CN103952302A - Novel substrate for single-cell gel eletrophoresis experiments - Google Patents
Novel substrate for single-cell gel eletrophoresis experiments Download PDFInfo
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Abstract
The invention relates to a novel substrate for single-cell gel eletrophoresis experiments. The novel substrate comprises a slide, coronal plane necks, horizontal plane baffles and horizontal plane necks, wherein the coronal plane necks and the horizontal plane necks are arranged above the slide in an alternate and superposition manner, and gaps are reserved between the horizontal plane necks and the slide and are used for laying gel; the horizontal plane baffles are arranged at the two sides of the coronal plane necks and the horizontal plane necks; and hydrophobic hard cards can be inserted in the coronal plane necks, and hydrophobic hard transparent cards can be inserted in the horizontal plane necks. The novel substrate can be used for solving the problem of large difference between experiments results caused by difference of gel preparation methods due to different research groups or experimenters in single-cell gel eletrophoresis experiments and ensuring that the size and thickness of the gel prepared in the single-cell gel eletrophoresis experiments are standardized. The novel substrate can be used for rapidly preparing the gel and is relatively simple in preparation steps; the prepared gel is uniform in thickness, so that the problem that the follow-up comet image analysis is affected by the non-uniform of thickness of the gel is solved; and after gel eletrophoresis, the repeatability and comparability of comet images obtained by the different experimenters are relatively good.
Description
Technical field
The present invention relates to experiment detection fields such as belonging to cytobiology, the detection of environment genotoxicity, biological monitoring, medicine genetoxic, is specifically related to a kind of novel substrate for unicellular gel electrophoresis experiment.
Background technology
Single cell gel electrophoresis is analyzed (single cell gel eletrophoresis, SCGE) be that (1984) such as Ostling are pioneering, further improve and the experimental technique of the unicellular DNA damage of a kind of rapid detection that grows up gradually by (1988) such as Singh, because its cell is after electrophoresis, damaged cell core DNA dyeing shape, quite like comet, claims again comet test (cometassay).
The principle of this technology is that the DNA molecular amount based on karyocyte is very large, 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, is then diffused in lysate, only core DNA still keeps being wound around Huan district and is attached on remaining nuclear skeleton, and stays original position.If cell is without damage, electrophoresis center DNA, because its molecular weight rests in nuclear matrix greatly, presents circular fluorophore, without conditions of streaking after fluorescent dye.If cell is impaired, in alkaline electrophoresis liquid (pH>13), the uncoiling of DNA double chain and alkaline denaturation were strand before this, little can the entering in gel of fragment molecular weight of single-strand break, in the time of 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, and the distance of migration shows as the long increase of tail and afterbody fluorescence intensity and strengthens.Therefore, just can quantitative assay individual cells DNA damage degree by optical density(OD) or the migration length of measuring DNA migration part.
In SCGE experiment, the sepharose of laying on common slide glass, while carrying out multiple steps such as lysis, washing, electrophoresis, gel very easily drifts about, ruptures on common slide glass, even comes off.Index of this experimental analysis need to be carried out multiple sample supports, need to be at multiple common slide glasss upper berth glue, and process is very loaded down with trivial details.At common slide glass upper berth glue, thickness often can not be controlled, the thickness difference due to sepharose of sample room, and while examining under a microscope, the form of cell easily has difference, affects quantitative analysis results.Contain cell in laying low melting-point agarose gel time, often there is part low melting-point agarose gel to exceed the first layer plain agar sugar gel, directly contact with glass surface, due to the adsorption of glass, cause the cell directly contacting with glass surface to produce false positive results.
In order to solve the above-mentioned inconvenience being brought by common slide glass and error, we have invented a kind of novel substrate for SCGE experiment.This invention can be guaranteed SCGE experiment process middle berth glue even thickness, consistent, accelerates experiment process, alleviates artificial paving glue workload; Ensure that follow-up quantitative analysis 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, formed by 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 in microscope slide 1 top, between horizontal plane draw-in groove 4 and microscope slide 1, leave space, for laying gel; 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; When use, hydrophobic hard card inserts in paving adhesive dispenser both sides coronal-plane draw-in groove 2, prepare the first layer gel: transparent hydrophobic hard card is inserted in second layer horizontal plane draw-in groove 4, slowly inject in the first layer horizontal plane draw-in groove 4 socket one sides the high-melting-point agarose melting completely, room temperature leaves standstill, and slowly pulls out the transparent card of hydrophobic hard after solidifying completely.Prepare second layer gel: a transparent card of new hydrophobic hard is inserted in the 3rd layer of horizontal plane draw-in groove 4, slowly inject the low melting-point agarose of melting state in second layer horizontal plane draw-in groove 4 socket one sides, room temperature leaves standstill, and slowly pulls out the transparent card of hydrophobic hard after solidifying completely.Prepare the 3rd layer of gel: a transparent card of new hydrophobic hard is inserted in the 4th layer of horizontal plane draw-in groove 4, slowly inject the low melting-point agarose of melting state in the 3rd layer of horizontal plane draw-in groove 4 socket one sides, 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 has solved SCGE experiment because the very large problem of experimental result difference that different seminars or experimenter's gel process for preparing difference cause, gel size, the thickness calibration that can make SCGE experiment prepare; The common slide glass used with traditional SCGE experiment or prevent that after treatment the slide glass of DNA absorption from comparing, use described novel substrate can prepare more fast gel, preparation process is more simple, prepared gel thicknesses homogeneous, avoids follow-up comet image analysis affected by gel became uneven one; After gel electrophoresis, comet image repeatability and comparability that different experiments operator obtain are better.
Brief description of the drawings
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 substrate middle part coronal-plane figure.
Fig. 5 is comet image, (A) 100 μ M H after hydrogen peroxide is processed rat pulmonary alveolus macrophage
2o
2cell, (B) H after processing
2cell after O processes.
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
Further illustrate the present invention below by embodiment.
Embodiment 1: for the novel substrate of SCGE experiment.As showing this substrate, Fig. 1 mainly formed by 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 composition paving adhesive dispensers.The material of preparing substrate is the plastics such as quartz or 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 that coronal-plane draw-in groove 2, horizontal plane baffle plate 3 and horizontal plane draw-in groove 4 form 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 can be 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: transparent hydrophobic hard card is inserted in second layer horizontal plane draw-in groove 4, slowly inject in the first layer horizontal plane draw-in groove 4 socket one sides the high-melting-point agarose melting completely, room temperature leaves standstill, and slowly pulls out the transparent card of hydrophobic hard after solidifying completely.Prepare second layer gel: a transparent card of new hydrophobic hard is inserted in the 3rd layer of horizontal plane draw-in groove 4, slowly inject the low melting-point agarose of melting state in second layer horizontal plane draw-in groove 4 socket one sides, room temperature leaves standstill, and slowly pulls out the transparent card of hydrophobic hard after solidifying completely.Prepare the 3rd layer of gel: a transparent card of new hydrophobic hard is inserted in the 4th layer of horizontal plane draw-in groove 4, slowly inject the low melting-point agarose of melting state in the 3rd layer of horizontal plane draw-in groove 4 socket one sides, room temperature leaves standstill, and slowly pulls out the transparent card of hydrophobic hard after solidifying completely.
Above-mentioned substrate is detected to the degree of impairment of hydrogen peroxide to rat pulmonary alveolus macrophage for single cell gel electrophoresis, step:
(1) 1 of healthy adult SD rat, body weight 218g, male, chroma of hair light, without depilation, four limbs and afterbody are without incompleteness, and neck is soft without crooked.
(2) press 35 mg/kg dosage intraperitoneal injection of anesthesia 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 pours into physiological saline 10ml, physiological saline after syringe pump lavation at every turn.
(5) repeat (4) step 9 time, fully lavation rat pulmonary alveolus macrophage.
(6) physiological saline of centrifugal results, 4 DEG C, the centrifugal 5min of 800rpm, removes supernatant, and cold 1 × PBS is resuspended, counting cells, adjusting cell concn is 1.0 × 10
6individual/ml, the aseptic centrifuge tube packing of 1.5ml cell, makes 10
5individual cell/pipe.
(7) by cell and hydrogen peroxide (H
2o
2) in 1.5ml centrifuge tube, act on 5 min on ice.Use adds H
2o
2, make H in PBS
2o
2final concentration is respectively 100 μ M.Control group uses H
2o.4 ° of C, the centrifugal 5min of 800rpm, removes supernatant, adds 37 DEG C of low melting-point agaroses of equal-volume resuspended, under 37 DEG C of conditions, is incubated, and uses as early as possible.
(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 37 DEG C of low melting-point agaroses of 830 μ l and prepare second layer gel, gets 850 μ l low melting-point agaroses and prepares the 3rd layer of gel.
(9) substrate for preparing gel is dipped in to cell pyrolysis liquid (2.5M NaCl, the 100mM Na of 4 DEG C of precoolings of fresh preparation
2eDTA, 10mM Tris, 1M NaOH adds 1% Triton X-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 (0.3M NaOH, 1mM Na
2eDTA, pH>12) in the 40min that untwists, then 25 V(1V/cm under 4 DEG C of conditions, 300 mA) electrophoresis 30 min.
Result:
100 μ M 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 is significantly damage not, and nucleus DNA is rounded, has no comettail phenomenon (Fig. 5 A).Meanwhile, observe dyeing DNA in substrates of different substantially in an elevation plane under fluorescent microscope, in substrates of different, DNA fluorescence intensity is even.Traditional method is prepared gel and is existed three layers of gel thickness to differ, and dyeing DNA is not usually in same plane height, and fluorescence intensity size is subject to gel thickness and DNA dyeing depth distribution influence, thereby has affected comet imaging and analysis.Novel substrate, for SCGE experiment, has well been solved to the problems referred to above.
Claims (1)
1. the novel substrate for SCGE experiment, formed by 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, between horizontal plane draw-in groove (4) and microscope slide (1), leave space, for laying gel; 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; When use, hydrophobic hard card inserts in paving adhesive dispenser both sides coronal-plane draw-in grooves (2); Prepare the first layer gel: transparent hydrophobic hard card is inserted in second layer horizontal plane draw-in groove (4), slowly inject in the first layer horizontal plane draw-in groove (4) socket one side the high-melting-point agarose melting completely, room temperature leaves standstill, and slowly pulls out the transparent card of hydrophobic hard after solidifying completely; Prepare second layer gel: a transparent card of new hydrophobic hard is inserted in the 3rd layer of horizontal plane draw-in groove (4), slowly inject the low melting-point agarose of melting state in second layer horizontal plane draw-in groove (4) socket one side, room temperature leaves standstill, and slowly pulls out the transparent card of hydrophobic hard after solidifying completely
;prepare the 3rd layer of gel: a transparent card of new hydrophobic hard is inserted in the 4th layer of horizontal plane draw-in groove (4), slowly inject the low melting-point agarose of melting state in the 3rd layer of horizontal plane draw-in groove (4) socket one 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.
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| CN201410149239.5A CN103952302B (en) | 2014-04-15 | 2014-04-15 | A kind of novel substrate of testing for unicellular gel electrophoresis |
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| CN103952302B CN103952302B (en) | 2016-02-17 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113405881A (en) * | 2021-06-10 | 2021-09-17 | 华东理工大学 | Rapid detection method and kit for single cell DNA damage |
| CN114397349A (en) * | 2022-01-14 | 2022-04-26 | 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 |
Citations (3)
| 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 |
| US20130177903A1 (en) * | 2007-12-05 | 2013-07-11 | Ge Healthcare Uk Limited | Apparatus and method for detecting dna damage |
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2014
- 2014-04-15 CN CN201410149239.5A patent/CN103952302B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130177903A1 (en) * | 2007-12-05 | 2013-07-11 | Ge Healthcare Uk Limited | Apparatus and method for detecting dna damage |
| 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 |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113405881A (en) * | 2021-06-10 | 2021-09-17 | 华东理工大学 | Rapid detection method and kit for single cell DNA damage |
| CN114397349A (en) * | 2022-01-14 | 2022-04-26 | 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 |
| 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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