阅读说明：本技术 一种微藻总核糖核酸样品制备方法 (Preparation method of microalgae total ribonucleic acid sample ) 是由 薛松 范旭冉 曹旭鹏 朴海龙 于 2019-04-22 设计创作，主要内容包括：本发明涉及一种微藻总核糖核酸(RNA)样品制备技术,具体的说是对于高糖能源微藻特点而开发的一种总RNA提取纯化方法。本发明以微藻生物质为原料,经过液氮研磨、均相试剂提取和纯化,最终获得满足高通量测序所需要的总RNA样品。本发明操作简单,尤其适用高糖微藻总RNA样品制备。(The invention relates to a microalgae total ribonucleic acid (RNA) sample preparation technology, in particular to a total RNA extraction and purification method developed for the characteristics of high-sugar energy microalgae. According to the method, microalgae biomass is used as a raw material, and a total RNA sample required by high-throughput sequencing is finally obtained through liquid nitrogen grinding, homogeneous reagent extraction and purification. The method is simple to operate, and is particularly suitable for preparing the total RNA sample of the high-sugar microalgae.)

1. A preparation method of microalgae total ribonucleic acid samples is characterized by comprising the following steps:

extracting the freshly collected or low-temperature stored microalgae cells according to the following extraction process to obtain a purified microalgae total ribonucleic acid (RNA) sample, wherein the process comprises the following steps of:

1) placing 60-300 mg of fresh microalgae cells or cryopreserved microalgae cells into a precooled mortar at the temperature of-70 to-80 ℃, and grinding the cells into powder under the protection of liquid nitrogen;

2) adding TRIzol (Ambion-Life technologies Co., Carlsbad, Calif., USA) according to a proportion of 80-120 mg microalgae cells/1 mL TRIzol, soaking all algae powder in TRIzol, and then adding 5-10 mL liquid nitrogen to freeze the mixture into solid;

3) placing the mixture in a mortar at room temperature, melting the biomass powder and the TRIzol, and grinding and mixing the mixture uniformly by using a pestle in the melting process;

4) after the mixed solution is completely melted, transferring the mixed solution into centrifugal test tubes by using a pipettor, wherein the volume of the mixed solution in each test tube is 0.75-1 mL;

5) centrifuging the mixed solution at 10000-12000 g for 4-6 min at 4 ℃;

6) collecting the supernatant to a new centrifugal tube to avoid sucking sediment as much as possible;

7) adding 150-450 mu L of chloroform into 750 mu L of supernatant according to the proportion, after adding the chloroform, shaking and mixing for 15-20 s, and standing for 5-8 min at room temperature;

8) centrifuging the mixed solution at 10000-12000 g for 4-6 min at 4 ℃;

9) taking out the supernatant, transferring the supernatant into a new centrifugal test tube, adding 1/2 volumes of isopropanol of the volume of the current supernatant, mixing for 15-20 s in a shaking manner, and standing for 9-12 min at room temperature;

10) centrifuging the mixed solution at 10000-12000 g for 8-12 min at 4 ℃, and removing supernatant;

11) adding 1-1.2 mL of 75% ethanol in volume concentration, slightly reversing the volume of the ethanol to wash the tube wall of the centrifuge tube, centrifuging the solution at the temperature of 4 ℃ for 4-6 min at 10000-12000 g, and removing all liquid;

12) drying the precipitate at room temperature for 3-5min, adding 10-100 mu L of water to dissolve the precipitate, and obtaining the extracted total RNA solution.

2. The method of claim 1, wherein:

the liquid transfer device is a liquid transfer device without RNase enzyme; the centrifugal test tube is a centrifugal test tube without RNase; the water is RNase-free water.

3. The method of claim 1, wherein:

in step (2), a homogeneous extraction reagent containing phenol having a composition similar to that of TRIzol may be used instead of TRIzol, such as RNAisso reagent or Beyozol reagent.

4. The method of claim 1, wherein:

in the step (7), NaCl with the final concentration of 0.8-1M can be added.

5. The method of claim 1, wherein:

the used reagents and consumables are RNase-free reagents and consumables.

6. The method of claim 1, wherein:

the microalgae is one or more than two of microalgae in Chlorophyta and Chrysophyta.

Technical Field

The invention belongs to the technical field of biology, relates to a method for extracting total RNA of microalgae, and is particularly suitable for extracting total RNA from a high-sugar microalgae sample for research of transcriptomics.

Background

The microalgae can grow rapidly, and can directionally enrich energy storage substances by simply culturing and regulating metabolic pathways, so that the microalgae become a new generation of natural, low-cost and green and environment-friendly energy raw materials, industrial raw materials or food raw materials. In the research on the regulation mechanism of the metabolic pathway of microalgae, transcriptome sequencing is an efficient and economic technical means, and has been widely applied in the field of microalgae research in recent years. The transcription condition of genes in microalgae cells under specific environment and time can be qualitatively and quantitatively determined through transcriptome information, but transcriptome sequencing needs a high-quality RNA sample to accurately show the transcription condition of the cells at a specific time stage.

The high-sugar microalgae cells are rich in various polysaccharides. Polysaccharide and RNA have similar physical and chemical properties, and when RNA is precipitated, polysaccharide and RNA are often precipitated together to form gel-like RNA precipitate rich in polysaccharide and a large amount of wall hanging is formed. In the process of removing polysaccharide, partial RNA is easy to be removed together, so that the yield of RNA is reduced, and the added steps increase great degradation risk for the total RNA. Especially, in microalgae cultured in a stress environment, RNA of cells begins to degrade, the quality of a sample can be further reduced through long-time extraction and purification steps, and the generated difference can be further amplified in a PCR multiplication step in a subsequent sequencing process, so that a significant error occurs in the quantitative determination of transcriptome sequencing. In addition, the polysaccharide can inhibit the activity of partial enzyme, and can also interfere with subsequent library construction and sequencing.

The invention is based on the technology of extracting total RNA of cells by using reagents such as phenols, guanidinium isothiocyanate and the like, extracts the total RNA from newly collected or low-temperature stored high-sugar microalgae cells, can be directly used for establishing a library for transcriptome sequencing, and is an efficient and simple method for obtaining a high-quality RNA sample.

Disclosure of Invention

The invention provides a microalgae total RNA sample preparation technology aiming at solving the technical problems of low total RNA extraction quality and low extraction rate in high-sugar microalgae cells.

The invention provides a microalgae total RNA sample preparation technology, which comprises the following steps:

adding liquid nitrogen with the volume of 2/3 into a mortar to be used, and waiting for the liquid nitrogen to be completely evaporated; placing the algae mud sample in a mortar before melting, adding a small amount of liquid nitrogen, grinding the algae precipitate in the liquid nitrogen into powder, and adding the liquid nitrogen for 1-2 times in the process; ③ adding TRIzol (Ambion-Life Technologies Co., Carlsbad, Calif., USA) according to the proportion of 100mg algae precipitation/1 mL TRIzol to cover the algae powder, then adding a small amount of liquid nitrogen to make it become solid, melting at room temperature, and fully stirring with a pestle when in paste state during melting; fourthly, after complete melting, the sample is mixed with a pestleTransferring the reagent into a new 1.5mL centrifugal test tube every 1000. mu.L, centrifuging for 5min at 12000g, taking 750. mu.L supernatant, and completely not sucking broken substances; adding 150 mu L of chloroform into each centrifuge tube, shaking by hand for 15s, standing for 5min at room temperature, and then centrifuging for 15min at 12000 g; sixthly, taking the supernatant, completely not touching the middle layer, leaving a large distance to prevent inhalation, adding 375 mu L of isopropanol, uniformly mixing, standing at room temperature for 10min, centrifuging at 12000g for 10min, and removing the supernatant; seventhly, adding 1mL of 75% ethanol, upside down, cleaning and precipitating, centrifuging for 5min at 12000g, and removing the ethanol as much as possible; drying the mixture at room temperature for 2-5min with 100 μ L DEPC-H₂And dissolving O for 20min in sequence, and then respectively quantifying and verifying by agarose gel electrophoresis.

The key operation content notice items of the invention comprise:

(1) the operating environment is clean and dustless, DNA & RNase Away treatment is carried out in advance, and a mask, a head cover and gloves are strictly worn;

(2) thoroughly treating a pipettor rod and an experiment table, wherein consumable materials and reagents such as a gun head, a centrifuge tube and the like all adopt consumable materials and reagents without RNase;

(3) the freeze thawing process after adding TRIzol to the sample can improve the RNA extraction rate;

(4) in the step of removing the ethanol, 1000 mu L, 100 mu L and 10 mu L pipettes are used for removing the ethanol as much as possible in sequence, so that the salt content in the RNA sample can be obviously reduced.

According to the method, microalgae biomass is used as a raw material, and a total RNA sample required by high-throughput sequencing is finally obtained through liquid nitrogen grinding, homogeneous reagent extraction and purification. The method is simple to operate, and is particularly suitable for preparing the total RNA sample of the high-sugar microalgae.

Drawings

FIG. 1 is an agarose gel electrophoresis chart of a total RNA sample of dinoflagellate such as Zhanjiang, wherein M is Marker, and the rightmost 1 strip is a sample with the serial number 15;

FIG. 2 is an agarose gel electrophoresis of a total RNA sample of Tetraselmis subcordiformis, on the left hand side of which is Marker;

FIG. 3 is a diagram of Total RNA quality analysis of algal fluid after dark induction;

Detailed Description

The invention discloses a micro-meterThe preparation method of the total RNA sample of algae can be realized by appropriately improving technical parameters including the species of microalgae, the sample amount, the room temperature drying time and DEPC-H when the final RNA sample is dissolved by taking the contents of the sample as reference by the technical personnel in the field₂The amount of O added. If the sample is subsequently applied to quantitative PCR, a step of degrading residual DNA by adding Dnase is required. The change of the technical parameters has little influence on the result and is considered to be included in the invention. The method and application of the present invention have been described in detail by way of example, and the person skilled in the art can easily apply the method described herein. Compared with the prior art, the invention has the following advantages and effects: the method is simple and easy to implement, and can quickly extract the total RNA from the high-sugar microalgae cells with high quality.