Detection method for dose of ionizing radiation on human peripheral blood lymphocytes
A lymphocyte and ionizing radiation technology, applied in the field of radiation biological dose estimation, can solve the problems of fast quantitative detection of biological radiation dose that cannot meet high-throughput detection, and the technology is complicated, and achieves the effect of saving time, good repeatability and stability
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[0019] Example 1: TaqMan-MGB probe and primer pair design
[0020] 1) Design the upstream and downstream primers and probe sequences according to the expression sequence of lymphocyte gdf15 gene.
[0021] The inventors used gene chip technology to screen the differential expression of 45200 genes in the three dose groups of 0.5, 3 and 8Gy. Among them, the dose change trend of the lymphocyte gdf15 positive regulatory gene is obvious. It is useful to explore the expression changes of the peripheral blood level of the gdf15 gene. The important practical value is closer to the application.
[0022] The expression sequence of lymphocyte gdf15 gene SEQ ID NO:1 is:
[0023] GACTTGTTAGCCAAAGACTGCCACTGCATATGAGCAGTCCTGGTCCTTCCACTGTGCACCTGCGCGGAGGACGCGACCTCAGTTGTCCTGCCCTGTGGAATGGGCTCAAGGTTCCT.
[0024] The upstream primer SEQ ID NO: 2 is: 5'-GTTAGCCAAAGACTGCCACTG-3'.
[0025] The downstream primer SEQ ID NO: 3 is: 5'-CCTTGAGCCCATTCCACA-3'.
[0026] The probe nucleotide sequence SEQ ID NO: 4 is: CCG...
Example Embodiment
[0027] Example 2: Construction of standard material recombinant pUC57 plasmid
[0028] 1) Separate single lymphocytes from human peripheral blood by conventional methods;
[0029] 2) The RNA of a single lymphocyte is extracted by conventional methods and reverse transcribed into cDNA;
[0030] 3) PCR amplification of lymphocyte gdf15 gene with specific primers;
[0031] 4) After the PCR product is purified, follow figure 1 Schematic diagram of the construction of the standard material recombinant pUC57 plasmid. The PCR product was ligated to the pUC57 plasmid (provided by Shanghai Shenggong Company) to construct the recombinant pUC57 plasmid. figure 2 Agarose gel electrophoresis diagram of the recombinant pUC57 plasmid of standard material.
[0032] 5) The recombinant pUC57 plasmid was sequenced to verify that the lymphocyte gdf15 gene expression sequence SEQ ID NO:1 was correctly inserted into the pUC57 plasmid.
Example Embodiment
[0033] Example 3: Preparation of standard curve
[0034] 1) The standard material recombinant pUC57 plasmid is diluted in a 10-fold series. The NanoDrop 2000 ultraviolet spectrophotometer was used to determine the concentration of the standard material recombinant pUC57 plasmid, and the standard material was diluted to 10 with double distilled water. 8 Copy / μl. Add 9μl of double distilled water into 5 0.5ml centrifuge tubes, and draw 1μl10 8 Copy / μl standard substance, add 9μl double-distilled water and mix thoroughly, and then perform gradient dilution successively, dilute the standard substance to 10 7 , 10 6 , 10 5 , 10 4 And 10 3 Copy / μl for use in 96-well plates.
[0035] 2) Perform fluorescent quantitative PCR with the 10-fold diluted standard substance recombinant pUC57 plasmid as a template.
[0036] The real-time fluorescent quantitative PCR instrument is the 7500fast Real Time PCR measuring instrument from ABI, the optimized reaction system is 20μl, of which 10μl Master Mi...
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