Kit and method for extracting plant RNA (Ribonucleic Acid) of polysaccharide and polyphenol based on magnetic beads

Abstract

The invention is applicable to the technical field of nucleic acid extraction, and provides a kit and a method for extracting plant RNA (Ribonucleic Acid) of polysaccharide and polyphenol based on magnetic beads, the kit comprises a lysis solution, a composite remover, a functionalized magnetic bead suspension, a stepped washing solution and an eluent; the surface of the functionalized magnetic bead is covalently modified with a poly (N-isopropylacrylamide-co-vinyl imidazole) copolymer brush (having RNA adsorption and temperature / pH response functions) and a phenylboronic acid (PBA) functional group (specifically capturing polyphenol); the pH value of the matched second washing liquid is controlled to be 5.8-6.8, reversible bond breakage of PBA and polyphenol can be triggered, and selective removal of polyphenol is achieved; meanwhile, the lysate and the composite remover have a synergistic effect, polyphenol oxidation is inhibited from the source, and polysaccharide is precipitated; according to the method disclosed by the invention, the problems of low purity and poor yield in extraction of the RNA of the high-polysaccharide polyphenol plant are solved through a synergistic strategy of'source inhibition-synchronous capture-accurate sorting '.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid extraction technology, and particularly relates to a kit and method for extracting polysaccharide and polyphenol plant RNA based on magnetic beads. Background Technology

[0002] Obtaining high-quality total RNA from plants is a prerequisite for molecular biology research such as gene expression analysis and transcriptome sequencing. However, many plant tissues with significant economic or scientific value, such as tea leaves, pine needles, mature fruits (mangoes, grapes), tubers, and various traditional Chinese medicinal materials, are rich in polysaccharides, polyphenols, pigments, and other secondary metabolites, posing a significant challenge to RNA extraction. Polysaccharides co-precipitate with RNA during alcohol precipitation, forming a viscous gel that severely affects RNA solubility and downstream enzyme activity. Polyphenols are easily oxidized to quinones, which covalently bind to RNA, leading to RNA degradation, pigment residue, and severe interference with spectrophotometer readings (especially the A260 / A230 ratio, which reflects polysaccharide / polyphenol contamination).

[0003] Currently, conventional methods such as the CTAB-lithium chloride method, the Trizol method, and commercially available kits based on silica membranes or ordinary magnetic beads all have significant limitations when processing such samples: the CTAB method is cumbersome and difficult to completely remove polysaccharides; the Trizol method uses toxic phenol-chloroform and has limited ability to remove polyphenols; ordinary silica membrane or magnetic bead methods mainly rely on electrostatic interactions or hydrogen bonding under high-salt conditions to adsorb nucleic acids, lacking specific recognition and separation capabilities for polysaccharides and polyphenols, resulting in severe co-adsorption of impurities and low product purity.

[0004] In existing technologies, although some studies have attempted to add cross-linked polyvinylpyrrolidone (PVPP) to the lysis buffer to adsorb polyphenols, or to use polyethylene glycol (PEG) and metal ions to precipitate polysaccharides, these are mostly auxiliary methods to address a single type of impurity and have not formed a systematic solution. Although the reversible covalent binding properties of phenylboronic acid (PBA) and ortho-dihydroxy compounds have applications in glycoprotein separation, they have never been introduced into the design of nucleic acid extraction magnetic beads to achieve simultaneous capture and selective release of RNA and polyphenols at the same interface. In addition, environmentally responsive polymer brushes are widely used in drug controlled release, but their use in regulating the surface properties of magnetic beads to assist in nucleic acid purification and impurity separation is also a technological gap. Therefore, the development of an integrated magnetic bead extraction system that can actively and intelligently separate RNA from polysaccharides and polyphenols has an urgent technological need and broad industrial application prospects. Summary of the Invention

[0005] This invention provides a kit and method for extracting polysaccharide and polyphenol plant RNA based on magnetic beads, aiming to solve the above-mentioned problems.

[0006] The present invention is implemented as follows: a kit for extracting polysaccharide and polyphenol plant RNA based on magnetic beads, comprising a lysis buffer, a composite removal agent, a functionalized magnetic bead suspension, a first washing solution, a second washing solution, and an RNA elution solution;

[0007] The lysis buffer contains 3-5 M of a ionizing salt (such as at least one of guanidine isothiocyanate, guanidine hydrochloride, and guanidine thiocyanate, preferably guanidine isothiocyanate), 20-100 mM of an acidic buffer with a pH of 3.5-5.0 (such as at least one of sodium citrate buffer and sodium acetate buffer), and 1-5% (w / v) of cross-linked polyvinylpyrrolidone (PVPP). This lysis buffer provides a strong denaturing environment to inhibit RNase, and the acidic pH synergistically with PVPP to effectively complex polyphenols and inhibit their oxidation.

[0008] The composite removal agent comprises 1-3M of a monovalent salt (such as at least one of sodium chloride and sodium acetate), 5-15% (w / v) of polyethylene glycol (PEG, number average molecular weight 4000-8000), and 10-30mM of a divalent metal ion salt (such as zinc acetate); this agent is added after pyrolysis, utilizing the divalent metal ions (Zn... 2+ It neutralizes the negative charge of polysaccharides and reduces their solubility. In synergy with polyethylene glycol (PEG), it promotes the formation of insoluble complex precipitates of polysaccharides through the size exclusion effect, thereby reducing the polysaccharide background in the sample from the source.

[0009] The magnetic beads in the functionalized magnetic bead suspension have their surfaces covalently modified with temperature- and pH-responsive copolymer brushes and phenylboronic acid (PBA) functional groups. The copolymer brushes contain copolymer units of N-isopropylacrylamide and vinylimidazole, with a molar ratio of N-isopropylacrylamide units to vinylimidazole units of (10:1) to (3:1).

[0010] Preferably, the copolymer brush is poly(N-isopropylacrylamide-co-vinylimidazole)(P(NIPAM-co-VI)); wherein, the N-isopropylacrylamide unit (NIPAM unit) in the copolymer chain makes it hydrophilic and expandable at room temperature, which is beneficial for RNA adsorption; in subsequent steps, its hydrophobic contraction can generate a steric compression effect, which helps to remove impurities; the vinylimidazole unit (VI unit) is protonated under acidic conditions to enhance RNA adsorption, and deprotonated under near-neutral conditions to facilitate RNA elution;

[0011] The PBA functional group is preferably attached to the surface of the magnetic beads via flexible polyethylene glycol (PEG) linkers (number average molecular weight 2000-5000) to increase the spatial freedom and efficiency of its binding with polyphenols. The phenylboronic acid (PBA) functional group can form a reversible cyclic borate ester with the ortho-dihydroxy structure of the polyphenol molecule. This bond remains stable in the acidic environment (pH 3.5-5.0) of the supernatant during the binding stage, thereby capturing polyphenols. However, it dissociates under the weakly acidic conditions (pH 5.8-6.8) of the second washing solution, thereby achieving selective elution of polyphenols and specifically 'capturing' them.

[0012] The combination of this copolymer brush and PBA functional groups forms an active interface that can simultaneously capture RNA and polyphenols and achieve precise separation through environmental changes (pH).

[0013] The pH of the second washing solution is 5.8-6.8, preferably 6.0-6.3; this pH range is a "chemical switch" that triggers the specific breaking of the reversible bond between PBA and polyphenol, thereby achieving selective dissociation and removal of polyphenol, while the binding of the copolymer brush to RNA remains stable under these conditions.

[0014] Optionally, the kit also includes a third washing solution containing 1-2M NaCl and 0.05-0.2% anionic surfactant (such as sodium lauroyl sarcosinate) at a pH of 6.5-7.5 for deep removal of residual nonspecific adsorbed impurities; more preferably, it also contains 3-10mM histidine, whose zwitterionic properties stabilize the RNA-magnetic bead complex and competitively bind residual metal ions.

[0015] Preferably, the lysis buffer further comprises 1-3% (w / v) of anionic surfactant (such as sodium dodecyl sarcosinate) and 20-100 mM of reducing antioxidant (such as sodium ascorbate).

[0016] Preferably, the RNA elution buffer is a low-salt, weakly alkaline buffer (such as Tris-EDTA at pH 8.5).

[0017] Preferably, the functionalized magnetic beads have a core-shell structure, with the core being superparamagnetic nanoparticles (such as Fe3O4), and from the core surface outwards, the following are arranged in sequence:

[0018] Inner connecting layer: flexible polyethylene glycol chains (number average molecular weight 2000-5000) covalently linked to carboxyl groups on the surface of the magnetic core via amide bonds, serving as spatial arms;

[0019] First functional layer: phenylboronic acid functional groups located at the ends of the polyethylene glycol chains, connected by amide bonds;

[0020] The second functional layer consists of a temperature- and pH-responsive copolymer brush grafted onto the surface of a magnetic core via surface-initiated atom transfer radical polymerization. This copolymer brush is composed of N-isopropylacrylamide and vinylimidazole, and its chains can reversibly expand and contract in solution with varying temperatures and pH.

[0021] PBA functional groups and copolymers are spatially distributed on the surface of magnetic beads, together forming an active interface that can simultaneously capture polyphenols and RNA.

[0022] Preferably, the method for preparing the functionalized magnetic beads includes the following steps:

[0023] S1. Preparation of magnetic bead substrate: Provide a suspension of magnetic nanoparticles with carboxyl groups on the surface, wherein the particle size of the magnetic nanoparticles is preferably 50-500 nm, more preferably 100-300 nm.

[0024] S2. Linker Arm Modification: The magnetic beads from step S1 are coupled with bifunctional polyethylene glycol linker arms in the presence of a condensing agent, causing one end of the linker arm to form an amide bond with the carboxyl group on the surface of the magnetic beads. Preferably, the bifunctional polyethylene glycol linker arm is an amino-polyethylene glycol-carboxyl group with a number-average molecular weight (Mn) of 2000-5000. The condensing agent is preferably a combination of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The coupling reaction is preferably carried out in a buffer solution with a pH of 5.5-6.5 for 2-8 hours at a temperature of 15-30°C (room temperature). The mass ratio of the magnetic beads to the linker arm molecules can be adjusted according to the surface carboxyl group density, with a typical ratio of 1:1-5 (magnetic bead solid mass: linker arm molecular mass).

[0025] S3. Introduction of Phenylboronic Acid (PBA) Functional Group: The linker-modified magnetic beads obtained in step S2 are reacted with aminophenylboronic acid (APBA) in the presence of a condensing agent, causing the amino group of APBA to form an amide bond with the carboxyl group at the end of the linker, thereby covalently anchoring the PBA functional group to the surface of the magnetic beads through the linker. This reaction is preferably carried out in a weakly alkaline buffer solution with a pH of 8.0-9.0, and the reaction is carried out in the dark for 4-12 hours. The amount of APBA used is usually 1.2-2 times the molar amount of the linker to ensure sufficient reaction. After the reaction, it is necessary to wash thoroughly to remove unreacted boric acid compounds, for example, until the washing solution does not show a red color when tested with turmeric paper.

[0026] S4. Grafting of copolymer brushes: A copolymer brush of N-isopropylacrylamide (NIPAM) and vinylimidazole (VI) is grafted onto the surface of the magnetic beads obtained in step S3 by surface-initiated atom transfer radical polymerization (SI-ATRP).

[0027] Specifically, the magnetic beads, NIPAM monomer, VI monomer, catalyst (such as cuprous bromide) and ligand (such as pentamethyldiethylenetriamine) are placed in an anhydrous organic solvent (such as N,N-dimethylformamide) and polymerized under the protection of an inert gas (such as argon or nitrogen).

[0028] The preferred molar ratio of NIPAM to VI monomers is (10:1) to (3:1), the preferred total monomer concentration is 0.5-2.0 mol / L, the preferred polymerization temperature is 40-70℃, and the preferred reaction time is 8-36 hours. The reaction system needs to be thoroughly deoxygenated before the reaction, for example, by performing at least three "vacuum-inert gas" cycles. After the reaction, the system is separated by magnetic separation and thoroughly washed with organic solvent, alcohol and aqueous buffer solution in sequence to finally obtain functionalized magnetic beads with P(NIPAM-co-VI) copolymer brushes and PBA functional groups on the surface.

[0029] This invention also provides a method for extracting total RNA using the above-described kit. This method fully utilizes the synergistic effect of the lysis buffer, the composite removal agent, the functionalized magnetic beads, and the specific pH washing solution, and includes the following steps:

[0030] (a) Mix the plant tissue with the lysis buffer and the composite removal agent, homogenize, incubate, centrifuge, and collect the supernatant;

[0031] (b) Add the functionalized magnetic bead suspension to the supernatant and incubate at room temperature for 10-20 minutes to allow RNA and polyphenols to bind to the magnetic beads;

[0032] (c) Perform magnetic separation, discard the supernatant, and wash the magnetic beads bound to RNA and polyphenols sequentially with the first washing solution and the second washing solution; optionally, the magnetic beads may also be washed with a third washing solution.

[0033] (d) Dry the magnetic beads and elute them with the RNA elution buffer to obtain purified total RNA.

[0034] Compared with the prior art, the embodiments of this application have the following main advantages:

[0035] Employing a dual mechanism of "divalent metal ion / PEG composite precipitation of polysaccharides" and "PBA reversible capture / pH-triggered release of polyphenols", this invention can efficiently remove two main types of impurities. The A260 / A230 ratio of the extracted RNA is usually greater than 2.0, and the A260 / A280 ratio is stable at 1.9-2.1.

[0036] The high concentration of ionizing salt and antioxidants in the lysis buffer work synergistically to rapidly inhibit RNase activity and protect RNA from polyphenol oxidative damage; the temperature- and pH-responsive copolymer brush on the surface of the functionalized magnetic beads hydrophilically expands at room temperature, significantly improving RNA adsorption capacity, and combined with an efficient elution strategy, improving RNA yield.

[0037] The conformational response of the copolymer brush integrated on the surface of the magnetic beads is combined with the chemical recognition of PBA. The copolymer brush expands during the acid binding phase to enhance RNA adsorption, and shrinks during the washing phase by temperature / salinity regulation to assist in the removal of impurities. The reversible binding of PBA and polyphenols enables precise regulation of "capture-release", achieving synergistic impurity removal of "physical assistance" and "chemical precision" and avoiding RNA loss.

[0038] It requires no toxic reagents such as phenol or chloroform, is easy to operate, safe and environmentally friendly, and has strong compatibility. Attached Figure Description

[0039] Figure 1 This is a process flow diagram of the RNA extraction method of the present invention.

[0040] Figure 2 This is a flowchart of the preparation method of the functionalized magnetic beads of the present invention. Detailed Implementation

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used herein in the specification of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "comprising" and "having," and any variations thereof, in the specification, claims, and foregoing drawings of this application are intended to cover non-exclusive inclusion. The terms "first," "second," etc., in the specification, claims, or foregoing drawings of this application are used to distinguish different objects, not to describe a particular order.

[0042] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0043] Example 1: As Figure 2 As shown, a copolymer brush modified with dual temperature and pH responses and magnetic beads made of PBA were prepared.

[0044] Raw materials and pretreatment: Carboxyl-modified Fe3O4 magnetic nanoparticles (particle size 200nm, 10mg / mL), amino-polyethylene glycol-carboxyl (NH2-PEG5000-COOH), 3-aminophenylboronic acid (APBA), N-isopropylacrylamide (NIPAM), vinylimidazole (VI), cuprous bromide (CuBr), pentamethyldiethylenetriamine (PMDETA), EDC, NHS, all solvents were anhydrous, and the monomers were subjected to alkaline alumina column to remove polymerization inhibitors.

[0045] Linker modification (S2): Take 10 mL of carboxyl magnetic beads, magnetically separate them, and wash them three times with pH 6.0 MES buffer (50 mM); resuspend them in 10 mL of MES buffer, add 100 mg EDC and 150 mg NHS, and activate them by shaking at room temperature for 30 minutes; magnetically separate and discard the supernatant, resuspend the magnetic beads in 10 mL of MES buffer containing 200 mg NH2-PEG5000-COOH, and react them by shaking at room temperature for 4 hours; after the reaction, magnetically separate them, and wash them three times each with MES buffer and deionized water to obtain the intermediate MB-PEG.

[0046] PBA modification (S3): MB-PEG was resuspended in 10 mL of pH 8.5 borate buffer (50 mM), and 100 mg EDC and 150 mg NHS were added for activation for 30 minutes. After magnetic separation, the beads were resuspended in 10 mL of borate buffer containing 150 mg APBA and reacted at room temperature in the dark for 6 hours. After the reaction, the beads were magnetically separated, and the magnetic beads were repeatedly washed with deionized water. The washing solution was tested using the turmeric test paper method until the test paper did not turn red, confirming that there was no free boric acid residue, thus obtaining MB-PEG-PBA.

[0047] Copolymer brush grafting (S4): MB-PEG-PBA was washed three times with anhydrous DMF; resuspended in 50 mL of deoxygenated DMF, and NIPAM (5.0 g, 44.2 mmol), VI (0.42 g, 4.47 mmol), CuBr (63 mg, 0.44 mmol), and PMDETA (152 μL, 0.73 mmol) were added sequentially; the reaction system was subjected to three cycles of "vacuuming-purifying high-purity argon" to thoroughly deoxygenate; under argon protection, the reaction was vigorously stirred in an oil bath at 50 °C for 16 hours; after the reaction, the magnetic beads were magnetically separated and washed thoroughly with DMF (3 × 50 mL), anhydrous ethanol (2 × 50 mL), and PBS containing 1 mM EDTA (pH 7.4, 3 × 50 mL); finally, the magnetic beads were resuspended in 20 mM Tris-HCl buffer containing 0.02% NaN3 (pH 7.4). In step 7.0), the solid content was adjusted to 10 mg / mL to obtain functionalized magnetic bead suspension C (abbreviated as FMB), which was stored at 4℃.

[0048] Example 2: Preparation of the reagent kit

[0049] Prepare each component of the reagent kit of this invention according to the following formula (taking the amount of one standard extraction as an example, which can be scaled up proportionally as needed):

[0050] Lysis buffer: 4M guanidine isothiocyanate, 50mM sodium citrate (pH 4.0), 2% (w / v) sodium dodecyl sarcosinate, 2% (w / v) cross-linked polyvinylpyrrolidone (PVPP, average molecular weight 40k), 40mM sodium ascorbate. Store at room temperature and add 1% (v / v) β-mercaptoethanol immediately before use.

[0051] Composite removal agent: 2M NaCl, 10% (w / v) PEG 6000, 15mM zinc acetate, store at room temperature.

[0052] Functionalized magnetic bead suspension C (FMB): Take the magnetic beads prepared in Example 1 and dilute them with 20mM Tris-HCl (pH 7.0) buffer containing 0.1% Tween-20 and 0.02% NaN3 to a solid content of 2mg / mL, ensuring that the adsorption capacity of magnetic beads in each 1mL extraction system is ≥5μgRNA, and store at 4℃.

[0053] First washing solution: 70% (v / v) ethanol, 0.5M NaCl, 10mM EDTA (pH 8.0), store at room temperature.

[0054] Second washing solution: 80% (v / v) ethanol, 20 mM sodium citrate, pH adjusted to 6.3 with hydrochloric acid, stored at room temperature.

[0055] Third washing solution: 1.5M NaCl, 0.1% (w / v) sodium lauroyl sarcosinate, 5mM histidine, pH adjusted to 7.0 with HCl, stored at room temperature.

[0056] RNA elution buffer: 10mM Tris-HCl, 1mM EDTA, pH 8.5, store at room temperature.

[0057] Example 3: As Figure 1 As shown, total RNA was extracted from young tea leaves.

[0058] Sample preparation: Weigh about 100mg of fresh tea bud and leaf, and grind them into fine powder quickly in liquid nitrogen.

[0059] Pyrolysis and clarification: Transfer the powder to a pre-cooled 2mL centrifuge tube, immediately add 1mL of pre-cooled lysis buffer (with added β-mercaptoethanol) and 200μL of composite removal agent, vortex vigorously for 30 seconds; incubate in a 65℃ water bath for 5 minutes, inverting and mixing twice during the incubation period; after cooling to room temperature, centrifuge at 13000g for 10 minutes.

[0060] Magnetic bead binding: Carefully pipette 800 μL of supernatant into a new 1.5 mL RNase-free centrifuge tube, add 150 μL of LMB suspension, vortex to mix, and incubate at room temperature (approximately 25 °C) on a rotary mixer for 15 minutes.

[0061] washing:

[0062] a. Magnetic separation for 2 minutes, then completely discard the supernatant.

[0063] b. Add 500 μL of the first washing solution, vortex to resuspend the magnetic beads, transfer the entire suspension to a new 1.5 mL centrifuge tube, magnetically separate for 1 minute, and discard the supernatant.

[0064] c. Add 500 μL of second washing buffer (pH 6.3), vortex to resuspend, magnetically separate for 1 minute, and discard the supernatant.

[0065] d. Add 300 μL of third washing buffer, gently resuspend by blowing, let stand at room temperature for 2 minutes, magnetically separate for 1 minute, and discard the supernatant.

[0066] Elution: Open the cap and let stand at room temperature for 3 minutes until there is no liquid luster on the surface of the magnetic beads. Add 50 μL of RNA elution buffer preheated to 65°C, resuspend by pipetting, and incubate at 65°C for 5 minutes. Immediately separate magnetically for 2 minutes and transfer the supernatant (i.e., RNA solution) to a new tube.

[0067] Testing: Concentration and purity were determined using NanoDrop One: concentration 312 ng / μL, A260 / A280 = 2.07, A260 / A230 = 2.18. Integrity was tested using Agilent 2100 Bioanalyzer: RIN = 8.7.

[0068] Example 4: Extraction of total RNA from ripe mango pulp

[0069] The operating steps are the same as in Example 3, and the sample is approximately 80 mg of ripe mango pulp (Tainong No. 1).

[0070] Results: The concentration was 168 ng / μL, A260 / A280 = 2.03, A260 / A230 = 2.06. Compared with a commercially available plant RNA magnetic bead kit (the results were: concentration 95 ng / μL, A260 / A280 = 1.88, A260 / A230 = 1.52), the present invention has significant advantages in yield and purity.

[0071] Example 5: Optimizing the pH value of the second washing solution (using mango as a sample)

[0072] The procedure was followed as described in Example 3, except that the pH value of the second washing solution was changed systematically (5.8, 6.0, 6.1, 6.3, 6.5, 6.8). Each group was repeated three times, and the average value was taken. The specific data are shown in Table 1.

[0073] Table 1. Effect of pH of the second washing solution on mango RNA extraction efficiency.

[0074]

[0075]

[0076] As shown in Table 1, when the pH of the second washing solution is in the range of 6.0-6.3, the RNA yield and the A260 / A230 purity ratio both reach the optimal and stable plateau. When the pH is below this range, the polyphenol removal is incomplete (low A260 / A230, solution staining); when the pH is above this range, the RNA yield decreases, indicating that the binding may be weakened. Therefore, 6.0-6.3 is the optimal pH range.

[0077] Example 6: Extraction of total RNA from pine needles of Pinus tabuliformis

[0078] The operation steps are the same as in Example 3, and the sample is about 50mg of fresh pine needles.

[0079] Results: At a concentration of 189 ng / μL, A260 / A280 = 2.09, A260 / A230 = 2.10, the obtained RNA solution was subjected to a full-band (220-350 nm) UV scan. No abnormal absorption peak was observed at 230 nm, and the curve smoothly decreased from 260 nm to 230 nm. The A260 / A230 ratio was as high as 2.10, which was significantly higher than 1.85 of Comparative Example 3 (whose spectrum showed a distinct shoulder peak at 230 nm), directly demonstrating that polysaccharide / polyphenol impurities were effectively removed.

[0080] Example 7: Effect of extreme values ​​of PVPP concentration in the lysis buffer on extraction efficiency

[0081] Using young tea leaves with high polyphenol content as samples, RNA was extracted according to the method described in Example 3, with only the concentration of PVPP (average molecular weight 40,000) in the lysis buffer being systematically changed to 1%, 2%, and 5% (w / v), respectively. Each group was repeated 3 times, and the extraction results (mean ± standard deviation) are shown in Table 2 below:

[0082] Table 2. Effect of different PVPP concentrations in the lysis buffer on extraction efficiency.

[0083]

[0084] Experiments show that high-purity, high-yield RNA can be effectively extracted within a concentration range of 1% to 5%.

[0085] Comparative Example 1 (Ordinary magnetic beads): Carboxyl magnetic beads modified only with ordinary linear PEI (molecular weight 10k) were used instead of the functionalized magnetic beads of the present invention. Other reagents and steps were the same as in Example 3.

[0086] Results: At a concentration of 105 ng / μL, A260 / A280 = 1.91, A260 / A230 = 1.45, RIN = 6.2, the Ct value of the internal reference gene UBQ detected by qPCR was 28.5, which was 3.8 cycles higher than that of Example 3 (Ct = 24.7), but the amplification efficiency was only 75%.

[0087] Comparative Example 2 (Washing at Incorrect pH): The functionalized magnetic beads (FMB) and kit of the present invention were used, but the second wash solution was replaced with the same formulation as the first wash solution (high-salt ethanol at pH 8.0), otherwise the same as in Example 3.

[0088] Results: Concentration 280 ng / μL, A260 / A280 = 2.05, A260 / A230 = 1.62, RNA solution was light brown.

[0089] The RNA integrity of Example 3, Comparative Example 1, and Comparative Example 2 was visually assessed by 1.2% agarose gel electrophoresis, and the main band characteristics are summarized in Table 3:

[0090] Table 3 Comparison of gel electrophoretic characteristics of total RNA extracted from tea leaves using different methods

[0091]

[0092] Comparative Example 3 (composite removal agent omitted): The functionalized magnetic beads of the present invention and the complete steps were used, but the composite removal agent (PPAR) was not added in the pyrolysis step. Otherwise, it was the same as Example 6 (pine needle sample).

[0093] Results: At a concentration of 175 ng / μL, A260 / A280 = 2.06, A260 / A230 = 1.85; a full-band (220-350 nm) UV scan of the obtained RNA solution showed a distinct shoulder peak at 230 nm, resulting in a significant decrease in the A260 / A230 ratio to 1.85. This directly confirms that omitting the composite removal agent leads to incomplete removal of polysaccharide / polyphenol impurities; when constructing the RNA-Seq library, the effective fragment ratio was only 68%, 12% lower than that of Example 6 (80%).

[0094] Comparative Example 4 (third washing liquid omitted): In step 4 of Example 3, step d is omitted (i.e., the third washing liquid is not used), and everything else is exactly the same.

[0095] Results: The concentration was 305 ng / μL, A260 / A280 = 2.06, A260 / A230 = 2.05; the purity was slightly lower than that of the sample in Example 3 after the third wash (A260 / A230 = 2.18). When this RNA was used to construct a small RNA-Seq library, the effective library yield (the proportion of fragments >200bp) was about 12% lower than that of the sample using the third wash, indicating that the W3 wash has an optimizing effect on removing trace impurities that affect advanced applications.

[0096] It should be noted that, for the sake of simplicity, the foregoing embodiments are all described as a series of actions. However, those skilled in the art should understand that the present invention is not limited to the described order of actions, as some steps may be performed in other orders or simultaneously according to the present invention. Furthermore, those skilled in the art should also understand that the embodiments described in the specification are preferred embodiments, and the actions and modules involved are not necessarily essential to the present invention.

[0097] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on these embodiments, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still combine, add, delete, or otherwise adjust the features of the various embodiments of the present invention according to the circumstances without conflict or creative effort, thereby obtaining different technical solutions that do not fundamentally depart from the concept of the present invention. These technical solutions also fall within the scope of protection of the present invention.

Claims

1. A kit for extracting polysaccharide-polyphenol plant RNA based on magnetic beads, characterized in that, include: The lysis buffer contains 3-5 M of ionizing salt, 20-100 mM of acidic buffer at pH 3.5-5.0, and 1-5% (w / v) of cross-linked polyvinylpyrrolidone. The composite removal agent contains 1-3M monovalent salt, 5-15% (w / v) polyethylene glycol, and 10-30mM divalent metal ion salt; A functionalized magnetic bead suspension, wherein the surface of the magnetic beads is covalently modified with a temperature- and pH-responsive copolymer brush and phenylboronic acid functional groups; The first washing solution is a 70-75% (v / v) ethanol solution containing 0.3-0.8M NaCl, with a pH of 7.5-8.5; The second washing solution has a pH value of 5.8-6.8; and RNA elution buffer.

2. The kit for extracting polysaccharide-polyphenol plant RNA based on magnetic beads as described in claim 1, characterized in that, The functionalized magnetic bead suspension contains magnetic beads whose surface copolymer brushes comprise copolymer units of N-isopropylacrylamide and vinylimidazole, with a molar ratio of N-isopropylacrylamide units to vinylimidazole units of (10:1) to (3:1).

3. The kit for extracting polysaccharide-polyphenol plant RNA based on magnetic beads as described in claim 1, characterized in that, The phenylboronic acid functional group is connected to the magnetic bead substrate through a flexible connecting arm, which is a polyethylene glycol chain with a number average molecular weight of 2000-5000.

4. The kit for extracting polysaccharide-polyphenol plant RNA based on magnetic beads as described in claim 1, characterized in that, It also contains a third washing solution, which contains 1-2M NaCl, 0.05-0.2% anionic surfactant and 3-10mM histidine, with a pH of 6.5-7.

5.

5. The kit for extracting polysaccharide-polyphenol plant RNA based on magnetic beads as described in claim 1, characterized in that, The lysis solution also contains 1-3% (w / v) of anionic surfactant and 20-100mM of reducing antioxidant; the divalent metal ion salt in the composite removal agent is a zinc salt.

6. A method for preparing functionalized magnetic beads for the kit according to any one of claims 1-5, characterized in that, Includes the following steps: S1: Provides a suspension of carboxyl-modified magnetic nanoparticles; S2: React the magnetic beads from step S1 with polyethylene glycol linker molecules containing dual-terminal functional groups, so that one end of the linker is coupled to the carboxyl group on the surface of the magnetic beads to obtain a linker-modified magnetic bead intermediate. S3: The intermediate obtained in step S2 is reacted with aminophenylboronic acid, so that the phenylboronic acid functional group is covalently linked to the surface of the magnetic bead through the linker arm to obtain phenylboronic acid modified magnetic beads. S4: The functionalized magnetic beads are obtained by grafting a copolymer of N-isopropylacrylamide and vinylimidazole onto the surface of the magnetic beads obtained in step S3 through surface-initiated atom transfer radical polymerization.

7. The method for preparing functionalized magnetic beads as described in claim 6, characterized in that, In step S2, the polyethylene glycol linker molecule with dual-terminal functional groups is an amino-polyethylene glycol-carboxyl group with a number-average molecular weight of 2000-5000; the reaction is carried out in the presence of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in a buffer solution at pH 5.5-6.5 for 2-6 hours at room temperature.

8. The method for preparing functionalized magnetic beads as described in claim 6, characterized in that, In step S4, the reaction conditions for the surface-initiated atom transfer radical polymerization are as follows: using cuprous bromide and pentamethyldiethylenetriamine as the catalytic system, the molar ratio of N-isopropylacrylamide to vinylimidazolium monomer is (10:1) to (3:1), and the reaction is carried out at 40-60°C under inert gas protection for 12-24 hours; before the reaction, the system is subjected to at least three "vacuuming-inert gas filling" cycles to completely remove oxygen.

9. A method for extracting RNA from plant tissues rich in polysaccharides and polyphenols, characterized in that, Using the kit according to any one of claims 1-5, and comprising the following steps: (a) Mix the plant tissue with the lysis buffer and the composite removal agent, homogenize, incubate, centrifuge, and collect the supernatant; (b) Add the functionalized magnetic bead suspension to the supernatant and incubate at room temperature for 10-20 minutes to allow RNA and polyphenols to bind to the magnetic beads; (c) Perform magnetic separation, discard the supernatant, and wash the magnetic beads containing RNA and polyphenols sequentially with the first washing solution, the second washing solution, and optionally the third washing solution described above; (d) Dry the magnetic beads and elute them with the RNA elution buffer to obtain purified total RNA.

10. The method as described in claim 9, characterized in that, In step (c), when washing with the second washing solution, the pH of the washing environment is adjusted to 5.8-6.8 to trigger the breakage of the reversible covalent bond between the phenylboronic acid functional group and the polyphenol, thereby selectively dissociating the polyphenol from the magnetic beads and eluting it.

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