Alzheimer's marker fam170a
A FAM170A and substance technology, applied in DNA/RNA fragments, biological testing, recombinant DNA technology, etc., can solve the problems of unsatisfactory sensitivity and specificity, and few types of biomarkers
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[0053] Examples of specific binding agents are peptides, peptidomimetics, aptamers, spiegelmers, darpins, ankyrin repeat proteins, Kunitz-type domains, antibodies, single domain antibodies and monovalent antibody fragments. As a preferred embodiment, the specific binding agent is a FAM170A specific antibody.
[0054] Illustrative, non-limiting examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. Those of ordinary skill in the art will recognize that RNA is typically reverse transcribed into DNA prior to sequencing because RNA is less stable in cells and more susceptible to nuclease attack in experiments.
[0055] Chips, preparations, nucleic acid membrane strips, kits
[0056] In the present invention, the chip includes a gene chip and a protein chip; the gene chip includes a solid phase carrier; and oligonucleotide probes fixed on the solid phase carrier in an orderly manner, and t...
Embodiment 1
[0078] Example 1 Screening for Gene Markers Related to Alzheimer's Disease
[0079] 1. Sample collection
[0080] 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.
[0081] 2. Preparation of RNA samples
[0082] RNA samples were extracted using Invitrogen's Blood RNA Extraction Kit. For details, see the instruction manual.
[0083] 3. Quality analysis of RNA samples
[0084] 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.
[0085] 4. Remove rRNA ...
Embodiment 2
[0095] Example 2 QPCR sequencing to verify the differential expression of the FAM170A gene
[0096] 1. According to the detection results of high-throughput sequencing, the FAM170A gene was selected for large-sample QPCR verification.
[0097] 2. RNA extraction
[0098] RNA samples were extracted using Invitrogen's Blood RNA Extraction Kit. For details, see the instruction manual.
[0099] 3. Reverse transcription:
[0100] 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.
[0101] 4. QPCR amplification
[0102] (1) Primer design
[0103] QPCR amplification primers were designed according to t...
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