Application method of serum Exosomes-derived long non-coding RNA PRKAG2-AS1

A RNAPRKAG2-AS1, long-chain non-coding technology, applied in the field of tumor molecular biology, can solve the problems that the survival rate of glioma patients has not been significantly improved, and cannot adapt to the screening and early diagnosis of glioma high-risk groups

Active Publication Date: 2014-08-06
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, although the diagnosis and treatment methods of glioma are in the stage of continuous improvement, the survival rate of glioma patients has not been significantly improved.
The diagnosis of glioma is still in the empirical stage based on clinical, pathological and imaging information, and once diagnosed, most of them are in the middle and late stages, which is far from being suitable for screening and early diagnosis of high-risk groups for glioma needs

Method used

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  • Application method of serum Exosomes-derived long non-coding RNA PRKAG2-AS1
  • Application method of serum Exosomes-derived long non-coding RNA PRKAG2-AS1
  • Application method of serum Exosomes-derived long non-coding RNA PRKAG2-AS1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Preparation of a long-chain non-coding RNA PRKAG2-AS1 kit for screening, early diagnosis or prognosis of glioma patients at high risk of glioma (50 reactions)

[0026] 1. RNA stabilization solution 50ml

[0027] 2. Isopropanol 100ml

[0028] 3. Chloroform 100ml

[0029] 4. Trizol 50ml

[0030] 5. Enzyme-free water 10ml

[0031] 6.1μM random reverse transcription primer 50ul

[0032] 7.5× reverse transcription buffer 200ml

[0033] 8.10mM base triphosphate deoxyribonucleotides 100ul

[0034] 9.40U / μl RNase inhibitor 500ul

[0035] 10.200U / μl MMLV reverse transcriptase 50ul

[0036] 11.Premix Ex Taq 50ul

[0037] 12.10μM LncRNA PRKAG2-AS1 real-time fluorescence quantitative PCR specific primer 30ul

[0038] LncRNA PRKAG2-AS1 forward primer: 5'-CAGTTTCTCATCAAATAGGGTGT-3',

[0039] LncRNA PRKAG2-AS1 reverse primer: 5'-TATTGTCCCACTGAATGCTC-3';

[0040] 13. 10μM U6snRNA specific primer 30ul

[0041] The forward primer is 5′-ATTGGAACGATACAGAGAAGATT-3′

[0...

Embodiment 2

[0044] Verification of the expression difference of lncRNA PRKAG2-AS1 in glioma tissue and normal brain tissue

[0045] 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.

[0046] 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 7500...

Embodiment 3

[0063] Specific and sensitive detection of LncRNA PRKAG2-AS1 derived from serum Exosomes for glioma diagnosis

[0064] 1. Isolation of Exosomes in Serum

[0065] Collect the peripheral blood of the individual to be tested

[0066] 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.

[0067] 1.2 Isolation 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)

[0068] 2. Extraction and purification of RNA in Exosomes (select the commercialized Exosomes separation and purification R...

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Abstract

The invention discloses an application method of serum Exosomes-derived long non-coding RNA (long no-coding RNA, LncRNA) PRKAG2-AS1, namely, the serum Exosomes-derived LncRNA PRKAG2-AS1 is used for preparing drugs for the screening and early diagnosis of high-risk population with gliomas or prognosis preparations for patients with gliomas. Through researching, a situation that RNA is extracted after Exosomes are separated from serums of patients with gliomas, then the RNA is subjected to reverse transcription and real-time fluorescent quantitative analysis, and the expression level of LncRNA PRKAG2-AS1 drops is verified. The specificity of the preparation disclosed by the invention and for the early diagnosis of gliomas can reach 85.7%, and the sensitivity reaches 88.2%. Through detecting the expression level of LncRNA PRKAG2-AS1 in serum Exosomes of patients with gliomas, the early and rapid noninvasive diagnosis on the patients with gliomas is implemented.

Description

technical field [0001] The invention belongs to the field of tumor molecular biology, and in particular relates to the application method of LncRNA PRKAG2-AS1 derived from serum Exosomes in the preparation of prognostic reagents for glioma patients. Background technique [0002] Glioma accounts for about 40.49% of intracranial tumors. Glioma has the highest incidence rate in brain tumors, and gliomas in the cerebral hemisphere account for 51.4% of all gliomas. Because most gliomas grow infiltratingly and have unclear boundaries with the surrounding brain tissue, even the most modern neurosurgical techniques are difficult to achieve pathological total resection, so the recurrence rate of gliomas is very high, and With the increase of operation and recurrence times, the degree of malignancy tends to increase. At present, although the diagnosis and treatment methods of glioma are in the stage of continuous improvement, the survival rate of glioma patients has not improved sig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6886C12Q2600/118C12Q2600/158C12Q2600/178
Inventor 武明花刘长红余志斌徐刚李桂源
Owner CENT SOUTH UNIV
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