Technical Field

The present invention relates to an RNA preservation medium comprising cathode-electrolyzed(cathodic electrolyzed) water and a method of preserving RNA using the same.

Background Art

When direct current voltage is applied to water, electrons move between the electrodes to generate electrolyzed water that changes pH values. At the anode, H+ ions are generated so as to produce oxidative water having a low pH and high oxidation-reduction potential (ORP) , while reductive water having a high pH is produced at the cathode due to the motion of OH- thereto (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 ORP of electrolyzed water is more pH-dependent than that of other aqueous solutions. In general, electrolyzed water of pH 2 exhibits an oxidative potential of +120OmV or more whereas acidic solutions have an oxidative potential of about +60OmV. On the other hand, a reductive potential of as low as -85OmV is measured in electrolyzed water having a pH of 11 while the reductive potential of alkaline solutions is no more than +2OmV. Electrolyzed water, used as a clearer, has great economical and environmental adventages compared to conventional alkaline or acidic cleaning solutions in that it produces no pollution, is recovered into neutral water over time, and may be produced at a low cost. Additionally, electrolyzed water is found to have germicidal activity against microorganisms (Fabrizio, K. A. et al., J. Food Prot., 66(8), pp.1384-1397, 2003; Sharma, R. R. et al., Int. J. Food Microbiol., 86(3), pp.231-237, 2003), show therapeutic effects on injuries and burns in mice (Xin, H. et al., Chin. J. Traumatol., 6(4), pp.234-237, 2003), and has positive effects in the treatment of diabetes mellitus (Huang, K. C, et al., Kidney Int., 64(2), pp.704-714, 2003). Further, it is reported that reductive electrolyzed water inhibits oxidative damage to DNA thanks to its anti-oxidative activity (Sanetaka, S. et al., Biochemical and Biophysical Research Communications, 234, pp. 269-274, 1997) . In biochemical, molecular biological and bioengineering fields, RNA finds a variety of applications, including the analysis of gene expression with DNA chips, RT- PCR, cDNA library construction, Northern blotting, etc. Difficulty in isolating or preserving RNA, compared to DNA, is attributed to the fact that most RNases are quite stable and have activity even in the absence of cofactors. A particular caution that must be taken to isolate or preserve RNA is to protect RNA from degradation by RNase. Due to their ubiquity throughout cells, RNases must be inhibited from acting in, or be removed from, the tools used in the isolation or preservation of RNA. Thus, the tools used must be sterilized or heated at 180°C or higher and the reagents used must be treated with potent protein modifying or degrading agents (e.g., DEPC (diethylpyrocarbonate) ) . DEPC is widely used to inhibit RNase activity and can be added to almost all solutions except for Tris buffers. However, DEPC must be handled with caution and removed by heating under high pressure after treatment therewith because it induces adenines of single stranded DNA into carboxymethylation and thus is inferred to act as a carcinogen. Also, it is difficult to treat the waste water of DEPC. Therefore, there is an urgent need for an RNA preservation medium that is more convenient, safe, potent, environment-friendly and harmless. As alternatives to DEPC, an ultrafiltration system which can produce deionized water free of RNase by removing ions and organic matter to a high degree of purification (Nucleic Acids Symp Ser. 1995; (33) :129-33) , and RNase removers having functions of adsorption, deionization, UV oxidation, and ultrafiltration have been developed, but they are expensive. U. S. Pat. Unexamined Publication No. 2003-114651 discloses an RNA preservation medium that precipitates RNA with a sulfate solution so as to render RNA inaccessible to nucleases. Thus far, the preservation of RNA using alkaline, reductive electrolyzed water has not been explored because RNA is known to be susceptible to alkali. Nonetheless, leading to the present invention, intensive study on RNA preservation, conducted by the present inventors, resulted in the finding that alkaline electrolyzed water has no influence on enzymatic reactions using RNA and provides excellent protection from RNA degradation and thus can be used as an RNA preservation medium which is environmentally friendly and harmless to the body, with a significant, economical profit.

Disclosure of the Invention

Therefore, the object of the present invention is to solve the problems encountered in prior arts and to provide an RNA preservation medium that is convenient and safe for conducting reactions with RNA in addition to being environment-friendly and harmless to the body, thus being highly economically profitable, and a method for preserving RNA using the same.

Brief Description of Drawings FIG. 1 is an electrophorogram of the total RNA isolated from plant cells, which is dissolved in: sterilized ultrapure tertiary distilled water (lane 1) , DEPC-treated ultrapure water (lane 2), reductive electrolyzed water immediately after preparation (lane 3) , reductive electrolyzed water stored at 40C for two days after preparation (lane 4) , reductive electrolyzed water stored at 40C for four days after preparation (lane 5) , reductive electrolyzed water stored at -200C and thawed (lane 6) , reductive electrolyzed water stored at 250C for two days after preparation (lane 7) , and reductive electrolyzed water stored at 25°C for four days after preparation (lane 8) . FIG. 2 is electrophorograms of the total plant RNA which has been preserved at 40C for 1, 3 and 7 days in sterilized, ultrapure water (lane 1) , DEPC-treated, ultrapure water (lane 2) , and the reductive electrolyzed water stored under the same temperature and time conditions as in FIG. 1 (lanes 3 to 8) . FIG. 3 is electrophorograms of the total plant RNA which has been preserved at 250C for 1, 3 and 7 days in sterilized, ultrapure water (lane 1), DEPC-treated, ultrapure water (lane 2) , and the reductive electrolyzed water stored under the same temperature and time conditions as in FIG. 1 (lanes 3 to 8) . FIG. 4 is electrophorograms of the total RNA (isolated from animal cells) which has been preserved for 0, 1, 3, 5, 10, 14 and 16 days in: sterilized ultrapure water at 250C (lane 1) , sterilized ultrapure water at 40C (lane 2), sterilized ultrapure water at -2O0C (lane 3) , sterilized ultrapure water at -7O0C (lane 4), DEPC-treated ultrapure water at 250C (lane 5), DEPC-treated ultrapure water at 40C (lane 6), DEPC-treated ultrapure water at -2O0C (lane 7) , DEPC-treated ultrapure water at -700C (lane 8), reductive electrolyzed water at 250C (lane 9) , reductive electrolyzed water at 40C (lane 10), reductive electrolyzed water at -2O0C (lane 11), and reductive electrolyzed water at -700C (lane 12) . FIG. 5 is an electrophorogram of the RT-PCR products amplified in reductive electrolyzed water (lanes 1 and 2) and in DEPC-treated, ultrapure water (lanes 3 and 4), along with a 100 bp DNA ladder mark (M) .

Best Mode for Carrying Out the Invention

In accordance with an embodiment of the present invention, an RNA preservation medium comprising reductive electrolyzed water is provided. The term "electrolyzed water", as used herein, means an aqueous solution which is controlled in pH or oxidation- reduction potential by electrolysis. The electrolysis of water, an electrochemical process, yields new water which has quite different characteristics, with the concomitant production of alkaline electrolyzed water in a cathode chamber and acidic electrolyzed water in an anode chamber. The term "reductive electrolyzed water" or "cathode electrolyzed water" as used herein means the water obtained around the cathode upon the electrolysis of water. The cathode electrolyzed water included in the RNA preservation medium of the present invention can be prepared using various apparatus and well-known methods. A typical electrolyzed water generator comprises two electrolytic cells, an anode and a cathode, with a membrane disposed therebetween. When an electric field is applied across the electrodes, alkaline electrolyzed water is generated in the cathode cell while acidic electrolyzed water is generated in the anode cell. For instance, an electrolysis device comprises a septum or an ion exchange membrane for separating an anode from a cathode, and an electrolyte (e.g., NaCl, KCl, etc.) for promoting electrolysis, and is supplied with a voltage higher than a decomposition voltage of 2 to 3 volts. On the market, there are various devices for directly electrolyzing tap water to produce cathode electrolyzed water. The continuous production of electrolyzed water is achieved by continuously supplying into an electrolytic cell tap water filtered through an absorbing medium, such as active charcoal. In addition, batch type electrolyzed water generators comprising anode and cathode chambers are also well known. Apparatuses for producing cathode-electrolyzed water suitable for various applications have been developed. For example, a voltage is applied between an anode and a cathode to electrolyze a pure water-based electrolyte comprising a predetermined molar concentration of NH4OH or HCl to obtain reductive water having pH values of interest at the cathode. In addition, a device for the continuous mass production of electrolyzed water at high yield has been developed, in which a water flow regulating means equipped in an electrolytic cell is structured such that a cathode plate extends alongside an anode plate with a gap from 0.1 to 3 mm therebetween. It is known that cathode electrolyzed water can be used to preserve DNA (Biochemical and Biophysical Research Communications, 234, pp. 269-274, 1997) . RNA is physiochemically different from DNA. In detail, DNA generally exists in the form of a double strand, whereas RNA exists as a single strand. RNA employs uracil, instead of thymine, as a base, in contrast to DNA. In the skeleton of RNA, a ribose is linked to a' base, whereas a 2'-deoxyribose plays the role in DNA. That is, the pentose of RNA has an OH (hydroxyl) group at position 2. The 2'-0H of the ribose is chemically very unstable, so that it is readily hydrolyzed in water. Due to the lack of stability in comparison to DNA, RNA is highly susceptible to alkali. In contrast, DNA is resistant to alkali and thus the double strand may be separated into single strands by alkaline treatment for use in experiments. Generally, RNA experiences a secondary structural change into, e.g., a hairpin structure, with bases forming hydrogen bonds between themselves in the single strand. Therefore, reductive electrolyzed water, even though it can be used as a DNA preservation medium, is not readily inferred to be useful to preserve RNA. In the present invention, the term "RNA (ribonucleic acid)" indicates a polymer consisting of nucleotides, each having a base, a ribose and a phosphate linked with each other, exemplified by non-limitative antisense RNA, catalytic RNA, cRNA (complementary RNA) , mRNA (messenger RNA) , rRNA (ribosomal RNA), siRNA (small interfering RNA), snRNA (small nuclear RNA), tRNA (transfer RNA), and their derivatives. The RNA preservation medium according to the present invention is useful for the preservation not only of the respective RNAs but also their combinations (e.g., total RNA). Also, the RNA preservation medium of the present invention is effectively applicable for the preservation of various RNAs regardless of their lengths and concentrations. As used herein, the term "total RNA" means a combination of RNA in which various kinds of RNA from various origins are mixed without discrimination. Antisense RNA refers to an RNA molecule which can hybridize with a complementary sequence of RNA or DNA of interest, thereby changing the function of the corresponding DNA. Catalytic RNA has an intron sequence, showing catalytic activity like an enzyme. cRNA is 'a synthetic RNA that is transcribed from a single strand template of DNA of interest. mRNA is a class of RNA that transmits information about protein synthesis from a gene to a ribosome. rRNA is a structural/functional ■ constituent of a ribosome, which is a sub-cellular unit essential for protein synthesis. siRNA is short antisense RNA resulting from the cutting of a double-stranded RNA, with the purpose of decomposing an mRNA of interest. snRNA is a class of short RNA (100 to 300 nucleotides long) found in the nucleus, frequently forming a complex with an snRNP (small nuclear ribonucleoprotein) . tRNA (transfer RNA) is single stranded RNA about 70-90 nucleotides long, carrying an amino acid coincident with an anticodon of mRNA during protein synthesis. These RNA may be naturally occurring in various origins, including microorganisms, animals, and plants, or may be artificially synthesized. In electrolyzed water, there is a relationship between oxidation-reduction potential and pH. As the ORP takes on negative values, electrolyzed water increases in reducing power. For example, electrolyzed water has a pH of 6 to 7 at an ORP of about -200 mV and the pH value is elevated to 8.5 to 10 at an ORP of -800 mV. In accordance with the present invention, the reductive electrolyzed water suitable for RNA preservation has an ORP of -20OmV or less, preferably -100OmV to -20OmV, more preferably -90OmV to -80OmV, and most preferably -84OmV to -86OmV, with a corresponding pH value of 6 or higher, preferably 6 to 11, more preferably 8.5 to 11, and most preferably 10 to 11. When the ORP exceeds -20OmV, RNA is likely to degrade in the electrolyzed water. An examination was made of the effect of the reductive electrolyzed water on the preservation of RNA. For this, total RNA was isolated from a plant using a TRI reagent and dissolved in sterilized, ultrapure water, DEPC-treated ultrapure water, and the reductive electrolyzed water stored at various temperature for various time periods. Electrophoresis showed good isolation of total RNA in all of the conditions (FIG. 1) . Also, in order to examine the influence of the electrolyzed water on RNA stability according to temperature and time, total RNA of plant origin was measured for quantitative change after being preserved at 40C and 250C for 1, 3 and 7 days in sterilized ultrapure water, DEPC-treated ultrapure water, and the reductive electrolyzed water, stored at various temperatures for various time periods (FIGS. 2 and 3) . As for total RNA of animal origin, its preservation was measured after storage at 4°C, 250C, -200C and -700C for 1, 3, 5, 10, 14 and 16 days in sterilized, ultrapure water, DEPC- treated ultrapure water, and the reductive electrolyzed water (FIG. 4). In FIG. 2, there are electrophorograms of the total plant RNA that has been preserved at 40C for 1, 3 and 7 days in sterilized, ultrapure water, DEPC-treated ultrapure water, and the electrolyzed water stored at various temperatures for various time periods. The electrophoretic pattern of the total RNA preserved in sterilized ultrapure water shows the initiation of RNA degradation 3 days after the preservation (lane 1) . The RNA in DEPC-treated ultrapure water also underwent degradation, although to a lesser degree than in sterilized ultrapure water, as identified in the electrophoretic pattern (lane 2) . In contrast, no degradation can be detected in the electrophoretic pattern of the total RNA that has been preserved in the reductive electrolyzed water for as long as one week (lanes 3-8) . FIG. 3 provides electrophorograms of total plant RNA that has been preserved at 250C for 1, 3 and 7 days in sterilized, ultrapure water, DEPC-treated ultrapure water, and the electrolyzed water stored at various temperatures for various time periods. At 250C, RNA starts to degrade one day after preservation in DEPC-treated ultrapure water as well as sterilized, ultrapure water (lanes 1 and 2) . However, the total RNA preserved in the reductive electrolyzed water is found to undergo no degradation at all until three days after preservation. One week after preservation, DEPC-treated ultrapure water, as well as sterilized, ultrapure water, shows electrophoretic patterns of complete degradation of total plant RNA, whereas the total plant RNA in reductive electrolyzed water undergoes degradation only to a significantly lesser extent. In FIG. 4, there are electrophorograms of the total animal RNA that has been preserved at 40C, 250C, -200C and - 7O0C for 1, 3, 5, 10, 14 and 16 days in sterilized, ultrapure water, DEPC-treated ultrapure water, and electrolyzed water. At 250C, as seen in FIG. 4, total animal RNA starts to degrade three days after preservation in DEPC-treated ultrapure water as well as ultrapure water (lanes 1 and 5) , whereas no degradation is found at all in reductive electrolyzed water until after three days of preservation (lanes 9 to 12) . After preservation for 16 days, RNA is found to be degraded in sterilized, ultrapure water and DEPC- treated ultrapure water at both 250C (lanes 1 and 5) and 40C (lanes 2 and 6) , but remains almost intact in reductive electrolyzed water at those temperatures, as revealed by the electrophoretic patterns. These experimental data imply that reductive electrolyzed water can preserve RNA for 16 days, at 250C as well as at 40C, without degradation. As verified by the experimental data, reductive electrolyzed water, even if not further treated with additional processes, such as autoclaving, can preserve RNA for a long period of time regardless of temperature. Suitable as an RNA preservation medium, reductive electrolyzed water may be used to preserve RNA preferably for two weeks or more, and more preferably for four weeks or more, at 30°C or less, preferably at 25°C or less, and more preferably at 40C to 250C. In addition to preserving RNA without degradation, reductive electrolyzed water has no negative influence on subsequent enzymatic reactions with RNA. FIG. 5 is an electrophorogram showing the results of an enzymatic reaction with RNA in reductive electrolyzed water and DEPC-treated ultrapure water. To be used as an effective RNA preservation medium, reductive electrolyzed water should be able to not only preserve RNA at low and high temperatures for long periods of time without causing degradation, but also be helpful in, or at least not inhibitory of, subsequent reactions with RNA. As indicators for examining the influence of reductive electrolyzed water on enzymatic reactions with RNA, reverse transcriptase and Taq DNA polymerase are used because they are susceptible to reaction conditions. To this end, RT-PCR (Reverse transcriptase-Polymerase chain reactions) may be carried out. Reductive electrolyzed water, instead of DEPC- treated ultrapure water, was used for cDNA synthesis while PCR employed DEPC-treated ultrapure water and reductive electrolyzed water to amplify DNA. The PCR products thus obtained were quantitatively analyzed (FIG. 5) . There is no difference between the PCR products amplified in DEPC-treated ultrapure water and reductive electrolyzed water. Also, both the PCR products were found to have the desired 352 bp size. As described above, the reductive electrolyzed water of the present invention can be useful as an RNA preservation medium, effectively performing a sensitive enzymatic reaction such as cDNA synthesis and a high temperature enzymatic reaction such as PCR in addition to preserving RNA without degradation. In another embodiment of the present invention, a method of preserving RNA using the reductive electrolyzed water is provided. In the method, RNA is dissolved in an RNA preservation medium including reductive electrolyzed water. The RNA preservation medium used in the RNA preservation method of the present invention is as described above. The kinds of RNA and the ORP and pH of the reductive electrolyzed water used in the method are as described above. A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention. EXAMPLE 1: RNA Preservation Ability of Reductive Electrolyzed Water

Total RNA was isolated from corn seeding cells (Zea Mays L.) using a TRI reagent (FIG. 1) (Sambrook, J and D. W. Russel : Molecular cloning; A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 2001) . The total RNA was dissolved in sterilized, ultrapure water, DEPC(Sigma- Aldrich Co.)-treated ultrapure water, and the reductive electrolyzed water (ORP -850 mV, pH 11) stored at various temperatures for various periods of time, followed by immediate formaldehyde gel electrophoresis, with 3 μg of RNA loaded per lane. Electrophoretic patterns showed good isolation of total RNA without degradation (FIG. 1) . After being preserved at 4°C for 1 to 7 days in sterilized, ultrapure water, DEPC-treated ultrapure water, and the electrolyzed water stored at various temperatures for various time periods, the total RNA was run on formalhehyde gel in the presence of an electric field. As shown in the electrophorogram of FIG. 2, the total RNA started to degrade 3 days after preservation in sterilized, ultrapure water (lane 1) , and degradation, although to a lesser extent, also occurred in the RNA preserved in DEPC-treated ultrapure water (lane 2) . On the contrary, the total RNA preserved at 40C in reductive electrolyzed water did not undergo degradation at all (lanes 3 to 8) . When preserved at 25°C (FIG. 3) , the plant total RNA was found to start to degrade one day after preservation in DEPC-treated distilled water as well as sterilized ultrapure water, as. shown in the electrophoretic patterns (lanes 1 and 2) . However, no degradation was found in the RNA preserved in reductive electrolyzed water until after three days. One week after preservation at 250C, the total RNA was completely degraded in DEPC-treated ultrapure water as well as ultrapure water, but degraded to a significantly lesser extent in reductive electrolyzed water (lanes 3 to 8) . Therefore, reductive electrolyzed water can be used in all RNA experiments as an RNA preservation medium that is not only more convenient, safe and effective in RNA preservation than DEPC, but also is environment-friendly and harmless to the body.

EXAMPLE 2: RNA Preservation Ability of Reductive Electrolyzed Water

1 ml of TRIzol (Invitrogen) was added to a population of 5χlO6 to IxIO7 NIH 3T3 cells (mouse fibroblast) to isolate total RNA. Immediately after the isolation, the total RNA was dissolved in sterilized, ultrapure water, DEPC-treated ultrapure water, and the reductive electrolyzed water (ORP - 850 mV, pH 10.1), and stored at 250C, 4°C, -2O0C, and -70°C for predetermined periods of time, followed by electrophoresis on 1.5% formaldehyde gel for 30 min with the application of 50 volts, with 9 μg of RNA samples loaded per lane. Good total RNA isolation was confirmed in the electrophoretic patterns of FIG. 4. However, the animal total RNA started to degrade slightly three days after preservation at 250C in sterilized, ultrapure water and DEPC- treated ultrapure water, while no degradation was found in the RNA preserved in reductive electrolyzed water. The degradation was found to become serious 10 days after preservation at 250C in sterilized, ultrapure water and DEPC- treated ultrapure water, but the RNA still remained intact in the reductive electrolyzed water, even 14 days after the preservation at 25°C. On the 16th day after preservation at 40C as well as 250C, RNA degradation was detected in both the sterilized ultrapure water and the DEPC-treated ultrapure water. Notably, the animal RNA hardly degraded in the reductive electrolyzed water during the 16 days of preservation at 40C and 250C (FIG. 4) .

EXAMPLE 3: Effect of Reductive Electrolyzed Water on RT-PCR

To be useful as a RNA preservation medium, reductive electrolyzed water is required to allow smooth operation of enzymatic reactions with RNA as well as preserve RNA at low and high temperatures for a long period of time without causing degradation. To test for this, the reductive electrolyzed water was applied to RT-PCR (Reverse transcriptase-Polymerase chain reaction) (Sambrook, J and D. W. Russel: Molecular cloning; A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 2001), because reverse transcriptase and Taq DNA polymerase are sensitive to reaction conditions. For cDNA synthesis, first, reductive electrolyzed water (ORP -850 mV, pH 11) was used, instead of DEPC (Sigma- Aldrich Co.)-treated ultrapure water, in a typical reaction (oligo-(dT) primer, 1OmM dNTP, RNA sample, 5X first strand buffer, 0.1M DTT, reverse transcriptase, DEPC-treated ultrapure water) . In PCR, DEPC-treated ultrapure water, and, separately, the reductive electrolyzed water, instead of DEPC-treated ultrapure water, were used, in addition to a cDNA template, a 10 μM sense primer, a 10 μM antisense-primer and a premix (Taq DNA polymerase mixture) . The PCR products thus obtained under the two conditions were quantitatively analyzed and compared to each other (FIG. 5) . Using a set of the sense primer 5'-CCATATGGTTGCACCAAGTAACATGGAGA-S' (SEQ. ID. NO. 1) and the antisense primer δ'-CTCGAGGGGTTCCAGTCCTCTATGTTCAGA-S' (SEQ. ID. NO. 2), the PCR was started with 940C pre- denaturation for 3 min and was carried out with 30 cycles of denaturing temperature at 940C for 1 min, annealing temperature at 550C for 1 min, and elongating temperature at 720C for 1 min, finally followed by 720C extension for an additional 5 min and then by cooling at 40C. With a 100 bp DNA (2,000, 1,600, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100 bp) ladder serving as a marker, electrophoresis of the PCR products was conducted, showing no difference between the amplification in reductive electrolyzed water and DEPC-treated ultrapure water. Both in the DEPC-treated ultrapure water and in the reductive electrolyzed water, a DNA sequence 352 bp long was amplified accurately (FIG. 5) . For all RNA reactions including RT-PCR, it is very important to use highly pure water. Water of high purity, free of DNases or RNases, has been developed and is commercially available, but is expensive. Taken together, the data of the Examples demonstrate that reductive electrolyzed water can preserve RNA for a longer period of time at higher temperatures without causing degradation than can DEPC-treated ultrapure water, and allows the effective amplification of DNA, with no influence on enzymatic reactions with RNA, effectively performing a sensitive enzymatic reaction such as cDNA synthesis and a high temperature enzymatic reaction such as PCR.

Industrial Applicability

Since RNA is highly susceptible to alkali, temperature and RNase, the preservation of RNA without degradation is very important in biotechnology and related fields. Therefore, the reductive electrolyzed water of the present invention can be used as a novel RNA preservation medium. With the advantage of being highly superior in RNA preservation ability and having no influence on enzymatic reactions with RNA, the reductive electrolyzed water in accordance with the present invention can be used as an RNA preservation medium that is convenient, safe, economically profitable, and environment-friendly.