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Application method of long non-coding RNA (ribonucleic acid) CASC2 originated from serum exosomes

A technology of long-chain non-coding and application methods, applied in the application field of LncRNA CASC2 in the diagnosis of glioma, which can solve the problems of early diagnosis and unsatisfactory standardized treatment

Active Publication Date: 2014-08-20
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the early diagnosis and standardized treatment of glioma are still unsatisfactory. Therefore, it is necessary to find specific early diagnostic markers for glioma to screen high-risk groups in order to conduct early diagnosis and early treatment of glioma patients. , Improving the survival rate of patients 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 (ribonucleic acid) CASC2 originated from serum exosomes
  • Application method of long non-coding RNA (ribonucleic acid) CASC2 originated from serum exosomes
  • Application method of long non-coding RNA (ribonucleic acid) CASC2 originated from serum exosomes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Preparation of long-chain non-coding RNA CASC2 kit for screening, early diagnosis or prognosis of glioma patients with high-risk groups of glioma (50 reactions)

[0025] 1. RNA stabilization solution 50ml

[0026] 2. Isopropanol 100ml

[0027] 3. Chloroform 100ml

[0028] 4. Trizol 50ml

[0029] 5. Enzyme-free water 10ml

[0030] 6.1μM random reverse transcription primer 50ul

[0031] 7.5× reverse transcription buffer 200ml

[0032] 8.10mM base triphosphate deoxynucleotide 100ul

[0033] 9.40U / μl RNase inhibitor 500ul

[0034] 10.200U / μl MMLV reverse transcriptase 50ul

[0035] 11.Premix Ex Taq50ul

[0036] 12.10μM LncRNA PRKAG2 real-time fluorescence quantitative PCR specific primer 30ul

[0037] CASC2 forward primer 5′-TTGACCCTTCCAGCTTCC-3′,

[0038] CASC2 Reverse Primer 5′-CCATCCGCACATCACAAT-3′,

[0039] 13. 10μM U6snRNA specific primer 30ul

[0040] The forward primer is 5′-ATTGGAACGATACAGAGAAGATT-3′,

[0041] The forward primer was 5'-GGAACGC...

Embodiment 2

[0043] Verification of the expression difference of lncRNA CASC2 in glioma tissue and normal brain tissue

[0044] 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.

[0045]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

[0057] Specific and sensitive detection of LncRNA CASC2 derived from serum Exosomes for glioma diagnosis

[0058] 1. Isolation of Exosomes in Serum

[0059] Collect the peripheral blood of the individual to be tested

[0060] 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.

[0061] 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)

[0062] 2. Extraction and purification of RNA in Exosomes (select the commercialized Exosomes separation and purification RNA k...

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Abstract

The invention discloses an application method of long non-coding RNA (ribonucleic acid) (LncRNA) CASC2 originated from serum exosomes, namely the LncRNA CASC2 originated from the serum exosomes is used for preparing preparations for screening and early diagnosis of a high-risk group of gliomas or prognosis of patients with the gliomas. Studies prove that the expression level of the LncRNA CASC2 is reduced by extracting the RNA after separating the exosomes in serum of the patients with the gliomas, performing reverse transcription and performing real-time fluorescence quantitative analysis. By utilizing the LncRNA CASC2 originated from the serum exosomes, the specificity for early diagnosis of the gliomas can achieve 85.7% and the sensitivity can achieve 97.1%. By detecting the expression level of the LncRNA CASC2 in the serum exosomes of the patients with the gliomas, early and fast non-invasive diagnosis can be performed on the patients with the gliomas.

Description

technical field [0001] The invention belongs to the field of tumor molecular biology, and in particular relates to an application method of LncRNA CASC2 derived from serum Exosomes in glioma diagnosis. Background technique [0002] Gliomas account for 40.49% of intracranial tumors and can occur anywhere in the central nervous system. Except for a small number of astrocytomas and pilocytomas, which can be considered benign and have a good prognosis, most patients have a poor prognosis, with an average survival of 3-5 years. However, traditional surgery is difficult to accurately identify the tumor border and perform a complete surgical resection. At present, the diagnosis of glioma is still in the empirical stage based on clinical, pathological and imaging information, and the diagnosis and treatment of glioma have not made significant progress in general. At present, the early diagnosis and standardized treatment of glioma are still unsatisfactory. Therefore, it is necessa...

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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