CN106191055A - A kind of non-small cell lung carcinoma marker, detectable and test kit - Google Patents
A kind of non-small cell lung carcinoma marker, detectable and test kit Download PDFInfo
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- CN106191055A CN106191055A CN201510227774.2A CN201510227774A CN106191055A CN 106191055 A CN106191055 A CN 106191055A CN 201510227774 A CN201510227774 A CN 201510227774A CN 106191055 A CN106191055 A CN 106191055A
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Abstract
The invention discloses a kind of blood miRNA marker relevant to human non-small cell lung cancer and application thereof.This mark is the miRNA-375 secreting body source outside blood plasma.Present invention also offers the detectable of the above-mentioned blood miRNA marker relevant to Human Lung Cancer and contain its test kit.Another further aspect, present invention also offers a kind of method detecting described mark miRNA-375.The mark of the present invention and detectable thereof can be used for preparing detection kit, and the early stage anticipation for nonsmall-cell lung cancer detects.Different from traditional biological mark, secrete outward the miRNA marker in body source not only stable, Micro-operation, be prone to detection, and can accurate quantification, greatly improve the Sensitivity and Specificity of medical diagnosis on disease.
Description
Technical field
The invention belongs to biology field, be specifically related to a kind of miRNA mark and application thereof.
Background technology
Pulmonary carcinoma is one of the most modal malignant tumor.Immediate and mid-term many countries all report that the M & M of pulmonary carcinoma the most substantially increases, and male lung cancer M & M all accounts for first of all malignant tumor, and women sickness rate accounts for second, and mortality rate accounts for second.Pulmonary carcinoma leaps to become the first reason of China's urban population mortality of malignant tumors the most.Nonsmall-cell lung cancer accounts for the 80% of all pulmonary carcinoma, including squamous cell carcinoma (scale cancer), adenocarcinoma etc..Comparing small cell carcinoma, non-small cell lung cancer cell growth division is relatively slow, and diffusion transfer is the most later.The Patients with Non-small-cell Lung of about 75% has been in middle and advanced stage when finding, and within 5 years, survival rate is the lowest.
The cause of disease of pulmonary carcinoma is the clearest and the most definite, and great mass of data shows, long-term a large amount of smokings, environment contact and chronic pulmonary inflammatory etc. are all closely related with the generation of pulmonary carcinoma.The diagnosis of pulmonary carcinoma at present depends on the inspection of iconography, including X-ray, bronchoscope, ECT inspection, mediastinoscopy etc., in combination with phlegm cytology checking.But due to the restriction of this type of method, the Sensitivity and Specificity of detection itself is not the most highly satisfactory.Pulmonary carcinoma early symptom is inconspicuous, and when late period makes a definite diagnosis, owing in cancerous cell pleura and/or pleural reflection shifts, patient has lost the chance of excision focus.
Secrete outward the capsule balloon-shaped structure that body (Exosome) is a kind of a diameter of 30-150 nanometer, be widely present in the biological fluids such as blood, saliva, urine, cerebrospinal fluid.It is formed at various kinds of cell, such as the exocytosis of dendritic cell, tumor cell etc..The adventitia secreting outward body is lipid bilayer structure, is enclosed with special albumen, messenger RNA and non-coding RNA etc. in it.Secrete outward the non-coding RNA in body based on miRNA, and abundant species, it can affect stem cell differentiation (let-7), orga-nogenesis (miR-1), hemoposieis (miR-181), tumor generation many physiological activities such as (miR-17, miR-18, miR-19a, miR-20, miR-19b-1, miR-93-1) and metabolism.
MiRNA is the non-coding RNA of a class short-movie section, and they all played an important role in propagation, differentiation and the apoptotic process of cell.Suppress translation on the specific site of 3 '-noncoding region that miRNA is combined in messenger RNA by targeting or promote the degraded of messenger RNA.The exception of miRNA level may result in the disorder of interior environment, ultimately results in the generation of tumor.The miRNA gene being positioned at the relevant region of oncogene or fragile site can expand in tumor cell or lack, it is meant that miRNA has played important function during malignant proliferation of tumor.Almost all of human tumor cell has had been found that the imbalance of miRNA.Secreting outward the miRNA in body not to be degraded in the body fluid such as serum, blood plasma of the mankind presented in stable, can be detected by specific isolated and purified means, this Novel heavy also making it possible to become Non-invasive detection examination wants mark.
Summary of the invention
An object of the present invention is to provide a kind of can be used in detect the novel miRNA mark of nonsmall-cell lung cancer and the application in preparing nonsmall-cell lung cancer detection kit thereof.
The two of the purpose of the present invention are the detectable providing above-mentioned miRNA mark and the application in preparing nonsmall-cell lung cancer detection kit thereof.
The three of the purpose of the present invention are to provide a kind of method detecting above-mentioned miRNA mark.
To achieve these goals:
On the one hand, the invention provides a kind of blood miRNA mark relevant to human non-small cell lung cancer, described mark is the miRNA-375 that body source is secreted by blood China and foreign countries, and its sequence information is as shown in SEQ ID NO:1.
Present invention also offers the blood miRNA mark relevant to Human Lung Cancer application in preparation detection human non-small cell lung cancer's test kit, described mark is the miRNA-375 that body source is secreted by blood China and foreign countries, and its sequence information is as shown in SEQ ID NO:1.
On the other hand, present invention also offers the detectable of the above-mentioned blood miRNA mark relevant to Human Lung Cancer.Described reagent includes reverse transcription primer and/or amplimer；Described reverse transcription primer is neck ring structure primer special for miRNA, sequence such as SEQ
Shown in ID NO:2；The forward primer sequence of described amplimer is as shown in SEQ ID NO:3, and the downstream primer of described amplimer is general reverse primer, and sequence is as shown in SEQ ID NO:4.Described primer generally uses in QPCR tests.
Another further aspect, present invention also offers the application in the test kit of preparation detection human non-small cell lung cancer of the detectable of the blood miRNA mark relevant to human non-small cell lung cancer, described reagent includes reverse transcription primer and/or the amplimer used in QPCR experiment；Described reverse transcription primer is miRNA specific neck ring structure primer, sequence such as SEQ
Shown in ID NO:2；The forward primer sequence of described amplimer is as shown in SEQ ID NO:3, and the downstream primer of described amplimer is general reverse primer, and sequence is as shown in SEQ ID NO:4.Described detection kit comprises above-mentioned primer sequence.
Another further aspect, present invention also offers a kind of test kit detecting human non-small cell lung cancer, and described test kit comprises the detectable of the blood miRNA mark relevant to human non-small cell lung cancer, and described reagent includes reverse transcription primer and/or amplimer；Described reverse transcription primer is miRNA specific neck ring structure primer, and sequence is as shown in SEQ ID NO:2；The forward primer sequence such as SEQ of described amplimer
Shown in ID NO:3, the downstream primer of described amplimer is general reverse primer, and sequence is as shown in SEQ ID NO:4.Described primer generally uses in QPCR tests.
Preferably, the detection kit of the present invention also comprises reagent conventional for QPCR and enzyme, it is also possible to include standard substance and/or reference substance.Described common agents comprises PCR reaction buffer, ribonuclease inhibitor, dNTP etc.；Described enzyme comprises M-MLV
Reverse transcriptase, Genomic DNA digestion enzyme (gDNA Eraser) etc..
Another further aspect, present invention also offers a kind of method detecting described mark miRNA-375, including:
(1) outer in isolated and purified blood secretes body；
(2) sample total serum IgE is extracted；
(3) the RNA reverse transcription that step (2) obtains is become cDNA；
(4) on fluorescence real-time quantitative PCR instrument, miRNA and reference gene are carried out augmentation detection；
(5) determine that purpose band, Δ Δ CT method carry out relative quantification by melt curve analysis analysis and electrophoresis.
Preferably, the primer that RNA reverse transcription described in step (3) uses when becoming cDNA, sequence is as shown in SEQ ID NO:2；Carrying out in step (4) in the amplimer used during described amplification, the sequence of forward primer is as shown in SEQ ID NO:3, and the sequence of downstream primer is as shown in SEQ ID NO:4.
Preferably, in step (4), reference gene is snRNA U6.
As example, the operation that step (1) is concrete is:
The peripheral blood of the freshest collection or the frozen state. plasma at room temperature 2000*g of thawing
Centrifugal 20min, removes cell and cell debris；
B. taking supernatant and move to new centrifuge tube, under room temperature, 10000*g is centrifuged 20min, removes cell debris further；
C. take supernatant liquid and move into centrifuge tube, add the 1X of 0.5 times of volume
PBS solution, vortex mixes；
D. body separation agent (Invitrogen) is secreted outside the most always adding, mixing；Preferably 500 μ l initial plasma add 350 μ l separation agents；
E. under mixed liquor carries room temperature, 10000*g is centrifuged 5min
(now, secreting outward body to be enriched in bottom pipe)；
F. all supernatants are removed in sucking-off；
G. with 25 μ l 1X
The resuspended whole precipitations of PBS solution ,-20 DEG C of preservations.
As example, the specific implementation method of step (2) is:
A. adding 700 μ l TRIzol solution, firmly concussion is to presenting homogeneous liquid phase, and room temperature stands 5min；
B. adding chloroform 350 μ l, use forced oscillation centrifuge tube, fully mix, room temperature stands 5min；
DEG C c.4 under the conditions of, 12000rpm high speed centrifugation 15min
Rear absorption upper strata aqueous phase, in new centrifuge tube, is careful not to the protein layer being drawn onto between two-layer aqueous phase；
D. isopyknic isopropanol is added, the most reverse mixing, under the conditions of-20 DEG C, stand 20min；
DEG C e.4 under the conditions of, 12000rpm high speed centrifugation 15min
The most carefully discard supernatant, add 75% washing with alcohol precipitation of 1ml pre-cooling, 7500rpm high speed centrifugation 5min at 4 DEG C；
F. discarding liquid, ambient temperatare puts 5min fully to dry precipitation, adds the sterilized water dissolution precipitation without RNase；
G. measuring RNA purity and concentration with Nanodrop2000 ultraviolet spectrophotometer, sample is frozen in-80 DEG C.
As example, RNA is transcribed into cDNA by step (3) can use that stem is around-France becomes cDNA by the miRNA reverse transcription in total serum IgE.
The around-France reverse transcription of stem is the use of a special loop-stem structure primer, and primer 3 ' end has the nucleotide of 6 complementary pairings with miRNA 3 ' end, is combined by 3 ' ends of primer with purpose miRNA molecule, reverse transcriptase can by reverse transcription become cDNA the first chain.The stem circulus of cDNA the first chain adds the length of chain after opening, then can carry out traditional quantitative fluorescent PCR with it for template.
In a specific embodiment of the present invention, use neck ring method that miRNA reverse transcription is become cDNA.Concrete operation step is: the total serum IgE template of 10pg-1 μ g mixed with 2 μ l 5* buffer, 1 μ l Genomic DNA digestion enzyme and the distilled water (RNase-free water) without ribonuclease, final volume is 10 μ l, hatches 5min to remove genomic DNA for 42 DEG C.Then in reaction tube, add 20 μMs of specific reverse transcription primers of 0.5 μ l, 65 DEG C hatch 10min after hatch at least 2min the most on ice, to interrupt the secondary structure of RNA and primer.Subsequently, by above-mentioned reactant mixture and 4 μ l 5* buffer, 1 μ l dNTP (10mM), 0.5 μ l M-MLV reverse transcriptase, water (RNase-free water) mixing of 0.5 μ l ribonuclease inhibitor and 3.5 μ l deoxyribonucleases, hatch 1h for 42 DEG C.5 seconds are reacted with the enzyme in inactivation reaction system under 85 DEG C of high temperature.Synthesis cDNA template be saved in-20 DEG C standby.
As example, the specific implementation method of step (4) is: use 25 μ l reaction systems, and each sample arranges 3 parallel pipes, and all amplified reactions are above to ensure the reliability of result.Prepare following reaction system: SYBR
Green polymerase chain reaction mixed system 12.5 μ l, forward primer (20 μMs) 0.5 μ l, reverse primer (20 μMs) 0.5 μ l, template cDNA 2 μ l, distilled water 9.5 μ l.Operations is all carried out on ice.Amplification program is: 95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 30s) * 45 circulations.Using SYBR Green as fluorescent marker, at ABI
7500 real-time fluorescence quantitative PCR instrument enterprising performing PCR reactions.The forward primer sequence such as SEQ of amplification miRNA-375
Shown in ID NO:3, reverse primer is general reverse primer, and sequence is as shown in SEQ ID NO:4.Using snRNA U6 as reference gene, its primer is purchased from GeneCopoeia brand.
The method of described detection mark miRNA-375, can carry out quantitative analysis, other quantitative analysis methods in large sample crowd, such as RT-PCR, high throughput sequencing technologies, Taqman low density chip (TLDA) detection, also comprise in the present invention.The method carrying out quantitative analysis in large sample crowd includes:
(1) standard compliant blood sample, demographic data that systematic collection is complete and clinical data are gathered with S.O.P.；
(2) the miRNA differential expression analysis of spectrum in body source is secreted by blood China and foreign countries: select nonsmall-cell lung cancer case, pneumonia and the blood sample of healthy individuals, collect blood China and foreign countries and secrete the miRNA in body source, carry out chip analysis, the miRNA of screening differential expression；
(3) the differential expression miRNA screened is carried out quantitative analysis in large sample crowd.
The beneficial effect comprise that
(1) miRNA in body source secretes in blood China and foreign countries is a kind of new biomarkers.Different from traditional biological mark, secrete outward the miRNA marker in body source not only stable, Micro-operation, be prone to detection, and can accurate quantification, greatly improve the Sensitivity and Specificity of disease detection.The successful exploitation of the RNA biomarker secreting outward body source contributes to the auxiliary detection of pulmonary carcinoma, and also the exploitation for other diseases biomarker provides pattern.
(2) test kit predicting human non-small cell lung cancer by the outer expression secreting body RNA in tumor source in detection blood is a kind of system, comprehensively detection and kit for screening, can be used for the auxiliary diagnosis of patients with lung cancer, contribute to reflecting the morbid state of patients with lung cancer, quick and precisely grasp conditions of patients for clinician, take the diagnosis and treatment scheme of personalization to provide support in time.
(3) using tight design and appraisement system, the present inventor's initial stage uses miRNA chip to be analyzed miRNAs, and the method for application qRT-PCR is verified in great amount of samples；The application acceleration of above method and strategy secretes body miRNA biomarker and the application of detection kit, also for the reference developed on offer method and strategy of other diseases biomarker with ensure that outside tumor source in blood.
Accompanying drawing explanation
Fig. 1 shows that utilizing tumor in QPCR checking blood to originate secretes outward the expression of body miRNA-375.
Fig. 2 shows the outer miRNA-375 secreting and dissociating in body source and blood
Expression in patients with lung cancer, patients with pneumonia and healthy individuals, wherein Fig. 2 A: secrete outward the miRNA-375 in body source；Fig. 2 B: miRNA-375 free in blood.
Fig. 3 shows the Sensitivity and Specificity utilizing ROC tracing analysis miRNA-375 to distinguish pulmonary carcinoma group and healthy group.
Detailed description of the invention
Further illustrating the present invention below in conjunction with specific embodiment, embodiments of the invention are only used for explaining the present invention, are not intended to limit protection scope of the present invention.
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
The screening of the miRNA that embodiment 1 is relevant to Human Lung Cancer
The collection of 1.1 samples and the arrangement of data
Inventor have collected substantial amounts of patients with lung cancer in November, 2014 in Affiliated Hospital of Wenzhou Medical University, peripheral blood sample (the sample collection of different batches of patients with pneumonia and healthy individuals, pretreatment, subpackage, preservation conditions etc. all keep consistent), by the arrangement to sample data, inventor therefrom have selected 30 Li Fu assigned hospitals, as far as possible with nonsmall-cell lung cancer patient's blood plasma of section office's random collecting, 20 examples are with the blood plasma (include 18 examples have smoking history but be shown as normal healthy individuals under the conditions of test in laboratory) of the blood plasma of hospital and the patients with pneumonia of experimental group contemporaneity random collecting and 40 examples and the healthy individuals of experimental group contemporaneity random collecting, avoid taking the sample of the blood plasma of pulmonary carcinoma family history people the most as far as possible, sample above is carried out the detection of miRNA chip.
1.2 miRNA chip detection
1.2.1 the outer isolation and purification secreting body
(1) peripheral blood of fresh collection or the frozen state. plasma at room temperature 2000*g of thawing
Centrifugal 20min, removes cell and cell debris；
(2) taking supernatant and move to new centrifuge tube, under room temperature, 10000*g is centrifuged 20min, removes cell debris further；
(3) take supernatant liquid and move into centrifuge tube, add the 1X of 0.5 times of volume
PBS solution, vortex mixes；
(4) body separation agent (Invitrogen) is secreted outside the most always adding, mixing；Preferably 500 μ l initial plasma add 350 μ l separation agents；
(5) under mixed liquor carries room temperature, 10000*g is centrifuged 5min
(now, secreting outward body to be enriched in bottom pipe)；
(6) all supernatants are removed in sucking-off；
(7) with 25 μ l 1X
The resuspended whole precipitations of PBS solution ,-20 DEG C of preservations.
1.2.2
The extraction of total serum IgE
A. adding 700 μ l TRIzol solution, firmly concussion is to presenting homogeneous liquid phase, and room temperature stands 5min；
B. adding chloroform 350 μ l, use forced oscillation centrifuge tube, fully mix, room temperature stands 5min；
DEG C c.4 under the conditions of, 12000rpm high speed centrifugation 15min
Rear absorption upper strata aqueous phase, in new centrifuge tube, is careful not to the protein layer being drawn onto between two-layer aqueous phase；
D. isopyknic isopropanol is added, the most reverse mixing, under the conditions of-20 DEG C, stand 20min；
DEG C e.4 under the conditions of, 12000rpm high speed centrifugation 15min
The most carefully discard supernatant, add 75% washing with alcohol precipitation of 1ml pre-cooling, 7500rpm high speed centrifugation 5min at 4 DEG C；
F. discarding liquid, ambient temperatare puts 5min fully to dry precipitation, adds the sterilized water dissolution precipitation without RNase；
G. measuring RNA purity and concentration with Nanodrop2000 ultraviolet spectrophotometer, sample is frozen in-80 DEG C.
1.2.3miRNA chip operation
MiRNA chip is purchased from ABI company, and the operation scheme pointed out to specifications carries out the detection of miRNA express spectra.
1.2.4 result
According to the testing result of miRNA chip, find the outer of Patients with Non-small-cell Lung tumor source secretes in body how group miRNA expressions exist significant difference, wherein miRNA-375
Expression less than pneumonia sample and the level of healthy individuals.
The miRNA-375 of embodiment 2 QPCR checking differential expression
Testing result according to above-mentioned miRNA chip selects miRNA-375 to carry out the QPCR checking of greater amount sample.Nonsmall-cell lung cancer group 95 example sample and healthy group 120 example samples is selected according to the Collator Mode of the sample collection in embodiment 1 and sample data.
2.1 total serum IgE extract process with embodiment 1.
2.2 reverse transcriptions: by water (RNase-free water) mixing of the total serum IgE template of 10pg-1 μ g with 2 μ l 5* buffer, 1 μ l Genomic DNA digestion enzyme and deoxyribonuclease, final volume is 10 μ l, hatches 5min to remove genomic DNA for 42 DEG C.Then in reaction tube, add 20 μMs of specific reverse transcription primers of 0.5 μ l, 65 DEG C hatch 10min after hatch at least 2min the most on ice, interrupt the secondary structure of RNA and primer.Finally, by above-mentioned reactant mixture and 4 μ l 5* buffer, 1 μ l dNTP (10mM), 0.5 μ l
M-MLV reverse transcriptase, water (RNase-free water) mixing of 0.5 μ l ribonuclease inhibitor and 3.5 μ l deoxyribonucleases, hatch 1h for 42 DEG C.
2.3QPCR reacts: using 25 μ l reaction systems, each sample arranges 3 parallel pipes, and all amplified reactions are above to ensure the reliability of result.Prepare following reaction system: SYBR
Green polymerase chain reaction mixed system 12.5 μ l, forward primer (20 μMs) 0.5 μ l, reverse primer (20 μMs) 0.5 μ l, template cDNA 2 μ l, distilled water 9.5 μ l.Operations is all carried out on ice.Amplification program is: 95 DEG C of 10min, (95 DEG C of 15s, 60 DEG C of 30s) * 45 circulations.Using SYBR Green as fluorescent marker, at ABI
7500 real-time fluorescence quantitative PCR instrument enterprising performing PCR reactions.The forward primer sequence such as SEQ of amplification miRNA-375
Shown in ID NO:3, reverse primer is general reverse primer, and sequence is as shown in SEQ ID NO:4.Using snRNA U6 as reference gene, its primer is purchased from GeneCopoeia brand.Purpose band is determined by melt curve analysis analysis and electrophoresis, Δ Δ CT method carries out relative quantification, and result is as shown in Fig. 1, compared with healthy individuals, it is relatively low that miRNA-375 secretes the content in body outside Patients with Non-small-cell Lung blood, and the result with the prompting of embodiment 1 chips is consistent.
Embodiment 3 is secreted body outside analyzing and is carried out the prediction that nonsmall-cell lung cancer is fallen ill by miRNAs
The miRNA-375 secreting body source in addition is object of study, and makes comparisons in terms of distinguishing matched group and pulmonary carcinoma group Sensitivity and Specificity with the miRNA of plasma free.With Patients with Non-small-cell Lung as experimental group, with patients with pneumonia and normal person as a control group, first with total outer secrete that body separation agent (Invitrogen) extracts in blood plasma outer secrete body (step is substantially with embodiment 1).Select 40 example nonsmall-cell lung cancer plasma samples, 20 example pneumonia patients and 40 example human normal plasma's samples to carry out outer secreting respectively and outside body miRNA-375(, secrete body group) and plasma free miRNA-375(blood plasma group) (method is substantially with embodiment 2 in QPCR experiment, but the blood plasma group free miRNA-375 that is extracting directly rather than secrete body from outward and separate), try to achieve each specimen CT value.To sample miRNA-375
The ratio of CT value and U6 CT value does scatterplot (Fig. 2).Result prompting is outer, and to secrete body group aggregation more preferable than blood plasma group, secrete outward body group experimental result also with cancerous lung tissue before and miRNA-375 in cancer beside organism
Expression trend identical.Blood plasma China and foreign countries secrete the miRNA-375 in body source and have statistical significance (p < 0.05) at differentiation patients with lung cancer on normal person.ROC curve analysis shows, miRNA-375 is when distinguishing pulmonary carcinoma group and normal group, and its AUC value is 0.8052, and sensitivity during best cut point is 69%, specificity is 64% (Fig. 3).Meanwhile, in blood plasma, free miRNA-375 is distinguishing pulmonary carcinoma group and normal group without notable significant difference.These results show that outer body of secreting carrys out miRNAs and is better than in blood plasma free rna level as susceptiveness and the specificity of potential detection mark.
The explanation of above-described embodiment is only intended to understand the method for the present invention and core concept thereof.It should be pointed out that, for the person of ordinary skill of the art, under the premise without departing from the principles of the invention, it is also possible to the present invention is carried out some improvement and modification, these improve and modify also by the protection domain falling into the claims in the present invention.
<110>Shang Hairun rises bio tech ltd
<120>a kind of non-small cell lung carcinoma marker, detectable and test kit
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<213>homo sapiens (Homo sapiens)
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uuuguucguu
cggcucgcc gu 22
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ctcaactggt
gtcgtggagt cggcaattca gttgagtcac gcga 44
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acactccagc
tgggtttgtt cgttcggctc 30
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Claims (8)
1. a blood miRNA marker relevant to human non-small cell lung cancer, it is characterised in that described miRNA marker is the outer miRNA-375 secreting body source, and its sequence is as shown in SEQ ID NO:1.
2. the detectable of the miRNA mark described in claim 1, it is characterised in that described reagent includes reverse transcription primer and amplimer；Described reverse transcription primer, sequence is as shown in SEQ ID NO:2；The sequence of the forward primer of described amplimer is as shown in SEQ ID NO:3, and the sequence of the downstream primer of described amplimer is as shown in SEQ ID NO:4.
3. the detectable of the miRNA marker described in claim 2 application in preparation human non-small cell lung cancer's detection kit.
4. human non-small cell lung cancer's detection kit, it is characterised in that described detection kit comprises claim 2
Described reagent.
5. according to the detection kit described in claim 4, it is characterised in that described detection kit also comprises the conventional reagent of quantitative PCR reaction and enzyme.
6. the method that a test right requires the miRNA marker described in 1, it is characterised in that said method comprising the steps of:
(1) in isolated and purified human peripheral blood, the outer of tumorigenic secretes body；
(2) sample total serum IgE is extracted；
(3) the RNA reverse transcription that step (2) obtains is become cDNA；
(4) cDNA and reference gene step (3) obtained on quantitative fluorescent PCR instrument carries out augmentation detection；
(5) determine that purpose band, Δ Δ CT method carry out relative quantification by melt curve analysis analysis and electrophoresis.
7. according to the method described in claim 6, it is characterised in that the primer that RNA reverse transcription described in step (3) uses when becoming cDNA, sequence such as SEQ
Shown in ID NO:2；Carrying out in step (4) in the amplimer used during described amplification, the sequence of forward primer is as shown in SEQ ID NO:3, and the sequence of downstream primer is as shown in SEQ ID NO:4.
8. according to the method described in claim 6, it is characterised in that in step (4), reference gene is snRNA U6.
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Cited By (5)
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| CN108802389A (en) * | 2018-06-04 | 2018-11-13 | 郭伟 | A kind of kit for Early stage NSCLC diagnosis |
| CN108949961A (en) * | 2018-08-10 | 2018-12-07 | 广州欣源生物科技有限公司 | For detecting kit and its screening of adenovirus pneumonia |
| CN109423517A (en) * | 2017-08-28 | 2019-03-05 | 中国医学科学院肿瘤医院 | Purposes of the excretion body in diagnosing tumor, treatment and prognosis evaluation |
| CN110799648A (en) * | 2017-06-29 | 2020-02-14 | 东丽株式会社 | Kit, device and method for detecting lung cancer |
| CN112280846A (en) * | 2020-10-12 | 2021-01-29 | 东南大学 | Abnormal miRNA marker of benzene induced blood system and application thereof |
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2015
- 2015-05-07 CN CN201510227774.2A patent/CN106191055A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110799648A (en) * | 2017-06-29 | 2020-02-14 | 东丽株式会社 | Kit, device and method for detecting lung cancer |
| CN110799648B (en) * | 2017-06-29 | 2024-03-22 | 东丽株式会社 | Kit, device and method for detecting lung cancer |
| CN109423517A (en) * | 2017-08-28 | 2019-03-05 | 中国医学科学院肿瘤医院 | Purposes of the excretion body in diagnosing tumor, treatment and prognosis evaluation |
| CN108802389A (en) * | 2018-06-04 | 2018-11-13 | 郭伟 | A kind of kit for Early stage NSCLC diagnosis |
| CN108949961A (en) * | 2018-08-10 | 2018-12-07 | 广州欣源生物科技有限公司 | For detecting kit and its screening of adenovirus pneumonia |
| CN112280846A (en) * | 2020-10-12 | 2021-01-29 | 东南大学 | Abnormal miRNA marker of benzene induced blood system and application thereof |
| CN112280846B (en) * | 2020-10-12 | 2022-05-31 | 东南大学 | Abnormal miRNA marker of benzene induced blood system and application thereof |
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