An early hypoxia detection kit using miRNA-210 as a marker

A miRNA-210 and kit technology, applied in the field of molecular biomedicine, can solve the problems of tolerance and poor prognosis

Inactive Publication Date: 2011-12-07
INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

On the other hand, hypoxia is also one of the important characteristics of the microenvironment of solid tumors, and it is an important reason why solid tumors are resistant to radiotherapy and chemotherapy and have poor prognosis in clinical practice.

Method used

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  • An early hypoxia detection kit using miRNA-210 as a marker
  • An early hypoxia detection kit using miRNA-210 as a marker
  • An early hypoxia detection kit using miRNA-210 as a marker

Examples

Experimental program
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Effect test

Embodiment 1

[0025] Example 1 sample preparation

[0026] Twenty-four SD rats with an average weight of 250 g (provided by the Animal Center of the Academy of Military Medical Sciences) were divided into 4 groups, namely the control group, the hypoxia 0.5 hour group, the hypoxia 1 hour group and the hypoxia 2 hour group. Animals in the experimental group were given 5000-meter low-pressure hypoxic treatment for different time in the hypoxic chamber. The hypoxic chamber rose to 5000 meters at a constant speed of 20 m / s, and dropped to normal oxygen at a constant speed of 20 m / s after the experiment. Immediately after the hypoxic treatment, the rats were anesthetized with sodium pentobarbital, and brain tissue, cerebrospinal fluid and blood were collected for the detection of miRNA-210.

[0027] Wistar rats on day 13.5 of pregnancy were weighed and anesthetized with pentobarbital sodium (60-65 mg / kg) intraperitoneally. The abdomen was disinfected with 75% alcohol and placed in an ultra-clea...

Embodiment 2

[0030] Example 2 Extraction of RNA in rat brain tissue, cerebrospinal fluid, blood, neural stem cells and neural stem cell culture fluid

[0031] (1) Samples (rat brain tissue, cerebrospinal fluid, blood, neural stem cells, and neural stem cell culture fluid) were left at room temperature for 5 minutes, and samples taken out of a -80 degree refrigerator were kept at room temperature for 15 minutes until they melted.

[0032] (2) Add 200 microliters of chloroform to every milliliter of TRIZOL, shake vigorously for 15 seconds, and let stand at room temperature for 3 minutes.

[0033] (3) Centrifuge at 12000g at 4 degrees for 15 minutes, the upper layer is RNA

[0034] (4) Transfer about 500 microliters of the upper aqueous phase to a new EP tube, add an equal volume of isopropanol, mix up and down for 10 seconds, and place in a -20 refrigerator for two hours or overnight.

[0035] (5) Take out the sample, centrifuge at 12000g at 4 degrees for 15 minutes, pour off the supernatan...

Embodiment 3

[0038] Example 3 reverse transcription

[0039] Using RevertAid TM First Strand cDNA Synthesis Kit (Fermentas) kit was used for reverse transcription.

[0040] For neural stem cell culture fluid, blood and rat cerebrospinal fluid RNA samples: each sample takes 10 microliters of RNA samples and the specificity of mir-210 (SEQ ID No.3) and cel-54 (SEQ ID No.4) respectively Invert primer mix, 70°C water bath for 10 minutes, and place on ice for two minutes. Then add 4 microliters of buffer, 2 microliters of dNTP, 1 microliter of reverse transcriptase, and 1 microliter of RNAase inhibitor to each sample.

[0041]For neural stem cells and rat brain tissue RNA samples: take 2 micrograms of RNA from each sample and mix with the specific reverse primers of miRNA-210 (SEQ ID No.3) and U6 (SEQ ID No.5), in a 70-degree water bath for 10 minutes, and place on ice for two minutes. Then add 4 microliters of buffer, 2 microliters of dNTP, 1 microliter of reverse transcriptase, and 1 mic...

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Abstract

The invention discloses an early hypoxia detection kit using miRNA-210 as a marker, which belongs to the technical field of molecular biomedicine. The present invention designs miRNA-210-specific primers, and uses quantitative PCR to detect changes in the content of miRNA-210 in rat cerebrospinal fluid, blood, brain tissue, neural stem cells, and neural stem cell culture fluid in different concentrations of hypoxic environments, and the content is significant. Increased for hypoxic stress tissue. The method of the invention is simple, rapid and sensitive, and is suitable for detecting whether animals and cells are in hypoxic state.

Description

technical field [0001] The invention belongs to the technical field of molecular biomedicine, and in particular relates to an early hypoxia detection kit and method using miRNA-210 as a marker. Background technique [0002] Hypoxia is a hotspot in current pathological research, including plateau hypoxia and organ hypoxia caused by various diseases. Plateau hypoxia means that as the altitude increases, the oxygen concentration gradually decreases, forming a plateau low-pressure hypoxic environment. Plateau hypoxia can lead to a series of physiological and pathological changes in the human body, namely altitude sickness. The hypoxia in the body caused by the disease is due to the low local oxygen concentration in the body caused by insufficient blood supply, such as myocardial infarction, stroke, atherosclerosis and other cardiovascular and cerebrovascular diseases, hypoxia is also one of the important therapeutic factors. On the other hand, hypoxia is also one of the import...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 王飞朱玲玲范明赵彤刘朝晖黄欣吴丽颖吴奎武
Owner INST OF BASIC MEDICAL SCI ACAD OF MILITARY MEDICAL SCI OF PLA
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