A kind of extraction method of fish oocyte total RNA

An oocyte and extraction method technology, applied in the field of molecular biology, can solve the problems of sample transfer loss, cumbersome extraction steps, low extraction concentration, etc., reduce RNA degradation and RNase contamination, improve RNA purity and quality, and less loss effect

Inactive Publication Date: 2017-05-10
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The RNAisoPlus reagent extraction method is also a relatively mature method for extracting total RNA, but this method has certain limitations, especially when extracting total RNA samples from fish oocytes, the extraction steps are cumbersome and the loss of sample transfer is a direct result. Problems such as low extraction concentration and poor purity

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  • A kind of extraction method of fish oocyte total RNA
  • A kind of extraction method of fish oocyte total RNA
  • A kind of extraction method of fish oocyte total RNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Total RNA extraction experiment of tongue sole oocytes:

[0027] 1) Ultrasonic disruption of oocyte samples of tongue sole

[0028] ①Take about 30 mature oocytes of tongue sole from semi-smooth tongue sole stored in RNase-free EP tubes (1.5mL) at -80°C, quickly place the EP tubes in an EP tube box pre-cooled with liquid nitrogen, and add 1ml RNAiso Plus .

[0029] ②Before using the ultrasonic processor, soak the horn, especially the ultrasonic crushing head, in diethyl pyrocarbonate (DEPC) aqueous solution for 24 hours, and then sterilize by autoclaving to eliminate RNase contamination.

[0030] ③Transfer the RNase-free EP tube containing the mature oocytes of tongue sole of half-smooth tongue sole to the EP tube box mixed with ice and water, and immerse the horn of the ultrasonic processor into the RNAiso Plus to a depth of about 1.0 cm. The probe should be centered and not attached to the wall.

[0031] ④ Connect the power supply, set the ultrasonic mode of the proc...

Embodiment 2

[0041] Zebrafish oocyte total RNA extraction experiment

[0042] 1) Ultrasonic disruption of zebrafish oocyte samples

[0043]① Take about 30 oocytes from zebrafish late stage of yolk production stored in RNase-free EP tubes (1.5mL) at -80°C, and quickly place the EP tubes in an EP tube box pre-cooled with liquid nitrogen. Add 1ml RNAiso Plus.

[0044] ②Before using the ultrasonic processor, soak the horn, especially the ultrasonic crushing head, in DEPC water for 24 hours, and then sterilize it by autoclaving to eliminate RNase contamination.

[0045] ③Transfer the RNase-free EP tube containing the zebrafish oocytes to the EP tube box mixed with ice and water, and immerse the horn of the ultrasonic processor into the RNAiso Plus to a depth of about 1.0 cm. The probe should be centered and not attached to the wall.

[0046] ④Connect the power supply, set the ultrasonic mode of the processor, the Pattern key is to select the ultrasonic mode key, press the Pattern key to set 8...

Embodiment 3

[0056] Extraction experiment of total RNA from oocytes of semi-smooth tongue sole—ordinary grinding method:

[0057] 1) Grinding and homogenizing the oocyte samples of tongue sole

[0058] ① Take about 60 mature oocytes of tongue sole frozen in RNase-free EP tubes (1.5 mL) at -80°C, transfer them quickly to a mortar pre-cooled with liquid nitrogen, grind the eggs with a pestle, and continuously Add liquid nitrogen until ground into powder;

[0059] ②Scrape the powdered sample from the wall of the mortar with a small iron spoon without RNase, then transfer it into a glass homogenate tube containing 1mL RNAiso Plus, and place the homogenate tube in an ice bath Homogenize until the homogenate is transparent without particles.

[0060] ③ Transfer the homogenized sample to a new RNase-free EP tube and let it stand at room temperature for 5 minutes; then centrifuge at 12000g for 5 minutes at 4°C.

[0061] ④Absorb about 500ul of the supernatant, and transfer it into a new RNase-fr...

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Abstract

The invention relates to an extraction method of total RNA of fish oocytes, belonging to the field of molecular biology. According to the extraction method, total RNA contained in the oocytes is obtained by virtue of combination of a chemical method and an ultrasonic disruption physical method, and the completeness of RNA is guaranteed; the extraction method can be applied to researches including next gene cloning, mRNA expression, high-throughput sequencing and the like; by adding 1ml of RNAiso Plus into trace oocytes, components such as ribosome in the oocytes can be effectively removed; and by respectively adding chloroform and isopropanol, the centrifugation time is prolonged for 5 minutes, the proteins and the like are beneficially removed, and the purity and quality of obtained RNA are improved.

Description

technical field [0001] The invention belongs to the field of molecular biology, in particular to a method for extracting fish oocyte total RNA. [0002] technical background [0003] Oocyte is an important kind of reproductive cells, and its good development and maturation is an important indicator of the reproductive ability of female animals. In order to obtain more high-quality oocytes, it is necessary to understand the molecular regulation mechanism of the oocyte development process in female animals. High purity and good integrity of oocyte total RNA is a very important prerequisite for the analysis of the molecular mechanism of oocytes using real-time fluorescent quantitative RT-PCR technology or high-throughput sequencing technology based on trace RNA. RNA extraction involves crushing and lysing cells, using related reagents to remove pollution such as polysaccharides, phenols, proteins, and DNA, and finally obtaining pure RNA through a series of extraction, washing, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 史宝柳学周徐永江王滨徐涛孙中之
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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