CN103981271B - The application method of the long-chain non-coding RNA LINC00470 that serum Exosomes originates - Google Patents
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Abstract
The invention discloses long-chain non-coding RNA (the long no-coding RNA secreting body (Exosomes) outside a kind of serum and originate, LncRNA) application method of LINC00470, namely the LncRNA LINC00470 that originates of serum Exosomes is for the preparation of the prognosis preparation of the examination of glioma high risk population, early diagnosis or patients with gliomas.Extract RNA after confirming to be separated Exosomes in patients with gliomas serum by research, reverse transcription also carries out the downward of Real time PCR discovery LncRNA LINC00470 expression level.The LncRNA LINC00470 that the present invention utilizes serum Exosomes to originate can reach 85.7% to the specificity of glioma early diagnosis, and sensitivity reaches 94.1%.By detecting the expression level of LncRNA LINC00470 in patients with gliomas serum Exosomes, thus patients with gliomas is made in early days, Noninvasive diagnosis fast.
Description
Technical field
The invention belongs to oncomolecularbiology field, be specifically related to the application method of LncRNA LINC00470 in diagnosis of glioma that serum Exosomes originates.
Background technology
Glioma accounts for 60% of all primary nervous system tumours, can betide any position of central nervous system and any age, and excision is first-selected methods for the treatment of.Be that infiltrative growth is without clear and definite border because glioma, operation is difficult to real thoroughly excision, and Local Recurrence rate is high, although imaging diagnosis technology development in recent years, Micro-neurosurgical technology is applied, but the Diagnosis and Treat of glioma is significantly not progressive generally, and after making a definite diagnosis, the several years is dead mostly.At present, to glioma make a definite diagnosis in early days and Canonical management also unsatisfactory, therefore, the glioma early diagnosis marker finding non-invasive carries out examination to high risk population, to carry out early diagnosis, early treatment to patients with gliomas, improve survival, be the main task of neuroscience field research always.
Exosomes is a class diameter is the vesica of 30-150nm, containing 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 shuttle in body fluid, transport genetic material and protein between cell.Exosomes constantly can be discharged in surrounding environment and go by tumour 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 by being separated Exosomes in serum and directly utilize blood to extract molecule to carry out the impact of non-detection material in the blood of diagnosing tumor, make assay more true, accurately.These features make Exosomes contribute to early diagnosis and the Index for diagnosis of tumour.In the Exosomes in transitional cell bladder carcinoma source, PCA-3, TMPRSS2 two biomarkers have been found by research; In melanoma patients blood plasma Exosomes, tumor related marker thing CAV1 expression level obviously increases, and with this diagnosable melanoma, finds that the miRNA in Exosomes can be used as the mark of lung cancer in lung cancer model.The LncRNALINC00470 utilizing Exosomes to originate not only can better diagnose patients with gliomas to diagnose cerebral glioma, ahead of time treatment, and makes assay sensitiveer, special.More than demonstrate the great potential of LncRNA LINC00470 in glioma early diagnosis and examination that serum Exosomes originates.
Summary of the invention
The LncRNA LINC00470 (long intergenicnon-protein coding RNA470) that the object of the present invention is to provide a kind of serum Exosomes to originate is positioned karyomit(e) 18p11, the application method of GeneBank accession number: NR-023925, especially a kind of can be used in preparing glioma high risk population examination, early diagnosis or patients with gliomas the application method of prognosis preparation.Research confirms 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 the examination of glioma high risk population and early diagnosis and prognosis.
The application method of the long-chain non-coding RNA LINC00470 that serum Exosomes originates, the long-chain non-coding RNA LINC00470 that described serum Exosomes originates is for the preparation of the prognosis preparation of the examination of glioma high risk population, 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 carrying 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, comprise RNA stabilizing solution, Trizol reagent, trichloromethane, Virahol, without enzyme water; (2) take total serum IgE as template be cDNA agents useful for same by LncRNA LINC00470 reverse transcription, comprise RT Buffer, 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 primers, 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 primers:
Forward primer is 5 '-ATTGGAACGATACAGAGAAGATT-3 ',
Forward primer is 5 '-GGAACGCTTCACGAATT TG-3 '.
By quantitative fluorescence analysis, applicant finds that Lnc RNA LINC00470 property of there are differences in the cerebral tissue of 52 routine samples of human glioma and 12 routine normal peoples expresses (P=0.0449) (Fig. 1), and in samples of human glioma down-regulated expression, the detection of Exosomes for LncRNA LINC00470 is separated further in serum, find that the expression of LncRNA LINC00470 and the expression of normal people 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 risk of prediction patients with gliomas, examination high risk population, and patients with gliomas is made in early days, Noninvasive diagnosis fast.The present invention is to glioma early diagnosis specificity good (Fig. 4), and can reach 85.7%, sensitivity can reach 94.1%, only need extract at Exosomes the expression level that RNA can detect LncRNA LINC00470, simple to operate, good stability.The prediction of the early diagnosis that not only can be used for glioma but also the extensive examination that may be used for patients with gliomas and risk, for the early diagnosis of glioma and prediction provide strong technical support, has far-reaching clinical meaning and generalization.
Accompanying drawing explanation
Fig. 1 is that real-time fluorescence quantitative PCR analyzes 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 analyzes Exosomes source;
Fig. 3 is that Roc analyzes the LncRNA LINC00470 in Exosomes source to the specificity of glioma early diagnosis, 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 the examination of glioma high risk population, 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
6.1 μMs of random reverse transcriptase primer 50ul
7.5 × RT Buffer 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 μMs of 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 μMs of 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 filling RNA stabilizing solution, put to-80 DEG C of refrigerators for subsequent use.
2, the extracting of RNA in organizing: get appropriate sample and add liquid nitrogen grinding sample in the mortar after 180 DEG C of baking 6-8h, be ground to Powdered after in mortar, add 1ml Trizol mortar sample, grind to form liquid rear with moving to tube pipe, add chloroform 200 μ l/mlTrizol in Tube, 15-30s is shaken with hand, place 5min on ice, 4 DEG C of centrifugal 15min of 12000g; Carefully get upper strata aqueous phase to enter in new tube, the Virahol 0.5ml/mlTrizol adding precooling mixes, and-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 and mix, 4 DEG C of centrifugal 5min of 7500g, abandon supernatant, drying at room temperature 5-10min as far as possible, add DEPC water 10-20 μ l and dissolve RNA.The spectrophotometric measurement concentration of RNA and quality, OD260/280 ratio between 1.8-2.0 ,-80 DEG C of preservations.
3, LncRNA LINC00470 reverse transcription: the Reverse Transcriptase kit using Thermo company.The system of 20 μ L reverse transcription reactions is as follows:
| Composition | Dosage/pipe |
| Random reverse transcriptase primer (1 μM) | 1μl |
| RNA sample | 2μg |
| Without enzyme water | To12μl |
Reverse transcription the first step condition: 65 DEG C 5 minutes
| Composition | Dosage/pipe |
| 5 × RT Buffer | 4μl |
| Triphosphoric acid base deoxynucleotide (10mM) | 2μl |
| RNA enzyme inhibitors (40 Μ/μ l) | 1μl |
| MMLV reversed transcriptive enzyme (200 Μ/μ l) | 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 LINC00470 Auele Specific Primer of Shanghai Sheng Gong biotechnology company limited synthesis carries out real-time quantitative PCR: first reverse transcription product is diluted 5 times, mixing.20 μ L reaction systems are as follows:
| Composition | Dosage/pipe |
| SYBR Premix Ex Taq | 10μl |
| LINC00470 Auele Specific Primer (10 μMs) | 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 separate software GraphPad Prism analyzes.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 that serum Exosomes originates is used for the specificity of diagnosis of glioma, the detection of susceptibility
1, the separation of Exosomes in serum
Collect the peripheral blood of test individual
The separation of 1.1 peripheral blood serum: adopt the short solidifying pipe of blood, gather test individual blood 5ml.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: the Total ExosomeIsolation Reagent adding 100 μ l in each serum sample 500 μ l, vortex mixes, and 4 DEG C are reacted 30 minutes.Centrifugal 10 minutes of 10000g under room temperature.Exosomes is had, with the resuspended Exosomes of 200 μ lPBS bottom EP pipe.(selecting commercial Exosomes separating kit)
2, the extraction purification (selecting commercial Exosomes separation and purification RNA test kit) of RNA in Exosomes
The extraction of RNA in 2.1Exosomes: the 2X Denaturing Solution adding 200 μ l mixes, hatches 5 minutes on ice, then adds the acid-Phenol:Chloroform of 400 μ l, vortex 60 seconds.Centrifugal 10 minutes of 12000g under room temperature, containing 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 mixings.Added by mixed solution in Filter column, centrifugal 15 seconds of 10000g, outwells the mixed solution in collection tube.Add the miRNA Wash Solution1 of 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.Filter column is put into 10000g in collection tube centrifugal 1 minute.Filter column is put into the Elution Solution that new collection tube adds 35 μ l, under room temperature, centrifugal 30 seconds of 10000g, can obtain the RNA of purifying.
3, the method adopting 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 separate software GraphPad Prism analyzes.Analyze and find: compared with the expression of normal people LncRNA LINC00470, in 20 routine patients serum Exosomes, the differential expression of LncRNA LINC00470 obviously (P=0.0455), down-regulated expression in patients with gliomas, this result organize with embodiment 2 in detected result consistent, the detection of the LncRNALINC00470 expression level of being originated by serum exosome is described, can judges whether this patient suffers from glioma.Simultaneously, being analyzed by Roc finds to utilize LncRNA LINC00470 in serum Exosomes for area (the AreaUnder Roc Curve below the early stage diagnosis ROC curve of glioma, AUC) can reach 0.908, specificity can reach 85.7%, and sensitivity reaches 94.1%.Therefore the LncRNA LINC00470 that serum Exosomes originates can preferably for the early diagnosis of glioma.
Detection method 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 purposes of the reagent detecting the long-chain non-coding RNA LINC00470 in serum Exosomes source in the preparation of the prognosis for the preparation of the examination of glioma high risk population, early diagnosis or patients with gliomas, it is characterized in that, the sequence of this long-chain non-coding RNA LINC00470 is as shown in SEQ NO:1.
2. purposes according to claim 1, is characterized in that, described preparation comprises real-time fluorescence quantitative PCR detection reagent.
3. purposes according to claim 2, is characterized in that, described real-time fluorescence quantitative PCR detection reagent comprises the Auele Specific Primer carrying out real-time fluorescence quantitative PCR:
Lnc RNA LINC00470 forward primer: 5 '-AAACGGTCAAGAAGAAGTCA-3 ',
Lnc RNA LINC00470 reverse primer: 5 '-CTGTTGCTCAGCGTGTAGGA-3 '.
4. purposes 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, comprise RNA stabilizing solution, Trizol reagent, trichloromethane, Virahol, without enzyme water; (2) take total serum IgE as template be cDNA agents useful for same by LncRNA LINC00470 reverse transcription, comprise RT Buffer, triphosphoric acid base deoxynucleotide, RNA enzyme inhibitors, MMLV reversed transcriptive enzyme and reverse transcription 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 primers, 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 primers:
Forward primer is 5 '-ATTGGAACGATACAGAGAAGATT-3 ',
Forward primer is 5 '-GGAACGCTTCACGAATT TG-3 '.
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