not affect the results.