METHOD FOR DETERMINATION OF ATHEROGENIC MICRORNAS IN PATIENTS WITH CAROTID ATHEROSCLEROSIS Russian patent published in 2021 - IPC C12N15/10 

FIELD: medical biotechnology.

SUBSTANCE: invention relates to the field of medical biotechnology, in particular to a method for the isolation and determination of atherogenic microRNA from biological fluids in patients with atherosclerosis. Isolation of atherogenic microRNA from blood is performed in three stages. At the first stage, erythrocyte lysis is carried out by vortexing a blood sample for 10 seconds with Lysis buffer RBC. Then incubated for 3-5 minutes at room temperature, and then centrifuged for 3 minutes at 1000 rpm to pellet the cells. Lysis buffer RBC is added to the resulting precipitate, after which RL buffer is added to the resulting leukocyte suspension and shaken on a vortex until a homogeneous state is obtained – lysate. In the second step, the lysate is passed through the column and centrifuged for 3 minutes at 14,000 rpm. 70% alcohol is added to the resulting suspension-mixture. It is passed through the column again and centrifuged for one minute at 6000 rpm. The resulting suspension is once again introduced into the column and wash solution A is added, centrifuged for one minute at 14 rpm, then loaded onto the DNase I column, centrifuged again for one minute at 14 rpm. The resulting mixture is incubated at room temperature for 15 minutes. At the third stage, wash solution A is again introduced into the column twice and centrifuged twice for one minute at 14000 rpm. Then the column with the sample is air-dried for 2 minutes and Elution solution A is added to it to elute microRNA, after which it is centrifuged for one minute at 2000 rpm and then for two minutes at 14000 rpm. Next, the number of copies in the obtained microRNA sample is determined on a CFX Touch amplifier. The resulting sample is added to a mini Eppendorf tube in amounts of 2 mcl, 3 mcl of Polly A buffer, 3 mcl of ATP, 1.8 mcl of Polly A enzyme and 10.2 mcl of distilled water are added to the sample, incubated at 65°C for 10 minutes , after which 18 mcl of ligase buffer, 27 mcl of PEG 800 A, 3.6 mcl of ligation adapter, 9 mcl of RNA ligase, 2.4 mcl of distilled water are added and the newly obtained mixture is incubated at 16°C for 1 hour. Then the following is added: 36 mcl of 5x BufferRT, 7.2 mcl of dNTP mix, 9 mcl of Universal RT primer, 15 mcl of Enzyme mix, 20 mcl of distilled water, incubated at 85°C for 5 minutes to obtain an extracted sample. Next, a stirring solution is prepared with 300 mcl of MiR Amp master Mix, 30 mcl of MiR Amp primer Mix and 210 mcl of distilled water. Then 45 mcl of the resulting solution is added to 5 mcl of the extracted sample and PCR is carried out, starting from the stage of reverse transcription according to the following RNA-1 program: 1 cycle - 5 minutes at a temperature of 95°C, 14 cycles - 30 seconds at a temperature of 60°C, 10 minutes at a temperature of 99°C, then holding at a temperature of 4°C. Next, the sample is added to 5 clean mini Eppendorf tubes, 5 mcl in each, into which 4 mcl of distilled water and microRNA primers-probes in an amount of 2 mcl are then added in the following order of introduction of the microRNA primers-probes: first - 126-5p; in the second - 126-3p; in the third - 33-5p; in the fourth - 21-5p; in the fifth - 21-3p. Then 10 mcl of a stock solution containing TaqMan Fast Advanced and TaqMan Fast Advanced Buffer is added to each tube in a ratio of 1:100, respectively. The RNA-2 amplification program is carried out: 1 cycle - 20 min at a temperature of 95°C, 40 cycles for 1 minute at a temperature of 95°C, then holding at a temperature of 4°C, while obtaining the number of copies of atherogenic microRNA.

EFFECT: invention allows isolating and determining atherogenic microRNAs with high accuracy from the blood of patients with carotid atherosclerosis.

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