Application of c14orf129 as a molecular target in the diagnosis and treatment of Alzheimer's disease
A technology for Alzheimer's disease and reagents, applied in the biological field, can solve problems such as unsatisfactory sensitivity and specificity, and few types of biomarkers, and achieve the effect of changing proliferation
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0072] Example 1 Screening for Gene Markers Related to Alzheimer's Disease
[0073] 1. Sample collection
[0074] Blood samples from 45 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. The blood samples of 5 normal people and Alzheimer's patients were selected for high-throughput sequencing, and all samples were used for verification.
[0075] 2. Preparation of RNA samples
[0076] RNA samples were extracted using Invitrogen's Blood RNA Extraction Kit. For details, see the instruction manual.
[0077] 3. Quality analysis of RNA samples
[0078] 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.
[0079] 4. Remove rRNA ...
Embodiment 2
[0089] Example 2 QPCR sequencing to verify the differential expression of C14orf129 gene
[0090] 1. According to the detection results of high-throughput sequencing, the C14orf129 gene was selected for large-sample QPCR verification.
[0091] 2. The RNA extraction steps are the same as in Example 1.
[0092] 3. Reverse transcription:
[0093] 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.
[0094] 4. QPCR amplification
[0095] (1) Primer design
[0096] QPCR amplification primers were designed according to the coding sequences of C14orf129 gene and GAPDH gene in Genbank, and synthesized by...
Embodiment 3
[0111] Example 3 Overexpression of C14orf129 gene
[0112] 1. Cell culture
[0113] 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.
[0114] 2. Transfection
[0115] 1) Treatment of cells before transfection
[0116] The day ...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More