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Application method of long non-coding RNA LINC00470 derived from serum exosomes

A long-chain non-coding, source technology, applied in the application of LncRNA LINC00470 in glioma diagnosis, can solve the problems of no obvious progress in glioma diagnosis and treatment, unsatisfactory early diagnosis and high local recurrence rate , to achieve far-reaching clinical significance and the effect of promotion, good specificity and good stability

Active Publication Date: 2015-08-26
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Because gliomas mostly show infiltrative growth without clear boundaries, it is difficult to completely remove them by surgery, and the local recurrence rate after surgery is high. There is no significant progress in the diagnosis and treatment in general, and most of them die a few years after diagnosis
At present, the early diagnosis and standardized treatment of glioma are still unsatisfactory. Therefore, it is necessary to search for non-invasive early diagnostic markers of glioma to screen high-risk groups in order to carry out early diagnosis and early diagnosis of glioma patients. Treatment, improving patient survival, has always been the main task of research in the field of neuroscience

Method used

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  • Application method of long non-coding RNA LINC00470 derived from serum exosomes
  • Application method of long non-coding RNA LINC00470 derived from serum exosomes
  • Application method of long non-coding RNA LINC00470 derived from serum exosomes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Preparation of long-chain non-coding RNA LINC00470 kit for screening, early diagnosis or prognosis of glioma patients at high risk of glioma (50 reactions)

[0024] 1. RNA stabilization solution 50ml

[0025] 2. Isopropanol 100ml

[0026] 3. Chloroform 100ml

[0027] 4. Trizol reagent 50ml

[0028] 5. Enzyme-free water 10ml

[0029] 6.1μM random reverse transcription primer 50ul

[0030] 7.5× reverse transcription buffer 200ml

[0031] 8.10mM base triphosphate deoxyribonucleotides 100ul

[0032] 9.40U / μl RNase inhibitor 500ul

[0033] 10.200U / μl MMLV reverse transcriptase 50ul

[0034] 11. Premix Ex Taq 50ul

[0035] 12.10μM LncRNA LINC00470 real-time fluorescence quantitative PCR specific primer 30ul

[0036] LINC00470 Forward primer 5'-AAACGGTCAAGAAGAAGTCA-3'

[0037] LINC00470 reverse primer 5'-CTGTTGCTCAGCGTGTAGGA-3'

[0038] 13.10μM U6snRNA specific primer 30ul

[0039] The forward primer is 5′-ATTGGAACGATACAGAGAAGATT-3′,

[0040] The forward...

Embodiment 2

[0042] Verification of expression difference of lncRNA LINC00470 in glioma tissue and normal brain tissue

[0043] 1. Collect the glioma tissue to be tested, put it into a cryopreservation tube filled with RNA stabilization solution, and put it in a -80°C refrigerator for later use.

[0044]2. Extraction of RNA in tissues: Take an appropriate amount of specimen, add liquid nitrogen to the mortar after baking at 180°C for 6-8 hours, grind the specimen, grind to powder, add 1ml Trizol mortar specimen to the mortar, and grind into After liquid, transfer to tube, add 200μl / ml Trizol in chloroform, shake by hand for 15-30s, place on ice for 5min, centrifuge at 12000g at 4°C for 15min; carefully take the upper aqueous phase into a new tube, add pre-cooled Mix with isopropanol 0.5ml / ml Trizol, put it in a refrigerator at -20°C for 20min, centrifuge at 12000g at 4°C for 10min; discard the supernatant, add 1-2ml of ethanol diluted with 75% DEPC water, mix well, centrifuge at 7500g at 4...

Embodiment 3

[0056] Specificity and sensitivity of LncRNA LINC00470 derived from serum Exosomes for the diagnosis of glioma

[0057] 1. Isolation of Exosomes in Serum

[0058] Collect the peripheral blood of the individual to be tested

[0059] 1.1 Separation of peripheral blood serum: 5 ml of blood from the individual to be tested is collected using a blood coagulation tube. After blood collection, centrifuge at 1000rpm for 6min, absorb serum and store it in EP tube at -80°C.

[0060] 1.2 Separation of Exosomes in serum: 100 μl of Total Exosome Isolation Reagent was added to 500 μl of each serum sample, vortexed and mixed, and reacted at 4°C for 30 minutes. Centrifuge at 10,000g for 10 minutes at room temperature. Exosomes are stored at the bottom of the EP tube, and the Exosomes are resuspended with 200 μl PBS. (Choose the commercialized Exosomes separation kit)

[0061] 2. Extraction and purification of RNA in Exosomes (select the commercialized Exosomes separation and purification...

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Abstract

The invention discloses an application method of serum Exosomes derived long no-coding RNA (long no-coding RNA, LncRNA) LINC00470, namely, serum Exosomes derived long no-coding RNA LINC00470 is used for preparing prognosis preparations for the screening and early diagnosis of high-risk groups with glioma and patients with glioma. Reaches prove that RNA is extracted after Exosomes are separated from serums of patients with glioma, and through carrying out reverse transcription and real-time fluorescent quantitative analysis on the RNA, a situation that the expression level of LncRNA LINC00470 is lowered is found. According to the invention, the specificity of the serum Exosomes LncRNA LINC00470 to the early diagnosis of glioma can reach 85.7%, and the sensitivity can reach 94.1%. By testing the expression level of LncRNA LINC00470 in serum Exosomes of patients with glioma, an early and rapid noninvasive diagnosis on the patients with glioma is implemented.

Description

technical field [0001] The invention belongs to the field of tumor molecular biology, and in particular relates to an application method of LncRNA LINC00470 derived from serum Exosomes in glioma diagnosis. Background technique [0002] Glioma accounts for about 60% of all primary nervous system tumors, and can occur in any part of the central nervous system and at any age. Surgical resection is the preferred treatment. Because gliomas mostly show infiltrative growth without clear boundaries, it is difficult to completely remove them by surgery, and the local recurrence rate after surgery is high. There has been no significant progress in diagnosis and treatment in general, and most of them died a few years after diagnosis. At present, the early diagnosis and standardized treatment of glioma are still unsatisfactory. Therefore, it is necessary to search for non-invasive early diagnostic markers of glioma to screen high-risk groups in order to carry out early diagnosis and ea...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q1/6886C12Q2600/118C12Q2600/158C12Q2600/178C12Q2531/113C12Q2545/101C12Q2561/113
Inventor 武明花刘长红余志斌徐刚李桂源
Owner CENT SOUTH UNIV
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