CN104031910A - High-efficiency extraction method of total RNA (ribonucleic acid) in freshwater crab haemolymph - Google Patents

Abstract

The invention discloses a method for efficiently extracting total RNA in freshwater crabs, especially the hemolymph of river crabs. The steps include: (1) collection of hemolymph: sucking the hemolymph of river crabs from the base of the crab legs with a sterilized needle with a total volume of 1mL (2) Extraction of total RNA: Treat the absorbed hemolymph with Trizol reagent, centrifuge to get the supernatant, add chloroform, then centrifuge to get the supernatant, add isopropanol, centrifuge again, then remove the supernatant and add 75% ethanol Wash the RNA, centrifuge and dry the pellet for storage. The method of the invention can simply, quickly and efficiently extract the RNA in the hemolymph of river crabs. The extracted RNA is large in quantity and high in quality, free from protein contamination, and can be used in various molecular biological analyzes with strong application value.

Description

A high-efficiency extraction method of total RNA in freshwater crab hemolymph

technical field

The invention relates to a simple, rapid and high-quality method for extracting total RNA in the hemolymph of river crabs.

Background technique

River crab, scientific name Eriocheir sinensis , belongs to Arthropoda, Crustacca, Decapo, Grapsidae, Eriocheir , endemic to China The aquatic economic animals are widely distributed in the Liaohe River, Yangtze River, Oujiang River, Minjiang River and other water systems. Since the breakthrough in the artificial breeding technology of Chinese mitten crab in the 1970s, the Chinese mitten crab industry has gradually developed into one of the pillar industries of my country's aquaculture industry, with an annual output value of nearly 40 billion yuan. The best and most widely farmed industry. In the past few decades of industrial development, with the rapid development of Chinese mitten crab breeding, phenomena such as mixed germplasm resources, precocious puberty, individual miniaturization, and explosive diseases have frequently occurred in the Chinese mitten crab population, which has a great impact on my country. The river crab industry has caused huge impact and loss. Along with this, the scientific research on Eriocheir sinensis is constantly expanding and deepening, especially the research on molecular biology is the most in-depth. Therefore, the extraction and application of total RNA of Eriocheir sinensis is becoming more and more extensive.

On the other hand, due to the needs of experiments, it is necessary to collect RNA from river crab samples multiple times or at different time periods for various experimental studies, requiring the development of a non-damaging or non-death RNA collection technology. The collection of RNA from hemolymph can realize multiple or multi-time sampling without taking dead crab samples, effectively ensuring the smooth progress of related experiments.

Eriocheir sinensis belongs to an open circulatory system, and its blood is colorless, composed of lymphocytes, blood cells (agranular cells, small granular cells and large granular cells) and related body fluids, so it is collectively called hemolymph. The characteristics of the open hemolymph circulation of river crabs are interlinked with body tissues, which makes it difficult to sample hemolymph, and it is difficult to collect a sufficient amount of hemolymph, which affects subsequent RNA extraction and other work. However, high-quality RNA samples are often required in related research on the germplasm, genetics, and diseases of river crabs. In addition, during the RNA extraction process, the hemolymph tissue of river crabs is easy to coagulate, and it is easy to react with Trizol reagent to form lumps, which makes it difficult to extract high-quality RNA, which seriously affects subsequent related research work. Inventing a simple and efficient method for extracting RNA from river crab hemolymph is very important for scientific research and industrial development.

Contents of the invention

The purpose of the present invention is to provide a method for efficiently extracting total RNA from river crab hemolymph, which is a stable technology developed by the inventors in the research practice. This method can ensure that the total RNA extracted from river crab hemolymph is large in quantity and high in quality , can be used for a variety of downstream analyzes to meet the needs of experimental RNA samples, and can ensure that crabs can continue to survive after hemolymph extraction, and can achieve multiple or multiple time periods of RNA extraction.

The technical scheme provided by the invention is: a method for extracting total RNA from river crab hemolymph, the steps of which are as follows:

(1) Take live crab samples for analysis, preferably with an individual size of more than 10g, to ensure that enough hemolymph can be extracted;

(2) Take ice, take the centrifuge tube and add 1mL Trizol reagent, put it on crushed ice;

(2) Extract hemolymph from river crab;

(3) Extract total RNA, the steps are as follows:

a. Add the crab hemolymph extracted in step (2) into a centrifuge tube containing Trizol reagent, mix the hemolymph tissue with Trizol reagent, and let stand at room temperature for 3-10 minutes, preferably 5 minutes;

b. Centrifuge, take the supernatant and add chloroform;

c. Centrifuge, take the supernatant and add isopropanol;

d. Discard the supernatant after centrifugation, add 75% ethanol to precipitate RNA;

e. Discard the supernatant after centrifugation; dry the precipitated RNA;

f. Add RNase-free water to dissolve the precipitate.

Among them, in step (2), in the process of extracting the hemolymph of the river crab, the oblique section of the tip of the needle is upward, the insertion depth is 2mm, and the river crab is inclined towards the insertion end, and extracted at a uniform speed.

Wherein, in step a of step (3), the mixing of hemolymph tissue and Trizol reagent is vigorously shaken by hand first, and then shaken on a vortex shaker to form a uniform cloudy solution.

The invention is also applicable to the RNA extraction of hemolymph of other freshwater crabs, so the invention also claims the extraction of RNA of hemolymph of freshwater crabs.

The present invention has the following advantages: the method of the present invention is the rapid collection of hemolymph and the design of experimental steps by the inventor during the continuous groping process for the extraction of the hemolymph RNA of river crabs, using the hemolymph of river crabs as a material, and extracting the hemolymph of river crabs by the Trizol method The total RNA solves the problem that the hemolymph RNA of river crab is not easy to extract, including the operation of the collection method and extraction steps. This method is simple, fast and effective for the extraction of total RNA in the hemolymph of river crabs. The extracted RNA is sufficient in quantity and high in quality, and is almost free from protein contamination. Survival can realize multiple or multiple time periods of RNA extraction, providing an effective technical means for the study of genetic diversity, germplasm protection and resource utilization of river crabs, which has great scientific significance and practical value.

Description of drawings

Fig. 1 is the electrophoresis detection result of the total RNA extracted in the present invention.

Detailed ways

The specific embodiment of the present invention is as follows:

1. Preparation of experimental reagents

1mL sterile syringe and several 1.5mL centrifuge tubes. Wipe the test bench with 70% ethanol solution. Using crushed ice produced by an ice machine, add 1 mL of Trizol reagent to the centrifuge tube and place it on ice.

Second, crab material preparation

Take living mitten crabs, in order to improve the total amount of total RNA extraction, it is best to take individuals with a body weight of about 10 g or more as experimental materials. Wipe off the moisture on the crab body surface with gauze.

3. Hemolymph collection

Hemolymph collection was performed with a sterilized 1 mL syringe. The needle tip of the syringe is obliquely cut upwards, and is inserted into the crab at the base of the foot for about 2mm, and the crab is inclined towards the insertion point, and extracted at a uniform speed. If it is necessary to ensure that the river crab can continue to survive, the extraction volume should not be too large. When extracting RNA, each test tube needs about 300 μL of hemolymph. Note that if the collection method is not correct, enough hemolymph cannot be collected, thereby affecting the amount of RNA extraction.

4. Total RNA Extraction

1. Quickly add the extracted hemolymph into the centrifuge tube containing Trizol reagent, shake vigorously by hand for 15 seconds, then shake on a vortex shaker for 1 minute to make it into a suspension state, and let it stand at room temperature for 5 minutes. (This is the second key step. Since the crab hemolymph will react with the Trizol reagent to form a solid block, which will affect the amount of RNA extraction, the shaking must be uniform and the duration should be 1 minute.)

2. Place in a centrifuge at 4°C, 12,000 rpm, and centrifuge for 5 minutes.

3. Carefully extract the supernatant, pour it into another centrifuge tube, then add 200 μL of chloroform, shake vigorously for 15 seconds, and let stand at room temperature for 5 minutes.

4. Place in a centrifuge at 4°C, 12,000 rpm, and centrifuge for 15 minutes.

5. Carefully extract the supernatant, pour it into another centrifuge tube, add an equal volume of isopropanol and mix gently up and down for 15 seconds, then let stand at room temperature for 10 minutes.

6. Place in a centrifuge at 4°C, 12,000 rpm, and centrifuge for 10 minutes.

7. Discard the supernatant, add 1 mL of 75% ethanol to the centrifuge tube to wash the precipitated RNA, and pipette the RNA pellet up with a pipette tip to obtain a better cleaning effect.

8. Place in a centrifuge at 4°C, 12000 rpm, and centrifuge for 5 minutes.

9. Carefully discard the supernatant and let stand at room temperature for 2-3 minutes to dry the RNA pellet. (This is the third key step, and the drying time should not exceed 3 minutes to avoid RNA degradation caused by excessive drying)

10. Add 30-50 μL of RNase-free ddH ₂ o to dissolve, and immediately store in a low-temperature refrigerator at -80°C.

4. RNA Detection

1. Detection by UV spectrophotometer

Take 2 μL of the RNA solution, add 98 μL of deionized water to dilute it 50 times, and use a 752-type ultraviolet spectrophotometer to detect the absorbance value of the sample at OD values of 260 and 280 nm. The purity of RNA was determined according to the value of OD ₂₆₀ /OD ₂₈₀ . The OD ₂₆₀ / OD ₂₈₀ ratio of high-quality RNA solution is between 1.8-2.1, and a ratio of 2.0 is a sign of high-quality RNA.

After testing, the OD ₂₆₀ / OD ₂₈₀ ratio of the RNA sample extracted by this method is between 1.80-2.05, which shows that the total RNA extracted by this method from the hemolymph of Chinese mitten crab has good quality and high purity.

The above-mentioned main test is the purity of RNA, and its amplification effect and use efficiency need further testing, such as electrophoresis testing.

2. Electrophoretic detection

Take 3 μL of RNA solution, and use 1.5% agarose gel for electrophoresis (voltage 110V, time 30min) for detection, and observe the clarity of 28S, 18S, and 5S three RNA bands in the gel imaging analysis system.

After electrophoresis detection, the RAN extracted by this method can clearly distinguish 28S, 18S and 5S three RNA bands, indicating that the quality of RNA extraction is good and can be used for other follow-up studies. Specifically shown in Figure 1.

Claims (5)

1. a highly effective extraction method of total RNA in fresh water crab hemolymph, is characterized in that comprising the steps:

(1) get aseptic centrifuge tube and add 1mL Trizol reagent, being placed on trash ice;

(2) hemolymph extracts;

(3) extract total RNA, step is as follows:

The river crab hemolymph that (2) step is extracted adds in the centrifuge tube of (1) step, hemolymph tissue and Trizol reagent is mixed to the standing 3-10 minute of room temperature;

Centrifugal, get supernatant liquor and add chloroform;

Centrifugal, get supernatant liquor and add Virahol;

After centrifugal, abandon supernatant liquor, precipitated rna adds 75% alcohol;

After centrifugal, abandon supernatant liquor; Drying precipitated RNA;

In precipitation, add RNase-free water dissolution.

2. the method for extraction fresh water crab hemolymph RNA according to claim 1, is characterized in that: in (2) step, in the process that extracts river crab hemolymph, upwards, depth of penetration 2mm, tilts river crab needle tip diagonal plane to insertion end, at the uniform velocity extracts.

3. the method for extraction fresh water crab hemolymph RNA according to claim 1, is characterized in that: a step in (3) step, hand concuss is first used in mixing of hemolymph tissue and Trizol reagent, then concussion is uniform dirty solution on vortex oscillation device.

4. the method for extraction fresh water crab hemolymph RNA according to claim 1, is characterized in that: in (3) step a step, and standing 5 minutes of room temperature.

5. according to the method for the extraction fresh water crab hemolymph RNA described in claim 1 to 5 any one, it is characterized in that: described fresh water crab is river crab.