Application of gene marker to multiple myeloma
A multiple myeloma, genetic technology, applied in the field of biomedicine, can solve problems that need further research
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Embodiment 1
[0069] Example 1 Screening for Gene Markers Related to Multiple Myeloma
[0070] 1. Sample collection
[0071] Bone marrow fluid samples were collected from 6 patients with multiple myeloma and 6 normal subjects, including 3 males and 3 females with confirmed multiple myeloma, with a median age of 55 years. The diagnosis of multiple myeloma was based on WHO criteria for diagnosing multiple myeloma. All the above samples were obtained with the informed consent of the patient and the consent of the organizational ethics committee.
[0072] 2. Preparation of RNA samples
[0073] Extract 5-10ml of human bone marrow fluid from the case group and the control group, and put them into anticoagulant tubes. After the bone marrow cells were extracted, 1ml Trizol reagent (Invitrogen Company) was added, mixed thoroughly, and the specimen was stored at -80°C for RNA extraction.
[0074] The RNA in the sample was extracted by the Trizol method, the purity and concentration of the RNA were...
Embodiment 2
[0083] Example 2 QPCR sequencing verification of differential expression of IFFO1 gene
[0084] 1. Large-sample QPCR verification of differential expression of IFFO1 gene. According to the sample collection method in Example 1, 50 cases of multiple myeloma tissues and 50 cases of normal bone marrow tissues were selected.
[0085] 2. The RNA extraction steps are as described in Example 1.
[0086] 3. Reverse transcription
[0087] 1) Reaction system:
[0088] Add 1 μl of RNA template, 1 μl of random primers, add double distilled water to 12 μl, mix well, centrifuge at low speed at 65°C for 5 min, then place on ice to cool.
[0089] Continue to add the following components to the 12 μl reaction solution:
[0090] 5× reaction buffer 4μl, RNase inhibitor (20U / μl) 1μl, 10mM dNTP mixture 2μl, AMV reverse transcriptase (200U / μl) 1μl; mix well and centrifuge;
[0091] 2) Reverse transcription reaction conditions
[0092] 5 minutes at 25°C, 60 minutes at 42°C, and 5 minutes at 70...
Embodiment 3
[0107] Example 3 Overexpression of IFFO1
[0108] 1. Cell culture
[0109] Take out the cryopreserved human multiple myeloma cell line RPMI8226 and resuscitate it in a culture flask, and use RPMI1640 medium containing 10% fetal bovine serum and 1% P / S at 37°C, 5% CO 2 , Cultivated in an incubator with a relative humidity of 90%. Change the medium once every 2-3 days, and use 0.25% EDTA-containing trypsin for routine digestion and passage.
[0110] Digest the cells in the culture flask with trypsin, press 1×10 6 cells / well were seeded in a 24-well culture plate.
[0111] 2. Construction of gene overexpression vector
[0112] Synthesize specific PCR amplification primers, the primer sequences are as follows:
[0113] Forward primer: 5'-CCGAAGCTTGCCACCATGAATCCGTTATTCG-3' (SEQ ID NO.7)
[0114] Reverse primer: 5'-CGGCTCGAGTCTCATGGAGCTGTCAGATGAG-3' (SEQ ID NO.8)
[0115] Two restriction enzyme sites, HindIII and XhoI, were added to the 5' end primer and the 3' end primer, re...
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