KR100658605B1 - High-efficiency rna extraction method from cyanobacteria - Google Patents

Abstract

A method for extracting RNA from cyanobacteria is provided to avoid employment of lysis solution, lysozyme and protenase K in the extraction, and improve rapidness and yield of extraction. The method for extracting RNA from cyanobacteria comprises the steps of: adding distilled water, phenol and glass or ceramic beads into cyanobacteria cells; and adding the cell-bead solution into a bead-beating machine and pulverizing the cyanobacteria cells at 1500-7000 rpm for 5-300 seconds, wherein the phenol has the temperature of 10-35 deg. C; and the method further comprises a step of sequentially adding phenol and chloroform into the pulverized cyanobacteria cell.

Description

High-efficiency RNA extraction method from cyanobacteria

1 is a process chart showing the step of extracting RNA from cyanobacteria with high efficiency.

Figure 2 is a picture of the electrophoresis of the RNA extract of cyanobacteria when the RNA was extracted by the BPC method of Example with 1.6% agarose gel and 0.5 X T buffer.

Figure 3 is a picture of the electrophoresis of the RNA extract of cyanobacteria when extracted RNA by the XSP method of Comparative Example 1 with 1.6% agarose gel and 0.5 X TI buffer.

Figure 4 is a picture of the electrophoresis of the RNA extract of cyanobacteria when the RNA was extracted by the method of Comparative Example 2 with 1.6% agarose gel and 0.5 X TI buffer.

Figure 5 is a photoelectrophoresis of the RNA extract of cyanobacteria by the method of Comparative Example 3 with 1.6% agarose gel and 0.5 X TI buffer.

The present invention relates to a method for extracting RNA from cyanobacteria, and more particularly, to distilled water in cyanobacteria cells; phenol; And adding a glass or ceramic bead and injecting the cell-bead solution into a bead beating machine, thereby crushing the cells, which is simpler than the conventional method. The RNA extraction method is fast and very efficient and can be used for sample preparation such as Northern blot, reverse transcription-polymerase chain reaction (PCR) and microarray analysis.

In the analysis of the entire microbial community, rDNA is analyzed by rDNA, which contains the DNA of all individuals in the population. However, when analyzing the metabolic activity in the community, it is preferable to analyze by rRNA rather than rDNA. CDNA synthesis, gene cloning and sequencing are required to analyze the metabolic activity and properties of each population. The first step for this analysis is the extraction of RNA. For example, in technologies such as Northern blot, reverse transcription-polymerase chain reaction (PCR), and microarray analysis, fast RNA isolation without damage, contamination of DNA, and high purity is essential for accurate results. to be.

Separation and purification of RNA from cyanobacteria cells is different from Gram-negative or Gram-positive bacteria with only cell membranes, and has a cell wall and various cell walls, and many components such as proteins, mucus and polysaccharides are mixed. It is known that RNA is not easily collected without damage, and it is reported that a large amount of sample is required when using the existing RNA extraction method, and it has a low recovery rate.

In the conventional cyanobacteria, RNA extraction method uses Xanthogenate, Sodium dodecylsulfate and Phenol to liquefy the cyanobacteria, and then centrifuge and accumulate the integrated cells. Dissociation to separate RNA (hereinafter abbreviated as 'XSP method') [Journal of Phycology, 2000; 36: 251-258. However, the method takes about 10 to 30 minutes to completely liquefy the cyanobacteria, and the extraction process is complicated and a lot of reagents are used, which is disadvantageous in terms of economics, and the extraction efficiency is low, and the DNA is extracted together. Had the disadvantage of being.

The present invention provides a method for extracting RNA in a simple, fast and highly efficient manner without using a lysis solution, lysozyme, proteinase k, or the like as an RNA extraction method for cyanobacteria. It is about.

The present invention is a method for extracting cyanobacteria RNA, distilled water in cyanobacteria cells; phenol; And adding glass or ceramic beads and injecting the cell-bead solution into a bead beating machine to disrupt the cells.

In another aspect, the present invention provides a method for RNA extraction of cyanobacteria further comprising the step of adding phenol and chloroform simultaneously or sequentially after the cell disruption.

The present invention is a method of extracting RNA of cyanobacteria, distilled water in cyanobacteria; phenol; And crushing the cells by adding glass or ceramic beads and introducing the cell-bead solution into a bead beating machine.

In another aspect, the present invention is characterized in that it further comprises the step of adding phenol and chloroform simultaneously or sequentially after the cell disruption.

Hereinafter, the RNA extraction method of cyanobacteria of the present invention will be referred to as a bead-phenol-chloroform (Bead-Phenol-Chloroform) method (hereinafter referred to as 'BPC method'), which will be described in detail as follows.

Cyanobacterial cells can be recovered from the precipitates by normal centrifugation or the like from the culture of cyanobacteria for RNA extraction.

In order to extract RNA from the cells, the cells must be crushed, and methods for cell crushing may include bead beating [ Int . J. Syst. Evol . Microbiol ., 50, 931-936, Cardinali et al. 2000], cell wall lysis through enzymes [ J. Clin . Microbiol., 29, 810-812, Varma and Kwon-Chung. 1991], and methods via complex reagents such as surfactants [ Gene, 42, 169-173, Holm et al. 1986; Appl . Environ. Microbiol ,. 53, 2588-2589, Lippke et al. 1987] and the like have been used. However, these methods require a large amount of cells, costly and time-consuming to extract RNA, and the traditional method of separating RNA and DNA from cyanobacteria requires many cells. Have a low recovery rate [ Phycol . Res., 48, 15-17, Nakajima et al. 2000].

In the present invention, distilled water in the cyanobacterial cells for cell disruption; phenol; And glass or ceramic beads are added.

The distilled water is preferably sterilized, and distilled water used together with phenol serves to dissolve DNA in the crushed cells and preserve RNA.

The phenol serves to inhibit RNA degrading enzymes present in the cells, including the cytoplasm, while lysing the cell wall and crushing the cells. It is preferable to use phenol at 10 to 35 ° C that is not warmed. The use of heated phenol over the above range may cause the phenol to expand and come out of the container, such as a tube, and even through the heat generated naturally in the bead beating stage, even if the heated phenol is not used separately. Dissolution effect can be obtained.

The glass or ceramic beads may use conventional beads used for bead beating, for example, glass beads, silica beads, zirconia beads, zirconia / silica beads.

Distilled water into the cells; phenol; And the cell-bead solution to which the glass or ceramic beads are added is added to a bead beating machine to disrupt the cells.

The bead beating device may be used in general, and can be adjusted according to the particle size of the bead, but cell crushing for 5 to 300 seconds, preferably 10 to 40 seconds at 1500 ~ 7000 rpm, preferably 2000 ~ 5000 rpm Is carried out. In the case where the rotational speed or the crushing time is lower than the lower limit, the cell crushing is insufficient to reduce the RNA extraction efficiency, and when the upper limit is exceeded, the RNA is crushed. In addition, the extraction may be performed at room temperature without maintaining the constant temperature during the bead beating process.

The cell crushing step of the present invention is bead beating after addition of distilled water, phenol and zirconia / silica bead without the use of lysis solution, lysozyme, protease k. It is characterized by completing all cell disruption steps in one step.

When the bead beating step is completed, nucleic acids including RNA are dissolved in a small amount in the distilled water layer of the cell lysate, and may further comprise the step of removing impurities other than RNA from the distilled water layer.

In order to remove the nonspecific impurities from the distilled water layer including the nucleic acid including the RNA, phenol and chloroform may be sequentially extracted. This is done by centrifugation in order to remove cell wall debris from the cell debris, and the supernatant is sequentially added with phenol, centrifugation, distilled water layer obtained, chloroform and centrifuged to obtain a distilled water layer containing high concentration of RNA.

The phenol is used to remove the phenol of 10 ~ 35 ℃ not warmed the same as the cell wall disruption, the chloroform was used to remove the trace phenol remaining in the separated supernatant, and serves to increase the purity of the extracted RNA Done.

In addition to the above phenol and chloroform treatment, 3M sodium acetate which can concentrate the nucleic acid, isopropyl alcohol which precipitates RNA, 70% ethanol (no RNAase) and the like can be used in any combination.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the following Examples are intended to illustrate the present invention and do not limit the present invention.

Example : BPC  RNA Extraction of Cyanobacteria by the Method

Referring first to the process of separating RNA from cyanobacteria samples with reference to FIG. 1, first, the cyanobacteria samples are collected (1), and sterilized distilled water and phenol and zirconia / silica beads are added to the collected samples. Shake for 20 seconds using a bead beating machine (2). Next, after centrifugation, the supernatant is collected, phenol is added to the collected supernatant, mixed, centrifuged once more, the supernatant is transferred to a clean tube, chloroform reagent is injected, mixed, and centrifuged to collect the RNA layer on the upper layer. (3). RNA was precipitated by addition of isopropyl alcohol and 3M sodium acetate, the supernatant was removed, washed with 70% ethanol and finally centrifuged to remove ethanol and diethylpyrocarbonate-DW was added to RNA Dissolve (4).

The RNA separation process will be described in more detail step by step.

(1) collecting cyanobacterial cells

Cyanobacterial cells are harvested from cyanobacterial cell culture and centrifuged to collect cyanobacterial cells.

(2) Cell disruption using distilled water, phenol and beads

To the concentrated cyanobacterial cells, 800 μl of distilled water and 600 μl of pH 4.3 phenol at room temperature are added and pre-weighed zirconia / silica beads (0.5 mm, 0.5 g / 0.1 mm, 0.2 g) are added. The tube containing this cell-bead solution is placed in a bead beating machine and the cells are crushed by shaking for 20 seconds at 3800 rpm.

(3) isolation of RNA

Crushed cells and RNA can be separated by centrifugation at 10,000 x g for 10 minutes. The mixed sample is centrifuged and the supernatant is transferred to a 1.5 ml tube.

600 µl of pH 4.3 phenol at room temperature was added to the collected supernatant, followed by mixing, followed by centrifugation at 10,000 x g for 5 minutes to remove impurities.

After adding 600 μl of chloroform to the collected supernatant, the cap was closed and vigorously stirred for 15 seconds using a vortex, followed by centrifugation at 10,000 × g for 5 minutes.

(4) RNA enrichment

After centrifugation, the supernatant is transferred to a new test tube, an equal amount of isopropyl alcohol and 1/10 of 3M sodium acetate of the supernatant are added, mixed slowly by hand, and stored briefly at -20 ° C. Samples stored at −20 ° C. are centrifuged at 10,000 × g for 5 minutes to precipitate RNA. At this time, the RNA precipitate looked like a gel-like pellet before centrifugation.

The supernatant was removed and mixed with 1 ml of 70% ethanol, followed by centrifugation at 10,000 x g for 5 minutes.

The sample was centrifuged to remove the supernatant and the RNA pellet was dried by vacuum dryer for 5 minutes. Be careful not to dry completely. Add 60 µl of distilled water without RNAse (RNase), carefully mix with a pipette, and store at -70 ° C.

Comparative example  One

RNA was extracted by XSP method which is mainly used to extract RNA from cyanobacteria.

(1) collecting cyanobacterial cells

Cyanobacterial cells are harvested from cyanobacterial cell culture and centrifuged to collect cyanobacterial cells.

(2) Cell disruption using lysis solution

The collected cells are resuspended in 50 μl Ti buffer and to this cell solution are added 1.3 ml of 65 ° C. preheated XSP buffer (1: 1 volume ratio of xanthogenate-SDS buffer and phenol). After addition of the XSP buffer, the sample is left to stand at 65 ° C. for 5 minutes while stirring to mix well.

(3) isolation of RNA

Next, the mixture was mixed for 10 seconds using a vortex, and 200 μl of chloroform: isoamyl alcohol (24: 1 by volume) was added. Centrifuge at 10,000 x g for 5 minutes to separate the phenol and aqueous layers, and then transfer the supernatant to a new tube.

500 μl of phenol: chloroform: isoamyl alcohol (25: 24: 1 volume ratio) is added to the new tube. Mix for 10 seconds using a vortex and centrifuge at 10,000 × g for 5 minutes to separate the phenol and aqueous layers. The separated aqueous layer is transferred to a new tube, and 500 µl of phenol: chloroform: isoamyl alcohol (25: 24: 1 volume ratio) is added to the tube to separate the phenol layer from the aqueous layer twice.

(4) RNA enrichment

Isopropyl alcohol is added to the separated water layer in the same amount, and it is left to stand on cold ice for 15 minutes. RNA is isolated by centrifugation at 10,000 x g for 10 minutes.

Remove the supernatant, add 1 ml of 70% ethanol, mix, and centrifuge at 10,000 x g for 5 minutes.

The sample centrifuged is supernatant removed and the RNA pellet is air dried. At this time, be careful not to dry completely, add 50 μl of RNase-free distilled water, carefully mix with a pipette, and store at -70 ° C.

Comparative example  2

In the (2) cell disruption step of the BPC method of the above example, the same amount of distilled water was further added without adding phenol, and the rest of the RNA was extracted in the same manner as in the example.

Comparative example  3

RNA was extracted from cyanobacteria using Oh et al. Described in the Korean Journal of Biotechnology (vol 17, 311-313, 2002) as a method of extracting RNA from Lactobacillus spp. Microorganisms.

(1) collecting cyanobacterial cells

For RNA isolation, 3 ml cyanobacterial culture was centrifuged (15,000 rpm, 5 min) to concentrate the cyanobacterial cells.

(2) Cell membrane disruption using decomposition solution, glass beads and phenol

To the concentrated cyanobacterial cells, 800 μl of a dissolution solution (2.7 g / L sodium acetate, 5 g / L SDS, 0.34 g / L EDTA, pH 5.5) was added, and 0.4 mm of glass beads were used to destroy the cell membrane. 0.8 g was added (vortex) for 3 minutes after addition.

Each sample was centrifuged and 500 μl of the supernatant was mixed with saturated phenol (pH 5.5) stored at 68 ° C. in the same volume, and then slowly mixed for 5 minutes while maintaining 68 ° C.

(3) isolation of RNA

After each sample was centrifuged (15,000 rpm, 5 min), 450 μl of the supernatant was taken, the same volume of chloroform was mixed, vortexed for 20 seconds, and centrifuged.

(4) RNA enrichment

After taking 400 μl of the upper layer of each layered sample, 45 μl of 3 M sodium acetate (1/10 vol.) And double volume of 100% ethanol were added to precipitate RNA at −20 ° C. for 10 minutes. After each sample was centrifuged (15,000 rpm, 10 min), the supernatant was removed, washed with 70% ethanol, and then centrifuged (15,000 rpm, 10 min).

The supernatant is removed, mixed with 1 ml of 70% ethanol and centrifuged at 15,000 rpm for 5 minutes.

The sample centrifuged is supernatant removed and the RNA pellet is air dried. At this time, be careful not to dry completely, add 50 μl of RNase-free distilled water, carefully mix with a pipette, and store at -70 ° C.

Experimental Example  One: Comparative example  Comparison with 1

Table 1 shows the cyanobacteria used to compare the BPC method of Example and the XSP method of Comparative Example 1.

Sample Number group Taxonomic name Strain name One I Merismopedia tenuissima NIES ^{1 )} 230 2 I Synechococcus leopoliensis UTEX ^{2 )} 625 3 Ⅲ Arthrospira platensis NIES 39 4 Ⅲ Oscillatoria tenuis NIES 33 5 Ⅲ Phormidium sp. KCTC ^{3 )} AG10164 6 Ⅲ Synechocystis sp. PCC ^{4 )} 6803 7 Ⅳ Anabaena flos -aquae UTEX 2557 8 Ⅳ Nodularia spumigena UTEX 2092 9 Ⅳ Nostoc sp. PCC 7120 10 I Microcystis aeruginosa UTEX 2666

¹ NIES: National Institute for Environmental Studies

² UTEX: The Culture Collection of Algae at the University of Texas at Austin

³ KCTC: Korean Collection for Type Cultures

⁴ PCC: Pasteur Culture Collection of Cyanobacterial Strains

RNA was extracted by the BPC method of Example 1 and the XSP method of Comparative Example 1, respectively, using the 10 kinds of cyanobacteria of Table 1, and the time, extraction concentration, and RNA purity for extraction were measured and shown in Table 2. . The extraction efficiency of RNA by BPC method was superior to that of RNA by XSP method in most samples.

The concentration of extracted RNA was measured by UV method, and the purity of RNA was expressed as the ratio of absorbance between 260 nm and 280 nm. When comparing the electrophoresis results of FIG. 2 and FIG. 3, it was confirmed that there was no contamination of DNA when RNA was extracted using the BPC method, and a large amount of DNA was contaminated in all samples during RNA extraction by the XSP method of FIG. 3. It is shown. In addition, although Nodaria spumigena UTEX 2092 ( Nodularia spumigena UTEX 2092), sample 8, was not extracted using XSP method, the RNA extraction method of cyanobacteria by BPC method was very effective. It is an extraction method.

Sample number Strain name BPC XSP RNA concentration (ng / ul) Total extracted RNA (ng) A260 / A280 ratio RNA concentration (ng / ul) Total extracted RNA (ng) A260 / A280 ratio One NIES 230 514.60 30.88 1.95 399.75 23.99 2.01 2 UTEX 625 569.68 34.18 1.94 635.01 38.10 1.97 3 NIES 39 1123.00 67.38 2.27 564.15 33.85 1.99 4 NIES 33 1327.24 79.63 1.94 217.14 13.03 2.12 5 AG 10164 303.96 18.24 1.92 231.18 13.87 2.07 6 PCC 6803 823.48 49.41 1.93 336.99 20.22 1.95 7 UTEX 2557 202.28 12.14 2.10 314.70 18.88 2.14 8 UTEX 2092 370.84 22.25 2.00 101.01 6.06 1.92 9 PCC 7120 146.68 8.80 1.94 120.36 7.22 2.02 10 UTEX 2666 1327.12 79.63 1.93 533.46 32.01 2.06

The BPC method of Example obtained an economic effect by minimizing the use of reagents of phenols.

In addition, the BPC method of Example took about 1 hour and 30 minutes to extract RNA, but the XSP method of Comparative Example 2 took about 3 hours, which is twice that. In the present invention, the RNA extraction step was minimized and the time required was minimized. In addition, the method of the present invention minimizes the extraction step of the RNA, thereby preventing the loss of RNA by minimizing the time required and maximizing the extraction amount of the RNA (Table 2, Figure 2 and Figure 3).

In addition, in the RNA extracted by the BCP method of the present invention, unlike the XSP method of Comparative Example 2, even if the step for removing a separate DNA it was found that the purity of the RNA (A260 / 280) is almost equal or rather superior ( Table 2, Figures 2 and 3). Commercially available kits have already become common for DNA contamination to occur, and an additional step must be taken to eliminate it [ Journal of Microbiology Methods, 44, 235-238, Nuyts et al. 2001]. However, the present invention had the advantage of removing RNA without a separate additional DNA removal step.

In addition, the BCP method of the present invention was able to more completely crush the cells than the chemical treatment of the XSP method of the comparative example by using bead beating as a physical treatment of cell crushing.

Experimental Example  2: Comparative example  Comparison with 2

As cyanobacteria, PCC 6803 ( Synechocystis sp. PCC 6803, No. 1) and Microcystis aeruginosa UTEX 2385 (No. 2) were used.

Agarose gel electrophoresis of RNA extracted by the method of Comparative Example 2 is shown in FIG. 4. In FIG. 4, no RNA was detected in PCC 6803 of the genus Cynecosis, and slightly disrupted RNA was detected in Microcistis aeruginosa UTEX 2385. In addition, the purity and amount of RNA by UV method was measured and shown in Table 3. Both samples showed low concentrations and purity incomparable to RNA extracted by the BPC method of Example. Therefore, the use of a mixture of distilled water, zirconia / silica bead, phenol in cell disruption in the BPC method of the present invention demonstrates that one of the key techniques in the present invention.

Sample Number Strain name RNA concentration (ng / ul) Total extracted RNA (ng) A260 / A280 ratio One Synechocystis sp. PCC 6803 3.22 161 1.69 2 Microcystis aeruginosa UTEX 2385 86.87 4323.5 1.99

Experimental Example  3: Comparative example  Comparison with 3

Comparative Example 3 compares the RNA extraction method by the method published in the Korean Journal of Biotechnology, Vol. 17, No. 311-313, 2002, and the BPC method of the present invention. Cyanobacteria were extracted RNA according to the Korean Society for Biotechnology Engineering using the two species used in Comparative Example 2, the agarose gel electrophoresis picture is shown in FIG.

In FIG. 5, RNA of the microcistis aeruginosa UTEX 2385 was extracted, but the DNA was contaminated, and it was confirmed that the PCC 6803 in the cinecocytis was hardly extracted.

Table 4 shows the results of determining the amount and purity of the RNA extracted by the UV method.

Sample Number Strain name RNA concentration (ng / ul) Total extracted RNA (ug) A260 / A280 ratio One Synechocystis sp. PCC 6803 38.56 1.92 1.72 2 Microcystis aeruginosa UTEX 2385 266.48 13.30 2.01

As also shown in Table 4, the amount of RNA extraction of PCC 6803 of the genus Cynecosis was very low.

Therefore, when the method of the biotechnological journal was applied to cyanobacteria, it showed very low RNA extraction rate and DNA contamination, and also it was confirmed that it was difficult to apply to cyanobacteria overall species.

The RNA extraction method of cyanobacteria according to the present invention is a simpler, faster, and more efficient extraction method of RNA than the conventional method. Northern blot, reverse transcription PCR (reverse transcription-polymerase chain reaction) and microarray It can be used for sample preparation such as analysis.

Claims (4)

Distilled water on cyanobacterial cells; phenol; And adding glass or ceramic beads, and injecting the cell-bead solution into a bead beating machine to disrupt the cells. The method according to claim 1, Said phenol is cyanobacterial RNA extraction method, characterized in that the phenol is not heated 10 ~ 35 ℃. The method according to claim 1, The method of RNA extraction of cyanobacteria, characterized in that the cells are disrupted for 5 to 300 seconds at 1500 ~ 7000 rpm in the bead beating machine (bead beating machine). The method according to any one of claims 1 to 3, The method of extracting RNA of cyanobacteria further comprises the step of adding the phenol and chloroform simultaneously or sequentially after the cell disruption.

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