CN107937540A - Diagnosis of glioma marker circ17:47618350 | 47619164 and application - Google Patents
Diagnosis of glioma marker circ17:47618350 | 47619164 and application Download PDFInfo
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- 206010018338 Glioma Diseases 0.000 title claims abstract description 37
- 208000032612 Glial tumor Diseases 0.000 title claims abstract description 29
- 238000003745 diagnosis Methods 0.000 title claims abstract description 23
- 239000003550 marker Substances 0.000 title claims abstract description 8
- 230000029142 excretion Effects 0.000 claims abstract description 26
- 210000002966 serum Anatomy 0.000 claims abstract description 26
- 239000003153 chemical reaction reagent Substances 0.000 claims description 13
- 238000010839 reverse transcription Methods 0.000 claims description 7
- 238000003753 real-time PCR Methods 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 238000013211 curve analysis Methods 0.000 abstract 1
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- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000011282 treatment Methods 0.000 description 4
- 108091028075 Circular RNA Proteins 0.000 description 3
- 102100034343 Integrase Human genes 0.000 description 3
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000000034 method Methods 0.000 description 3
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- 241000282414 Homo sapiens Species 0.000 description 2
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- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 2
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- 238000004393 prognosis Methods 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 241000713869 Moloney murine leukemia virus Species 0.000 description 1
- 206010029260 Neuroblastoma Diseases 0.000 description 1
- 208000005890 Neuroma Diseases 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
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- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 1
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- 239000000090 biomarker Substances 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 201000007983 brain glioma Diseases 0.000 description 1
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
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- 238000013399 early diagnosis Methods 0.000 description 1
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- 210000004881 tumor cell Anatomy 0.000 description 1
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Abstract
The invention belongs to biological technical field, discloses diagnosis of glioma marker circ17:47618350 | 47619164 and application.In the present invention, circ17 in patients with gliomas serum excretion body is found first:47618350 | 47619164 expression is significantly raised compared to control group (p=0.0218), and ROC curve analysis then shows that it has higher diagnostic value (AUC=0.875, P to glioma<0.001, sensitivity and specificity are respectively 96.3% and 72.7%).Therefore by detecting circ17 in patients with gliomas serum excretion body:47618350 | 47619164 expression, can make patients with gliomas early stage, quick Noninvasive diagnosis.
Description
Technical field
The invention belongs to biological technical field, is related to a kind of serum circRNA markers for diagnosis of glioma
circ17:47618350 | 47619164 and detect the marker reagent be used to prepare the application of diagnosis of glioma preparation,
Also kit.
Background technology
Glioma originates from brain neuroblastoma spongiocyte, is common intracranial tumors, accounts for central nerve neuroma
40%~50%, wherein glioblastoma (World Health Organization classify III, IV grade) accounts for the 77.5% of all gliomas.Brain
Glioma has the characteristics that " three high and one low " that is, high incidence, postoperative high recurrent, high case fatality rate and cure rate are low, maximum
Biological characteristics is that tumour cell is in infiltrative growth, Chang Wufa complete resections of performing the operation.Although its treatment method is via single
Operative treatment develop into the operation of today and place the complex treatments such as chemotherapy, but in the past few decades in Patients with gliomas
Prognosis not be improved significantly.Therefore, the index for finding glioma early diagnosis, therapeutic evaluation and judging prognosis is extremely closed
It is important.
Circular rna (circular RNA, circRNA) is a kind of emerging endogenous non-coding RNA (non-coding
RNA, ncRNA), gradually received significant attention in recent years, be the hot spot of current RNA researchs.CircRNA passes through extron ring
Change or introne cyclisation connects 3 ' and 5 ' ends to form complete loop configuration, thus it is more more stable than linear rna, more
There is conservative, can largely exist with polytype in organism.More and more researchers have found that circRNA is in gene table
Play an important roll up in regulation and control, this enriches understanding of the people to endogenous non-coding RNA, prompts circRNA to be examined in clinic
Have broad prospects in terms of disconnected and treatment.Gradually find also to contain substantial amounts of circRNA in excretion body in recent years, may play
Important function.At present, since circRNA has the characteristics that rich, stability, high conservative and Space-time speciality, in tumour
Just play a greater and greater role in terms of diagnosis marker.
The content of the invention
The present invention first purpose be：A kind of serum excretion body circRNA markers for diagnosis of glioma are provided.
Main contents include：A kind of serum excretion body circRNA markers circ17 for diagnosis of glioma:
47618350 | 47619164, its sequence such as SEQ NO:Shown in 1.The circRNA is located on No. 17 chromosome of the mankind, total length
264bp。
Second object of the present invention is to provide detection circRNA markers expression quantity in serum excretion body
Application of the reagent in diagnosis of glioma preparation is prepared.
Third object of the present invention is to provide a kind of diagnosis of glioma kit, it can be measured in serum excretion body
Circ17:47618350 | 47619164 content.
The diagnosis of glioma kit, contains detection circ17:47618350 | the PCR primer of 47619164 contents.
It is preferred that the sequence of primer such as SEQ NO:Shown in 2 and 3.
The diagnosis of glioma kit, except circ17:47618350 | outside 47619164 primer, also contain from blood
Excretion body is extracted in clear, by extracting RNA in excretion body and carrying out all reagents of reverse transcription and quantitative fluorescent PCR.
Including：
(1) reagent needed for serum excretion body is extracted：Total Exosome Isolation Reagent(from
Serum), can be bought by Invitrogen companies, article No. 4478360；
(2) reagent needed for excretion body RNA is extracted：Trizol reagents, chloroform, isopropanol, 75% ethanol, without enzyme water；
(3) reagent needed for reverse transcription：Random primer (Random Primer), without enzyme water, 5 × RT Buffer, three phosphorus
Soda acid base deoxynucleotide, RNase inhibitor, MMLV reverse transcriptases；
(4) reagent needed for quantitative fluorescent PCR：circ17:47618350 | in 47619164 upstream and downstream primers, GAPDH internal references
Anti-sense primer, SYBR dyestuffs, without enzyme water.
The beneficial effects of the present invention are：
Circ17 in the serum excretion body of patients with gliomas is found first:47618350 | 47619164 compare normal serum
Excretion body control group significantly raises (p=0.0218).ROC curve is analysis shows that circ17:47618350 | 47619164 as life
Substance markers thing has glioma higher diagnostic value, and (AUC=0.875, sensitivity and specificity are respectively 96.3% He
72.7%).By application of the circular rna in diagnosis of glioma analysis, it can cause the more convenient standard of diagnosis of glioma
Really, conditions of patients is quick and precisely grasped for clinician, laid the foundation to improve clinical therapeutic efficacy, and it is potential to find to have
The new small molecule drug targets of therapeutic value provide help.
Brief description of the drawings
Fig. 1 analyzes circ17 for real-time fluorescence quantitative PCR:47618350 | 47619164 in glioma serum and normal blood
Differential expression in clear excretion body；
Fig. 2 is the circ17 that ROC curve analyzes serum excretion body source:47618350 | 47619164 pairs of glioma early stages
The specificity of diagnosis, sensitivity.
Embodiment
The present invention is intended to further illustrate below in conjunction with embodiment, is not intended to limit the present invention.
First, research object
The serum sample of 27 patients with gliomas is provided by Xiang Ya hospitals, and 11 normal serum samples carry out community for the same period
The healthy individuals of disorder in screening.Sample for research is collected for the same period, sample, dispense, preservation condition it is consistent.
2nd, research method
1. the extracting of RNA in glioma/normal serum excretion body
Take 200 μ l of serum to be centrifuged 30 minutes in 2000g room temperature, supernatant liquor is extracted to 600 new μ l with micropipettor
Centrifuge tube, adds 40 μ l excretion bodies extracts reagent (Total Exosome Isolation Reagent (from serum), goods
Number 4478360, Invitrogen companies) gently turning upside down shakes up, and 4 DEG C are incubated 45 minutes.10000g room temperature after incubation
Centrifugation 10 minutes, discards supernatant, and gained precipitation is the excretion body in serum.200 μ lTrizol (MRC are added in precipitation
Company) precipitation is resuspended, suspension is moved to new 1.5mltube manages, and mends Trizol to 1ml.15min is cracked on ice,
12000rpm centrifuges 10min, and supernatant moves to new tube pipes.Chlorination imitates 200 μ l in Tube, shakes 15-30s, ice with hand
Upper placement 5min, 4 DEG C, 12000rpm centrifugations 15min；Carefully take upper strata aqueous phase to enter in new tube, add the isopropanol of precooling
0.5ml is mixed, and stands be more than 20min on ice, 4 DEG C, 12000rpm centrifugations 10min；Supernatant is abandoned, it is water-reducible to add 75%DEPC
Ethanol 1ml is mixed, 4 DEG C, 7500rpm centrifugation 5min, abandons supernatant as far as possible, drying at room temperature 5-10min, adds the 10 μ l dissolvings of DEPC water
RNA.- 80 DEG C of preservations, refrigerator temperature is recorded by laboratory technician daily.
It is prepared by 2.cDNA
Reverse transcription reaction is carried out according to Reverse Transcriptase kit (Thermo companies) specification.Reaction cumulative volume is (total for 20 μ l
10 μ l, Random primer of RNA 1 μ l, no 1 μ l, 5 × Reaction Buffer of enzyme water 4 μ l, RI 1 μ l, RT 1 μ l and
10mM dNTP 2μl)。
| Component | Dosage/pipe |
| Random reverse transcriptase primer (1 μM) | 1μl |
| RNA samples | 10μl |
| Without enzyme water | To 12μl |
Reverse transcription first step condition：65 DEG C 5 minutes
Reverse transcription second step program：25 DEG C 5 minutes, 42 DEG C 60 minutes, 70 DEG C 5 minutes.
3. real-time fluorescence quantitative PCR
(primer sequence is shown in SEQ NO to the specific primer synthesized using Han Heng biotechnologies (Shanghai) Co., Ltd.：2 and 3)
Carry out real-time quantitative PCR：Reverse transcription product is first diluted 10 times, is mixed.20 μ l reaction systems are as follows：
Real-time fluorescence quantitative PCR response procedures：95 DEG C 3 minutes, 40 circulation, 95 DEG C 10 seconds, 60 DEG C 30 seconds.
4. data analysis：Using 2-ΔΔCTRepresent the circ17 of glioma serum excretion body:47618350 | 47619164 phases
For the expression multiple of normal serum excretion body, wherein △ CT=CTSample–CTInternal reference, Δ Δ CT=Δs CTGlioma–ΔCTNormally.This reality
The analysis method that data use relative quantification is tested, (primer sequence is shown in SEQ NO to GAPDH as reference gene：4 and 5), data profit
Analyzed with software GraphPad Prism and SPSS 17.0.
3rd, result of study
Circ17 in the serum excretion body of patients with gliomas:47618350 | 47619164 compare normal serum excretion body pair
According to group significantly up-regulation (p=0.0218).Specific data are as shown in Figure 1.ROC curve is analysis shows that circ17:47618350|
47619164 are used as biomarker to have higher diagnostic value (AUC=0.875, p to glioma<0.001, sensitivity and spy
The opposite sex is respectively 96.3% and 72.7%), and detailed results are shown in Fig. 2.
Sequence table
<110>Xiangya Hospital, Central-South China Univ.
<120>Diagnosis of glioma marker circ17:47618350 | 47619164 and application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 264
<212> RNA
<213>Homo sapiens (Homo sapiens)
<400> 1
cuucauaaac aagcagauau gcaagaagag aaaaaccgaa ucgaaagagu ccuuggcgcu 60
acucuuuugc cugaccugau ucaaaaaguc cucacguuug cacuuucaga agagguacgu 120
ccacaggaca cuguaucggu aauuggugga guagcuggag gcagcaagca ugguaggaaa 180
gcugcuugga aauucauaaa ggacaacugg gaagaacuuu auaaccgaua ccagggagga 240
uucuuaauau ccagacuaau aaag 264
<210> 2
<211> 19
<212> DNA
<213>Unknown (Unknown)
<400> 2
cagcaagcat ggtaggaaa 19
<210> 3
<211> 19
<212> DNA
<213>Unknown (Unknown)
<400> 3
tagcgccaag gactctttc 19
<210> 4
<211> 19
<212> DNA
<213>Unknown (Unknown)
<400> 4
atcatcagca atgcctcct 19
<210> 5
<211> 18
<212> DNA
<213>Unknown (Unknown)
<400> 5
catcacgcca cagtttcc 18
Claims (6)
- A kind of 1. diagnosis of glioma marker circ17:47618350 | 47619164, its sequence such as SEQ NO:Shown in 1.
- 2. the reagent of marker expression quantity in serum excretion body described in test right requirement 1 is preparing diagnosis of glioma preparation In application.
- 3. a kind of diagnosis of glioma kit, it is characterised in that the circ17 in serum excretion body can be measured:47618350| 47619164 content.
- 4. diagnosis of glioma kit according to claim 3, it is characterised in that contain detection circ17:47618350| The PCR primer of 47619164 contents.
- 5. diagnosis of glioma kit according to claim 4, it is characterised in that the sequence of primer such as SEQ NO:2 and 3 It is shown.
- 6. according to the diagnosis of glioma kit described in claim 3 or 4 or 5, it is characterised in that except circ17:47618350| Outside 47619164 primer, also contain and excretion body is extracted from serum, by extracting RNA in excretion body and carrying out reverse transcription and fluorescence All reagents of quantitative PCR.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108893451A (en) * | 2018-07-24 | 2018-11-27 | 郑州伊美诺生物技术有限公司 | The method of human parainfluenza I type virus large-scale culture |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108893451A (en) * | 2018-07-24 | 2018-11-27 | 郑州伊美诺生物技术有限公司 | The method of human parainfluenza I type virus large-scale culture |
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