CN109593857A - In blood circ-DLG1 molecular marker diagnosis esophageal squamous cell carcinoma by stages in application - Google Patents
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Abstract
The invention discloses circ-DLG1 molecular marker in blood diagnosis esophageal squamous cell carcinoma by stages in application, the nucleotides sequence of circ-DLG1 molecular marker is classified as SEQ ID NO.1, it is related to the discovery, detection, application of circ-DLG1 molecular marker, designs and synthesizes out the detection primer for being specifically used for real-time quantitative PCR.It is poor to cancer of the esophagus Endoscopic Screening compliance for target group, and the patients with esophageal squamous cell carcinoma overwhelming majority clinically made a definite diagnosis is the status proposition of middle and advanced stage, ideal molecule pre-warning signal is found in esophageal squamous cell carcinoma change process, targetedly carry out endoscopy, definitive pathological diagnosis, to mitigate patient's pain, over-treatment is avoided, medical resource is saved.
Description
Technical field
The present invention relates to oncomolecularbiology field, circ-DLG1 molecular marker is being examined in specially a kind of blood
Disconnected esophageal squamous cell carcinoma by stages in application.
Background technique
The cancer of the esophagus is to seriously threaten one of most common malignant tumour of human health, and there are mainly two types of pathologicals: squamous carcinoma
And gland cancer.90% cancer of the esophagus is squamous cell carcinoma, and distribution has apparent areal variation.China ESCC disease incidence Zhan Quanshi
70% or more boundary, prognosis is poor, and overall survival only has 15%-25% within 5 years.Clinician's Journal of Cancer report in 2016, food
Pipe cancer has become one of highest five kinds of tumours of Chinese disease incidence, and disease incidence ranked third position in male, and is arranged in women
Five.
Circular rna (circRNAs) is a kind of new endogenous non-coding RNA, is confirmed as in early stage the 1990s
The transcript of out-of-order exon, structure, function and mechanism are reported in succession, become later 20 years research hotspots.It is different from
The special closed loop of 5' end cap and the end 3' poly (A) tail is the absence of using the end 5' and the end 3' as the conventional linear RNA, circRNAs of end
Structure, and compared with Microrna in mammalian cell (miRNAs) and long-chain non-coding RNA (lncRNAs), have higher
Stability and sequence conservation are because they have nuclease resistant.Recent numerous studies report, in different plant species
CircRNAs has expression.Rapid development based on bioinformatic analysis and high throughput sequencing technologies, researcher have found
Ten hundreds of circRNAs has found that they participate in the disease developments such as vascular lesion, the nervous system disease, tumour.
CircRNAs chip analysis technology has been applied to many tumours, including digestive system tumor, nervous system neoplasm, and urinary system swells
Tumor, head and neck neoplasm etc., such as hypopharyngeal squamous cell carcinoma, squamous carcinoma of larynx, basal-cell carcinoma, ductal adenocarcinoma of pancreas, ESCC, liver
Cell cancer, thyroid papillary carcinoma etc..However, circRNAs is rarely reported in esophageal squamous cell carcinoma.
ESCC still assesses the state of an illness without tumor marker at present, and scope is goldstandard, but wound is big, patient is not
It is easily accepted by.
Summary of the invention
To solve the above problems, the invention discloses circ-DLG1 molecular markers in a kind of blood in diagnosis esophageal squamous cell
Cancer by stages in application, for target group to cancer of the esophagus Endoscopic Screening compliance esophageal squamous cell carcinoma that is poor, and clinically making a definite diagnosis
The patient overwhelming majority is that the status of middle and advanced stage proposes, ideal molecule early warning letter is found in esophageal squamous cell carcinoma change process
Number, endoscopy is targetedly carried out, definitive pathological diagnosis avoids over-treatment, save medical resource to mitigate patient's pain.
Circ-DLG1 in blood plasma can be used as the blood plasma marker object of ESCC patient, this inspection method is easy to be received by subject, more may be used
Effective means as the early diagnosis of ESCC patient, the judgement of disease progression and prognosis evaluation.
In order to reach the goals above, the present invention supplies following technical solution:
In a kind of blood circ-DLG1 molecular marker diagnosis esophageal squamous cell carcinoma by stages in application, the circ-DLG1
The nucleotides sequence of molecular marker is classified as SEQ ID NO.1.The ID number of circ-DLG1 is circ_0007203, sequence at shearing
For CGATGAGGTCGGAGTGATTCCCAGTAAACESCCAGAGTTGAGAAGAAAGAACGAES CCCCGATTAAAAACAGTG
AAATTCAATTCTAAAACGAGAGATAAAGGESCCTTCTTCTCAESCCCTGTTGATAACCATGTTAESCCCCATCTTC
CTTCTTGGESCCCAGACACCAESCCATCTCCAESCCCAGATACTCCCCAGTTTCTAAAESCCAGTACT。
Further, for diagnose esophageal squamous cell carcinoma by stages in reagent be real-time quantitative PCR kit.
The present invention devises 2 pairs of primers according to the circ-DLG1 molecular marker that nucleotides sequence is classified as SEQ ID NO.1
For detecting the sequence, primer nucleotide sequences are SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID
NO.5。
Specific primer difference is as follows:
The invention also discloses a kind of detection methods of circ-DLG1 molecular marker, including sample Total RNAs extraction, RNA
Reverse transcription, real-time quantitative PCR amplification.
The first step, Total RNAs extraction
A. blood plasma Total RNAs extraction
Blood plasma, serum Total RNAs extraction use mir Vana PARIS Kit (Ambion1566, USA) kit, specific to walk
It is rapid as follows:
All reagents are both needed to be restored to and use at room temperature, take out and melt spare, eluent 95 in plasma/serum sample ice face
DEG C pre- stand-by heat；
400 μ l plasma/serum samples are added in the EP pipe of added isometric 2X Denaturing Solution,
Mixing fullys shake；
Isometric Acid-Phenol:Chloroform is added, concussion mixes 1min, and then 10000g is centrifuged at room temperature
5min；Careful transfer upper strata aqueous phase is managed to new EP, and records transfer water phase volume, then adds 1.25 times of 100% second of volume
Alcohol shifts lysate/alcohol mixture into EP pipe, after mixing well and passes through filter (10000g is centrifuged 30s)；
Add 700 1 cleaning filter of μ l mi RNA Wash Solution 1 time (10000g is centrifuged 15s), 500 μ are then added
2/3 cleaning filter of l Wash Solution 2 times (10000g is centrifuged 15s)；
RNA (10000g is centrifuged 30s) on the deoxyribonuclease washing filter apparatus of 50 95 DEG C of μ l preheating, it is -80 DEG C long-term
It saves；
Ultraviolet specrophotometer measures concentration of specimens and purity.
B. total tissue RNA extracts Trizol method and extracts tissue and cell total rna, and specific step is as follows:
After cleaning porcelain grinding body, with DEPC liquid soaked overnight (at least 12h), ultralow temperature then is put into after high pressure steam sterilization
Refrigerator cold-storage；
Prepare sterile no enzyme pipette tips, EP pipe, 4 DEG C of centrifuge it is spare to the cold
Cryopreserved tissue (about 50mg) is taken out out of ultra low temperature freezer, surface frozen stock solution is wiped, is filled in mortar (- 40 DEG C of pre-coolings)
Divide grinding, adds liquid nitrogen in grinding to prevent tissue RNA from degrading.Powdery is ground into tissue samples；
Every part of sample adds 1ml Trizol lysate, blows even rear transfer lysate with pipette tips and manages to EP, stands in ice face
5min decomposes it completely；
Chlorination imitates 0.2ml, acutely shakes 15s, stands 2min at room temperature, 4 DEG C, 12000g is centrifuged 15min；
Careful transfer upper strata aqueous phase avoids drawing cotton-shaped middle layer as far as possible into new EP pipe.Then add 0.5ml isopropyl
Alcohol, concussion mix, stand 10min at room temperature, and subsequent 4 DEG C, 12000g is centrifuged 10min.It is now spare with 75% ethyl alcohol；
It carefully discards supernatant, 75% ethyl alcohol 1ml concussion cleaning precipitating, subsequent 7500g, 4 DEG C of centrifugation 5min is added；
It finally discards supernatant, sucks residual liquid as far as possible with pipette tips, be air-dried at room temperature, according to precipitating size, 30-60 is added
μ l DEPC water dissolves RNA precipitate；
Ultraviolet specrophotometer measures concentration of specimens and purity.
Second step, RNA reverse transcription and real-time quantitative PCR
Using Prime-Script by RNA reverse transcription at cDNA TM One step RT-PCR kit, glyceraldehyde 3 phosphate is de-
Hydrogen enzyme (GAPDH) is used as internal contrast.Owned using ABI7500 system and SYBR Green PCR Master Mix
QRT-PCR reaction.For the multiple variation that circ-DLG1 is expressed in accurate validation ESCC, by the Ct value of calculating relative to from phase
With sample amplification GAPDH (Δ Ct=Cttested-CtGAPDH) standardize, and use-Δ Ct method come estimate variation
Value.In triplicate, all reactions are independent in triplicate to ensure the repeatabilities of all data for each sample.circ-DLG1
Cutoff value be -13.22, when detect-Δ Ct be greater than this value when for ESCC, specificity is 74.55%, sensibility
It is 74.55%.
Data analysis: being examined using t and the method circ-DLG1 and ESCC patient clinical of Chi-square Test and survival analysis point
Phase data and the correlation of prognosis.Receiver Operating Characteristics' (ROC) curve is carried out to assess its diagnostic value.All statistical analysis
V.17.0 carried out using SPSS for Windows.For all as a result, P < 0.05 is considered statistically significant.
The invention also discloses a kind of for diagnosing the real time quantitative PCR detecting reagent kit of esophageal squamous cell carcinoma by stages, including root
It is designed and synthesized out according to the circ-DLG1 molecular marker that nucleotides sequence is classified as SEQ ID NO.1 and is specifically used for real-time quantitative
The detection primer of PCR；SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID in specific primer such as sequence table
NO.5。
The purpose of the present invention is to provide the circ-DLG1 molecular markers that a kind of nucleotides sequence is classified as SEQ ID NO.1.
The present invention has the advantage that
(1) purposes of circ-DLG1 is proposed.(2) circular rna has mechanism stable, abundance degree and sample specificity table
Up to etc. features, and circ-DLG1 expression quantity in ESCC patient and normal human blood sample has differences, and with phase by stages
It closes, therefore circ-DLG1 has the application prospect as esophageal squamous cell carcinoma biomarker.
The present invention be directed to target groups to cancer of the esophagus Endoscopic Screening compliance esophageal squamous cell carcinoma that is poor, and clinically making a definite diagnosis
The patient overwhelming majority is that the status of middle and advanced stage proposes, ideal molecule early warning letter is found in esophageal squamous cell carcinoma change process
Number, endoscopy is targetedly carried out, definitive pathological diagnosis avoids over-treatment, save medical resource to mitigate patient's pain.
Circ-DLG1 in blood plasma can be used as the blood plasma marker object of ESCC patient, this inspection method is easy to be received by subject, more may be used
Effective means as the early diagnosis of ESCC patient, the judgement of disease progression and prognosis evaluation.
Detailed description of the invention
The expression of circ-DLG1 expression is significant in Fig. 1, ESCC tissue is higher than adjacent non-cancer tissue；
The expression of circ-DLG1 level is significant in Fig. 2, ESCC blood plasma is higher than normal plasma；
Fig. 3, area is 0.7861 under ROC curve；
In Fig. 4, ESCC blood plasma circ-DLG1 level be positively correlated by stages.
Specific embodiment
With reference to the accompanying drawings and detailed description, the present invention is furture elucidated, it should be understood that following specific embodiments are only
For illustrating the present invention rather than limiting the scope of the invention.
The invention discloses circ-DLG1 molecular marker in a kind of blood diagnosis esophageal squamous cell carcinoma by stages in application,
The nucleotides sequence of circ-DLG1 molecular marker is classified as SEQ ID NO.1.
Further, for diagnose esophageal squamous cell carcinoma by stages in reagent be real-time quantitative PCR kit.
The present invention devises 2 pairs of primers according to the circ-DLG1 molecular marker that nucleotides sequence is classified as SEQ ID NO.1
For detecting the sequence, primer nucleotide sequences are SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID
NO.5。
In the present invention, the detection method of circ-DLG1 molecular marker, including sample Total RNAs extraction, RNA reverse transcription, reality
When quantitative pcr amplification.
The invention also discloses a kind of for diagnosing the real time quantitative PCR detecting reagent kit of esophageal squamous cell carcinoma by stages, including root
It is designed and synthesized out according to the circ-DLG1 molecular marker that nucleotides sequence is classified as SEQ ID NO.1 and is specifically used for real-time quantitative
The detection primer of PCR；SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID in specific primer such as sequence table
NO.5。
The present invention also provides the circ-DLG1 molecular markers that a kind of nucleotides sequence is classified as SEQ ID NO.1.
Specific embodiment is as follows:
1. experimental subjects
Using the attached Nanjing hospital Oncological Surgery of Nanjing Medical University as study base, Oncological Surgery accepts food for medical treatment throughout the year for this research
Pipe squamous cell carcinoma patients, and sample storehouse is established, case-data abundant can be provided for this research.Study patient tissue and blood sources in
Receive the patients with esophageal squamous cell carcinoma of radical cure or palliative operation excision during 2013 in Augusts, 2017 in our hospital.Normal control blood
Liquid sample standard deviation derives from Nanjing No.1 Hospital's people taking physical examination, and normal healthy controls crowd excludes malignant tumour medical history and oesophagus
Related disease medical history.All samples of this experiment obtain patient or its trustee signs informed consent, related to human body specimen
Research obtain Ethics Committee, Nanjing No.1 Hospital approval.
It is as follows that research patient is included in standard:
A. esophageal squamous cell carcinoma is turned out to be (according to the 7th edition (2009) cancer of the esophagus TNM stage standard through postoperative pathological
Carry out clinical stages)；
B. receive the patient of radical cure or palliative resction, while the person that excludes visiting before operation between hospital stay；
C. present illness history, family history, personal history and various inspections of data are perfect.
ESCC cancer patient's blood sample 35 in this research, normal human blood sample 28, ESCC tissue and cancer beside organism 55 are right.
2. clinical data acquires
The collection of clinical data is broadly divided into patients with esophageal squamous cell carcinoma and normal person's two parts, is both needed to comprising general information: packet
Include name, gender, age, occupation, medical history, family history, feminine menstrual history and the obsterical history etc. of patient.Esophageal squamous cell carcinoma is suffered from
Person is mainly from the data to arrange in hospital of patients with esophageal squamous cell carcinoma.Mainly include outside above-mentioned general information, should also include clinical special
Sign, whether there is or not transfer, tumor cell differentiation degree to swell for histological type, position, size, infiltration degree, lymph node including tumour
Tumor TNM stage, operating time, postoperative whether there is or not the data such as chemicotherapy and conventional auxiliary examination.Proprietary follow-up is obtained simultaneously
Agree to, inspection result data, which is improved, to be saved.
3. sample is collected
It is divided into two parts, a part is the collection of blood, preoperative primary (5ml/ pipe) use of taking a blood sample of every patients with esophageal squamous cell carcinoma
In subsequent experimental research.Generation haemolysis should be avoided in whole blood sample.It is ethylenediamine tetrem that wherein plasma sample, which collects heparin tube,
Acid disodium (Ethylenediaminetetraacetic acid, EDTA) anticoagulant tube, whole blood sample should complete blood plasma in 0.5h
Be collected by centrifugation and freeze；It is that routine biochemistry promotees solidifying pipe that serum sample, which collects heparin tube, and sample should complete the precipitation of serum in 2h
Centrifugation freezes.Whole blood sample removes cell component influence by two step centrifugal process, first in 2000g, 4 DEG C, is centrifuged 10min,
Then careful transfer supernatant is into new EP pipe, and then 12000g, is centrifuged 10min, finally completes blood plasma and serum sample by 4 DEG C
It dispenses (400 μ l/ pipe), is put into -80 DEG C of refrigerator long-term preservations.
Some is the collection of tissue specimen.Clinic, which is collected, receives radical cure or palliative operation excision because of esophageal squamous cell carcinoma
Patient tumor tissues and cancer by normal esophageal tissue specimen.Control group sample from same patient cancer beside organism (away from
From borderline tumor at least 5cm), pathologic examination after operation does not find that incisxal edge cancer cell remains.Normal group by fresh tumor tissue and cancer
It is cleaned twice with pre- cold saline as early as possible first after knitting in vitro, subsequent clip about soya bean size tissue has been put into RNA protection liquid
Cryopreservation tube in mix well.All samples after obtaining it is first quick-frozen in liquid nitrogen container for 24 hours, be then put into -80 DEG C of refrigerators in time
Medium-term and long-term preservation, to be used to subsequent experimental.
4. main primer
5. Total RNAs extraction
A. blood plasma Total RNAs extraction
Blood plasma, serum Total RNAs extraction use mir Vana PARIS Kit (Ambion1566, USA) kit, specific to walk
It is rapid as follows:
All reagents are both needed to be restored to and use at room temperature, take out and melt spare, eluent 95 in plasma/serum sample ice face
DEG C pre- stand-by heat；
400 μ l plasma/serum samples are added in the EP pipe of added isometric 2X Denaturing Solution,
Mixing fullys shake；
Isometric Acid-Phenol:Chloroform is added, concussion mixes 1min, and then 10000g is centrifuged at room temperature
5min；
Careful transfer upper strata aqueous phase is managed to new EP, and records transfer water phase volume, then adds 1.25 times of volumes 100%
Ethyl alcohol shifts lysate/alcohol mixture into EP pipe, after mixing well and passes through filter (10000g is centrifuged 30s)；
Add 700 1 cleaning filter of μ l mi RNA Wash Solution 1 time (10000g is centrifuged 15s), 500 μ are then added
2/3 cleaning filter of l Wash Solution 2 times (10000g is centrifuged 15s)；
RNA (10000g is centrifuged 30s) on the deoxyribonuclease washing filter apparatus of 50 95 DEG C of μ l preheating, it is -80 DEG C long-term
It saves；
Ultraviolet specrophotometer measures concentration of specimens and purity.
B. total tissue RNA extracts Trizol method and extracts tissue and cell total rna, and specific step is as follows:
After cleaning porcelain grinding body, with DEPC liquid soaked overnight (at least 12h), ultralow temperature then is put into after high pressure steam sterilization
Refrigerator cold-storage；
Prepare sterile no enzyme pipette tips, EP pipe, 4 DEG C of centrifuge it is spare to the cold
Cryopreserved tissue (about 50mg) is taken out out of ultra low temperature freezer, surface frozen stock solution is wiped, is filled in mortar (- 40 DEG C of pre-coolings)
Divide grinding, adds liquid nitrogen in grinding to prevent tissue RNA from degrading.Powdery is ground into tissue samples；
Every part of sample adds 1ml Trizol lysate, blows even rear transfer lysate with pipette tips and manages to EP, stands in ice face
5min decomposes it completely；
Chlorination imitates 0.2ml, acutely shakes 15s, stands 2min at room temperature, 4 DEG C, 12000g is centrifuged 15min；
Careful transfer upper strata aqueous phase avoids drawing cotton-shaped middle layer as far as possible into new EP pipe.Then add 0.5ml isopropyl
Alcohol, concussion mix, stand 10min at room temperature, and subsequent 4 DEG C, 12000g is centrifuged 10min.It is now spare with 75% ethyl alcohol；
It carefully discards supernatant, 75% ethyl alcohol 1ml concussion cleaning precipitating, subsequent 7500g, 4 DEG C of centrifugation 5min is added；
It finally discards supernatant, sucks residual liquid as far as possible with pipette tips, be air-dried at room temperature, according to precipitating size, 30-60 is added
μ l DEPC water dissolves RNA precipitate；
Ultraviolet specrophotometer measures concentration of specimens and purity.
5.RNA reverse transcription and real-time quantitative PCR
Using Prime-Script by RNA reverse transcription at cDNA TM One step RT-PCR kit, glyceraldehyde 3 phosphate is de-
Hydrogen enzyme (GAPDH) is used as internal contrast.Owned using ABI7500 system and SYBR Green PCR Master Mix
QRT-PCR reaction.For the multiple variation that circ-DLG1 is expressed in accurate validation ESCC, by the Ct value of calculating relative to from phase
With sample amplification GAPDH (Δ Ct=Cttested-CtGAPDH) standardize, and use-Δ Ct method come estimate variation
Value.In triplicate, all reactions are independent in triplicate to ensure the repeatabilities of all data for each sample.circ-DLG1
Cutoff value be -13.22, when detect-Δ Ct be greater than this value when for ESCC, specificity is 74.55%, sensibility
It is 74.55%.
6, data are analyzed:
As shown in Figures 1 to 4, using t inspection and Chi-square Test and the method circ-DLG1 and ESCC patient of survival analysis
Clinical stages data and the correlation of prognosis.Receiver Operating Characteristics' (ROC) curve is carried out to assess its diagnostic value.All systems
V.17.0, meter analysis is carried out using SPSS for Windows.For all as a result, P < 0.05, which is considered, statistics meaning
Justice.
The technical means disclosed in the embodiments of the present invention is not limited only to technological means disclosed in above embodiment, further includes
Technical solution consisting of any combination of the above technical features.It should be pointed out that for those skilled in the art
For, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also considered as
Protection scope of the present invention.
Sequence table
<110>Jiangsu ten thousand is at Co., Ltd, biomedical research institute
<120>in blood circ-DLG1 molecular marker diagnosis esophageal squamous cell carcinoma by stages in application
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 740
<212> DNA
<213> Homo sapiens
<400> 1
cttcttctca gcctgttgat aaccatgtta gcccatcttc cttcttgggc cagacaccag 60
catctccagc cagatactcc ccagtttcta aagcagtact tggagatgat gaaattacaa 120
gggaacctag aaaagttgtt cttcatcgtg gctcaacggg ccttggtttc aacattgtag 180
gaggagaaga tggagaagga atatttattt cctttatctt agccggagga cctgctgatc 240
taagtggaga gctcagaaaa ggagatcgta ttatatcggt aaacagtgtt gacctcagag 300
ctgctagtca tgagcaggca gcagctgcat tgaaaaatgc tggccaggct gtcacaattg 360
ttgcacaata tcgacctgaa gaatacagtc gttttgaagc taaaatacat gatttacggg 420
agcagatgat gaatagtagt attagttcag ggtcaggttc tcttcgaact agccagaagc 480
gatccctcta tgtcagagcc ctttttgatt atgacaagac taaagacagt gggcttccca 540
gtcagggact gaacttcaaa tttggagata tcctccatgt tattaatgct tctgatgatg 600
aatggtggca agccaggcag gttacaccag atggtgagag cgatgaggtc ggagtgattc 660
ccagtaaacg cagagttgag aagaaagaac gagcccgatt aaaaacagtg aaattcaatt 720
ctaaaacgag agataaaggg 740
<210> 2
<211> 20
<212> DNA
<213> Homo sapiens
<400> 2
tgcaccacca actgcttagc 20
<210> 3
<211> 21
<212> DNA
<213> Homo sapiens
<400> 3
ggcatggact gtggtcatga g 21
<210> 4
<211> 22
<212> DNA
<213> Homo sapiens
<400> 4
aaacgagaga taaagggctt ct 22
<210> 5
<211> 22
<212> DNA
<213> Homo sapiens
<400> 5
actgctttag aaactgggga gt 22
Claims (9)
1. in a kind of blood circ-DLG1 molecular marker diagnosis esophageal squamous cell carcinoma by stages in application, the circ-DLG1
The nucleotides sequence of molecular marker is classified as SEQ ID NO.1.
2. in blood as described in claim 1 circ-DLG1 molecular marker diagnosis esophageal squamous cell carcinoma by stages in application,
It is characterized by: for diagnose esophageal squamous cell carcinoma by stages in reagent be real-time quantitative PCR kit.
3. in a kind of blood as claimed in claim 1 or 2 circ-DLG1 molecular marker diagnosis esophageal squamous cell carcinoma by stages in
Application, it is characterised in that: devise 2 pairs according to the circ-DLG1 molecular marker that nucleotides sequence is classified as SEQ ID NO.1 and draw
Object is SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID for detecting the sequence, primer nucleotide sequences
NO.5。
4. in a kind of blood as described in claim 1 circ-DLG1 molecular marker diagnosis esophageal squamous cell carcinoma by stages in answer
With, it is characterised in that: the detection method of circ-DLG1 molecular marker, including sample Total RNAs extraction, RNA reverse transcription, in real time
Quantitative pcr amplification.
5. in a kind of blood as claimed in claim 4 circ-DLG1 molecular marker diagnosis esophageal squamous cell carcinoma by stages in answer
With, it is characterised in that: the detection method specific steps of circ-DLG1 molecular marker include that blood plasma Total RNAs extraction uses mir
Vana PARIS Kit kit, total tissue RNA extract Trizol method and extract tissue and cell total rna, and second step uses
For Prime-Script by RNA reverse transcription at cDNA TM One step RT-PCR kit, glyceraldehyde 3 phosphate dehydrogenase is used as inside
Control, carries out all qRT-PCR reactions using ABI7500 system and SYBR Green PCR Master Mix.
6. in a kind of blood as claimed in claim 5 circ-DLG1 molecular marker diagnosis esophageal squamous cell carcinoma by stages in answer
Include with, it is characterised in that: specific steps,
The first step, Total RNAs extraction,
A. blood plasma Total RNAs extraction, blood plasma, serum Total RNAs extraction use mir Vana PARIS Kit, Ambion1566, USA examination
Agent box, the specific steps are as follows:
All reagents are both needed to be restored to and use at room temperature, take out plasma/serum sample ice face on melt it is spare, 95 DEG C of eluent
Pre- stand-by heat；
400 μ l plasma/serum samples are added in the EP pipe of added isometric 2X Denaturing Solution, sufficiently
Concussion mixes；
Isometric Acid-Phenol:Chloroform is added, concussion mixes 1min, and then 10000g is centrifuged 5min at room temperature；
It shifts upper strata aqueous phase and manages to new EP, and record transfer water phase volume, then add 1.25 times of 100% ethyl alcohol of volume to EP
Guan Zhong shifts lysate/alcohol mixture by filter after mixing well, 10000g is centrifuged 30s；
Add 700 μ l mi RNA Wash Solution 1 cleaning filter 1 time, 10000g is centrifuged 15s, and 500 μ l are then added
2/3 cleaning filter of Wash Solution 2 times, 10000g are centrifuged 15s；
RNA on the deoxyribonuclease washing filter apparatus of 50 95 DEG C of μ l preheating, 10000g are centrifuged 30s, -80 DEG C of long-term preservations；
Ultraviolet specrophotometer measures concentration of specimens and purity；
B. total tissue RNA extracts Trizol method and extracts tissue and cell total rna, and specific step is as follows:
After cleaning porcelain grinding body, with DEPC liquid soaked overnight, then at least 12h is put into ultra low temperature freezer after high pressure steam sterilization
Refrigeration；
Prepare sterile no enzyme pipette tips, EP pipe, 4 DEG C of centrifuge it is spare to the cold；
Cryopreserved tissue is taken out out of ultra low temperature freezer, surface frozen stock solution is wiped, is fully ground in mortar, adds liquid nitrogen in grinding
To prevent tissue RNA from degrading；
Powdery is ground into tissue samples；
Every part of sample adds 1ml Trizol lysate, blows even rear transfer lysate with pipette tips and manages to EP, stands in ice face
5min decomposes it completely；
Chlorination imitates 0.2ml, acutely shakes 15s, stands 2min at room temperature, 4 DEG C, 12000g is centrifuged 15min；
Careful transfer upper strata aqueous phase avoids drawing cotton-shaped middle layer as far as possible into new EP pipe；Then add 0.5ml isopropanol, shake
It swings mixing, stand 10min at room temperature, subsequent 4 DEG C, 12000g is centrifuged 10min；It is now spare with 75% ethyl alcohol；
It discards supernatant, 75% ethyl alcohol 1ml concussion cleaning precipitating, subsequent 7500g, 4 DEG C of centrifugation 5min is added；
It finally discards supernatant, sucks residual liquid with pipette tips, be air-dried at room temperature, according to precipitating size, 30-60 μ l DEPC water is added
Dissolve RNA precipitate；
Ultraviolet specrophotometer measures concentration of specimens and purity；
Second step, RNA reverse transcription and real-time quantitative PCR,
Using Prime-Script by RNA reverse transcription at cDNA TM One step RT-PCR kit, glyceraldehyde 3 phosphate dehydrogenase
(GAPDH) it is used as internal contrast；All qRT-PCR are carried out using ABI7500 system and SYBR Green PCR Master Mix
Reaction.
7. a kind of for diagnosing the real time quantitative PCR detecting reagent kit of esophageal squamous cell carcinoma by stages, it is characterised in that: including according to nucleosides
Acid sequence designs and synthesizes out for the circ-DLG1 molecular marker of SEQ ID NO.1 and is specifically used for real-time quantitative PCR
Detection primer.
8. as claimed in claim 7 for diagnosing the real time quantitative PCR detecting reagent kit of esophageal squamous cell carcinoma by stages, feature exists
In: SEQ ID NO.4, SEQ ID NO.5 in specific primer such as sequence table.
9. the circ-DLG1 molecular marker that a kind of nucleotides sequence is classified as SEQ ID NO.1.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110093419A (en) * | 2019-04-26 | 2019-08-06 | 北京大学深圳医院 | Application, kit and the pharmaceutical composition of circular rna and its application |
| CN111269985A (en) * | 2020-03-20 | 2020-06-12 | 安阳市肿瘤医院 | Application of hsa _ circRNA6448-14 in diagnosis and prognosis prediction of esophageal squamous cell carcinoma |
| CN111304322A (en) * | 2019-12-19 | 2020-06-19 | 徐州市肿瘤医院 | Preparation method of kit for joint detection of esophageal cancer by four novel circRNAs |
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| CN103923984A (en) * | 2014-03-27 | 2014-07-16 | 南京市第一医院 | Development and application of esophageal squamous carcinoma detection kit |
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| CN103923984A (en) * | 2014-03-27 | 2014-07-16 | 南京市第一医院 | Development and application of esophageal squamous carcinoma detection kit |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110093419A (en) * | 2019-04-26 | 2019-08-06 | 北京大学深圳医院 | Application, kit and the pharmaceutical composition of circular rna and its application |
| CN110093419B (en) * | 2019-04-26 | 2023-10-27 | 北京大学深圳医院 | Application of circular RNA, kit and pharmaceutical composition and application thereof |
| CN111304322A (en) * | 2019-12-19 | 2020-06-19 | 徐州市肿瘤医院 | Preparation method of kit for joint detection of esophageal cancer by four novel circRNAs |
| CN111304322B (en) * | 2019-12-19 | 2022-04-08 | 王强 | Preparation method of kit for joint detection of esophageal cancer by four novel circRNAs |
| CN111269985A (en) * | 2020-03-20 | 2020-06-12 | 安阳市肿瘤医院 | Application of hsa _ circRNA6448-14 in diagnosis and prognosis prediction of esophageal squamous cell carcinoma |
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