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Hydroxyl nanometer magnetic ball method RNA extraction kit and extraction method thereof

A nano-magnetic bead and kit technology, applied in the field of molecular biology, can solve the problems of test system interference, test result interference, test interference, etc., achieve high RNA extraction rate, increase RNA concentration, and reduce interference

Active Publication Date: 2018-06-22
HANGZHOU BIGGER FISH BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the hydroxyl nano-magnetic bead method is easy to operate for most fungal RNA extraction, its shortcoming is that the influence of polysaccharides on RNA adsorption cannot be ruled out.
[0007] In addition, although there are many classic methods for the RNA extraction of organisms, these traditional methods cannot meet the needs of some tests. Some tests require RNA with high purity and do not want to contain any other impurities, such as proteins, Peptides, DNA, polysaccharides, or other non-RNA substances, as these can interfere with the test
Sometimes, although there is RNA in the sample, the target RNA cannot be detected. This may be caused by the interference of the impurities contained in the test system. Although these impurities are small in content, they still interfere with the detection results. Sometimes, Even let the detection fail

Method used

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  • Hydroxyl nanometer magnetic ball method RNA extraction kit and extraction method thereof
  • Hydroxyl nanometer magnetic ball method RNA extraction kit and extraction method thereof
  • Hydroxyl nanometer magnetic ball method RNA extraction kit and extraction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1: RNA Extraction Kit for Sclerotinia-producing Fungi Using Hydroxy Nano Magnetic Beads

[0069] 1.1. Hydroxy Nano Magnetic Bead Method Sclerotia-producing Fungus RNA Extraction Kit includes:

[0070] Step 1: Clamp the cultured Rhizoctonia solani sclerotia into a 1.5ml centrifuge tube under sterile conditions, the mass of sclerotia should not be less than 300mg, add 100μL solution Ⅰ, freeze and grind with liquid nitrogen, and then Add 100 μL solution Ⅰ, shake and mix;

[0071] Step 2: Add 250 μL of solution II, mix well, centrifuge at 12000 rpm at 4 °C for 1 min, and transfer the supernatant to a new tube;

[0072] Step 3: Add 500 μL solution III, gently turn up and down 5-6 times; centrifuge at 12000 rpm at 4 °C for 1 min, and take the supernatant into a new tube;

[0073] Step 4: Add solution Ⅲ, turn it up and down gently, so that the bacteria solution is fully lysed into a transparent solution. Centrifuge at 12000rpm at 4°C for 1min, and transfer the super...

Embodiment 2

[0101] Example 2: Extraction of RNA from Samples Using Treated Hydroxyl Nano Magnetic Beads

[0102] According to the extraction process described in Example 1, the difference is: before using hydroxyl nano-magnetic beads to extract RNA, the purchased hydroxyl nano-magnetic beads are processed, and the processed hydroxyl nano-magnetic beads are used to extract RNA. Other methods The practical use of the reagent is the same as the method for extracting hydroxyl nano-magnetic beads in Example 1, and the steps are the same.

[0103] The hydroxyl nano magnetic beads are processed, and the processing method is as follows:

[0104] The hydroxyl nano-magnetic beads are purchased from Shanghai Carboxyphene Biomedical Technology Co., Ltd. (the same batch number as Example 1), and are specially used to extract the hydroxyl nano-magnetic beads of RNA. The hydroxyl nano-magnetic beads are treated with Trizol solution (5mol / L, purchased from Thermo Fisher) (Trizol solution is a method of...

Embodiment 3

[0115] Example 3: Determination of RNA adsorption capacity by treated hydroxyl nano-magnetic beads

[0116] Use an ultrasonic cell disruptor to disrupt the cell wall of the bacteria to be tested (Staphylococcus aureus), and the specific method of disruption is as follows:

[0117] Bacteria were scraped and made into a suspension with distilled water (10 7 spore / mL), and then place the beaker containing the suspension in the crushing instrument, put the probe of the crushing instrument into the beaker and insert it into the spore suspension, set the crushing time to 10 min in an ice bath environment, and take out the sample for use after cell crushing The crude RNA extract was obtained by the guanidinium isothiocyanate-phenol method, and then the crude extract was subjected to RNA detection by hydroxyl nano-magnetic beads adsorption.

[0118] Add a certain weight of hydroxyl nano-magnetic beads of the present invention (but 2-5 grams) (the processed hydroxyl nano-magnetic bead...

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Abstract

The invention discloses a hydroxyl nanometer magnetic ball method RNA extraction kit, and a method for fast and simply extracting sclerotium-producing fungus RNA. The kit comprises a solution I, a solution II, a solution III, a solution IV, a washing solution, an eluant and a hydroxyl nanometer magnetic ball suspension. The solution II is creatively used for removing polysaccharide substances of sclerotium-producing fungus, so that in the RNA extraction process, the interference of exopolysaccharides is avoided. In the experiment process, the hydroxyl nanometer magnetic balls used by the kit are also creatively pretreated; the RNA is used as a molecular target; the adsorption capability of the hydroxyl nanometer magnetic balls on DNA and proteins is reduced; the operation has the obvious characteristic of high specificity. The kit is applicable to RNA extraction of the sclerotium-producing fungus; the detection is simple, convenient and fast; the interference of the exopolysaccharideson the RNA extraction is reduced.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a kit for extracting RNA from sclerotinia-producing fungi by a hydroxyl nanometer magnetic bead method, and a method for quickly extracting RNA from sclerotia-producing fungi with strong specificity. Background technique [0002] Sclerotia is a dormant body composed of closely intertwined hyphae, and its main function is to resist adverse environments. Common sclerotia-producing fungi include Sclerotinia and Rhizoctonia, which contain a large amount of exopolysaccharides. The fungal exopolysaccharide has the characteristics of high adsorption and high viscosity, which is one of the difficulties in the separation and extraction of high-purity RNA from sclerotia-producing fungi. With the rapid development of molecular biology, techniques such as RNA-related transcriptomics analysis, cDNA library construction, and Northern blot have attracted much attention, and RNA is ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1013
Inventor 谢廉毅
Owner HANGZHOU BIGGER FISH BIOTECHNOLOGY CO LTD
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