CN103952302B - A kind of novel substrate of testing for unicellular gel electrophoresis - Google Patents

Abstract

The invention relates to a new type of substrate for single-cell gel electrophoresis experiments, which is composed of an object slide, a coronal plane slot, a horizontal plane baffle and a horizontal plane slot, and the coronal plane slots and the horizontal plane slots are alternately stacked and arranged on the Above the slide, there is a gap between the horizontal card slot and the slide for laying the gel; there are horizontal baffles on both sides of the coronal card slot and the horizontal card slot; the coronal card slot can insert hydrophobic hard cards , the horizontal card slot can be inserted into a hydrophobic hard transparent card; the invention solves the problem that the experimental results of the single-cell gel electrophoresis experiment are very different due to the differences in the gel preparation methods of different research groups or experimenters, and can make the single-cell gel electrophoresis The size and thickness of the gel prepared by the electrophoresis experiment are standardized; the gel can be prepared more quickly by using the present invention, the preparation steps are simpler, and the thickness of the prepared gel is uniform, which avoids the subsequent comet image analysis from being affected by the uneven thickness of the gel; After gel electrophoresis, comet images obtained by different experimental operators are more reproducible and comparable.

Description

A new substrate for single-cell gel electrophoresis experiments

technical field

The invention relates to the experimental detection fields of cell biology, environmental genotoxicity detection, biological monitoring, drug genotoxicity, etc., and specifically relates to a novel substrate for single-cell gel electrophoresis experiments.

Background technique

Single cell gel electrophoresis (single cell geleletrophoresis, SCGE) is an experimental method for rapid detection of DNA damage in single cells, pioneered by Ostling et al. (1984) and further improved by Singh et al. (1988). Finally, the DNA staining shape of the damaged nucleus resembles a comet, also known as the cometassay.

The principle of this technology is based on the fact that the DNA molecular weight of nucleated cells is very large. The DNA superhelical structure is attached to the nuclear matrix, and the cells are embedded on a glass slide with agarose gel. Under the action of cell lysate, the cell membrane, nucleus Disruption of membranes and other biofilms, allowing intracellular RNA, proteins, and other components to enter the gel and then diffuse into the lysate, except that the loops of nuclear DNA remain attached to the remaining nuclear skeleton and remain in place . If the cells are not damaged, the nuclear DNA stays in the nuclear matrix due to its large molecular weight during electrophoresis, and after fluorescent staining, it presents a round fluorophore without tailing. If the cells are damaged, in the alkaline electrophoresis solution (pH>13), the DNA double-strand unwinds first and is denatured into a single-strand by alkali. Fragments migrate away from the DNA towards the anode, forming a smear. The heavier the DNA damage in the nucleus, the more broken chains or alkali-variable fragments will be produced, and the smaller the broken chains or short pieces will be. The amount of DNA migrating under the action of an electric field will be larger, and the migration distance will be longer, which is manifested by increased tail length and Increased fluorescence intensity at the tail. Therefore, the degree of DNA damage in a single cell can be quantitatively determined by measuring the optical density or migration length of the DNA migration part.

In the single-cell gel electrophoresis experiment, when the agarose gel laid on the ordinary glass slide is subjected to multiple steps such as cell lysis, washing, and electrophoresis, the gel is very easy to drift, break, or even fall off on the ordinary glass slide. The analysis of an indicator in this experiment requires multiple samples to be supported, and glue needs to be spread on multiple ordinary glass slides. The process is very cumbersome. The thickness of the gel on ordinary glass slides is often uncontrollable. Due to the difference in the thickness of the agarose gel between samples, the morphology of the cells is likely to be different when observed under a microscope, which affects the quantitative analysis results. When laying the low-melting point agarose gel containing cells, some low-melting point agarose gels often exceed the first layer of ordinary agarose gel and are in direct contact with the glass surface. Due to the adsorption of the glass, they directly contact the glass surface. cells yield false positive results.

In order to solve the above-mentioned inconvenience and errors caused by ordinary glass slides, we invented a new type of substrate for single-cell gel electrophoresis experiments. The invention can ensure that the thickness of the gel is uniform and consistent during the single-cell gel electrophoresis experiment, speed up the experimental process, reduce the workload of manual gel laying, and ensure the reliability of subsequent quantitative analysis results.

Contents of the invention

The invention relates to a novel substrate for single-cell gel electrophoresis experiments.

The novel substrate for single-cell gel electrophoresis experiments proposed by the present invention is composed of a slide 1, a coronal plane slot 2, a horizontal plane baffle plate 3 and a horizontal plane slot 4, wherein: the coronal plane slot 2, the horizontal plane slot The board 3 and the horizontal plane slot 4 form a laying device, the coronal plane slot 2 and the horizontal plane slot 4 are alternately stacked and arranged above the loading sheet 1, and there is a gap between the horizontal slot 4 and the loading sheet 1 for laying Gel; both sides of the coronal slot 2 and the horizontal slot 4 are provided with horizontal baffles 3; the coronal slot 2 can be inserted with a hydrophobic hard card, and the horizontal slot 4 can be inserted with a hydrophobic hard transparent card; when in use, the hydrophobic Insert the hard card into the coronal slot 2 on both sides of the glue laying device, and prepare the first layer of gel: insert the hydrophobic hard transparent card into the slot 4 of the second horizontal plane, on the side of the slot 4 of the first horizontal plane Slowly inject completely melted high-melting point agarose, let it stand at room temperature, and slowly pull out the hydrophobic hard transparent card after it is completely solidified. Preparation of the second layer of gel: insert a new hydrophobic hard transparent card into the card slot 4 of the third layer of horizontal plane, slowly inject low-melting point agarose in molten state on the socket side of the card slot 4 of the second layer of horizontal plane, and let it stand at room temperature. After it is completely solidified, slowly pull out the hydrophobic hard transparent card. Preparation of the third layer of gel: Insert a new hydrophobic hard transparent card into the slot 4 of the fourth horizontal plane, slowly inject molten low-melting point agarose into the socket side of the third horizontal plane slot 4, and let it stand at room temperature. After it is completely solidified, slowly pull out the hydrophobic hard transparent card, and so on to prepare several gels.

The beneficial effect of the present invention is that: the novel substrate solves the problem that the experimental results of the single-cell gel electrophoresis experiment are very different due to differences in the gel preparation methods of different research groups or experimenters, and can make the single-cell gel electrophoresis experiment The prepared gel size and thickness are standardized; compared with ordinary slides used in traditional single-cell gel electrophoresis experiments or slides that have been treated to prevent DNA adsorption, the use of the new substrate can prepare gels more quickly. gel, the preparation steps are simpler, the thickness of the prepared gel is uniform, and the follow-up comet image analysis is avoided from being affected by the uneven thickness of the gel; after gel electrophoresis, the repeatability and comparability of the comet images obtained by different experimental operators are better. it is good.

Description of drawings

Figure 1 is a schematic diagram of a new type of substrate structure used in single-cell gel electrophoresis experiments.

Figure 2 is a top view of the substrate.

Figure 3 is a sagittal side view of the substrate.

Figure 4 is a coronal view of the middle part of the substrate.

Figure 5 is the comet image of rat alveolar macrophages treated with hydrogen peroxide, (A) cells after 100 μM H ₂ O ₂ treatment, (B) cells after H ₂ O treatment.

Numbers in the figure: 1 is the slide, 2 is the coronal plane slot, 3 is the horizontal plane baffle, and 4 is the horizontal plane slot.

detailed description

The present invention is further illustrated below by way of examples.

Example 1: A new type of substrate for single-cell gel electrophoresis experiments. As shown in FIG. 1 , the substrate is mainly composed of an object slide 1 , a coronal plane slot 2 , a horizontal plane baffle 3 and a horizontal plane slot 4 . Among them: the coronal surface card slot 2, the horizontal plane baffle plate 3 and the horizontal plane card slot 4 form the glue laying device. The material for preparing the substrate is quartz or glass or plastics such as PVC and PP, or a variety of materials are properly matched and mixed. Figure 2, Figure 3, and Figure 4 show the size of the substrate: the size of the slide 1 is 76.0mm × 26.0mm × 2.0mm, and the coronal plane slot 2, the horizontal plane baffle plate 3 and the horizontal plane slot 4 are composed of The size of the glue device is 26.0mm×26.0mm×5.0mm, the size of the slot 2 on the coronal plane is 26.0mm×5.0mm×1.2mm, the size of the baffle plate 3 on the horizontal plane is 26.0mm×1.2mm×0.5mm, the slot on the horizontal plane The size of 4 is 26.0mm×26.0mm×1.2mm. Card slot 2 on the coronal plane can insert a hydrophobic hard card of 26.0mm×(≥5.0mm)×(≤1.2mm), card slot 4 on the horizontal plane can insert a hydrophobic hard transparent card of 26.0mm×26.0mm×(≤1.2mm) .

The substrate can be used to quickly prepare three-layer agarose gel. Insert the hydrophobic hard card into the coronal slot 2 on both sides of the glue laying device to prepare the first layer of gel: Insert the hydrophobic hard transparent card into the slot 4 of the second horizontal plane, and insert it into the slot 4 of the first horizontal plane Slowly inject completely melted high-melting point agarose into one side, let it stand at room temperature, and slowly pull out the hydrophobic hard transparent card after it is completely solidified. Preparation of the second layer of gel: insert a new hydrophobic hard transparent card into the card slot 4 of the third layer of horizontal plane, slowly inject low-melting point agarose in molten state on the socket side of the card slot 4 of the second layer of horizontal plane, and let it stand at room temperature. After it is completely solidified, slowly pull out the hydrophobic hard transparent card. Preparation of the third layer of gel: Insert a new hydrophobic hard transparent card into the slot 4 of the fourth horizontal plane, slowly inject molten low-melting point agarose into the socket side of the third horizontal plane slot 4, and let it stand at room temperature. After it is completely solidified, slowly pull out the hydrophobic hard transparent card.

The above-mentioned substrate is used for single-cell gel electrophoresis to detect the damage of hydrogen peroxide to rat alveolar macrophages, the steps are:

(1) One healthy adult SD rat, weighing 218g, male, with bright hair color, no hair loss, no deformity of limbs and tail, soft neck and no skew.

(2) Anesthetized by intraperitoneal injection of 20mg/ml pentobarbital sodium injection at a dose of 35mg/kg.

(3) Bleeding from the abdominal aorta, and the rat's eyeballs were pale, indicating that the bloodletting was sufficient.

(4) Free the trachea, fix the PE hose, infuse 10ml of normal saline each time, and aspirate the normal saline after lavage with the syringe.

(5) Repeat step (4) 9 times to fully lavage rat alveolar macrophages.

(6) Centrifuge the harvested saline, centrifuge at 800rpm at 4°C for 5min, remove the supernatant, resuspend in cold ¹ ×PBS, count the cells, adjust the cell concentration to 1.0×106 cells/ml, and aliquot them in 1.5ml sterile centrifuge tubes For cells, make 10 ⁵ cells/tube.

(7) React the cells with hydrogen peroxide (H ₂ O ₂ ) in a 1.5ml centrifuge tube for 5 minutes on ice. Use the addition of H ₂ O ₂ to make the final concentration of H ₂ O ₂ in PBS 100 μM, respectively. The control group used _H2O . Centrifuge at 800 rpm for 5 minutes at 4°C, remove the supernatant, add an equal volume of 37°C low-melting point agarose to resuspend, keep warm at 37°C, and use as soon as possible.

(8) To prepare three-layer gel, take 850 μl high melting point agarose with a pipette gun to prepare the first layer of gel, take 20 μl of cells and add it to 830 μl 37°C low melting point agarose to prepare the second layer of gel, take 850 μl low melting point agarose Prepare a third layer of gel.

(9) Soak the prepared gel substrate in freshly prepared 4°C pre-cooled cell lysate (2.5M NaCl, 100mM Na ₂ EDTA, 10mM Tris, 1M NaOH to adjust the pH to 10.0, then add 1% TritonX-100), 4°C Crack for 1h.

(10) Put it in the electrophoresis tank, soak in 4°C electrophoresis solution (0.3M NaOH, 1mMNa ₂ EDTA, pH>12) for 40min, then electrophoresis at 25V (1V/cm, 300mA) for 30min at 4°C.

result:

Under the action of 100 μM H ₂ O ₂ , the DNA of rat alveolar macrophages was significantly damaged, and the DNA of the nucleus showed an obvious elliptical comet tailing phenomenon (Figure 5B ₎ . Round shape, no comet tailing observed (Fig. 5A). At the same time, it was observed under a fluorescence microscope that the stained DNA on different substrates was basically at the same height plane, and the fluorescence intensity of DNA on different substrates was uniform. The traditional method of gel preparation has three layers of gel with different thicknesses, and the stained DNA is often not at the same plane height. The fluorescence intensity is affected by the thickness of the gel and the distribution of DNA staining depth, which affects comet imaging and analysis. The new substrate is used in single-cell gel electrophoresis experiments, which solves the above problems well.

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.