Application of gene in diagnosis of Alzheimer's disease
A reagent and expression level technology, applied in the field of biomedicine, can solve the problems of few types of biomarkers, unsatisfactory sensitivity and specificity, etc.
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Embodiment 1
[0038] Example 1 Screening for Gene Markers Related to Alzheimer's
[0039] 1. Sample collection
[0040] Blood samples from 4 healthy people and Alzheimer's patients were collected and mixed with anticoagulant for high-throughput sequencing.
[0041] 2. RNA sample preparation and quality analysis
[0042] Use Takara's blood extraction kit to extract RNA, the steps are as follows:
[0043] 1) Transfer 0.25ml blood sample to a centrifuge tube, add 0.75ml RNAiso Blood, pipette up and down repeatedly until the cells are completely lysed;
[0044] 2) Add chloroform (1 / 5 volume of sample solution + RNAiso Blood volume), close the cap of the centrifuge tube tightly, mix until the solution emulsifies and turns milky white, and let stand at room temperature for 5 minutes;
[0045] 3) Centrifuge at 12,000×g, 4°C for 15 minutes, absorb the supernatant and transfer to a new centrifuge tube;
[0046] 4) Add an equal volume of isopropanol to the supernatant, invert the centrifuge tube ...
Embodiment 2
[0069] Example 2 QPCR sequencing verification of differential expression of CREG2 gene
[0070] 1. According to the collection method in Example 1, blood samples of 31 cases of Alzheimer's patients and blood samples of 24 cases of healthy people were collected for QPCR verification.
[0071] 2. RNA extraction
[0072] Use Takara RNA extraction kit to extract RNA in blood, see Example 1 for specific steps.
[0073] 3. QPCR
[0074] 1) Design of primers
[0075] Primers were designed according to the gene sequences of CREG2 and GADPH, and the primer sequences are as follows.
[0076] CREG2 gene:
[0077] SEQ ID NO.1 (forward primer): 5'-TTATGAAGATGAGGATAGAACA-3'
[0078] SEQ ID NO.2 (reverse primer): 5'-TGGGAACTGCTTTGAAAT-3'
[0079] GAPDH gene:
[0080] SEQ ID NO.3 (forward primer): 5'-AATCCCATCACCATCTTCCAG-3'
[0081] SEQ ID NO.4 (reverse primer): 5'-GAGCCCCAGCCTTTCTCAT-3'
[0082] 2) Real-time quantitative PCR
[0083] Using TaKaRa One Step TB Green TM Prime Scrip...
Embodiment 3
[0092] The functional verification of embodiment 3 CREG2
[0093] 1. Cell culture
[0094] Culture PC12 cells in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C, 5% CO 2 , cultured in an incubator with a relative humidity of 90%, passaged at a ratio of 1:3, and after 24 hours, the cells entered the logarithmic growth phase to replace the culture medium, and different interventions were given according to the experimental requirements.
[0095] 2. Transfection
[0096] 2.1 Treatment of cells before transfection
[0097] The day before transfection, 5×10 5 Cells / well were cultured in antibiotic-free medium for one day, and the cell density was 60% at the time of transfection, and replaced with serum-free medium before transfection.
[0098] 2.2 Construction of gene overexpression vector
[0099] The overexpression vector of CREG2 was designed and synthesized by Shanghai Jikai Gene. The experiment was divided into three groups: blank cont...
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