Invention content
The object of the present invention is to provide in situ hybridization probe, reagent and its applications of long-chain non-coding RNA AFAP1-AS1
Method can prepare the system for Computer-aided Diagnosis of Breast Cancer and Index for diagnosis using long-chain non-coding RNA AFAP1-AS1 sequences
Agent.
The application process of LncRNA AFAP1-AS1 is used to prepare the preparation of breast cancer outcome prediction, the long-chain non-coding
The sequence of RNA AFAP1-AS1 such as SEQ NO:Shown in 1.
The preparation of the breast cancer outcome prediction is in situ hybridization detection reagent.
The in situ hybridization detection reagent includes the oligonucleotides spy in situ hybridization detection AFAP1-AS1 expression
Needle, sequence are as follows:
AFAP1-AS1 probes 1:5 '-ATTCCTTTATTTTATGGGATGTTCTGTAGGGAGTT-3 ', SEQ NO:2,
AFAP1-AS1 probes 2:5 '-TAGAAATGAGAAAAGAATCACCAAGAGAGTAAGCA-3 ', SEQ NO:3,
AFAP1-AS1 probes 3:5 '-TCCCTACAGCTAGTTTCCTCTTCATTTATTCATTT-3 ', SEQ NO:4.
The in situ hybridization detection reagent is kit.
Contain the above-mentioned oligonucleotide probe in situ hybridization detection AFAP1-AS1 expression in kit:
AFAP1-AS1 probes 1:5’-ATTCCTTTATTTTATGGGATGTTCTGTAGGGAGTT-3’
AFAP1-AS1 probes 2:5’-TAGAAATGAGAAAAGAATCACCAAGAGAGTAAGCA-3’
AFAP1-AS1 probes 3:5’-TCCCTACAGCTAGTTTCCTCTTCATTTATTCATTT-3’
Positive control probe (detection house-keeping gene GAPDH):
GAPDH probes 1:5'-CCACTTTACCAGAGTTAAAAGCAGCCCTGG-3', SEQ NO:5
GAPDH probes 2:5'-CAGTAGAGGCAGGGATGATGTTCTGGAGAG-3', SEQ NO:6
GAPDH probes 3:5'-GTCAGAGGAGACCACCTGGTGCTCAGTGTA-3', SEQ NO:7.
Also contain in kit:Digoxin oligonucleotides tailing reagent, anti-digoxin-horseradish peroxidase complex inspection
Test agent, DAB staining reagents;
Other conventional biochemical reagents, including 20x sodium citrate buffers (saline sodium citrate,
SSC), dextran sulfate (Dextran sulphate), deionized formamide (Deionized Formamide), polyadenosine
Sour (polyadenylic acid, Poly A), poly deoxyadenylic acid (polydeoxyadenylic acid, Poly dA),
It is denaturalized the frog essence DNA (denatured and sheared salmon sperm DNA, ssDNA) of shearing, yeast transfer RNA
(yeast t-RNA, tRNA), dithiothreitol (DTT) (DTT), Deng 50x Han Shi buffer solutions (Denhardts ' s solution), phosphoric acid
Buffer solution (PBS buffer), pepsin K, bovine serum albumin(BSA) (BSA), triethanolamine (TEA), acetic anhydride block reagent
(Blocking reagent agent), TNB buffer solutions are (i.e.:The 0.1M Tris-Cl+0.15M NaCl+0.5% resistances of pH7.5
Disconnected reagent), TNT buffer solutions are (i.e.:The 0.1M Tris-Cl+0.15M NaCl+0.05%Tween 20 of pH7.5).
We have found expression and the mammary gland of AFAP1-AS1 by situ hybridization in the breast cancer paraffin organization sample of archive
The prognosis of cancer patient is closely related, and it is shorter that AFAP1-AS1 expresses the high survival of patients time, and AFAP1-AS1 is prompted to can be used as breast
The molecular marker of gland cancer prognosis prediction.The present invention provides strong molecule life for the auxiliary diagnosis and prognosis prediction of breast cancer
Object tool has far-reaching clinical meaning and important popularizing application prospect.
Embodiment 2, in situ hybridization detection find that expression of the AFAP1-AS1 in breast cancer is related to patient's prognosis
1. MATERIALS METHODS
1.1 design and synthesize hybridization probe
In order to detect the expression of AFAP1-AS1 using in-situ hybridization method, we devise examines in situ hybridization
Survey the oligonucleotide probe and each 3 of two groups of in situ hybridization oligonucleotide probes of positive control of AFAP1-AS1 expression.
Oligonucleotide probe in situ hybridization detection AFAP1-AS1 expression:
AFAP1-AS1 probes 1:5’-ATTCCTTTATTTTATGGGATGTTCTGTAGGGAGTT-3’
AFAP1-AS1 probes 2:5’-TAGAAATGAGAAAAGAATCACCAAGAGAGTAAGCA-3’
AFAP1-AS1 probes 3:5’-TCCCTACAGCTAGTTTCCTCTTCATTTATTCATTT-3’
Positive control probe (detection house-keeping gene GAPDH):
GAPDH probes 1:5'-CCACTTTACCAGAGTTAAAAGCAGCCCTGG-3'
GAPDH probes 2:5'-CAGTAGAGGCAGGGATGATGTTCTGGAGAG-3'
GAPDH probes 3:5'-GTCAGAGGAGACCACCTGGTGCTCAGTGTA-3'
Each gene specific oligonucleotides probe sequence of above-mentioned design is synthesized using chemical synthesis process.
1.2 oligonucleotide probe labelling kits and in situ hybridization detection reagent
Digoxin oligonucleotides tailing reagent (Dig Oligonucleitide Tailing Kit 2ndGeneration,
Roche companies), anti-digoxin-horseradish peroxidase complex detection kit (Anti-Digoxigenin-POD, Fab
Fragments, Roche company), enhance the TSA signal amplifying systems (TSA of detection of expression signal in situTM Biotin
System, NEL700 kit, PerkinElmer companies), DAB staining kits (Beijing Zhong Shan companies), 20x sodium citrates
Buffer solution (saline sodium citrate, SSC), dextran sulfate (Dextran sulphate), deionized formamide
(Deionized Formamide), polyadenylic acid (polyadenylic acid, Poly A), poly deoxyadenylic acid
(polydeoxyadenylic acid, Poly dA) is denaturalized frog essence DNA (the denatured and sheared of shearing
Salmon sperm DNA, ssDNA), yeast transfer RNA (yeast t-RNA, tRNA), dithiothreitol (DTT) (DTT), 50x Deng Han
Family name's buffer solution (Denhardts ' s solution), phosphate buffer (PBS buffer), pepsin K, bovine serum albumin(BSA)
(BSA), triethanolamine (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, resistance
Disconnected reagent (Blocking reagent agent, Roche companies).
1.3 other main agents and material
Absolute ethyl alcohol, 90% alcohol, 70% alcohol, 50% alcohol, turpentine oil, distilled water, PBS buffer solution (pH7.2~
7.4, NaCl 137mmol/L, KCl 2.7mmol/L, Na2HPO44.3mmol/L, KH2PO41.4mmol/L);3% methanol-bis-
Oxygen aqueous solution (80% methanol and the configuration of 30% hydrogen peroxide);0.01mol/L citrate buffers (citrate buffer, CB,
Provisional configuration in 450ml distilled water is added in pH6.0 ± 0.1,9ml 0.1M citric acid solutions and 41ml 0.1M sodium citrate solutions
Correction work liquid pH value again afterwards);0.1% trypsase;Haematoxylin;1% hydrochloride alcohol (70% alcohol of 1ml concentrated hydrochloric acids+99ml
Configuration);Mounting glue (PTS Cure Mount II);Special coverslip (480 × 240mm2) customize in Zhengzhou Glassware Factory.
Leica low melting points (58 DEG C) paraffin, domestic beeswax, absolute alcohol, dimethylbenzene, 10% neutrality paraformaldehyde (0.01mol/L,
PH7.4, DEPC distilled water and PBS buffer solution are prepared), haematoxylin, Yihong, neutral mounting natural gum, coverslip, glass slide.
1.4 label probe
Oligonucleotide probe label is carried out using 3-tailing DIG Olignucleutide Kit, reaction system is such as
Under.
100pmol oligonucleotide+ddH2O=9 μ l (control:control oligonucleutide 5μ
l+ddH2O 4μl)
Mixing slightly centrifuges.37 DEG C of water-bath 30min add 2 μ l EDTA (0.2M, PH 8.0) stopped reactions.
It is purified after 1.5 oligonucleotide probes label
In order to increase the purity of label probe, marked probe need to be purified, concrete operations are as follows:
1) 100% cold ethyl alcohol (- 20 DEG C) of+2.5 μ l 4M LiCL+75 μ l of probe reaction mixture (22 μ l)
2) -70 DEG C of precipitations 60min, or-20 DEG C of 2h.
3) 13.000 × g, 4 DEG C of centrifugation 15min.
4) supernatant is abandoned, is washed with 70% ice-cold (V/V) ethyl alcohol of 50 μ l.
5) 4 DEG C of 13.000xg centrifuge 5min.
6) supernatant, 4 DEG C of dryings of vacuum are abandoned.
7) with the molten probe of aseptic double-distilled water weight.
1.6 in situ hybridizations detection achieves the expression of AFAP1-AS1 in paraffin section
Paraffin section hybridizes pre-treatment
1) paraffin section of 4 DEG C of preservations is placed in 58 DEG C of roasting piece 30min, melted surface paraffin.
2) dimethylbenzene dewaxes 3 × 5min successively.
3) step ethanol wash, 100% 1 × 5min of the alcohol of 2 × 2min of alcohol → 95% alcohol of 1 × 5min → 70% →
50% 1 × 5min of alcohol → 2 × 3min of DEPC water washings → DEPC-PBS washs 2 × 5min.
4) 300 μ l pepsins K (10 μ g/ml) are added dropwise on slice, 37 DEG C digest 20min.
5) it is sliced and washs 1min, stopped reaction into PBS (0.1M PBS+2mg/ml glutamic acid).
6) it is sliced into 0.2N HCL, reacts 20-30min in 37 DEG C, increase the permeability of tissue.
7) slice fixes 10min, room temperature afterwards with 4% paraformaldehyde (0.1M PBS dissolvings).
8) in order to increase tissue positive intensity for hybridization, acetyl processing is carried out to slice.It is sliced into 0.25% acetic anhydride
Buffer I (0.1M triethanolamines), room temperature 10min.
9) 1M PBS wash 2 × 5min.
Prehybridization and hybridization
Prehybridization:The prehybridization solution of -20 DEG C of preservations is first placed in 37 DEG C of incubation 60min, and the dosage of prehybridization solution is 50 μ l,
Parafilm is carried out lid and is sliced, prehybridization 2 hours in 37 DEG C of wet box.(prehybridization solution composition includes: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) parafilm is removed, prehybridization solution is got rid of, slice is placed in 5min in 2 × SSC.
2) hybridization reaction:37 DEG C of hybridized overnights (18-20h).Each slice is added 250 μ l hybridization solutions and is carried out with parafilm
Lid.Corresponding probe is added in prehybridization solution just becomes hybridization solution.Hybridization solution is prepared in prehybridization, is placed 37 DEG C of incubations, is made
Probe is completely dissolved in hybridization solution, this experiment is mixed with a plurality of oligonucleotide probe, is matched by each probe 500ng/ml concentration
Probe hybridization solution is made.Digoxin tailing labelling kit label probe concentration calculation basis:The concentration of each probe by its with
Colour developing is compared and the label reaction theory spy of the naked probe of 30 bases of 100pmol when detection reaction when the positive quantifies probe
Needle yield is that two kinds of standards of 900ng carry out the concentration that COMPREHENSIVE CALCULATING goes out label probe.
3) post-hybridization washing, slice immerse 2 × SSC, 10min, throw off parafilm.It is washed successively in shake on shaking table, 2 ×
SSC (0.5%SDS), 2 × 15min → 0.25 × SSC (0.5%SDS), 2 × 15min.
Color developing detection is reacted after hybridization
1) Anti-Digoxigenin-POD detection digoxigenin-probes and mRNA combination compounds are used;TSA amplification systems
Enhance the positive signal of in situ hybridization reaction solution reaction, DAB colour developings.
2) slice is gone in TNT buffer solutions, 3 × 5min.
3) TNB is added dropwise and blocks buffer solution, 300 μ l/TMAs, room temperature, 30min.
4) extra blocking agent is sucked, 1:100 diluted Anti-Digoxigenin-POD (TBS+0.1%Triton X-
100+1% blocking agents), room temperature 4 hours.
5) TNT Buffer (0.1M Tris-CL, pH7.5,0.15M NaCL, 0.05%Tween 20) are washed,
3x5min。
6) signal amplification reagent Biotinyl Tyamid, 300 μ l/TMAs, (Biotinyl Tyramid are added dropwise on slice
Store liquid:Biotinyl Tyramid are dissolved in 0.2ml DMSO, Biotinyl Tyramid working solutions:1 × dilution, 1:50
Dilute Biotinyl Tyramid and store liquid), room temperature 10 minutes.
7) TNT is washed, 3 × 5min.
8) SA-HRP (strepto- avidin-horseradish peroxidase), 300 μ l/TMAs, room temperature 30min are added dropwise in slice.
9) TNT is washed, 3 × 5min.
10) distilled water washing, 1 × 1min.
11) DAB develops the color, and chromogenic reaction is controlled under microscope.
12) haematoxylin is redyed,
13) alcohol step is dehydrated, chip drying.
14) mounting glue, the coverslip cover plate of dimension, crosslinking slice 1min under ultraviolet lamp is added dropwise.
1.7 results judge and standard
Application Optics microscope is observed under low power and high power lens respectively, looks first at the positive expression of target RNA
The signal positioning intracellular in object observing:Positioned at nucleus, cytoplasm or cell membrane.
It is carried out respectively with two kinds of standards of the intensity of the detection rna expression position positive signal and the cell number of positive expression again
Comprehensive score, criterion are:(1) judge according to positive cell dyeing intensity:A. cell dye-free remembers 0 point;B. cell is dyed
Light brown is weakly positive, remembers 1 point;C. cell dyes brown and dyes dark-brown without background coloration or cell and have the light brown back of the body
Scape is moderate positive, remembers 2 points;D. cell dyes dark-brown and is strong positive without background coloration, remembers 3 points.(2) according to positive cell
Express number score:A. no positive cell expression, remembers 0 point;B. positive expression cell number≤25% remembers 1 point;C.25% < is positive thin
Born of the same parents' number < 50% remembers 2 points;D. positive expression cell number >=50% remembers 3 points.
In order to reduce the subjective factor of appraisal result as possible, one of above-mentioned standard is pressed respectively respectively by two pathology experts
Judged and scored, then the two is scored and is multiplied, result is:1. 0 point of person is finally calculated as 0 point, it is believed that feminine gender expression;2. 1 point
It is finally calculated as 1 point with 2 points of persons, it is believed that weakly positive is expressed;3. 3 points and 4 points of persons are finally calculated as 2 points, it is believed that moderate positive is expressed;④
6, which assign to 9 points of persons, is finally calculated as 3 points, it is believed that strong positive is expressed.
1.8 analyses and statistical software
Statistical analysis is carried out to experimental result using SPSS13.0 statistical softwares, compares use χ two-by-two2Test or Fisher
Exact test, correlation analysis use Spearmen correlation methods;P < 0.05 are that difference is statistically significant.
Survivorship curve analysis uses Kaplan-Meier method and log-rank test;Multi-variables analysis uses Cox ' s
proportional hazards model;P < 0.05 are that difference is statistically significant.
2 results
Expressions of 2.1 AFAP1-AS1 in breast cancer
AFAP1-AS1 (61/77) in 79.22% breast cancer tissue has expression (Fig. 2 is left), remaining 16 mammary gland
The expression of AFAP1-AS1 is relatively low (Fig. 2 is right) in cancerous tissue.
Patient with breast cancer's prognosis of 2.2 AFAP1-AS1 high expression is poor
We have carried out Effect of follow-up visit by telephone to 77 patient with breast cancers, inquired in detail they start time, treatment,
Whether there is or not recurrence, whether there is or not suffering from other diseases, recurrence and death time etc. again, and register life span and state, and to breast cancer
The survival analysis that the expression of AFAP1-AS1 and the life span of patient and state carry out in tissue finds AFAP1-AS1 high expression
Patient is significantly shorter than AFAP1-AS1 low expressions or the patient not expressed (Fig. 3) mean survival time.Illustrate that AFAP1-AS1 is one
A and relevant molecular labeling of Prognosis in Breast Cancer, lncRNA expression is high, patient's poor prognosis.