Gene chip used for researching action of Alzheimer disease-controlling medicament
An Alzheimer's disease and gene chip technology, applied in the field of preparation of the chip, can solve problems such as adverse drug side effects, difficulty in obtaining satisfactory results, etc., achieve uniform concentration of synthetic products, and prevent amplification failure from affecting experiments and hybridization temperature. uniform effect
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Embodiment 1
[0046] Example 1: Fabrication of gene chip for drug research on prevention and treatment of Alzheimer's disease
[0047] 1. Construction of DNA square matrix: 200 kinds of genes listed in Table 1 and negative controls (such as bacterial control probes), positive controls (such as eukaryotic housekeeping genes GAPDH, β-Actin) are arranged according to functional classification (or in random order). Each substrate is divided into 4 regions of equal size, and 200 molecules are spotted in each region, and each molecule is spotted twice.
[0048] 2. Probe design and synthesis: according to the three important parameters of the probe, namely sensitivity, specificity and the stability of forming a double strand with the target gene, the probe sequence is designed with a length of 60mer (or greater than 60mer). The oligonucleotides of 200 genes listed in Table 1 were synthesized by off-line solid-phase chemical method, the synthesized oligonucleotides were purified by PAGE method, an...
Embodiment 2
[0050] Example 2: Application of gene chip for drug research on prevention and treatment of Alzheimer's disease
[0051] 1. Total RNA extraction: TRIzol reagent, chloroform, isopropanol and 75% ethanol were used to extract the total RNA in the cells, tissues and patients' serum, cerebrospinal fluid and tissues before and after drug (or different drugs) treatment. The concentration and purity of total RNA were measured by ultraviolet spectrophotometer, and the integrity of total RNA was detected by formaldehyde-denatured agarose gel electrophoresis. last use Total RNA was purified by RNA Clean-up (740948.250, MN).
[0052] 2. Synthesis of cRNA
[0053] (1) Use the MessageAmpTM II-96aRNA Amplification kit (1819, Ambion) to reverse transcribe the total RNA into the first strand of cDNA;
[0054] (2) Use the MessageAmpTM II-96aRNA Amplification kit (1819, Ambion) to reverse transcribe the total RNA into the second strand of cDNA;
[0055] (3) use Extract II (740609.250, MN)...
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