FFPE reference substance as well as preparation method and application thereof
A reference material and cell technology, applied in the field of its preparation and FFPE reference material, can solve the problem of not being able to play an accurate reference role, and achieve the effect of improving accuracy
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[0024] In a typical embodiment of the present application, a method for preparing FFPE reference products is provided. The preparation method includes: selecting a target cell line; after culturing the cells of the target cell line to a predetermined concentration, collecting the cells for formal Lin fixed and made paraffin blocks; sliced the paraffin blocks to obtain FFPE reference products.
[0025] The preparation method of the FFPE reference product is to select a suitable cell line, then culture and prepare the same FFPE sample as the clinical sample, so as to provide the clinical FFPE sample with a reference comparison function from nucleic acid extraction to test results. It is an FFPE sample The entire testing process plays a quality control role, thereby improving the accuracy of the testing results. It is suitable for mass preparation of FFPE reference materials for tumor cell lines containing specific gene mutation ratios, which solves the problem that enterprises do...
Embodiment 1
[0043] For the EGFR gene, prepare FFPE positive reference samples for 4 mutation sites of EGFR gene p.G719S, Exon19Del, p.S768I, and p.L858R, including the following steps:
[0044] (1) Purchasing cell lines, the specific information is as follows:
[0045] Table 1:
[0046]
[0047] (2) Cell culture, DNA extraction of cultured cell lines.
[0048] (3) Use QuantStudio for the extracted cell line TM 3D Digital PCR performs verification to determine the mutation frequency of the site.
[0049] As shown in the following table:
[0050] Table 2:
[0051] Serial number Cell line name Mutation name Digital PCR detection frequency Digital PCR scatter plot 1 SW48 p.G719S27.43% figure 1 2H1650 E746_A750del60.07% figure 2 3 KYSE450 p.S768I49.70% image 3 4H1975 p.L858R67.85% Figure 4
[0052] (4) The cells were diluted by the limiting dilution method, and the monoclonal cells containing the target gene mutation were selected for culture, and counted at 12h, 24h, 36h, 48h and 60h respectively, a...
Embodiment 2
[0064] Preparation of wild-type FFPE cell line reference:
[0065] (1) Purchasing cell lines, the specific information is as follows:
[0066] table 5:
[0067]
[0068]
[0069] (2) Cell culture, DNA extraction of the cultured cell line, Sanger sequencing of the extracted genomic DNA to verify the gene mutation.
[0070] (3) After the verification is passed, the wild-type cell line will be enlarged and cultivated, and the number of cells will reach 4×10 6 ~5×10 6 When cells / mL, digest the adherent cells and collect the cells by centrifugation.
[0071] (4) Cell embedding and paraffin roll preparation
[0072] The collected cells were washed 3 times with PBS and then fixed with 10% formalin for 2 hours, and processed according to the conventional dehydration procedure. After the dehydration procedure is completed, the tissue block is taken out from the automatic dehydrator and embedding in conventional paraffin. Put the embedded cell line wax blocks on the freezing table and freeze the...
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