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Application method of long-chain non-coding RNA CASC2 derived from serum exosomes

A long-chain non-coding, application method technology, applied in the application field of LncRNACASC2 in the diagnosis of glioma, can solve the problems of unsatisfactory early diagnosis and standardized treatment

Active Publication Date: 2016-01-06
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-chain non-coding RNA CASC2 derived from serum exosomes
  • Application method of long-chain non-coding RNA CASC2 derived from serum exosomes
  • Application method of long-chain non-coding RNA CASC2 derived from serum exosomes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Preparation of long-chain non-coding RNACASC2 kit for screening, early diagnosis or prognosis of glioma patients at high risk of glioma (50 responses)

[0024] 1. RNA stabilization solution 50ml

[0025] 2. Isopropyl alcohol 100ml

[0026] 3. Trichloromethane 100ml

[0027] 4. Trizol 50ml

[0028] 5. 10ml of enzyme-free water

[0029] 6.1μM random reverse transcription primer 50ul

[0030] 7.5× reverse transcription buffer 200ml

[0031] 8.10mM triphosphate base deoxynucleotide 100ul

[0032] 9.40U / μl RNase inhibitor 500ul

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

[0034] 11.PremixExTaq50ul

[0035] 12.10 μM ncRNAPRKAG2 real-time quantitative PCR specific primers 30ul

[0036] CASC2 forward primer 5′-TTGACCCTTCCAGCTTCC-3′,

[0037] CASC2 reverse primer 5'-CCATCCGCACATCACAAT-3',

[0038] 13.10μM 6snRNA specific primer 30ul

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

[0040] The forward primer was 5'-GGAACGCTTCACGAATTTG...

Embodiment 2

[0042] Validation of differential expression of LncRNACASC2 in glioma tissue and normal brain tissue

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

[0044]2. Extraction of RNA from tissue: Take an appropriate amount of the sample and add liquid nitrogen to the mortar after baking at 180°C for 6-8 hours to grind the sample, grind it to a powder, add 1ml Trizol mortar sample to the mortar, and grind it into a liquid Transfer to a tube, add 200 μl / ml of chloroform Trizol to the tube, shake by hand for 15-30 s, place on ice for 5 min, and centrifuge at 12,000g at 4°C for 15 min; carefully take the upper aqueous phase into a new tube, add pre-cooled isopropyl alcohol Mix well with 0.5ml / ml Trizol of propanol, let stand at -20°C for 20min, centrifuge at 12000g for 10min at 4°C; discard the supernatant, add 1-2ml of ethanol diluted with 75% DEPC water, ...

Embodiment 3

[0055] Specific and sensitive detection of serum Exosomes-derived LncRNACASC2 for glioma diagnosis

[0056] 1. Isolation of Exosomes in Serum

[0057] Collection of peripheral blood from individuals to be tested

[0058] 1.1 Separation of peripheral blood serum: use a blood coagulation tube to collect 5ml of blood from the individual to be tested. The blood was centrifuged at 1000 rpm for 6 min after blood collection, and the serum was drawn and stored in an EP tube at -80°C.

[0059] 1.2 Separation of Exosomes in serum: Add 100 μl of TotalExosomeIsolationReagent to 500 μl of each serum sample, mix by vortexing, and react at 4°C for 30 minutes. Centrifuge at 10,000g for 10 minutes at room temperature. There are Exosomes at the bottom of the EP tube, resuspend the Exosomes in 200 μl PBS. (Choose a commercial Exosomes isolation kit)

[0060] 2. Extraction and purification of RNA from Exosomes (select a commercialized Exosomes isolation and purification RNA kit)

[0061] 2....

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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 particularly relates to an application method of serum Exosomes-derived LncRNACASC2 in glioma diagnosis. Background technique [0002] Glioma accounts for 40.49% of intracranial tumors and can occur in any part of the central nervous system. Except for a few astrocytomas and pilocytic tumors that 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 boundary and perform 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 generally improved significantly. At present, the early diagnosis and standard treatment of glioma are still unsatisfactory. Therefore, it is necessary to find specific e...

Claims

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Application Information

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Patent Type & Authority Patents(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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