CN100587451C - Method for trapping tissue special region and special equipment - Google Patents
Method for trapping tissue special region and special equipment Download PDFInfo
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- CN100587451C CN100587451C CN200610045852A CN200610045852A CN100587451C CN 100587451 C CN100587451 C CN 100587451C CN 200610045852 A CN200610045852 A CN 200610045852A CN 200610045852 A CN200610045852 A CN 200610045852A CN 100587451 C CN100587451 C CN 100587451C
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Abstract
The present invention discloses a method for trapping specific region of tissue and its special-purpose equipment. Said special-purpose equipment includes the following several portions: calibration pen and electric cutting knife, in which said calibration pen has a pen core, the upper end of said pen core is equipped with microscope standard interface, its middle portion is equipped with elasticdye bottle and its lower portion is equipped with calibration head connected with the elastic dye bottle, and the exterior of said pen core is equipped with a cap of pen. Besides, said invention alsoprovides the concrete operation method and steps for trapping specific region of tissue by using said special-purpose equipment.
Description
Technical field:
The present invention relates to a kind of method and specialized equipment of specific region of from tissue, catching, especially a kind of simple to operate, cost is low, can effectively prevent the catching method and the specialized equipment of the tissue special area of cell cross pollution.
Background technology:
Cellular elements science of heredity especially genomics research is the important ingredient of life science.The key problem that it will solve comprises: genomic formation, genetic development and diversity, genomic expression characteristics under different physiology and the pathological state and the adjusting on space-time and store, process and analyze by the means of the bioinformatics data to above-mentioned analysis.Thereby the closely-related gene of generation, development and differentiation at genome and comprehensive announcement of proteomics level and life.
Though this research field progress is very rapid both at home and abroad, but also face some technical matters, wherein very noticeablely be: different with clone in vitro culture, that form by the single type cell, there is the various kinds of cell composition in the sample of taking from in-vivo tissue/organ, even if the zones of different of same sample, the formation of tissue also has evident difference.All have the various kinds of cell composition to exist in all kinds of tissues, this is easy to cause the nucleic acid and the albumen sample that extract intercellular cross pollution to occur.Because different gene expression of cells characteristics have notable difference, therefore be easy to cause experimental result deviation to occur.In addition, present tissue treatment methods also is difficult to guarantee to be used for the tissue samples that the upstream gene group detects with the downstream candidate gene is examined and derives from (substantially) identical cell colony, cause experimental result to lack correspondence, obviously increased the workload that candidate gene is examined.For this reason, people feel that all the more the tissue of original position and molecular pathology are observed and the nucleic acid and the protein extraction of tissue special area are to complement one another, and the relation that brings out the best in each other should be finished on a piece of tissue synchronously.Only in this way, could improve the preciseness of experimental design, guarantee science, credibility and the high efficiency of subject study.
For the capture tissue specific region, people have done all effort.Wherein, rather effective and efficient manner be people such as the scientist Emmert-Buck of NIH (NIH) invention laser capture microdissection technology (Laser Capture Microdissection, LCM).Its ultimate principle is, the specific region in microscopically is selected histotomy, with laser beam with itself and body portion from, through special filter membrane it is moved to and to do further processing (as DNA, RNA and protein extraction) in the test tube.The accuracy of this technology is very high, but complicated operation, consuming time longer, particularly processing ease causes the RNA degraded at normal temperatures, and, the maximum of LCM is caught diameter only 0.6mm, so each tissue/cell quantity of gathering is very limited, even if through repeatedly catching the basic consumption that also may not necessarily satisfy such as experiments such as gene expression spectrum analysis, Northern or the analyses of the Western marking.If adopt high efficiency method that the sample of nucleic acid that extracts is increased, then be easy to cause false-positive result, further increase the workload of finishing the fruit stone reality.In addition, the specialized equipment of LCM very expensive (ten thousand yuan of 60-80), its experiment assistant product and consumable material are also expensive, thereby have obviously increased the experimental cost of project research.So though this equipment comes out year surplus in the of ten, the researchist is " stepping back of prestige valency " mostly, the sample size that can catch is less in addition, causes its range of application very limited.
Summary of the invention:
The present invention is in order to solve technical matterss such as existing in prior technology complicated operation, length consuming time, cost height, sample quantum of output are few, and the catching method and the specialized equipment of the big tissue special area of a kind of economical and practical, simple and easy to do, high efficiency and sample quantum of output is provided.
Technical solution of the present invention is: a kind of catching method of tissue special area is characterized in that having the following steps:
A. getting section from the frozen tissue piece places microscopically to select the purpose zone;
B. demarcate the purpose zone with calibration pen in the front of frozen tissue section;
C. be coated with information transfer film liquid and use pen at the back side of frozen tissue section and go out the purpose zone at information transfer film image scale on show;
E. the information transfer face with frozen tissue section pastes mutually with the frozen tissue piece is identical, and then frozen tissue section is taken off;
D. cut along the purpose area flag on the frozen tissue with cutter, after slice separation under the freezing condition, collect the purpose zone.
Described information transfer film liquid is that when method is formulated by following component, weight: coconut oil 18～22, castor oil 5～9, tallow 11～15, sugar 12～14, NaOH 20～24, alcohol 8～12, soda 0.3～0.7, water 28～30, coconut oil, castor oil, tallow are dropped into vessel in heating to 75～85 ℃, slowly add NaOH, the water-soluble back of soda in the oil plant that is heated in addition, stirring makes it saponification, treat that soap lye is transparent, add sugar and alcohol.
A kind of catching method calibration pen of tissue special area has pen core 1, and the upper end of pen core 1 is provided with microscope standard interface 2, and the middle part is provided with elastic dye bottle 3, and the lower end is the demarcation of joining with elastic dye bottle 34, is circumscribed with the pen cap 5 with pen core 1.
The middle part of described elastic dye bottle 3 is a screw-like elastic shrinkage body; Described mark 4 is metal-cored for being provided with, and metal-coredly is with length and slightly is longer than metal-cored elasticity pressure reducing sleeve outward.
The electronic cutter of a kind of catching method of tissue special area, pen type housing 6 is arranged, and pen type housing 6 Inner Front Ends are equipped with motor 7, and the output shaft of motor 7 joins with placing the milling cutter 8 outside the pen type housing 6, also be provided with battery 9 in pen type housing 6, battery 9 joins by switch 10 and motor 7.
Flexible coil 11 between described motor 7 and the motor 9, switch 10 is arranged on the rear end of pen type housing 6, flexible coil 12 between switch 10 and the battery 9.
The present invention can realize the accurate location in purpose zone, adopt unique method the locating information on the histotomy to be transferred to reliably the corresponding site of piece of tissue, then use the special-purpose electronic cutter of organizing that the purpose tissue regions is cut, make its in the process of section with organize body portion from and under freezing fully state, it is collected fast and carries out sample extraction in the test tube in the mode of craft.Have following advantage:
1. the piece of tissue long-time operation at normal temperatures that can avoid other experimental technique to occur dissolves sample to cause sample phenomenon rotten or degraded to take place;
2. very little to the destruction of maternal tissue, satisfy accurately extract the nucleic acid and protein sample of high-quality, high production from the particular organization zone in, the residue tissue can also be used for other research purposes;
3. high efficiency, the sample size obtained are big, and experimental cost obviously reduces;
4. the device therefor cost is low, economical and practical, easy and simple to handle, be easy to promote, can be used in such as high-end area researches such as genome and proteomics, also can in daily scientific research and clinical molecular diagnosis, use, improve science, credibility, advance and the high efficiency of scientific research.
Description of drawings:
Fig. 1 is the structural representation of embodiment of the invention calibration pen.
Fig. 2 is the structural representation of embodiment of the invention cutter.
Embodiment:
Below in conjunction with description of drawings the specific embodiment of the present invention.
Embodiment 1:
A kind of catching method of tissue special area is characterized in that having the following steps:
A. in liquid nitrogen, get section from the frozen tissue piece, techtology dyeing, the size of section is advisable to be fit to desk-top microscopic examination.Frozen tissue can be fresh frozen tissue, also can be the fixing sample of paraffin-embedded tissue sample or paraformaldehyde;
B. section is placed microscopically to observe, selected point of destination (spot) or purpose zone (reqion);
C. in the front of frozen tissue section, with pen point of destination (spot) or purpose zone (reqion) are located paintedly, promptly demarcate the purpose zone;
D. be coated with information transfer film liquid at the back side of frozen tissue section, after treating information transfer film liquid air-dry (about 20 seconds), the use pen goes out point of destination or purpose zone at information transfer film image scale on show, information transfer film liquid must be can be in section film forming and can being transferred on the frozen tissue when pasting mutually with frozen tissue.
E. the information transfer face with frozen tissue section pastes mutually with frozen tissue piece (25～-30 ℃) is identical, and then frozen tissue section is taken off;
F. use cutter to do width less than 0.2mm along the outer rim of the purpose area flag on the frozen tissue (30～-50 ℃), the degree of depth is the cutting of 0.2～0.3mm, the serial section of about 5 μ m, the purpose zone just with organize body portion from, collect the purpose zone.The purpose zone can be placed the centrifuge tube that contains sample extraction liquid, (DNA is RNA) with the albumen sample to prepare nucleic acid with conventional method.
Embodiment 2:
The embodiment of the invention 2 used calibration pens are as shown in Figure 1: pen core 1 is arranged, and the upper end of pen core 1 is provided with microscope standard interface 2, and the middle part is provided with elastic dye bottle 3, and the lower end is the demarcation of joining with elastic dye bottle 34, is circumscribed with the pen cap 5 with pen core 1.Elastic dye bottle 3 can be made with resilient material, also the middle part can be processed into screw-like elastic shrinkage body, mark 4 is metal-cored for being provided with, and the metal-cored outer diameter that is with is that 0.5mm, length are than the metal-cored slightly elasticity pressure reducing sleeve of length, as the rigid plastic cover, play the buffering decompression.The used electronic cutter of the invention process power are as shown in Figure 2: pen type housing 6 is arranged, pen type housing 6 Inner Front Ends are equipped with motor 7, the output shaft of motor 7 joins with placing the milling cutter 8 outside the pen type housing 6, also is provided with battery 9 in pen type housing 6, and battery 9 joins by switch 10 and motor 7.Can be flexible coil 11 between motor 7 and motor 9, switch 10 be arranged on the rear end of pen type housing 6, flexible coil 12 between switch 10 and the battery 9.
The method concrete steps are as follows:
A. prepare the information transfer film, stand-by.Get coconut oil 18～22, castor oil 5～9, tallow 11～15, sugar 12～14, NaOH 20～24, alcohol 8～12, soda 0.3～0.7, water 28～30 by weight; Coconut oil, castor oil, tallow are dropped into vessel in heating to 75～85 ℃, again NaOH, the water-soluble back of soda are added in the oil plant that is heated slowly, stir and make it saponification, treat that soap lye is transparent, add sugar and alcohol, stir, insert in the soft squeeze receptacle stand-by;
B. from liquid nitrogen, take out frozen tissue and make section, techtology dyeing, the size of section is advisable to be fit to desk-top microscopic examination.Frozen tissue can be fresh frozen tissue, also can be the fixing sample of paraffin-embedded tissue sample or paraformaldehyde;
C. section is placed microscopically to observe, selected point of destination (spot) or purpose zone (reqion);
D. in the front of frozen tissue section, locate with the calibration pen aligning point of destination (spot) or purpose zone (reqion) that are installed on the microscope, the demarcation 4 of calibration pen is contacted with point of destination or purpose zone, compressing elastic dye bottle 3, dyestuff in the elastic dye bottle 3 just at point of destination or purpose area coloring, is promptly demarcated the purpose zone;
E. be coated with information transfer film liquid and use doctor blade at the back side of frozen tissue section, treat information transfer film liquid air-dry (about 20 seconds) after, the use pen goes out point of destination or purpose zone at information transfer film image scale on show;
F. the information transfer face with frozen tissue section pastes mutually with frozen tissue piece (25～-30 ℃) is identical, and then frozen tissue section is taken off, and at this moment, the information transfer film is attached on the frozen tissue piece, demonstrates point of destination or purpose zone significantly;
G. press electronic cutter switch, motor and power supply join, and milling cutter rotates, and cut along the purpose area flag on the frozen tissue with electronic cutter, and depth of cut can be controlled by the length of regulating milling cutter.Purpose regional organization just separates with organizing main body when serial section automatically, collects the purpose zone.The purpose zone can be placed the centrifuge tube second month in a season that contains sample extraction liquid, ((DNA is RNA) with the albumen sample to prepare nucleic acid with conventional method.
Claims (2)
1. the catching method of a tissue special area is characterized in that having the following steps:
A. getting section from the frozen tissue piece places microscopically to select the purpose zone;
B. demarcate the purpose zone with calibration pen in the front of frozen tissue section;
C. be coated with information transfer film liquid and use pen at the back side of frozen tissue section and go out the purpose zone at information transfer film image scale on show;
D. the information transfer face with frozen tissue section pastes mutually with the frozen tissue piece is identical, and then frozen tissue section is taken off;
E. cut along the purpose area flag on the frozen tissue with cutter, after slice separation under the freezing condition, collect the purpose zone.
2. the catching method of tissue special area according to claim 1, it is characterized in that described information transfer film liquid is that when method is formulated by following component, weight: coconut oil 18～22, castor oil 5～9, tallow 11～15, sugar 12～14, NaOH 20～24, alcohol 8～12, soda 0.3～0.7, water 28～30, coconut oil, castor oil, tallow are dropped into vessel in heating to 75～85 ℃, slowly add NaOH, the water-soluble back of soda in the oil plant that is heated in addition, stirring makes it saponification, treat that soap lye is transparent, add sugar and alcohol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200610045852A CN100587451C (en) | 2006-02-14 | 2006-02-14 | Method for trapping tissue special region and special equipment |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200610045852A CN100587451C (en) | 2006-02-14 | 2006-02-14 | Method for trapping tissue special region and special equipment |
Related Child Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200910179568 Division CN101710034A (en) | 2006-02-14 | 2006-02-14 | Calibration pen for tissue special area capturing method |
| CN 200910179398 Division CN101672730A (en) | 2006-02-14 | 2006-02-14 | Electric cutter used in capture method of tissue location |
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| CN1811369A CN1811369A (en) | 2006-08-02 |
| CN100587451C true CN100587451C (en) | 2010-02-03 |
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| CN200610045852A Expired - Fee Related CN100587451C (en) | 2006-02-14 | 2006-02-14 | Method for trapping tissue special region and special equipment |
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Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8744163B2 (en) * | 2010-02-09 | 2014-06-03 | International Genomics Consortium | System and method for laser dissection |
| EP2629078B1 (en) * | 2010-11-19 | 2019-04-17 | Olympus Corporation | Method for preparing biological sample |
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2006
- 2006-02-14 CN CN200610045852A patent/CN100587451C/en not_active Expired - Fee Related
Non-Patent Citations (6)
| Title |
|---|
| Cytopathological evaluations combined RNA andproteinanalyseson defined cell regions using single frozentissue block. HONG LI等.Cell Research,Vol.12 No.2. 2002 |
| Cytopathological evaluations combined RNA andproteinanalyseson defined cell regions using single frozentissue block. HONG LI等.Cell Research,Vol.12 No.2. 2002 * |
| Three-dimensional distribution of collateral bloodflowwithinthe anatomic area at risk after circumflex coronaryarteryocclusion in dogs. K. A. Reimer等.Basic Research in Cardiology,No.82. 1987 |
| Three-dimensional distribution of collateral bloodflowwithinthe anatomic area at risk after circumflex coronaryarteryocclusion in dogs. K. A. Reimer等.Basic Research in Cardiology,No.82. 1987 * |
| 冰冻组织特定区域的RNA快速提取及其RT-PCR分析. 江岩等.中华医学遗传学杂志，第17卷第1期. 2000 |
| 冰冻组织特定区域的RNA快速提取及其RT-PCR分析. 江岩等.中华医学遗传学杂志，第17卷第1期. 2000 * |
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| CN1811369A (en) | 2006-08-02 |
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