EP0360822A1 - Process for investigating an acellular biological fluid for cellular oncogenic transcripts or fragments thereof - Google Patents
Process for investigating an acellular biological fluid for cellular oncogenic transcripts or fragments thereofInfo
- Publication number
- EP0360822A1 EP0360822A1 EP88904461A EP88904461A EP0360822A1 EP 0360822 A1 EP0360822 A1 EP 0360822A1 EP 88904461 A EP88904461 A EP 88904461A EP 88904461 A EP88904461 A EP 88904461A EP 0360822 A1 EP0360822 A1 EP 0360822A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- rna
- labeled
- oncogene
- dna
- product
- 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.)
- Ceased
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
Definitions
- the invention relates to a method for the detection of cellular oncogene transcripts, or their fragments, according to the preamble of claim 1, it relates to a malignancy test.
- RNA transcripts Methods for detecting cellular oncogene (RNA) transcripts, including separation of RNA, mRNA, denaturation and hybridization, and the like are known in the field of molecular biological research. Reproducibility, reliability, specificity and extreme sensitivity of the in vitro hybridization between DNA and DNA, as well as between RNA and DNA are known from the literature, which has found application as a standard method for specific problem solutions.
- RNA oncogene
- RNA hybridization with DNA using a solid substrate for immobilization has been known since 1965 (J. Mol. Biol. 12: 829-842; 1965). Since then, several improvements to this method and the methods of separating RNA from cells and tissues have been published.
- RNA or poly (A) RNA (iriRNA) by the living malignant cells, as the experiments with in vitro grown malignant cell cultures have already proven.
- RNA in particular the ⁇ lRNA from the cell nucleus
- cancer-specific products such as "cancer-associated protein etc.
- RNA form (ds for double-stranded RNA).
- the causes and circumstances mentioned above are responsible for the changed RNA content in the blood plasma in different combinations and different dimensions in the different types and stages of malignant states.
- the increased transcription of cellular oncogenes is the basic requirement, but not enough.
- precancerous conditions such as polyposis of the colon, for example, the transcription of one of the cellular oncogenes can even be increased 90-fold, but no RNA transcript of the highly activated oncogene appears in the blood plasma.
- Others and several of the circumstances noted above that are characteristic of the malignant states must be present and cooperative at the same time.
- RNA hybridization in vitro is not only extremely specific, but also highly sensitive. 1 pg of RNA can already be detected in a complex of RNA molecules.
- the normal values showed a large spread and so there can be a significant overlap of the normal and pathological values.
- studies with monoclonal antibodies against an oncogene polypeptide showed positive values in 25-29% of the malignant and in 6-9% of the non-malignant cases.
- a very significant disadvantage of this like any other method, which is based on the immunological detection of the proteins or polypeptides of cancer cell origin, is that the presence of specific antibodies which are formed by the host or are introduced for therapeutic purposes has unrealistic results cause and can be a significant disruptive factor at all.
- the invention is based on the object of specifying a method for the detection of the RNA transcripts, or their fragments, of cellular oncogenes in an acellular biological fluid, such as blood plasma, as a malignancy test. Since it is necessary for this to first separate the RNA from the blood plasma, it is a further object of the invention to provide a reliable method for separating the RNA from the acellular body fluid, e.g. B. to indicate the blood plasma.
- This object is achieved by a method according to claim 1.
- reliable, effective, potent RNase inhibitors by avoiding the mixing of the ubiquitous, highly resistant RNase enzyme from exogenous sources throughout the process, by treating the blood plasma to separate the RNA Content, by immobilizing the RNA in denatured form on a solid substrate and by contacting the solid substrate with the labeled denatured oncogene DNA around them when the plasma RNA and oncogene DNA have complementary sequence (s) bind (to hybridize), a reliable detection method is achieved. The fact of hybridization is determined by the detection of the labeling substance, or in the case of radioisotopic labeling by autoradiography.
- the total plasma RNA such as the RNA, mRNA, or fragments thereof which have entered the bloodstream, are isolated from the blood plasma of patients with malignant and non-malignant diseases and the presence of the RNA transcripts of cellular oncogenes with in vitro mole ⁇ molecular hybridization examined.
- RNA transcripts, or their fragments, of cellular oncogenes from human blood plasma is a universal malignancy test for the detection of malignant processes, (as a confirmation test), for early detection of malignancy ( as an addiction test) and for early detection of recurrences and metastases after therapeutic measures (as a follow-up test).
- RNA RNA that can differentiate between malignancy and non-malignancy origin.
- Messencjer activity of the RNA, tested in the cell-free protein-synthesizing system has proven to be unsuitable for this, because this activity could be observed in both malignant and non-malignant states.
- this differentiation could be achieved by the presence of the oncogene transcripts or their fragments in the plasma RNA.
- plasma RNAs from malignant cases were found to contain oncogene transcripts or their fragments, while practically none of them were detectable from plasma RNAs from non-malignant states. Further research confirmed this tendency and indicated that this method is capable of smaller amounts of
- the most important advantage of this invention is that it specifies a method that can serve as a universal malignancy test: This test is based on the basic principles of the malignant transformation by activating cellular oncogenes and the special features of the malignant cells and tumors.
- the second significant advantage of this method is the high sensitivity (1-5 pg / ml).
- Another major advantage of the invention in contrast to II tests, is that the specific antibodies which have been formed by the host or which have been introduced into the bloodstream for therapeutic purposes cannot influence the results.
- the method according to the invention has the advantage that there is no interference with the result or specific antibodies.
- the basic character of malignant processes and their behavior in the host is largely determined by which oncogenes have been activated (genetic scripture, cell oncogene profile). Up to now, this could only be determined histologically in tumor tissues by in vitro nucleic acid hybridization.
- the malignancy test according to the invention can provide this information at least partially from the blood plasma (plasma oncogene profile) long before the relatively small tumor cell mass can be visualized and reached for histological examinations.
- New oncogenes can be activated during the malignant processes in vitro as well as during the existence of the malignant disease in patients, which can lead to a significant progression of the malignancy. This risk can be detected early with the malignancy test during the follow-up by identifying a new oncogene transcript from the blood plasma that was not previously present in the plasma.
- an amplification amplification the activated oncogenes occur, which can cause a substantially increased aggressiveness and progression of the malignant cells.
- the occurrence of such an enhancement can be observed early on during the follow-up with the quantitative maglignity test.
- This subclone can be recognized at an early stage before it has been able to spread and could worsen the prognosis.
- This subclone can be influenced in the early stage by specific immunotherapy, immunochemotherapy, or by preventing the activity of the oncogene RNA transcripts, etc. and possibly destroyed.
- a - amplification of the cellular oncogenes occurs either in the primary tumor or during the progression and diversification of the malignant cells and in approximately 30% to 50% of the malignant diseases.
- Gene products of the activated cellular oncogenes play an important, even vital function in the transformed malignant cells, many of which act as protein kinase enzymes in the cell membrane.
- the change or impairment of this function by attacking the malignant cells in an immunospecific manner, targeting their oncogene products with monoclonal antibodies alone or in conjunction with substances having a radioactive or chemotherapeutic effect, can lead to a healing effect before the tumor cell mass has been able to spread.
- a plasma oncogene profile with early detection shows the specific therapy options.
- the monitoring of the course with new oncogene samples makes it possible to recognize newly occurring oncogene transcripts.
- a quantitative malignancy test during the course of the disease offers an early detection of a possible amplification of a cellular oncogene and gives specific information on how to suppress or destroy this new subclone of malignant cells before a larger progression or aggressiveness is caused.
- the freshly obtained blood is immediately mixed with an RNase enzyme-inhibiting substance and the plasma quickly separated.
- the blood plasma is mixed in the presence of the RNase inhibitor with a detergent such as sodium duodecyl sulfate (SDS) and a proteolytic enzyme such as Proteinase K (Boehringer, Mannheim) with an aqueous medium and is then at 37 degrees C for 1 to 3 h in one Buffer solution (pH 7.5) incubated. It is then mixed with 4 M guanidinium isothiocyanate to effect protein denaturation and the cleavage of the proteins from the RNA. The mixture is then extracted with organic solvents such as phenol and chloroform at 60 ° C.
- RNA is precipitated by mixing with two vol ethanol at -20 degrees C.
- the RNA is dissolved in Tris.Cl buffer (pH 7.4) with EDTA and SDS and treated with proteinase K at 37 degrees C for one hour in the presence of RNase heaters .
- the RNA precipitated with ethanol washed and stored in 70% ethanol at -70 degrees C.
- Either the poly (A) RNA is selected from the RNA by affinity adsorption and elution methods with oligo (dT) cellulose, or the RNA is subjected to a DNase enzyme treatment in order to degrade and remove the DN that may be present.
- the denatured RNA or the poly (A) RNA is applied to pure nitrocellulose paper as a solid substrate and is treated for 2 hours at 80 ° C. in a vacuum oven in order to cause the RNA to bind on the solid substrate.
- the baked solid substrate is then treated to prevent further binding of nucleic acids to the solid substrate.
- An amount of purified molecularly cloned DNA containing the sequence as a code for oncogene is labeled either radioisotopically or non-radioisotopically.
- the radioisotopic labeling is carried out by nick translation according to Rigby et al. (J. Mol. Biol. 113: 237-251, 1977) performed to achieve high specific activity. For this
- Radioisotopes that can be used in procedures are, for example,
- Oncogene DNA samples labeled or unlabeled, are commercially available (ONCOR, Inc. Gaithersburgh, Ma. USA 20877; ONCOGENE SCIENCE Inc. Mineola, NY. 11501 USA).
- the labeled oncogene DNA sample is denatured and contacted in a solution with the solid substrate on which the plasma RNA is immobilized to hybridize therewith. This causes the labeled oncogene DNA sample to be linked (hybridized) by hydrogen compounds to the complementary sequence (s) of the plasma RNA which is immobilized on the solid substrate.
- the solid substrate is washed to remove non-specifically bound labeled oncogene DNA sample.
- the solid substrate is then subjected to analysis to detect the hybridization between the plasma RNA and the labeled oncogene DNA sample.
- this is assaulted by autoradiography, in the case of biotin labeling by biotin detection using an enzyme affinity test by color reaction.
- a mixture of 0.2 M Tris.Cl (pH.7.5); 25 mM ethylenediaminetetraacetic acid (EDTA); 0.3 M NaCl; 2% (wt / full sodium duodecyl sulfate (SDS) and proteinase K (final concentration 200 micrograms / ml) were added and incubated for 1 to 3 h in the presence of RNase inhibitors at 37 ° C. Then mixed with 4 M guanidinium isothiocyanate. The slimy mixture is drawn into a syringe equipped with an 18 gr. Needle and expelled vigorously until the viscosity is significantly reduced at 60 degrees C.
- RNA is dissolved in Tris.Cl buffer (0.1 M, pH 7.4) buffer solution which still contains 50 mM NaCl, 10 mM EDTA, 2% SDS and RNase inhibitor and with Proteinase K (final concentration 200 micrograms) / ml) incubated at 37 degrees C for 1 h. Then it is extracted twice with phenol and chloroform at 60 degrees C and with chloroform at room temperature. The RNA is precipitated from the aqueous phase with ethanol, washed with 70% ethanol and each dissolved in the necessary solution. The extraction of the RNA is essential and significantly facilitated by the use of the "Nucleic Acid Extractor" instrument (Applied Biosystem, Foster City, Ca. USA). The procedure written above is easily adaptable for this instrument.
- Either the poly (A) RNA is selected from the isolated plasma RNA with oligo (dT) cellulose by "batch" absorption and elution, or we subject the plasma RNA to a DNase enzyme treatment in order to degrade the DNA which may be present.
- the plasma RNA is dissolved in sterile water, incubated at 65 degrees C for 10 min and mixed with 2 x loading buffer and cooled to temperature (loading buffer: 20 mM Tris. Cl, pH 7.6; 0.5 M NaCl; 1 mM EDTA;. 0.1% SDS) then is mixed with 0.3 g (Trok- kensolv) "of oligo (dT) -cellulose for each 0.5 mg of RNA, at 1500 g for 4 Centrifuged min at 15 degrees C and the oligo (dT) cellulose washed 4-5 times with 5 ml loading buffer (at room temperature). The poly (A) + RNA is washed with 1 ml of sterile 10 mM Tris. Cl (pH 7.5; 1mM EDTA; 0.05% SDS. Eluted.
- Na acetate (3 M pH 5.2) is mixed to 0.3 M and the RNA precipitated with 2.2 vol ethanol at -20 ° C. and the precipitate washed with 70% ethanol.
- the plasma RNA is subjected to a DNase enzyme treatment.
- RNase-free DNase enzyme and MgCl_ are mixed to the plasma RNA solution (50 mM Tris. Cl, pH 7.5 in 1 mM EDTA) and in the presence of RNase inhibitors at 37 degrees C for 30 min incubated, then extracted with phenol / chloroform and in the presence of Na acetate (pH 5.2, 0.3 M) the RNA is precipitated with 70% ethanol at -20 degrees C.
- Biotin-labeled DNA can be isolated with 2-butanol extraction with ethanol precipitation and can be stored as a stable labeled sample for half a year in 0.1 M EDTA at -15 degrees C.
- the plasma RNA or the poly (A) RNA is subjected to a treatment, the so-called prehybridization, before the hybridization.
- the RNA solution or the RNA dilutions are denatured first by incubating the 65 degrees C for 15 min and then by rapid cooling.
- the RNA is then applied to the solid substrate, such as nitrocellulose paper, which was previously equilibrated with 20 x NaCl / Cit and then dried.
- a corresponding volume of the denatured RNA solution i is a well of the microfilter plate (Bio-Dot Microfiltration Apparatus, Bio-Rad Richmond, ca. 94804 USA; Minifold Filtration & Incubation Plate, Schleicher & Schuel, Keene, New Hampshire 03431, USA and filtered under gentle vacuum to form a spot of 3.0 mm in diameter, quantity of RNA applied: 10 micrograms, the poly (A) RNA: 2 micrograms
- nitrocellulose paper is then dried in a vacuum oven at 80 ° C. for 2 hours. After baking, in a solution (prehybridization buffer): formamides (50% vol / vol); 5 x NaCl / Cit; 50 M sodium phosphate pH 6.5; sonicated, denatured Salmon sperm DNA (250 micrograms / ml) and 0.02% per bovine serum albumin (BSA), Ficoll and polyvinylpyrrolidone, incubated for 8-20 h at 42 ° C.
- prehybridization buffer formamides (50% vol / vol); 5 x NaCl / Cit; 50 M sodium phosphate pH 6.5; sonicated, denatured Salmon sperm DNA (250 micrograms / ml) and 0.02% per bovine serum albumin (BSA), Ficoll and polyvinylpyrrolidone
- the radioisotopically labeled human oncogene DNA sample (2 x 10 cpm / ml) is only denatured at 100 degrees C for 5-10 min, cooled rapidly and becomes the solution (prehybridization buffer) which immobilized the nitrocellulose paper with the immobilized product Contains mixed plasma RNA.
- the hybridization is effected at 42 degrees C for 20 hours.
- the nitrocellulose paper is inserted between clear acetate foils and brought close to a film sensitive to the X-rays.
- An intensifying screen is attached to the opposite side and packed light-tight. After a certain time, the film is developed. The presence of the RNA transcripts or their fragments that hybridized with the oncogene DNA sample is determined by the presence of a spot on the film.
- the DNA samples are applied in 0.1 M EDTA, otherwise the prehybridization is carried out as with radioisotopically labeled DNA samples, but the The duration of the pre-hybridization is shorter: 4-8 h.
- the hybridization is carried out as with radioactive samples, but at a higher temperature (55 degrees C) for 20 h in a solution: 4 vol.
- Prehybridization buffer 1 volume of about 0.5 g / ml sodium dextran sulfate and 20 ng / ml biotin-labeled DNA sample (oncogene DNA sample).
- the dried nitrocellulose papers are at 42 degrees C for 30 min in STMT buffer (1 M NaCl; 0.1 M Tris. Cl. PH 7.5; 2 mM MgCl 2 0.05% v / v Triton X-100) with 30 mg of Bovine serum albumin incubated. After the nitrocellose paper has dried, it is incubated at room temperature for 10 min in SIMT buffer with 1 microgram / ml Sigma Avidin-alkaline phosphatase complex, then it is shaken frequently in STMT buffer (3 x 10 min) STM buffer (1 M NaCl, Tris. Cl pH 9.5, 5 mM MgCl incubated for 2 x 5 min
- the nitrocellulose paper is darkened at room temperature with substrate solution (STM buffer but only with 0.1 M NaCl) with 0. mg / ml nitro-blue-tetrazolium, 0.17 mg / ml 5-br ⁇ no-4-chloro-3-indolyl Phophate and 0.33% v / v N, N-dimethylformamide mixed.
- substrate solution STM buffer but only with 0.1 M NaCl
- the reaction is carried out by washing the nitrocellulose paper with 10 mM Tris.Cl pH 7.5; -1 mM EDTA terminated.
- the detection of the presence of oncogene RNA transcripts or fragments is positive if the plasma RNA or the poly (A) RNA has a positive signal at least with one of the oncogene DNA samples (above the background or above negative control).
- the radioactive sample a dark spot appears on the film according to autoradiography in the case of biotin-labeled samples as a spot with a color reaction.
- D Malignancy test is negative if no RNA is isolated from the blood plasma or if the blood plasma RNA does not give a positive signal with any of the oncogene samples.
- the hybridized DNA sample can be washed at 65 degrees C for 1 - 2 h with 0.1 - 0.05 x wash buffer (1 x wash buffer: 50 mM Tris.Cl. pH 8.0; 0.2 mM EDTA; 0.5% sodium pyrophosphate and 0.02% per BSA, Ficoll , Polyvinyl pyrrolidone) can be removed.
- the treatment of the nitrocellulose paper, that is to say the prehybridization and the re-hybridization is carried out as above (original).
- RNA transcripts, or their fragments, of cellular oncogenes in human blood plasma comprises several favorable reaction methods and steps in order to result in a high reliability and reproducibility.
- the use of reliable RNase-inhibiting substances already prevents the possible degradation of RNA when the blood is drawn.
- the risk of mixing (admixing) the ubiquitous, highly resistant RNase from exogenous sources is mitigated throughout the entire process by using baked glassware, autoclaved solutions, baked spatulas, tools, dry chemical preparations, and glass distilled Autoclave-sterilized water and wearing gloves reduced during all phases and stages of the preparation and examination process.
- Proteinase K facilitates the removal of the proteins, the guanidinite-isothiocyanate treatment, the denaturation of the proteins and the cleavage of the RNA-protein complexes.
- the rest of the proteins are removed by organic extraction.
- the degradation and removal of the DNA is achieved by DNase treatment, because otherwise the DNA could interfere with the hybridization.
- Baking ensures the reliability of the tight binding of the RNA to the nitrocellulose filter. Treating the nitrocellulose filter after baking prevents unspecific binding and also ensures reliability. Washing the nitrocellulose filter after hybridization removes unnecessary, or non-specifically bound, labeled oncogene DNA samples and also contributes to reliability.
- a semi-quantitative evaluation can also be carried out.
- the intensity of the black spot on the X-ray film is measured by densitometry and quantitative comparisons can be made. (Reflectance Densitcmeter, Bio-Rad, Richmond CA 94804, USA). Quantitative test:
- the relative amount can be determined by the dilution method.
- a 1: 2 serial dilution is made from the blood plasma RNA and each dilution is tested by hybridization.
- the highest dilution from which a positive signal is still emitted is the titer.
- quantitative comparisons can be made during the observation period. In this way you can e.g. B. observe the amplification of one of the activated oncogenes: If the titer of an oncogene is increased disproportionately compared to other oncogenes during the follow-up, this means the amplification of this oncogene.
- Radioactive ogenogen samples with higher activity or according to Leary et al . (Proc . Natl Acad. Sci. USA 80: 4045 4049, 1983) use oncogene samples and enzyme affinity tests labeled with biotin and use them - if possible - with from the plasma RNA selek
- Hybridized poly (A) RNA In this way a sensitivity of pg / ml RNA can be achieved. Otherwise the use for hybridization is
- a positive test in the case of a malignant disease is usually obtained if the plasma RNA hybridizes only with oncogene group A (see above), because these oncogenes are activated in all or almost all types of malignancy. It is less common to extend hybridization to Group B.
- oncogenes are particularly often activated in this cancer. New oncogenes are still being discovered. This can also be used as a sample if necessary use, as well as oligonucleotide samples for the detection of oncogenes activated by point mutation.
- RNA in denatured form, free in solution is mixed with the labeled oncogene DNA sample, incubated for 1-2 hours at 70 ° C. in the presence of the nuclease inhibitors. Then hydroxyapatite is added for 5 min. incubated at 70 degrees C to adsorb the product of the hybridization (RNA-labeled oncogene-DNA complexes).
- the hydroapatite fraction thus formed is separated as sediment by centrifugation.
- the sediment is washed (for 5 min, at 70 degrees C) and the product in the hydroxyapatite fraction is determined based on the label.
Abstract
Selon un procédé d'examen de fluides biologiques acellulaire afin d'y dépister la présence de transcripts oncogènes cellulaires ou de fragments de ceux-ci, indicateurs de malignité, a) on concentre ou sépare l'ARN de l'ensemble du fluide biologique acellulaire en présence permanente d'un inhibiteur efficace de la RNase, puis on dénature l'ARN et on l'immobilise sous cette forme dans un support ou on le laisse libre dans une solution, b) on met l'ARN en contact avec des échantillons marqués d'ADN oncogène afin d'hybrider ce dernier avec l'ARN dans le cas où une séquence complémentaire serait présente, ce qui élimine l'ADN oncogène marqué excédentaire, lié de manière non spécifique, et c) on examine le produit afin d'y dépister la présence d'ADN marqué.According to a process for examining acellular biological fluids in order to detect there the presence of cellular oncogenic transcripts or fragments thereof, indicators of malignancy, a) the RNA is concentrated or separated from the whole of the acellular biological fluid in the permanent presence of an effective RNase inhibitor, then denature the RNA and immobilize it in this form in a support or leave it free in a solution, b) put the RNA in contact with samples labeled with oncogenic DNA in order to hybridize the latter with RNA in the event that a complementary sequence is present, which eliminates the excess labeled oncogenic DNA, linked in a non-specific manner, and c) the product is examined in order to '' detect the presence of labeled DNA.
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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DE19873717212 DE3717212A1 (en) | 1987-05-22 | 1987-05-22 | METHOD FOR THE EXAMINATION OF A CELLULAR BIOLOGICAL LIQUID FOR CELLULAR ONCOGEN TRANScripts or THEIR FRAGMENTS |
DE3717212 | 1987-05-22 |
Publications (1)
Publication Number | Publication Date |
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EP0360822A1 true EP0360822A1 (en) | 1990-04-04 |
Family
ID=6328136
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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EP88904461A Ceased EP0360822A1 (en) | 1987-05-22 | 1988-05-17 | Process for investigating an acellular biological fluid for cellular oncogenic transcripts or fragments thereof |
Country Status (4)
Country | Link |
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EP (1) | EP0360822A1 (en) |
JP (1) | JPH03502518A (en) |
DE (1) | DE3717212A1 (en) |
WO (1) | WO1988009385A1 (en) |
Families Citing this family (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5582970A (en) * | 1982-11-03 | 1996-12-10 | City Of Hope | Competitive hybridization technique |
WO1990009456A1 (en) * | 1989-02-16 | 1990-08-23 | Viktor Balazs | Malignancy test (cancer test) using the polymerase chain reaction |
US5824476A (en) * | 1996-02-28 | 1998-10-20 | City Of Hope | Competitive hybridization technique |
US6607898B1 (en) | 1996-03-26 | 2003-08-19 | Oncomedx, Inc. | Method for detection of hTR and hTERT telomerase-associated RNA in plasma or serum |
EP0938320B2 (en) | 1996-03-26 | 2014-06-18 | Michael S. Kopreski | Method enabling use of extracellular rna extracted from plasma or serum to detect, monitor or evaluate cancer |
US6759217B2 (en) | 1996-03-26 | 2004-07-06 | Oncomedx, Inc. | Method enabling use of extracellular RNA extracted from plasma or serum to detect, monitor or evaluate cancer |
US8043835B1 (en) | 1996-03-26 | 2011-10-25 | Oncomedx, Inc. | Methods for detecting and monitoring cancer using extracellular RNA |
US7785842B2 (en) | 1996-03-26 | 2010-08-31 | Oncomedx, Inc. | Comparative analysis of extracellular RNA species |
US8440396B2 (en) | 1997-03-14 | 2013-05-14 | Oncomedx, Inc. | Method enabling use of extracellular RNA extracted from plasma or serum to detect, monitor or evaluate cancer |
DE19736691A1 (en) * | 1997-08-22 | 1999-02-25 | Michael Prof Dr Med Giesing | Characterising and identifying disseminated metastatic cancer cells |
US8163524B2 (en) | 1998-09-22 | 2012-04-24 | Oncomedx, Inc. | Comparative analysis of extracellular RNA species |
US7767422B2 (en) | 1998-09-22 | 2010-08-03 | Oncomedx, Inc. | Detection of 5T4 RNA in plasma and serum |
EP1158055A1 (en) | 2000-05-26 | 2001-11-28 | Xu Qi University of Teaxs Laboratoire de Leucémie Chen | Method for diagnosing cancers |
DE10126572B4 (en) | 2001-05-31 | 2006-10-26 | Infineon Technologies Ag | Polyhydroxyamides and highly crosslinked chemically and thermally stable polymers obtainable therefrom |
US20100159464A1 (en) * | 2001-11-05 | 2010-06-24 | Oncomedx, Inc. | Method for Detection of DNA Methyltransferase RNA in Plasma and Serum |
US7767390B2 (en) | 2001-11-20 | 2010-08-03 | Oncomedx, Inc. | Methods for evaluating drug-resistance gene expression in the cancer patient |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
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US4446237A (en) * | 1981-03-27 | 1984-05-01 | Life Technologies, Inc. | Method for detection of a suspect viral deoxyribonucleic acid in an acellular biological fluid |
US4483920A (en) * | 1982-05-17 | 1984-11-20 | Hahnemann University | Immobilization of message RNA directly from cells onto filter material |
US4588682A (en) * | 1982-12-13 | 1986-05-13 | Integrated Genetics, Inc. | Binding nucleic acid to a support |
JPS6091999A (en) * | 1983-10-25 | 1985-05-23 | Fujirebio Inc | Measurement of polynucleotide |
FR2588383B1 (en) * | 1985-10-04 | 1988-01-08 | Inst Nat Sante Rech Med | NOVEL PROCESS FOR RECOVERING AND DETERMINING MICROQUANTITES OF DEOXYRIBONUCLEIC ACID IN AN ACELLULAR BIOLOGICAL LIQUID |
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1987
- 1987-05-22 DE DE19873717212 patent/DE3717212A1/en not_active Ceased
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1988
- 1988-05-17 EP EP88904461A patent/EP0360822A1/en not_active Ceased
- 1988-05-17 JP JP63504274A patent/JPH03502518A/en active Pending
- 1988-05-17 WO PCT/DE1988/000287 patent/WO1988009385A1/en not_active Application Discontinuation
Non-Patent Citations (1)
Title |
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See references of WO8809385A1 * |
Also Published As
Publication number | Publication date |
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JPH03502518A (en) | 1991-06-13 |
WO1988009385A1 (en) | 1988-12-01 |
DE3717212A1 (en) | 1988-12-08 |
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