CN103981271A - Application method of serum Exosomes derived long no-coding RNA LINC00470 - Google Patents
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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
The invention belongs to oncomolecularbiology field, the application method of the LncRNA LINC00470 that is specifically related to serum Exosomes source in diagnosis of glioma.
Background technology
Glioma accounts for 60% of all primary nervous system neoplasms, can betide any position and any age of central nervous system, and excision is first-selected methods for the treatment of.Because glioma is infiltrative growth without clear and definite border more, operation is difficult to real thoroughly excision, and Local Recurrence rate is high, although imaging diagnosis technology development in recent years, microneurosurgery technology is applied, but the diagnosis of glioma and treatment are significantly not progressive generally, several years death after making a definite diagnosis mostly.At present, also unsatisfactory to making a definite diagnosis in early days with specification treatment of glioma, therefore, the glioma early diagnosis marker of finding non-invasive carries out examination to high risk population, to patients with gliomas is carried out to early diagnosis, early treatment, improve survival, be the main task of neuroscience area research always.
Exosomes is the vesica that a class diameter is 30-150nm, contains the compositions such as RNA, lipid and protein.At blood, saliva, urine, all has existence in the multiple body fluid such as cerebrospinal fluid and breast milk, can in body fluid, shuttle, and transports genetic material and protein between cell.Tumour can constantly be discharged into Exosomes in surrounding environment and go in process of growth, Exosomes can preserve at 96 hours or-70 DEG C and preserve the longer time under 4 DEG C of conditions simultaneously, and carry out diagnosis of glioma and overcome and directly utilize blood to extract molecule to carry out the impact of non-detection material in the blood of diagnosing tumor by separate Exosomes in serum, make assay truer, accurately.These features make Exosomes contribute to early diagnosis and the prognosis judgement of tumour.In the Exosomes in transitional cell bladder carcinoma source, find PCA-3, two biomarkers of TMPRSS2 by research; In melanoma patients blood plasma Exosomes, tumor related marker thing CAV1 expression level obviously increases, and with this diagnosable melanoma, finds that in lung cancer model the miRNA in Exosomes can be used as the mark of lung cancer.Utilize the LncRNA LINC00470 in Exosomes source to diagnose cerebral glioma not only can better diagnose patients with gliomas, treatment ahead of time, and make assay sensitiveer, special.The great potential of the LncRNA LINC00470 that more than demonstrates serum Exosomes source in glioma early diagnosis and examination.
Summary of the invention
The object of the present invention is to provide the LncRNA LINC00470 (long intergenic non-protein coding RNA470) in a kind of serum Exosomes source to be positioned karyomit(e) 18p11, the application method of GeneBank accession number: NR-023925, especially a kind of application method of prognosis preparation of the examination, early diagnosis or the patients with gliomas that can be used in preparation glioma high risk population.Studies confirm that the LncRNA LINC00470 in serum Exosomes source can be used for the diagnosis of patients with gliomas, LncRNA LINC00470 down-regulated expression and the samples of human glioma the result in Exosomes source have consistence, and the susceptibility of assay is high, specificity is good.Therefore Exosomes can be used for glioma high risk population's examination and early diagnosis and prognosis.
The application method of the long-chain non-coding RNA LINC00470 in serum Exosomes source, the long-chain non-coding RNA LINC00470 in described serum Exosomes source is for the preparation of the prognosis preparation of glioma high risk population's examination, early diagnosis or patients with gliomas, and the sequence of this long-chain non-coding RNA LINC00470 is shown in SEQ NO:1.
The prognosis preparation of the described examination for the preparation of glioma high risk population, early diagnosis or patients with gliomas comprises real-time fluorescence quantitative PCR detection reagent.
Described real-time fluorescence quantitative PCR detection reagent comprises the Auele Specific Primer that carries out real-time fluorescence quantitative PCR:
Lnc RNA LINC00470 forward primer: 5 '-AAACGGTCAAGAAGAAGTCA-3 ',
Lnc RNA LINC00470 reverse primer:: 5 '-CTGTTGCTCAGCGTGTAGGA-3 '.
Described real-time fluorescence quantitative PCR detection reagent is test kit,
This test kit comprises: (1) is extracted total RNA agents useful for same from samples of human glioma, comprises RNA stabilizing solution, Trizol reagent, trichloromethane, Virahol, without enzyme water; (2) be cDNA agents useful for same taking total RNA as template by LncRNA LINC00470 reverse transcription, comprise reverse transcription damping fluid, triphosphoric acid base deoxynucleotide, RNA enzyme inhibitors, MMLV reversed transcriptive enzyme and LncRNA LINC00470 random primer used; (3), by cDNA real-time quantitative PCR agents useful for same, comprise LncRNA LINC00470 real-time fluorescence quantitative PCR Auele Specific Primer, U6snRNA internal reference specific PCR primer, real time fluorescent quantitative SYBR dyestuff, without enzyme water;
LncRNA LINC00470 real-time fluorescence quantitative PCR Auele Specific Primer:
LINC00470 forward primer 5'-AAACGGTCAAGAAGAAGTCA-3'
LINC00470 reverse primer 5'-CTGTTGCTCAGCGTGTAGGA-3'
U6snRNA internal reference specific PCR primer:
Forward primer is 5 '-ATTGGAACGATACAGAGAAGATT-3 ',
Forward primer is 5 '-GGAACGCTTCACGAATT TG-3 '.
Applicant by quantitative fluorescence analysis find Lnc RNA LINC00470 in 52 routine samples of human glioma and 12 routine normal peoples' cerebral tissue the property of there are differences express (P=0.0449) (Fig. 1), and down-regulated expression in samples of human glioma, further in serum, separate the detection of Exosomes for LncRNA LINC00470, find that the expression of LncRNA LINC00470 and normal people's expression exist obvious difference (P=0.0455) (Fig. 2), and consistent with the detected result in tissue.This method can detect the expression level of LncRNA LINC00470 in each crowd, thus the ill risk of prediction patients with gliomas, examination high risk population, and to patients with gliomas make in early days, Noninvasive diagnosis fast.The present invention, to glioma early diagnosis specificity good (Fig. 4), can reach 85.7%, and sensitivity can reach 94.1%, only need extract RNA and can detect at Exosomes the expression level of LncRNA LINC00470, simple to operate, good stability.Not only can be used for the early diagnosis of glioma but also can, for the extensive examination of patients with gliomas and the prediction of ill risk, for early diagnosis and the prediction of glioma provide strong technical support, there is far-reaching clinical meaning and generalization.
Brief description of the drawings
Fig. 1 is that real-time fluorescence quantitative PCR is analyzed the differential expression of LncRNA LINC00470 in samples of human glioma and normal cerebral tissue;
Fig. 2 is the differential expression of LncRNA LINC00470 in patients with gliomas and normal people that real-time fluorescence quantitative PCR is analyzed Exosomes source;
Fig. 3 is the specificity of LncRNA LINC00470 to glioma early diagnosis that Roc analyzes Exosomes source, susceptibility.
Embodiment
Be intended to further illustrate the present invention below in conjunction with embodiment, and unrestricted the present invention.
Embodiment 1 prepares the test kit (50 secondary response) of long-chain non-coding RNA LINC00470 for glioma high risk population's examination, early diagnosis or patients with gliomas prognosis
1.RNA stabilizing solution 50ml
2. Virahol 100ml
3. trichloromethane 100ml
4.Trizol reagent 50ml
5. without enzyme water 10ml
The random reverse transcriptase primer 50ul of 6.1 μ M
7.5 × reverse transcription damping fluid 200ml
8.10mM triphosphoric acid base deoxynucleotide 100ul
9.40U/ μ l RNA enzyme inhibitors 500ul
10.200U/ μ l MMLV reversed transcriptive enzyme 50ul
11.Premix?Ex?Taq?50ul
12.10 μ M LncRNA LINC00470 real-time fluorescence quantitative PCR Auele Specific Primer 30ul
LINC00470 forward primer 5'-AAACGGTCAAGAAGAAGTCA-3'
LINC00470 reverse primer 5'-CTGTTGCTCAGCGTGTAGGA-3'
13.10 μ M U6snRNA Auele Specific Primer 30ul
Forward primer is 5 '-ATTGGAACGATACAGAGAAGATT-3 ',
Forward primer is 5 '-GGAACGCTTCACGAATT TG-3 '.
Embodiment 2
The checking of the differential expression of LncRNA LINC00470 in samples of human glioma and normal cerebral tissue
1, collect samples of human glioma to be measured and put into the cryopreservation tube that fills RNA stabilizing solution, put to-80 DEG C of refrigerators for subsequent use.
2, the extracting of RNA in tissue: get appropriate sample and add liquid nitrogen grinding sample in the mortar after 180 DEG C of baking 6-8h, be ground to after Powdered and in mortar, add 1ml Trizol mortar sample, grind to form liquid rear use and move to tube pipe, add chloroform 200 μ l/mlTrizol in Tube, shake 15-30s with hand, place 5min on ice, 4 DEG C of centrifugal 15min of 12000g; Carefully get upper strata water and enter in new tube, add the Virahol 0.5ml/mlTrizol of precooling to mix ,-20 DEG C of refrigerators leave standstill 20min, 4 DEG C of centrifugal 10min of 12000g; Abandon supernatant, add the water-reducible ethanol 1-2ml of 75%DEPC to mix, 4 DEG C of centrifugal 5min of 7500g abandon supernatant as far as possible, and drying at room temperature 5-10min adds DEPC water 10-20 μ l to dissolve RNA.Concentration and the quality of spectrophotometric instrumentation RNA, OD260/280 ratio between 1.8-2.0 ,-80 DEG C of preservations.
3, LncRNA LINC00470 reverse transcription: the reverse transcription test kit that uses Thermo company.The system of 20 μ L reverse transcription reactions is as follows:
Composition | Dosage/pipe |
Random reverse transcriptase primer (1 μ M) | 1μl |
RNA sample | 2μg |
Without enzyme water | To12μl |
Reverse transcription the first step condition: 65 DEG C 5 minutes
Composition | Dosage/pipe |
5 × reverse transcription damping fluid | 4μl |
Triphosphoric acid base deoxynucleotide (10mM) | 2μl |
(40 Μ/μ l) for RNA enzyme inhibitors | 1μl |
(200 Μ/μ l) for MMLV reversed transcriptive enzyme | 1μl |
The product of the first step PCR | 12μg |
? | 20μl |
Reverse transcription second step program: 25 DEG C 5 minutes, 42 DEG C 60 minutes, 70 DEG C 5 minutes.
4, the synthetic LINC00470 Auele Specific Primer of Shanghai Sheng Gong biotechnology company limited carries out real-time quantitative PCR: first, by 5 times of reverse transcription product dilutions, mix.20 μ L reaction systems are as follows:
Composition | Dosage/pipe |
SYBR?Premix?Ex?Taq | 10μl |
LINC00470 Auele Specific Primer (10 μ M) | 0.5μl |
CDNA product | 1μl |
Without enzyme water | To20μl |
Real-time fluorescence quantitative PCR response procedures: 95 DEG C 3 minutes, 40 circulations, 95 DEG C 10 seconds, 60 DEG C 30 seconds.
5 ,-2
Δ Δ CTthe mensuration of index: this experimental data adopts the analytical procedure of relative quantification, and U6 is as reference gene, and data utilize software GraphPad Prism to analyze.Analyze and find, compared with the expression of LncRNA LINC00470 in normal cerebral tissue, in 52 routine Patients with gliomas, the expression of LncRNA LINC00470 is obviously lowered, and difference has significance (P=0.0449).
Embodiment 3
The LncRNA LINC00470 in serum Exosomes source is for the specificity of diagnosis of glioma, the detection of susceptibility
1, the separation of Exosomes in serum
Collect the peripheral blood of individuality to be measured
The separation of 1.1 peripheral blood serum: adopt the short solidifying pipe of blood, gather individual blood 5ml to be measured.The centrifugal 6min of 1000rpm after blood sampling, draws serum-80 DEG C of preservations in EP pipe.
The separation of Exosomes in 1.2 serum: add the Total Exosome Isolation Reagent of 100 μ l in each serum sample 500 μ l, vortex mixes, 4 DEG C are reacted 30 minutes.Centrifugal 10 minutes of 10000g under room temperature.There is Exosomes EP pipe bottom, with the resuspended Exosomes of 200 μ lPBS.(selecting commercial Exosomes separating kit)
2, the extraction purifying of RNA (selecting commercial Exosomes separation and purification RNA test kit) in Exosomes
The extraction of RNA in 2.1Exosomes: add the 2X Denaturing Solution of 200 μ l to mix, hatch 5 minutes on ice, then add the acid-Phenol:Chloroform of 400 μ l, vortex 60 seconds.Under room temperature, centrifugal 10 minutes of 12000g, contains RNA in supernatant.
The purifying of 2.2RNA: 300 μ l supernatant liquors are drawn in the EP pipe without enzyme, add the dehydrated alcohol of 375 μ l, both mix.Mixed solution is added in Filter column, and centrifugal 15 seconds of 10000g, outwells the mixed solution in collection tube.The miRNA Wash Solution1 that adds 700 μ l, under room temperature, the centrifugal 15s of 10000g, outwells the mixed solution in collection tube.Add the Wash Solution2/3 of 500 μ l, under room temperature, centrifugal 15 seconds of 10000g, repeats this step.Put in collection tube Filter column into 10000g centrifugal 1 minute.Filter column is put into the Elution Solution that new collection tube adds 35 μ l, and centrifugal 30 seconds of 10000g under room temperature, can obtain the RNA of purifying.
3, the method that adopts step 3 in embodiment 2 to carry out reverse transcription and real-time quantitative detects LncRNA LINC00470
4 ,-2
Δ Δ CTthe mensuration of index: this experimental data adopts the analytical procedure of relative quantification, and U6 is as reference gene, and data utilize software GraphPad Prism to analyze.Analyze and find: compared with the expression of normal people LncRNA LINC00470, obviously (P=0.0455) of the differential expression of LncRNA LINC00470 in 20 routine patients serum Exosomes, down-regulated expression in patients with gliomas, this result is consistent with the detected result in embodiment 2 tissues, the detection of the LncRNALINC00470 expression level of originating by serum exosome is described, can judges whether this patient suffers from glioma.Simultaneously, analyze and find to utilize area (the Area Under Roc Curve for the early stage diagnosis ROC curve below of glioma of LncRNA LINC00470 in serum Exosomes by Roc, AUC) can reach 0.908, specificity can reach 85.7%, and sensitivity reaches 94.1%.Therefore the LncRNA LINC00470 in serum Exosomes source can be preferably for the early diagnosis of glioma.
Detection method of the present invention only need 500 μ l serum just separable enough Exosomes of going out detect for the expression level of LncRNA LINC00470, illustrate that the method has operability preferably.
Claims (4)
1. the application method of the long-chain non-coding RNA LINC00470 in serum Exosomes source, it is characterized in that, the long-chain non-coding RNA LINC00470 in described serum Exosomes source is for the preparation of the prognosis preparation of glioma high risk population's examination, early diagnosis or patients with gliomas, and the sequence of this long-chain non-coding RNA LINC00470 is shown in SEQ NO:1.
2. the application method of long-chain non-coding RNA LINC00470 according to claim 1, it is characterized in that, the prognosis preparation of the described examination for the preparation of glioma high risk population, early diagnosis or patients with gliomas comprises real-time fluorescence quantitative PCR detection reagent.
3. the application method of long-chain non-coding RNA LINC00470 according to claim 2, is characterized in that, it is characterized in that, described real-time fluorescence quantitative PCR detection reagent comprises the Auele Specific Primer that carries out real-time fluorescence quantitative PCR:
Lnc RNA LINC00470 forward primer: 5 '-AAACGGTCAAGAAGAAGTCA-3 ',
Lnc RNA LINC00470 reverse primer:: 5 '-CTGTTGCTCAGCGTGTAGGA-3 '.
4. the application method of long-chain non-coding RNA LINC00470 according to claim 3, is characterized in that, described real-time fluorescence quantitative PCR detection reagent is test kit,
This test kit comprises: (1) is extracted total RNA agents useful for same from samples of human glioma, comprises RNA stabilizing solution, Trizol reagent, trichloromethane, Virahol, without enzyme water; (2) be cDNA agents useful for same taking total RNA as template by LncRNA LINC00470 reverse transcription, comprise reverse transcription damping fluid, triphosphoric acid base deoxynucleotide, RNA enzyme inhibitors, MMLV reversed transcriptive enzyme and LncRNA LINC00470 random primer used; (3), by cDNA real-time quantitative PCR agents useful for same, comprise LncRNALINC00470 real-time fluorescence quantitative PCR Auele Specific Primer, U6snRNA internal reference specific PCR primer, real time fluorescent quantitative SYBR dyestuff, without enzyme water;
LncRNA LINC00470 real-time fluorescence quantitative PCR Auele Specific Primer:
LINC00470 forward primer 5'-AAACGGTCAAGAAGAAGTCA-3'
LINC00470 reverse primer 5'-CTGTTGCTCAGCGTGTAGGA-3'
U6snRNA internal reference specific PCR primer:
Forward primer is 5 '-ATTGGAACGATACAGAGAAGATT-3 ',
Forward primer is 5 '-GGAACGCTTCACGAATT TG-3 '.
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CN107619868A (en) * | 2017-10-27 | 2018-01-23 | 中南大学湘雅医院 | Application of Glioma Prognostic Marker Circ3:129880309|129880559 |
CN107828779A (en) * | 2017-10-25 | 2018-03-23 | 武汉大学 | Prostatic cancer specific excretion body, lncRNA and its preparation method and application |
CN110358834A (en) * | 2019-07-12 | 2019-10-22 | 深圳大学 | The application of lncRNA a kind of and kit and drug |
WO2020216385A3 (en) * | 2019-04-22 | 2020-12-17 | 中山大学孙逸仙纪念医院 | Application of serum exosome has_circ_0004771 in preparing reagent for alcohol dependence syndrome diagnosis |
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CN107828779A (en) * | 2017-10-25 | 2018-03-23 | 武汉大学 | Prostatic cancer specific excretion body, lncRNA and its preparation method and application |
CN107828779B (en) * | 2017-10-25 | 2020-04-21 | 武汉大学 | Prostate cancer specific exosome, lncRNA, preparation method and application thereof |
CN107604074A (en) * | 2017-10-27 | 2018-01-19 | 中南大学湘雅医院 | Glioma prognostic marker circ15:101235082|101235577 and application thereof |
CN107619868A (en) * | 2017-10-27 | 2018-01-23 | 中南大学湘雅医院 | Application of Glioma Prognostic Marker Circ3:129880309|129880559 |
CN107604074B (en) * | 2017-10-27 | 2020-06-30 | 中南大学湘雅医院 | Glioma prognostic marker circ15:101235082|101235577 and application thereof |
WO2020216385A3 (en) * | 2019-04-22 | 2020-12-17 | 中山大学孙逸仙纪念医院 | Application of serum exosome has_circ_0004771 in preparing reagent for alcohol dependence syndrome diagnosis |
CN110358834A (en) * | 2019-07-12 | 2019-10-22 | 深圳大学 | The application of lncRNA a kind of and kit and drug |
CN113533728A (en) * | 2021-06-03 | 2021-10-22 | 中国科学院生物物理研究所 | Biomarker of brain glioma and application thereof |
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