CN104878010A - Application of long-chain non-coding RNA AFAP1-AS1 in preparation of auxiliary diagnosis and prognosis judgment preparations for breast cancer - Google Patents
Application of long-chain non-coding RNA AFAP1-AS1 in preparation of auxiliary diagnosis and prognosis judgment preparations for breast cancer Download PDFInfo
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Abstract
The invention discloses application of long-chain non-coding RNA AFAP1-AS1 in preparation of auxiliary diagnosis and prognosis judgment preparations for breast cancer. The long-chain non-coding RNA can be used for preparing the auxiliary diagnosis and prognosis judgment preparations of patients with breast cancer, particularly for preparing kits of predicting the prognosis of patients with breast cancer with in-situ hybridization detecting method. Through study, AFAP1-AS1 expression is proved to be up-regulated in breast cancer tissues, and the prognosis of patients with breast cancer with high expression of AFAP1-AS1 is poorer than that of patients with breast cancer with low expression of AFAP1-AS1, so that using the expression of AFAP1-AS1 for the prognosis prediction of patients with breast cancer can provide powerful molecular biological foundation for the prognosis of patients with breast cancer, and has profound clinical significance and an important popularization and application prospect.
Description
Technical field
The invention belongs to oncomolecularbiology field, be specifically related to the in situ hybridization probe of long-chain non-coding RNA AFAP1-AS1, detection reagent and application method thereof.
Background technology
The Human Genome Project and follow-up DNA element encyclopedia plan (The Encyclopedia of DNA Elements Project thereof, ENCODE) achievement in research shows, protein coding gene sequence only accounts for the 1-3% of human genomic sequence, and the transcribed sequence of the overwhelming majority is long-chain non-coding RNA (Long non-coding RNA, lncRNA) in human genome.LncRNA is present in various biology widely, and along with the rising of biological complex degree, in genome, the ratio of lncRNA sequence also correspondingly increases, and lncRNA is significant in organic evolution process in prompting.Along with lncRNA is constantly found, their function is annotated gradually, scientists finds that lncRNA extensively and actively participates in the function controlling of vital movement different aspects, as a brand-new field, has become new forward position, international life science field and focus.
LncRNA can be used as the various ways such as signal (signal), induction (guide), bait (decoy) or support (scaffold) molecule of functional protein, in the expression of chromatin reconstruct, genetic transcription, translation and the multiple levels regulatory gene such as protein modified, and play irreplaceable role comprising in the basic physiological processes such as growth, immunity, reproduction, the more important thing is, expression and the functional disorder of lncRNA be closely connected with the mankind's various diseases comprising malignant tumour together with.Therefore, the function of further investigation lncRNA, disclose the genetic information transfer mode and expression regulation network that are mediated by lncRNA, not only can again annotate from the angle beyond protein coding gene and illustrate genomic structure and fuction, deeply find essence and the rule of vital movement, also be expected to the pathogeny comprising multiple mankind's common disease of tumour from a new visual angle understanding, and provide new molecular marker and therapy target for the Clinics and Practices of these diseases.
The sickness rate of mammary cancer occupies first of female malignant, its sickness rate is started always in rising trend from late 1970s, the U.S. 8 women just have 1 people in life and suffer from breast cancer, and the also sustainable growth always in recent years of China's breast cancer incidence, become the great public health problem of current social, mammary cancer easily shifts, and patients with terminal prognosis is poor.Research shows, the generation development of this tumour is polygene participation, multi-step, multistage complex process, and LncRNA may also play an important role in the generation evolution of mammary cancer.We utilize lncRNA chip recently, construct the express spectra of lncRNA in breast cancer biopsy tissue and normal reference sample, therefrom screen the lncRNAs of some differential expressions in mammary cancer, verify through enlarged sample real-time fluorescence quantitative PCR, confirm that lncRNA AFAP1-AS1 (NCBI accession number:NR_026892) significantly raises at breast carcinoma, the detection preparation for this lncRNA may be used for the auxiliary diagnosis of mammary cancer.Subsequently, we increase sample further, have in the mammary cancer paraffin file sample of Clinical Follow-up data in 77 examples, the expression level of AFAP1-AS1 is have detected by the method for in situ hybridization (in situ hybridization), find that AFAP1-AS1 expresses high patient and more easily shifts, its survival time is shorter than this lncRNA and expresses low patient, therefore also may be used for the Index for diagnosis of mammary cancer for the detection preparation of this lncRNA.
Summary of the invention
The object of this invention is to provide the in situ hybridization probe of long-chain non-coding RNA AFAP1-AS1, reagent and application method thereof, utilize long-chain non-coding RNA AFAP1-AS1 sequence can for the preparation of the preparation of Computer-aided Diagnosis of Breast Cancer and Index for diagnosis.
The application method of LncRNA AFAP1-AS1, for the preparation of the preparation of mammary cancer outcome prediction, the sequence of this long-chain non-coding RNA AFAP1-AS1 is as shown in SEQ NO:1.
The preparation of described mammary cancer outcome prediction is in situ hybridization detection reagent.
Described in situ hybridization detection reagent comprises the oligonucleotide probe detecting AFAP1-AS1 expression in situ hybridization, and sequence is as follows:
AFAP1-AS1 probe 1:5 '-ATTCCTTTATTTTATGGGATGTTCTGTAGGGAGTT-3 ', SEQ NO:2,
AFAP1-AS1 probe 2:5 '-TAGAAATGAGAAAAGAATCACCAAGAGAGTAAGCA-3 ', SEQ NO:3,
AFAP1-AS1 probe 3:5 '-TCCCTACAGCTAGTTTCCTCTTCATTTATTCATTT-3 ', SEQ NO:4.
Described in situ hybridization detection reagent is test kit.
Containing the above-mentioned oligonucleotide probe detecting AFAP1-AS1 expression in situ hybridization in test kit:
AFAP1-AS1 probe 1:5 '-ATTCCTTTATTTTATGGGATGTTCTGTAGGGAGTT-3 '
AFAP1-AS1 probe 2:5 '-TAGAAATGAGAAAAGAATCACCAAGAGAGTAAGCA-3 '
AFAP1-AS1 probe 3:5 '-TCCCTACAGCTAGTTTCCTCTTCATTTATTCATTT-3 '
Positive control probe (detecting house-keeping gene GAPDH):
GAPDH probe 1:5'-CCACTTTACCAGAGTTAAAAGCAGCCCTGG-3', SEQ NO:5
GAPDH probe 2:5'-CAGTAGAGGCAGGGATGATGTTCTGGAGAG-3', SEQ NO:6
GAPDH probe 3:5'-GTCAGAGGAGACCACCTGGTGCTCAGTGTA-3', SEQ NO:7.
Also contain in test kit: digoxin oligonucleotide tailing reagent, anti-digoxin-horseradish peroxidase complex detection reagent, DAB staining reagent;
Other conventional biochemical reagent, comprise 20x sodium citrate buffer (saline sodium citrate, SSC), T 500 (Dextran sulphate), deionized formamide (Deionized Formamide), polyadenylic acid (polyadenylic acid, Poly A), poly deoxyadenylic acid (polydeoxyadenylic acid, Poly dA), frog essence DNA (the denatured and sheared salmon sperm DNA that sex change is sheared, ssDNA), yeast transfer RNA (yeast t-RNA, tRNA), dithiothreitol (DTT) (DTT), Deng 50x Han Shi damping fluid (Denhardts ' s solution), phosphoric acid buffer (PBS buffer), stomach en-K, bovine serum albumin (BSA), trolamine (TEA), acetic anhydride, block reagent (Blocking reagent agent), TNB damping fluid (that is: the 0.1M Tris-Cl+0.15M NaCl+0.5% of pH7.5 blocks reagent), TNT damping fluid (that is: the 0.1M Tris-Cl+0.15M NaCl+0.05%Tween 20 of pH7.5).
By in situ hybridization, we find that in the mammary cancer paraffin organization sample filed the prognosis of the expression of AFAP1-AS1 and patient with breast cancer is closely related, it is shorter that AFAP1-AS1 expresses the high survival of patients time, and prompting AFAP1-AS1 can be used as the molecular marker of Prognosis in Breast Cancer prediction.The present invention is that the auxiliary diagnosis of mammary cancer and prognosis prediction provide strong biology tool, has far-reaching clinical meaning and important popularizing application prospect.
Accompanying drawing explanation
Fig. 1 is the expression of Real-Time Fluorescent Quantitative PCR Technique checking lncRNA AFAP1-AS1 in mammary cancer and normal galactophore tissue, significantly improves in the expression ratio normal galactophore tissue (N) of AFAP1-AS1 in mammary cancer (T);
Fig. 2 is that in situ hybridization detects the expression of AFAP1-AS1 in mammary cancer;
The high expression level (left side) of AFAP1-AS1 is detected, the expression lower (right side) of AFAP1-AS1 in remaining 16 routine breast cancer tissue in mammary cancer (61 examples in 77 routine breast cancer tissues) 79.22%;
Fig. 3 is the expression of AFAP1-AS1 in mammary cancer and the relation of patient with breast cancer's prognosis;
In mammary cancer, the high expression level of AFAP1-AS1 is relevant with the poor prognosis of patient with breast cancer, namely survival time (the Over-all survival that the patient of AFAP1-AS1 high expression level is total, left) and the patient that will be starkly lower than the low expression of AFAP1-AS1 without recurrence survival time (Relapse-free sruvival, right) or not express.
Embodiment
The present invention is further illustrated below in conjunction with embodiment, and unrestricted the present invention.
Embodiment 1, quantitative real-time PCR detects and confirms that AFAP1-AS1 raises at breast carcinoma
1. materials and methods:
Collect 17 routine normal galactophore tissues and 104 routine breast cancer tissues, extracted total RNA, 2 μ g RNA become after cDNA through reverse transcription, carry out real-time fluorescence quantitative PCR.AFAP-AS1 primer is 5 '-AATGGTGGTAGGAGGGAGGA-3 ', SEQ NO:8 and 5 '-CACACAGGGGAATGAAGAGG-3 ', SEQ NO:9.
GAPDH primer for contrasting is 5 '-ACCACAGTCCATGCCATCAC-3 ', SEQ NO:10 and 5 '-TCCACCACCCTGTTGCTGTA-3 ', SEQ NO:11,
Real-time fluorescence quantitative PCR reaction system
Real-time fluorescence quantitative PCR reactions steps
Reaction terminates amplification curve and the melting curve of rear confirmation real-time fluorescence quantitative PCR, the expression intensity of each gene, according to after CT value (threshold cycle values), reference gene (GAPDH) markization, adopts group t-test inspection to calculate P value.
2. result
AFAP1-AS1 does not express or expresses very low in normal control tissue, and in breast cancer tissue high expression level P=0.006 (Fig. 1)
Embodiment 2, in situ hybridization detects and finds that the expression of AFAP1-AS1 in mammary cancer is relevant to patient's prognosis
1. MATERIALS METHODS
1.1 design and synthesize hybridization probe
In order to the expression adopting in-situ hybridization method to detect AFAP1-AS1, we devise oligonucleotide probe and each 3 of positive control two groups of in situ hybridization oligonucleotide probes of detecting AFAP1-AS1 expression in situ hybridization.
The oligonucleotide probe of AFAP1-AS1 expression is detected in situ hybridization:
AFAP1-AS1 probe 1:5 '-ATTCCTTTATTTTATGGGATGTTCTGTAGGGAGTT-3 '
AFAP1-AS1 probe 2:5 '-TAGAAATGAGAAAAGAATCACCAAGAGAGTAAGCA-3 '
AFAP1-AS1 probe 3:5 '-TCCCTACAGCTAGTTTCCTCTTCATTTATTCATTT-3 '
Positive control probe (detecting house-keeping gene GAPDH):
GAPDH probe 1:5'-CCACTTTACCAGAGTTAAAAGCAGCCCTGG-3'
GAPDH probe 2:5'-CAGTAGAGGCAGGGATGATGTTCTGGAGAG-3'
GAPDH probe 3:5'-GTCAGAGGAGACCACCTGGTGCTCAGTGTA-3'
Chemical synthesis process is adopted to synthesize each gene specific oligonucleotides probe sequence of above-mentioned design.
1.2 oligonucleotide probe labelling kits and in situ hybridization detection reagent
Digoxin oligonucleotide tailing reagent (Dig Oligonucleitide Tailing Kit 2
ndgeneration, Roche company), anti-digoxin-horseradish peroxidase complex detection kit (Anti-Digoxigenin-POD, Fab fragments, Roche company), strengthen the TSA signal amplifying system (TSA of expressed in situ detection signal
tMbiotin System, NEL700 test kit, PerkinElmer company), DAB staining kit (Beijing Zhong Shan company), 20x sodium citrate buffer (saline sodium citrate, SSC), T 500 (Dextran sulphate), deionized formamide (Deionized Formamide), polyadenylic acid (polyadenylic acid, Poly A), poly deoxyadenylic acid (polydeoxyadenylic acid, Poly dA), frog essence DNA (the denatured and sheared salmon sperm DNA that sex change is sheared, ssDNA), yeast transfer RNA (yeast t-RNA, tRNA), dithiothreitol (DTT) (DTT), Deng 50x Han Shi damping fluid (Denhardts ' s solution), phosphoric acid buffer (PBS buffer), stomach en-K, bovine serum albumin (BSA), trolamine (TEA), TNB Buffer (0.1M Tris-HCl, pH7.5, 0.15M NaCL, 0.5%Blocking Reagent), TNT Buffer (0.1M Tris-HCl, pH7.5, 0.15M NaCL, 0.05%Tween 20), acetic anhydride, block reagent (Blocking reagent agent, Roche company).
1.3 other main agents and materials
Dehydrated alcohol, 90% alcohol, 70% alcohol, 50% alcohol, turps, distilled water, PBS damping fluid (pH7.2 ~ 7.4, NaCl 137mmol/L, KCl 2.7mmol/L, Na
2hPO
44.3mmol/L, KH
2pO
41.4mmol/L); 3% methyl alcohol-hydrogen peroxide solution (80% methyl alcohol and the configuration of 30% hydrogen peroxide); 0.01mol/L citrate buffer (citrate buffer, CB, pH6.0 ± 0.1,9ml 0.1M citric acid solution and 41ml 0.1M sodium citrate solution to add in 450ml distilled water after provisional configuration correction work liquid pH value again); 0.1% trypsinase; Hematorylin; 1% hydrochloride alcohol (configuration of 1ml concentrated hydrochloric acid+99ml 70% alcohol); Mounting glue (PTS Cure Mount II); Special cap slide (480 × 240mm
2) customize in Zhengzhou Glassware Factory.Leica low melting point (58 DEG C) paraffin, domestic beeswax, raw spirit, dimethylbenzene, 10% neutral paraformaldehyde (0.01mol/L, pH7.4, DEPC distilled water and PBS buffer), Hematorylin, Yihong, neutral mounting natural gum, cover glass, slide glass.
1.4 label probe
Utilize 3-tailing DIG Olignucleutide Kit to carry out oligonucleotide probe mark, reaction system is as follows.
100pmol oligonucleotide+ddH
2O＝9μl(control:control oligonucleutide 5μl+ddH
2O 4μl)
Mixing, slightly centrifugal.37 DEG C of water-bath 30min, add 2 μ l EDTA (0.2M, PH 8.0) stopped reactions.
Purifying after 1.5 oligonucleotide probe marks
In order to increase the purity of label probe, need carry out purifying to the probe marked, concrete operations are as follows:
1) probe reaction mixture (22 μ l)+2.5 μ l 4M LiCL+75 μ cold ethanol of l 100% (-20 DEG C).
2)-70 DEG C of precipitations 60min, or-20 DEG C of 2h.
3) 13.000 × g, 4 DEG C of centrifugal 15min.
4) supernatant is abandoned, by 70% (V/V) washing with alcohol that 50 μ l are ice-cold.
5) 13.000xg 4 DEG C, centrifugal 5min.
6) supernatant is abandoned, vacuum 4 DEG C of dryings.
7) with the heavy molten probe of aseptic double-distilled water.
1.6 in situ hybridizations detect the expression of AFAP1-AS1 in file paraffin section
Paraffin section hybridization pre-treatment
1) 4 DEG C of paraffin sections preserved are placed in 58 DEG C of roasting sheet 30min, melted surface paraffin.
2) dimethylbenzene dewaxes 3 × 5min successively.
3) step ethanol wash, 100% alcohol 2 × 2min → 95% alcohol 1 × 5min → 70% alcohol 1 × 5min → 50% alcohol 1 × 5min → DEPC water washing 2 × 3min → DEPC-PBS washs 2 × 5min.
4) 300 μ l stomach en-K (10 μ g/ml) are dripped in section, 37 DEG C of digestion 20min.
5) cut into slices and wash 1min, stopped reaction into PBS (0.1M PBS+2mg/ml L-glutamic acid).
6) cut into slices into 0.2N HCL, in 37 DEG C of reaction 20-30min, increase the permeability of tissue.
7) section fixes 10min, room temperature afterwards with 4% paraformaldehyde (0.1M PBS dissolves).
8) organize positive intensity for hybridization to increase, acetyl process is carried out to section.Cut into slices into 0.25% diacetyl oxide Buffer I (0.1M trolamine), room temperature 10min.
9) 1M PBS washs 2 × 5min.
Prehybridization and hybridization
Prehybridization :-20 DEG C of prehybridization solutions preserved, be first placed in 37 DEG C and hatch 60min, the consumption of prehybridization solution is 50 μ l, and Parafilm carries out lid section, prehybridization 2 hours in 37 DEG C of wet boxes.(prehybridization solution composition comprises: 2XSSC, 10%Dextran sulphate, 1X Denhardt ' s solution, 50mM Phosphate Buffer (PH 7.0), 50mM DTT, 250 μ l, 100 μ g/ml poly A, 5 μ g/ml poly dA, 250 μ g/ml yeast t-RNA, 500 μ g/ml ssDNA, 47%Deionized formamide).
1) remove Parafilm, get rid of prehybridization solution, section is placed in 2 × SSC 5min.
2) hybridization: 37 DEG C of hybridized overnight (18-20h).Each section adds 250 μ l hybridization solutions and carries out lid with Parafilm.Add corresponding probe in prehybridization solution and just become hybridization solution.Hybridization solution is prepared when prehybridization, and place 37 DEG C and hatch, probe is fully dissolved in hybridization solution, this experiment many oligonucleotide probes mix, and is mixed with probe hybridization liquid by each probe 500ng/ml concentration.Digoxin tailing labelling kit label probe concentration basis: the concentration of each probe is compared by developing the color during detection reaction when itself and the positive quantitative probe and the naked probe mark reaction theory probe output of 100pmol 30 bases is that 900ng two kinds of standards carry out the concentration that COMPREHENSIVE CALCULATING goes out label probe.
3) post-hybridization washing, section immersion 2 × SSC, 10min, throws off Parafilm.Shake washing on shaking table successively, 2 × SSC (0.5%SDS), 2 × 15min → 0.25 × SSC (0.5%SDS), 2 × 15min.
Color developing detection reaction after hybridization
1) Anti-Digoxigenin-POD is adopted to detect digoxigenin-probe with mRNA in conjunction with mixture; TSA amplification system strengthens the positive signal of in situ hybridization reaction solution reaction, and DAB develops the color.
2) section goes in TNT damping fluid, 3 × 5min.
3) drip TNB and block damping fluid, 300 μ l/TMAs, room temperature, 30min.
4) unnecessary blocker is sucked, the Anti-Digoxigenin-POD (TBS+0.1%Triton X-100+1% blocker) of 1:100 dilution, room temperature 4 hours.
5) TNT Buffer (0.1M Tris-CL, pH7.5,0.15M NaCL, 0.05%Tween 20) washing, 3x5min.
6) section drips signal and amplify reagent Biotinyl Tyamid, 300 μ l/TMAs, (Biotinyl Tyramid stock solution: Biotinyl Tyramid is dissolved in 0.2ml DMSO, Biotinyl Tyramid working fluid: 1 × diluent, 1:50 dilutes Biotinyl Tyramid stock solution), room temperature 10 minutes.
7) TNT washes, 3 × 5min.
8) section drips SA-HRP (strepto-avidin-horseradish peroxidase), 300 μ l/TMAs, room temperature 30min.
9) TNT washes, 3 × 5min.
10) aquae destillata washing, 1 × 1min.
11) DAB colour developing, controls color reaction under microscope.
12) Hematorylin is redyed,
13) alcohol step dehydration, chip drying.
14) mounting glue is dripped, the cover glass cover plate of dimension, crosslinked section 1min under ultraviolet lamp.
1.7 results judge and standard
Applied optics microscope is observed respectively under low power and high power lens, and first the positive expression signal of object observing RNA is in the intracellular location of object observing: be positioned at nucleus, cytoplasm or cytolemma.
Carry out comprehensive grading with cell count two kinds of standards of the intensity of this detection rna expression position positive signal and positive expression respectively again, judging criterion is: (1) judges according to positive cell dyeing intensity: a. cell dye-free, remembers 0 point; B. cell dyes light brown is the weak positive, remembers 1 point; C. cell is dyed brown and without background coloration, or cell is dyed dark-brown and has light brown background to be moderate positive, remembers 2 points; D. cell is dyed dark-brown and is strong positive without background coloration, remembers 3 points.(2) express number score according to positive cell: the no positive cell expressing of a., remember 0 point; B. positive expression cell count≤25%, remembers 1 point; C.25% < positive cell number < 50%, remembers 2 points; D. positive expression cell count >=50%, remembers 3 points.
In order to reduce the subjective factor of appraisal result as far as possible, carried out separately judging and marking by one of above-mentioned standard respectively by two pathology experts, then both scorings be multiplied, result is: 1. 0 point of person finally counts 0 point, thinks negative and expresses; 2. 1 point and 2 points of persons finally count 1 point, think weak positive expression; 3. 3 points and 4 points of persons finally count 2 points, think that moderate positive is expressed; 4. 6 assign to 9 points of persons and finally count 3 points, think that strong positive is expressed.
1.8 analyze and statistical software
Application SPSS13.0 statistical software carries out statistical analysis to experimental result, compares between two and uses χ
2test or Fisher exact test, correlation analysis adopts Spearmen correlation method; P < 0.05 i.e. difference has statistical significance.Survival curve analysis adopts Kaplan-Meier method and log-rank test; Multivariate analysis adopts Cox ' s proportional hazards model; P < 0.05 i.e. difference has statistical significance.
2 results
The expression of 2.1 AFAP1-AS1 in mammary cancer
AFAP1-AS1 (61/77 example) in the breast cancer tissue of 79.22% has expression (Fig. 2 is left), the expression of AFAP1-AS1 lower (Fig. 2 is right) in remaining 16 routine breast cancer tissue.
Patient with breast cancer's prognosis of 2.2 AFAP1-AS1 high expression levels is poor
We have carried out Effect of follow-up visit by telephone to 77 routine patient with breast cancers, detailed inquired them start time, treatment situation, with or without recurrence, with or without suffering from other diseases, recurrence and death time etc. again, and register survival time and state, and to the survival analysis that the expression of AFAP1-AS1 in breast cancer tissue and the survival time of patient and state are carried out, find the patient (Fig. 3) that AFAP1-AS1 high expression level patient is significantly shorter than AFAP1-AS1 low expression the mean survival time or does not express.Illustrate that AFAP1-AS1 is a molecule marker relevant to Prognosis in Breast Cancer, this lncRNA expresses high, patient's poor prognosis.
Claims (10)
1. the application method of long-chain non-coding RNA AFAP1-AS1, is characterized in that, for the preparation of the preparation of Computer-aided Diagnosis of Breast Cancer or outcome prediction, the sequence of this long-chain non-coding RNA AFAP1-AS1 is as shown in SEQ NO:1.
2. application method according to claim 1, is characterized in that, described Computer-aided Diagnosis of Breast Cancer or the preparation of outcome prediction are in situ hybridization detection reagent.
3. application method according to claim 2, is characterized in that,
Described in situ hybridization detection reagent comprises the oligonucleotide probe detecting AFAP1-AS1 expression in situ hybridization, and sequence is as follows:
AFAP1-AS1 probe 1:5 '-ATTCCTTTATTTTATGGGATGTTCTGTAGGGAGTT-3 '
AFAP1-AS1 probe 2:5 '-TAGAAATGAGAAAAGAATCACCAAGAGAGTAAGCA-3 '
AFAP1-AS1 probe 3:5 '-TCCCTACAGCTAGTTTCCTCTTCATTTATTCATTT-3 '.
4. the application method according to Claims 2 or 3, is characterized in that,
Described in situ hybridization detection reagent is test kit.
5. application method according to claim 4, is characterized in that,
Containing the oligonucleotide probe detecting AFAP1-AS1 expression in situ hybridization in test kit:
AFAP1-AS1 probe 1:5 '-ATTCCTTTATTTTATGGGATGTTCTGTAGGGAGTT-3 '
AFAP1-AS1 probe 2:5 '-TAGAAATGAGAAAAGAATCACCAAGAGAGTAAGCA-3 '
AFAP1-AS1 probe 3:5 '-TCCCTACAGCTAGTTTCCTCTTCATTTATTCATTT-3 ';
Also containing positive control probe in test kit,
GAPDH probe 1:5'-CCACTTTACCAGAGTTAAAAGCAGCCCTGG-3'
GAPDH probe 2:5'-CAGTAGAGGCAGGGATGATGTTCTGGAGAG-3'
GAPDH probe 3:5'-GTCAGAGGAGACCACCTGGTGCTCAGTGTA-3'.
6. application method according to claim 4, is characterized in that,
Also contain in test kit: digoxin oligonucleotide tailing reagent, anti-digoxin-horseradish peroxidase complex detection reagent, DAB staining reagent;
Other conventional biochemical reagent, comprises 20x SSC, T 500, deionized formamide, polyadenylic acid, poly deoxyadenylic acid, the frog essence DNA that sex change is sheared, yeast transfer RNA, dithiothreitol (DTT), 50xDenhardts ' s solution, PBS buffer, stomach en-K, bovine serum albumin, trolamine, acetic anhydride
The 0.1M Tris-Cl+0.15M NaCl+0.5% of TNB damping fluid: pH7.5 blocks reagent;
The 0.1M Tris-Cl+0.15M NaCl+0.05%Tween 20 of TNT damping fluid: pH7.5.
7., for the preparation of the oligonucleotide probe of the in situ hybridization detection reagent of Computer-aided Diagnosis of Breast Cancer or outcome prediction, sequence is as follows:
AFAP1-AS1 probe 1:5 '-ATTCCTTTATTTTATGGGATGTTCTGTAGGGAGTT-3 '
AFAP1-AS1 probe 2:5 '-TAGAAATGAGAAAAGAATCACCAAGAGAGTAAGCA-3 '
AFAP1-AS1 probe 3:5 '-TCCCTACAGCTAGTTTCCTCTTCATTTATTCATTT-3 '.
8., for the in situ hybridization detection reagent of Computer-aided Diagnosis of Breast Cancer or outcome prediction, be the test kit including oligonucleotide probe according to claim 7.
9. in situ hybridization detection reagent according to claim 8, is characterized in that, also containing positive control probe in test kit:
GAPDH probe 1:5'-CCACTTTACCAGAGTTAAAAGCAGCCCTGG-3'
GAPDH probe 2:5'-CAGTAGAGGCAGGGATGATGTTCTGGAGAG-3'
GAPDH probe 3:5'-GTCAGAGGAGACCACCTGGTGCTCAGTGTA-3'.
10. in situ hybridization detection reagent according to claim 8 or claim 9, is characterized in that,
Also contain in test kit: digoxin oligonucleotide tailing reagent, anti-digoxin-horseradish peroxidase complex detection reagent, DAB staining reagent;
Other conventional biochemical reagent, comprises 20x SSC, T 500, deionized formamide, polyadenylic acid, poly deoxyadenylic acid, the frog essence DNA that sex change is sheared, yeast transfer RNA, dithiothreitol (DTT), 50xDenhardts ' s solution, PBS buffer, stomach en-K, bovine serum albumin, trolamine, acetic anhydride
The 0.1M Tris-Cl+0.15M NaCl+0.5% of TNB damping fluid: pH7.5 blocks reagent;
The 0.1M Tris-Cl+0.15M NaCl+0.05%Tween 20 of TNT damping fluid: pH7.5.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106222242A (en) * | 2016-01-29 | 2016-12-14 | 复旦大学 | Long-chain non-coding RNA-APOC1P1-3 gene and for preparing the purposes in target spot mark |
| CN106222169A (en) * | 2015-12-13 | 2016-12-14 | 周平 | Long-chain non-coding RNA APOC1P1-3 gene and application thereof |
| CN109762821A (en) * | 2019-02-28 | 2019-05-17 | 中山大学孙逸仙纪念医院 | Inhibit the RNA interfering of AFAP1-AS1 expression and increases the application in radiotherapy in breast cancer sensibility |
-
2015
- 2015-05-22 CN CN201510266789.XA patent/CN104878010B/en active Active
Non-Patent Citations (4)
| Title |
|---|
| ANDREA ET AL: "AFAP-110 Is Required for Actin Stress Fiber Formation and Cell Adhesion in MDA-MB-231 Breast Cancer Cells", 《CELL. PHYSIOL》 * |
| HAO ET AL: "Upregulated long non-coding RNA AFAP1-AS1 expression is associated with progression and poor prognosis of nasopharyngeal carcinoma", 《ONCOTARGET》 * |
| LIPOVICH ET AL: "NR_026892.1", 《GENBANK》 * |
| TANG ET AL: "Long Noncoding RNAs-Related Diseases, Cancers, and Drugs", 《THE SCIENTIFICWORLD JOURNAL》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106222169A (en) * | 2015-12-13 | 2016-12-14 | 周平 | Long-chain non-coding RNA APOC1P1-3 gene and application thereof |
| CN106222242A (en) * | 2016-01-29 | 2016-12-14 | 复旦大学 | Long-chain non-coding RNA-APOC1P1-3 gene and for preparing the purposes in target spot mark |
| CN106222242B (en) * | 2016-01-29 | 2019-10-29 | 复旦大学 | Long-chain non-coding RNA-APOC1P1-3 gene and its in the purposes being used to prepare in target spot marker |
| CN109762821A (en) * | 2019-02-28 | 2019-05-17 | 中山大学孙逸仙纪念医院 | Inhibit the RNA interfering of AFAP1-AS1 expression and increases the application in radiotherapy in breast cancer sensibility |
| CN109762821B (en) * | 2019-02-28 | 2021-10-01 | 中山大学孙逸仙纪念医院 | Interfering RNA for inhibiting expression of AFAP1-AS1 and application of interfering RNA in increasing sensitivity of breast cancer radiotherapy |
| CN113999846A (en) * | 2019-02-28 | 2022-02-01 | 中山大学孙逸仙纪念医院 | Interfering RNA for inhibiting AFAP1-AS1 expression and application of interfering RNA in increasing breast cancer radiotherapy sensitivity |
| CN113999846B (en) * | 2019-02-28 | 2023-06-09 | 中山大学孙逸仙纪念医院 | Interference RNA for inhibiting AFAP1-AS1 expression and application thereof in increasing breast cancer radiotherapy sensitivity |
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