CN106701742A - Blood collection tube for protecting and stabilizing free RNA (Ribonucleic Acid) - Google Patents
Blood collection tube for protecting and stabilizing free RNA (Ribonucleic Acid) Download PDFInfo
- Publication number
- CN106701742A CN106701742A CN201611214321.7A CN201611214321A CN106701742A CN 106701742 A CN106701742 A CN 106701742A CN 201611214321 A CN201611214321 A CN 201611214321A CN 106701742 A CN106701742 A CN 106701742A
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- CN
- China
- Prior art keywords
- heparin tube
- free rna
- blood
- blood collection
- rna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
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- 210000004369 blood Anatomy 0.000 title claims abstract description 25
- 239000008280 blood Substances 0.000 title claims abstract description 25
- 229920002477 rna polymer Polymers 0.000 title abstract 6
- 230000000087 stabilizing effect Effects 0.000 title abstract 2
- 238000012360 testing method Methods 0.000 claims abstract description 12
- 229920001971 elastomer Polymers 0.000 claims abstract description 5
- 239000003223 protective agent Substances 0.000 claims abstract description 5
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims description 29
- 229960002897 heparin Drugs 0.000 claims description 29
- 229920000669 heparin Polymers 0.000 claims description 29
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 8
- 230000006641 stabilisation Effects 0.000 claims description 7
- 238000011105 stabilization Methods 0.000 claims description 7
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims description 6
- -1 Formic acid alcohol ester Chemical class 0.000 claims description 5
- 239000011324 bead Substances 0.000 claims description 5
- 239000004471 Glycine Substances 0.000 claims description 4
- MGSKVZWGBWPBTF-UHFFFAOYSA-N aebsf Chemical compound NCCC1=CC=C(S(F)(=O)=O)C=C1 MGSKVZWGBWPBTF-UHFFFAOYSA-N 0.000 claims description 4
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 claims description 4
- 239000000463 material Substances 0.000 claims description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 4
- JZBRFIUYUGTUGG-UHFFFAOYSA-J tetrapotassium;2-[2-[bis(carboxylatomethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical compound [K+].[K+].[K+].[K+].[O-]C(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O JZBRFIUYUGTUGG-UHFFFAOYSA-J 0.000 claims description 4
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 claims description 3
- 239000002342 ribonucleoside Substances 0.000 claims description 3
- 229920005549 butyl rubber Polymers 0.000 claims description 2
- 235000019253 formic acid Nutrition 0.000 claims description 2
- 230000002209 hydrophobic effect Effects 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 2
- 238000007789 sealing Methods 0.000 abstract 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 32
- 238000000605 extraction Methods 0.000 description 11
- 108020004707 nucleic acids Proteins 0.000 description 10
- 102000039446 nucleic acids Human genes 0.000 description 10
- 150000007523 nucleic acids Chemical class 0.000 description 10
- 108010067316 Catenins Proteins 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 5
- 238000001514 detection method Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 230000001605 fetal effect Effects 0.000 description 4
- 238000003752 polymerase chain reaction Methods 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 3
- 102000006382 Ribonucleases Human genes 0.000 description 3
- 108010083644 Ribonucleases Proteins 0.000 description 3
- 238000003793 prenatal diagnosis Methods 0.000 description 3
- 102000016362 Catenins Human genes 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 210000001124 body fluid Anatomy 0.000 description 2
- 239000010839 body fluid Substances 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 206010000234 Abortion spontaneous Diseases 0.000 description 1
- 108060000903 Beta-catenin Proteins 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 208000002151 Pleural effusion Diseases 0.000 description 1
- 238000011530 RNeasy Mini Kit Methods 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- 238000002669 amniocentesis Methods 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000004252 chorionic villi Anatomy 0.000 description 1
- 238000012631 diagnostic technique Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 208000015994 miscarriage Diseases 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 239000011814 protection agent Substances 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 208000000995 spontaneous abortion Diseases 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Classifications
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a blood collection tube for protecting and stabilizing free RNA (Ribonucleic Acid). The blood collection tube is composed of a test tube, a sealing rubber plug and a protective agent. According to the blood collection tube, after blood is collected in the tube, the tube is overturned up and down for 10 times, so that the blood is fully and uniformly mixed, and the blood is stored at room temperature. The free RNA can be protected for three days, and the blood collection tube can be used for protecting and storing the free RNA.
Description
Technical field
The present invention relates to a kind of protection and the heparin tube of the free RNA of stabilization.
Background technology
Free RNA refer to be present in it is extracellular free in the body fluid such as serum, blood plasma, bronchoalveolar lavage fluid, urine, Pleural effusions
RNA。
With the exhibition of not stopping paying out of diagnostic techniques, the effect of the RNA that dissociates is increasing.
Studies have found that, expression and body fluid middle reaches in the tumor-related gene of the patients such as lung cancer, breast cancer, melanoma
Expression from cancer-related gene RNA is closely related, and free RNA can be as tumor markers, to differentiating Malignant and benign lesions
There are preferably specificity and sensitiveness.
In Non-invasive Prenatal Diagnosis, free RNA also has the effect that can not be substituted.Free fetal rna molecule mainly comes
Placenta is come from, and is stable in the presence of in parent blood circulation, it is easy to detected.Though Fetal genetic information is obtained to can be by obtaining tire
Youngster's cell is carried out, if but obtaining the invasive of fetal cell with the tradition pre-natal diagnosis of such as amniocentesis and chorionic villi sampling etc.
Mode of operation, the risk of miscarriage for having about 1%, this is greatly narrow diagnostic area.And carried out using female blood middle reaches isolated human fetal cell
Pre-natal diagnosis, though non-invasive feature can be reached, its is cumbersome, is related to relatively costly cell enrichment process, it is impossible to general
And in clinic.But, detected using the free RNA of parent, non-invasive effect can be reached, without excessively complicated mistake
Journey, it is easy to clinic popularization, there is very big prospect.
But, substantial amounts of RNase is contained in the blood of people, free RNA is very easy to be degraded by RNase, and in blood
Free RNA concentration it is very low.Clinically, most of blood for being to be gathered using heparin tube people.
And it is current, various heparin tubes are there are on the market, most of these heparin tubes all simply play anti-hemostasis
The effect of liquid solidification, without playing a part of the free RNA of protection.And in CC, due to condition or the limit of time
System, usually cannot after blood is obtained the very first time just carry out the extraction of free RNA.Cell rupture, substantial amounts of RNase, short
Just most free RNA is degraded in short several hours, great obstacle is caused to subsequent experimental, experimental result is easy
False negative is produced, so that valuable information cannot be provided for clinic.
It is therefore, a kind of that can to protect and stablize the heparin tube of free RNA be necessary.
The content of the invention
A kind of heparin tube it is an object of the invention to prepare protection and the free RNA of stabilization, the heparin tube can not only be prevented
Blood clotting, and can protect and stablize, collect wherein free RNA.The heparin tube is by test tube, seal rubber plug and protection
Agent composition.By blood collection to Guan Zhonghou, turning upside down 10 times, it is uniform to be sufficiently mixed.At room temperature, the free RNA in blood
Can preserve 3 days, -20 DEG C to -80 DEG C can preserve for a long time.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of protection and the heparin tube of the free RNA of stabilization, it is characterised in that described test tube is by the poly- to benzene two of hydrophobic property
Formic acid alcohol ester material is fabricated by, and described seal rubber plug is fabricated by by butyl rubber material.
It is a kind of protect and the free RNA of stabilization heparin tube, it is characterised in that the protective agent be by EDTA-3K, IDU,
Bestain, glycine, AEBSF, ribonucleoside complex, guanidinium isothiocyanate, magnetic bead are dissolved in DEPC according to certain ratio
Water is formulated, and wherein concentration of each component in the solution is respectively：EDTA-3K（20g-80g/L）、IDU（250-
700g/L）、bestain（0.06-0.38g/L）、glycine（15-68g/L）、AEBSF（0.01-0.34g/L）, ribonucleotide
Compound（0.2ml-1ml/L）, guanidinium isothiocyanate（30-100g/L）, magnetic bead（0.5-4mg/ml）.
Preferably, the free protectant EDTA-3K of RNA of blood of the present invention, IDU, bestain, glycine,
AEBSF, ribonucleoside complex, guanidinium isothiocyanate, the concentration of magnetic bead be respectively 40g/L, 500g/L, 0.2g/L, 35g/L,
0.162g/L、0.7ml/L、60g/L、1mg/ml。
Brief description of the drawings
Fig. 1 be whole blood sample in it is of the present invention it is a kind of protect and the heparin tube of the free RNA of stabilization in preserve 0 respectively,
1st, nucleic acid extraction is carried out after 2,3 days, the nucleic acid that extraction is obtained carries out β -- the testing result of catenin mRNA real-time fluorescence PCRs.
Fig. 2 is whole blood in conventional EDTA-Na2Nucleic acid extraction is carried out after preserving 0,1,2,3 days in heparin tube respectively, is extracted
To nucleic acid carry out β -- the testing result of catenin mRNA real-time fluorescence PCRs.
Specific implementation method
Following case study on implementation is only used for explaining the present invention, rather than the limitation present invention.
Example：
With heparin tube of the present invention and routine EDTA-Na2Heparin tube randomly selects blood sample simultaneously, and each blood sample extracts 4 pipes respectively, often
Temperature place 0 day, 1 day, 2 days, after 3 days carry out nucleic acid extraction（RNeasy Mini Kit (the 50)-article No. of Qiagen companies
74104 kits）, enter pedestrian β by sample of the nucleic acid of extraction -- the real-time PCR detection of catenin mRNA genes.
Testing result：
It is placed in conventional EDTA-Na2Whole blood sample in the heparin tube of heparin tube and the free RNA of protection of the present invention, normal
Temperature place different time after, nucleic acid extraction is carried out respectively, the nucleic acid of extraction enters pedestrian β -- catenin mRNA genes it is real-time glimmering
Light PCR detections, the Ct values such as table 1 of its testing result.
Table 1
As shown in Table 1, the heparin tube of the free RNA of protection of the present invention can effectively protect free RNA in 3 days, only
There is extremely slight degraded；And routine EDTA-Na2Heparin tube to the stability maintenance time of RNA less than 1 day and degraded situation ratio
It is more serious.
Fig. 1 ~ Fig. 2 is the corresponding people β of table 1 -- the real-time PCR detection result figure of catenin mRNA genes, wherein scheming
1 carries out nucleic acid extraction for whole blood is placed in the heparin tube of protection free nucleic acid RNA as herein described after preservation different time, extracts
The nucleic acid for obtaining carries out β -- the testing result figure of catenin mRNA gene real-time fluorescence PCRs, as seen from the figure, in the inspection of 0 ~ 3 day
In the survey time, all detection samples are all correctly detected, and are had good stability, and free RNA is effectively protected；Fig. 2 is
Whole blood is placed in conventional EDTA-Na2Nucleic acid extraction is carried out after different time is preserved in heparin tube, the nucleic acid that extraction is obtained carries out β --
The testing result figure of catenin mRNA gene real-time fluorescence PCRs, testing result shows, conventional EDTA-Na2Trip in heparin tube
Can not all be maintained from the RNA stability of 1 day, degraded situation is extremely serious.
The heparin tube of the free RNA of protection of the present invention is a kind of heparin tube that can efficiently protect free RNA.
Claims (6)
1. it is a kind of protect and the free RNA of stabilization heparin tube, it is characterised in that the heparin tube be by test tube, seal rubber plug and
Protective agent composition.
2. the heparin tube according to claim 1, it is characterised in that described test tube is by the poly- to benzene two of hydrophobic property
Formic acid alcohol ester material is fabricated by.
3. the heparin tube according to claim 1, it is characterised in that described seal rubber plug is by butyl rubber material system
Make and form.
4. the heparin tube according to claim 1, it is characterised in that described protective agent be by EDTA-3K, IDU,
Bestain, glycine, AEBSF, ribonucleoside complex, guanidinium isothiocyanate, magnetic bead are dissolved in DEPC according to certain ratio
Water, concentration is respectively 40g/L, 500g/L, 0.2g/L, 35g/L, 0.162g/L, 0.7ml/L, 60g/L, 1mg/ml.
5. the heparin tube according to claim 1, it is characterised in that the magnetic bead in the protective agent can be enriched with blood
Free RNA.
6. the heparin tube according to claim 1 ~ 5, it is characterised in that in the application, by blood collection to Guan Zhonghou, up and down
It is sufficiently mixed uniform, room temperature preservation for reverse 10 times.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611214321.7A CN106701742A (en) | 2016-12-26 | 2016-12-26 | Blood collection tube for protecting and stabilizing free RNA (Ribonucleic Acid) |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611214321.7A CN106701742A (en) | 2016-12-26 | 2016-12-26 | Blood collection tube for protecting and stabilizing free RNA (Ribonucleic Acid) |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106701742A true CN106701742A (en) | 2017-05-24 |
Family
ID=58903277
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611214321.7A Pending CN106701742A (en) | 2016-12-26 | 2016-12-26 | Blood collection tube for protecting and stabilizing free RNA (Ribonucleic Acid) |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106701742A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108261332A (en) * | 2018-01-22 | 2018-07-10 | 无锡百泰克生物技术有限公司 | A kind of blood rna preserves pipe |
| CN110747193A (en) * | 2019-09-25 | 2020-02-04 | 广州市达瑞生物技术股份有限公司 | Lung cancer polygene joint detection kit quality control product based on second-generation sequencing platform |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010134246A1 (en) * | 2009-05-20 | 2010-11-25 | オリンパス株式会社 | Method for preparation of nucleic acid-containing sample |
| CN202458389U (en) * | 2012-02-07 | 2012-10-03 | 山东侨牌集团有限公司 | Disposable vacuum blood collecting tube |
| CN103789202A (en) * | 2014-01-26 | 2014-05-14 | 付士明 | Container for collecting nucleic acid preserved at normal temperature |
| CN104673623A (en) * | 2015-02-05 | 2015-06-03 | 广州新诚生物科技有限公司 | Blood sample collecting device |
-
2016
- 2016-12-26 CN CN201611214321.7A patent/CN106701742A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010134246A1 (en) * | 2009-05-20 | 2010-11-25 | オリンパス株式会社 | Method for preparation of nucleic acid-containing sample |
| CN202458389U (en) * | 2012-02-07 | 2012-10-03 | 山东侨牌集团有限公司 | Disposable vacuum blood collecting tube |
| CN103789202A (en) * | 2014-01-26 | 2014-05-14 | 付士明 | Container for collecting nucleic acid preserved at normal temperature |
| CN104673623A (en) * | 2015-02-05 | 2015-06-03 | 广州新诚生物科技有限公司 | Blood sample collecting device |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108261332A (en) * | 2018-01-22 | 2018-07-10 | 无锡百泰克生物技术有限公司 | A kind of blood rna preserves pipe |
| CN110747193A (en) * | 2019-09-25 | 2020-02-04 | 广州市达瑞生物技术股份有限公司 | Lung cancer polygene joint detection kit quality control product based on second-generation sequencing platform |
| CN110747193B (en) * | 2019-09-25 | 2021-08-31 | 广州市达瑞生物技术股份有限公司 | Lung cancer polygene joint detection kit quality control product based on second-generation sequencing platform |
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|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170524 |
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| RJ01 | Rejection of invention patent application after publication |