CN107478625A - Cancerous lung tissue lncRNA fluorescent in situ hybridization detecting method - Google Patents
Cancerous lung tissue lncRNA fluorescent in situ hybridization detecting method Download PDFInfo
- Publication number
- CN107478625A CN107478625A CN201710659278.3A CN201710659278A CN107478625A CN 107478625 A CN107478625 A CN 107478625A CN 201710659278 A CN201710659278 A CN 201710659278A CN 107478625 A CN107478625 A CN 107478625A
- Authority
- CN
- China
- Prior art keywords
- lncrna
- lung tissue
- cancerous lung
- fluorescent
- detecting method
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Optics & Photonics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention provides a kind of cancerous lung tissue lncRNA fluorescent in situ hybridization detecting method, including：Lung cancer frozen section, tissue is fixed, digests and penetrating, lncRNA probe hybridization checks, DNA dyeing, mounting, fluoroscopic examination imaging.The present invention can accurately, intuitively detect cancerous lung tissue lncRNA expression, Subcellular Localizations of the original position displaying lncRNA in cancerous lung tissue.The cancerous lung tissue lncRNA of present invention fluorescence in situ hybridization technique, it is simple to operate, it is easy to spread, there is important medical application to be worth.
Description
Technical field
The present invention relates to technical field of molecular biology, and in particular to a kind of cancerous lung tissue lncRNA FISHs inspection
Survey method.
Background technology
Lung cancer is the most common malignant tumour in China.Research shows that lncRNA participates in the generation of human malignant tumor, development.
Effects of the LncRNA in lung cancer is that the focus of medical research is also difficult point.How target lncRNA table in cancerous lung tissue is detected
Up to being matter of utmost importance that scholars face.
LncRNA quantitative analysis common method is RT-PCR in cancerous lung tissue；Caryoplasm separation RT-PCR determines for lncRNA
Position analysis.But these experimental procedures are cumbersome, complex operation.Cancerous lung tissue RNA is more importantly extracted, often comprising mesenchyma stroma of tumors
The RNA of cell, it is impossible to lncRNA expression completely in reaction tumor parenchymal cells.
In situ hybridization detection has important technical advantage.At present in lung cancer tumor cell line lncRNA in situ detection technology
Comparative maturity, but lncRNA fluorescence in situ hybridization technique is immature always in cancerous lung tissue.Lung cancer histological type is complicated, group
It is obvious to knit atypia.The poor hybridization in situ technique inspection of sensitiveness does not measure lncRNA expression.Lung Cancer Stroma internal probe remains
Excessively, background stainings are poor, and essence dyeing distinguishes unobvious with interstitial residual.Therefore, cancerous lung tissue lncRNA fluorescent in situ is miscellaneous
Detection technique is handed over to be further improved optimization.
Chinese patent 201611041669.0, publication date 2017 year 01 month are disclosed in a kind of esophageal squamous cell carcinoma tissue on the 25th
LncRNA fluorescent in situ hybridization detecting method, including：Frost esophageal squamous cell carcinoma tissue section is prepared, is fixed, digests with leading to
Thoroughly, detected using lncRNA probes, carry out DNA dyeing, mounting, imaging or fluorescence microscopy are detected using Laser Scanning Confocal Microscope
Microscopy surveys imaging.But cancerous lung tissue is very different with esophageal squamous cell carcinoma tissue, esophageal squamous cell carcinoma is based on reality tissue, lung cancer group
The cavity and obstructive emphysema of interior often residual gassiness are knitted, and surrounding is wrapped by alveolar tissue.It is therefore desirable to for lung cancer group
The specificity knitted, further Optimal Experimental condition, experimental procedure is improved, design cancerous lung tissue lncRNA fluorescence in situ hybridization detection
Method.But in the prior art, on the cancerous lung tissue lncRNA fluorescent in situ hybridization detecting method of the present invention, yet there are no
Report.
The content of the invention
First purpose of the present invention is to be directed to deficiency of the prior art, there is provided a kind of cancerous lung tissue lncRNA fluorescence is former
Position hybridization detection method, expression and Subcellular Localization for lncRNA in cancerous lung tissue.
To achieve the above object, the present invention adopts the technical scheme that：
A kind of cancerous lung tissue lncRNA fluorescent in situ hybridization detecting method, including：Prepare frost cancerous lung tissue section, group
Knit fixed, digestion and penetrating, lncRNA probe hybridization checks, DNA dyeing, mounting, fluoroscopic examination imaging.
Further, described lncRNA probes are marked using Cy3.
Further, described cancerous lung tissue lncRNA fluorescent in situ hybridization detecting method also includes：According to lncRNA
Image space, judge that lncRNA is positioned at nucleus or cytoplasm, or caryoplasm is uniformly expressed.
Further, described cancerous lung tissue lncRNA fluorescent in situ hybridization detecting method also includes：According to lung cancer group
The fluorescence optical density of middle lncRNA imagings is knitted, calculates relative expression quantities of the lncRNA in cancerous lung tissue.
Further, the method detected using lncRNA probes is comprised the following steps：
(1) prehybridization solution, 37 DEG C of closing 40min are added in cancerous lung tissue section；
(2) under the conditions of lucifuge, lncRNA probes are added in 37 DEG C of hybridization solution, obtain the hybridization of lncRNA probes
Liquid；
(3) prehybridization solution in histotomy is discarded, adds lncRNA probe hybridization solutions, room temperature hybridized overnight, lucifuge bar
Cleansing tissue under part.
Further, described DNA dyeing is redyed using DAPI.
Further, the prehybridization solution in the step 1 is by Blocking Solution and Pre-hybridization
Buffer is according to 1:99 ratios are mixed.
Further, the hybridization solution in the step 2 is by Blocking Solution and Hybridization Buffer
According to 1:99 ratios are mixed.
The present invention is according to ATCG complementation rules, specific probe and the complementary combinations of lncRNA, and lncRNA is in lung cancer group for detection
The expression knitted.DAPI is combined with double-stranded DNA, for conventional nucleus fluorescent dye.Under fluorescence excitation, Cy3 marks
The aobvious red of probe；The aobvious bluenesss of DAPI；Color contrast is obvious, does not interfere with each other.According to target lncRNA fluorescence optical density, calculate
Expression quantity of the lncRNA in cancerous lung tissue.By detecting target lncRNA image space, judge target lncRNA in lung cancer
It is cell nuclear expression or cytoplasmic expression in tissue.
Compared with prior art, the beneficial effects of the invention are as follows：
1st, cancerous lung tissue lncRNA fluorescence in situ hybridization technique of the invention, can quickly and accurately detect lncRNA's
Expression, laid the foundation for effects of the research lncRNA in lung cancer.
2nd, the present invention carries out high quality imaging, visual inspection lncRNA expression, and original position to FISH image
Show lncRNA Subcellular Localization.
3rd, expressions of the accurate detection lncRNA of the present invention in Parenchyma of Lung Carcinoma, avoids the interference of mesenchyma stroma of tumors, detects
As a result it is more accurate.
4th, the present invention is directed to the particularity of cancerous lung tissue structure, systemic modified test method, effectively detects lung cancer group
LncRNA expression is knitted, this method also can detect lncRNA expression in the tumor tissues of other residual cavity structures.
5th, the present invention is simple to operate, convenient and swift, can be applied to biological basis research and clinical detection.
Brief description of the drawings
Expression figures of the application lncRNA fluorescence in situ hybridization technique detection target lncRNA of accompanying drawing 1 in cancerous lung tissue
(×600)。
Embodiment
The invention will be further elucidated with reference to specific embodiments.It should be understood that these embodiments are merely to illustrate this hair
Bright rather than limitation the scope of the present invention.In addition, it is to be understood that after the content of the invention recorded has been read, art technology
Personnel can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited
Fixed scope.
Embodiment 1：Cancerous lung tissue lncRNA fluorescent in situ hybridization detecting method
The present embodiment is tested by taking cancerous lung tissue as an example, is concretely comprised the following steps：
1. prepare frost cancerous lung tissue section：
Film-making, 8 μm of thickness (- 20 DEG C of operation temperature) are carried out to fresh cancerous lung tissue using freezing microtome.
2. tissue is fixed, digest with it is penetrating：
A.4% the fixed 10min of 37 DEG C of paraformaldehyde；
B.1 × PBS 3 times, each 3min；
C. Collagenase IV (sigma companies, article No.:C5138) 37 DEG C of incubation 0.3min；
D.1 × PBS 3 times, each 3min；
DEG C e.4 under the conditions of, add 100 μ l 0.5%TritonX-100 and be incubated 3min；
F.1 × PBS 3 times, each 3min.
3.LncRNA hybridization is detected：
A. 100 μ l prehybridization solutions are added in tissue block, the prehybridization solution is by Blocking Solution (sharp rich biologies
Company, article No.：C10903) with 1 × Pre-hybridization Buffer (Rui Bo biotech firms, article No.：C10901) according to
1:99 mixing, 40min is incubated in 37 DEG C；
B. in darkroom, by 2.5 μ l 20 μM of lncRNA FISH Probe Mix storing liquid (Rui Bo biotech firms, article No.s：
C10902, the probe are marked using cy3) with 100 μ l hybridization solutions (by 1 μ l Blocking Solution and 99 μ l 1 ×
Hybridization Buffer are formed) mix, prepare lncRNA probe hybridization solutions；
C. prehybridization solution is discarded, adds 100 μ l lncRNA probe hybridization solutions, ambient temperature overnight；
D. under the conditions of lucifuge, 45 DEG C hybridization washing lotion I (4 × SSC, 0.1%Tween-20) cleansing tissue 3min, totally 3 times；
E. under the conditions of lucifuge, 45 DEG C hybridization washing lotion II (2 × SSC) cleansing tissue 3min, totally 3 times；
F. under the conditions of lucifuge, 45 DEG C hybridization washing lotion III (1 × SSC) cleansing tissue 3min, totally 3 times；
G. under the conditions of lucifuge, 1 × PBS tissue 3min, totally 3 times；
4.DNA is dyed
A. under the conditions of lucifuge, 1 × DAPI (green skies biotechnology research institute, article No.:C1005) stained tissue 5min；
B. under the conditions of lucifuge, 1 × PBS tissue 3min, totally 3 times；
5. mounting
Under the conditions of lucifuge, fluorescence anti-quencher (green skies biotechnology research institute, article No. is added dropwise:P0126), mountant is used
Cover glass is fixed on slide by (such as nail polish), carries out fluoroscopic examination.
6. fluorescent microscopic imaging：
LncRNA probe imagings launch wavelength 570nm, excitation wavelength 550nm；DAPI is imaged excitation wavelength 364nm, transmitting
Wavelength 454nm.
As a result as shown in figure 1, Fig. 1 is to detect target lncRNA in cancerous lung tissue using lncRNA fluorescence in situ hybridization technique
Expression figure (× 600).Display DAPI is primarily targeted for nucleus, and in blueness, Cy3 mark target lncRNA are in red
Color, it is primarily targeted for cytoplasm.
Detection imaging is carried out using Laser Scanning Confocal Microscope in the present embodiment, passes through LEICA Qwin V3 image analysis softwares
(German Leica companies) calculates the fluorescence optical density of lncRNA in cancerous lung tissue, and red fluorescence optical density is stronger, and lncRNA is in lung
Relative expression quantity in cancerous tissue is higher.According to lncRNA image spaces, judge that lncRNA is positioned at nucleus or cell
Matter, or caryoplasm are uniformly expressed, and are specifically parsed according to Fig. 1.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, on the premise of the inventive method is not departed from, can also make some improvement and supplement, and these are improved and supplement also should be regarded as
Protection scope of the present invention.
Claims (8)
- A kind of 1. cancerous lung tissue lncRNA fluorescent in situ hybridization detecting method, it is characterised in that including：Prepare frost lung cancer group Section is knitted, tissue is fixed, digests and dyes, mounting, be imaged using fluoroscopic examination with penetrating, lncRNA probe hybridization checks, DNA.
- 2. cancerous lung tissue lncRNA as claimed in claim 1 fluorescent in situ hybridization detecting method, it is characterised in that described LncRNA probes are marked using Cy3.
- 3. cancerous lung tissue lncRNA as claimed in claim 1 fluorescent in situ hybridization detecting method, it is characterised in that described Cancerous lung tissue lncRNA fluorescent in situ hybridization detecting method also includes：According to lncRNA image spaces, it is fixed to judge lncRNA Uniformly expressed positioned at nucleus or cytoplasm, or caryoplasm.
- 4. cancerous lung tissue lncRNA as claimed in claim 1 fluorescent in situ hybridization detecting method, it is characterised in that described Cancerous lung tissue lncRNA fluorescent in situ hybridization detecting method also includes：It is close according to the fluorescence light that lncRNA in cancerous lung tissue is imaged Degree, calculate relative expression quantities of the lncRNA in cancerous lung tissue.
- 5. cancerous lung tissue lncRNA as claimed in claim 1 fluorescent in situ hybridization detecting method, it is characterised in that described The method detected using lncRNA probes is comprised the following steps：(1) prehybridization solution, 37 DEG C of closing 40min are added in cancerous lung tissue section；(2) under the conditions of lucifuge, lncRNA probes are added in 37 DEG C of hybridization solution, obtain lncRNA probe hybridization solutions；(3) discard the prehybridization solution in histotomy, add lncRNA probe hybridization solutions, room temperature hybridized overnight, under the conditions of lucifuge Cleansing tissue.
- 6. cancerous lung tissue lncRNA as claimed in claim 1 fluorescent in situ hybridization detecting method, it is characterised in that described DNA dyeing is redyed using DAPI.
- 7. cancerous lung tissue lncRNA as claimed in claim 5 fluorescent in situ hybridization detecting method, it is characterised in that the step Prehybridization solution in rapid 1 is by Blocking Solution and Pre-hybridization Buffer according to 1:99 ratios mix It is made.
- 8. cancerous lung tissue lncRNA as claimed in claim 5 fluorescent in situ hybridization detecting method, it is characterised in that the step Hybridization solution in rapid 2 is by Blocking Solution and Hybridization Buffer according to 1:99 ratios are mixed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710659278.3A CN107478625A (en) | 2017-08-04 | 2017-08-04 | Cancerous lung tissue lncRNA fluorescent in situ hybridization detecting method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710659278.3A CN107478625A (en) | 2017-08-04 | 2017-08-04 | Cancerous lung tissue lncRNA fluorescent in situ hybridization detecting method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107478625A true CN107478625A (en) | 2017-12-15 |
Family
ID=60597499
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710659278.3A Pending CN107478625A (en) | 2017-08-04 | 2017-08-04 | Cancerous lung tissue lncRNA fluorescent in situ hybridization detecting method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107478625A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111218499A (en) * | 2020-02-20 | 2020-06-02 | 南京林业大学 | Frozen slice-based 3D fluorescence in situ hybridization method for poplar root tips |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103382499A (en) * | 2012-05-04 | 2013-11-06 | 长庚医疗财团法人林口长庚纪念医院 | Specimen pretreatment method for reducing autofluorescence interference in fluorescence in situ hybridization method and reagent kit thereof |
| CN106282385A (en) * | 2016-10-14 | 2017-01-04 | 天津医科大学肿瘤医院 | Long-chain non-coding RNA XLOC_000090 qualification in pulmonary carcinoma and purposes |
| CN106350604A (en) * | 2016-11-22 | 2017-01-25 | 复旦大学附属中山医院 | Fluorescence in situ hybridization detection method of IncRNA in esophageal squamous carcinoma tissues |
-
2017
- 2017-08-04 CN CN201710659278.3A patent/CN107478625A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103382499A (en) * | 2012-05-04 | 2013-11-06 | 长庚医疗财团法人林口长庚纪念医院 | Specimen pretreatment method for reducing autofluorescence interference in fluorescence in situ hybridization method and reagent kit thereof |
| CN106282385A (en) * | 2016-10-14 | 2017-01-04 | 天津医科大学肿瘤医院 | Long-chain non-coding RNA XLOC_000090 qualification in pulmonary carcinoma and purposes |
| CN106350604A (en) * | 2016-11-22 | 2017-01-25 | 复旦大学附属中山医院 | Fluorescence in situ hybridization detection method of IncRNA in esophageal squamous carcinoma tissues |
Non-Patent Citations (3)
| Title |
|---|
| 杨晓琨: "荧光原位杂交技术检测非小细胞肺癌患者现场细胞学制片表皮生长因子受体基因扩增状态的研究", 《中国优秀硕士论文电子期刊网》 * |
| 王晓敏等: "荧光原位杂交技术检测非小细胞肺癌EGFR基因扩增", 《临床与实验病理学杂志》 * |
| 陈顺平等: "非小细胞肺癌EGFR荧光原位杂交与免疫组化检测的比较", 《实用癌症杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111218499A (en) * | 2020-02-20 | 2020-06-02 | 南京林业大学 | Frozen slice-based 3D fluorescence in situ hybridization method for poplar root tips |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| ES2538123T3 (en) | Method for chromogenic detection of two or more target molecules in a single sample | |
| Stack et al. | Multiplexed immunohistochemistry, imaging, and quantitation: a review, with an assessment of Tyramide signal amplification, multispectral imaging and multiplex analysis | |
| Karstensen et al. | Endoscopic ultrasound-guided fine needle aspiration: from the past to the future | |
| CN101226118B (en) | Cytochemical staining method being compatible with immunofluorescence analysis and uses thereof | |
| Jain et al. | Implementation of fluorescence confocal mosaicking microscopy by “early adopter” Mohs surgeons and dermatologists: recent progress | |
| Hartmann et al. | Immunofluorescence and confocal microscopy for ex‐vivo diagnosis of melanocytic and non‐melanocytic skin tumors: A pilot study | |
| Luo et al. | Phasor–FLIM as a Screening tool for the differential diagnosis of actinic keratosis, Bowen’s disease, and basal cell carcinoma | |
| CA2365097A1 (en) | Method for increasing clinical specificity when detecting tumours and their precursor stages by simultaneously measuring at least two different molecular markers | |
| Ravi et al. | Surgical margins and its evaluation in oral cancer: a review | |
| US20220389523A1 (en) | Image acquisition methods for simultaneously detecting genetic rearrangement and nuclear morphology | |
| CN102203283A (en) | Methods for the cytological analysis of cervical cells | |
| CN104350376A (en) | Characterization of biological tissues at cellular level using red and far-red fluorescent dyes | |
| CN112014562A (en) | Marker combinations, methods and systems for dynamic monitoring of immune checkpoint PD-1/PD-L1 | |
| Peng et al. | Discrimination of cirrhotic nodules, dysplastic lesions and hepatocellular carcinoma by their vibrational signature | |
| US8969100B2 (en) | Method for detecting afferent lymph vessel inflow regions and method for identifying specific cells | |
| KR101559798B1 (en) | method for normalizing image in digital pathology | |
| Liu et al. | High-resolution microscopy for imaging cancer pathobiology | |
| CN107478625A (en) | Cancerous lung tissue lncRNA fluorescent in situ hybridization detecting method | |
| CN106350604A (en) | Fluorescence in situ hybridization detection method of IncRNA in esophageal squamous carcinoma tissues | |
| CN106995837A (en) | Early diagnosing mammary cancer kit | |
| Levenson et al. | Digital spectral imaging for histopathology and cytopathology | |
| Kawakubo et al. | Feasibility of using an enzymatically activatable fluorescence probe for the rapid evaluation of pancreatic tissue obtained using endoscopic ultrasound-guided fine needle aspiration: a pilot study | |
| Caspersson et al. | Cytochemical changes in the nucleus during tumour development | |
| Chen et al. | Label-free identification of early stages of breast ductal carcinoma via multiphoton microscopy | |
| Titze et al. | Feasibility study for ex vivo fluorescence confocal microscopy (FCM) on diagnostic prostate biopsies |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20171215 |