WO2022139709A1 - Modified method for high quality and pure rna isolation from pomegranate - Google Patents
Modified method for high quality and pure rna isolation from pomegranate Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 41
- 241000219991 Lythraceae Species 0.000 title claims abstract description 16
- 235000014360 Punica granatum Nutrition 0.000 title claims description 15
- 238000002123 RNA extraction Methods 0.000 title abstract description 20
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- 235000019441 ethanol Nutrition 0.000 claims description 32
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims description 31
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- 238000005119 centrifugation Methods 0.000 claims description 11
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 claims description 10
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- 229940063673 spermidine Drugs 0.000 claims description 6
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- 235000017281 sodium acetate Nutrition 0.000 claims description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
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- 229910052573 porcelain Inorganic materials 0.000 claims description 2
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 claims 1
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- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims 1
- 229930000044 secondary metabolite Natural products 0.000 abstract description 10
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 abstract description 9
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- 210000001519 tissue Anatomy 0.000 description 21
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- 239000013614 RNA sample Substances 0.000 description 17
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- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 8
- 238000005406 washing Methods 0.000 description 6
- 229960004592 isopropanol Drugs 0.000 description 5
- 108091070501 miRNA Proteins 0.000 description 5
- 239000011543 agarose gel Substances 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 238000000227 grinding Methods 0.000 description 4
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 description 4
- 239000007858 starting material Substances 0.000 description 4
- 108020004463 18S ribosomal RNA Proteins 0.000 description 3
- 150000004676 glycans Chemical class 0.000 description 3
- 238000010841 mRNA extraction Methods 0.000 description 3
- 239000002679 microRNA Substances 0.000 description 3
- 229920001282 polysaccharide Polymers 0.000 description 3
- 239000005017 polysaccharide Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000002689 soil Substances 0.000 description 3
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- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000002798 spectrophotometry method Methods 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- 108020005096 28S Ribosomal RNA Proteins 0.000 description 1
- 101100283604 Caenorhabditis elegans pigk-1 gene Proteins 0.000 description 1
- 238000007399 DNA isolation Methods 0.000 description 1
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- 108090000790 Enzymes Proteins 0.000 description 1
- 241000209510 Liliopsida Species 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 244000294611 Punica granatum Species 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 239000012163 TRI reagent Substances 0.000 description 1
- 238000012233 TRIzol extraction Methods 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
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- 108020004999 messenger RNA Proteins 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
Abstract
The invention relates to a modified new method for RNA isolation from the Pomegranate plant, which has high phenolic content and secondary compounds, and RNA isolation is very difficult.
Description
MODIFIED METHOD FOR HIGH QUALITY AND PURE RNA ISOLATION FROM POMEGRANATE
TECHNICAL AREA
The invention relates to a modified novel method for RNA isolation from the pomegranate peel tissue, which has a lot of phenolic content and secondary compounds where RNA isolation is very difficult.
PREVIOUS TECHNIC
In many areas of molecular biology, the RNA isolation is the most fundamental and most important step. Obtaining the high quality, pure and high concentration RNA from plant tissue is very important. Various problems are encountered in the RNA isolation stages using plant-derived tissues.
Plants contain secondary metabolites with very different composition such as phenol, flavonoid, glycoside and alkaloid. This secondary metabolite content in plants varies at the level of species and variety, and also creates differences between plant tissues. The main problem in RNA isolation is these secondary metabolites that plants have, and these metabolites cause problems in RNA isolation and reduce the quality and purity of the isolated RNA samples. Different isolation methods have been developed in order to eliminate these problems. TRIzol (TRI reagent), CTAB (Cetyltrimethylammonium Bromide), Phenol: Chloroform methods are among these methods developed. In addition to the developed methods, commercial RNA isolation kits are also available. However, the isolation methods developed or the existing commercial kits are not sufficient to solve the problems experienced in isolation. For this reason, existing methods should be changed or used by modification.
Commercial kits and the TRIzol method are among the methods that are not suitable for replacement. The phenol: chloroform method is not preferred because it contains some harmful chemicals. The CTAB method is suitable for modification and can be optimized for each plant and tissue. The CTAB method takes its name from the chemical Cetyltrimethylammonium bromide, which is a detergent. Especially it is a preferred method to isolate RNA from tissues of plant origin. Although the CTAB method is used extensively, there is no CTAB method optimized for every plant or every tissue. Researchers working in the field of molecular biology research and use
the appropriate method for the plant or tissue they are working on or try to create protocols suitable for them by making changes to existing protocols. Establishing suitable protocols for the study is time consuming and requires a certain amount of knowledge. Extensive waste of chemicals and consumables used in establishing appropriate protocols is one of the most important cost factors. In addition, the absence of intense chemicals and consumables in every laboratory is another reason why researchers avoid optimization studies. For this reason, it is very important to develop isolation methods suitable for plant species and varieties or plant tissues.
Patent application numbered CN103740698A is a protocol developed for the isolation of micro RNA from tropical plants with high polysaccharide content. Micro RNAs are usually 20-25 nucleotides in length and originate from regions that are noncoded in DNA. Micro RNAs and mRNAs are very different molecules. Although both groups are included in the total RNA pool, mRNA isolation is used in gene expression-based studies. The protocol we modified in our study is intended for mRNA isolation and differs from patent application CN103740698A.
CTAB (Cetyltrimethylammonium bromide) chemical is a herbal detergent and is frequently used in DNA and RNA isolations. Although it has been used jointly in the method subject to our invention and in the patent document numbered CN103740698A, PEG8000 (polyetyleneglicol) was used in addition to CTAB in the patent document numbered CN103740698A to remove phenolic compounds and polysaccharides from the environment. PEG chemical is not available in the method we modified. In patent document numbered CN103740698A, guanidinium isothiocyanate chemical was used for degradation of proteins in CTAB solution. The solutions of the method of our invention do not contain guanidinium isothiocyanate. In addition, in our invention, there is a spermidine chemical in the CTAB solution. Spermidine is a polyamine with the closed formula C7H19N3. Besides having important functions in monocot and dicot plants, it is a substance used in RNA isolation studies. It is a substance that allows spermidine, DNA and proteins to be removed from the RNA molecule to be precipitated, used for RNA and nuclear membrane stabilization, and inhibits the activity of RNAase enzymes. It also has a function that reduces the negative charge of phosphate bonds by binding to the RNA molecule and increases the stability of the single-stranded RNA molecule.
In addition, washing solution was used in patent document numbered CN103740698A. The content of the washing solution used and the chemicals used in
the solution create differences in terms of the content of the solutions of the method we offer and the chemicals used in the solution. In application no CN103740698A, PEG8000 chemical was used in the extraction solution. PEG8000 is used in studies that allow the isolation of RNA from plant tissues with high polysaccharide content. The tissue we used in our study is pomegranate peel and the secondary metabolite composition is very high. Therefore, the solution used in the protocol we present is the CTAB solution. In patent application numbered CN103740698A, CTAB lysis buffer, PEG8000 buffer, EDC crosslinking buffer, miRNA Northern blotting hybridization buffer, 17% DPAGE buffer, Trizol extraction buffer and Washing Buffer solutions were used. They are costly solutions in terms of the solutions used and the chemicals they contain. The protocol we present includes extraction 1 and 2 solutions. The scarcity of the solutions used creates cost-reducing factors and saves time. In patent application CN103740698A, Washing Buffer was used as the washing solution. Washing Buffer content; 2 x SSC is 0.1 % SDS. Washing solution has not used in our protocol. Only ethyl alcohol was used as the washing solution. Using only ethyl alcohol is an important cost reduction factor.
In patent document CN103740698A, there is a step in which the TRIzol reagent is used, but in our invention there is no such step. Patent document number CN103740698A is a protocol that combines the CTAB and TRIzol methods and aims to isolate micro RNA from tropical plants. The solutions and steps used in our invention differ from patent document CN103740698A. The method we use is a method for mRNA isolation.
The patent document numbered JP2009082066A aims to isolate RNA from microorganisms in the soil. This application contains different steps and different solutions for RNA isolation. The first difference is the starting material. The soil has used as the starting material in this protocol. In the protocol we present, our starting material is pomegranate peel, which is a herbal tissue.
The patent document numbered JP2009082066A is a protocol that includes different steps from our invention. The first difference that exists in the patent document numbered JP2009082066A is the stage of removing humic substances from the soil and obtaining an extract for RNA isolation. The solution used in this step (200 mM EDTA 1200 mMNa 2 HPO 4 (pH 8.6) is also another difference. This phase is a phase belonging to the patent document numbered JP2009082066A and is not included in our invention. The second difference is the proportional differences of the solution used
for RNA isolation and the chemicals in the solution (2% SDS, 100 mM Tris-HCI (pH 8.0), 50 mM EDTA (pH 8.0)). This solution used in the patent document numbered JP2009082066A is not included in our invention. The third difference is the chemicals used in the CTAB solution and the proportional volumes of chemicals. The content of the CTAB solution used in the patent document numbered JP2009082066A is different from the CTAB solution of the protocol we have presented. In the patent document numbered JP2009082066A, the CTAB concentration in the CTAB solution has preferred as 10%.And this CTAB solution contains 10% CTAB, 10% polyvinylpyrrolidone, 4M CH 3 COONa. In the same solution, the guanidinium isothiocyanate chemical has used. In our invention, the proportion of CTAB in the CTAB solution is 2% and it does not contain guanidinium isothiocyanate chemical. In addition, our invention contains spermidine chemical and this chemical is not included in patent document JP2009082066A. In our invention, the ratios specified in the CTAB solution and the 4M CH 3 COONa chemical shows differences. The fourth difference that exists between the methods is that the PEG8000 solution and the PEG8000 solutions of used. Although the PEG8000 solution was used in the application numbered JP2009082066A to precipitate RNA, the PEG8000 solution has not used in our invention. In addition to the differences listed, there are also differences in the additional solutions and stages used in document JP2009082066A. In summary, the document number JP2009082066A and our invention including differences in terms of starting material, purpose, solutions used and the amount of chemicals in the solutions.
DETAILED DESCRIPTION OF THE INVENTION
Fruit peel tissue of pomegranate (Punica granatum L.) plant has used in our study. Pomegranate peel is a tissue with a very high secondary metabolite content. In our study, the CTAB method was modified to isolate high quality and pure RNA from pomegranate peel tissue. 2 different solutions were used in the modified CTAB method. The main solutions used were named Extraction 1 and Extraction 2. In addition to the two different solutions used in isolation, solutions with different concentrations were also used. 10 M LiCI2 (Lithium chloride) solution, 3 M Sodium Acetate solution and Chloroform: Isoamylalcohol (24/1 v / v) solution are the other solutions used. In addition, 2-Propanol, ethyl alcohol with 96% concentration, and ethyl alcohol with 70% concentration were used for washing the samples.
Preparation of Solutions
Extraction Solution 1: 2% CTAB (Cetyltrimethylammonium bromide), 25 mM EDTA (Ethylenediamine tetraacetic acid), 100 mM Tris-HCI (Tris-hydrochloric acid, pH: 8), 2 M NaCI (Sodium chloride), 1.05 g / L Spermidine, The solution was prepared using 2% PVP (Polyvinylpyrrolidone) chemicals. The prepared solution was sterilized by autoclaving at 121 degrees at 1 .05 atm pressure for 15 minutes. The sterilized solution has been stored at ± 4 degrees until the isolation stage.
Extraction Solution 2: 1 M NaCI (Sodium chloride), 10 mM Tris-HCI (Tris- Hydrochloric acid, pH: 8), 1 mM EDTA (Ethylenediamine tetraacetic acid, pH: 8), 0.5% SDS (Sodium dodecylsulfate) using chemicals, extraction 2 solution was prepared. The prepared solution was sterilized by autoclaving at 121 degrees at 1.05 atm pressure for 15 minutes. The sterilized solution has been stored at ± 4 degrees until the isolation stage.
Lithium Chloride (LiCk) Solution: 10 M Lithium chloride solution was prepared. The prepared solution was sterilized by autoclaving at 121 degrees at 1.05 atm pressure for 15 minutes. The sterilized solution has been stored at ± 4 degrees until the extraction stage.
Sodium Acetate (NaOAc, pH: 5.2) Solution: 3M Sodium Acetate solution has been prepared. The prepared solution was sterilized by autoclaving at 121 degrees at 1.05 atm pressure for 15 minutes. The sterilized solution has been stored at ± 4 degrees until the isolation stage.
Chloroform: Isoamylalcohol Solution: Chloroform and isoamylalcohol solvents were used for this solution. The solution was prepared by mixing 24 parts of chloroform with
1 part of isoamylalcohol. The prepared mixture has volumetric ratios of 24: 1 (v I v). The prepared mixture has been stored at -20 ° C until the isolation stage.
70% Ethyl Alcohol (EtOH): In order to prepare ethyl alcohol with 70% concentration, 30 ml of autoclaved pure water was added to 70 ml of 96% concentration of ethyl alcohol and the volume was completed to 100 ml. The prepared mixture has been stored at -20 ° C until the isolation stage.
96% Ethyl Alcohol (EtOH): Ethyl alcohol with a purity value of 96% was used and it was stored at -20 until the isolation stage.
2 - Propanol (Isopropanol): Isopropanol with a purity value of 99% was used and it was stored at -20 until the isolation stage.
RNA Isolation Stages
Before starting the isolation phase, -mercaptoethanol is added to the Extraction 1 solution as much as 3% of the solution volume and the prepared mixture is incubated at 65 ° C for 15 minutes. Preheated extraction 1 solution is used for isolation.
- The pomegranate peel tissues to be used in isolation are ground in a porcelain mortar with the help of liquid nitrogen. In the grinding process, the grinding is continued until the samples turn into powder. In the grinding process, liquid nitrogen is added and care is taken not to break the cold chain of the samples. If nitrogen is not supplemented during grinding, the tissues will be degraded and the isolated RNA sample cannot be used.
- 100 mg of powdered pomegranate peel is taken from the tissues and transferred to 2 mL eppendorf tubes. 150 mg powder PVP is added into the same tube. 1.5 ml of the extraction 1 solution that was previously incubated at 65 degrees on the eppendorf tube with powder tissue and PVP is added to the tube. The tissue is mixed rapidly with the help of an eppendorf tube vortex containing PVP and solution. The well mixed tube is placed in a heat block or water bath at 65 degrees and left for incubation. The samples are allowed to incubate for 30-40 minutes. During the incubation, the samples are mixed with a vortex every 5 minutes. At the end of the incubation period, the samples are centrifuged at 20000g for 30 minutes at +4 degrees.
- After centrifugation, samples are taken from the centrifuge device. After centrifugation, 3 phases are formed in the tubes. The light-colored and homogeneous liquid phase from the formed phases is taken with a 1000 pl automatic pipette and transferred to a new 2 ml Eppendorf tube. The liquid phase taken is approximately 1000p. 1000 pl of chloroform: isoamylalcohol (24: 1 v / v) stored at -20 ° C is taken on the liquid part taken into the tube and transferred to the tube. The mixture in the tube is gently mixed and the samples are placed in the Centrifuge. Tubes are centrifuged at 20000 g for 30 minutes at +4 degrees. After the centrifuge process, the samples are taken from the centrifuge. Two different phases are formed in the tubes. The supernatant should be open and clear. The upper phase is carefully removed with an automatic pipette and transferred to a new 1.5 ml Eppendorf tube. If the transferred liquid part is cloudy or dirty, the chloroform: isoamylalcohol step can be repeated. Approximately 350-400 pl of LiCI (Lithium chloride) solution, which
is kept at +4 degrees, is taken on the liquid part taken into 1.5 ml Eppendorf tube and transferred to the Eppendorf tube. The tube is gently mixed. After mixing, the tube is incubated at +4 degrees overnight.
- Samples taken from the incubation are centrifuged at 20000 g for 30 minutes at +4 degrees. After the centrifuge process is completed, the Eppendorf tubes are taken from the centrifuge. Two phases are formed in the tubes. The first phase is the liquid phase (supernatant) at the top, and the second phase is the solid phase (pellet) part under the tube. The liquid part is poured carefully. Care is taken not to lose pellets during the pouring process. The pellet may sometimes not be in solid state, so the removal of the liquid part should be done carefully.
- 1 ml of 70% ethyl alcohol is added to the tubes where the liquid phase is removed. The pellets in the tubes are carefully floated in the liquid part with the help of alcohol. While the pellet is separated from the tube and floated in the liquid, harsh mixing operations should be avoided. Hard mixing can degrade RNA samples. After alcohol addition, tubes are centrifuged at +4 degrees for 5 minutes at 150000 g. Alcohol in the tubes is removed after centrifugation. Care should be taken not to lose pellets during alcohol removal.
- 750pl of Extraction 2 solution, pre-heated at 65 degrees, is added to the tubes purified from alcohol. The tubes are mixed gently and softly so that the pellets in the tubes float in the added liquid part. The tubes in which the pellet floats in the liquid part are placed in a 65-degree heat block and left to incubate for 15 minutes. The tubes are gently mixed every 5 minutes during the incubation step. After the incubation, 750pl chloroform: isoamylalcohol (24: 1 v/v) is added to the tubes. Tubes are gently mixed. After the mixing process, the tubes are centrifuged. Centrifugation is performed at +4 degrees for 30 minutes at 20000g
- After the centrifugation process, the tubes are taken from the centrifuge. After centrifugation, two phases are formed in the tubes. The light-colored liquid part in the upper part is taken and transferred to the clean 1.5 ml Eppendorf tube. 100 pl of 3M NaOAc (Sodium acetate, pH: 5.2) solution is added onto the transferred liquid part. 1 ml of 2-Propanol kept at -20 degrees is added to the same tube and the tubes are mixed gently. After mixing, the tubes are incubated for a minimum of 4 hours at -20 degrees. Extending the time will allow further precipitation of the RNA samples in the liquid.
- Tubes taken from the incubation are centrifuged at +4 degrees for 40 minutes at 20000g. After centrifugation, the liquid part in the tube is poured and the solid part at the bottom of the tube is left. 1 ml of 96% ethyl alcohol is added to the tube and the tubes are gently mixed. Tubes are centrifuged at +4 degrees for 20 minutes at 20000g. After centrifugation, the alcohol in the tubes is poured. Care is taken not to lose the pellet during alcohol removal. 70% ethyl alcohol preserved at 1 ml -20 degrees is added to the tubes and the tubes are centrifuged. Tubes are centrifuged at +4 degrees for 20 minutes at 20000g. After centrifugation, alcohol in the tubes is carefully poured. The pellet is let to dry so that the alcohol is removed from the pellet. The drying process can be done in an open fume hood, speed vacuum device or at room temperature.
- After making sure that the tubes and pellets are well dried, 50pl of T: E (Tris I EDTA, pH: 8) solution or autoclaved distilled water can be added to the isolated RNA samples to dissolve the samples. During the thawing process, one should be very careful and avoid hard movements to avoid degrading RNA samples.
- After the thawing process, the samples should be stored at - 80 degrees to be used in molecular studies.
Determining the Quality and Concentration of the Samples
It is very important that the quality and concentration of the isolated RNA samples match certain parameters. The quality, purity levels, and concentrations of the isolated RNA samples are not suitable for certain parameters, causing the isolated RNA samples to be unusable. Confirmation of 28S rRNA and 18S rRNA bands of RNA samples in an agarose gel is among the quality parameters. The values of the 260/280 and 260/230 parameters of the RNA samples isolated in spectrophotometric measurements should be between 1.8-2.2. It is understood that RNA samples with spectrophotometric values of 1.8-2.2 have appropriate purity values in terms of secondary metabolite and protein content.
RNA samples isolated using the RNA isolation protocol we presented were loaded on agarose gel with a concentration of 1 .5%. 5pl of isolated RNA samples were taken and transferred to 0.2 ml eppendorf tubes. 1 pl of loading dye was added to the eppendorf tube containing the RNA sample and the prepared mixture was loaded on agarose gel. The agarose gel on which the samples were loaded was subjected to electrophoresis at 100 volts for 60 minutes. The 18S rRNA and 28S RNA bands of the
RNA samples were confirmed in the gel imaged under UV light after electrophoresis. No damage, fracture, or degradation has not observed in the 18S rRNA and 28S rRNA bands displayed on the gel. In addition, it was confirmed that the values obtained after spectrophotometric measurements of the samples were in accordance with the quality parameters and that the spectrophotometric values of the RNA samples were in accordance with 1 .8-2.2 values. As a result, the method has been applied successfully.
The method modified in our invention is for the isolation of total RNA from herbal tissue with high secondary metabolite content. Although it has been used in pomegranate peels, RNA isolation is a method that can be used in other problematic plant species. As a result; The RNA isolation method, which we have developed by modifying different RNA isolation methods, is recommended as a successful method in the isolation of pure, high quality and sufficient amount of RNA for plant species with high phenolic and secondary compounds such as pomegranate plant in plant molecular biology studies.
Claims
CLAIMS A modified method for the isolation of high quality and pure RNA from pomegranate, characterized by the steps below;
- Add 5 mercaptoethanol up to 3% of the solution volume to the extraction 1 solution and allowing the prepared mixture to incubate for 15 minutes at 65 degrees,
- Grind the pomegranate peel tissues to be used in insulation with the help of liquid nitrogen in a porcelain mortar until the samples become powdery,
- Transfer to 100 mg of powdery pomegranate peel tissue into 2 mL eppendorf tubes,
- Add 150 mg of PVP into the tube,
- Add 1 .5 ml of from Extraction 1 solution that was previously incubated at 65 degrees to the tube,
Mix rapidly the eppendorf tube containing tissue, PVP, and solution with the help of vortex,
- Let the mixed tube to a heat block or water bath at 65 degrees to incubate for 30-40 minutes, mix the samples every 5 minutes with the help of a vortex,
- Centrifuge the samples at +4 degrees for 30 minutes at 20000g at the end of the incubation period,
- Take the light colored and homogeneous liquid phase out of the 3 phases formed in the tubes after the centrifuge with the help of a 1000 pl automatic pipette and transfert to a new 2 ml eppendorf tube,
- Transfer 1000 pl of chloroform: isoamylalcoholl (24:1 v/v) which stored at - 20 degrees to the liquid part taken into the tube,
- Centrifuge at 20000 g for 30 minutes at +4 degrees,
- Renewal of the chloroform: isoamylalcohol step until the upper liquid (supernatant) part in two different phases to be formed in the tubes becomes clear and clear,
- When the upper phase is clear and clean, it should be carefully taken with an automatic pipette and transferred to a clean 1.5 ml eppendorf tube,
- Add approximately 350-400pl of LiCI (Lithium chloride) solution stored at +4 degrees and transfer to the eppendorf tube,
- Stir the tube gently and leave the tube to incubate at +4 degrees overnight after mixing,
- Centrifuge the samples taken from incubation at +4 degrees for 30 minutes at 20000 g,
- The upper liquid first phase (supernatant) of the two phases formed in the tubes is carefully poured without losing the pellet,
- Add 1 ml of ethyl alcohol with 70% concentration to the tubes where the liquid phase is removed,
- Floating the pellets in the tubes carefully in the liquid part with the help of alcohol,
- Centrifuge the tubes at +4 degrees for 5 minutes at 150000 g,
- Removal of alcohol in tubes after centrifugation,
- Add 750pl of the extraction solution pre-heated at 65 degrees to the tubes that are purified from alcohol,
- Lightly and softly mixing of the tubes so that the pellets in the tubes float in the added liquid part,
- Take the tubes in which the pellet floats in the liquid part to the heat block at 65 degrees and leave it to incubate for 15 minutes,
- Slightly mix the tubes every 5 minutes during the incubation step,
- After the incubation step, add 750pl chloroform: isoamylalcohol (24: 1 v / v) to the tubes and mixing the tubes slightly,
- Centrifuge the tubes at +4 degrees for 30 minutes at 20000 g after mixing,
- From the two phases formed in the tubes after the centrifuge process, the light colored liquid part in the upper part is taken and transferred to the clean 1.5 ml eppendorf tube,
- Add 100pl 3M NaOAc (Sodium acetate, pH: 5.2) solution onto the transferred liquid part,
- Adding 1 ml of 2-Propanol preserved at -20 degrees to the same tube and mixing the tubes slightly,
- Allow the tubes to incubate at -20 degrees for a minimum of 4 hours,
- Centrifuge the tubes taken from incubation at +4 degrees for 40 minutes at 20000g,
- After the centrifugation process, the liquid part in the tube is poured and the solid part is left at the bottom of the tube,
- Adding 1 ml of 96% ethyl alcohol to the tube and mixing the tubes slightly,
- Centrifuge the tubes at +4 degrees for 20 minutes at 20000g and pouring the alcohol in the tubes,
- Add 70% ethyl alcohol kept at 1 ml -20 degrees to the tubes and centrifuging the tubes,
- Centrifuge the tubes at +4 degrees for 20 minutes at 20000g, and then pour the alcohol in the tubes carefully,
- Leaving the tubes open to dry, at speed-vacuum device or room temperature to remove alcohol from the pellet.
2. The extraction 1 mentioned in claim 1 , characterized by comprising 2% CTAB (Cetyltrimethylammonium bromide), 25 mM EDTA (Ethylenediamine tetraacetic acid), 100 mM Tris-HCI (Tris-hydrochloric acid, pH: 8), 2 M NaCI (Sodium chloride), 1.00 g / L Spermidine, 2% PVP (Polyvinylpyrrolidone).
3. The extraction 1 mentioned in claim 2, characterized by being sterilized by autoclaving at 121 degrees at 1.05 atm pressure for 15 minutes.
4. The extraction 2 mentioned in claim 1 , characterized by comprising chemicals 1 M NaCI (Sodium dodecyl sulfate), 25 10 mM Tris-HCI (Tris-Hydrochloric acid, pH: 8), 1 mM EDTA (Ethylenediamine tetraacetic acid, pH: 8), 0.5% SDS (Sodium dodecylsulfate).
5. The extraction 2 mentioned in claim 4, characterized in that it has been sterilized by autoclaving at 121 degrees at 1 .05 atm pressure for 15 minutes.
6. The chloroform: isoamylalcohol solution mentioned in Claim 1 characterized in that being prepared by mixing with 24 units of chloroform and 1 unit of isoamylalcohol.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| TR2020/21063A TR202021063A2 (en) | 2020-12-21 | 2020-12-21 | MODIFIED METHOD FOR NARDAN QUALITY AND PURE RNA ISOLATION |
| TR2020/21063 | 2020-12-21 |
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| WO2022139709A1 true WO2022139709A1 (en) | 2022-06-30 |
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| PCT/TR2020/051496 WO2022139709A1 (en) | 2020-12-21 | 2020-12-31 | Modified method for high quality and pure rna isolation from pomegranate |
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| Country | Link |
|---|---|
| TR (1) | TR202021063A2 (en) |
| WO (1) | WO2022139709A1 (en) |
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2020
- 2020-12-21 TR TR2020/21063A patent/TR202021063A2/en unknown
- 2020-12-31 WO PCT/TR2020/051496 patent/WO2022139709A1/en active Application Filing
Non-Patent Citations (2)
| Title |
|---|
| ELÇI EMINUR, GAZEL MONA, ÇAĞLAYAN KADRIYE: "Asma ve Narlardan İzole Edilen Grapevine leafroll-associated virus-1 İzolatlarının Kısmi Sekanslarının Karşılaştırmalı Genomik Analizleri", TURKISH JOURNAL OF AGRICULTURE - FOOD SCIENCE AND TECHNOLOGY, vol. 5, no. 10, 1 October 2017 (2017-10-01), pages 1136 - 1141, XP055954442, DOI: 10.24925/turjaf.v5i10.1136-1141.1202 * |
| ZAREI ABDOLKARIM, MOUSAVI AMIR, FATAHI REZA: "An Effective Protocol for Isolation of High-Quality RNA from Pomegranate Seeds", THE ASIAN AND AUSTRALASIAN JOURNAL OF PLANT SCIENCE AND BIOTECHNOLOGY, 1 November 2012 (2012-11-01), pages 32 - 37, XP055954441, [retrieved on 20220824] * |
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