Method for extracting total ribonucleic acid (RNA) from paraffin-embedded tissues

A paraffin embedding and tissue technology, applied in the field of RNA extraction, can solve the problems of clinical research application limitations, severe degradation, unsatisfactory concentration and purity, etc., and achieve the effect of broad application value

Inactive Publication Date: 2011-09-14
DALIAN MEDICAL UNIVERSITY
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Problems solved by technology

The extraction methods reported in the literature are mostly Trizol cleavage method, protease digestion method or kit extraction method, but the concentration and purity of the extracted RNA are not ideal, the degradation is serious, and only small fragments of RNA can be amplified, let alone further. Research on real-time quantitative PCR (q RT-PCR) and gene chip (microarray)
Moreover, most of the FFPE samples used by the existing technology are samples within one year, which also brings great limitations to the application of clinical research.

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  • Method for extracting total ribonucleic acid (RNA) from paraffin-embedded tissues
  • Method for extracting total ribonucleic acid (RNA) from paraffin-embedded tissues
  • Method for extracting total ribonucleic acid (RNA) from paraffin-embedded tissues

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Embodiment 1

[0049] Sample RNA extraction

[0050] The material of this example is: formalin-fixed paraffin-embedded tissue of human ovarian cancer, 1M, 2M, 3M, 4M, 5M, 8M, 10M, 11M, 2Y, 3Y, 4Y, 5Y, 6Y in this example , respectively representing the storage time of the tissue from the beginning of fixed embedding to the experiment is 1 month, 2 months, 3 months, 4 months, 5 months, 8 months, 10 months, 11 months, 2 years, 3 years, 4 years, 5 years, 6 years.

[0051] 21 specimens within one year: numbered 1-21 in sequence.

[0052] Specimens aged 2 to 6 years: 21: numbered 22 to 42 in sequence.

[0053] This numbering system is also applicable to other embodiments of this specification.

[0054] Extract RNA from above-mentioned 42 specimens with the method of the present invention, through nucleic acid trace tester (brand: NanoVue Plus TM , GE Healthcare product, U.S., model: 28923216) the measured concentration and purity are as follows table 1:

[0055] Table 1

[0056]

Embodiment 2

[0058] RNA agarose electrophoresis analysis.

[0059] Experimental method and conditions: Clean the electrophoresis tank and sample comb with 0.1% DEPC water (Invitrogen product, article number: inv-750024), select a spotting comb with a suitable aperture size, and vertically place it on one end of the offset plate. Weigh 1g of agarose (Biowest agarose, product of GENE TECH Co., Ltd., 111760), add 100ml of 1×TAE (50x TAE, product of Beyuntian Biotechnology Research Institute, product number: ST716) into the electrophoresis buffer, heat in a microwave oven to dissolve the agarose evenly . Add 4 μl of EB (10 mg / ml) (ethidium bromide, product of Beyotime Institute of Biotechnology, product number: ST059). After the gel is cooled to about 50°C, pour the gel onto the electrophoresis gel plate gently to remove air bubbles. After the gel has solidified, carefully remove the spotting comb. Add 1×TAE electrophoresis buffer into the electrophoresis tank (DYCP-31F type, Beijing Liuyi ...

Embodiment 3

[0062] RNA quality analysis

[0063] Experimental method and conditions: Agilent 2100 (Agilent) bioanalyzer (Agilent 2100bioanalyzer) is used to fully quantify the RNA integrity index, which can more accurately measure RNA samples and quantitatively evaluate the quality of RNA samples, and the loading volume is only 1ul.

[0064] Experimental results such as figure 2 shown. It can be seen that the results are basically consistent with the results of agarose electrophoresis, the RNA ladder used ranges from 218 to 281.3, and the No. 12 specimen within one year ( figure 2 .C) and No. 14 specimen ( figure 2 .E) Obvious peak shapes can be seen at 18S and 28S, indicating that RNA degradation is less, and specimen No. 21 ( figure 2 .B) Although slightly degraded, the 18S peak disappears, but the 28S peak is clear; RNA extracted from samples aged 2 to 6, some samples (such as No. 22, figure 2 .G) The extracted RNA also has obvious peak shapes at 18S and 28S, suggesting less d...

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Abstract

The invention discloses a method for extracting total ribonucleic acid (RNA) from paraffin-embedded tissues, which is an improvement method provided on the basis of a kit. The method comprises the following steps: deparaffinating, digesting, extracting and purifying. By adopting the method, the RNA in a tissue sample stored by being embedded by paraffin can be extracted, wherein, the longest storage time reaches up to 6 years; the RNA is analyzed by a nucleic acid trace tester and an internationally recognized Agilent 2100bioanalyzer; the extraction concentration is 211-3116ng / mu l, and the extraction purity is A260 / 280:1.75-2.04, which are all very ideal; and the expression research of microarray high-abundance genes of qRT-PCR and miRNA in mRNA and miRNA level can be carried out, which provides an experimental basis for developing a large amount of research on formalin fixed paraffin-embedded (FFPE) genes, and has wide application value.

Description

technical field [0001] The invention relates to an RNA extraction method, in particular to a method for extracting total RNA from long-term formalin-fixed paraffin-embedded tissues. Background technique [0002] Formalin-fixed paraffin-embedded tissue (FFPE) is the most commonly used method for preserving pathological tissues in medical institutions. Due to the difficulty in obtaining fresh specimens, retrospective studies on existing cases can only be performed in order to conduct molecular biology research on solid tumors. Gene extraction is performed from paraffin-embedded tissue, making paraffin-embedded tissue the most abundant source of precious resources. The method of extracting DNA from paraffin-embedded tissues is mature, but the research on DNA sometimes cannot accurately reflect the changes in the transcription process of disease-related genes to some extent, so extracting sufficient and effective methods from pathological archive specimens Amplified RNA is of g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 崔世英丁艳芳赵瑾瑶
Owner DALIAN MEDICAL UNIVERSITY
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