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Method for detecting miRNA (micro ribonucleic acid) in stomach juice

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

Inactive Publication Date: 2011-07-20
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
[0005] The content of miRNA in gastric juice is very low, so it is very difficult to extract and detect miRNA in gastric juice, and there is no specific extraction and detection method for miRNA in gastric juice

Method used

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  • Method for detecting miRNA (micro ribonucleic acid) in stomach juice
  • Method for detecting miRNA (micro ribonucleic acid) in stomach juice
  • Method for detecting miRNA (micro ribonucleic acid) in stomach juice

Examples

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Effect test

Embodiment 1

[0028] A method for detecting miRNA in gastric juice includes the following main steps in sequence:

[0029] a. Gastric juice extraction and neutralization: Use a gastric tube to extract 5ml of two fasting gastric juice samples (different parts), put them in a centrifuge tube, place them on ice, and then add 0.5% sodium hydroxide solution with a concentration of 20% by mass. ml, place in a shaker at room temperature and shake for 20 minutes to liquefy the mucus in the gastric juice. Adjust the pH value to 7.0±0.5 with 20% by mass sodium hydroxide solution, and then add 1% by mass Trypsin (commercially available) solution 20μl, vortex and mix, place in 37℃ water bath for 15 minutes to hydrolyze the protein in gastric juice and homogenize the gastric juice, centrifuge at 1000rpm for 20 minutes at 4℃;

[0030] b. RNA release: Take 200μl of the supernatant from step a and place it in a sterile centrifuge tube, add 50μl of sterile DEPC (commercially available) treated double distilled w...

Embodiment 2

[0037] The method is basically the same as in Example 1, except that in step g, the upstream primer for amplification is replaced by has-miR-21 specific amplification upstream primer instead of has-miR-17, and the expression level of miR-21 in gastric juice is detected. Amplification curve (attached image 3 ) And melting curve (attached Figure 4 ), from the amplification curve, the Ct values ​​of the two gastric juice samples tested are 29.04 and 32.03, respectively; from the melting curve, it can be seen that the curve is a narrow single peak, and the miR of each gastric juice sample can be seen -21 is specifically amplified, without interference from primer dimers and bands.

Embodiment 3

[0039] The method is basically the same as in Example 1, except that in step g, the upstream primer for amplification is replaced by the upstream primer specific for small RNA U6 amplification instead of piR-651, and the amplification curve of the expression level of small RNA U6 in gastric juice is detected ( Attached Figure 5 ) And melting curve (attached Image 6 ), from the amplification curve, the Ct values ​​of the two gastric juice samples tested are 29.60 and 32.04, respectively; from the melting curve, it can be seen that the curve is a narrow single peak, and it can be seen that the small value of each gastric juice sample RNA U6 is specifically amplified without interference from primer dimers and bands.

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Abstract

The invention discloses a method for detecting miRNA (micro ribonucleic acid) in stomach juice, which comprises stomach juice extraction and neutralization, RNA release, chloroform extraction, isopropanol precipitation, ethanol washing, RNA reverse transcription, PCR (Polymerase Chain Reaction) detection and other steps. In the detection method, a sodium hydroxide solution is used to liquefy viscous components in the stomach juice, so that mucoproteins and polysaccharides are suspended in the solution, and the stomach juice is neutralized; trypsinase is added to carry out digestion, so that the mucoproteins and other proteins can be hydrolyzed, and cell components in the stomach juice are loosened at the same time, thereby facilitating centrifugal separation; and a Trizol solution is added to carry out repeated blow and beat, so that the RNA is completely released and separated from the polysaccharides, the mucoproteins and other impurities, thereby solving the problem that the RNA extraction can be disturbed by the acidity of the stomach juice as well as polysaccharides, mucoproteins and the like in the stomach juice. Thus, the invention can eliminate the disturbance caused by polysaccharides, proteins and other impurities in the stomach juice, complete the efficient extraction of miRNA in the stomach juice and carry out fluorescent quantitative PCR detection on the extracted miRNA.

Description

Technical field [0001] The invention relates to a detection method of miRNA, in particular to a detection method of miRNA in gastric juice. Background technique [0002] RNA in organisms can be divided into two categories: coding RNA and non-coding RNA. Non-coding RNA can be divided into long-chain non-coding RNA and short-chain non-coding RNA; the latter is more important including microRNA (microRNA, miRNA), which is 19-24 nucleotides in length. In the normal development of organisms, miRNAs play a pivotal role. They play an important negative regulatory role in gene expression through the cleavage or translation inhibition of target mRNA, and have important functions such as affecting cell growth, differentiation and apoptosis. In the physiological abnormal variation of different tissues and cells, some miRNAs have obvious abnormal expression. [0003] The analysis of miRNA requires small RNA extraction and detection. For small RNA extraction, such as Chinese patent applicatio...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 肖丙秀崔龙郭俊明宋皓军
Owner NINGBO UNIV
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