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Method for extracting and detecting IncRNA in gastric juice

A detection method, gastric juice technology, applied in the field of lncRNA extraction and detection, can solve the problems of low lncRNA abundance, difficult extraction and detection of lncRNA in gastric juice, incomplete extraction and detection of lncRNA, etc., and achieve the effect of simplifying the experimental operation steps

Inactive Publication Date: 2014-06-04
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In addition, the abundance of lncRNA in gastric juice is low, and it is difficult to extract and detect lncRNA in gastric juice. Up to now, there is still no perfect method for extracting and detecting lncRNA in gastric juice

Method used

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  • Method for extracting and detecting IncRNA in gastric juice
  • Method for extracting and detecting IncRNA in gastric juice
  • Method for extracting and detecting IncRNA in gastric juice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] A method for extracting and detecting lncRNA in gastric juice, comprising the steps of:

[0045] a. Gastric juice extraction: extract 5ml of fasting gastric juice with a gastric tube, put it into an RNase-free centrifuge tube, centrifuge at 3000rpm at room temperature for 3 minutes, transfer the supernatant to another RNase-free centrifuge tube, and put it on ice;

[0046] b. Gastric juice pretreatment: Take 300 μl of the gastric juice from the previous step and place it in a 2ml RNase-free centrifuge tube, add 900 μl of Trizol LS reagent, vortex for 10 seconds, let stand at room temperature for 5 minutes, and centrifuge at 12,000 rpm for 10 minutes at 4°C. Two layers, the upper layer is a pink homogenized liquid, the lower layer is a relatively dark viscous impurity layer, transfer 1000 μl of the upper layer liquid to a new RNase-free centrifuge tube, discard the precipitate to remove polysaccharides and mucin other impurities;

[0047] c. Chloroform extraction: add 0...

Embodiment 2

[0054] The method is basically the same as in Example 1, except that in step h, the amplified upstream and downstream primers are replaced by AA174084 specific amplification upstream and downstream primers for RMRP, and the amplification curve and melting curve of the expression level of AA174084 in gastric juice are detected, separately as attached image 3 and 4 , it can be obtained from the amplification curve that the Ct values ​​of the two gastric juice specimens detected are 31.42 and 32.65 respectively; It is specifically amplified without the interference of primer dimers and heterogeneous bands.

Embodiment 3

[0056] The method is basically the same as in Example 1, except that in step h, the upstream and downstream primers for amplification are replaced by GAPDH-specific amplification upstream and downstream primers for RMRP, and the amplification curve of the expression level of GAPDH in gastric juice is detected, as shown in the attached Figure 5 and 6 , it can be obtained from the amplification curve that the Ct values ​​of the two gastric juice samples detected are 28.26 and 32.45 respectively; from the melting curve, it can be known that the curve is a narrow single peak, and it can be seen that the GAPDH of each gastric juice sample is It is specifically amplified without the interference of primer dimers and heterogeneous bands. The Ct of the sample cDNA in this example GAPDH If the value is ≤34, the quality of RNA (ie, cDNA) is qualified. In addition, through 130 cases of gastric juice Ct GAPDH Value analysis showed that the level of GAPDH in the gastric juice of health...

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Abstract

The invention discloses a method for extracting and detecting IncRNA in gastric juice. The method comprises the following steps: gastric juice extraction, gastric juice pretreatment, trichloromethane extraction, isopropanol participation, ethanol washing, drying a ribonucleic acid (RNA), RNA reverse transcription, polymerase chain reaction (PCR) detection and the like. Compared with the prior art, the method has the advantages that the impurities such as polysaccharide, mucoprotein and the like in the gastric juice are removed by the steps of gastric juice pretreatment, trichloromethane extraction, isopropanol participation, ethanol washing, drying a ribonucleic acid, RNA reverse transcription, and PCR detection. High-quality and high-yield extraction of the IncRNA in the gastric juice is achieved on the basis of simplifying experimental operation steps; fluorescent quantitative RT-PCR detection of the target IncRNA is achieved; reduced glyceraldehyde-phosphate dehydrogenase (GAPDH) is found out to be a good exterior parameter gene in the gastric juice by Ct value analysis of the GAPDH on 130 cases of gastric samples; a GAPDH reference Ct value range for judging the total RNA mass of the gastric juice is also proposed on the basis; and the method has great significance on further research of the occurrence regularity of IncRNA in the gastric juice and the relationship between the occurrence regularity of IncRNA in the gastric juice and the physiological status and the disease.

Description

technical field [0001] The invention relates to a method for extracting and detecting lncRNA, in particular to a method for extracting and detecting lncRNA in gastric juice. Background technique [0002] There are about 20,000 to 30,000 genes that encode proteins in the human body, accounting for only 2% of the human genome, and the remaining 98% of genomic DNA that does not encode proteins was initially considered to be non-functional and garbage in organisms, often referred to as "Junk DNA". However, current research shows that most of these junk DNA can be transcribed to produce noncoding RNA (noncoding RNA, ncRNA). for ncRNA. Among these ncRNA molecules, there are well-known "housekeeping" ncRNAs (such as tRNA and rRNA, etc.), small ncRNAs (such as microRNA and piRNA, etc.), and more are long-term ncRNAs that have yet to be further studied. Strand non-coding RNA (long noncoding RNA, lncRNA) molecules. [0003] LncRNA refers to a class of RNA molecules whose transcrip...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/10
CPCC12Q1/686C12Q1/6886C12Q2600/158C12Q2600/178C12Q2531/113
Inventor 郭俊明邵永富肖丙秀
Owner NINGBO UNIV
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