Application of biomarker scfd2 in the diagnosis and treatment of Alzheimer's disease
A material and composition technology, applied in the field of Alzheimer's diagnosis and treatment, can solve the problems of unsatisfactory sensitivity of mild dementia, mild patients with insignificant brain atrophy are not enough to provide diagnosis basis, etc.
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Embodiment 1
[0073] Example 1 Screening for Gene Markers Related to Alzheimer's Disease
[0074] 1. Sample collection
[0075] Blood samples from 5 normal people and patients with Alzheimer's disease were collected respectively. All patients gave their informed consent, and all the above samples were obtained with the consent of the ethics committee.
[0076] 2. Preparation of RNA samples
[0077] RNA samples were extracted using Invitrogen's Blood RNA Extraction Kit. For details, see the instruction manual.
[0078] 3. Quality analysis of RNA samples
[0079] Nanodrop2000 was used to detect the concentration and purity of the extracted RNA, agarose gel electrophoresis was used to detect RNA integrity, and Agilent2100 was used to determine the RIN value. Concentration ≥ 200ng / μl, OD260 / 280 between 1.8 and 2.2.
[0080] 4. Remove rRNA
[0081] Ribosomal RNA was removed from total RNA using the Ribo-Zero kit.
[0082] 5. Construction of cDNA library
[0083] The Illumina TruseqTM RNA ...
Embodiment 2
[0090] Example 2 QPCR sequencing to verify the differential expression of the SCFD2 gene
[0091] 1. According to the detection results of high-throughput sequencing, the SCFD2 gene was selected for large-sample QPCR verification. According to the sample collection method in Example 1, 45 cases of blood from Alzheimer's patients and 45 cases of normal people's blood were selected.
[0092] 2. The RNA extraction steps are the same as in Example 1.
[0093] 3. Reverse transcription:
[0094] Take 2 μg of total RNA for reverse transcription, add 2 μl of Oligo(dT), and mix well. After bathing in water at 70°C for 5 minutes, immediately ice-bath for 2-3 minutes; continue to add 5 μl of 5× reverse transcription buffer, 5 μl of dNTP (2.5mM), 40U / μl of RNasin, 200U / μl of M-MLV, and make up to the expected volume with nuclease-free water , 42 ° C water bath for 60 minutes, 95 ° C water bath for 5 minutes to inactivate M-MLV.
[0095] 4. QPCR amplification
[0096] (1) Primer desig...
Embodiment 3
[0112] Overexpression of embodiment 3SCFD2 gene
[0113] 1. Cell culture
[0114] Dopamine neuronal cells SH-SY5Y were prepared in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin (pH 7.2-7.4), at 37°C and 5% CO 2 , Cultivated in an incubator with a relative humidity of 90%. Change the medium once every 2 days, and subculture when the cells grow to 90% contact. After washing with PBS, add 0.25%-EDTA trypsin to separate the cells from the bottle wall, and terminate with DMEM medium containing fetal bovine serum. Trypsin digestion reaction, centrifuged at 1000g for 2min, discarded the supernatant, resuspended with the newly prepared culture medium, and passaged at a ratio of 1:3 to 1:4. After 24 hours, the cells entered the logarithmic growth phase and replaced the culture medium according to the experimental requirements. give different interventions.
[0115] 2. Transfection
[0116] 1) Treatment of cells before transfection
[0117] The day be...
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