RU2803798C1 - Method of isolating total ribonucleic acid from plant tissues of fruit crops - Google Patents

Abstract

FIELD: biotechnology; molecular biology.

SUBSTANCE: following is proposed: a method of isolating total ribonucleic acid from fruit crops plant tissues, including lysis of cell membranes and extraction of nucleic acids from plant cells, while 200 mM Tris-HCl pH 8.5, 50 mM EDTA, 500 mM NaCl, 1% 2-mercaptaethanol, 2% SDS, 2% PVP 40 are used as an extracting buffer; for purification and precipitation of total ribonucleic acid at the second stage of isolation, isopropyl alcohol (94%) is used, in the washing buffer sodium acetate at a concentration of 160 mm and 23 mm Tris-HCl pH 8.0, 0.1 mm EDTA are used; for selective precipitation of ribonucleic acid and enrichment of the total RNA preparation with microribonucleic acid (miRNA) molecules a mixture of LiCl/urea at a concentration of 8 M is used.

EFFECT: invention improves the quality of the total RNA preparation from various tissues of fruit plants.

1 cl, 1 dwg, 1 tbl, 3 ex

Description

The invention relates to the field of biochemistry and molecular biology, and can be used to isolate ribonucleic acid (RNA) from plant material.

Isolation of nucleic acids (NA) from plant tissues is a complex procedure, which is associated with the presence of associated biochemical substances in the tissues of fruit plants: esters, sugars, polyphenols and pigments. Moreover, the process of NK purification is usually multi-stage and time-consuming. In most laboratory methods, rather toxic substances, such as phenol, are used as denaturing agents, high concentrations of which have a negative effect on human health [Macedo N.J., Ferreira T.L. Maximizing total RNA yield from TRIzol reagent protocol: a feasibility study //ASEE zone I conference. - 2014. - P. 1-8].

One of the methods for isolating viroid RNA is based on treating plant material with phenol solutions, followed by enriching the preparation with small RNAs by chromatography on nonionic cellulose [Pallis V., Navarro A., Flores R. Isolation of a viroid-like RNA from hop different from hop stunt viroid // J Gen Virol. 1987. - Vol. 68. - P. 3201-3205.].

The disadvantages of this method are: the complexity of execution associated with the presence of a chromatography step, as well as the use of toxic substances such as phenol.

There is a known method for isolating RNA using two extraction buffers. In the first step, a 400 μL aliquot of extraction buffer I (100 mM tris(hydroxymethyl)aminomethane (Tris), pH 8.0, 150 mM LiCl, 50 mM ethylenediaminetetraacetic acid (EDTA), 1.5% sodium dodecyl sulfate (SDS) was added to the seed powder ), 1.5% 2-mercaptoethanol). After mixing the contents, 250 μL of phenol-chloroform (1:1, pH 4.7) was added by vigorous vortexing and the samples were mixed well by inversion. After centrifugation, the resulting upper aqueous phase was transferred to a new 1.5-ml tube containing 250 μl of extraction buffer II (70% guanidinium sulfate (w/v), 0.75 M sodium citrate, 10% lauryl sarcosine, 2 M sodium acetate, pH 4.0). To the recovered supernatant (about 450 μl) 300 μl of isopropanol and 250 μl of 1.2 M sodium chloride were added. The resulting RNA sediments were dried and suspended in an appropriate volume of water.

The disadvantage of this method is the use of a large amount of substances toxic to the operator: phenol, guanidinium sulfate and chloroform.

The closest method is the express method, which consists of sequential extraction in a buffer solution, SDS and 5 M potassium acetate with further precipitation of NC with ethanol. The plant sample was ground in liquid nitrogen and then 0.1 g aliquots were used for each extraction method. A sample of plant tissue was homogenized in 2.5 ml of extraction buffer (100 mM Tris (hydroxymethyl) aminomethane + HCl (Tris-HCl), 50 mM EDTA, pH 8.0, 500 mM NaCl, 10 mM denaturing agent 2-mercaptoethanol), 0.5 ml of the homogenate was transferred to a 1.5 ml tube and incubated with 25 μl of 20% SDS at 65°C for 20 min. Then 125 μl of 5 M potassium acetate was added to the extract and the mixture was kept at 0°C for 20 min. The tubes were then centrifuged at 10 K for 15 min to remove unwanted plant debris. Nucleic acids were recovered from the supernatant by ethanol precipitation and resuspended in 50 μl of sterile water.

The disadvantages are the presence of total RNA, double-stranded deoxyribonucleic acid (DNA) and single-stranded DNA in the preparation and unsatisfactory quality in terms of extinction ratio 260 nm/280 nm, indicating the presence of protein contamination.

The technical result of the proposed invention is to increase the yield and improve the quality of the total RNA preparation from various tissues of fruit plants.

The technical result is achieved by the fact that at the stage of extraction of nucleic acids (NA) from plant tissues of fruit crops rich in, first of all, phenolic substances, in addition to 200 mM Tris-HCl pH 8.5, 50 mM EDTA, 500 mM NaCl, 1 % 2-mercaptaethanol, add a chaotropic agent - 2% sodium dodecyl sulfate (SDS) and an antioxidant that increases the viscosity of the solution - 2% polyvinylpyrrolidone 40 (PVP 40); to purify and precipitate total ribonucleic acid at the second stage of isolation, isopropyl alcohol (94%) is used; to wash the NC, a saline buffer is used: 160 mM sodium acetate, 23 mM Tris-HCl pH 8.0, 0.1 mM EDTA; For selective precipitation of ribonucleic acid and enrichment of the total RNA preparation with microribonucleic acid (siRNA) molecules, a LiCl/urea mixture at a concentration of 4 M is used.

The method for isolating total ribonucleic acid includes the following sequential steps:

1) preparation of buffer solutions;

2) sample preparation;

3) lysis of cell membranes and extraction of ribonucleic acid from tissue cells of fruit crops in an extraction buffer;

4) centrifugation to remove unextracted cellular structures;

5) precipitation of NC;

6) washing in a saline buffer solution (wash solution)

7) selective RNA precipitation;

8) washing in a 70% ethanol solution;

9) dissolution of the RNA sediment in Tris-EDTA (TE).

1 - 2 ml of extraction buffer (200 mM Tris-HCl pH 8.5, 50 mM EDTA, 500 mM NaCl, 1% 2-mercaptaethanol, 2% SDS, 2% PVP 40) is added to the crushed plant tissue. The resulting solution of destroyed cells (lysate) was incubated on a solid-state thermostat at 56°C for 20 minutes, then cooled to room temperature and 300 μl of 5 M sodium acetate was added. Then incubation was carried out at a temperature close to 0°C for 20 minutes. The tubes were centrifuged at 12,000g and 4°C for 15 min. Next, using a dispenser, trying not to touch the interphase and sediment, we collected the supernatant and transferred 1.5 ml into new tubes and added 800 μl of isopropyl alcohol to each. After sedimentation of the nucleic acid extract, the sediment was resuspended in 200 μl of washing saline buffer (160 mM sodium acetate, 23 mM Tris-HCl pH 8.0, 0.1 mM EDTA), after complete dissolution of the sediment, 400 μl of 96% ethanol was added, mixed and centrifuged for 16 000g at 4°C 10 min. The supernatant liquid was drained and the sediment was waited for drying without allowing it to dry out. Then the sediment was dissolved in 133 μl of water, 167 μl of 8 M LiCl/urea was added and incubated for 60 min at a temperature of 0 to 4°C. The samples were centrifuged for 30 min at 16,000 g and 4°C, the supernatant liquid was drained, and 150 μl of 70% chilled ethanol was added to the sediment. The sediment was resuspended. The tubes were centrifuged at 4°C in short centrifugation mode (shot-spin) for 20-30 seconds. We waited for the sediment to dry without allowing overdrying, then dissolved the sediment in 60 μl of TE buffer.

As a result of the studies and comparisons with the analogue, it was determined that the addition of PVP 40 total ribonucleic acid to the extraction buffer at the first stage of extraction made it possible to remove impurities of organic pollutants and preserve their structure from the action of oxidizing agents, and the inclusion of the stages of precipitation with isopropanol and washing in the extraction process in buffers containing sodium acetate increases the yield of total RNA. Precipitation of RNA in the presence of LiCl/urea at a final concentration of 4 M allowed us to get rid of DNA impurities in the isolated sample.

Research was carried out on more than 100 samples of plant material from various fruit crops. The materials used were leaves, roots and stems of fruit plants.

The samples were analyzed on a NanoPhotometer® NP80 nanophotometer according to the optical density of the solution, according to the wavelength ratios of 260/280 nm, 260/230 nm. The amount of isolated RNA was calculated at a wavelength of 260 nm. The results are shown in Table 1.

The quality of the isolated RNA was assessed by gel electrophoresis in a 2% agarose gel, after denaturation of the isolated RNA sample. And it was determined by the ratio of the 28S and 18S RNA bands (Fig. 1). The ratio of stripes was on average 1:1. The fluorescence intensity of the 28S band is higher than that of the 18S band. No DNA bands were observed.

Examples of specific execution

Example 1. To isolate a total RNA preparation from the leaves of the Alaya cherry variety, sample preparation was carried out using liquid nitrogen and a ceramic mortar and pestle. The material was crushed to a homogeneous powder and quantitatively, 70 - 100 mg, transferred into a plastic tube with a volume of 1.5 ml and 1 ml of extraction buffer was added (200 mM Tris-HCl pH 8.5, 50 mM EDTA, 500 mM NaCl, 1% 2 -mercaptaethanol, 2% SDS, 2% PVP 40). The resulting solution of destroyed cells (lysate) was incubated on a solid-state thermostat for 20 minutes, then cooled to room temperature and 300 μl of 5 M sodium acetate was added. Then incubation was carried out at a temperature close to 0°C for 20 minutes. The tubes were centrifuged at 12,000 g and 4°C for 15 minutes, without touching the interphase and sediment, the supernatant was collected with a dispenser, 1.5 ml was transferred to new tubes, and 800 μl of isopropyl alcohol was added to each. After sedimentation of NC, the sediment was resuspended in 200 μl of washing saline buffer (160 mM sodium acetate, 23 mM Tris-HCl pH 8.0, 0.1 mM EDTA), after complete dissolution of the sediment, 400 μl of 96% ethanol was added, mixed and centrifuged at 16000g at 4 °C 10 min. The supernatant liquid was drained and the sediment was allowed to dry without allowing it to dry out. Then the sediment was dissolved in 133 μl of water, 167 μl of 8 M LiCl/urea was added and incubated for 60 min at a temperature of 0 to 4°C. The samples were centrifuged for 30 min at 16,000g and 4°C, the supernatant was drained and 150 μl was added to the sediment 70 % refrigerated ethanol. The sediment was resuspended. The tubes were centrifuged at 4°C in short centrifugation mode (shot-spin) for 20-30 seconds. We waited for the precipitate to dry without allowing overdrying, then dissolved the precipitate in 60 μl of TE buffer. The RNA content in the sample was determined spectrophotometrically.

As a result, 250-375 ng of RNA was isolated from 70 - 100 mg of material. The purity in terms of extinction coefficient ratios was 2.07 for (260/280 nm) and 2.03 for (260/230 nm).

Example 2. Isolation of a total RNA preparation from the skin of the Geneva Earley apple tree using manual homogenization using a plastic micropestle and a 2 ml microtube. 70 mg of apple fruit skin of the Geneva Earley variety and 1 ml of extraction buffer were added to the test tube. Conducted according to Example 1.

The qualitative parameters of the resulting total RNA preparation were studied by spectrophotometry. As a result, 50-120 ng of RNA was isolated from 70 mg of material. The purity in terms of extinction coefficient ratios was 2.05 for (260/280 nm) and 1.94 for (260/230 nm).

Example 3. Isolation of a total RNA preparation from the roots of the rootstock of stone fruit crops PK SK 1.

1 g of roots and 2.5 ml of extraction buffer were added to a test tube, crushed until homogeneous and incubated at room temperature for 5 minutes. The liquid phase was taken from the resulting homogenate and transferred into 1.5 ml test tubes. The resulting lysate was manipulated as described in example 1.

The resulting total RNA preparation was studied by spectrophotometry. As a result, 250-400 ng of RNA was isolated from 1 g of material. The purity in terms of extinction coefficient ratios was 2.15 for (260/280 nm) and 2.01 for (260/230 nm).

Claims (1)

A method for isolating total ribonucleic acid from plant tissues of fruit crops, including lysis of cell membranes and extraction of nucleic acids from plant cells, characterized in that an extraction buffer of the following composition is used: 200 mM Tris-HCl pH 8.5, 50 mM EDTA, 500 mM NaCl, 1 % 2-mercaptaethanol, 2% SDS, 2% PVP 40; to purify and precipitate total ribonucleic acid at the second stage of isolation, isopropyl alcohol (94%) is used; in the washing buffer, sodium acetate is used at a concentration of 160 mM and 23 mM Tris-HCI pH 8.0, 0.1 mM EDTA; For selective precipitation of ribonucleic acid and enrichment of the total RNA preparation with microribonucleic acid (siRNA) molecules, a LiCl/urea mixture at a concentration of 8 M is used.