CN1358866A - Novel technology of peripheral blood white cell hybridization in situ experiment - Google Patents
Novel technology of peripheral blood white cell hybridization in situ experiment Download PDFInfo
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- CN1358866A CN1358866A CN 01135273 CN01135273A CN1358866A CN 1358866 A CN1358866 A CN 1358866A CN 01135273 CN01135273 CN 01135273 CN 01135273 A CN01135273 A CN 01135273A CN 1358866 A CN1358866 A CN 1358866A
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Abstract
The present invention relates to a simple and convenient in-situ hybridization experimental technique for detecting several biological informational molecules mRNA of peripheral blood leukocyte. Said invention is directed against expression of several specific informational moleculus mRNA, and makes exploration of the in-situ hybridization experimental technique of peripheral blood leukocyte, improves the in-situ hybridization process and provides a simple, convenient, stable and practical research technique.
Description
The present invention relates to the hybridization in situ experiment technology that a kind of messenger RNA(mRNA) (mRNA) of easy, the stable multiple informational molecule of detection peripheral blood leucocyte is expressed.
At present, the detection that mRNA expresses in the cell still lacks stable in situ hybridization working method, because result's poor stability makes the reliability of the data that obtain be subjected to very big influence, even can not do the result sometimes, causes the waste of time, human and material resources.Therefore, the hybridization in situ experiment of detection mRNA is difficult to be widely used in molecular biology experiment and clinic study.In addition, hybridization in situ experiment also is subjected to organizing the difficult restriction of drawing materials clinically.Peripheral blood is a window of understanding human body diseases, and it is easy to get sample, easy to utilize.We have carried out the exploration of peripheral blood white cell hybridization in situ experiment method at the expression of customizing messages molecule mRNA.By experiment repeatedly, set up the in situ hybridization flow process, and made detection method obtain success.
The objective of the invention is for molecular biology, basis and clinic study etc. provide a kind of easy, stable, practical investigative technique, for the detection that is used for multiple informational molecule mRNA.
The used probe of the present invention is three probes of oligonucleotide of 35 bases of digoxigenin labeled; Antibody is anti-digoxin alkaline phosphatase; Chromogenic substrate is nitro blue tetrazolium/5-bromo-4-chloro-3-indoles phosphoric acid salt (NBT/BCIP).Specific implementation method is as follows:
Aseptic taking heparin anticoagulation 0.5ml places the centrifuge tube of handling with 0.1% diethylpyrocarbonate (DEPC) distilled water, leaves standstill 2 hours.Get and contain leukocytic blood plasma 0.1ml, fix 3 hours at 10: 1, centrifugal 4000 rev/mins, 5 minutes, abandon supernatant with 4% Paraformaldehyde 96.0.05mol/L PBS washing 2 minutes, centrifugal 4000 rev/mins, 5 minutes, abandon supernatant, repeat 3 times.0.05mol/L PBS 0.2ml mixing precipitation, smear, 37 ℃ of dried overnight.
Detect the in situ hybridization flow process of white corpuscle mRNA: 1. 2. 0.1mol/L PBS (pH7.2) 3 minutes * 2 (flush away vitamins Cs of 0.05mol/L vitamins C 30 minutes (deactivation endogenous alkaline phosphatase and peroxidase), adjust pH to 7.2), the hybridization solution 10ul (concentration and probe concentration is 2ng/ul) that (flush away PBS) 3. dry smear (has just done to spending on the surface) 4. contains probe is washed once in distillation, 5. 37 ℃ in wet box spends the night in the 2 * SSC 30-37 ℃ of water-bath, and 6. flushing 10 minutes is (5. in 0.2 * SSC 30-37 ℃ of water-bath in flushing 5 minutes * 3,6. the 7. confining liquid room temperature 20 minutes probe of the non-specific combination of flush away), do not wash anti digoxin antibody-alkaline phosphatase [Anti-DIG-AP (antibody is with confining liquid dilution in 1: the 25)] 10ul that (the non-specific combination site of blocking antibody) 8. dilutes, 37 ℃ of 9. 0.05mol/L PBS (pH7.2) flushings in 1 hour of wet box, 5 minutes * 4 (antibody of the non-specific combination of flush away); Buffer1 washes 5 minutes (for alkaline phosphatase provides magnesium ion and suitable osmotic pressure); Buffer3 washes NBT/BCIP (diluting by 1: 100 with the buffer3) 50ul that 5 minutes (for alkaline phosphatase provides suitable pH environment) 10. dilutes, and 37 ℃ of lucifuges developed the color 10-30 minute, washing, and the nuclear fast red is redyed, washing, water-soluble mountant mounting.To detect the example that is expressed as of diabetics's peripheral blood leucocyte inducible nitric oxide synthase mRNA (iNOS-mRNA) and diabetics's structure type nitricoxide synthase mRNA (cNOS-mRNA), detected result is seen (Fig. 1).
Fig. 1. the expression of healthy people and diabetics's peripheral blood leucocyte smear iNOS-mRNA and diabetics cNOS-mRNA.
A: healthy human peripheral blood white cell hybridization in situ sheet, no iNOS-mRNA positive hybridization signal in the endochylema, the nuclear fast red redyes 100 * 10
B: the positive sheet of diabetic subject's peripheral blood white cell hybridization in situ, navy blue positive hybridization signal is iNOS-mRNA in the endochylema, the nuclear fast red redyes 100 * 10
C: the positive sheet of diabetic subject's peripheral blood white cell hybridization in situ, navy blue positive hybridization signal is cNOS-mRNA in the endochylema, the nuclear fast red redyes 100 * 10
Claims (5)
1. hybridization in situ experiment technology that detects the multiple biological information molecule mRNA of peripheral blood leucocyte, used probe is the oligonucleotide probe of digoxigenin labeled.
2. hybridization in situ experiment technology according to claim 1, the deactivation of endogenous alkaline phosphatase and peroxidase in its sample is adopted the 0.05mol/L vitamins C to soak and was handled in 30 minutes.
3. experimental technique according to claim 1, steps such as increasing probe permeability and protease digestion is omitted in the processing before its hybridization.
4. experimental technique according to claim 1, its hybridization back with 2 * SSC in 30-37 ℃ of water-bath, wash 5 minutes * 3 and 0.2 * SSC in 30-37 ℃ of water-bath, washed 10 minutes.
5. according to the hybridization in situ experiment technology under the claim 1, anti digoxin antibody-alkaline phosphatase directly adds in the confining liquid that drips in advance on the slide after diluting at 1: 25 with confining liquid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011352736A CN1155724C (en) | 2001-12-12 | 2001-12-12 | Novel technology of peripheral blood white cell hybridization in situ experiment |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011352736A CN1155724C (en) | 2001-12-12 | 2001-12-12 | Novel technology of peripheral blood white cell hybridization in situ experiment |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1358866A true CN1358866A (en) | 2002-07-17 |
| CN1155724C CN1155724C (en) | 2004-06-30 |
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| Application Number | Title | Priority Date | Filing Date |
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| CNB011352736A Expired - Fee Related CN1155724C (en) | 2001-12-12 | 2001-12-12 | Novel technology of peripheral blood white cell hybridization in situ experiment |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108169474A (en) * | 2018-01-12 | 2018-06-15 | 中国科学院成都生物研究所 | A kind of novel cell fixative |
| CN111579798A (en) * | 2020-05-29 | 2020-08-25 | 深圳市锦欣医疗科技创新中心有限公司 | Kit for evaluating endometrial receptivity and using method thereof |
-
2001
- 2001-12-12 CN CNB011352736A patent/CN1155724C/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108169474A (en) * | 2018-01-12 | 2018-06-15 | 中国科学院成都生物研究所 | A kind of novel cell fixative |
| CN108169474B (en) * | 2018-01-12 | 2020-09-25 | 中国科学院成都生物研究所 | Novel cell fixing agent |
| CN111579798A (en) * | 2020-05-29 | 2020-08-25 | 深圳市锦欣医疗科技创新中心有限公司 | Kit for evaluating endometrial receptivity and using method thereof |
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| Publication number | Publication date |
|---|---|
| CN1155724C (en) | 2004-06-30 |
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