Atherosclerosis-related serum miRNA (microribonucleic acid) marker group, and specific primers and application thereof
A technology of atherosclerosis and specific primers, applied in the field of medical molecular biology, to achieve the effects of accurate quantitative detection, strong complementarity, and improved sensitivity and specificity
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Embodiment 1
[0029] 1. 54 cases of patients with coronary heart disease and 35 cases of normal healthy controls received physical examination, 5ml of peripheral blood was extracted using coagulation tubes, centrifuged in two steps at 4°C, and finally the upper serum was taken into RNase / DNase-free centrifuge tubes.
[0030] 2. Serum total RNA extraction: TRI Reagent BD was used to extract RNA from serum.
[0031] (1) Lysis: 0.75ml TRI Reagent BD+0.25ml serum;
[0032] (2) Two-phase separation: lysate + 0.1ml bromochloropropane or 0.2ml chloroform;
[0033] (3) RNA precipitation: aqueous phase layer + 0.5ml isopropanol;
[0034] (4) Wash RNA: 1ml 75% ethanol;
[0035] (5) RNA dissolution: dissolve RNA with FORMAzol, water or 0.5% SDS solution prepared by pipetting and mixing with a pipette tip.
[0036] 3. miRNA chip hybridization: using Exiqon miRCURY LNA TM The 11.0 chip includes all miRNAs from the three species of human, mouse, and rat in the miBase database. The capture probes on ...
Embodiment 2
[0047] Using the specific primers of miR-135b and miR-499a-3p (as shown in Table 3), hsa-miR-135b and hsa-miR- There are significant differences in the expression of 499a-3p between the atherosclerosis case group and the healthy control group, such as Figure 4 shown.
[0048] Table 3. Primer information for related miRNAs (Note: GSP is the corresponding specific primer, R is the primer that matches the RT primer)
[0049]
[0050] According to the above results, by analyzing the expression levels of miRNAs in two groups of serum samples (case group and healthy control group), the 90th percentile of the expression levels of miR-135b and miR-499a-3p in the healthy control group was used as the threshold. Grouped by miR-135b and miR-499a-3p, the expression level was less than 90 percentile, scored as 0, divided into healthy group, and the expression level was greater than or equal to 90 percentile, scored as 1 point, divided into case group, Then evaluate the ability of hig...
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