CN109266751A - Biomarker combination for nasopharyngeal carcinoma diagnosis and application - Google Patents
Biomarker combination for nasopharyngeal carcinoma diagnosis and application Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
The invention discloses a biomarker combination for nasopharyngeal carcinoma diagnosis and application thereof, and relates to the technical field of tumor diagnosis. The biomarker combination disclosed by the invention comprises the following microRNAs: has-miR-30b-3p, has-miR-1202, has-miR-1321, has-miR-3612, has-miR-4478 and has-miR-4730. The biomarker combination is used for diagnosing nasopharyngeal carcinoma and has good sensitivity and specificity.
Description
Technical field
The present invention relates to diagnosing tumor technical fields, in particular to a kind of biological marker for nasopharyngeal carcinoma diagnosis
Object combination and application.
Background technique
Nasopharyngeal carcinoma (nasopharyngeal carcinoma, NPC) is a kind of cancer as caused by nasopharyngeal epithelial cell, in world's most area phase
When rare, disease incidence is lower than every 100,000 people 1 year.However, it is fairly common in SOUTHERN CHINA, Southeast Asia and north African.It unites from population
From the point of view of counting the trend learned, a possibility that male suffers from the disease is three times of women, and the onset peak age is between 50-60 years old.Nose
Pharynx cancer and Epstein-Barr viral (EBV) infection, genetic predisposition smoke and drink and environmental factor is also highly relevant.
For the early stage patient of nasopharyngeal carcinoma, radiotherapy is usually a kind of effective treatment method, and the prognosis of patients with terminal is usually poor.Cause
This, early detection is conducive to improve the healing of NPC patient.
With the rapid development of nasopharyngeal carcinoma Molecular pathogenesis, there are many biomarkers relevant to diagnosis and prognosis
It is reported, including EBV DNA, circulation microRNAs (miRNAs), the miRNAs of EBV virus, certain cell factors and several
Methylated genes.However, the detection of these molecules depends on invasive sample, the fixed paraffin packet of such as fresh or formalin
(FFPE) tissue, blood plasma or serum are buried, patient's discomfort is easily caused.Previous research reports that the specific molecular in body fluid can
It can be closely related with surrounding tissue function.Saliva is a kind of body fluid being easily obtained of pharynx nasalis adjacent tissue, is that one kind has very much
The Noninvasive sample for being used to detect nasopharyngeal carcinoma biomarker of future.Since the substance of blood and tissue in the circulating cycle is handed over
It changes and a large amount of blood supplies of the blood to salivary gland, the molecule in tissue and blood plasma may exist in saliva.Therefore, saliva
In molecule can be used for detecting Whole Body disease, the especially disease of salivary gland tissue around.
MicroRNA (miRNA) is the non-coding RNA that raw, length is about 18-25nt in one kind, is had in the cell
A variety of important adjustment effects, generally believing has close ties with human diseases.Evidence suggests miRNA is in tumour
The important function similar to oncogene or tumor suppressor gene is played in generation, can be applied in the diagnosing and treating monitoring of tumour.
MiRNA is very stable in vitro, and research thinks that miRNA is mainly released into blood by cell secretion in circulation, with excretion body
(exosome) or the form of protein complexes exists, and the digestion and exacting terms of RNA enzyme can be resisted, thus thin
Biological information is transmitted between born of the same parents.Existing document report serum or specific miRNA in blood plasma or miRNA combination are used for various cancers
In the detection of disease.But miRNA molecule is carried out slowly for the detection research of cancer markers in saliva, and does not report saliva
Middle miRNA molecule is used for the detection of nasopharyngeal carcinoma.
In consideration of it, the present invention is specifically proposed.
Summary of the invention
The purpose of the present invention is to provide a kind of biomarker combinations for nasopharyngeal carcinoma diagnosis, the biomarker groups
The diagnosis that can be used for nasopharyngeal carcinoma is closed, there are preferable sensitivity and specificity.
Another object of the present invention is to provide the reagent for detecting above-mentioned biomarker combinations, which can detecte sample
Biomarker in this.
Another object of the present invention is to provide the applications of above-mentioned reagent.
Another object of the present invention is to provide a kind of nasopharyngeal carcinoma diagnosis kit and nasopharyngeal carcinoma diagnosis chips.
The present invention is implemented as follows:
On the one hand, the present invention provides a kind of biomarker combinations for nasopharyngeal carcinoma diagnosis comprising as follows
MicroRNA molecule (miRNA molecule):
Has-miR-30b-3p, has-miR-1202, has-miR-1321, has-miR-3612, has-miR-4478 and
has-miR-4730；
Wherein, the nucleotide sequence of the has-miR-30b-3p is as shown in SEQ ID NO.1；The has-miR-1202
Nucleotide sequence as shown in SEQ ID NO.4；The nucleotide sequence of the has-miR-1321 is as shown in SEQ ID NO.7；
The nucleotide sequence of the has-miR-3612 is as shown in SEQ ID NO.10；The nucleotide sequence of the has-miR-4478
As shown in SEQ ID NO.13；The nucleotide sequence of the has-miR-4730 is as shown in SEQ ID NO.16.
Research discovery of the invention is several with differential expression in the saliva of Nasopharyngeal Carcinoma Patients and normal control
MicroRNA molecule, i.e. has-miR-30b-3p, has-miR-1202, has-miR-1321, has-miR-3612, has-
MiR-4478 and has-miR-4730.And by experimental verification, using these types of microRNA molecule as the life of nasopharyngeal carcinoma diagnosis
Object marker, not only each individual microRNA molecule shows good specificity and sensitivity (Fig. 2-Fig. 7), and will
A little microRNA molecule combinations (or being interpreted as combining) equally also show preferably specificity and sensitivity (Fig. 8).It is based on
This, above-mentioned microRNA molecule, i.e. has-miR-30b-3p, has-miR-1202, has-miR-1321, has-miR-
3612, any one or the combination of several of them in has-miR-4478 and has-miR-4730 or joint can be used as nasopharynx
The biomarker of cancer diagnosis, and the reagent such as primer or probe that detect these microRNA molecules may be incorporated for preparing nose
Pharynx cancer diagnostic kit or chip.
On the other hand, the present invention provides a kind of for detecting the reagent of above-mentioned biomarker combinations.
Further, in some embodiments of the present invention, the reagent is with mutual with the microRNA molecule
The probe of the sequence of benefit；
Alternatively, the reagent is the primer for detecting microRNA molecule.
Further, in some embodiments of the present invention, the primer includes reverse transcription primer, quantitative PCR leading
Primer after object and quantitative PCR；
Preferably, the reverse transcription primer sequence of has-miR-30b-3p is detected as shown in SEQ ID NO.2, before quantitative PCR
Primer sequence is as shown in SEQ ID NO.3, and primer sequence is as shown in SEQ ID NO.21 after quantitative PCR；
Preferably, the reverse transcription primer sequence of has-miR-1202 is detected as shown in SEQ ID NO.5, quantitative PCR leading
Object sequence is as shown in SEQ ID NO.6, and primer sequence is as shown in SEQ ID NO.21 after quantitative PCR；
Preferably, the reverse transcription primer sequence of has-miR-1321 is detected as shown in SEQ ID NO.8, quantitative PCR leading
Object sequence is as shown in SEQ ID NO.9, and primer sequence is as shown in SEQ ID NO.21 after quantitative PCR；
Preferably, the reverse transcription primer sequence of has-miR-3612 is detected as shown in SEQ ID NO.11, before quantitative PCR
Primer sequence is as shown in SEQ ID NO.12, and primer sequence is as shown in SEQ ID NO.21 after quantitative PCR；
Preferably, the reverse transcription primer sequence of has-miR-4478 is detected as shown in SEQ ID NO.14, before quantitative PCR
Primer sequence is as shown in SEQ ID NO.15, and primer sequence is as shown in SEQ ID NO.21 after quantitative PCR；
Preferably, the reverse transcription primer sequence of has-miR-4730 is detected as shown in SEQ ID NO.17, before quantitative PCR
Primer sequence is as shown in SEQ ID NO.18, and primer sequence is as shown in SEQ ID NO.21 after quantitative PCR.
On the other hand, the present invention provides mentioned reagents in preparing nasopharyngeal carcinoma diagnosis kit or nasopharyngeal carcinoma diagnosis chip
Application.
Further, in some embodiments of the present invention, the detection sample of the nasopharyngeal carcinoma diagnosis kit is saliva
Liquid.
On the other hand, the present invention provides a kind of nasopharyngeal carcinoma diagnosis kits comprising above-mentioned reagent.
Further, in some embodiments of the present invention, the kit further include one of following ingredient or
It is a variety of:
Reverse transcriptase, buffer, dNTPs, Mgcl2、ddH2O, fluorescent dye, Taq enzyme and standard items and control.
On the other hand, the present invention provides a kind of nasopharyngeal carcinoma diagnosis chip, the above-mentioned biology of detection is fixed on the chip
The miRNA probe of marker.
Further, in some embodiments of the present invention, the miRNA probe has and the microRNA molecule
Complementary sequence.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 is the expression map of the miRNA of 12 differential expressions in embodiment 1.
Fig. 2 is the ROC curve of the has-miR-30b-3p miRNA molecule in embodiment 2.
Fig. 3 is the ROC curve of the has-miR-1202miRNA molecule in embodiment 2.
Fig. 4 is the ROC curve of the has-miR-1321miRNA molecule in embodiment 2.
Fig. 5 is the ROC curve of the has-miR-3612miRNA molecule in embodiment 2.
Fig. 6 is the ROC curve of the has-miR-4478miRNA molecule in embodiment 2.
Fig. 7 is the ROC curve of the has-miR-4730miRNA molecule in embodiment 2.
Fig. 8 be embodiment 2 in 6 miRNAs (has-miR-30b-3p, has-miR-1202, has-miR-1321,
Has-miR-3612, has-miR-4478 and has-miR-4730) united ROC curve.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Feature and performance of the invention are described in further detail with reference to embodiments.
Embodiment 1
The cDNA microarray experiment of differential expression miRNA in saliva
1, after obtaining patient's informed consent, 10 is collected from Jiangsu Prov. Tumour Hospital and is diagnosed as Nasopharyngeal Carcinoma Patients for the first time
The saliva sample of saliva sample and 10 Healthy Peoples as normal control.Saliva sample time is subject to 1 hour after meal, leaves and takes
2ml is stored in -20 DEG C of refrigerators.
2, using miRNeasy Serum/Plasma Kit kit (#217184, Qiagen, Hilden, Germany)
The RNA in Nasopharyngeal Carcinoma Patients and healthy human saliva is extracted, operating procedure is carried out according to handbook, and has been made part and modified.
(1) the QIAzol lysate of the 2ml saliva sample collected and 4ml is thoroughly mixed, is placed at room temperature for 5 minutes；
(2) chloroform of 1.1ml is added, thoroughly mixes, is placed at room temperature for 5 minutes；
(3) under conditions of 12000g, 4 degree are centrifuged 15 minutes；
(4) honest and upright and thrifty 2ml in suction, is added 100% ethyl alcohol (3ml) of 1.5 times of volumes, and tenderness mixes；
(5) mixed liquor being added in centrifugal column, 700 μ l is added every time, room temperature is centrifuged 15 seconds under conditions of 8000g, and
It goes to separate out waste liquid；
(6) mixed liquor, has been centrifuged by the operation for repeating (5) repeatedly several times；
(7) RWT of 700 μ l is added in centrifugal column, room temperature is centrifuged 15 seconds under conditions of 8000g, and goes to separate out useless
Liquid；
(8) RPE of 500 μ l is added in centrifugal column, room temperature is centrifuged 15 seconds under conditions of 8000g, and goes to separate out useless
Liquid；
(9) 80% ethyl alcohol (ready-to-use) of 500 μ l is added in centrifugal column, room temperature is centrifuged under conditions of 8000g
2 minutes, and go to separate out waste liquid；
(10) centrifugal column is put into new collecting pipe, is uncapped, centrifuge room temperature centrifugation 5 minutes at full speed, for dry from
Organic solvent on stem film；
(11) centrifugal column is put into new 1.5ml collecting pipe, film center, centrifuge is added in the RNase-free water of 27 μ l
Room temperature centrifugation 2 minutes at full speed, collect and have dissolved the 25 μ l of solution (having 2 μ l dead volumes) of RNA, be put into -20 DEG C of refrigerators collections to
With.
3, in discovery experiment, mankind's UnitagTM miRNA chip of expression spectrum detection platform can be optionally selected, it is right
The miRNA of differential expression is analyzed in sample.The chip contains 2017 miRNA of the mankind from Sanger database
v.19.0。
(1) will extract obtained 20 μ l RNA samples be added in the hybridization solution of 80 μ l (5 × SSC, 0.2%SDS,
The dedicated fluorescence report probe of 100nM, RNase-free water are mended to 80 μ l), and piping and druming mixes；
(2) 100 μ l of aforesaid liquid is all added on UnitagTM miRNA chip, covers 4 hole Agilent silication lid glass
Piece (Agilent, G2534-60011), and be put into chip hybridization box；
(3) hybridizing box is put into 15rpm in hybrid heater (Agilent, 2545A), 44 DEG C of hybridized overnights, usual hybridization time
At 16 hours；
(4) after hybridizing, chip and cover plate are first opened in 37 DEG C of 400ml of 5 × SSC solution, then in 400ml
Wash 2 times in 37 DEG C of 5 × SSC solution containing 0.02%SDS, 3 minutes every time, later in 27 DEG C of 400ml of 0.2 × SSC
It is washed in solution 2 times, 3 minutes every time, four washings, were all to rinse liquid with 100rpm on shaking table altogether；
(5) chip is taken out from washing lotion, is dried on slide centrifuge；
(6) chip is scanned with LuxScan 10K twin-channel laser scanner (CapitalBio company), and data mention
It takes 3.0 image analysis of LuxScan (CapitalBio company) to analyze chip image, picture signal is converted into number
Word signal.
4, after microarray data background correction signal, Quantile normalization is carried out to data in R language, is used
19.0 software of SPSS carries out pairing t- and examines acquisition p < 0.01, and difference is greater than 2 times of difference expression gene, and uses Cluster
3.0 carry out clustering.
5, Differential expression analysis is carried out using saliva of the miRNA chip to 10 pairs of Nasopharyngeal Carcinoma Patients and normal control, sieved altogether
The miRNA for selecting 12 differential expressions is has-miR-30b-3p, has-miR-575, has-miR-650, has- respectively
MiR-937-5p, has-miR-1202, has-miR-1203, has-miR-1321, has-miR-3612, has-miR-
3714, has-miR-4259, has-miR-4478 and has-miR-4730, and compared with normal control, in nasopharyngeal carcinoma saliva
Above-mentioned miRNA lowers expression (Fig. 1).
Embodiment 2
The qRT-PCR experiment of miRNA in saliva
1, according to miRNA chip results, 6 miRNA qRT-PCR sides of the signal value greater than 500 in chip results are selected
Method is further tested.Selected has-miR-30b-3p, has-miR-1202, has-miR-1321, has-miR-
The primer of 3612, has-miR-4478 and has-miR-4730 see the table below 1.It is single to the saliva of Nasopharyngeal Carcinoma Patients and normal control
Individual carries out the qRT-PCR detection of miRNA, implements strict quality control in entire research, each sample continuously detects three times.
Table 1
2, after obtaining patient's informed consent, 80 salivas for being diagnosed as Nasopharyngeal Carcinoma Patients are collected from Jiangsu Prov. Tumour Hospital
The saliva sample of sample and 80 Healthy Peoples as normal control.
3, using miRNeasy Serum/Plasma Kit kit (#217184, Qiagen, Hilden, Germany)
The RNA in Nasopharyngeal Carcinoma Patients and healthy human saliva is extracted, operating procedure is carried out according to handbook, and has been made part and modified.
(1) the QIAzol lysate of the 1ml saliva sample collected and 2ml is thoroughly mixed, is placed at room temperature for 5 minutes；
(2) chloroform of 0.54ml is added, thoroughly mixes, is placed at room temperature for 5 minutes；
(3) under conditions of 12000g, 4 degree are centrifuged 15 minutes；
(4) honest and upright and thrifty 1ml in suction, is added 100% ethyl alcohol (1.5ml) of 1.5 times of volumes, and tenderness mixes；
(5) mixed liquor being added in centrifugal column, 700 μ l is added every time, room temperature is centrifuged 15 seconds under conditions of 8000g, and
It goes to separate out waste liquid；
(6) mixed liquor, has been centrifuged by the operation for repeating (5) repeatedly several times；
(7) RWT of 700 μ l is added in centrifugal column, room temperature is centrifuged 15 seconds under conditions of 8000g, and goes to separate out useless
Liquid；
(8) RPE of 500 μ l is added in centrifugal column, room temperature is centrifuged 15 seconds under conditions of 8000g, and goes to separate out useless
Liquid；
(9) 80% ethyl alcohol (ready-to-use) of 500 μ l is added in centrifugal column, room temperature is centrifuged under conditions of 8000g
2 minutes, and go to separate out waste liquid；
(10) centrifugal column is put into new collecting pipe, is uncapped, centrifuge room temperature centrifugation 5 minutes at full speed, for dry from
Organic solvent on stem film；
(11) centrifugal column is put into new 1.5ml collecting pipe, film center, centrifuge is added in the RNase-free water of 20 μ l
Room temperature centrifugation 2 minutes at full speed, collect and have dissolved the 18 μ l of solution (having 2 μ l dead volumes) of RNA, be put into -20 DEG C of refrigerators collections to
With.
4, using PrimeScriptTM RT Master Mix (Cat#RR037A, TAKARA) and reverse transcription primer by behaviour
Make handbook and carries out reverse transcription reaction.
(1) configuration of reaction solution is carried out according to the following table, until 20 μ l of total volume；
| Ingredient | Final concentration | Volume |
| 5×Reverse Transcription Buffer | 1× | 4μl |
| Has U6/microRNA RT Primer(2uM) | 1pmol | 1μl |
| RNA Template | ---- | 5μl |
| PrimeScript RT Enzyme Mix 1 | ---- | 1μl |
| DEPC ddH2O | To 20 μ l |
(2) after the reaction solution of above-mentioned preparation being mixed gently of short duration centrifugation with pipettor, 37 DEG C are reacted 15 minutes, and 85 DEG C anti-
It answers 5 seconds, completion is placed on ice, and the dd H2O dilution of 20 μ l of addition is stand-by.
5, qPCR reaction, instrument are carried out using 480SYBR Green I Master (Cat#04707516001, Roche)
It uses Roche LightCycler 480 (Roche company).
(1) configuration of reaction solution is carried out according to the following table, until 20 μ l of total volume；
| Ingredient | Final concentration | Volume |
| 2×Real-time PCR Master Mix | 1× | 10μl |
| Has U6/microRNA F Primer(10uM) | 0.5μM | 1μl |
| Has U6/microRNA R Primer(10uM) | 0.5μM | 1μl |
| cDNA Template | ---- | 1μl |
| dd H2O | To 20 μ l |
(2) by after the of short duration centrifugation of the reaction solution of above-mentioned preparation, PCR reaction is carried out, is reacted by following programs；
6, PCR amplification result is indicated with Ct value, i.e. recurring number when fluorescence signal reaches set threshold value in PCR reaction.
MiRNA expression quantity in saliva sample can indicate with equation 2- △ Ct, wherein △ Ct=CtmiRNA-CtU6。
7, data processing is carried out using 19.0 software of SPSS, Mann-Whitney U is examined for comparing nasopharyngeal cancer patient
The differential expression of saliva miRNA in group and Normal group, p < 0.01 thinks statistically significant.With 15.8 software of MedCalc
ROC curve analysis is carried out, and calculates sensitivity and specificity.
8, data analysis result is shown, has-miR-30b-3p, has-miR-1202, has-miR-1321, has-miR-
The differential expression of 3612, has-miR-4478 and has-miR-4730 has conspicuousness (p < 0.01), and the ROC of 6 miRNA
Area under the curve (AUC) is as follows respectively:
Has-miR-30b-3p, 0.883 (95% confidence interval, 0.755-0.958), sensitivity 81.82%, specificity
88.00%；
Has-miR-1202,0.865 (95% confidence interval, 0.733-0.947), sensitivity 81.82%, specificity
88.00%；
Has-miR-1321,0.831 (95% confidence interval, 0.693-0.924), sensitivity 77.27%, specificity
80.00%；
Has-miR-3612,0.855 (95% confidence interval, 0.722-0.941), sensitivity 77.27%, specificity
80.00%；
Has-miR-4478,0.823 (95% confidence interval, 0.684-0.919), sensitivity 72.73%, specificity
80.00%；
Has-miR-4730,0.852 (95% confidence interval, 0.718-0.938), sensitivity 72.73%, specificity
92.00% (Fig. 2-7).
The AUC value that this 6 miRNAs join together can reach 0.941, and (95% confidence interval, 0.831-0.989) is sensitive
Degree and specificity are respectively 95.45% and 80.00% (Fig. 8), are better than single miRNA.
These results indicate that has-miR-30b-3p, has-miR-1202, has-miR-1321, has-miR-3612,
Has-miR-4478 and has-miR-4730 joins together to detect nasopharyngeal carcinoma saliva, has to the diagnosis of nasopharyngeal carcinoma non-
Often high sensitivity and specificity.
Embodiment 3: human nasopharyngeal carcinoma diagnosis qPCR kit
Above-described embodiment shows has-miR-30b-3p, has-miR-1202, has-miR-1321, has-miR-
3612, has-miR-4478 and has-miR-4730 to nasopharyngeal carcinoma saliva detect diagnostic value with higher (it is highly sensitive and
Specificity), therefore it can be based on has-miR-30b-3p, has-miR-1202, has-miR-1321, has-miR-3612,
Has-miR-4478 and has-miR-4730 producer's nasopharyngeal carcinoma diagnosis kit.The kit includes has-miR-30b-
3p primer, probe；Has-miR-1202 primer, probe；Has-miR-1321 primer, probe；Has-miR-3612 primer is visited
Needle；Has-miR-4478 primer, probe；Has-miR-4730 primer, probe.Primer specifically includes reverse transcription primer, quantifies
Primer after primer and quantitative PCR before PCR, as shown in table 1.
It should also include conventional enzyme and reagent needed for corresponding PCR reaction in certain kit, such as reverse transcriptase, buffering
Liquid, dNTPs, MgCl2、ddH2O, fluorescent dye, Taq enzyme and standard items and control.The design of primer is this field routine techniques hand
Section, may be designed as other sequences.The value of this kit is only to need saliva sample, without organizing, blood sample,
It may be used for the diagnosis of nasopharyngeal carcinoma.
Embodiment 4: nasopharyngeal carcinoma diagnosis chip agent box
Similarly, the miRNA detection based on chip can also be used for the diagnosis of nasopharyngeal carcinoma saliva.By the core for making a small amount of probe
Piece can be detected has-miR-30b-3p, has-miR-1202, has-miR-1321, has-miR-3612, has-miR-
The expression of 4478 and has-miR-4730, to carry out the diagnosis of nasopharyngeal carcinoma to saliva sample.
1, the overall length maturation miRNA of the miRNA probe sequence of all designs detection corresponding with them is complete in miRNA chip
It is complementary.The miRNA of detection includes has-miR-30b-3p, has-miR-1202, has-miR-1321, has-miR-3612,
Has-miR-4478 and has-miR-4730.In addition, devise 2 short oligonucleotides, they and known miRNA sequence
It is not homologous.In order to arrive aldehyde group modified surface of glass slide for probe-immobilized, 5 ' ends of these probe sequences add 15 bases
PolyA, and have that C6's is 5 '-amido modified.Oligonucleotide probe is synthesized in TAKARA, is dissolved in EasyArrayTM spotting solution
In, concentration is 20 μM.Using SmartArrayTM point sample instrument (Bo Ao biotech firm), each probe is repeated point sample 3 times.
2, the extraction of the total serum IgE of saliva Trizol reagent, tiny RNA are mentioned with the miRNA Isolation Kit of Ambion
It takes.Tiny RNA is marked using T4 RNA ligase labelling technique, i.e., by the 5 '-of the tiny RNA of 1 μ g and 500ng
Phosphate-cytidyl-uridyl-cy3-3 ' (Dharmacon, Lafayette) mixing, in T4 RNA ligase (New
England Biolabs) effect under be marked.Label reaction carries out 2 hours at 4 DEG C.0.3M sodium acetate and 2.5 are used later
Times volume ethanol precipitating, then ethanol washing is used, 50 μ l, which are suspended in, after drying contains 5 × SSC, the hybridization buffer of 0.2%SDS
In.
3, chip hybridization carries out in Agilent hybridizing box (Agilent, G2534A), using 8 hole silication coverslips
(Agilent, G2534A-60014), hybridizing box 15rpm, 44 DEG C of hybridized overnights in hybrid heater (Agilent, 2545A) are miscellaneous
Handing over the time is 16 hours.
4, chip opens chip and cover plate in 37 DEG C of 5 × SSC solution, washs solution with 5 × SSC of 0.02%SDS
Washing 3 minutes, 2 times, is washed 3 minutes, 2 times in 27 DEG C of 0.2 × SSC solution later.After the completion, chip is taken from washing lotion
Out, it is dried on slide centrifuge.
5, chip is with LuxScan 10K scanner scanning (CapitalBio company), with 3.0 image analysis of LuxScan
The image that (CapitalBio company) analysis obtains.After microarray data background correction signal, Quantile normalization is carried out,
It determines the expression of these miRNAs, and is compared with control sample, according to Logistic regression analysis equation to offer
People's saliva sample diagnose.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
SEQUENCE LISTING
<110>Suzhou Institute of Nano-tech. and Nano-bionics, Chinese Academy of Sciences
<120>for the biomarker combinations of nasopharyngeal carcinoma diagnosis and application
<160> 21
<170> PatentIn version 3.5
<210> 1
<211> 22
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cugggaggug gauguuuacu uc 22
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gtcgggatcc agagcaggtc cgagggtaca cgttcgctct ggatcccgac gaagta 56
<210> 3
<211> 18
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<213>artificial sequence
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<400> 5
gtcgggatcc agagcaggtc cgagggtaca cgttcgctct ggatcccgac ctcccc 56
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<210> 9
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<400> 9
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Claims (10)
1. a kind of biomarker combinations for nasopharyngeal carcinoma diagnosis, which is characterized in that it includes following microRNA molecule:
Has-miR-30b-3p, has-miR-1202, has-miR-1321, has-miR-3612, has-miR-4478 and has-
miR-4730；
Wherein, the nucleotide sequence of the has-miR-30b-3p is as shown in SEQ ID NO.1；The core of the has-miR-1202
Nucleotide sequence is as shown in SEQ ID NO.4；The nucleotide sequence of the has-miR-1321 is as shown in SEQ ID NO.7；It is described
The nucleotide sequence of has-miR-3612 is as shown in SEQ ID NO.10；The nucleotide sequence of the has-miR-4478 such as SEQ
Shown in ID NO.13；The nucleotide sequence of the has-miR-4730 is as shown in SEQ ID NO.16.
2. a kind of for detecting the reagent of biomarker combinations described in claim 1.
3. reagent according to claim 2, which is characterized in that the reagent is with complementary with the microRNA molecule
Sequence probe；
Alternatively, the reagent is the primer for detecting microRNA molecule.
4. reagent according to claim 3, which is characterized in that the primer includes primer before reverse transcription primer, quantitative PCR
With primer after quantitative PCR；
Preferably, the reverse transcription primer sequence of has-miR-30b-3p is detected as shown in SEQ ID NO.2, primer before quantitative PCR
Sequence is as shown in SEQ ID NO.3, and primer sequence is as shown in SEQ ID NO.21 after quantitative PCR；
Preferably, the reverse transcription primer sequence of has-miR-1202 is detected as shown in SEQ ID NO.5, primer sequence before quantitative PCR
Column are as shown in SEQ ID NO.6, and primer sequence is as shown in SEQ ID NO.21 after quantitative PCR；
Preferably, the reverse transcription primer sequence of has-miR-1321 is detected as shown in SEQ ID NO.8, primer sequence before quantitative PCR
Column are as shown in SEQ ID NO.9, and primer sequence is as shown in SEQ ID NO.21 after quantitative PCR；
Preferably, the reverse transcription primer sequence of has-miR-3612 is detected as shown in SEQ ID NO.11, primer sequence before quantitative PCR
Column are as shown in SEQ ID NO.12, and primer sequence is as shown in SEQ ID NO.21 after quantitative PCR；
Preferably, the reverse transcription primer sequence of has-miR-4478 is detected as shown in SEQ ID NO.14, primer sequence before quantitative PCR
Column are as shown in SEQ ID NO.15, and primer sequence is as shown in SEQ ID NO.21 after quantitative PCR；
Preferably, the reverse transcription primer sequence of has-miR-4730 is detected as shown in SEQ ID NO.17, primer sequence before quantitative PCR
Column are as shown in SEQ ID NO.18, and primer sequence is as shown in SEQ ID NO.21 after quantitative PCR.
5. the described in any item reagents of claim 2-4 are preparing answering in nasopharyngeal carcinoma diagnosis kit or nasopharyngeal carcinoma diagnosis chip
With.
6. application according to claim 5, which is characterized in that the detection sample of the nasopharyngeal carcinoma diagnosis kit is saliva
Liquid.
7. a kind of nasopharyngeal carcinoma diagnosis kit, which is characterized in that it includes the described in any item reagents of claim 2-4.
8. the tumor diagnosis kit according to claim 7 for nasopharyngeal carcinoma, which is characterized in that the kit also wraps
Include one of following ingredient or a variety of:
Reverse transcriptase, buffer, dNTPs, Mgcl2、ddH2O, fluorescent dye, Taq enzyme and standard items and control.
9. a kind of nasopharyngeal carcinoma diagnosis chip, which is characterized in that be fixed with detection biology mark described in claim 1 on the chip
The miRNA probe of will object combination.
10. according to claim 9 be used for nasopharyngeal carcinoma diagnosis chip, which is characterized in that the miRNA probe has and institute
State the sequence of microRNA molecule complementation.
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