Lung cancer detection quality control product and preparation method thereof
A technology for quality control products and lung cancer, which is applied in biochemical equipment and methods, measuring devices, microbiological determination/inspection, etc., can solve the problems of lack of gaps in tissue structures, inability to completely resemble tissues, and limited sources of quality control products. Short test cycle, easy operation, good repeatability and consistency
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Embodiment 1
[0056] Example 1: Preparation of quality control products for clinical detection of EML4-ALK fusion gene in heterogeneous tumor tissue
[0057] 1. Method
[0058] 1. Culture of Lung Cancer Cell Lines
[0059] Lung cancer cells H3122, H2228 and H1299 were cultured with RPIM-1640 medium containing 10% fetal bovine serum, 100IU / ml penicillin and 100IU / ml streptomycin in the medium. Culture in a constant temperature cell incubator with 5% CO2 at 37°C, digest with 0.25% trypsin for cell passage, and expand the three cell lines to the required number of 10 7 -10 8 and in the logarithmic growth phase.
[0060] 2. Verify the EML4-ALK gene fusion of the cell line by PCR method
[0061] (1) RNA extraction: ① Pour off the old medium of H1299, H2228 and H3122, wash twice with PBS, directly add 2.5ml lysate MZ to the culture bottle to lyse the cells, and pipette several times with a sampler.
[0062] ②Place the sample at room temperature for 5 minutes to completely separate the nuclei...
Embodiment 2
[0113] Example 2: Using a commercially available FISH kit to verify the preparation of the quality control product EML4-ALK fusion gene
[0114] 1. Method: Abbott EML4-ALK fusion gene FISH kit was used for verification
[0115] 1. Immerse the sample slice in a Coplin bottle filled with xylene for 5 minutes. Repeat this step for a total of 3 times.
[0116] 2. Dehydrate the sample slices in absolute ethanol for 1 minute. Repeat this step once, then remove the slices and allow them to air dry (2-5 minutes).
[0117] 3. Place the slices in the Abbott pretreatment solution preheated to 80±2°C for 12±3 minutes. The slices were then immersed in deionized water for 3 minutes.
[0118] 4. Take the slice out of the water, wipe off the excess water on the edge, and digest the slice in a protease solution preheated at 37±1°C for 20±2 minutes. The slices were then immersed in deionized water for 3 minutes.
[0119] 5. Dehydrate the slices in 70%, 85% and 100% ethanol for 1 minute, d...
Embodiment 3
[0130] Embodiment 3: Use the real-time fluorescent RT-PCR method to verify the EML4-ALK fusion gene of preparation quality control product
[0131] 1. Method: Verified by Aide’s EML4-ALK fusion gene real-time fluorescent RT-PCR kit
[0132] 1. RNA extraction: (1) Randomly take one slice of each of the three EML4-ALK paraffin-embedded samples obtained in Example 1, and add 1 ml of xylene solution into the tube. Vortex for 10 seconds and then centrifuge at full speed for 2 minutes.
[0133] 2) Aspirate and discard the supernatant, being careful not to aspirate the lower pellet.
[0134] 3) Add 1ml of absolute ethanol to the precipitate, vortex to mix, and centrifuge at full speed for 2min.
[0135] 4) Suck off the supernatant, be careful not to suck out the lower part of the sediment, use a new tip to suck out the added ethanol.
[0136] 5) Open the lid and incubate at room temperature for 10 minutes until all residual ethanol evaporates.
[0137] 6) Add 150 μL BufferPKD, sh...
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