KR100572721B1 - RNA preservation medium comprising cathodic electrolyzed water and method of preserving RNA using the same - Google Patents

Abstract

The present invention relates to an RNA preservation solution containing cathodic electrolyzed water and a method for preserving RNA using the same. RNA preservation solution including the reduced ionizing water according to the present invention can be used as a simple, safe, economical and environmentally friendly preservation solution is excellent in RNA preservation capacity and does not affect subsequent enzymatic reaction.

Cathodic electrolyzed water, RNA preservatives, DEPC (diethylpyrocabonate)

Description

RNA preservation solution containing reduced ionized water and the method of preserving RNA using the same {RNA preservation medium comprising cathodic electrolyzed water and method of preserving RNA using the same}             

1 is a result of electrophoresis by separating total RNA from plant cells.

* Description of drawing symbols

         Lane 1: sterile tertiary distilled water

         Lane 2: DEPC treated tertiary distilled water

         Lane 3: Reduction ionization just after manufacture

         Lane 4: reduced ionized water stored at 4 ° C. for 2 days

         Lane 5: reduced ionized water stored at 4 ° C. for 4 days

         Lane 6: Reduced ionized water melted after freezing at -20 ℃

         Lane 7: reduced ionized water stored at 25 ° C. for 2 days

         Lane 8: reduced ionization water stored at 25 ° C. for 4 days

FIG. 2 shows the results of electrophoresis on changes in total RNA in sterile tertiary distilled water, DEPC treated tertiary distilled water, and reduced ionized water at various storage temperatures and times at 4 ° C. at 1, 3, and 7 day intervals. (The symbols are the same as in FIG. 1).

FIG. 3 shows the results of electrophoresis on changes in total RNA in sterilized tertiary distilled water, DEPC treated tertiary distilled water, and reduced ionized water at various storage temperatures and times at 25 ° C. at 1, 3, and 7 day intervals. (Drawing symbols are the same as in FIG. 1)

Figure 4 shows the results of the enzymatic reaction in the case of using reduced ionized water instead of DEPC-treated tertiary distilled water in the subsequent enzymatic reaction with RNA.

* Description of drawing symbols

          Marker: 100 bp DNA ladder

          Lanes 1 and 2: amplified DNA products reacted with reduction ionized water

          Lanes 3 to 4: amplified DNA products reacted with DEPC-treated tertiary distilled water

The present invention relates to an RNA preservation solution containing a reduced ionized water and an RNA preservation method using the same.

Applying a direct voltage to the water creates electrolyzed water that can change pH by the movement of ions. The water produced at the anode decreases in pH due to the increase of H ⁺ ions, and the oxidation-reduction potential (ORP) increases, resulting in a strong oxidative state. ^­ The pH rises due to the increase of ions and becomes a reducing state (Ryoo, K., Kang, B. and Sumida, S., Electrolyzed water as an alternative for environmentally-benign semiconductor cleaning. Material Research Society, 17 (6), pp. 1298-1304, 2002). The redox potential of ionized water shows a much stronger pH dependence than other aqueous solutions. In general, the redox potential of the ionized water at pH 2 shows a high oxidation potential of +1200 mV or more, while the acidic aqueous solution shows a redox potential of about +600 mV. The redox potential of the ionized water at pH 11 has a reduction potential of -850 mV or less, but +20 mV for alkaline aqueous solutions.

When the ionizing water is used as a cleaner, it does not emit pollutants more than the use of conventional alkali or acid, and after a certain time of use, it is not only reduced to natural water but also cheaper in manufacturing cost, which is very economical and economical. Is advantageous. In addition, the ionizing water has a bactericidal activity against microorganisms (Fabrizio, KA and Cutter, CN, Stability of electrolyzed oxidizing water and its efficacy against cell suspensions of Salmonella typhimurium and Listeria monocytogenes. J. Food Prot., 66 ( 8), pp. 1384-1397, 2003; Sharma, RR and Demirci, A., Treatment of Escherichia coli O157: H7 inoculated alfalfa seeds and sprouts with electrolyzed oxidizing water.Int. J. Food Microbiol., 86 (3), Interestingly, it is interesting to treat the wounds and burns of mice (Xin, H., Zheng, YJ, Hajime, N. and Han, ZG, Effect of electrolyzed oxidizing water and hydrocolloid). occlusive dressings on excised burn-wounds in rats.Chin.J. Traumatol., 6 (4), pp.234-237, 2003) It has been reported to have a positive effect on the treatment of diseases such as diabetes (Huang, KC, Yang, CC, Lee, KT and Chien, CT, Reduced hemodialysis-induced oxidative stress in end-stage r enal disease patients by electrolyzed reduced water.Kidney Int., 64 (2), pp.704-714, 2003). In addition, reducing ionization can inhibit oxidative damage of DNA because it has been reported to have antioxidant activity (Sanetaka, S., Shigeru K., Mariko, N., Takumi, M., Kenichi, K., Miho, G., Hidemitsu, H., Kazumichi, O., Shinkatsu, M. and Yoshinori, K., Electrolyzed-reduced water scavenges active oxygen species and protects DNA from oxidative damage.Biochemical and Biophysical Research Communications, 234, pp. 269-274, 1997).

On the other hand, compared to DNA, the difficulty in separating or preserving RNA is that most RNA degrading enzymes are very stable and active without cofactors. The most caution when separating or preserving RNA is to minimize RNA degradation by protecting RNA from RNA degrading enzymes. To protect RNA from the action of RNA degrading enzymes present in all cells, RNA degrading enzymes are removed or inhibited from all the instruments and reagents used in RNA isolation and preservation. Therefore, all instruments must be sterilized or baked at 180 ° C, and all solutions must be treated with DEPC (diethylpyrocabonate), a potent protein modifier or digester. DEPC can be added to any solution other than Tris. However, DEPC should be removed by high-pressure heating after DEPC treatment, as it carries out carboxymethylation of adenine on a single strand of DNA. In addition, DEPC is considered a carcinogen and requires attention. Therefore, it is difficult to properly preserve RNA that is easily degraded, temperature sensitive, and easily degraded by RNA degrading enzymes everywhere. In addition, DEPC, which is widely used to inhibit RNA degrading enzymes, is toxic to humans, difficult to treat wastewater, and highly carcinogenic, thus making it easier, safer and more powerful, and environmentally friendly and harmless to human RNA. This is urgently requested.

Ultrafiltration device (Nucleic Acids Symp Ser. 1995; (33): 129-33), which manufactures ultrapure water without RNA degrading enzyme by removing ions and organic matter with high purity as a technology that can replace the DEPC, adsorption, desorption RNA degrading enzyme removal device composed of ionization, UV oxidation, ultrafiltration, etc. has been developed, but has a disadvantage of high price. US Patent No. 2003-114651 discloses RNA from RNA degrading enzyme by precipitating RNA using a sulfate solution. An RNA preservation liquid which prevents degradation of a compound is disclosed. However, since RNA is known to be unstable to alkali, no attempt has been made to preserve RNA by using an alkaline reducing ionized water.

Nevertheless, the inventors of the present invention show that the alkaline reducing ion does not affect the performance of the enzyme reaction using RNA as well as an excellent protection against RNA degradation, so that it can be used as an economical, environmentally friendly and harmless RNA preservative. The surprising fact that it can be found, and came to complete the present invention.

The present invention has been made to solve the conventional problems as described above, an object of the present invention is to provide an RNA preservation solution containing a reduced ionized water and an RNA preservation method using the same.

The present invention provides an RNA preservation solution including reduced ionized water.

As used herein, the term "electrolyzed water" is an aqueous solution in which pH or oxidation-reduction potential is adjusted by electrolysis. Electrolysis of water is one of the electrochemical phenomena, which changes the properties of water to provide new chemicals, and ionizing alkaline water (cathode ionizing water) and ionizing water, which are different ionizing products at the anode and the cathode. Create an anode ionizer. Electrolysis phenomena can be handled quantitatively and are used in a variety of applications in the industry. As used herein, "cathodic electrolyzed water" means reduced water collected from a cathode when electrolyzing water.

The reduced ionized water contained in the RNA preservation solution of the present invention can be prepared using various apparatuses and known methods for electrolyzing water. The ionizer generator for generating ionized water has an electrolytic cell divided into two areas in which an anode and a cathode are disposed by a separator. When an electric current flows through the anode, ionized alkaline water is generated at the cathode and ionized at the anode. Acidic water is produced. Typically, a voltage of at least 2 to 3 volts or higher is supplied, a diaphragm or ion exchange membrane is installed to separate the anode and the cathode, and an electrolytic material (eg, NaCl, KCl, etc.) is added to promote electrolysis. . Many devices are commercially available for producing electrolyzed tap water by directly electrolyzing tap water. The continuous generation of ionizing water is filtered by tap water using an adsorption medium such as activated charcoal, and then purified water is continuously supplied to the electrolytic cell so that the ionizing water is continuously generated. In addition, batch devices for producing ionized water comprising an anode and a cathode chamber are also well known.

Apparatuses for producing reduced water for various applications have been researched and developed. As a specific example, after supplying pure water to an electrolyte preparation tank, an electrolyte having a predetermined molar concentration of NH ₄ OH or HCl is prepared, and then an anode electrode. By applying a voltage having a predetermined magnitude between the cathode and the cathode, electrolytic solution of the electrolytic cell may be electrolyzed to produce reduced water having a desired pH from the cathode. In addition to the above-described method, a device capable of continuously generating a large amount of ionized water with high efficiency, comprising a flow restricting means in an electrolytic cell so that the raw water can maintain a long contact between the negative electrode plate and the positive electrode plate. There is known a device for generating ionized water with a very narrow interval.

In addition to the above-described method, a device capable of continuously generating a large amount of ionized water with high efficiency, comprising a flow restricting means in an electrolytic cell so that the raw water can maintain a long contact between the negative electrode plate and the positive electrode plate. There are known ionizing water generating apparatuses which have a very narrow interval of 0.1 mm to 3 mm.

It is known that reduced ionized water can be used as a DNA preservative (Electrolyzed-reduced water scavenges active oxygen species and protects DNA from oxidative damage. Biochemical and Biophysical Research Communications, 234, pp. 269-274, 1997). However, RNA is physicochemically different from DNA. Specifically, in the case of DNA, nucleic acids are present in double strands, but RNA is present in nucleic acids in single strands, and uracil is used as a base instead of thymine in RNA. In particular, RNA has a saccharide of 5, called ribose, linked to a base, and in the case of DNA, 2'-deoxyribose is linked to a base. In other words, the OH (hydroxyl) group is bound to the second carbon of the pentose in RNA. The 2'-OH group of ribose pentose is chemically very unstable and hydrolyzes well in water. Therefore, RNA lacks much stability compared to DNA and is easily destroyed by alkali. On the other hand, DNA is not destroyed well even by alkali treatment, so it is often treated by alkaline treatment to make double strands into single strands. RNA present in the outer strands hydrogen bonds with the bases in itself, causing secondary structural changes such as hairpin structure. Therefore, it can not be easily inferred that the reduced ionizing water can be used as a DNA preservation solution.

In the present invention, "RNA (ribonucleic acid)" refers to a polymer organic material in which nucleotides (nucleotides) in which a base, ribose, and phosphoric acid are bonded to each molecule are connected. For example, messenger RNA, transfer RNA , Ribosome RNA (ribosomal RNA), catalytic RNA (ribozyme), snRNA (small nuclear RNA) and the molecule comprising a derivative thereof, and the like, but is not limited thereto.

The redox potential and pH of the reduced ionization water are correlated and the reducing power is increased as the redox potential becomes negative. The pH is 6-7 when the redox potential is about -200 mV, and the pH is 8.5-10 when the redox potential is -800 mV. In the present invention, the redox potential of the reduced ionized water used for RNA preservation is -200 mV or less, preferably -1000 mV to -200 mV, more preferably -900 mV to -800 mV, and most preferably -840 mV to -860 mV. , pH is 6 or more, preferably 6 to 11, more preferably 8.5 to 11, most preferably 10 to 11. When the redox potential of the reduced ionized water is -200 mV or more, a problem arises in that RNA is degraded and RNA retention is reduced.

In order to investigate the effect of the reduced ionization water of the present invention on the preservation of RNA, total RNA was separated from plant cells by TRI reagent method, and the total RNA was isolated from sterile tertiary distilled water, DEPC treated tertiary distilled water, and storage temperature and time. As a result of the electrophoresis after melting in various reducing ionized water, it was confirmed that the separation well without RNA degradation under all conditions (Fig. 1).

In addition, in order to determine whether the reduced ionization water affects RNA stability over temperature and time, sterilized tertiary distilled water, DEPC treated tertiary distilled water, and at 1, 3, and 7 days intervals at 4 ° C. and 25 ° C. The change of total RNA in reducing ionized water of varying storage temperature and time was investigated (FIGS. 2 and 3).

FIG. 2 shows the results of electrophoresis on changes in total RNA in sterile tertiary distilled water, DEPC treated tertiary distilled water, and reduced ionized water at various storage temperatures and times at 4 ° C. at 1, 3, and 7 day intervals. . Total RNA stored in sterile tertiary distilled water began to degrade slowly from day 3 of storage (lane 1), while total RNA stored in DEPC treated tertiary distilled water was slightly degraded than sterile tertiary distilled water. It was possible to visually confirm the decomposition of (lane 2). In contrast, the total RNA stored in the reduced ionized water did not degrade at all during the 1 week storage period (lanes 3-8).

FIG. 3 shows the results of electrophoresis on changes in total RNA in sterilized tertiary distilled water, DEPC treated tertiary distilled water, and reduced ionized water at various storage temperatures and times at 25 ° C. at 1, 3, and 7 day intervals. . When RNA was stored at 25 ° C., RNA was decomposed from sterile tertiary distilled water as well as DEPC treated tertiary distilled water after 1 day (lanes 1, 2). However, the RNA stored in the reduced ionized water was not degraded at all until 3 days of storage. When RNA was stored for 1 week, RNA was completely decomposed in sterile tertiary distilled water as well as DEPC treated tertiary distilled water, but the degree of degradation of RNA stored in reduced ionized water was very weak.

Through the experiments, it was confirmed that the reduced ionization water preserved the RNA well without degradation of the RNA at 25 ° C. as well as at 4 ° C. during the storage period for a total of 7 days. Accordingly, the reduced ionized water is an RNA preservative having an excellent RNA preservation effect that prevents degradation of RNA without being influenced by the storage period and storage temperature, and is suitable for long-term storage, preferably 4 weeks or more, and more preferably 7 days or more. It can be used for, preferably 30 ° C or less, more preferably 25 ° C or less, even more preferably 4 to 25 ° C RNA storage.

Reduction ionization water does not adversely affect RNA preservation as well as subsequent enzymatic reaction with RNA. Figure 4 shows the results of the enzymatic reaction in the case of using reduced ionized water instead of DEPC-treated tertiary distilled water in the subsequent enzymatic reaction with RNA. In order to be used as an effective RNA preservative, the reduced ionized water of the present invention should be able to be stored for a long time at low temperature or room temperature without degradation of RNA, and also be able to smoothly trigger the enzymatic reaction used in subsequent experiments using RNA. Therefore, RT-PCR (Reverse transcriptase-Polymerase chain reaction) reaction was used to investigate whether the enzyme reaction of reverse transcriptase and Taq DNA polymerase sensitive to the reaction conditions proceed effectively in the presence of reducing ionization. . In the cDNA synthesis reaction, the reduced ionized water was reacted instead of DEPC-treated tertiary distilled water.In the PCR reaction, the reacted DEPC-treated tertiary distilled water was reacted. The amount of DNA amplified was compared with each other (FIG. 4). As a result, the amplified DNA product using reduced ionized water was not different from the amplified DNA product using DEPC-treated tertiary distilled water, and the desired gene of 352 bp was amplified properly in both reactions.

Therefore, the reduced ionization water of the present invention is sensitive enzyme reactions such as cDNA synthesis reaction and high temperature enzyme reactions such as PCR reaction even when using reduced ionization water instead of DEPC-treated tertiary distilled water without RNA degrading enzyme in cDNA synthesis reaction and PCR reaction. Because it can effectively perform and amplify the DNA efficiently, it can be used as an excellent RNA preservative that can facilitate the subsequent enzymatic reaction using RNA.

Another aspect of the present invention provides a method for preserving RNA using reduced ionization water. The redox potential of the reducing ion used in the RNA preservation method is -200 mV or less, preferably -1000 mV to -200 mV, more preferably -900 mV to -800 mV, most preferably -840 mV to -860 mV, and the pH is 6 or more, preferably 6-11, more preferably 8.5-11, most preferably 10-11.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the examples.

Example 1 RNA Preservation of Reduction Ionized Water

To investigate the effect of reducing ionization on the preservation of RNA, total RNA was isolated from corn seedlings ( Zea Mays L.) cells by TRI reagent (FIG. 1) (Sambrook, J and DW Russel: Molecular cloning; A laboratory manual.Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 2001) The total RNA isolated from sterile tertiary distilled water, DEPC (Sigma-Aldrich Co.) treated tertiary distilled water, and reduced ionizing water with varying storage temperature and duration (ORP After dissolving in -850 mV, pH 11), formaldehyde gel electrophoresis was performed immediately. At this time, 3 ug of RNA was injected for each lane. As a result of the electrophoresis it was confirmed that well separated without RNA degradation in all conditions (Fig. 1). In FIG. 2, formaldehyde gel electrophoresis was performed while total RNA was stored in sterile tertiary distilled water and DEPC treated tertiary distilled water at 4 ° C., and reduced ionized water having various storage temperatures and periods from 1 day to 1 week. As a result, total RNA stored in sterile tertiary distilled water began to degrade slowly from the third day of storage (lane 1). Total RNA stored in DEPC-treated tertiary distilled water is also slightly degraded than sterile tertiary distilled water, but it can be seen visually that some degradation occurs (lane 2). In contrast, the total RNA stored in the reduced ionized water at 4 ° C. did not degrade at all during the 1 week storage period (lane 3-8).

When RNA was stored at 25 ° C. (FIG. 3), RNA was decomposed after 1 day in sterile tertiary distilled water as well as in DEPC treated tertiary distilled water (lanes 1,2). However, the RNA stored in the reduced ionized water was not degraded at all until 3 days of storage. At this time, the storage period and storage temperature of the reduced ionized water itself did not affect the degradation of RNA. When RNA was stored at 25 ° C. for one week, RNA was completely decomposed in sterile tertiary distilled water as well as DEPC treated tertiary distilled water, but the degree of degradation of RNA stored in reduced ionized water was very weak.

Therefore, reducing ionized water is not only simpler, safer, and stronger than DEPC, but also can be used in all experiments using RNA as an environmentally friendly and harmless RNA preservative.

Example 2 Influence of Reduced Ion Number on the RT-PCR Reaction

In order to be used as an effective RNA preservative, the reducing ion should not only be able to be stored for a long time at low temperature or room temperature without degradation of RNA, but also be able to smoothly trigger the enzymatic reaction used in subsequent experiments using RNA. Therefore, RT-PCR (Reverse transcriptase-Polymerase chain reaction) reaction using reduction ionization to examine whether the enzyme reaction of reverse transcriptase and Taq DNA polymerase sensitive to reaction conditions proceeds effectively in the presence of reduction ionization , J and DW Russel: Molecular cloning; A laboratory manual.Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 2001). RT-PCR reaction consists of cDNA synthesis reaction and PCR reaction.

First, in cDNA synthesis reaction, DEPC (Sigma-Aldrich) was reacted with a conventional reaction (oligo (dT) primer, 10 mM dNTP, RNA sample, 5X first strand buffer, 0.1M DTT, reverse transcriptase, and DEPC treated tertiary distilled water). Treated with reduced ionized water (ORP -850 mV, pH 11) instead of Co. In the PCR reaction, DEPC-treated tertiary distilled water was reacted with a template cDNA, 10 μM sense primer, 10 μM antisense primer, and premix (Taq DNA polymerase mixture), followed by reduction ionization instead of amplified DNA and DEPC-treated tertiary distilled water. After the reaction, the amplified DNA amounts were compared with each other (FIG. 4). PCR amplification conditions were incubation at 94 ° C for 3 minutes, 30 cycles of amplification reaction (denature 94 ° C for 1 minute, annealing 55 ° C for 1 minute, elongation 72 ° C for 1 minute), final extension 72 ° C for 5 minutes, cooling 4 It was set to ° C.

As the primer used above, the antisense primer was 5'-CTCGAGGGGTTCCAGTCCTCTATGTTCAGA-3 'and the sense primer was 5'-CCATATGGTTGCACCAAGTAACATGGAGA-3'. As a marker for measuring the size of DNA, 100 bp DNA (2,000, 1,600, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100 bp) ladder was used. As a result, the amplified DNA product using reduced ionized water was not different from the amplified DNA product using DEPC-treated tertiary distilled water, and the desired gene of 352 bp was properly amplified in both reactions (FIG. 4).

In all reactions using RNA, including RT-PCR, the use of high purity water is particularly important. High purity water such as DNA degrading enzymes and RNA degrading enzymes has been developed and used, but the price is high. The results of this experiment show that the use of reducing ionized water instead of DEPC-treated tertiary distilled water without RNA degrading enzymes in cDNA synthesis and PCR can efficiently amplify DNA without affecting the enzymatic reaction at all. In addition, it shows that the reduced ionized water can effectively perform high temperature enzyme reactions such as PCR and sensitive enzyme reactions such as cDNA synthesis.

Since RNA is very sensitive to alkali, temperature and RNase, and is easily degraded, storing RNA without degradation is a core technology of biotechnology, so reducing ionization can be used as a new concept of RNA preservation.

RNA preservation solution including the reduced ionizing water of the present invention is excellent in RNA preservation ability and does not affect subsequent enzymatic reaction can be used as a simple, safe, economical and environmentally friendly preservation solution.

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Claims (6)

RNA preservation solution containing a reduced ionization water. The RNA preservation liquid according to claim 1, wherein the redox potential of the reduced ionization water is -1000 mV to -200 mV. The RNA stock solution of claim 1, wherein the pH of the reduced ionized water is 6 to 11. 11. RNA preservation method using reduced ionization water. The method according to claim 4, wherein the redox potential of the reduced ionization water is -1000 mV to -200 mV. The method of claim 4 wherein the pH of the reduced ionized water is 6-11.

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