CN110272899A - A kind of method that breast tissue RNA is extracted - Google Patents
A kind of method that breast tissue RNA is extracted Download PDFInfo
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- CN110272899A CN110272899A CN201910628508.9A CN201910628508A CN110272899A CN 110272899 A CN110272899 A CN 110272899A CN 201910628508 A CN201910628508 A CN 201910628508A CN 110272899 A CN110272899 A CN 110272899A
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- rna
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- breast tissue
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
Abstract
The present invention provides the preprocess methods that a kind of breast tissue RNA is extracted, it is first to make breast tissue frozen section material, then dye corpus mamma tissue in identification sliced materials by HE, cuts corpus mamma tissue.Aforementioned corpus mamma tissue is directly used in cell cracking and RNA is extracted, can be obtained the RNA of high quality, high concentration.The present invention be able to solve the breast tissue extracted as RNA it is rare caused by RNA extract low-quality problem.
Description
Technical field
The present invention relates to RNA extraction purification fields.
Background technique
With the development of modern medicine, biomarker is increasingly becoming the important reference indicator of medical diagnosis on disease and treatment.
In the detection of breast cancer, need to extract and detect canceration and normal corpus mamma tissue (and it is non-fat
And connective tissue) in RNA.Canceration corpus mamma tissue is relatively easy to get, but normal corpus mamma tissue is then very dilute
It lacks.The reason is that: need to be the patient with breast cancer of Breast-consering surgery, normal galactophore tissue is originally less, it is impossible to a large amount of samplings；And it needs
Do entirely cut mammary gland and new adjuvant chemotherapy cut full mammary gland case it is also rare.
The normal breast gland tissue extracted for RNA is rare, and people often obtain quality using conventional RNA extraction method
RNA low, concentration is low is even extracted less than RNA sometimes.
Summary of the invention
To solve the above-mentioned problems, the present invention provides a kind of breast tissue RNA to extract preprocess method and the extraction side RNA
Method.
Technical solution of the present invention includes:
A kind of sample preprocessing method that breast tissue RNA is extracted, it is first to make breast tissue frozen section, then pass through
Corpus mamma tissue in HE dyeing identification frozen section, cuts corpus mamma tissue.
A kind of breast cancer tissue's RNA extraction method, comprising:
1) sample preprocessing is carried out with preceding method；
2) sliced materials are placed in cell pyrolysis liquid；
3) RNA is extracted.
RNA extraction method as the aforementioned, the cell pyrolysis liquid of step 2) are the cracking of QIAGAN RNeasy Mini Kit
Liquid.
RNA extraction method as the aforementioned, step 3) are to extract RNA using QIAGAN RNeasy Mini Kit.
RNA extraction method as the aforementioned, step 3) are finally molten for 10~40 microlitres of water or dissolution buffer using volume
Solve RNA.
RNA extraction method as the aforementioned, the water or dissolution buffer solution that step 3) is finally 30 microlitres using volume
RNA。
The invention has the following beneficial effects:
1) pre-treating method of the invention includes the frozen section for making breast tissue, can avoid the anti-of fresh breast tissue
Multiple freeze thawing；
2) present invention carries out RNA extraction using frozen section, it is possible to reduce tissue usage amount, and tissue can be accurately determined
Usage amount；
3) present invention carries out RNA extraction using frozen section, while tissue dosage is few, is avoided that blocking kit
Column is eluted, the quality of the RNA of extraction is improved；
4) present invention is directly used in the cleavage step of RNA extraction using frozen section, tissue can be made sufficiently to crack；
5) pre-treating method of the invention includes dyeing identification gland tissue, eliminates a large amount of fat and connective tissue,
It can guarantee the concentration of the RNA extracted.
Obviously, above content according to the present invention is not being departed from according to the ordinary technical knowledge and customary means of this field
Under the premise of the above-mentioned basic fundamental thought of the present invention, the modification, replacement or change of other diversified forms can also be made.
Above content of the invention is described in further detail again below by way of specific embodiment.But it should not be by this
The range for being interpreted as the above-mentioned theme of the present invention is only limitted to example below.All technologies realized based on above content of the present invention are equal
Belong to the scope of the present invention.
Specific embodiment
The breast tissue RNA extraction method of the invention of embodiment 1
1 Tissue Lysis pre-treatment: the identification of freezing microtome slice and slice sampling
1) breast tissue (containing corpus mamma tissue, adipose tissue and connective tissue) sample is taken out from -80 degree refrigerators；
2) sample is placed in freezing microtome and embeds, and makes frozen section；
3) slice identification: HE, which is dyed, determines region locating for mammary glandular cell in tissue, cuts cutting with corpus mamma tissue
Panel region.
4) it weighs, determines tissue mass used in single-trial extraction no more than 30mg.
The cracking of 2 tissue samples and RNA are extracted
Previous slice organization material is put into the cell pyrolysis liquid of RNA extraction (in QIAGAN RNeasy Mini Kit
Lysate) in, pipettor dispels.
Using RNA extracts kit (QIAGAN RNeasy Mini Kit) extract RNA, finally dissolve RNA water (or
Dissolution buffer) amount can determine that usual 10~40 microlitres, the present embodiment is with 30 microlitres on demand.
The content and quality of 3 detection RNA
The content and quality of RNA are detected using the NANODROP 2000 of thermo company.
The Contrast on effect that 1 breast tissue RNA of comparative example is extracted
1. method
The frozen section of breast cancer tissue and normal galactophore tissue is taken, breast cancer tissue is randomly divided into A1 group and A2 group, just
Normal breast tissue is randomly divided into B1 group and B2 group.
A1 group and B1 group slice extract RNA according to the method in embodiment 1 and detect the content and quality of RNA；A2 group and
The step of B2 group slice is identified without slice cuts the tissue no more than 30mg i.e. using 1 step 2~3 of embodiment at random
Method extracts RNA and detects the content and quality of RNA.
2. result
Absorbance (A280 and A260) of the RNA solution at 280,260nm has respectively represented nucleic acid, albumen (or other has
Machine object) content.A260/A280 is the main evaluation index of RNA mass.When A260/A280 value is greater than 2.2, it is likely that
RNA has been hydrolyzed as mononucleotide；When A260/A280 is less than 1.8, then the pollution of albumen (or other organic matters) is brighter
It is aobvious；The A260/A280 of pure RNA solution is 2.
As shown in Table 1, the A260/A280 of the extracted RNA of method of the invention is close to pure RNA solution, reliable in quality.
In addition, breast tissue extracting method of the invention compares conventional method (not sliced identification), can extract more
More RNA.
1 RNA mass of table and Concentration Testing result
To sum up, method of the present invention due to taking slice dyeing identification, accurately cuts corpus mamma tissue and mentions for RNA
It takes, the yield of RNA can be greatly improved.
Claims (6)
1. a kind of sample preprocessing method that breast tissue RNA is extracted, which is characterized in that it is that first the frost of production breast tissue is cut
Piece, then corpus mamma tissue in identification frozen section is dyed by HE, cut corpus mamma tissue.
2. a kind of breast cancer tissue's RNA extraction method characterized by comprising
1) sample preprocessing is carried out with method of claim 1；
2) sliced materials are placed in cell pyrolysis liquid；
3) RNA is extracted.
3. RNA extraction method as claimed in claim 2, which is characterized in that the cell pyrolysis liquid of step 2) is QIAGAN
The lysate of RNeasy Mini Kit.
4. RNA extraction method as claimed in claim 2 or claim 3, which is characterized in that step 3) is using QIAGAN RNeasy
Mini Kit extracts RNA.
5. RNA extraction method as claimed in claim 4, which is characterized in that step 3) is finally 10~40 microlitres using volume
Water or dissolution buffer solution RNA.
6. RNA extraction method as claimed in claim 5, which is characterized in that the water that step 3) is finally 30 microlitres using volume
Or dissolution buffer solution RNA.
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| CN201910628508.9A CN110272899A (en) | 2019-07-11 | 2019-07-11 | A kind of method that breast tissue RNA is extracted |
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Citations (4)
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|---|---|---|---|---|
| CN1940069A (en) * | 2005-09-30 | 2007-04-04 | 北京大学 | Ig light chain variable region sequence and CDR3 sequence from human cancers and their use |
| CN101633922A (en) * | 2009-08-24 | 2010-01-27 | 中山大学 | Molecular marker hsa-miR-374a of breast carcinoma and application thereof |
| CN104634626A (en) * | 2013-11-15 | 2015-05-20 | 上海交通大学 | Improved frozen slicing method and applications thereof |
| CN109000956A (en) * | 2018-08-06 | 2018-12-14 | 四川大学华西医院 | A kind of closely-spaced secondary serial sectioning production method of full tumor of breast |
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2019
- 2019-07-11 CN CN201910628508.9A patent/CN110272899A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1940069A (en) * | 2005-09-30 | 2007-04-04 | 北京大学 | Ig light chain variable region sequence and CDR3 sequence from human cancers and their use |
| CN101633922A (en) * | 2009-08-24 | 2010-01-27 | 中山大学 | Molecular marker hsa-miR-374a of breast carcinoma and application thereof |
| CN104634626A (en) * | 2013-11-15 | 2015-05-20 | 上海交通大学 | Improved frozen slicing method and applications thereof |
| CN109000956A (en) * | 2018-08-06 | 2018-12-14 | 四川大学华西医院 | A kind of closely-spaced secondary serial sectioning production method of full tumor of breast |
Non-Patent Citations (3)
| Title |
|---|
| 张嵘: "《生物化学与分子生物学实验 第3版》", 31 March 2019, 中国医药科技出版社, pages: 88 - 89 * |
| 班克罗夫特: "《病理切片染色技术》", 吉林医学院, pages: 21 * |
| 郭旭东: "芦丁对哺乳大鼠乳腺发育及相关激素水平与受体表达量关系的影响", 《中国农业大学学报》, vol. 16, no. 5, 31 December 2011 (2011-12-31), pages 88 - 95 * |
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