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Application of miR-182 as glioma generating molecular marker and detective reagent kit thereof

A technology of mir-182 and kit, which can be used in the determination/inspection of microorganisms, material excitation analysis, biochemical equipment and methods, etc., can solve problems such as inability to adapt to glioma screening and early diagnosis

Inactive Publication Date: 2012-05-23
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The diagnosis of glioma is still in the empirical stage based on clinical, pathological and imaging information, and once diagnosed, most of them are late, which is far from suitable for screening and early diagnosis of high-risk groups for glioma needs

Method used

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  • Application of miR-182 as glioma generating molecular marker and detective reagent kit thereof
  • Application of miR-182 as glioma generating molecular marker and detective reagent kit thereof
  • Application of miR-182 as glioma generating molecular marker and detective reagent kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Preparation of glioma peripheral blood diagnostic kit (50 reactions)

[0018] 1. Lymphocyte Separation Solution 300ml,

[0019] 2. Hank's solution or PBS 300ml,

[0020] 3. Isopropanol 100ml,

[0021] 4. Chloroform 100ml,

[0022] 5. Trizol 50ml,

[0023] 6. DEPC water or enzyme-free water 10ml, double distilled water 10ml,

[0024] 7.5× reverse transcription buffer 1ml,

[0025] 8.25mM magnesium chloride 1ml,

[0026] 9. 1ml of 10mM base triphosphate deoxyribonucleotides,

[0027] 10.5U / μl RANase inhibitor 500μl,

[0028] 11. 200U / μl MMLV reverse transcriptase 50μl or 25U / μl AMV enzyme 50μl,

[0029] 12.2× real-time quantitative PCR buffer 2ml,

[0030] 13.5U / μl Taq polymerase 50μl,

[0031] 14.5 μM hsa-miR-182 specific PCR primer 50 μl,

[0032] The forward primer is 5′-ACTTTTGGCAATGGTAGAACTCAC-3′,

[0033] Its reverse primer is 5'-AATCCATGAGAGATCCCTAGCG-3'.

[0034] 15.5 μM U6snRNA specific PCR primer 30 μl

[0035] The forward primer is 5′-ATTG...

Embodiment 2

[0039] Example 2 Detection of miR-182 in peripheral blood samples

[0040] 1. Extraction of peripheral blood RNA: Use EDTA anticoagulant tubes to collect 5ml of peripheral blood samples from the individual to be tested. After blood collection, the anticoagulant tube needs to be shaken back and forth to allow the blood to fully contact the anticoagulant on the tube wall, and the movement should be gentle to prevent hemolysis, and the total RNA of peripheral blood should be extracted immediately.

[0041] 1) Peripheral blood mononuclear cell extraction: Take 5ml of EDTA anticoagulant blood, centrifuge at 1000rpm for 6min, absorb the upper layer of plasma and store it in an EP tube at -70°C, add an equal volume of Hanks solution to the lower layer of peripheral blood cells and mix them upside down; take lymphocytes Put 6ml of separation solution in a centrifuge tube, slowly inject the mixed blood cells into the separation solution along the tube wall to keep a clear interface bet...

Embodiment 3

[0071] Example 3 Clinical verification of miR-182 detection method in peripheral blood samples

[0072] Using the above detection method, the expression of miR-182 in the peripheral blood of 22 normal persons and 106 suspected brain tumor patients (newly admitted initial patients, before surgery) was detected.

[0073] Compared with the expression of miR-182 in the peripheral blood of normal people, the expression of miR-182 in the peripheral blood of 33 patients was elevated, and their ΔCT were all ≤ 7.61, and 33 patients were confirmed by pathology after surgery: 31 cases of glioma, There was 1 case of pituitary adenoma, 1 case of schwannoma, and the other 73 patients. When miR-182 real-time quantitative PCR was detected in peripheral blood, miR-182 did not change significantly, and the ΔCT values ​​were all > 7.61, and the difference was not significant (p > 0.05 ). Pathological diagnosis confirmed after operation: 5 cases of craniopharyngioma, 4 cases of medulloblastoma, ...

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Abstract

The invention discloses application of a miR-182 as a glioma generating molecular marker and a detective reagent kit thereof. On the basis of providing the molecular marker as the early diagnosis of the glioma, the detective reagent kit is prepared, a method for detecting peripheral blood miR-182 is provided and comprises the steps of: (1) collecting a peripheral blood sample of an individual to be detected and extracting total RNA; (2) carrying out reverse transcription on miR-182 specificity into cDNA by using total RNA as a template; and (3) carrying out real-time quantitative PCR amplification by using a miR-182 specific primer for obtaining a relative quantitative delta CT value, and when delta CT <=7.61, promoting that miR-182 expresses positive. The method and the reagent kit can be used for detecting the expression condition of miR-182 in the peripheral blood of all people in order to forecast the suffering risk of the glioma, screening high-risk people of the glioma and carrying out early and quick non-invasive diagnosis on glioma patients.

Description

technical field [0001] The invention relates to molecular markers of tumor occurrence, in particular to the application of microRNA (miRNA) as molecular markers in the diagnosis of glioma. Background technique [0002] Glioma includes tumors of various neuroepithelial origins, and is the most common malignant tumor in the central nervous system, accounting for about 40% to 50% of all intracranial tumors. Because most gliomas grow infiltratively and have unclear borders with the surrounding brain tissue, even the most modern neurosurgical techniques are difficult to achieve pathological complete resection, so the recurrence rate of gliomas is very high after surgery, and With the increase in the number of operations and recurrence, the degree of malignancy tends to increase. After using modern microsurgical techniques, radiotherapy, chemotherapy and other comprehensive treatment measures, the prognosis of glioma patients is still unsatisfactory. The 1-year and 2-year surviva...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68G01N21/64
Inventor 李桂源武明花唐海林王泽友廖前进刘晓萍
Owner CENT SOUTH UNIV