Method and kit for extracting total RNA (Ribonucleic Acid) of fern rhizome

Abstract

The invention relates to the technical field of nucleic acid extraction, in particular to a method and a kit for extracting total RNA (Ribonucleic Acid) of fern rhizomes. The method comprises the following steps: grinding a pteridophyte rhizome sample, dissolving sample powder by using a reagent I, beta-mercaptoethanol and spermidine in a combined and matched manner, treating by using a first protein denaturant, precipitating by using lithium chloride to obtain a precipitate containing RNA, and further dissolving by using a reagent II to obtain the RNA-containing precipitate. And treating with a second protein denaturant and precipitating and purifying with an alcohol solvent to obtain the total RNA of the fern rhizome. The method is easy to implement, high-quality total RNA of the fern rhizomes can be obtained, and a good basis is provided for subsequent molecular biology related research.

Description

Technical Field

[0001] The present application belongs to the field of nucleic acid extraction technology, and in particular relates to a method and a kit for extracting total RNA from fern rhizomes. Background Art

[0002] Ferns are the second-largest group of vascular plants on Earth, second only to seed plants in species richness. They are a key plant group for studying the origin and evolution of genes and organs in Earth's plants. With the exception of species like the Alsophilaceae and Pteridaceae, which have tall, tree-like, upright stems, most ferns have root-like stems, either slender, horizontal or short, upright. These stems are called rhizomes. The rhizome of a fern is composed of thin-walled cells, a cortex, and a stele. The rhizome is a perennial organ that continuously elongates and grows along the apical meristem of the stem. The rhizomes of ferns have important medicinal and edible value. The perennial rhizomes of some ferns have become famous traditional Chinese medicinal materials because they contain natural active ingredients, such as Cibotium barometz (L.) J.Sm., Drynaria fortunei (Kunze) J.Sm., Dryopteris crassirhizoma Nakai, and Osmunda japonica Thunb.; the rhizomes of plants of the genus Achyranthes are rich in starch and are edible; the rhizomes of plants of the Pteridaceae family are often used to make fern root powder.

[0003] Because the rhizomes of ferns are rich in secondary metabolites and polysaccharides, it is difficult to obtain large amounts of high-quality plant total RNA using existing RNA extraction reagents, making it difficult to carry out subsequent molecular biology-related research. Summary of the Invention

[0004] The purpose of this application is to provide a method and a kit for extracting total RNA from fern rhizomes, aiming to solve the technical problem of how to extract high-quality total RNA from fern stems.

[0005] To achieve the above application objectives, the technical solutions adopted in this application are as follows:

[0006] In a first aspect, the present application provides a method for extracting total RNA from fern rhizomes, comprising:

[0007] Provide fern rhizome samples;

[0008] Grinding the fern rhizome sample into a powder and then dissolving it in a mixture with a reagent I, β-mercaptoethanol, and spermidine to obtain a first dissolving solution; wherein the reagent I contains hexadecyltrimethylammonium bromide, polyvinylpyrrolidone, sodium chloride, tris(hydroxymethyl)aminomethane hydrochloride, and ethylenediaminetetraacetic acid;

[0009] The first lysate is mixed with a first protein denaturant and then subjected to a first centrifugation to obtain a first supernatant; the first supernatant is then mixed with lithium chloride and then subjected to a second centrifugation to obtain a precipitate;

[0010] The precipitate is mixed and dissolved with a reagent II to obtain a second dissolution solution; wherein the reagent II contains sodium lauryl sulfate, sodium chloride, tris(hydroxymethyl)aminomethane hydrochloride and ethylenediaminetetraacetic acid;

[0011] The second dissolving liquid is mixed with a second protein denaturant and then subjected to a third centrifugation to obtain a second supernatant. The second supernatant is then mixed with an alcohol solvent and subjected to a fourth centrifugation to obtain total RNA from fern rhizomes.

[0012] In a second aspect, the present application provides a kit for extracting total RNA from fern rhizomes, comprising:

[0013] Reagent I, Reagent II, β-mercaptoethanol, spermidine, lithium chloride, protein denaturant and alcohol solvent;

[0014] in,

[0015] The reagent I contains: hexadecyltrimethylammonium bromide, polyvinylpyrrolidone, sodium chloride, tris(hydroxymethyl)aminomethane hydrochloride and ethylenediaminetetraacetic acid;

[0016] The reagent II contains sodium lauryl sulfate, sodium chloride, tris(hydroxymethylaminomethane) hydrochloride and ethylenediaminetetraacetic acid.

[0017] The method provided in the first aspect of the present application is to first combine reagent I with β-mercaptoethanol and spermidine to dissolve the fern rhizome sample powder. Based on the above combination, not only can the plant cells be lysed and the intracellular substances released be promoted, but the RNA structure can also be well stabilized, significantly reducing its risk of degradation. After treatment with a first protein denaturant and lithium chloride precipitation, an RNA-containing precipitate can be obtained, which is then further dissolved by reagent II, treated with a second protein denaturant, and purified by alcohol solvent precipitation. The method of the present application can obtain high-quality fern rhizome total RNA, providing a good foundation for subsequent molecular biology related research.

[0018] The kit provided in the second aspect of the present application includes reagent I, reagent II, β-mercaptoethanol, spermidine, lithium chloride, a protein denaturant and an alcohol solvent, wherein reagent I and reagent II contain certain specific components, based on the combination of reagent I and β-mercaptoethanol and spermidine, it can be used to dissolve and extract the fern rhizome sample first, and then the precipitate obtained by protein denaturant treatment and lithium chloride precipitation can be dissolved with reagent II, and further treated with a protein denaturant and precipitated with an alcohol solvent, ultimately achieving the extraction of total RNA from the fern rhizome. Based on the use of each reagent in the kit of the embodiment of the present application, high-quality total RNA can be extracted from the fern rhizome. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments or descriptions of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0020] Figure 1 This is an agarose gel electrophoresis diagram of total RNA obtained by the method for extracting total RNA from fern rhizomes in an embodiment of the present application (electrophoresis conditions: 1% agarose, 180V, 10 minutes); wherein, 1: DNA marker, with lengths of 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp, respectively; 2: Cibotium barometz; 3: Drynaria roosii; 4: Pyrrosia sheareri; 5: Dryopteris crassirhizoma; 6: Osmunda japonica; 7: Angiopteris fokiensis; 8: Sphaeropteris lepifera; 9: tobacco leaves (for control).

[0021] Figure 2 This is an agarose gel electrophoresis diagram of total RNA extracted from fern rhizomes using the method of this application and compared with other commercially available plant total RNA extraction kits (electrophoresis conditions: 1% agarose, 180V, 10 minutes); A: young leaves of tobacco; B: rhizomes of Cibotium barometz; 1 and 6: TRIzol TM Reagents (Invitrogen TM) extraction results; 2 and 7: Plant RNA Extraction Kit (Polysaccharide and Polyphenol) (Beijing Quanshijin Biotechnology Co., Ltd.) extraction results; 3 and 8: Enhanced Plant RNA Extraction Kit (Beijing Polymer Biotechnology Co., Ltd.) extraction results; 4 and 9: Magnetic Bead Total RNA Extraction Kit (Beijing Quanshijin Biotechnology Co., Ltd.) extraction results; 5: Extraction results of the method of the embodiment of the present application; 10: DNA marker, with lengths of 5000bp, 3000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp and 100bp, respectively. DETAILED DESCRIPTION

[0022] In order to make the technical problems, technical solutions and beneficial effects to be solved by this application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit this application.

[0023] In this application, the term "and / or" describes the relationship between associated objects, indicating that three possible relationships exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0024] In this application, "at least one" means one or more, "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0025] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution. Some or all of the steps can be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0026] The terms used in the embodiments of the present application are for the purpose of describing specific embodiments only and are not intended to limit the present application. The singular forms "a", "an", "the" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms unless the context clearly indicates otherwise.

[0027] The weights of the relevant components mentioned in the examples of this application may not only refer to the specific content of each component, but also represent the weight ratio between the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the examples of this application, it is within the scope disclosed in the examples of this application. Specifically, the mass described in the examples of this application may be a mass unit known in the chemical industry, such as μg, mg, g, kg, etc.

[0028] The terms "first" and "second" are used solely for descriptive purposes to distinguish objects, such as substances, from one another and should not be understood to indicate or imply relative importance or to implicitly specify the quantity of the technical features being referred to. For example, without departing from the scope of the embodiments of this application, a first XX may also be referred to as a second XX, and similarly, a second XX may also be referred to as a first XX. Thus, features defined as "first" or "second" may explicitly or implicitly include one or more of such features.

[0029] The fern rhizome mentioned in the embodiments of the present application refers to the plant's nutritional organ that plays a role in conducting, supporting and extending between the roots and leaves of the fern.

[0030] The rhizomes of ferns are rich in substances such as secondary metabolites and polysaccharides. It is difficult to obtain a large amount of high-quality plant total RNA using existing RNA extraction reagents, so it is difficult to carry out molecular biology related research. At present, there are no relevant literature related to research reports on methods for extracting total RNA from tissues such as fern rhizomes. Therefore, the present application embodiment is based on the mutual combination of different reagents, focusing on aspects such as cell lysis, removal of polysaccharides and proteins, inhibition of RNase and DNase activity, specific precipitation RNA, and inhibition of oxidation. Through steps such as unique lysis-phase separation-specific precipitation-impurity removal-reprecipitation, a relatively large amount of high-quality fern rhizome total RNA is finally obtained. By comparing with commercially available multiple RNA extraction kits, this method can be used to extract high-quality total RNA from the rhizomes (or stem trunks) of ferns such as golden dog, oak fern, and pen holder tree, and the extraction effect is significantly better than commercially available kits. The specific technical solution is as follows.

[0031] In a first aspect, the embodiments of the present application provide a method for extracting total RNA from fern rhizomes.

[0032] Specifically, the method of the embodiment of the present application includes the following steps:

[0033] S01: Provide fern rhizome samples;

[0034] S02: Grinding a fern rhizome sample into a powder and then dissolving the powder in a mixture with reagent I, β-mercaptoethanol, and spermidine to obtain a first dissolving solution; wherein reagent I contains cetyltrimethylammonium bromide, polyvinylpyrrolidone, sodium chloride, tris(hydroxymethyl)aminomethane hydrochloride, and ethylenediaminetetraacetic acid;

[0035] S03: mixing the first dissolving solution with a first protein denaturant and performing a first centrifugation to obtain a first supernatant, then mixing the first supernatant with lithium chloride and performing a second centrifugation to obtain a precipitate;

[0036] S04: mixing and dissolving the precipitate with reagent II to obtain a second dissolving solution; wherein reagent II contains sodium lauryl sulfate, sodium chloride, tris(hydroxymethyl)aminomethane hydrochloride, and ethylenediaminetetraacetic acid;

[0037] S05: The second dissolving solution is mixed with a second protein denaturant and subjected to a third centrifugation to obtain a second supernatant. The second supernatant is then mixed with an alcohol solvent and subjected to a fourth centrifugation to obtain total RNA from fern rhizomes.

[0038] Specifically, the reagents used in the above method act as follows:

[0039] In Reagent I: Cetyltrimethylammonium bromide (CTAB) as a cationic detergent can lyse plant cells and cell nuclei, promote the release of cellular contents, dissolve proteins on ribosomes, release nucleic acids, and remove polysaccharides from the surrounding environment; polyvinylpyrrolidone (PVP-K40) can remove polyphenol compounds and reduce the risk of sample oxidation, thereby improving RNA yield and quality; sodium chloride (NaCl) provides a high-salt environment, thereby increasing RNA solubility and promoting polysaccharide precipitation; tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) can both promote cell lysis and inhibit RNase activity; ethylenediaminetetraacetic acid (EDTA) can inhibit the activity of nucleases such as RNase, reducing the risk of RNA degradation.

[0040] In Reagent II, sodium dodecyl sulfate (SDS) acts as a protein detergent, disrupting cell walls and disrupting nucleic acid:protein complexes. The roles of sodium chloride (NaCl), tris-HCl (Tris-HCl), and ethylenediaminetetraacetic acid (EDTA) are discussed above.

[0041] β-Mercaptoethanol: It can inhibit the activity of nucleases such as RNase and reduce the risk of RNA degradation.

[0042] Spermidine: It can stabilize RNA structure, bind to RNA to reduce its risk of degradation, and form complexes with polyphenols to reduce the risk of polyphenols damaging RNA.

[0043] The first protein denaturant and the second protein denaturant can destroy the secondary structure of the protein and precipitate it, thereby separating the protein from the nucleic acid solution.

[0044] Lithium chloride: can selectively bind to RNA molecules in solution and precipitate them.

[0045] Alcohol solvent: used for precipitation and cleaning of nucleic acids.

[0046] Therefore, based on the effects of the above-mentioned reagents, the embodiment of the present application first combines reagent I with β-mercaptoethanol and spermidine to dissolve the fern rhizome sample powder. Based on the above-mentioned combination, not only can the fern rhizome plant cells be lysed and the release of intracellular substances be promoted, but the RNA structure can also be well stabilized, significantly reducing the risk of degradation, and separating substances such as polysaccharides to improve the purity. The first dissolved solution obtained by dissolving is mixed with a first protein denaturant and then subjected to a first centrifugation treatment, the first supernatant obtained is then mixed with lithium chloride and subjected to a second centrifugation treatment, and a precipitate containing RNA is obtained by phase separation-specific precipitation; further, in order to improve the quality of RNA, the precipitate is further dissolved with reagent II, the second dissolved solution obtained by dissolving is mixed with a second protein denaturant and then subjected to a second centrifugation treatment, and the second supernatant obtained is purified by precipitation with an alcohol solvent, and finally high-quality fern rhizome total RNA can be obtained.

[0047] In step S01, the fern rhizome samples provided may be various fern rhizome samples, such as Cibotium barometz, Drynaria roosii, Pyrrosia sheareri, Dryopteris crassirhizoma, Osmunda japonica, Angiopteris fokiensis, Sphaeropteris lepifera, etc.

[0048] In step S02, the fern rhizome sample may be ground by grinding with liquid nitrogen for 3-5 times.

[0049] In some embodiments, the ratio of the fern rhizome sample to the reagent I, β-mercaptoethanol, and spermidine is 0.2-0.3 g: 0.8 ml: 8 μl: 1 μl. Within this ratio range, fern cells can be better lysed.

[0050] In some embodiments, the concentrations of the components in reagent I are: cetyltrimethylammonium bromide mass volume percentage concentration is 1.8-2.3%, polyvinylpyrrolidone mass volume percentage concentration is 2.8-3.3%, sodium chloride molar concentration is 1.8-2.3M, tris(hydroxymethyl)aminomethane hydrochloride molar concentration is 180-230mM, and ethylenediaminetetraacetic acid molar concentration is 23-28mM.

[0051] Step S03 is a phase separation-specific precipitation process.

[0052] In some embodiments, the first protein denaturant is selected from a chloroform-isoamyl alcohol mixed solvent; chloroform, as a protein denaturant, can destroy the secondary structure of the protein and precipitate it, thereby separating the protein from the solution, while isoamyl alcohol helps enhance this effect while reducing the foam generated during the operation.

[0053] Specifically, the chloroform-isoamyl alcohol mixed solvent may be a mixed solvent consisting of chloroform and isoamyl alcohol in a volume ratio of 22 to 26: 1. The first dissolving solution and the first protein denaturant may be mixed in an equal volume ratio.

[0054] In some embodiments, the step of mixing the first lysate with the first protein denaturant and then performing a first centrifugation comprises: centrifuging at 11,000-13,000 rpm and 4-6° C. for 10-20 min.

[0055] In some embodiments, after the obtained first supernatant is mixed with lithium chloride, the final concentration of lithium chloride is 2.5-3.5M.

[0056] In some embodiments, the step of mixing the first supernatant with lithium chloride and then performing a second centrifugation comprises: centrifuging at 11,000-13,000 rpm and 4-6° C. for 30-40 min.

[0057] In step S04, the reagent II further dissolves the precipitate.

[0058] In some embodiments, the concentrations of the components in Reagent II are as follows: sodium lauryl sulfate mass volume percentage concentration is 0.8-1.3%, sodium chloride molar concentration is 1.2-1.5M, tris(hydroxymethyl)aminomethane hydrochloride molar concentration is 200-250mM, and ethylenediaminetetraacetic acid molar concentration is 1.0-1.5mM.

[0059] Step S05 is a process of impurity removal and reprecipitation cleaning.

[0060] In some embodiments, the second protein denaturant is selected from a chloroform-isoamyl alcohol mixed solvent; specifically, it can be a mixed solvent consisting of chloroform and isoamyl alcohol in a volume ratio of 22 to 26:1. The second dissolving solution and the second protein denaturant can be mixed in an equal volume ratio.

[0061] In some embodiments, the step of mixing the second lysate with the second protein denaturant and then performing a third centrifugation comprises: centrifuging at 11,000-13,000 rpm and 4-6° C. for 10-20 min.

[0062] In some embodiments, the alcohol solvent is selected from isopropanol and ethanol. The ethanol can be a 75-85% ethanol-water solution. Isopropanol can be used for precipitation first, and then ethanol can be used for further washing.

[0063] In some embodiments, the step of mixing the second supernatant with an alcohol solvent for a fourth centrifugation comprises: first mixing the second supernatant with isopropanol and centrifuging at 11,000-13,000 rpm and 4-6° C. for 30-40 min, and then mixing the resulting precipitate with an ethanol solution and centrifuging at 11,000-13,000 rpm and 4-6° C. for 10-20 min.

[0064] In some embodiments, the obtained total RNA from the fern rhizome is dissolved in TE buffer for storage. The TE buffer is a buffer prepared from Tris-HCl and EDTA.

[0065] In a second aspect, embodiments of the present application provide a kit. Specifically, the kit is a kit for extracting total RNA from a fern rhizome, comprising: (1) Reagent I, which contains: hexadecyltrimethylammonium bromide, polyvinylpyrrolidone (PVP-K40), sodium chloride, tris(hydroxymethylaminomethane) hydrochloride, and ethylenediaminetetraacetic acid; (2) Reagent II, which contains: sodium lauryl sulfate, sodium chloride, tris(hydroxymethylaminomethane) hydrochloride, and ethylenediaminetetraacetic acid; (3) β-mercaptoethanol; (4) spermidine; (5) lithium chloride; (6) a protein denaturant; and (7) an alcohol solvent.

[0066] In the kit of the present invention, the combination of reagent I, β-mercaptoethanol, and spermidine can be used to dissolve and extract a fern rhizome sample. The dissolved solution can then be treated with a protein denaturant and precipitated with lithium chloride to obtain a precipitate. Reagent II can then dissolve the precipitate, which is then further treated with a protein denaturant and precipitated with an alcohol solvent to ultimately obtain fern rhizome total RNA. Based on the combination of the reagents, high-quality total RNA can be extracted from fern rhizomes.

[0067] In some embodiments, the concentrations of the components in reagent I in the kit are as follows: cetyltrimethylammonium bromide mass volume percentage concentration is 1.8-2.3%, polyvinylpyrrolidone mass volume percentage concentration is 2.8-3.3%, sodium chloride molar concentration is 1.8-2.3M, tris(hydroxymethyl)aminomethane hydrochloride molar concentration is 180-230mM, and ethylenediaminetetraacetic acid molar concentration is 23-28mM.

[0068] In some embodiments, the concentrations of the components of Reagent II in the kit are as follows: sodium lauryl sulfate mass volume percentage concentration is 0.8-1.3%, sodium chloride molar concentration is 1.2-1.5M, tris(hydroxymethyl)aminomethane hydrochloride molar concentration is 200-250mM, and ethylenediaminetetraacetic acid molar concentration is 1.0-1.5mM.

[0069] In some embodiments, the protein denaturant in the kit is selected from a chloroform-isoamyl alcohol mixed solvent; specifically, it can be a mixed solvent consisting of chloroform and isoamyl alcohol in a volume ratio of 22 to 26:1.

[0070] In some embodiments, the alcohol solvent in the kit is selected from isopropanol and ethanol, wherein the ethanol can be a 75-85% ethanol aqueous solution. Isopropanol can be used for precipitation first, and then ethanol can be used for further washing.

[0071] In some embodiments, the kit further comprises TE buffer, which is a buffer prepared from Tris-HCl and EDTA.

[0072] The following is an explanation with reference to specific examples, wherein the mass volume percentage concentration is the percentage (%) of the mass of the solid substance (g) and the volume of the solution (mL), the molar concentration M is mol / L, and normal temperature refers to 24-30°C.

[0073] Example 1

[0074] 1. Reagents

[0075] 1) Reagent I, containing: 2% by mass volume of cetyltrimethylammonium bromide (CTAB), 3% by mass volume of polyvinylpyrrolidone (PVP-K40), 2 M sodium chloride (NaCl), 200 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl, pH = 8.0), and 25 mM ethylenediaminetetraacetic acid (EDTA, pH = 8.0); the remaining solvent is DEPC-treated ddH2O.

[0076] 2) Reagent II, containing: sodium dodecyl sulfate (SDS) at a mass volume percentage concentration of 1%, sodium chloride (NaCl) at a molar concentration of 1 M, tris (hydroxymethyl)aminomethane hydrochloride (Tris-HCl, pH = 8.0) at a molar concentration of 200 mM, and ethylenediaminetetraacetic acid (EDTA, pH = 8.0) at a molar concentration of 1 mM; the remaining solvent is DEPC-treated ddH2O.

[0077] 3) Other reagents

[0078] 3.1) β-mercaptoethanol (2-ME or β-mercaptoethanol), working concentration (V / V) 1%.

[0079] 3.2) Spermidine.

[0080] 3.3) Lithium chloride (LiCl) solution; wherein the molar concentration of LiCl is 10 M.

[0081] 3.4) Chloroform-isoamyl alcohol mixed solvent; wherein the volume ratio of chloroform:isoamyl alcohol (V / V) is 24:1.

[0082] 3.5) Isopropyl alcohol: volume fraction is 100%.

[0083] 3.6) Ethanol solution; wherein the volume fraction (V / V) of ethanol is 75%.

[0084] 3.7) Double distilled water (ddH2O) treated with diethyl pyrocarbonate (DEPC).

[0085] 3.8) TE buffer; containing 10 mM Tris-HCl and 1 mM EDTA, pH = 8.0.

[0086] 2. Reagent Preparation Method

[0087] 1) Diethylpyrocarbonate (DEPC)-treated ddH2O: Add 1 mL of DEPC (molecular weight 162.14) to 1 L of ddH2O, mix thoroughly, and let stand in a fume hood for 8-12 hours. Autoclave at 120°C for 20 minutes, let cool, and store refrigerated.

[0088] 2) EDTA stock solution (0.5 M, pH = 8.0): Weigh 73.06 g of EDTA (molecular weight 292.24) and completely dissolve it in 450 ml of DEPC-treated ddH2O. Adjust the pH to 8.0 with solid sodium hydroxide (NaOH) and finally make up to 500 ml. Refrigerate.

[0089] 3) Tris-HCl stock solution (1 M, pH = 8.0): Weigh 60.57 g of Tris (molecular weight 121.14) and completely dissolve it in 450 ml of DEPC-treated ddH2O. Adjust the pH to 8.0 with concentrated hydrochloric acid and finally make up to 500 ml. Refrigerate.

[0090] 4) LiCl Solution (10M): Weigh 21.20 g of anhydrous lithium chloride (molecular weight 42.39) and dissolve completely in DEPC-treated ddH2O. Dilution will release heat. Allow to cool and store at -20°C.

[0091] 5) Chloroform-isoamyl alcohol mixed solvent (chloroform:isoamyl alcohol = 24:1): Place 96 ml of chloroform in a reagent bottle. Use a pipette to add 4 ml of isoamyl alcohol. Tighten the cap and gently shake to mix. Refrigerate.

[0092] 6) Ethanol solution (75%): Measure 75 ml of anhydrous ethanol and 25 ml of DEPC-treated ddH2O, place in a reagent bottle, tighten the lid, and shake gently to mix. Store in a refrigerator.

[0093] 7) TE buffer: Mix all components according to the ratio in Table 1. Autoclave at 121°C for 20 min, let cool, and store at room temperature.

[0094] Table 1

[0095] Components volume Tris-HCl mother solution (1 M, pH = 8.0) 5ml EDTA stock solution (0.5M, pH=8.0) 1ml <![CDATA[DEPC-treated ddH2O]]> Adjust the volume to 500 ml

[0096] 8) Reagent I: Mix all components according to the ratios in Table 2, autoclave at 121°C for 20 minutes, and allow to cool until ready for use. Store at room temperature.

[0097] Table 2

[0098] Components 50ml system 100ml system Solid CTAB 1g 2g Solid PVP-K40 1.5g 3.0g Solid NaCl 5.84g 11.69g Tris-HCl mother solution (1 M, pH = 8.0) 10ml 20ml EDTA stock solution (0.5M, pH=8.0) 2.5 ml 5ml <![CDATA[ddH2O treated with DEPC]]> Dilute to a total volume of 50 ml Adjust the volume to 100 ml

[0099] 9) Reagent II: Mix all components according to the ratios in Table 3, autoclave at 120°C for 20 minutes, and allow to cool until ready for use. Store at room temperature.

[0100] Table 3

[0101] Components 50ml system 100ml system Solid SDS 0.5g 1.0g Solid NaCl 2.92g 5.84g Tris-HCl mother solution (1 M, pH = 8.0) 0.5ml 1.0ml EDTA stock solution (0.5M, pH=8.0) 0.1ml 0.2ml <![CDATA[ddH2O (DEPC-treated)]]> Dilute to a total volume of 50 ml Adjust the volume to 100 ml

[0102] 3. Preparation of Consumables

[0103] 1) Sterilize the mortar (hammer) and medicine spoon at 200℃ for 10-16 hours, seal with tin foil and set aside.

[0104] 2) RNase-free and DNase-free pipette tips and centrifuge tubes.

[0105] 4. RNA Extraction Steps

[0106] 1) Preheat reagent I to 65°C.

[0107] 2) Grind a fern rhizome sample (0.2-0.3 g) with liquid nitrogen until powdered (grind 3-5 times with liquid nitrogen). Transfer the sample to a 2 ml centrifuge tube and add 0.8 ml of Reagent I, 8 μl of β-mercaptoethanol, and 1 μl of spermidine. Vortex the tube and incubate at 65°C for 10 min, shaking twice.

[0108] 3) After cooling the ice bath to room temperature, add an equal volume of chloroform-isoamyl alcohol mixed solvent (chloroform:isoamyl alcohol = 24:1, which can be pre-cooled at 4°C), invert several times, let it stand and then separate the layers (no plant material is suspended in the upper layer), and then centrifuge at 12000 rpm and 4°C for 10 min (the centrifugation can be repeated once).

[0109] 4) Transfer the supernatant to a new centrifuge tube and add an appropriate volume of 10 M LiCl solution (final LiCl concentration is 2.5 M; see Table 4 for specific ratios). Shake well, centrifuge at 12,000 rpm, 4°C for 30 min, and place on ice.

[0110] Table 4

[0111]

[0112] 5) Discard the supernatant, add 600 μl of Reagent II (preheated at 65°C) to dissolve the precipitate, transfer to a 1.5 ml centrifuge tube, and place on ice to cool to room temperature.

[0113] 6) Add an equal volume of chloroform-isoamyl alcohol mixed solvent (chloroform:isoamyl alcohol = 24:1, which can be pre-cooled at 4°C), shake up and down several times, centrifuge at 12000 rpm, 4°C for 10 min, and place on ice.

[0114] 7) Transfer the supernatant to a new centrifuge tube, add 0.7 volumes of isopropanol (pre-cooled at 4°C), mix thoroughly, and centrifuge at 12,000 rpm at 4°C for 30 minutes. Place on ice.

[0115] 8) Discard the supernatant, add 500 μl of 75% ethanol solution, shake the tube, and centrifuge at 12,000 rpm and 4°C for 5 minutes. Repeat once.

[0116] 9) Centrifuge at 12,000 rpm at 4°C for 30 seconds. Remove any remaining alcohol with a pipette on the workbench. Allow to stand for 5-10 minutes to allow the alcohol to evaporate completely. Dissolve the total RNA in 50 μl of TE buffer and store at -80°C until ready for use.

[0117] Analysis of total RNA extracted from rhizomes of different ferns:

[0118] The total RNA of 7 kinds of fern rhizomes was extracted by the above extraction steps of this embodiment, and young leaves of tobacco were used as control materials. After the extraction, the RNA quality was detected by agarose gel electrophoresis. The results are shown in FIG. Figure 1 The RNA concentration and yield were determined by spectrophotometer. The results are shown in Table 5.

[0119] Table 5

[0120]

[0121] Compared with existing RNA extraction kits:

[0122] Four commonly used plant RNA extraction kits were randomly purchased from the market: TRIzol TM Reagents (Invitrogen TM ), Plant RNA Extraction Kit (Polysaccharide and Polyphenol) (Beijing Quanshijin Biotechnology Co., Ltd.), Enhanced Plant RNA Extraction Kit (Beijing Juhemei Biotechnology Co., Ltd.), and Magnetic Bead Total RNA Extraction Kit (Beijing Quanshijin Biotechnology Co., Ltd.) were used to extract total RNA from the rhizomes of the fern Canis familiaris L., and young leaves of tobacco were used as control materials. The extraction steps were carried out according to the product instructions. After the extraction, the RNA quality was detected by agarose gel electrophoresis. The results are shown in the figure. Figure 2 .

[0123] Depend on Figure 1-2 As shown in Table 5, the method provided in the examples of the present application can extract high-quality total RNA from the rhizomes (or stem trunks) of various ferns, and the extraction effect is significantly better than that of existing kits.

[0124] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present application should be included in the scope of protection of the present application.

Claims

1. A method for extracting total RNA from fern rhizomes, characterized in that: include: Provide fern rhizome samples; Grinding the fern rhizome sample into a powder and then dissolving it in a mixture with a reagent I, β-mercaptoethanol, and spermidine to obtain a first dissolving solution; wherein the reagent I contains hexadecyltrimethylammonium bromide, polyvinylpyrrolidone, sodium chloride, tris(hydroxymethyl)aminomethane hydrochloride, and ethylenediaminetetraacetic acid; The first lysate is mixed with a first protein denaturant and then subjected to a first centrifugation to obtain a first supernatant; the first supernatant is then mixed with lithium chloride and then subjected to a second centrifugation to obtain a precipitate; The precipitate is mixed and dissolved with a reagent II to obtain a second dissolution solution; wherein the reagent II contains sodium lauryl sulfate, sodium chloride, tris(hydroxymethyl)aminomethane hydrochloride and ethylenediaminetetraacetic acid; The second dissolving liquid is mixed with a second protein denaturant and then subjected to a third centrifugation to obtain a second supernatant. The second supernatant is then mixed with an alcohol solvent and subjected to a fourth centrifugation to obtain total RNA from fern rhizomes.

2. The method according to claim 1, wherein The concentrations of the components in the reagent I are as follows: cetyltrimethylammonium bromide mass volume percentage concentration is 1.8-2.3%, polyvinylpyrrolidone mass volume percentage concentration is 2.8-3.3%, sodium chloride molar concentration is 1.8-2.3M, tris (hydroxymethyl)aminomethane hydrochloride molar concentration is 180-230mM, ethylenediaminetetraacetic acid molar concentration is 23-28mM; And / or, the concentrations of the components in the reagent II are: sodium lauryl sulfate mass volume percentage concentration of 0.8-1.3%, sodium chloride molar concentration of 1.2-1.5M, tris(hydroxymethyl)aminomethane hydrochloride molar concentration of 200-250mM, and ethylenediaminetetraacetic acid molar concentration of 1.0-1.5mM.

3. The method according to claim 1, wherein The first protein denaturant and the second protein denaturant are selected from a chloroform-isoamyl alcohol mixed solvent; And / or, the alcohol solvent is selected from isopropyl alcohol and ethanol.

4. The method according to claim 1, wherein The ratio of the fern rhizome sample to the reagent I, the β-mercaptoethanol, and the spermidine is 0.2-0.3 g: 0.8 ml: 8 μl: 1 μl; And / or, the final concentration of lithium chloride after mixing the first supernatant with lithium chloride is 2.5-3.5M.

5. The method according to any one of claims 1 to 4, characterized in that The first centrifugation treatment, the second centrifugation treatment, and the third centrifugation treatment steps independently include: centrifugation at 11000-13000 rpm and 4-6° C. for 10-40 minutes.

6. The method according to any one of claims 1 to 4, characterized in that The step of mixing the second supernatant with an alcohol solvent and performing a fourth centrifugation treatment includes: first mixing the second supernatant with isopropanol and centrifuging at 11000-13000 rpm and 4-6° C. for 30-40 minutes, and then mixing the obtained precipitate with an ethanol solution and centrifuging at 11000-13000 rpm and 4-6° C. for 10-20 minutes.

7. The method according to any one of claims 1 to 4, characterized in that Also includes: The obtained total RNA from the fern rhizome was dissolved in TE buffer.

8. A kit for extracting total RNA from fern rhizomes, characterized in that: include: Reagent I, Reagent II, β-mercaptoethanol, spermidine, lithium chloride, protein denaturant and alcohol solvent; in, The reagent I contains: hexadecyltrimethylammonium bromide, polyvinylpyrrolidone, sodium chloride, tris(hydroxymethyl)aminomethane hydrochloride and ethylenediaminetetraacetic acid; The reagent II contains sodium lauryl sulfate, sodium chloride, tris(hydroxymethylaminomethane) hydrochloride and ethylenediaminetetraacetic acid.

9. The kit according to claim 8, wherein The concentrations of the components in the reagent I are as follows: cetyltrimethylammonium bromide mass volume percentage concentration is 1.8-2.3%, polyvinylpyrrolidone mass volume percentage concentration is 2.8-3.3%, sodium chloride molar concentration is 1.8-2.3M, tris (hydroxymethyl)aminomethane hydrochloride molar concentration is 180-230mM, ethylenediaminetetraacetic acid molar concentration is 23-28mM; And / or, the concentrations of the components in the reagent II are: sodium lauryl sulfate mass volume percentage concentration of 0.8-1.3%, sodium chloride molar concentration of 1.2-1.5M, tris(hydroxymethyl)aminomethane hydrochloride molar concentration of 200-250mM, and ethylenediaminetetraacetic acid molar concentration of 1.0-1.5mM.

10. The kit according to claim 8 or 9, wherein The protein denaturant is selected from a chloroform-isoamyl alcohol mixed solvent; and / or, the alcohol solvent is selected from isopropanol and ethanol; And / or, the kit further comprises TE buffer.

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