Medicinal preparation for treating Alzheimer disease
A technology for Alzheimer's disease and pharmaceutical preparations, which is applied in the field of biomedicine and can solve problems such as difficult and accurate diagnosis of patients
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Embodiment 1
[0025] Example 1 Obtaining peripheral blood mononuclear cells
[0026] (1) Collect peripheral blood from 45 cases of Alzheimer's disease (AD group) and 45 normal people (NC group);
[0027] (2) Take a 15ml centrifuge tube, add 5ml of human peripheral blood lymphocyte separation medium, and then use a pipette gun to carefully draw 5ml of peripheral blood and slowly add it along the wall of the centrifuge tube;
[0028] (3) Centrifuge at 800g in a centrifuge for 25 minutes. After centrifugation, the liquid is divided into 4 layers from top to bottom, namely: plasma layer, PBMCs layer, transparent separation layer, and red blood cell layer;
[0029] (4) Carefully pipette the PBMCs layer into a 15ml enzyme-free centrifuge tube, add 10ml PBS buffer solution to wash the PBMCs layer, and place it in a centrifuge at 250g for 10min;
[0030] (5) Discard the supernatant, add 5ml PBS buffer to resuspend the cells, place in a centrifuge, and centrifuge at 250g for 10min;
[0031] (6) Di...
Embodiment 2
[0032] Example 2 Peripheral Blood Mononuclear Cell RNA Extraction
[0033] (1) Add 1ml of Trizol lysis buffer to the peripheral blood mononuclear cells obtained in Example 1, and use a pipette to repeatedly pipette so that the cells can be fully lysed;
[0034] (2) Transfer the lysed cells to an RNase-free 1.5ml EP tube and let stand at room temperature for 5 minutes;
[0035] (3) Add 200ul of chloroform to the tube, shake vigorously for 15s, and let stand at room temperature for 2min;
[0036] (4) Place the EP tube in a pre-cooled 4°C high-speed centrifuge and centrifuge at 12000rpm for 15 minutes. After taking it out, the sample is divided into three layers. Carefully absorb the aqueous phase of the upper layer into a new EP tube. Be careful not to Inhale the middle layer;
[0037] (5) Add an equal volume of pre-cooled isopropanol, mix thoroughly by inversion, and place at room temperature for 10 minutes;
[0038] (6) Place the EP tube in a pre-cooled 4°C high-speed centr...
Embodiment 3
[0040] Example 3 Reverse transcription reaction of total RNA
[0041] (1) Genomic DNA removal reaction, the reagent system is as follows:
[0042] Table 1 Reaction system for removing genomic DNA
[0043] Reagent dosage 5×gDNA Eraser Buffer 2.0μl gDNA Eraser 1.0μl Total RNA 1.0μg RNase Free dH2O Up to 10μl
[0044] Set the reaction conditions: Gently blow and beat to mix, centrifuge for 15s, put into PCR instrument at 42°C for 2min, 4°C ∞;
[0045] (2) Reverse transcription reaction, the reagent system is as follows:
[0046] Table 2 Reverse transcription reaction system
[0047]
[0048] Set the reaction conditions: Gently pipette to mix, centrifuge for 15s, put into PCR instrument at 37°C for 15min, 85°C for 5s, 4°C∞.
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