CN103952302B - A kind of novel substrate of testing for unicellular gel electrophoresis - Google Patents
A kind of novel substrate of testing for unicellular gel electrophoresis Download PDFInfo
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- CN103952302B CN103952302B CN201410149239.5A CN201410149239A CN103952302B CN 103952302 B CN103952302 B CN 103952302B CN 201410149239 A CN201410149239 A CN 201410149239A CN 103952302 B CN103952302 B CN 103952302B
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- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
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- G01N27/447—Systems using electrophoresis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The present invention relates to a kind of novel substrate for SCGE experiment, be made up of microscope slide, coronal-plane draw-in groove, horizontal plane baffle plate and horizontal plane draw-in groove, coronal-plane draw-in groove and horizontal plane draw-in groove replace stacked arrangement above microscope slide, space is left, for laying gel between horizontal plane draw-in groove and microscope slide; Coronal-plane draw-in groove and horizontal plane draw-in groove both sides are provided with horizontal plane baffle plate; Coronal-plane draw-in groove can insert hydrophobic hard card, and horizontal plane draw-in groove can insert the transparent card of hydrophobic hard; The invention solves SCGE experiment because the very large problem of experimental result difference that causes of different seminar or experimenter's gel process for preparing difference, the gel size that SCGE experiment can be made to prepare, thickness normalized; Use the present invention can prepare gel faster, preparation process is more simple, and prepared gel thicknesses is homogeneous, avoids follow-up comet image analysis to affect by gel became uneven one; After gel electrophoresis, the comet image that different experiments operator obtain is repeated and comparability is better.
Description
Technical field
The present invention relates to experiment detection fields such as belonging to cytobiology, the detection of environment genotoxicity, biological monitoring, pharmacogenetic toxicity, is specifically related to a kind of novel substrate of testing for unicellular gel electrophoresis.
Background technology
Single cell gel electrophoresis analyzes (singlecellgeleletrophoresis, SCGE) be that (1984) such as Ostling initiate, the experimental technique of the unicellular DNA damage of a kind of rapid detection grown up gradually by further the improving such as Singh (1988), because its cell is after electrophoresis, damaged cell core DNA dyes shape quite like comet, also known as comet test (cometassay).
The principle of this technology is very large based on the DNA molecular amount of karyocyte, be attached in nuclear matrix at DNA superhelix, with sepharose by cell embedding on slide glass, under cell pyrolysis liquid effect, cytolemma, nuclear membrane and other biological film destroy, make intracellular RNA, protein and other compositions enter gel, be then diffused in lysate, only core DNA still keeps the ring district be wound around to be attached on remaining nuclear skeleton, and stays original position.If cell is without damage, electrophoresis center DNA rests in nuclear matrix because its molecular weight is large, presents circular fluorophore, without conditions of streaking after fluorescent dye.If cell damage, in alkaline electrophoresis liquid (pH>13), DNA double chain uncoiling before this and alkaline denaturation is strand, the fragment molecular weight of single-strand break is little can be entered in gel, when electrophoresis, chain rupture or fragment leave the migration of DNA anode, form hangover.Nuclear DNA damage is heavier, and just the more, its chain rupture or short-movie are also just little for the chain rupture of generation or alkali volatility fragment, and the DNA amount of moving under electric field action is many, the distance of migration, shows as that tail is long to be increased and the enhancing of afterbody fluorescence intensity.Therefore, optical density(OD) or migration length by measuring DNA migration part just can quantitative assay individual cells DNA damage degree.
In SCGE experiment, the sepharose that common slide glass is laid, when carrying out multiple steps such as lysis, washing, electrophoresis, gel very easily drifts about, ruptures on common slide glass, even comes off.This experimental analysis index needs to carry out multiple sample support, and need at multiple common slide glass upper berths glue, process is very loaded down with trivial details.At common slide glass upper berth glue, thickness often can not control, and the thickness difference due to sepharose of sample room, when examining under a microscope, the form of cell easily has difference, affects quantitative analysis results.When laying the low melting-point agarose gel containing cell, part low melting-point agarose gel is often had to exceed the first layer plain agar sugar gel, directly contact with glass surface, due to the adsorption of glass, cause the cell directly contacted with glass surface to produce false positive results.
In order to solve the above-mentioned inconvenience that brought by common slide glass and error, we have invented a kind of novel substrate for SCGE experiment.This invention can guarantee that SCGE experiment process middle berth glue thickness evenly, unanimously, accelerates experiment process, alleviates artificial paving glue workload; Ensure that subsequent quantitation analytical results is reliable simultaneously.
Summary of the invention
The present invention relates to a kind of novel substrate for SCGE experiment.
The novel substrate for SCGE experiment that the present invention proposes, be made up of microscope slide 1, coronal-plane draw-in groove 2, horizontal plane baffle plate 3 and horizontal plane draw-in groove 4, wherein: coronal-plane draw-in groove 2, horizontal plane baffle plate 3 and horizontal plane draw-in groove 4 form laying apparatus, coronal-plane draw-in groove 2 and horizontal plane draw-in groove 4 replace stacked arrangement above microscope slide 1, space is left, for laying gel between horizontal plane draw-in groove 4 and microscope slide 1; Coronal-plane draw-in groove 2 and horizontal plane draw-in groove 4 both sides are provided with horizontal plane baffle plate 3; Coronal-plane draw-in groove 2 can insert hydrophobic hard card, and horizontal plane draw-in groove 4 can insert the transparent card of hydrophobic hard; During use, hydrophobic hard card inserts in paving adhesive dispenser both sides coronal-plane draw-in groove 2, prepare the first layer gel: inserted by transparent for hydrophobic hard card in second layer horizontal plane draw-in groove 4, the high-melting-point agarose melted completely is slowly injected in the first layer horizontal plane draw-in groove 4 socket side, room temperature leaves standstill, after solidifying completely, slowly pull out the transparent card of hydrophobic hard.Prepare second layer gel: insert in third layer horizontal plane draw-in groove 4 by a transparent card of new hydrophobic hard, the low melting-point agarose of melting state is slowly injected in second layer horizontal plane draw-in groove 4 socket side, room temperature leaves standstill, after solidifying completely, slowly pull out the transparent card of hydrophobic hard.Preparation third layer gel: a new transparent card of hydrophobic hard is inserted in the 4th layer of horizontal plane draw-in groove 4, the low melting-point agarose of melting state is slowly injected in third layer horizontal plane draw-in groove 4 socket side, room temperature leaves standstill, the slow transparent card of pull-out hydrophobic hard after solidifying completely, the like be prepared into some one-tenth gels.
Beneficial effect of the present invention is: described novel substrate solves SCGE experiment because the very large problem of experimental result difference that causes of different seminar or experimenter's gel process for preparing difference, the gel size that SCGE experiment can be made to prepare, thickness normalized; The common slide glass used with traditional SCGE experiment or prevent DNA from adsorbing after treatment slide glass compared with, use described novel substrate can prepare gel more fast, preparation process is more simple, prepared gel thicknesses is homogeneous, avoids follow-up comet image analysis to affect by gel became uneven one; After gel electrophoresis, the comet image that different experiments operator obtain is repeated and comparability is better.
Accompanying drawing explanation
Fig. 1 is a kind of novel substrate structure schematic diagram for SCGE experiment.
Fig. 2 is substrate vertical view.
Fig. 3 is substrate sagittal plane side elevational view.
Fig. 4 is coronal-plane figure in the middle part of substrate.
Fig. 5 be hydrogen peroxide to comet image after rat pulmonary alveolus macrophage process, (A) 100 μMs of H
2o
2cell after process, (B) H
2cell after O process.
Number in the figure: 1 is microscope slide, 2 is coronal-plane draw-in groove, and 3 is horizontal plane baffle plate, and 4 is horizontal plane draw-in groove.
Embodiment
The present invention is further illustrated below by embodiment.
Embodiment 1: for the novel substrate of SCGE experiment.Form primarily of microscope slide 1, coronal-plane draw-in groove 2, horizontal plane baffle plate 3 and horizontal plane draw-in groove 4 as Fig. 1 shows this substrate.Wherein: coronal-plane draw-in groove 2, horizontal plane baffle plate 3 and horizontal plane draw-in groove 4 composition paving adhesive dispenser.Prepare the material of substrate for quartz or the plastics such as glass or PVC, PP, or multiple material is reasonably combined mixed.Fig. 2, Fig. 3, Fig. 4 show the size of this substrate: microscope slide 1 is of a size of 76.0mm × 26.0mm × 2.0mm, the paving adhesive dispenser of coronal-plane draw-in groove 2, horizontal plane baffle plate 3 and horizontal plane draw-in groove 4 composition is of a size of 26.0mm × 26.0mm × 5.0mm, coronal-plane draw-in groove 2 is of a size of 26.0mm × 5.0mm × 1.2mm, horizontal plane baffle plate 3 is of a size of 26.0mm × 1.2mm × 0.5mm, and horizontal plane draw-in groove 4 is of a size of 26.0mm × 26.0mm × 1.2mm.Coronal-plane draw-in groove 2 can insert the hydrophobic hard card of 26.0mm × (>=5.0mm) × (≤1.2mm), and horizontal plane draw-in groove 4 can insert the transparent card of hydrophobic hard of 26.0mm × 26.0mm × (≤1.2mm).
This substrate may be used for preparing three layers of sepharose fast.Hydrophobic hard card is inserted in paving adhesive dispenser both sides coronal-plane draw-in groove 2, prepare the first layer gel: inserted by transparent for hydrophobic hard card in second layer horizontal plane draw-in groove 4, the high-melting-point agarose melted completely is slowly injected in the first layer horizontal plane draw-in groove 4 socket side, room temperature leaves standstill, after solidifying completely, slowly pull out the transparent card of hydrophobic hard.Prepare second layer gel: insert in third layer horizontal plane draw-in groove 4 by a transparent card of new hydrophobic hard, the low melting-point agarose of melting state is slowly injected in second layer horizontal plane draw-in groove 4 socket side, room temperature leaves standstill, after solidifying completely, slowly pull out the transparent card of hydrophobic hard.Preparation third layer gel: a new transparent card of hydrophobic hard is inserted in the 4th layer of horizontal plane draw-in groove 4, the low melting-point agarose of melting state is slowly injected in third layer horizontal plane draw-in groove 4 socket side, room temperature leaves standstill, after solidifying completely, slowly pull out the transparent card of hydrophobic hard.
Above-mentioned substrate is used for single cell gel electrophoresis and is detected the degree of impairment of hydrogen oxide to rat pulmonary alveolus macrophage, step:
(1) healthy adult SD rat 1, body weight 218g, male, chroma of hair light, without depilation, four limbs and afterbody are without incompleteness, and neck is soft without crooked.
(2) 35mg/kg dosage intraperitoneal injection of anesthesia is pressed with 20mg/ml pentobarbital sodium inj.
(3) aorta abdominalis bloodletting, rat eye is pale is that bloodletting is abundant.
(4) free tracheae, fixing PE flexible pipe, each perfusion physiological saline 10ml, physiological saline after syringe pump lavation.
(5) (4) step 9 time is repeated, abundant lavation rat pulmonary alveolus macrophage.
(6) physiological saline of harvested by centrifugation, 4 DEG C, the centrifugal 5min of 800rpm, removes supernatant, and cold 1 × PBS is resuspended, counting cells, and adjustment cell concn is 1.0 × 10
6individual/ml, 1.5ml sterile centrifugation tube packing cell, makes 10
5individual cell/pipe.
(7) by cell and hydrogen peroxide (H
2o
2) in 1.5ml centrifuge tube, act on 5min on ice.Use adds H
2o
2, make H in PBS
2o
2final concentration is respectively 100 μMs.Control group uses H
2o.4 ° of centrifugal 5min of C, 800rpm, remove supernatant, and it is resuspended to add equal-volume 37 DEG C of low melting-point agaroses, is incubated, uses as early as possible under 37 DEG C of conditions.
(8) prepare three layers of gel, liquid-transfering gun is got 850 μ l high-melting-point agaroses and is prepared the first layer gel, gets 20 μ l cells and joins in 830 μ l37 DEG C low melting-point agaroses and prepare second layer gel, get 850 μ l low melting-point agaroses and prepare third layer gel.
(9) substrate preparing gel is dipped in cell pyrolysis liquid (2.5MNaCl, the 100mMNa of Fresh 4 DEG C of precoolings
2eDTA, 10mMTris, 1MNaOH add 1%TritonX-100 after regulating pH to reach 10.0) in, 4 DEG C of cracking 1h.
(10) put into electrophoresis chamber, be immersed in 4 DEG C of electrophoresis liquid (0.3MNaOH, 1mMNa
2eDTA, pH>12) in untwist 40min, then 25V(1V/cm, 300mA under 4 DEG C of conditions) electrophoresis 30min.
Result:
100 μMs of H
2o
2under effect, rat pulmonary alveolus macrophage DNA is obviously impaired, and obvious oval comettail phenomenon (Fig. 5 B) appears in nucleus DNA, and adds H
2o, cell DNA does not significantly damage, and nucleus DNA is rounded, has no comettail phenomenon (Fig. 5 A).Meanwhile, the dyeing DNA observed under fluorescent microscope in substrates of different is in an elevation plane substantially, and in substrates of different, DNA fluorescence intensity is even.Traditional method is prepared gel and then be there are three layers of gel thickness and differ, and dyeing DNA is in same plane height, and fluorescence intensity size to dye depth distribution influence by gel thickness and DNA, thus have impact on comet imaging and analysis.Novel substrate is used for SCGE experiment, well solves the problems referred to above.
Claims (1)
1. the substrate for SCGE experiment, be made up of microscope slide (1), coronal-plane draw-in groove (2), horizontal plane baffle plate (3) and horizontal plane draw-in groove (4), it is characterized in that: coronal-plane draw-in groove (2), horizontal plane baffle plate (3) and horizontal plane draw-in groove (4) composition laying apparatus, coronal-plane draw-in groove (2) and horizontal plane draw-in groove (4) replace stacked arrangement in microscope slide (1) top, space is left, for laying gel between horizontal plane draw-in groove (4) and microscope slide (1); Coronal-plane draw-in groove (2) and horizontal plane draw-in groove (4) both sides are provided with horizontal plane baffle plate (3); Coronal-plane draw-in groove (2) inserts hydrophobic hard card, and horizontal plane draw-in groove (4) inserts the transparent card of hydrophobic hard; During use, hydrophobic hard card inserts in paving adhesive dispenser both sides coronal-plane draw-in groove (2); Prepare the first layer gel: inserted by transparent for hydrophobic hard card in second layer horizontal plane draw-in groove (4), the high-melting-point agarose melted completely is slowly injected in the first layer horizontal plane draw-in groove (4) socket side, room temperature leaves standstill, after solidifying completely, slowly pull out the transparent card of hydrophobic hard; Prepare second layer gel: insert in third layer horizontal plane draw-in groove (4) by a transparent card of new hydrophobic hard, the low melting-point agarose of melting state is slowly injected in second layer horizontal plane draw-in groove (4) socket side, room temperature leaves standstill, after solidifying completely, slowly pull out the transparent card of hydrophobic hard
;preparation third layer gel: a new transparent card of hydrophobic hard is inserted in the 4th layer of horizontal plane draw-in groove (4), the low melting-point agarose of melting state is slowly injected in third layer horizontal plane draw-in groove (4) socket side, room temperature leaves standstill, the slow transparent card of pull-out hydrophobic hard after solidifying completely, the like be prepared into some layers of gel.
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| CN113405881A (en) * | 2021-06-10 | 2021-09-17 | 华东理工大学 | Rapid detection method and kit for single cell DNA damage |
| CN114397349B (en) * | 2022-01-14 | 2024-04-12 | 中国热带农业科学院热带生物技术研究所 | Protein electrophoresis separation method based on agarose stacking gel |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101570785A (en) * | 2009-06-10 | 2009-11-04 | 南京大学 | Method for detecting potential inherent toxicity of organic pollutants in water body |
| CN101788527A (en) * | 2010-03-09 | 2010-07-28 | 浙江大学 | Complete set of device for SCGE (single cell gel electrophoresis) test |
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| GB0723725D0 (en) * | 2007-12-05 | 2008-01-16 | Ge Healthcare Uk Ltd | Apparatus and method for detecting dna damage |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101570785A (en) * | 2009-06-10 | 2009-11-04 | 南京大学 | Method for detecting potential inherent toxicity of organic pollutants in water body |
| CN101788527A (en) * | 2010-03-09 | 2010-07-28 | 浙江大学 | Complete set of device for SCGE (single cell gel electrophoresis) test |
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