Method for detecting piwi-interacting ribonucleic acid (piRNA) in gastric juice

A detection method and gastric juice technology, applied in the field of piRNA detection in gastric juice, can solve the problems of low piRNA, no specific extraction piRNA detection method, difficulty in piRNA extraction and detection, etc., and achieve the effect of facilitating centrifugal separation

Inactive Publication Date: 2013-04-24
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, there are a lot of substances such as mucopolysaccharides and mucins in the gastric juice. During the process of extracting RNA by conventional methods, because the physical and chemical properties of polysaccharides are very similar to RNA, polysaccharides and RNA can be precipitated at the same time, resulting in a transparent gel rich in polysaccharides. Precipitation is not conducive to subsequent cDNA synthesis and PCR detection
[0006] The content of piRNA in gastric juice is very low, so it is very difficult to extract and detect piRNA in gastric juice, and there is no specific extraction and detection method for piRNA in gastric juice

Method used

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  • Method for detecting piwi-interacting ribonucleic acid (piRNA) in gastric juice
  • Method for detecting piwi-interacting ribonucleic acid (piRNA) in gastric juice
  • Method for detecting piwi-interacting ribonucleic acid (piRNA) in gastric juice

Examples

Experimental program
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Effect test

Embodiment 1

[0026] A method for detecting piRNA in gastric juice, comprising the following main steps in sequence:

[0027]a. Gastric juice extraction and neutralization: extract two fasting gastric juice samples (different parts) with a gastric tube, 5ml each, put them into a centrifuge tube, put them on ice, and then add 0.5% sodium hydroxide solution with a mass percentage concentration of 20%. ml, placed in a shaker at room temperature and shaken for 20 minutes to liquefy the mucus in gastric juice, adjust the pH value to 7.0±0.5 with 20% sodium hydroxide solution by mass percentage, and then add 1% Trypsin (commercially available) solution 20μl, vortex to mix, place in a 37℃ water bath for 15 minutes to digest, hydrolyze the protein in the gastric juice and homogenize the gastric juice, centrifuge at 1000rpm for 20 minutes at 4℃;

[0028] b. RNA release: Take 200 μl of the supernatant from step a and place it in a sterile centrifuge tube, add 50 μl of sterilized DEPC (commercially av...

Embodiment 2

[0035] The method is basically the same as in Example 1, except that in step g, the amplified upstream primer is replaced by the piR-823 specific amplification upstream primer for piR-651, and the amplification curve of the expression level of piR-823 in gastric juice is detected (attached image 3 ) and melting curve (with Figure 4 ), from the amplification curve, the Ct values ​​of the two gastric juice samples detected are 22.27 and 22.91 respectively; from the melting curve, it can be known that the curve is a narrow single peak, and it can be seen that the piR of each gastric juice sample -823 were amplified specifically without the interference of primer dimers and heterogeneous bands.

Embodiment 3

[0037] The method is basically the same as in Example 1, except that in step g, the amplified upstream primer is replaced by the small RNA U6 specific amplification upstream primer for piR-651, and the amplification curve of the expression level of small RNA U6 in the gastric juice is detected ( attached Figure 5 ) and melting curve (with Image 6 ), from the amplification curve, the Ct values ​​of the two gastric juice samples detected are 29.60 and 32.04 respectively; from the melting curve, it can be known that the curve is a narrow single peak, and it can be seen that each gastric juice sample has a small RNA U6 was amplified specifically without the interference of primer dimers and heterogeneous bands.

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Abstract

The invention discloses a method for detecting a piwi-interacting ribonucleic acid (piRNA) in gastric juice. The method comprises the following steps of: extracting and neutralizing the gastric juice; releasing the RNA; extracting with chloroform; precipitating with isopropyl alcohol; washing with ethanol; performing reverse transcription of the RNA; detecting with a polymerase chain reaction (PCR); and the like, wherein sodium hydroxide solution is used for liquefying viscous components in the gastric juice to suspend mucoprotein and polysaccharide in the solution and neutralizing the gastric juice in the method; trypsin is added for digestion, so that proteins such as the mucoprotein and the like can be hydrolyzed and cell components in the gastric juice are loosened to bring convenience to centrifugal separation; Trizol solution is added for repeated blowing and beating; the RNA is fully released and is separated from impurities such as the polysaccharide, the mucoprotein and the like, so that the problem of interference of acidity, the polysaccharide, the mucoprotein and the like in the gastric juice on the extraction of the RNA is solved; thus, the interference of the impurities such as the polysaccharide, the mucoprotein and the like in the gastric juice can be eliminated, the piRNA in the gastric juice is effectively extracted, and fluorescence quantitative PCR detection is performed on the extracted piRNA.

Description

technical field [0001] The invention relates to a method for detecting piRNA, in particular to a method for detecting piRNA in gastric juice. Background technique [0002] RNA in organisms can be divided into two categories: coding RNA and non-coding RNA. It has been found that non-coding RNA (non-coding RNA, ncRNA) participates in the highly complex RNA regulatory network in cells. They play important functions in the regulation of cell proliferation, developmental timing, cellular immunity, and disease occurrence. Among these non-coding RNAs, Piwi-interacting RNA (piRNA) has multiple functions such as regulating the formation of germ cells, sex determination, participating in the directed differentiation of stem cells, maintaining the repressed state of transposons, and preventing transposon outbreaks. . [0003] piRNA is a class of single-stranded small RNA molecules with a length of 25-31 nucleotides and no protein coding. Their production does not depend on the proce...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 郭俊明崔龙肖丙秀宋皓军
Owner NINGBO UNIV
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