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Marker for early diagnosis of cerebral infarction and application thereof

A technology for early diagnosis and cerebral infarction, which can be used in the determination/examination of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., and can solve problems such as heavy laboratory labor.

Inactive Publication Date: 2014-03-26
石磊
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The currently commonly used serum markers are almost all proteins, and the traditional methods of detecting them require heavy laboratory labor, and no serum-based detection is widely applicable to the diagnosis of diseases, especially for the early diagnosis of tumors

Method used

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  • Marker for early diagnosis of cerebral infarction and application thereof
  • Marker for early diagnosis of cerebral infarction and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Total RNA extraction (mirVanaTM RNA Isolation Kit (Applied Biosystem p / n AM1556))

[0023] 1. Take 100-200 μl of cryogenically refrigerated serum sample, put it on ice to thaw, and mix well. Add 10 times the volume of lysis / binding buffer to a homogenizer and mix thoroughly.

[0024] 2. Add 1 / 10 volume of Homogenate additive, vortex to mix, and place on ice for 10 minutes. All the above operations were performed on ice.

[0025] 3. Add the same volume of acid-phenol:chloroform (300ullysis / 300ul acid-phenol:chloroform) as the lysis (excluding Homogenate additive), vortex for 30-60 seconds, and centrifuge at 10000g for 5 minutes at room temperature. If the phase separation is not good, re- centrifugal. Take the supernatant into a new tube and record the volume.

[0026] 4. Add 1.25 times the volume of 100% ethanol, vortex to mix, repeat the purification column, the volume does not exceed 700ul, and centrifuge at 10000g for 15 seconds.

[0027] 5. Add 350ul wash1, cen...

Embodiment 2

[0033] Preparation of miRNA chip

[0034] 1. Sample RNA fluorescent labeling

[0035] The experiment uses the microarray hybridization chip miRCURYTM LNA Array (Exiqon, V10.0), which contains 677 miRNA probes that have been identified by humans, and each probe is repeated 4 times. Thaw each component of the miRCURYTM LNA Array PowerLabeling Kit on ice for 15-20 minutes, briefly centrifuge and vortex to mix. Add 1 μg of total RNA (dissolved in 3 μL of water), 0.5 μL of buffer and 0.5 μL of CIP enzyme into a centrifuge tube, mix well on ice, incubate on a PCR instrument at 37°C for 30 min, inactivate enzyme activity at 95°C and denature RNA, and quickly place on ice Cool, put the mixture on ice for 2 min, rotate briefly to mix the reaction, then add 3 μL of labeling buffer, 1.5 μL of fluorescent marker (Hy3TM), 2 μL of DMSO and 2 μL of labeled enzyme, and mix on ice; place the mixture on a PCR machine at 16 °C to avoid Light reaction 1h, 65 ℃ incubation 15min. After the react...

Embodiment 3

[0041] Bioinformatics Analysis

[0042]After data preprocessing, inter-chip correction was performed according to the global mean of each chip, so that the global mean of each chip was the same; differentially expressed genes were selected by chip significance analysis. The screening conditions are: the false discovery rate FDR is controlled within 5%, and the fold change is not less than 2 times. Cluster analysis was performed on the microarray data using Cluster3.0. The results of the analysis found that there were 42 miRNAs with pairwise differences among the three groups of samples (100 specimens in each group). For details, see figure 1 . figure 1 It is the miRNA microarray clustering map related to three groups of samples of normal people (ZC), MRI-cerebral infarction (MRI-) patients and MRI+ cerebral infarction (MRI+) patients.

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Abstract

The invention relates to a marker for early diagnosis of cerebral infarction and application thereof. The marker is composed of multiple nucleic acid molecules, and each nucleic acid molecule encodes at least one microRNA (ribonucleic acid) sequence; and the multiple nucleic acid molecules at least contain a nucleic acid molecule for encoding any one of hsa-miR-106B-5P, hsa-miR-4306, hsa-miR-320e and hsa-miR-320d. The invention also provides a kit for distinguishing at least one plasma of a patient with cerebral infarction and at least one plasma of a healthy individual, which contains the marker for early diagnosis of cerebral infarction, wherein the at least one control plasma is from the healthy individual. The invention has the advantages of being effectively used for observing early occurrence and development of cerebral infarction and providing the marker for early diagnosis of cerebral infarction.

Description

technical field [0001] The invention relates to a diagnostic marker for cerebral infarction. Background technique [0002] Cerebrovascular disease is a common disease that seriously threatens human health and life expectancy. In my country, vascular diseases, tumors and chronic respiratory diseases are the main causes of death. Unlike western countries, cerebrovascular diseases account for the vast majority in my country, and the number of deaths due to stroke is more than three times the number of deaths from coronary heart disease; while the age-adjusted incidence of first stroke is not significantly different from developed countries (Liu WP , Ng KC, Huang JJ. Carotid artery injury with cerebral infarction following head and nck blunt truma: report of a cas. The Yale Journal of Biology and Medicine, 2005, 78:149-153.). Ischemic cerebrovascular disease accounts for about 60%-80% of all cerebrovascular diseases. It is mainly caused by cerebral atherosclerosis to narrow the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q2600/158C12Q2600/178
Inventor 石磊王万华
Owner 石磊
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