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Rapid extraction method for total RNA of acaudina molpadioides digestive tract

An extraction method and digestive tract technology, applied in the direction of DNA preparation, recombinant DNA technology, etc., can solve the problems affecting real-time quantitative PCR detection work, the decline of total RNA extraction yield, and difficult sample processing, so as to meet the requirements of real-time quantitative PCR detection, Improve the effect of RNA precipitation and RNA integrity

Inactive Publication Date: 2012-02-08
OCEAN RES CENT OF ZHOUSHAN ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, it is difficult to process the samples before extraction of total RNA from the digestive tract of seabed flowers. Improper handling will reduce the extraction rate of total RNA, degrade the samples, and be easily contaminated, which will seriously affect the subsequent real-time quantitative PCR detection.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Step (1). The digestive tract of Haitian melon is dissected, and the digestive tract is cut longitudinally with a needle, washed with seawater filtered by membrane and sterilized by high temperature, and then frozen with liquid nitrogen;

[0024] Step (2). Grinding the frozen Haitian melon digestive tract into powder, taking 30 mg of the powder and adding it to a centrifuge tube pre-cooled by liquid nitrogen;

[0025] Step (3). Add 0.8ml TRIZOL lysate to the centrifuge tube, shake and mix well, let stand at room temperature for 5min, and centrifuge for 0.5min;

[0026] Step (4). Take the supernatant, add 200 μl of chloroform, shake and mix for 5 minutes, let stand for 5 minutes, centrifuge for 10 minutes, and take 200 μl of the upper aqueous phase;

[0027] Step (5). Add 200 μl of isopropanol pre-cooled at -30°C to the upper aqueous phase, mix well, let stand at -30°C for 10 minutes, centrifuge for 5 minutes, and precipitate RNA;

[0028] Step (6). Place the centrifuge...

Embodiment 2

[0039] Step (1). The digestive tract of Haitian melon is dissected, and the digestive tract is cut longitudinally with a needle, washed with seawater filtered by membrane and sterilized by high temperature, and then frozen with liquid nitrogen;

[0040] Step (2). Grinding the frozen Haitian melon digestive tract into powder, taking 50 mg of the powder and adding it to a centrifuge tube pre-cooled with liquid nitrogen;

[0041] Step (3). Add 1.2ml of TRIZOL lysate to the centrifuge tube, shake and mix well, let stand at room temperature for 10min, and centrifuge for 2min;

[0042] Step (4). Take the supernatant, add 300 μl of chloroform, shake and mix for 10 minutes, let stand for 10 minutes, centrifuge for 20 minutes, and take 300 μl of the upper aqueous phase;

[0043] Step (5). Add 300 μl of isopropanol pre-cooled at -10°C to the upper aqueous phase, mix well, let stand at -10°C for 20 minutes, centrifuge for 15 minutes, and precipitate RNA;

[0044] Step (6). Place the cen...

Embodiment 3

[0055] Step (1). The digestive tract of Haitian melon is dissected, and the digestive tract is cut longitudinally with a needle, washed with seawater filtered by membrane and sterilized by high temperature, and then frozen with liquid nitrogen;

[0056] Step (2). Grinding the frozen Haitian melon digestive tract into powder, taking 40 mg of the powder and adding it to a centrifuge tube pre-cooled by liquid nitrogen;

[0057] Step (3). Add 1.0ml of TRIZOL lysate to the centrifuge tube, shake and mix well, let stand at room temperature for 8 minutes, and centrifuge for 1 minute;

[0058] Step (4). Take the supernatant, add 250 μl of chloroform, shake and mix for 8 minutes, let stand for 8 minutes, centrifuge for 15 minutes, and take 250 μl of the upper aqueous phase;

[0059] Step (5). Add 250 μl isopropanol pre-cooled at -20°C to the upper aqueous phase, mix well, let stand at -20°C for 15 minutes, centrifuge for 10 minutes, and precipitate RNA;

[0060] Step (6). Place the ce...

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Abstract

The invention relates to a rapid extraction method for total RNA of an acaudina molpadioides digestive tract. At present, a sample is relatively difficult to process before the total RNA of the acaudina molpadioides digestive tract is extracted, the extraction yield of the total RNA is not high, and the total RNA is easy to pollute. The method comprises the following steps of: grinding a liquid nitrogen frozen acaudina molpadioides digestive tract tissue by using a mortar, putting the powder into TRIZOL lysis solution for lysis and homogenization; homogenizing and centrifuging the tissue, taking supernatant fluid, adding chloroform, shaking, blending, centrifuging and taking the supernatant fluid; adding low temperature isopropyl alcohol, blending, and centrifugating; washing by using 75 percent of ethanol; and dissolving precipitates by using RNA (Ribose Nucleic Acid) enzyme-free deionized water, and preserving at the temperature of between -90 and -70 DEG C. According to the method, the RNA degradation of the sample is effectively avoided, the RNA precipitation effect is improved, the extracting time is short, the RNA integrity is high, and the requirement of real-time quantitative PCR (polymerase chain reaction) detection is completely met.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for extracting total RNA from the digestive tract of Haitian melon, in particular to a Haitian melon digestion method with relatively simple steps and good extraction integrity, which is mainly used for real-time quantitative PCR (real-time PCR) detection. method for tracking total RNA. Background technique [0002] The extraction of RNA with good integrity from marine biological tissue cells is a necessary prerequisite and guarantee for molecular biology research. Different marine biological tissue components and structures are quite different. Therefore, for tissue cells of different species and tissue sources, corresponding methods should be used to extract total RNA. [0003] The digestive tract tissue of Haitian melon contains the content of the digestive tract, and the chemical components of the digestive tract wall are mainly mucopolysaccharides, digestive enzymes, mus...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 王天明邵庆均刘永芹肖金星郑刚马文明
Owner OCEAN RES CENT OF ZHOUSHAN ZHEJIANG UNIV
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