Biological Sciences, Vol. 3, Issue 1, Dec  2019, Pages 8-16; DOI: 10.31058/j.bs.2019.31002 10.31058/j.bs.2019.31002

Design of RNA Extraction System Based on Liquid Nitrogen Grinding

, Vol. 3, Issue 1, Dec  2019, Pages 8-16.

DOI: 10.31058/j.bs.2019.31002

Yinbing Zhang 1*

1 Chemical Engineering and Technology, Hubei University

Received: 1 December 2019; Accepted: 24 December 2019; Published: 31 December 2019

Abstract

In this thesis, a new RNA grinding extraction system is designed and provided for the shortcomings of the existing RNA grinding extraction system technology. The designed RNA grinding and extraction system uses mechanical grinding to extract and extract RNA. This method eliminates the manual grinding operation process and simplifies the operation. At the same time, when the liquid nitrogen is passed in, it can avoid the limbs directly contacting the liquid. Nitrogen contact avoids frostbite of hands due to liquid nitrogen, which greatly improves safety, while mechanical grinding adopts rotary grinding, and the grinding parts are pushed by the cylinder, so that the grinding head of the grinding parts abuts the grinding groove, which is driven by the motor. When the grinding part rotates, it can fully contact the grinding tank to ensure the quality and efficiency of grinding.

Keywords

Liquid Nitrogen Milling, RNA Extraction, System Design

1. Introduction

Liquid nitrogen milling is widely used in molecular biology experiments, especially related to the separation of RNA and DNA. [1] The temperature of liquid nitrogen is 196 °C. It can not only make various tissue components difficult to be destroyed or degraded, but also make the tissue hard, and increase brittleness and easy to grind. Because of the extremely low temperature of liquid nitrogen, special care must be taken during operation. [2] Liquid nitrogen grinding, one is to stop all biological reactions inside and outside the cell, such as to prevent the degradation reaction of RNA enzyme when extracting RNA; there is another reason is that the cells in liquid nitrogen are completely frozen and the grinding can reach Very good cell-breaking effect, grinding the cells into powder to release the contents inside. [3] In this paper, a new RNA grinding and extraction system is designed and provided for the shortcomings of the existing RNA grinding and extraction system technology. It has the advantages of convenient use and high grinding efficiency, and solves the existing RNA grinding and extraction efficiency is low and difficult to operate.

Molecular biology is a frontier subject in the field of life science, and its theory and technology have penetrated into various disciplines, and molecular biology experiments are very important in life science research. In actual work, many experimental materials such as muscle tissue, connective tissue, bone tissue, hair tissue, etc. of animals, the roots, stems, leaves, seeds, etc. of plants need to be ground, and then their components, genes, proteins, and Structure and function are studied. [4]

Liquid nitrogen milling is widely used in molecular biology experiments, especially related to the separation of RNA and DNA. The temperature of liquid nitrogen is 196 °C. It can not only make various tissue components difficult to be destroyed or degraded, but also make the tissue hard, and increase the brittleness and easily grind. [5]

Existing RNA grinding devices require manual grinding and extraction of RNA. To this end, the arm needs to be insulated, and antifreeze gloves must be worn to prevent the limb from being injured by liquid nitrogen. The overall operation is more complicated and difficult to use. The efficiency of RNA grinding extraction is low, and if the action is too large, the sample will be splashed and the sample will be easily wasted. [6]

Aiming at the problems in related technologies, no effective solution has been proposed at present. To this end, this paper proposes an RNA grinding extraction system.

2. Materials and Methods

2.1. Materials

The design structure of the RNA grinding extraction system in this paper is shown in Figures 1-7 below.

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Figure 1. The front view of the grinding extraction system design.

Figure 2. Shows the internal structure of the grinding extraction system.

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Figure 3. The side view of the rotating plate designed for the grinding extraction system.

Figure 4. The top view of the rotating plate designed for the grinding extraction system.

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Figure 5. The cross section of the rotating plate designed for the grinding extraction system.

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Figure 6. The structure diagram of the designed grinding parts of the grinding extraction system.

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Figure 7. The top view of the grinding plate designed for the grinding extraction system.

Above figures: 1. Box body; 2. Feet; 3. Box door; 4. Grip; 5. Pressure gauge; 6. Drain pipe; 7. Valve A; 8. Storage tank; 9. Liquid nitrogen tank 10, conduit A; 11, three-way joint; 12, valve B; 13, valve C; 14, cylinder; 15, motor; 16, abrasive parts; 1601, cross bar; 1602, fastening screw; 1603, connecting rod 1604, grinding head; 17, rotating plate; 1701, outer plate; 1702, inner plate; 1703, jack; 1704, ball; 18, butterfly screw; 19, gantry bracket; 20, sealing strip; 21, slide rail; 22, Grinding plate; 2201, ventilation hole; 2202, grinding groove; 23, magnet; 24, inclined plate; 25, sealing ring.

The working principle of the RNA grinding and extraction system designed in this paper is as follows: When in use, open the door 3, pour the RNA material to be ground into the grinding tank 2202, then close the door 3, open the liquid nitrogen tank 9, and open the valve B12. And the valve C13, the liquid nitrogen is introduced into the cabinet 1. Subsequently, the grinding member 16 is pushed by the cylinder 14 and the grinding head 1604 of the grinding member 16 is pressed into the grinding groove 2202. In order to ensure the tightness, after the grinding head 1604 of the grinding member 16 is pushed tightly by the cylinder 14 and the eye tank, a sealing ring is filled between the connecting rod 1603 and the socket 1703 to seal the two to ensure tightness. Subsequently, the motor 15 is started, and the grinding member 16 is driven by the motor 15 to rotate around the rotation axis of the motor 15. The connecting rod 1603 of the grinding member 16 is rotated in the rotating plate 17, thereby driving the grinding head 1604 to rotate in the grinding tank 2202 to perform RNA substances. Grinding, this kind of mechanical grinding method can achieve a good cell breaking effect, the cells are ground into powder, the rotating plate 17 of the desktop plays a certain blocking role, slowing down the rate of liquid nitrogen loss. During the grinding process, observe the pressure gauge 5. When the pressure of liquid nitrogen is too high, open the valve A7, and some of the liquid nitrogen will be discharged from the drain pipe 6 through the inclined plate 24. While reducing the pressure, it is easy to recycle and reuse. After the grinding is completed, remove the sealing ring between the connecting rod 1603 and the socket 1703, and pull the grinding piece 16 out of the grinding groove 2202 through the air cylinder 14. Open the door 3 and pull the entire grinding plate 22 from the slide rail 21. After grinding in grinding tank 2202, the product can be transferred.

2.2. Methods

2.2.1. RNA Grinding and Extraction System Design Technical Solution

In order to achieve the above purpose, the technical solution designed in this thesis is as follows: An RNA grinding extraction system includes a box body and a liquid nitrogen tank, the box body has a rectangular parallelepiped structure, the bottom of the box body is fixed with four feet, and the box body A drain pipe is connected to the lower part, and a valve A is installed on the drain pipe. A liquid storage tank is arranged below the liquid discharge tube. The liquid drain pipe is connected to the liquid storage tank. The front of the box is connected by a hinge activity. A box door on which a grip is installed, three strip magnets are embedded in the sides and vertical edges of the front of the box, and three iron pieces are fixed on the inner edge of the box door. Three strip-shaped magnets attract each other to attract the box door to the box body. A grinding plate is horizontally arranged in the box body, and the middle of the grinding plate is recessed downward to form a grinding groove. A pressure gauge is installed on the side of the box body. A gantry bracket is installed above the box, the cylinder is fixed upside down on the gantry bracket, a motor is fixed on the piston rod of the cylinder upside down, a grinding part is fixed on the rotating shaft of the motor, and an opening is opened on the box. Through the opening two butterfly screws are fixed to the rotating plate, and the opening at the top of the box is completely covered. The grinding member passes through the rotating plate and extends into the grinding groove of the grinding plate. The liquid nitrogen tank is connected with a pipe A, so one end of the pipe A is connected with two sub-pipes through a three-way joint, and both sub-pipes are inserted into the box. The sub-pipes are respectively located on both sides of the grinding plate. The plates are symmetrical to each other, and both are inclined toward the drainage pipe and communicate with the drainage pipe.

2.2.2. Specific Implementation Method of RNA Grinding Extraction System

The technical solution in the design example of the grinding and extraction system will be clearly and completely described below with reference to the drawings in the design example of the grinding and extraction system. Obviously, the described embodiment is only a part of the design of the grinding and extraction system. , Not all examples.

Please refer to Figure 1- : Figure 7. The design of the grinding extraction system provides a technical solution: an RNA grinding extraction system, including a box 1 and a liquid nitrogen tank 9, the box 1 has a rectangular parallelepiped structure, and the bottom of the box 1 Four feet 2 are fixed, and a drain pipe 6 is connected to the lower part of the tank 1. A valve A7 is installed on the drain pipe 6. A reservoir 8 and a drain pipe 6 are provided below the drain pipe 6. It is in communication with the liquid storage tank 8. The front side of the tank 1 is connected with a tank door 3 through a hinge activity. A grip 4 is installed on the tank door 3. Both sides of the front side of the tank 1 and the vertical edges are inside. Three strip-shaped magnets 23 are embedded, and three iron pieces are fixed on the inner edge of the box door 3. The three iron pieces and the three strip-shaped magnets 23 attract each other to attract the box door 3 to the box body 1, inside the box body 1. A grinding plate 22 is arranged horizontally, the grinding plate 22 is recessed downward in the middle to form a grinding groove 2202, a pressure gauge 5 is installed on the side of the cabinet 1, a gantry bracket 19 is installed above the cabinet 1, and the gantry bracket An air cylinder 14 is fixed upside down on 19, and a motor 15 is fixed upside down on the piston rod of the air cylinder 14, and a research shaft is fixed on the rotating shaft of the motor 15. Piece 16, the box 1 is provided with an opening, and the rotating plate 17 is fixed by two butterfly screws 18 on the opening, and the opening at the top of the box 1 is completely covered, and the grinding piece 16 passes through the rotating plate 17 And into the grinding groove 2202 of the grinding plate 22, the liquid nitrogen tank 9 is connected with a pipe A10, one end of the pipe A10 is connected with two sub-pipes through a three-way joint 11, and both sub-pipes are inserted into the box In the body 1, the sub-pipes are respectively located on both sides of the grinding plate 22, and two inclined plates 24 are arranged below the grinding plate 22, and the two inclined plates 24 are symmetrical to each other, and both are inclined toward the drainage pipe 6 and the drainage pipe 6 Connected.

As shown in Figure 3, Figure 4, and Figure 5, the rotating plate 17 includes an inner plate 1702 and an outer plate 1701. The edges of the inner plate 1702 are recessed inward, and the edges of the outer plate 1701 protrude outward. Balls 1704 are slidably disposed between the recessed surface and the protruding surface of the outer plate 1701. When the outer plate 1701 is fixed on the case 1, the inner plate 1702 can be rotated in the outer plate 1701 with the connecting rod 1603, and the inner plate 1702 is provided with a jack 1703. The grinding member 16 passes through the insertion hole 1703, and a sealing ring 25 is attached to the bottom of the outer plate 1701. When the rotating plate 17 is fixed on the box 1, the sealing ring 25 can play a certain sealing role and reduce the loss of liquid nitrogen Speed, the joint between the inner plate 1702 and the outer plate 1701 is filled with a sealant cap to improve the sealing performance of the rotating plate 17.

As shown in Figure 6, the grinding member 16 includes a cross bar 1601, a connecting bar 1603, and a grinding head 1604. The connecting bar 1603 is vertically fixed on the cross bar 1601 and is clamped and fixed by a fastening screw 1602. One end of the rod 1603 fixes the grinding head 1604 obliquely. The grinding head 1604 is close to the groove wall of the grinding groove 2202. The grinding head 1604 has a pestle-like structure. The upper part is provided with a vent hole 2201, and the grinding head 1604 of the grinding member 16 can be closely attached to the grinding groove 2202, and the grinding effect is better.

As shown in Figure 2, the two sides of the grinding plate 22 are fixed to the inner wall of the cabinet 1 through slide rails 21, and the surface of the grinding head 1604 of the inner box of the grinding groove 2202 is covered with frost. The frost makes the grinding finer. A sealing strip 20 is attached to the front edge of the box 1. After closing the box door, the sealing strip plays a sealing role.

As shown in Figure 1, two sub-conductors are respectively installed with a valve B12 and a valve C13 to control the opening and closing of the two sub-conductors. The two sub-conductors at one end of the duct A10 pass through the contact surface of the box 1. The sealant seals to further improve the sealability of the box 1.

The cylinder 14 is of the model MA20 * 300.

The motor 15 is a 70KTYZ type.

The electrical components appearing in this article are electrically connected to the external main controller and 220V mains electricity, and the main controller can be a conventional known device that is controlled by a computer or the like.

3. Results and Discussion

3.1. Results

Compared with the prior art, the design of the grinding extraction system has the following beneficial effects:

This kind of RNA grinding and extraction system has reasonable structure design. It adopts mechanical grinding to extract and extract RNA. This method eliminates the manual grinding operation and simplifies the operation. At the same time, it can avoid liquid nitrogen. The limbs are in direct contact with liquid nitrogen, which avoids freezing the hands with liquid nitrogen and greatly improves safety.

This RNA grinding and extraction system uses mechanical grinding to rotate the grinding method, and pushes the grinding part through the cylinder, so that the grinding head of the grinding part abuts the grinding groove, and can fully contact the grinding groove when the motor drives the grinding piece to rotate, ensuring that Grinding quality.

This kind of RNA grinding and extraction system can open valve A, which can discharge liquid nitrogen into the liquid storage tank through the drain pipe, and recycle it, which is convenient for later recycling.

3.2. Discussion

Now, with the development of science and technology, the team of scientific researchers is growing, and the requirements for scientific research technology are constantly improving and improving. [7] RNA extraction is the basic work of molecular biology research, and its successful extraction provides a guarantee for the smooth development of RT-PCR, transcriptome and other molecular biology test technologies. In RNA extraction, the grinding process is the key, that is, in the shortest time, the sample is quickly ground into powder with liquid nitrogen, and then the powder is transferred to the centrifuge tube. [8] Due to the widespread existence of RNA enzyme, the contact time between sample powder and air should be shortened as much as possible in this process. At the same time, the half-life of RNA is short and it is easy to degrade, which requires that there is always liquid nitrogen in the mortar to ensure a low temperature environment. [9] In general, the materials that need to extract RNA are ground in a mortar. If the amount of sample materials is relatively small (such as the extraction of RNA from fungi, bacteria, etc.), after grinding, most of the sample powder sticks to the mortar and grinding rod, and only a few of the powder can be transferred to the centrifuge tube, which greatly reduces the concentration of the final extracted RNA. At the same time, the sample powder is reduced, which increases its air content The contact area of air accelerates the degradation of RNA, greatly reduces the purity of RNA, and in the process of grinding, it cannot avoid the sample quantity reduction caused by the splashing of sample and liquid nitrogen and the frostbite of liquid nitrogen on human skin. [10] Once again, the boiling point of liquid nitrogen is low, and it is very volatile. It volatilizes faster in the mortar, which reduces the utilization rate of liquid nitrogen. [11] Finally, after each grinding, the sample is transferred to the centrifuge tube for subsequent extraction steps, which greatly reduces the experimental efficiency.

Most of the RNA in cells is in the form of nucleoprotein complex. Trizol contains guanidine isothiocyanate and phenol, in which guanidine isothiocyanate can split cells and promote the dissociation of ribosomes, that is, to separate RNA and the protein it binds to, and release RNA into the solution. When chloroform is added, acid phenol can be extracted, and acid phenol can promote RNA to enter the water phase. After centrifugation, water phase layer and organic layer can be formed. [12] The water phase layer is above, the organic layer is below, and the middle layer is DNA and protein. [13] That is to say, there will be three layers, and RNA is in the water phase layer, that is, in the upper layer. Isopropanol precipitates RNA. The function of ethanol is to wash off isopropanol. [14] in the process of liquid nitrogen grinding, the tissue block is directly put into the mortar, a small amount of liquid nitrogen is added, and the tissue is quickly ground until it becomes soft (it' s better not to wait for the tissue to become soft (it' s actually proved that it doesn' t matter once). [15] When it' s soft, press it thin, and then add liquid nitrogen to make it hard and easy to grind. In fact, adding liquid nitrogen not only prevents RNA degradation, but also is more conducive to grinding). When it' s soft, RNA is easy to degrade. If liquid nitrogen volatilizes, replenish liquid nitrogen quickly to keep the sample frozen. [16]

4. Conclusions

In summary, the structure of this grinding extraction system is reasonable, and the RNA is extracted by mechanical grinding. This method eliminates the manual grinding operation process and simplifies the operation. At the same time, when liquid nitrogen is introduced, it can avoid direct contact between limbs and liquid nitrogen, which can avoid frostbite of liquid nitrogen, which greatly improves safety; mechanical grinding adopts rotary grinding, and pushes the grinding piece 16 through the cylinder 14, so that the grinding head 1604 of the grinding piece 16 is tight. Leaning inside the grinding tank 2202, when the motor 15 drives the grinding piece 16 to rotate, it can fully contact the grinding tank 2202 to ensure the quality of the grinding; the valve A7 can be opened, and the liquid nitrogen can be discharged into the storage tank 8 through the drain pipe 6 Inside, it is recycled, which is convenient for later recycling.

Conflicts of Interest

The author declares that there is no conflict of interest regarding the publication of this article.

Funding

This work was supported by the National Natural Science Foundation of China, grant number 21575109.

Acknowledgments

The author would like to acknowledge the teacher for helping complete the design of the grinding extraction system, acknowledge the authors of the references listed in this paper, whose paper provided the theory for the research Support, and acknowledge the editors of this magazine for their suggestions for revision.

Copyright

© 2017 by the authors. Licensee International Technology and Science Press Limited. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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