The real-time fluorescence quantitative PCR detection technology is to use the change of fluorescence signal to detect the change of the growing amount of each cycle in the PCR amplification reaction in real-time and to quantitatively analyze the starting template through the analysis of Ct value and the standard curve. Real-time fluorescence quantitative PCR detection technology The global production of fluorescent quantitative PCR equipment is spread by brands. The more well-known imported brands include: ABI, Bio-Rad, Roche, etc., and several well-known brands have emerged in China, such as Heal Force, Bioer, etc.
There are two main ways to quantify real-time fluorescence quantitative PCR:
- Absolute quantification: you need a standard to make a straight line first, and then test your sample for absolute quantification;
- Relative quantification: no standard, usually add internal reference gene for relatively quantitative. The ideal internal reference should be constantly expressed under the conditions of various experimental factors studied.
The main function of adding an internal reference in the experiment is to avoid errors caused by RNA quantification error, sample addition error, uneven amplification efficiency in each polymerase chain reaction system, and the temperature difference between each well.
Current internal references commonly used in experiments include GAPDH, ACTB (β-actin, β-actin), 18srna. GAPDH (glyceraldehyde-3-phosphate dehydrogenase, glyceraldehyde-3 -phosphate dehydrogenase) is an enzyme in glycolysis, widely distributed in cells in various tissues. It composes of four 30-40kDa subunits, with a molecular weight of 146kDa, and is expressed at a high level in almost all tissues. The amount of protein expression in such cells or tissues is generally constant and is not affected by the induction of substances such as partial recognition sites, so it is widely used to extract total RNA, poly(A)+ RNA, standardized internal reference for experimental operations such as Western blot.
Relevant Experiment Measured by qPCR Instruments of Different Brands
Let’s take a look at a Cq value (also known as Ct value) experiment of GAPDH in mouse bone marrow cells measured by qPCR instruments of different brands. The situation is as follows:
- Experimental name: In different brands of fluorescent quantitative PCR instruments, the expression level of GAPDH Cq value in bone marrow cells of the same mouse.
- Experimental instruments: Heal Force CG-05 and Bio-Rad CFX96 fluorescence quantitative polymerase chain reaction, FAM detection channel
- Experimental location: Zhejiang University
- Subject: Mouse bone marrow cells
- Sample carrier: AXYGEN 96-well PCR plate
- Fluorescent dye: SYBR Green
The experimental results are as follows:
- GAPDH gene expression:
- A: Bio-Rad qPCR test results: Cq average: 15.83, Cq standard deviation: 0.328
- B: Heal Force qPCR test result: Cq average: 16.08, Cq standard deviation: 0.104
The average difference between Heal Force CG series and Bio-Rad CFX96 Cq: 0.25
2.Melting curve and temperature:
- A: Bio-Rad qPCR test result: Melting: 84.53℃, standard deviation: 0.135
- B: Heal Force qPCR test result: Melting: 84.09, standard deviation: 0.191
Heal Force CG series and Bio-Rad CFX96 average difference in melting temperature: 0.44
It can be seen from the experiment that GAPDH shows high expression in Bio-Rad and Heal Force’s fluorescence quantitative PCR detection, and the Cq value (Ct value) and the dissolution temperature value measured by the two machines are basically the same. GAPDH is widely popular in which researchers love it and choose it as the housekeeping gene in many experiments.