In addition to the cell proliferation and protein level assays, specific gene expression analysis is also provided to assess compound effects on target cells. Gene analysis mainly adopts real-time fluorescence quantitative PCR technology and gene chip technology for testing.
I.Real-Time Fluorescence Quantitative PCR Technology
Fluorescence Quantitative PCR (Real-time PCR) refers to adding fluorophores into the PCR reaction system, testing the whole PCR process in real-time by accumulating fluorescent signals, and finally quantitatively analyzing unknown templates by standard curves.
The change of the number of amplification products in each cycle of PCR amplification reaction is tested in real-time by the change of fluorescence signal, and the initial template is quantitatively analyzed by the relationship between Ct value and the standard curve.
Baseline: refers to the fact that the fluorescence signal does not change much in the first few cycles of PCR amplification reaction. Close to a straight line, which is the baseline. Ct value: The number of amplification cycles when the amplified product's fluorescence signal reaches the set threshold value during PCR amplification.
II. Gene Chip
Gene Chip (also called DNA chip and biochip)
Technology System means that a large number of probe molecules (usually with a reticular density higher than 400 per square centimeter) are fixed on substrate and hybridized with labeled sample molecules, and the number and sequence information of sample molecules are obtained by testing the hybridization signal intensity of each probe molecule.
Generally speaking, tens of thousands or even millions of DNA fragments (gene probes) with specific sequences are regularly arranged and fixed on substrates such as 2cm2 silicon wafers and slides by micro-machining technology to form a two-dimensional DNA probe array, which is very similar to the electronic chips of computers. Therefore, it is called a gene chip.
The gene chip analysis platform of HY BIOTECH uses the second-generation functional classification gene chip (Real-time PCR chip) developed by SABiosciences Company (now the QIAGEN Company) of the United States for high throughput gene marker analysis. By combining the advantages of real-time fluorescence quantitative PCR, this chip can accurately and quantitatively test mRNA levels of hundreds of genes in one experiment and improve the testing accuracy of gene chips to the same level as real-time fluorescence quantitative PCR. This platform can analyze miRNA of multiple samples at one time or multiple miRNA levels of a few samples by using miScript miRNA PCR Array of QIAGEN for high throughput testing of miRNA.
Gene chip technology can test and analyze a large number of sequences in a sample at one time due to a large number of probes fixed on the substrate at the same time, thus solving the disadvantages of a complex operation, low degree of automation, a small number of operation sequences and low testing efficiency of the traditional nucleic acid blotting hybridization (Southern Blotting and Northern Blotting, etc.). Moreover, by designing different probe arrays and using specific analysis methods, this technology can have many different application values, such as gene expression profile determination, mutation change , polymorphism analysis, genome library mapping, and hybridization sequencing etc.
Gene chip and second-generation sequencing
Biochip and second-generation sequencing are both important means of genomics study. Compared with second-generation sequencing, biochip has the advantages of low price and easy analysis. The disadvantage is the need to provide a reference sequence (because the biochip's probe design is based on the reference sequence).
Differences:
The essence of gene chip is nucleic acid hybridization. It is only tens of thousands of nucleic acid hybridization simultaneously; the second-generation sequencing is essentially PCR. Firstly, the sequencing library is constructed by PCR, and then the sequence information is obtained by "sequencing while synthesizing" or "connection-mediated sequencing"; Amplification is unnecessary because of nucleic acid hybridization. Therefore, gene chip is a relatively closed system that can only test the concentration of fragments with known sequences; In addition, because of no need of amplification, the fidelity is also good. With a essence of sequencing, the second-generation sequencing is an open system that can test those fragments without reference sequences and give their sequences.
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