Fish ovary RNA extraction method

Abstract

The invention discloses a fish ovarian RNA (Ribonucleic Acid) extraction method which comprises the following steps: taking out ovarian tissues from living fish, and soaking the ovarian tissues into liquid nitrogen for freezing; taking out the frozen ovarian tissue, and grinding the ovarian tissue in liquid nitrogen; transferring the ground ovarian tissue into a pre-cooled tube, adding pre-cooled Trazol, oscillating, standing, centrifuging, and taking a supernatant; and purifying, precipitating and purifying in sequence to obtain the fish ovary RNA. The fish ovary RNA extracted by adopting the extraction method disclosed by the invention is not easy to degrade, meets scientific research experiment standards, and is high in extraction efficiency, simple and convenient to operate and high in universality.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology. More specifically, the present invention relates to an efficient method for extracting RNA from fish ovarian tissue. Background Art

[0002] With the continuous development of molecular biology and genetic engineering technologies, the research on the reproductive physiology and genetic characteristics of fish has become increasingly in-depth. As an important part of the reproductive system, the function regulation and development process of fish ovaries have received extensive attention. Ovarian RNA plays a crucial role in fish reproduction, sex determination and differentiation, reproductive capacity assessment, breeding improvement, etc.

[0003] At present, the technology for extracting fish ovarian RNA mainly faces problems such as easy degradation of RNA, difficult removal of impurities, low extraction efficiency, long time consumption, and poor universality in practice. In the article "Research on the Mechanism of Fish Oil Promoting the Ovarian Development of Scatophagus argus", a method for extracting RNA from the ovaries of Scatophagus argus is disclosed. The method includes the steps of grinding the ovarian samples taken out from an ultra-low temperature freezer at -80°C with liquid nitrogen, adding Trazol and shaking, standing at 4°C for 2 - 10 minutes, pipetting until it becomes transparent, centrifuging at 4°C, and taking the supernatant, and then successively adding chloroform, isopropanol, and ice ethanol for treatment to obtain RNA. The ovarian RNA extracted by this method is also prone to degradation, resulting in extraction failure, and this method is mainly applicable to the extraction of ovarian RNA from Scatophagus argus, with poor universality. Summary of the Invention

[0004] Based on this, the purpose of the present invention is to provide a method suitable for extracting fish ovarian RNA, which can extract fish ovarian RNA that is not easily degraded, has high extraction efficiency, and high universality.

[0005] The specific technical solutions for achieving the above invention purposes are as follows.

[0006] A method for extracting fish ovarian RNA, comprising the following steps:

[0007] (1) Take out the ovarian tissue from a live fish and immerse it in liquid nitrogen for freezing; the time from taking out the ovarian tissue to freezing does not exceed 90 s;

[0008] (2) Take out the frozen ovarian tissue in step (1) and grind it in liquid nitrogen;

[0009] (3) Transfer the ground ovarian tissue to a pre-cooled tube, add Trazol pre-cooled to 0°C - 1°C, shake and then stand, centrifuge and take the supernatant; the time from transferring the ovarian tissue to starting to shake does not exceed 40 s;

[0010] (4) Successively carry out purification, precipitation, and purification to obtain fish ovarian RNA.

[0011] When the inventors of the present invention were researching the technology for extracting RNA from fish ovaries, they found that the time from starting to remove ovarian tissue from live fish to performing liquid nitrogen freezing, and the time from transferring the ground ovarian tissue into a tube to starting to oscillate with Trazol, are crucial for the successful extraction of fish ovarian RNA. By controlling the time of these two key steps and coordinating with other steps, the 28S and 18S bands in the gel electrophoresis detection of the fish ovarian RNA extracted are bright, and the 5S band is not bright. Therefore, the fish ovarian RNA extracted by the method of the present invention is not easily degraded, meets the scientific research experiment standards, and has a high extraction efficiency.

[0012] In addition, the method for extracting fish ovarian RNA of the present invention is simple to operate and has strong universality, and is suitable for the extraction of RNA from the ovaries of all fish. Brief Description of the Drawings

[0013] Figure 1 It is the gel electrophoresis diagram of the fish ovarian RNA extracted in Example 1 of the present invention.

[0014] Figure 2 It is the gel electrophoresis diagram of the fish ovarian RNA extracted in Example 2 of the present invention.

[0015] Figure 3 It is the gel electrophoresis diagram of the fish ovarian RNA extracted in Comparative Example 1 of the present invention.

[0016] Figure 4 It is the gel electrophoresis diagram of the fish ovarian RNA extracted in Comparative Example 2 of the present invention.

[0017] Figure 5 It is the gel electrophoresis diagram of the fish ovarian RNA extracted in Comparative Example 3 of the present invention.

[0018] Figure 6 It is the gel electrophoresis diagram of the fish ovarian RNA extracted in Comparative Example 4 of the present invention.

[0019] Figure 7 It is the gel electrophoresis diagram of the fish ovarian RNA extracted in Comparative Example 5 of the present invention. Detailed Description of the Invention

[0020] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosed content of the present invention more thorough and comprehensive.

[0021] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0022] In some embodiments of the present invention, a method for extracting fish ovarian RNA is disclosed, comprising the following steps:

[0023] (1) Take out ovarian tissue from a live fish and immerse it in liquid nitrogen for freezing; the time from taking out the ovarian tissue to freezing does not exceed 90 s;

[0024] (2) Take out the frozen ovarian tissue in step (1) and grind it in liquid nitrogen;

[0025] (3) Transfer the ground ovarian tissue to a pre-cooled tube, add pre-cooled Trazol, shake it and then let it stand, and centrifuge to take the supernatant; the time from transferring the ovarian tissue to starting to shake does not exceed 40 s, and ensure that Trazol does not freeze during shaking;

[0026] (4) Perform purification, precipitation, and purification in sequence to obtain fish ovarian RNA.

[0027] In some embodiments, the time from taking out the ovarian tissue to freezing in step (1) does not exceed 80 s.

[0028] In some embodiments, the time from taking out the ovarian tissue to freezing in step (1) does not exceed 60 s.

[0029] In some embodiments, the time from transferring the ovarian tissue to starting to shake in step (3) does not exceed 35 s.

[0030] In some embodiments, the time from transferring the ovarian tissue to starting to shake in step (3) does not exceed 30 s.

[0031] In some embodiments, in step (3), the Trazol is pre-cooled to 0 °C - 1 °C.

[0032] In some embodiments, in step (3), the volume-mass ratio of the Trazol to the ovarian tissue is 1.5 mL - 2 mL: 1 mg.

[0033] In some embodiments, when taking out the ovarian tissue in step (2), the ovarian tissue is kept frozen all the time.

[0034] In some of these embodiments, the volume-mass ratio of the liquid nitrogen to the ovarian tissue in step (2) is 2 mL to 3 mL: 1 mg.

[0035] In some of these embodiments, during the grinding process in step (2), the ovarian tissue is always immersed in the liquid nitrogen.

[0036] In some of these embodiments, during the oscillation in step (3), sterilized steel beads are also added.

[0037] In some of these embodiments, the oscillation time in step (3) is 2 min to 3 min, and the oscillation speed is 30 Frequency / S to 40 Frequency / S.

[0038] In some of these embodiments, in step (3), the temperature for standing is 2°C to 4°C, and the standing time is 4 min to 6 min.

[0039] In some of these embodiments, in step (3), the centrifugation conditions are: 2°C to 4°C, 15000 rpm / min to 18000 rpm / min, and centrifugation for 6 min to 8 min.

[0040] In some of these embodiments, the purification in step (4) is carried out using chloroform.

[0041] In some of these embodiments, the purification includes the steps of: adding chloroform pre-cooled to 2°C to 4°C and stored in the dark to the supernatant, shaking vigorously for 10 s to 15 s, then shaking slightly for 4 s to 5 s, and standing for centrifugation.

[0042] In some of these embodiments, the volume ratio of the chloroform to the supernatant is 3 to 4:1.

[0043] In some of these embodiments, the temperature for standing is 2°C to 4°C, and the standing time is 2 min to 3 min.

[0044] In some of these embodiments, the centrifugation conditions are: 2°C to 4°C, 15000 rpm / min to 18000 rpm / min, and centrifugation for 6 min to 8 min.

[0045] In some of these embodiments, the precipitation in step (5) is carried out using isopropanol.

[0046] In some of these embodiments, the precipitation includes the steps of: adding isopropanol pre-cooled to 2°C to 4°C to the supernatant, shaking slightly for 10 s to 12 s, standing for centrifugation, and discarding the supernatant.

[0047] In some of these embodiments, the volume ratio of the isopropanol to the supernatant is 0.8 to 1.2:1.

[0048] In some of these embodiments, the temperature for standing is 2°C to 4°C, and the time for standing is 120 s to 150 s.

[0049] In some of these embodiments, the conditions for centrifugation are: 2°C to 4°C, 15000 rpm / min to 18000 rpm / min, and centrifugation for 6 min to 8 min.

[0050] In some of these embodiments, in step (6), the purification is carried out using ethanol.

[0051] In some of these embodiments, the purification includes the steps of: adding 70% to 80% ethanol pre-cooled to 2°C to 4°C to the precipitate, shaking to suspend the precipitate, and standing and centrifuging.

[0052] In some of these embodiments, the temperature for standing is 2°C to 4°C, and the time for standing is 4 min to 6 min.

[0053] In some of these embodiments, the conditions for centrifugation are: 2°C to 4°C, 15000 rpm / min to 18000 rpm / min, and centrifugation for 2 min to 3 min.

[0054] In some of these embodiments, after the purification, there is also a drying step, and the drying includes: sucking off the purification solvent and drying in ventilation at 15°C to 20°C for 2 min to 4 min.

[0055] In some of these embodiments, the fish include fish such as tuna, Trachinotus ovatus, tilapia, and pink snapper whose ovarian RNA is easily degraded.

[0056] In the following embodiments of the present invention, the raw materials used, unless otherwise specified, are all conventional commercially available products; the methods used, unless otherwise specified, are all conventional methods in the art.

[0057] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0058] Example 1 Extraction of ovarian RNA from Scatophagus argus

[0059] In May 2023, adult female Scatophagus argus (n > 500) with a body weight of about 180 g were selected for the extraction of ovarian RNA. Before sampling, the adult female fish were aerated and temporarily cultured in a breeding pond for 3 days without feeding to maintain the vitality of the fish. The specific method is as follows:

[0060] (1) Sampling: Before sampling, the operating table was simply sterilized by spraying alcohol. Sampling personnel need to wear masks and disposable nitrile gloves. Small pieces of ovarian tissue obtained from dissection were quickly immersed in liquid nitrogen for quick freezing. The average time from in vivo sampling to freezing was 60 s. The samples after quick freezing treatment were transferred to cryotubes and stored in liquid nitrogen.

[0061] (2) Grinding: Take out the sample tube frozen and stored in liquid nitrogen and place it on dry ice. Use sterile forceps to quickly take out the sample from it and place it in a mortar pre-added with liquid nitrogen (which needs to be soaked in DEPC water for 2 hours before use, then autoclaved, and finally dried at 200 °C for 2 h). The amount of liquid nitrogen added was 3 mL / mg of sample. Grind quickly, and keep the sample immersed in liquid nitrogen during grinding.

[0062] (3) Fixation: After grinding, transfer the sample to a centrifuge tube with dry ice buried at the bottom (for pre-cooling). Add pre-cooled Trazol at 0 °C to the sample at a rate of 2 mL / mg of sample. Add sterilized steel beads and shake strongly for 2 min (the shaking speed is 30 Frequency / S). The average time from transferring the sample to the centrifuge tube to starting to shake does not exceed 30 s to ensure that Trazol does not freeze. After shaking, place the centrifuge tube in an environment at 4 °C and let it stand for 6 min. Then centrifuge at 4 °C and 18000 rpm / min for 6 min.

[0063] (4) Purification: Take 100 μL of the supernatant after centrifugation in step (3) and add 300 μL of pre-cooled chloroform (chloroform) at 2 - 4 °C and protected from light. Shake vigorously for 15 s, then reduce the shaking intensity and shake for 5 s (to prevent RNA fragmentation). Then let it stand in an environment at 4 °C for 3 min, and then centrifuge at 4 °C and 18000 rpm / min for 6 min.

[0064] (5) Precipitation: Take 300 μL of the supernatant and place it in a centrifuge tube pre-cooled to 4 °C. Add an equal volume of pre-cooled isopropanol at 2 - 4 °C. Shake gently for 10 s, let it stand in an environment at 4 °C for 120 s, and then centrifuge at 4 °C and 18000 rpm / min for 6 min.

[0065] (6) Purification: Discard the supernatant in step 5, add 1 mL of pre-cooled 75% ice ethanol at 4 °C, gently shake to suspend the particles, let it stand at 4 °C for 5 min, and then centrifuge at 18000 rpm / min for 3 min.

[0066] (7) Drying: Aspirate the ethanol and dry it in a fume hood at 15 - 20 °C for 4 min to obtain fish ovarian RNA.

[0067] (8) Storage: Add 40 μL of deionized water to dissolve the RNA, measure the concentration, adjust it to 600 μg / mL, and store it at -80 °C for subsequent use.

[0068] The RNA extracted from fish ovaries in this example was detected by gel electrophoresis. The results of some samples are as Figure 1 shown. As can be seen from Figure 1 , for the RNA extracted from fish ovaries in Example 1, the 28S and 18S bands are bright, and the 5S band is not bright, meeting the standards for scientific research experiments, indicating that the method used in this example for extracting RNA from fish ovaries is not easily degraded and has high extraction efficiency.

[0069] Extraction of RNA from the ovaries of Trachinotus ovatus in Example 2

[0070] In May 2023, adult female Trachinotus ovatus with a body weight of about 1800 g (n > 500) were selected for the extraction of ovarian RNA. Before sampling, the adult female fish were aerated and temporarily cultured in the breeding pond for 3 days without feeding to maintain the vitality of the fish. The specific method is as follows:

[0071] (1) Sampling: Before sampling, the operation table was simply sterilized by spraying alcohol. The sampling personnel needed to wear masks and disposable nitrile gloves. The small pieces of ovarian tissue obtained by dissection were quickly immersed in liquid nitrogen for quick freezing treatment. The average time from live sampling to freezing was 90 s. The samples after quick freezing treatment were transferred to cryotubes and stored in liquid nitrogen.

[0072] (2) Grinding: Take out the sample tube frozen and stored in liquid nitrogen and place it on dry ice. Use sterile forceps to quickly take out the sample and place it in a mortar pre-added with liquid nitrogen (which needs to be soaked in DEPC water for 2 hours before use, then autoclaved, and finally dried at 200 °C for 2 h). The amount of liquid nitrogen added is 2 mL / mg of the sample. Grind quickly, and keep the sample immersed in liquid nitrogen during grinding.

[0073] (3) Fixing: After grinding, transfer the sample to a centrifuge tube with dry ice buried at the bottom (pre-cooled). Add pre-cooled Trazol at 0 °C according to the amount of 2 mL / mg of the sample, add sterilized steel beads and shake strongly for 2 min (the shaking speed is 35 Frequency / S). The average time from transferring the sample to the centrifuge tube to starting to shake does not exceed 35 s to ensure that Trazol does not freeze. After shaking, place the centrifuge tube in a 4 °C environment and let it stand for 6 min. Then centrifuge at 4 °C and 18000 rpm / min for 6 min.

[0074] (4) Purification: The same as in Example 1.

[0075] (5) Precipitation: The same as in Example 1.

[0076] (6) Purification: The same as in Example 1.

[0077] (7) Drying: Absorb ethanol and dry in a fume hood at 15 - 20 °C for 5 min to obtain fish ovary RNA.

[0078] (8) Preservation: Add 50 μL of deionized water to dissolve the RNA, measure the concentration, adjust it to 700 μg / mL, and store at -80 °C for subsequent use.

[0079] The fish ovary RNA extracted in this example was detected by gel electrophoresis. The results of some samples are as Figure 2 shown. From Figure 2 it can be seen that for the fish ovary RNA extracted in Example 2, the 28S and 18S bands are bright, and the 5S band is not bright, meeting the scientific research experiment standards, indicating that the method of extracting fish ovary RNA in this example is not easily degraded and has high extraction efficiency.

[0080] Comparative Example 1

[0081] This comparative example provides a method for extracting RNA from the ovary of Scatophagus argus. The fish used is the same as in Example 1. Except that the time from transferring the sample to the centrifuge tube to starting oscillation in step (3) is 45 s, the other steps are the same as in Example 1.

[0082] The fish ovary RNA extracted in this comparative example was detected by gel electrophoresis. The results of some samples are as Figure 3 shown. From Figure 3 it can be seen that for the fish ovary RNA extracted in this comparative example, for some samples, although there are bands for 28S and 18S of RNA, they are not bright enough. For other samples, the 28S and 18S bands of RNA are not bright, while the 5S band is bright. This result indicates that the method of extracting fish ovary RNA in this comparative example is somewhat degraded or completely degraded, not meeting the scientific research experiment standards.

[0083] Comparative Example 2

[0084] This comparative example provides a method for extracting RNA from the ovary of Scatophagus argus. The fish used is the same as in Example 1. Except that the time from transferring the sample to the centrifuge tube to starting oscillation in step (3) is 60 s, the other steps are the same as in Example 1.

[0085] The fish ovary RNA extracted in this comparative example was detected by gel electrophoresis. The results of some samples are as Figure 4 shown. From Figure 4It can be seen that for the fish ovary RNA extracted using this comparative example, some samples had bands for 28S and 18S of RNA, but they were not bright enough. At the same time, there were also bands for 5S. For other samples, the bands for 28S and 18S of RNA were not bright, while the band for 5S was bright. This result indicates that the method of extracting fish ovary RNA using this comparative example will cause partial degradation or complete degradation, not meeting the standards of scientific research experiments.

[0086] Comparative Example 3

[0087] This comparative example provides a method for extracting RNA from the ovary of Scatophagus argus. The fish used is the same as in Example 1. Except that the time from taking the ovary tissue from the living body to freezing in step (1) is 120 s, the other steps are the same as in Example 1.

[0088] Gel electrophoresis was used to detect the fish ovary RNA extracted in this comparative example. The results of some samples are as Figure 5 shown. It can be seen from Figure 5 that for the fish ovary RNA extracted using this comparative example, the 28S and 18S of the sample RNA had faint bright bands, and at the same time, the 5S had a bright band. This result indicates that the sample RNA has all been degraded, not meeting the standards of scientific research experiments. Therefore, the efficiency of extracting fish ovary RNA using the method of this comparative example is low.

[0089] Comparative Example 4

[0090] This comparative example provides a method for extracting RNA from the ovary of Scatophagus argus. The fish used is the same as in Example 1. Except that the time from taking out the ovary tissue from the living body to freezing in step (1) is 180 s, the other steps are the same as in Example 1.

[0091] Gel electrophoresis was used to detect the fish ovary RNA extracted in this comparative example. The results are as Figure 6 shown. It can be seen from Figure 6 that for the fish ovary RNA extracted using this comparative example, none of the 28S and 18S of the sample RNA had bright bands, but the 5S had a bright band. This result indicates that all the sample RNA has been degraded, not meeting the standards of scientific research experiments. Therefore, the efficiency of extracting fish ovary RNA using the method of this comparative example is low.

[0092] Comparative Example 5

[0093] This comparative example provides a method for extracting RNA from the ovary of Scatophagus argus. The fish used is the same as in Example 1, and it includes the following steps:

[0094] (1) Sampling: Before sampling, the operating table was simply sterilized by spraying alcohol. The sampling personnel needed to wear masks and disposable nitrile gloves. Small pieces of ovarian tissue obtained from dissection were quickly immersed in liquid nitrogen for quick freezing treatment. The samples after quick freezing treatment were transferred to cryotubes and stored in liquid nitrogen.

[0095] (2) Grinding: The same as Example 1.

[0096] (3) Fixing: After grinding, the samples were transferred to centrifuge tubes with dry ice buried at the bottom (for pre-cooling). According to the amount of 2 mL / mg of the samples, pre-cooled Trazol at 4°C was added. Sterilized steel beads were added and strongly shaken for 2 min (the shaking speed was 30 Frequency / S). After the shaking ended, the centrifuge tubes were placed in an environment at 4°C and left standing for 6 min. It was pipetted until it became transparent, and then centrifuged at 4°C and 12,000 rpm / min for 15 min.

[0097] (4) - (8) The same as Example 1.

[0098] The fish ovarian RNA extracted in this comparative example was detected by gel electrophoresis, and the results are as Figure 7 shown. For the fish ovarian RNA extracted in this comparative example, in some batches (the fish had better activity), there were bright bands for 28S and 18S of the sample RNA, and no bright band for 5S, indicating that the RNA of these samples was not degraded and met the scientific research experiment standard ( Figure 7 A in). However, at the same time, in some batches (the fish had poor activity), the bands of 28S and 18S of the sample RNA were blurred, and there was a bright band for 5S, indicating that the RNA of these samples was partially degraded and did not meet the scientific research experiment standard ( Figure 7 B in). Therefore, it shows that whether the method for extracting fish ovarian RNA in this comparative example can succeed is limited by the activity of the fish, and the extraction efficiency is not high.

[0099] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of concise description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0100] The above-described embodiments only represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent should be subject to the appended claims.

Claims

1. A method for extracting RNA from fish ovaries, characterized in that: The following steps are involved: (1) Ovarian tissue was removed from living fish and frozen in liquid nitrogen; the time from the start of ovarian tissue removal to freezing should not exceed 90 seconds; (2) taking out the ovarian tissue frozen in step (1), and grinding it in liquid nitrogen; (3) Transfer the ground ovarian tissue to a pre-cooled tube, add pre-cooled Trazol, shake, let stand, centrifuge and collect the supernatant; the time from transferring the ovarian tissue to starting shaking should not exceed 40 seconds; (4) Purification, precipitation and purification are performed in sequence to obtain fish ovary RNA.

2. The method for extracting fish ovary RNA according to claim 1, characterized in that: In step (1), the time from the start of taking the ovarian tissue to freezing does not exceed 80 seconds, preferably does not exceed 60 seconds.

3. The method for extracting fish ovary RNA according to claim 1, characterized in that, In step (3), the time from transferring the ovarian tissue to starting oscillation does not exceed 35 seconds, preferably does not exceed 30 seconds.

4. The method for extracting fish ovary RNA according to claim 1, characterized in that The Trazol in step (3) is precooled to 0°C to 1°C; and / or the volume mass ratio of the Trazol to the ovarian tissue is 1.5 mL to 2 mL: 1 mg.

5. The method for extracting fish ovary RNA according to claim 1, characterized in that, When the ovarian tissue is removed in step (2), the ovarian tissue is kept in a frozen state; And / or, the volume mass ratio of liquid nitrogen to ovarian tissue in step (2) is 2 mL to 3 mL: 1 mg; And / or, during the grinding process in step (2), the ovarian tissue is always immersed in the liquid nitrogen.

6. The method for extracting fish ovary RNA according to claim 1, characterized in that: During the shaking in step (3), sterilized steel balls are added; And / or, the oscillation time in step (3) is 2 min to 3 min, and the oscillation speed is 30Frequency / S to 40Frequency / S; And / or, in step (3), the standing temperature is 2°C to 4°C, and the standing time is 4min to 6min; And / or, in step (3), the centrifugation conditions are: 2°C to 4°C, 15000rpm / min to 18000rpm / min, and centrifugation for 6min to 8min.

7. The method for extracting fish ovary RNA according to any one of claims 1 to 6, characterized in that: The purification in step (4) is carried out using chloroform; and / or, the volume ratio of chloroform to supernatant is 3-4:1; And / or, the purification comprises the steps of: adding chloroform precooled to 2°C to 4°C and stored away from light to the supernatant, vigorously shaking for 10s to 15s, then gently shaking for 4s to 5s, and centrifuging statically; Preferably, the standing temperature is 2°C to 4°C, and the standing time is 2min to 3min; Preferably, the centrifugal conditions are: 2°C to 4°C, 15000 rpm / min to 18000 rpm / min, and centrifugation for 6 min to 8 min.

8. The method for extracting fish ovary RNA according to any one of claims 1 to 6, characterized in that: The precipitation in step (5) is carried out using isopropanol; and / or, the volume ratio of the isopropanol to the supernatant is 0.8 to 1.2:1; And / or, the precipitation comprises the steps of: adding isopropanol precooled to 2°C to 4°C to the supernatant, gently shaking for 10s to 12s, standing and centrifuging; discarding the supernatant; Preferably, the standing temperature is 2°C to 4°C, and the standing time is 120s to 150s; Preferably, the centrifugal conditions are: 2°C to 4°C, 15000 rpm / min to 18000 rpm / min, and centrifugation for 6 min to 8 min.

9. The method for extracting fish ovary RNA according to any one of claims 1 to 6, characterized in that: The purification in step (6) is carried out using ethanol; And / or, the purification comprises the steps of: adding 70% to 80% ethanol precooled to 2°C to 4°C to the precipitate, shaking to suspend the precipitate, and centrifuging at rest; Preferably, the standing temperature is 2°C to 4°C, and the standing time is 4min to 6min; Preferably, the centrifugal conditions are: 2°C to 4°C, 15000 rpm / min to 18000 rpm / min, and centrifugation for 2 min to 3 min.

10. The method for extracting fish ovary RNA according to any one of claims 1 to 6, characterized in that: The purification step further includes a drying step, wherein the drying step includes: removing the solvent used in the purification step by suction, and drying under ventilation at 15° C. to 20° C. for 2 min to 4 min; And / or, the fish includes golden croaker, oval pomfret, octopus or tilapia.

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