阅读说明：本技术 一种草鱼肠系膜脂肪组织高质量总rna的提取方法 (Method for extracting high-quality total RNA of grass carp mesenteric adipose tissue ) 是由 姜鹏 李胜杰 樊佳佳 马冬梅 杜金星 于 2020-04-30 设计创作，主要内容包括：本发明公开了一种草鱼肠系膜脂肪组织高质量总RNA的提取方法,包括样品液氮速冻,TRIzol试剂裂解与机械匀浆,离心去除油脂层,氯仿分离含RNA水相,异丙醇沉淀和75％乙醇洗涤等步骤。在样品前处理阶段,本发明通过增加取样量至30mg,可提升产量以满足常规分子生物学实验对RNA量的需求；样品采用液氮速冻,无需人工研磨,并将冻样在TRIzol试剂中进行长时间机械匀浆,时长3min,可显著降低RNA降解发生,同时增加2次离心去除油脂层等操作步骤,获得的总RNA纯度和完整性较高。本发明的提取方法有效解决了草鱼脂肪组织RNA提取易降解,产量低等问题,且操作简单,批处理效果稳定,可满足转录组测序等高质量RNA要求的实验,也适用于其它鱼类脂肪组织总RNA的提取。(The invention discloses a method for extracting high-quality total RNA from grass carp intestinal system adipose tissue, which comprises the steps of quick freezing of a sample by liquid nitrogen, cracking by a TRIzol reagent, mechanical homogenization, centrifugal removal of an oil layer, separation of an RNA-containing water phase by chloroform, isopropanol precipitation, washing by 75% ethanol and the like. In the sample pretreatment stage, the yield can be improved to meet the requirement of a conventional molecular biology experiment on the RNA amount by increasing the sampling amount to 30 mg; the sample is quickly frozen by liquid nitrogen, manual grinding is not needed, the frozen sample is mechanically homogenized in a TRIzol reagent for a long time of 3min, the occurrence of RNA degradation can be obviously reduced, operation steps of centrifuging for 2 times to remove a grease layer and the like are added, and the purity and the integrity of the obtained total RNA are high. The extraction method effectively solves the problems of easy degradation, low yield and the like of the grass carp adipose tissue RNA extraction, has simple operation and stable batch processing effect, can meet the experiment of high-quality RNA requirements such as transcriptome sequencing and the like, and is also suitable for extracting the total RNA of other fish adipose tissues.)

1. A method for extracting high-quality total RNA from grass carp mesentery adipose tissue is characterized in that in a pretreatment stage, a grass carp mesentery adipose tissue sample is subjected to quick freezing by liquid nitrogen and mechanical homogenization in a TRIzol reagent for a long time.

2. The method for extracting the high-quality total RNA from the grass carp mesentery adipose tissue as claimed in claim 1, wherein the mechanical homogenization time of the grass carp mesentery adipose tissue in the TRIzol reagent is 3 min.

3. The method for extracting the high-quality total RNA from the grass carp mesentery adipose tissue as claimed in claim 1, wherein the grass carp mesentery adipose tissue is put into liquid nitrogen for quick freezing in a pretreatment stage, and the frozen sample is not required to be ground and crushed manually or mechanically before being cracked by the TRIzol reagent.

4. The extraction method of the grass carp mesenteric adipose tissue high-quality total RNA is characterized by comprising a pretreatment stage and a post-extraction stage, wherein the pretreatment stage comprises the following steps:

(1) turning the head of a cleaned homogenizer, wrapping the head with tinfoil, treating at the high temperature of 150 ℃ for 2h, cooling to room temperature, and placing in a low-temperature refrigerator at the temperature of-20 ℃ for precooling for later use;

(2) adding 1.3mL of TRIzol reagent into 1.5mL of a centrifuge tube, and placing the centrifuge tube into a low-temperature refrigerator at the temperature of 20 ℃ below zero for pre-cooling for later use;

(3) anaesthetizing grass carp, cutting off cervical vertebra, dissecting, quickly taking 30mg of mesenteric adipose tissue, and quickly freezing in liquid nitrogen;

(4) taking out the centrifuge tube and the rotating head of the homogenizer, placing the centrifuge tube and the rotating head of the homogenizer on an ice box, adding the frozen sample into the centrifuge tube, immediately mechanically grinding and homogenizing for 3min, and then carrying out centrifugal operation;

(5) centrifuging homogenized adipose tissue at 4 deg.C for 5min, carefully removing 200 μ L of upper layer oil;

(6) centrifuging at 4 deg.C for 3min, sucking off 100 μ L of upper layer oil, transferring the lower most liquid to another new centrifuge tube, and performing RNA extraction.

5. The method for extracting the high-quality total RNA from the adipose tissue of the mesentery of the grass carp as claimed in claim 4, wherein the post-treatment stage comprises the following steps:

(7) adding 300 mu L of chloroform into the new centrifuge tube, tightly covering the centrifuge tube, violently shaking up and down for 15s, and then standing at room temperature for 5 min;

(8) centrifuging the centrifuge tube at 4 deg.C for 15min, carefully sucking the upper aqueous phase, and transferring into another new centrifuge tube;

(9) adding 500 mu L of isopropanol into a new centrifuge tube, turning upside down and uniformly mixing, and incubating for 5min at room temperature;

(10) centrifuging the tube at 4 deg.C for 15min until white RNA precipitate is visible at the bottom of the tube, discarding the supernatant, centrifuging again briefly and carefully discarding the residue at the bottom of the tube;

(11) adding 1.3mL of 75% ethanol prepared by DEPC water into a centrifuge tube, inverting the centrifuge tube from top to bottom, washing white RNA precipitate, centrifuging at 4 ℃ for 5min, removing supernatant, centrifuging briefly again, and removing residual liquid at the bottom of the tube by suction;

(12) and (5) volatilizing and drying for 5min to obtain a white precipitate, namely the high-quality total RNA of the adipose tissues of the grass carp mesentery.

6. The method for extracting the high-quality total RNA from the adipose tissue of the mesentery of the grass carp as claimed in claim 5, wherein the centrifugation rotation speed in the steps (5), (6), (8) and (10) is 12000 g; and (3) in the step (11), the centrifugal rotating speed is less than 7500g during the centrifugal operation.

7. The method for extracting high-quality total RNA from the adipose tissue of the mesentery of the grass carp in the claim 5, wherein the lower layer liquid in the step (6) is sucked in an amount of 0.8-1.0 mL.

8. The method for extracting high-quality total RNA from adipose tissue of mesentery of grass carp as claimed in claim 5, wherein the chloroform in step (7) is ice-cooled in advance.

9. The method for extracting high-quality total RNA from adipose tissue of mesentery of grass carp as claimed in claim 5, wherein the number of times of transferring the upper aqueous phase into a new centrifuge tube in step (8) is 3, 200 μ L of the upper aqueous phase is transferred into the centrifuge tube for the first two times, and no more than 100 μ L of the upper aqueous phase is transferred into the centrifuge tube for the last time.

10. The method for extracting the high-quality total RNA from the adipose tissue of the mesentery of the grass carp as claimed in claim 5, wherein the post-treatment stage further comprises the following steps:

(13) adding 30-50 mu L DEPC water to dissolve RNA according to the amount of the white precipitate, flicking the tube bottom to promote dissolution, and centrifuging briefly;

(14) detecting the extraction effect, measuring OD value, and finally storing the total RNA sample at-80 ℃ for later use.

Technical Field

The invention relates to the field of fish RNA extraction, and in particular relates to a method for extracting high-quality total RNA of grass carp mesenteric adipose tissue.

Background

Animal adipose tissue is not only the energy reservoir of the body, but also the active endocrine organ, and various hormones and cytokines secreted by it play important roles in the metabolic balance of the body, immune response, etc. (Scherer, 2006; Wellen, hotemisligil, 2003). In the study of tissue function, the study of molecular level is indispensable, wherein the extraction and purification of RNA are the commonly involved molecular biological experimental techniques. However, fat tissues contain abundant oil and low abundance of RNA, so that high-quality total RNA extraction often has certain difficulty, and the efficient development of downstream experiments is affected.

Currently, RNA extraction is widely performed by classical acidic phenol-guanidinium isothiocyanate-chloroform one-step extraction (Chomczynski, Sacchi, 1987). The method has the advantages of convenient operation, no need of complex equipment assistance and the like, and is provided with mature instant commercial reagents such as TRIzol (Wilfinger et al, 2010). Aiming at the problems of low RNA content in adipose tissues, difficult extraction and the like, most scholars select to perform optimization and improvement on the basis of a one-step method. For example, Wu Jiangwei et al (2006) use pig fat tissue as a material, and found that the quality of RNA extraction can be improved by operations such as increasing the sample size, prolonging the standing lysis time, removing the contamination of the fat layer, and the like. Using similar optimization procedures, higher quality total RNA samples were also obtained in adipose tissue of beef cattle, sheep, etc. (Lina et al, 2018; Xuhongwei et al, 2008).

Grass carp (Ctenophagodon idella) is the highest freshwater economic fish in China, and the excessive deposition of body fat usually occurs in the culture process (Jianpeng et al, 2019). Experiments show that under the operation of the conventional method, the extraction of the RNA of the adipose tissues of the grass carps is easy to degrade, and the sequential promotion of the subsequent molecular biological experiments is interfered. Grass carp adipose tissue accumulates mainly around the abdominal intestinal tract, and there is a significant difference in texture compared to mammalian adipose tissue. Experiments also find that the optimization and improvement method or strategy disclosed at present can not stably obtain the grass carp adipose tissue RNA with high quality. Therefore, it is necessary to research and develop a method for extracting high-quality total RNA from adipose tissue of grass carp to solve the existing problems.

Disclosure of Invention

In order to solve the technical problems, the invention aims to provide a method for extracting high-quality total RNA of grass carp mesenteric adipose tissue, which realizes the high-efficiency extraction of the high-quality total RNA of the grass carp mesenteric adipose tissue by optimizing and improving a conventional acidic phenol-guanidinium isothiocyanate-chloroform one-step extraction method.

In order to achieve the purpose, the invention provides the following technical scheme:

a method for extracting grass carp mesenteric adipose tissue high-quality total RNA (ribonucleic acid), which comprises the following steps: in the pre-treatment stage, grass carp mesentery adipose tissue samples were snap frozen using liquid nitrogen and mechanically homogenized in TRIzol reagent for a long time.

Preferably, the grass carp mesenteric adipose tissue is mechanically homogenized in TRIzol reagent for 3 min.

Preferably, the grass carp mesenteric adipose tissues are quickly frozen in liquid nitrogen in the pretreatment stage, and the frozen samples are not ground manually or mechanically before being cracked by the TRIzol reagent.

The invention also provides a specific extraction method of the grass carp mesenteric adipose tissue high-quality total RNA, which comprises a pretreatment stage and a post-extraction stage, wherein the pretreatment stage comprises the following steps:

(1) wrapping the cleaned homogenizer head by tinfoil, treating at 150 ℃ for 2h, cooling to room temperature, and pre-cooling in a low-temperature refrigerator at-20 ℃ for later use to avoid RNA decomposition caused by overheating of the head due to long-time homogenization;

(2) adding 1.3mL of TRIzol reagent into 1.5mL of a centrifuge tube, and placing the centrifuge tube into a refrigerator at the temperature of 20 ℃ below zero for pre-cooling for later use;

(3) anaesthetizing grass carp, cutting off cervical vertebra, dissecting, quickly taking 30mg of mesenteric adipose tissue, and quickly freezing in liquid nitrogen;

(4) taking out the centrifuge tube and the rotating head of the homogenizer, placing the centrifuge tube and the rotating head of the homogenizer on an ice box, adding the frozen sample into the centrifuge tube, immediately mechanically grinding and homogenizing for 3min, and then carrying out centrifugal operation;

(5) centrifuging homogenized adipose tissue at 4 deg.C for 5min, carefully removing 200 μ L of upper layer oil;

(6) centrifuging at 4 deg.C for 3min, sucking off 100 μ L of upper layer oil, transferring the lower most liquid to another new centrifuge tube, and performing RNA extraction.

As a preferred solution, the post-extraction stage comprises the following steps:

(7) adding 300 mu L of chloroform into the new centrifuge tube, tightly covering the centrifuge tube, violently shaking up and down for 15s, and then standing at room temperature for 5 min;

(8) centrifuging the centrifuge tube at 4 deg.C for 15min, carefully sucking the upper aqueous phase, and transferring into another new centrifuge tube;

(9) adding 500 mu L of isopropanol into a new centrifuge tube, turning upside down and uniformly mixing, and incubating for 5min at room temperature;

(10) centrifuging the tube at 4 deg.C for 15min until white RNA precipitate is visible at the bottom of the tube, discarding the supernatant, centrifuging again briefly and carefully discarding the residue at the bottom of the tube;

(11) adding 1.3mL of 75% ethanol prepared by DEPC water into a centrifuge tube, inverting the centrifuge tube from top to bottom, washing white RNA precipitate, centrifuging at 4 ℃ for 5min, removing supernatant, centrifuging briefly again, and removing residual liquid at the bottom of the tube by suction;

(12) and (5) volatilizing and drying for about 5min to obtain a white precipitate, namely the high-quality total RNA of the adipose tissues of the grass carp mesentery.

As a preferable technical scheme, the rotation speed of the centrifugation in the steps (5), (6), (8) and (10) is 12000 g; and (3) in the step (11), the centrifugal rotating speed is less than 7500g during the centrifugal operation.

As a preferable technical scheme, the suction amount of the lower layer liquid in the step (6) is 0.8-1.0 mL.

As a preferable embodiment, the chloroform in the step (7) is previously subjected to ice-cooling.

As a preferable technical scheme, in the step (8), the upper-layer water phase is transferred into a new centrifugal tube for 3 times, 200 mu L of the upper-layer water phase is transferred into the new centrifugal tube for the first two times, and the last transfer time is not more than 100 mu L.

As a preferred technical solution, the post-treatment stage further comprises the following steps:

(13) adding 30-50 mu L DEPC water to dissolve RNA according to the amount of the white precipitate, flicking the tube bottom to promote dissolution, and centrifuging briefly;

(14) and (3) running the gel to detect the extraction effect, measuring the OD value, and finally storing the total RNA sample at-80 ℃ for later use.

Compared with the prior art, the invention has the following advantages:

the extraction method provided by the invention can improve the yield by increasing the sampling amount to about 30mg so as to meet the requirement of the conventional molecular biology experiment on the RNA amount; the sample is quickly frozen by liquid nitrogen, manual grinding is not needed, the frozen sample is mechanically homogenized in a TRIzol reagent for a long time, the time is about 3min, the occurrence of RNA degradation can be obviously reduced, 2 times of operation steps of centrifuging to remove a grease layer and the like are added, and the purity and the integrity of the obtained total RNA are high. The method effectively solves the problems of easy degradation, low yield and the like of the extraction of the grass carp adipose tissue RNA, has simple operation and stable batch processing effect, can meet the experiment of high-quality RNA requirements such as transcriptome sequencing and the like, and is also suitable for the extraction of the total RNA of the adipose tissues of other fishes.

Drawings

FIG. 1 is a diagram showing the results of agarose gel electrophoresis detection of groups T1-T8;

FIG. 2 is a gel sample image and an electrophoretogram peak image generated by an Agilent2200 bioanalyzer of group T8.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. The reagents, equipment and methods employed in the present invention are all reagents, equipment and methods conventionally available in the art and conventionally used methods, unless otherwise specified.

1. Materials and methods

1.1 Fish for testing

The grass carp used in the experiment is taken from the biotechnology culture base of the Zhujiang aquatic research institute of the Chinese aquatic science research institute. Healthy individuals are randomly selected from the normally raised breeding groups, and the body mass range is about 80-150 g.

1.2 test consumables and instruments

The TRIzol reagent was selected from the group consisting of TaKaRa RNAiso Plus (Code No.9108), DEPC water was purchased from Beyotime, chloroform and isopropanol reagents were purified for domestic analysis, PRO200 type precision homogenizer was purchased from PROscientific (USA), high-speed refrigerated centrifuge 5417R was purchased from Eppendorf (Germany), and TGem micro-spectrophotometer (OSE-260) was purchased from Tiangen Biochemical technology (Beijing) Ltd. RNase-free products were used for the centrifuge tube and pipette tip.

1.3 conventional Total RNA extraction method

The grass carp living body is slightly anesthetized by MS-222, dissected after cutting off spinal column exsanguination, and rapidly cuts off liver, spleen, intestine (without mesenteric fat) and mesenteric adipose tissues, wherein the number of a corresponding sample group is T1-T4. Extracting total RNA of tissues by a conventional method, and testing 3-5 fish in each group, wherein the specific operation steps are as follows:

taking 8-20 mg of a fresh tissue sample, putting the fresh tissue sample into a precooled 1.0mL TRIzol reagent, quickly homogenizing and homogenizing for about 30s, and then standing for 5min at room temperature; adding 300 μ L chloroform, shaking vigorously for 15s, standing at room temperature for 5min, centrifuging at 4 deg.C for 15min (centrifugal speed 12000g), carefully sucking the upper water phase, and transferring to another new centrifuge tube; adding 500 μ L isopropanol, turning upside down, mixing, standing at room temperature for 5min, and centrifuging at 4 deg.C for 15min (centrifugal speed 12000 g); removing supernatant to obtain white RNA precipitate, adding 75% ethanol prepared from 1.3mL DEPC water, performing reversed elution, and centrifuging at 4 deg.C for 5min (centrifugal rotation speed of 7500 g); the supernatant is discarded, the precipitate is dried at room temperature for about 5min, and 30-50 μ L of DEPC treated water is added to dissolve the RNA. After quality inspection, storing in a refrigerator at-80 deg.C.

1.4 optimized Total RNA extraction method

According to the results of preliminary experiments, the sample amount of the fat tissue of grass carp was increased to about 30mg, the TRIzol reagent amount was increased to 1.3 mL/sample, and the optimized operation comparison was performed for the conventional total RNA extraction procedure.

A. Optimization operation 1: taking a fresh sample, mechanically homogenizing in a TRIzol reagent for 30s, centrifuging at 4 ℃ for 5min (the centrifugal rotation speed is 12000g), absorbing and removing about 150 mu L of upper-layer grease, repeating the operation for 1 time, transferring most of residual liquid to a new tube, and performing the rest steps in the same way as the conventional method, wherein the number of the corresponding sample group is T5.

B. And (3) optimizing operation 2: and (3) rapidly freezing a fresh sample in liquid nitrogen, fully grinding the fresh sample in a mortar manually, adding the powdery sample into a TRIzol reagent, cracking the powdery sample by violent oscillation for 30s, and discarding the grease by suction, wherein the number of the corresponding sample group is T6.

C. Optimization operation 3: essentially the same as for optimization run 1, but increasing the time for mechanical homogenization of the sample to about 3min corresponds to sample set number T7.

D. And (4) optimization operation: and (3) taking a fresh sample to be quickly frozen by liquid nitrogen, transferring the frozen sample to a TRIzol reagent, and mechanically homogenizing for about 3min, wherein the number of the corresponding sample group is T8.

Optimization operation 4 the specific operation procedure is as follows.

Preparation work:

(1) cleaning a plurality of scissors, tweezers, a homogenizer rotor and other devices, and performing high-temperature treatment at 150 ℃; freezing in a refrigerator at-20 deg.C;

(2) making ice a little;

(3) preparing liquid nitrogen;

(4) 2.6mL of TRIzol reagent, 800 mu L of chloroform, 1.2mL of isopropanol, DEPC water and absolute ethyl alcohol reagent are prepared according to 2 samples;

(5) preparing a homogenizer, an RNase free centrifuge tube, 200 mu L and 1000 mu L of an RNase-free gun head; opening the low-temperature centrifuge for precooling.

The specific operation steps (taking 2 samples as an example):

(1) taking 2 centrifuge tubes with the volume of 1.5mL, adding 1.3mL of TRIzol reagent, and placing the centrifuge tubes into a refrigerator with the temperature of-20 ℃ for precooling for later use;

(2) after the test fish is anesthetized and the cervical vertebra is cut off for bloodletting, the fish is dissected, 30mg of mesenteric adipose tissue is quickly taken and put into liquid nitrogen for quick freezing;

(3) placing the centrifuge tube on an ice box, adding the frozen sample into the ice box, and immediately mechanically grinding and homogenizing for 3 min;

(4) centrifuging at 4 deg.C for 5min at 12000g, and carefully sucking off about 200 μ L of the oil layer;

(5) centrifuging at 4 deg.C for 3min at 12000g, and carefully sucking off about 100 μ L of the oil layer; carefully sucking most of the red liquid on the lower layer into a new centrifuge tube, wherein the amount of the red liquid is about 0.8-1.0mL, and discarding a small part of the bottom layer;

(6) adding 300 μ L chloroform (precooling on ice), covering the centrifuge tube, shaking vigorously up and down for 15s, and standing at room temperature for 5 min;

(7) centrifuging at 4 deg.C for 15min at 12000g, carefully sucking the upper water phase into another new tube after centrifuging, transferring for 3 times, transferring 200 μ L for the first 2 times, transferring not more than 100 μ L for the next time, and transferring with fine movement without collision of the gun head with the tube wall and the lower layer material;

(8) adding 500 μ L isopropanol, turning upside down, mixing, and incubating at room temperature for 5 min;

(9) centrifuging at 4 ℃ for 15min, wherein the centrifugal rotating speed is 12000 g; during the period, a 2-tube 75% ethanol (1mL ethanol +334 μ L DEPC water) is prepared for standby;

(10) white precipitate, namely RNA material, appears after centrifugation, supernatant is discarded, the centrifugation is carried out briefly, and residual liquid at the bottom of the tube is discarded without touching the precipitate during the discard;

(11) adding 1.3mL of 75% ethanol, and washing the white precipitate by turning upside down;

(12) centrifuging at 4 deg.C for 5min at a rotation speed of less than 7500g, discarding the supernatant, centrifuging briefly and discarding the residual liquid;

(13) volatilizing and drying for about 5min without over-drying to obtain high-quality total RNA;

(14) adding 30-50 mu L DEPC water to dissolve RNA according to the amount of the precipitate, flicking the tube bottom to promote dissolution, and centrifuging briefly;

(15) detecting extraction effect by running glue, measuring OD value, etc., and storing at-80 deg.C for use.

It should be noted that: the above-mentioned "brief centrifugation" means short-time centrifugation, and the centrifugation time is controlled to be varied from 10s to 1min depending on the specific situation.

3-5 fish of each group (T1-T8) are tested.

1.5RNA quality testing

2.0. mu.L of each RNA sample was taken, and the concentration and purity of the RNA sample were determined using a microspectrophotometer. Preparing 1.5% non-denaturing agarose gel, taking 1.0-2.0 μ L of proper amount of RNA, carrying out electrophoresis at 140V for 15min, observing the total RNA band condition under ultraviolet light, and evaluating the extraction quality.

From the T8 group with better extraction quality, 3 samples were randomly taken and sent to Jinwei Zhi Biotech Co., Ltd for high-throughput sequencing platform quality inspection, which mainly comprises detecting total RNA concentration and quality by using a NanoDrop 2000 spectrophotometer and evaluating RNA integrity by an Agilent2200 bioanalyzer.

2. Results

2.1 comparison of quality of Total RNA extracted from different tissues of grass carp by conventional method

The quality detection conditions of the total RNA of different tissues of the grass carp extracted by the conventional method are shown in table 1. Experiments show that compared with liver, spleen and intestinal tissues, the extraction amount of total RNA of the adipose tissues of the mesentery of the grass carp is less, which indicates that the RNA content of the adipose tissues of the grass carp is lower, and the tissue amount (25-35mg) is properly increased in the optimization operation, so that the extraction amount is improved, and the requirement of the conventional biomolecule test dosage can be met. Under the conventional extraction method, the A260/A280 ratio of the total RNA of the liver, spleen, intestine and mesenteric adipose tissues of the grass carp is basically kept at about 2.0 and accords with the standard range of 1.9-2.0, which indicates that the nucleic acid purity of different tissues extracted by the conventional method is higher. The A260/A230 ratio of the sample is slightly less than 2.0, which indicates that trace impurities such as guanidine salt or organic solvent can remain, and the practical experience has slight influence on downstream molecular tests.

TABLE 1 Total RNA quality by spectrophotometric assay

Note: T1-T4 are total RNA of liver, spleen, intestine and mesenteric adipose tissue of grass carp extracted by conventional method respectively; T5-T8 is total RNA of the mesenteric adipose tissue of the grass carp extracted by different optimization methods, and the method is as follows.

The samples were subjected to agarose gel electrophoresis and FIG. 1 shows the results of detection of representative samples of each group (M in FIG. 1 represents DL2000 DNA molecular weight standard). As can be seen from FIG. 1, total RNA of liver, spleen and intestine tissue samples of T1-T3 groups shows clear 28s and 18s rRNA characteristic bands on gel, the fat tissue samples of T4 group show severe dragging phenomenon, and the band brightness of 28s rRNA is obviously weaker than that of 18s rRNA, which indicates that the grass carp fat tissue RNA extracted by the conventional method has degradation phenomenon. The comparative tests among different tissues show that the content of RNA in the mesenteric adipose tissues of the grass carps is low, and the degradation is easy to occur.

2.2 comparison of total RNA extracted from fat tissue of grass carp by different optimization operations

The detection of a spectrophotometer shows that the purity of the total RNA of the fat tissue of the grass carp extracted by the 4 optimization methods is kept at a higher level. Further, from the agarose gel electrophoresis result (fig. 1), it is found that the RNA degradation problem of the T5 group sample is not effectively improved by simply removing the grease layer, while the RNA degradation is alleviated but still has a certain dragging phenomenon by changing the liquid nitrogen quick-freezing grinding mode to the pretreatment of the T6 group sample. When the mechanical homogenization time of the samples is increased only by the T7 group samples, the RNA degradation problem is remarkably improved, particularly, when the mechanical homogenization time of the samples is increased while the liquid nitrogen is adopted for quick freezing, the total RNA of the extracted T8 group adipose tissues shows electrophoresis band characteristics with higher quality.

Experiments show that in the TRIzol reagent cracking process, the RNA degradation degree can be obviously reduced by increasing the mechanical homogenization time (about 3min) of a sample, and the degradation problem possibly caused by sampling operation can be relieved by adopting liquid nitrogen quick-freezing treatment on the sample before homogenization.

2.3 quality control analysis of total RNA extracted from grass carp adipose tissue

To further specifically optimize the quality of total RNA samples extracted in procedure 4, T8 groups of 3 samples were randomly selected and sent to the sequencing company for quality control analysis. The detection shows that the RNA purity of 3 samples is high, the key RIN (RNA integrity number) value range is 8.7-9.0 (see table 2 for details), and according to the company rating standard, the 3 samples are all the best grade A (grade A standard: nucleic acid quality is more than or equal to 2 mug, A260/A280 is more than or equal to 2.0, RIN value is more than or equal to 7, 28S/18S is more than or equal to 1.0, nucleic acid concentration is more than or equal to 100mg/L, volume is more than or equal to 10 mug, 5S peak shape is normal, and the like), which indicates that the RNA integrity is good, and the quality meets the sequencing requirement of high-throughput transcriptome construction library. In addition, from the gel sample image and the electrophoresis chromatogram peak image (fig. 2) generated by the Agilent2200 bioanalyzer, the quality level of 3 total RNA samples is higher, the peak image base line is smoother, no obvious degradation impurity peak exists, and the peak shapes of 28S and 18S are normal.

Table 2T 8 quality of total RNA extracted from grass carp mesentery fat by improved method

3. Analysis of

Rnases are proteases that specifically hydrolyze RNA phosphodiester bonds, are widely present in the environment, and can withstand harsh treatment conditions such as high temperature boiling (Evans, Kamdar, 1990). Therefore, the need to obtain high quality RNA requires strict precautions against disruption by both endogenous and exogenous RNases. In order to avoid the interference of RNase in the external environment, RNase-free consumables, clean experimental environment and scientific standard operation are usually required. In addition, in order to effectively inhibit the endogenous RNase contained in the tissue cells of the sample, it is necessary to rely on a potent protein denaturing agent such as guanidine isothiocyanate among the cleavage reagents.

Obviously, in the scheme, the RNA degradation problem of the grass carp mesenteric adipose tissue is mainly caused by the fact that the conventional extraction method cannot effectively prevent the hydrolysis of endogenous RNA enzyme. In the preliminary analysis, RNA degradation may occur during the initial stages of sampling, during tissue lysis, or both. For this reason, in the sampling stage, the method of liquid nitrogen quick freezing artificial grinding is tried, and it is desired to suppress the activity of rnase in the tissue and shorten the air exposure time of the sample as much as possible by using an ultra-low temperature environment. However, multiple practical operations find that the liquid nitrogen quick-freezing grinding operation has certain relief on the RNA degradation of the sample, but gel electrophoresis still shows dragging phenomena of different degrees, even a severe degradation example frequently occurs, which indicates that the RNA degradation also occurs in the tissue lysis process. This means that the composition of guanidine isothiocyanate or the like in the TRIzol reagent cannot effectively inhibit RNase in mesenteric adipose tissue. Considering adipose tissue-specific factors, it is presumed that a large amount of oil contained in adipose tissue is likely to influence the efficacy of the protein denaturant.

How to break the influence of the grease is that scholars use the characteristic of low specific gravity of liquid oil drops to remove grease layers by simple centrifugation (Liu article, etc., 2013); there are also researchers who have been adjusting the formulation of the agents to enhance the separation of fats and oils by fat emulsifiers (Sharma et al, 2018). The method is not ideal in effect of verifying the mesentery fat of the grass carp. Based on the above practical situation, it is attempted to add physical means such as vigorous shaking during the cracking process. After repeated groping tests, the RNA degradation is obviously improved and the batch verification effect is stable when the sample is mechanically homogenized and the homogenization time is prolonged from the original time of about 30s to about 3 min. It is presumed that the continuous shearing impact force generated by mechanical homogenization for a long time may contribute to effective inhibition of endogenous rnase by breaking through the oil droplet barrier by the guanidine isothiocyanate and other components in the TRIzol reagent.

Specifically, the mesenteric adipose tissue of the grass carp is actually wrapped with pancreatic tissue which is difficult to separate. It is generally believed that the pancreatic tissue of animals is rich in rnases and RNA is difficult to extract (Griffin et al, 2012; Kiba et al, 2007), which may be one of the reasons for the degradation of RNA in the mesenteric adipose tissue of grass carps.

4. Conclusion

The method for extracting the high-quality total RNA from the adipose tissues of the grass carp mesentery is improved on the basis of the conventional method, the sampling amount is increased to about 30mg, and the RNA yield is favorably improved so as to meet the requirement of the conventional biomolecule test; the pretreatment of the sample adopts a liquid nitrogen quick freezing mode (non-artificial grinding), and the sample is homogenized in a TRIzol reagent for a long time to about 3min, so that the occurrence of RNA degradation is remarkably reduced, and operation steps of centrifuging for 2 times to remove an oil layer and the like are added. The total RNA obtained by the method has higher purity and integrity and stable batch processing effect, and can meet the test requirements of transcriptome library construction sequencing and the like.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and their concepts should be considered to be equivalent or modified within the technical scope of the present invention.