Real-time quantitative PCR (Real-time PCR, QPCR , FQ) technique (Real-time quantitative PCR), refers to a fluorescent gene (QPCR, FQ) in the reaction system, using a fluorescent signal accumulation time monitoring throughout (QPCR, Fluorescence quantification) A PCR process that ultimately quantifies an unknown template by a standard curve. Real-time PCR technology was introduced by Applied Biosystems in the United States in 1996. This technology not only realizes a qualitative leap to quantitative PCR, but also real-time PCR (Q-PCR , fluorescence quantification ) compared with conventional PCR. It has the characteristics of stronger specificity, high sensitivity, good repeatability, accurate quantitative, fast speed, fully enclosed reaction , effective solution to PCR pollution problems, high degree of automation, etc. It has become an internationally recognized standard method for quantitative determination of nucleic acid molecules. Currently (Real -time PCR , QPCR, fluorescence quantification has been widely used in gene expression research and clinical disease detection (such as transgenic animal and plant detection, RNAi gene inactivation rate detection, pathogenic microorganism or virus content detection, gene differential expression, gene division) type). The company uses the Sybrgreen method or Taqman method Real-time PCR , QPCR, fluorescence quantification to perform quantitative determination or type identification of DNA/RNA by real-time PCR. ☆ TaqMan fluorescent probe ( Real-time PCR , QPCR, fluorescence quantification ) ◠The main experimental steps are as follows: Real-time PCR consists of three steps: Real-time PCR | Real-time PCR | Some terms in QPCR 1  CT value: The number of cycles when the fluorescence signal has a statistically significant increase (ie, crossing the threshold line), or the number of cycles corresponding to the inflection point from baseline to exponential growth. 2  Threshold: The default setting for the threshold is 10 times the standard deviation of the 3-15 cycles of the fluorescent signal. 3  The CT value is linear with the amount of the starting template. Lactobacillus Fermentum,Fermentum Lactobacillus,Lactobacillus Fermentum Powder,Lactobacillus Fermentum Probiotic Jiangsu Biodep Biotechnology Co. ,Ltd. , https://www.mbioda.com
Real-time PCR (Q-PCR , fluorescence quantification ) classification:
    In the PCR amplification, a specific fluorescent probe is added while adding a pair of primers, and the probe is an oligonucleotide, and a reporter fluorophore and a quenching fluorophore are respectively labeled at both ends. When the probe is intact, the fluorescent signal emitted by the reporter group is absorbed by the quenching group; when PCR is amplified, the 5'-3' exonuclease activity of the Taq enzyme degrades the probe, allowing the reporter to fluoresce and quench The fluorophore is separated, so that the fluorescence monitoring system can receive the fluorescent signal, that is, one fluorescent molecule is formed for each DNA strand amplified, and the accumulation of the fluorescent signal is completely synchronized with the formation of the PCR product.
☆SYBR Green Fluorescent Dye Method: Real-time PCR |QPCR
    SYBR Green is a DNA-binding dye that binds non-specifically to double-stranded DNA. In the free state, it does not fluoresce, but once bound to double-stranded DNA, it can fluoresce. In the Real-time PCR | QPCR experiment, its biggest advantage is that it can be used in any reaction system in combination with any primers and templates. From an economic point of view, it is also much cheaper than other probes for Real-time PCR |QPCR . However, since it binds to all double-stranded DNA, once non-specific amplification occurs in the reaction system, it affects the reliability and repeatability of the quantitative results. To avoid this disadvantage, non-specific effects can be eliminated by selecting good primers and optimizing the reaction conditions.
Customer supply
(1) Real-time PCR | QPCR gene information
(2) Real-time PCR | QPCR experiments as fresh and sufficient amount of tissue as possible (≧100mg), cell sample (10) or serum, or directly provide purified total RAN (≧1ug)
After more than 10 years of development, real-time PCR (Q-PCR , fluorescence quantification ) has evolved from the initial simple qualitative development to the current Real-time PCR |QPCR , which allows PCR to collect one data per cycle. Establish a real-time amplification curve and accurately determine the CT value, so as to determine the starting DNA copy number based on the CT value, and achieve true DNA quantification. The maturity of technology has laid the foundation for its wide application.
Weiss Teng bio | Real-time quantitative PCR | Real-time PCR | QPCR | Services
(1) Design primers and primer synthesis according to Real-time PCR |QPCR target gene
(2) RNA extraction + reverse transcription
(3) RNA quantification and quality analysis
(4) Real-time PCR |QPCR (repeated three times) using the fluorescent dye SYBR Green method
(5) Data processing
?? real-time quantitative PCR | QPCR the raw data | Real-time PCR. The amplified product has a good melting curve and reliable repeatability.
â‘¡ real-time quantitative PCR | Real-time PCR | QPCR standard test procedures report
3 Real-time PCR | Real-time PCR | QPCR test results test report.
1. Reverse transcription: reverse transcription of RNA into cDNDA by reverse transcriptase;
2. Amplification: amplification of cDNA by PCR;
3. Detection: real-time detection and quantitative amplification of the product.