EP1434883A2 - Reseaux d'affichage differentiels specifiques - Google Patents

Reseaux d'affichage differentiels specifiques

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Publication number
EP1434883A2
EP1434883A2 EP02800000A EP02800000A EP1434883A2 EP 1434883 A2 EP1434883 A2 EP 1434883A2 EP 02800000 A EP02800000 A EP 02800000A EP 02800000 A EP02800000 A EP 02800000A EP 1434883 A2 EP1434883 A2 EP 1434883A2
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EP
European Patent Office
Prior art keywords
biological
biological material
gene
identifying
expression profile
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.)
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Application number
EP02800000A
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German (de)
English (en)
Inventor
Jens BITSCH-NORHAVE
Kenneth Thirstrup
Rene Hummel
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Azign Bioscience AS
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Azign Bioscience AS
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Publication date
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Publication of EP1434883A2 publication Critical patent/EP1434883A2/fr
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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
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1072—Differential gene expression library synthesis, e.g. subtracted libraries, differential screening
    • 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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection

Definitions

  • the invention relates to methods for identifying gene fragments and proteins that is differentially expressed, specific differential display arrays, methods for preparation of specific differential display arrays, and kits containing a specific differential display array, and further to the use of specific differential display arrays in methods for the determination of expression profiles in biological materials in which 10 there is an interest in the expression of polynucleotides.
  • polynucleotide spots deposited on a solid surface in form of an array.
  • Arrays of both polypeptides and polynucleotides have been developed and find use in a variety of applications.
  • One of the applications is differential gene expression, where expression of genes in different cells or tissues (normally a control sample and a sample of the cell or tissue of interest) is compared, and any difference in the mRNA expression profile is
  • an array of "probe" nucleotides is contacted with a nucleic acid sample of interest such as mRNA or mRNA concerted into cDNA or cRNA from a particular tissue or cell.
  • a nucleic acid sample of interest such as mRNA or mRNA concerted into cDNA or cRNA from a particular tissue or cell.
  • Contact is carried out under hybridisation conditions favourable for hybridisation of nucleic acids complementary to
  • the present invention provides methods for identifying gene fragments for a completely new array being capable of a more specific determation of the polynucleotide expression profile of polynucleotides in a biological material. Furthermore, the invention provides an array with an improved signal to noise ratio of specific polynucleotide expression profiles.
  • the invention relates to a method for identifying a gene fragment that is differentially expressed in a non-stressed and stressed state of a biological source, said method comprising:
  • the invention relates to a specific differential display array comprising a plurality of polynucleotide spots associated with a surface of a solid support, wherein the individual polynucleotide spot comprises a gene fragment as identified by the method according to the present invention.
  • the invention relates to a method for preparing a specific differential display array, a method for the determination of an expression profile in a biological material, a method for the determination of a difference in expression profiles from at least a first and a second biological material, a method for identifying a therapeutic, prophylactic and/or toxic agent involved in a direct or indirect action on the expression profile in a biological material, a diagnostic method for the determination of the differences of expression profiles between two biological materials, a database comprising a plurality of gene fragments and gene expression profiles of stressed and non-stressed states, and a kit for use in a hybridisation assay.
  • the present invention provides a method for identifying a gene fragment that is differentially expressed in a non-stressed and stressed state of a biological source, said method comprising:
  • the identification of a gene that is differentially expressed in a non-stressed and stressed state of a biological source comprises:
  • the stressed state of the second biological source is directly or indirectly related to a disease state, a chemical treatment, a biological treatment, environmental influence or any other physiological or pathophysiological change, or a combination thereof.
  • the disease state is selected from CNS related diseases and treatments, depression, diabetes type II, or obesity.
  • the chemical treatment is treatment with one or more chemicals selected from the group consisting of naturally occurring chemical entities or synthetically derived chemical entities.
  • the chemical treatment is a treatment with one or more antidepressants or CNS drugs.
  • the biological source is selected from the group consisting of tissues, organs, biological fluids or parts or combinations thereof.
  • the first and second biological sources are of the same kind, i.e. of the same kind of origin, such as coming from the same type of tissue, organ, biological fluid or part or combinations thereof, or of the same organism or the same type of organism or the same cell type etc.
  • the non-conserved region has less than 90% intraspecies identity. In a special embodiment, the non-conserved region has less than 85% intraspecies identity. In a further special embodiment, the non-conserved region has less than 80% intraspecies identity. In a still further special embodiment, the non-conserved region has less than 75% intraspecies identity.
  • the non- conserved region has at least 50% interspecies identity.
  • the non-conserved region has at least 60% interspecies identity, such as at least 65% interspecies identity.
  • the non-conserved region has at least 70% interspecies identity, such as at least 75% interspecies identity.
  • the non-conserved region has at least 80% interspecies identity, such as at least 85% interspecies identity.
  • the non- conserved region has at least 90% interspecies identity.
  • the identified fragment has a length of from 25 to 1000 nucleotides. In a special embodiment, the identified fragment has a length of from 25 to 750 nucleotides. In a further special embodiment, the identified fragment has a length of from 25 to 500 nucleotides.
  • the step of identifying a gene that is differentially expressed in the first and second biological source comprises using a differential display technique.
  • the differential display technique used is restriction fragment differential display.
  • the differential display technique used is the restriction fragment differential display (RFDD) PCR analysis as described in US Patent No. 6,261 ,770 (Azign Bioscience).
  • the differential display technique is a microarray analysis using any commercial available microarray based e.g. on oligonucleotides, polynucleotides, cDNA, LNA, PNA or INA.
  • the microarray used has 100-100.000 gene fragments (25-1000 nt) attached to the surface. Examples of microarrays suitable for use in the present invention are those disclosed in US 5,445,934 and US 5,744,305.
  • the step of identifying a fragment of the identified gene comprising a non-conserved region comprises comparing by alignment the identified gene with other intraspecies genes. Having identified a gene that is differentially expressed in the first and second biological source it is a possibility to identify two or more fragments of the identified gene, all said fragments comprising a non-conserved region.
  • the identification of a gene that is differentially expressed in a non-stressed and stressed state of a biological source comprises:
  • the identification of a protein that is differentially expressed in a non- stressed and stressed state comprises • providing a first protein sample from a first biological source being in a non-stressed state;
  • the stressed state of the second biological source is directly or indirectly related to a disease state, a chemical treatment, a biological treatment, environmental influence or any other physiological or pathophysiological change, or a combination thereof.
  • the disease state is selected from CNS related diseases and treatments, depression, diabetes type II, or obesity.
  • the chemical treatment is treatment with one or more chemicals selected from the group consisting of naturally occurring chemical entities or synthetically derived chemical entities.
  • the chemical treatment is a treatment with one or more antidepressants or CNS drugs.
  • the biological source is selected from the group consisting of tissues, organs, biological fluids or parts or combinations thereof.
  • the first and second biological sources are of the same kind, i.e. of the same kind of origin, such as coming from the same type of tissue, organ, biological fluid or part or combinations thereof, or of the same organism or the same type of organism or the same cell type etc.
  • the non-conserved region of the gene fragment has less than 90% intraspecies identity. In a special embodiment, the non-conserved region has less than 85% intraspecies identity. In a further special embodiment, the non-conserved region has less than 80% intraspecies identity. In a still further special embodiment, the non-conserved region has less than 75% intraspecies identity.
  • the non-conserved region has at least 50% interspecies identity.
  • the non-conserved region has at least 60% interspecies identity, such as at least 65% interspecies identity.
  • the non-conserved region has at least 70% interspecies identity, such as at least 75% interspecies identity.
  • the non-conserved region has at least 80% interspecies identity, such as at least 85% interspecies identity.
  • the non-conserved region has at least 90% interspecies identity.
  • the identified fragment has a length of from 25 to 1000 nucleotides. In a special embodiment, the identified fragment has a length of from 25 to 750 nucleotides. In a further special embodiment, the identified fragment has a length of from 25 to 500 nucleotides.
  • the step of identifying the protein that is differentially expressed in the first and second biological source comprises using a differential display technique.
  • the differential display technique used is MDLC-MSMS (Mu ⁇ ti- Dimensional-Liquid-Chromatograhy-Mass-Spectrometry MS), cf. Muticultural Chromatography and the Signature Peptide Approach to Proteomics", Regnier F. et al. LCGC, Vol. 19 number 2, February 2001.
  • the differential display technique used is technologies as the ICAT technology (available from Applied Biosystems), cf.
  • the step of identifying a gene fragment of the identified protein comprising a non-conserved region comprises: comparing by alignment the identified gene with other intraspecies genes.
  • the present invention provides a database comprising a plurality of gene fragments as identified by the method as described above.
  • the database further comprises additional expressed genes that are substantially relevant to the biological state.
  • the present invention provides a specific differential display array comprising a plurality of polynucleotide spots associated with a surface of a solid support, wherein the individual polynucleotide spot comprises a gene fragment as identified by the method as described above in the sections "Genomics” and “Proteomics".
  • the specific differential display array comprises polynucleotide spots identified partly by the method of described in the section "Genomics” and partly by the method described in the section "Proteomics".
  • the individual polynucleotide spot is in single stranded or double stranded form.
  • the individual polynucleotide spot is of DNA, RNA, cDNA, natural, synthetic, semisynthetic origin or is a chemical analogous such as LNA, PNA, INA and other MNA's (Modified Nucleic Acid) with DNA, RNA or protein backbone structure.
  • the solid support is made of a flexible or rigid material.
  • the array comprises from 2 to 50,000 polynucleotide spots.
  • the array comprises two or more spots comprising a gene fragment from the same differentially expressed gene or protein.
  • the spots comprising a gene fragment from the same differentially expressed gene comprise the same gene fragment.
  • the spots comprising a gene fragment from the same differentially expressed gene or protein comprise different gene fragments from the same gene.
  • all said fragments comprise a non-conserved region.
  • all said fragments are chosen in such a way that they are non-overlapping regions.
  • the number of the gene fragments to be chosen is dependent inter alia on the length of the expressed gene of the differentially expressed gene or protein and the degree of non-conserved regions.
  • the use of two or more gene fragments from the same differentially expressed gene ensures an improved specificity of the array. Furthermore, the use of two or more gene fragments from the same differentially expressed gene will allow a statistical evaluation of the signal from the individual gene fragment.
  • a specific differential display array has a multiplicity of individual polynucleotide spots, stably associated with a surface of a solid support.
  • the specific differential display array comprises spots comprising a polynucleotide composition, wherein the polynucleotide regions within the composition are of known identity, usually of known sequence, as described later on in detail.
  • the polynucleotide spots may be of convenient shape but most often circular, oval or any other suitable shape.
  • the polynucleotide spots may be arranged in any convenient pattern across the surface of the solid support, such as in row or columns to form a grid, in a circular pattern and the like.
  • the pattern of polynucleotide spots are arranged as a grid to facilitate the evaluation of the results obtained from the analyses in which the specific differential display array is used.
  • the specific differential display array according to the invention may be of a flexible or rigid solid support and the polynucleotide spots are stably associated thereto.
  • stably associated is meant that the polynucleotide spots will be associated in their position on the solid support during the analysis in which the specific differential display array is used, such as during different hybridisation, washing and detection conditions.
  • the polynucleotide regions contained in the spots may be covalently or non-covalently associated to the surface of the solid support. Methods how to covalently or non-covalently bind the polynucleotide regions to the surface of the solid support are well known for a person skilled in the art and may be found in Ausubel et al., Current protocols in Molecular Biology, Greene Publishing Co.
  • the solid support to which the individual polynucleotide spots are stably associated to is made of a flexible or rigid material.
  • flexible is meant that the support is capable of being bent or folded without breakage.
  • rigid is meant that the support is solid and does not readily bend, i.e. the support is not flexible.
  • the support may be fabricated from a variety of materials, including plastics, ceramics, metals, gels, nitrocellulose, nylon, glass and the like.
  • the array may be produced according to any convenient methodology, such as preparing or obtaining the polynucleotides and then stably associate them with the surface of the support or growing them directly on the support.
  • a number of array configurations and methods for their production are known to those skilled in the art and disclosed in US Patents: 5,445,934, 5,532,128, 5,556,752, 5,242,974, 5,384,261 , 5,405,783, 5,412,087, 5,424,186, 5,429,807, 5,436,327, 5,472,672, 5,527,681 , 5,529,756, 5,545,531 , 5,554,501 , 5,561 ,071 , 5,571 ,639, 5,593,839, 5,599,695, 5,624,711 , 5,658,734 and 5,700,637.
  • the solid support of the invention may have several configurations ranging from a simple to a more complex configuration depending on the intended use of the specific differential display array.
  • the size and thickness of the specific differential display array is not critical as long as the array will function in the expected way and as long as the results obtained after use of the array are not changed.
  • the number and amount of the polynucleotide spots is dependent on the intended use of the arrays as well as the detection system use to determine the expression profile of the biological material being evaluated by the aid of the specific differential display array.
  • the number of the polynucleotide spots may vary from about 2 to about 100,000 such as, e.g., from about 2 to about 50,000, from about 10 to about 25,000, from about 100 to about 10,000, from about 100 to about 5,000, from about 100 to about 1 ,000, from about 400 to about 600 or about 500 polynucleotide spots, or at least 2 such as, e.g. at least 10, at least 25, at least 50, at least 100, at least 300, at least 400, at least 500 or at least 600 spots, or even more than 100,000 spots.
  • the limitations of the number of the polynucleotide spots are dependent on the way in which the evaluation of the expression profile of the biological material is performed.
  • the amount of the polynucleotide regions present in the polynucleotide spot may vary and the amount will be sufficient to provide adequate hybridisation and detection of the target nucleic acid. Generally the polynucleotides will be present in each spot at a concentration corresponding to an amount of 1 pg - 100 ⁇ g or less than 100 ⁇ g of the polynucleotide. Normally, only 1 polynucleotide region is present in each spot.
  • the copy number of the polynucleotide present in each polynucleotide spot will be sufficient to provide enough hybridisation for a target nucleic acid to yield a detectable signal, and generally range from about 50 fmol or less.
  • control spots which may be present on the specific differential display array, include spots comprising genomic DNA, housekeeping genes, negative and positive control polynucleotides and the like. These polynucleotide spots comprise polynucleotides, which are not unique, i.e they are not polynucleotide regions corresponding to differentially expressed genes. They are used for calibration or as control polynucleotides, and the function of these polynucleotide spots are not to give information of the expression of these polynucleotides, but rather to provide useful information, such as background or basal level of expression to verify that the analysis and the expression profiles obtained are relevant or not. Furthermore these control spots may serve as orientation spots.
  • the polynucleotide composition also comprises an excipient.
  • Suitable excipients are solvents like e.g. water or any other aqueous medium, pH adjusting agents like buffering agents, stabilising agents, hybridising agents, coloring agents, labelling agents and the like.
  • the excipients used are inert, i.e. they do not have any polynucleotide related effect.
  • the present invention provides a method for preparing a specific differential display array as described above comprising
  • the plurality of compositions comprises compositions identified partly by the method for identifying a differentially expressed gene fragment as described in the section "Genomics” above and partly by the method for identifying a differentially expressed gene fragment as described in the section "Proteomics” above
  • the individual gene fragment is produced using one or more primers specific for said gene fragment.
  • the gene fragment preferably has a length of 25-500 nucleotides, more preferably 200-300 nucleotides.
  • a gene fragment of this length has the advantage that it has a high specificity.
  • the individual gene fragment consists of a synthetically synthesised oligonucletide of length 25-80 nucleotides.
  • a gene fragment of this length has the advantage that it may be prepared automatically by a suitable apparatus.
  • the specific differential display array may be prepared (produced) using any convenient method and several methods are well known for a person skilled in the art, such as standard procedures according to Sambrook et al., (Molecular cloning: A laboratory manual 2 nd edition. Cold Spring Harbour Laboratory Press, New York.).
  • One means of preparing the array is: i) synthesising or otherwise obtaining the above mentioned gene fragments, ii) preparing the polynucleotide compositions to be used in each spot and then iii) depositing in the form of spots the polynucleotide compositions comprising the gene fragment onto the surface of the solid support.
  • the gene fragments may be of DNA, RNA, cDNA, natural, synthetic, semisynthetic origin or chemical analogous such as LNA, INA or PNA or any other molecule with a DNA, RNA or protein backbone.
  • the gene fragments may be obtained from any biological material such as, e.g., tissues or cells and/or produced by a cell culture.
  • the biological material may be an organism, such as a microorganism, plant, fungus (e.g. yeast or mushrooms) or animal.
  • the gene fragments may be prepared using any conventional methodology such as automated solid phase synthesis protocols, PCR using one or more primers specific for the gene fragments and the like. In general, PCR is advantageous in view of the large numbers of gene fragments that must be generated for specific differential display array.
  • the amplified non-conserved gene fragments may further be cloned in any suitable plasmid vector to enable multiplication and storage of the amplified non- conserved gene fragments.
  • the prepared non-conserved gene fragments may be spotted onto the solid support using any convenient methodology, including manual and automated techniques, e.g. by micro-pipette, ink jet pins etc. and any other suitable automated systems.
  • An example of an automated system is the automated spotting device, Beckman Biomek 2000 (Beckman Instruments).
  • the ready arrays may then be stored at suitable conditions until use.
  • Fragment that is differential expresses and comprises a non-conserved region
  • the fragments for use in the specific differential display array according to the invention are unique by being fragments from genes differentially expressed in a stressed and non-stressed state and comprising a non-conserved region. Information on genes that are differentially expressed in a stressed and non- stressed state may be obtained in a number of different ways.
  • the identification of a gene that is differentially expressed in a non- stressed and stressed state of a biological source comprises:
  • the identification of a gene that is differentially expressed in the first and second biological source may be performed by a number of ways known by a person skilled in the art. Examples are the use of a differential display technique, such as restriction fragment differential display (RFDD-PCR), northern blot analysis, microarray analysis and Protein Differential Display analysis, such as MDLC-MSMS and 2D-gel electrophoresis.
  • RFDD-PCR restriction fragment differential display
  • northern blot analysis such as northern blot analysis
  • microarray analysis such as Protein Differential Display analysis
  • Protein Differential Display analysis such as MDLC-MSMS and 2D-gel electrophoresis.
  • the present invention provides a method for the determination of an expression profile in a biological material, said method comprising:
  • the present invention provides a method for the determination of a difference in expression profiles from at least a first and a second biological material, said method comprising:
  • the first and the second biological material are of the same kind of biological material.
  • the first biological material is in a non-stressed state and the second biological material is in a stressed state.
  • the stressed state of the second biological material is directly or indirectly related to a disease state, a chemical treatment, a biological treatment, environmental influence or any other physiological or pathophysiological change, or a combination thereof.
  • the chemical treatment is treatment with one or more chemicals selected from the group consisting of naturally occurring chemical entities or synthetically derived chemical entities.
  • the disease state is selected from depression, CNS related diseases, diabetes type II, or obesity.
  • the stressed state of the second biological material is directly or indirectly related to the stressed state of the second biological source used in the method for identifying a gene fragment as described above.
  • each material is labelled with a unique label (e.g. Cy3 and Cy5 for each sample, respectively).
  • the present invention provides a method for identifying a therapeutic, prophylactic and/or toxic agent involved in a direct or indirect action on the expression profile in a biological material, said method comprises: • obtaining a first expression profile of a first biological material according to the method for the determination of an expression profile in a biological material as described above,
  • the method further comprises:
  • the first and second biological material are of the same kind of biological material.
  • the first biological material is in a non-stressed state and the second biological material is in a stressed state.
  • the expression profile of the second biological material is direct or indirect measure of a disease state, a chemical treatment, a biological treatment, environmental influence or any other physiological or pathophysiological change, or a combination thereof.
  • the test compound is a chemical or biological derived compound such as compounds selected from the group consisting of therapeutic, prophylactic and/or toxic chemical entities, physiologically chemical entities, hormones, vitamins, nutrients, pesticides, fungicides, bacteriocides and any other organic chemical entity.
  • the method according to the above embodiment of the invention is used to identify potential therapeutic, prophylactic and/or toxic agents useful for the treatment of diseases caused by an alteration in the expression profile of the polypeptides.
  • a biological model such as a rat model in which a first, second, third and/or fourth group are used.
  • the first and third group is non-stressed and the second and fourth group stressed in such a way that the expression of one or more polynucleotides are influenced in such as way that an increase or a decrease of the gene expression is obtained.
  • the third and fourth groups are treated with a test compound. Determination of expression profiles typically means determination of the expression level of multiple mRNAs, all of them corresponding to the spotted gene fragments on the array.
  • the detection limit of the expression level of a mRNA may be approximately 0.2 ng or less of total RNA of the biological material used to hybridise each polynucleotide spot.
  • the expression profiles can be produced by any means known in the art, including but not limited to the methods disclosed by: Liang et al., (1992) Science 257: 967-971 ; Ivanova et al., (1995) Nucleic Acids Res 23: 2954-2958; Guilfoyl et al., (1997) Nucleic Acids Res 25(9): 1854-1858; Chee et al., (1996) Science 274: 610-614; Velculescu et al., (1995) Science 270: 484-487; Fiscker et al., (1995) Proc Natl Acad Sci USA 92(12): 5331.5335; and Kato (1995) Nucleic Acids Res 23(18): 3685-3690.
  • the specific differential display array will be used for the evaluation of the expression profile of one or more biological materials or a mixture of biological materials.
  • the method for the determination of an expression profile in a biological material or in a mixture of biological materials comprises obtaining a polynucleotide from the biological material(s), labelling said polynucleotide to obtain a labelled target polynucleotide sample, contacting at least one labelled target polynucleotide sample with an array as defined above under conditions which are sufficient to produce a hybridisation pattern and detecting said hybridisation pattern to obtain the polynucleotide expression profile of the biological material or the mixture of biological materials.
  • the expression profile in the biological material may be determined to correspond to the expression of specific genes.
  • the biological material or the mixture of biological materials may be in a non-stressed or a stressed stage.
  • the stress may directly or indirectly influence the expression profile and thereby the polynucleotides identified which react upon that type of stress.
  • the stress may be caused by any disease or condition.
  • the examples of the present application show the non-limiting diseases depression, obesity and diabetes (diabetes type II).
  • the analysis of the expression profile includes several steps of procedures in which well known techniques are used, such as those mentioned in Sambrook et al., Molecular Cloning: A Laboratory approach, Cold Spring Harbour Press, NY (1987), and in Ausubel et al., Current protocols in Molecular Biology, Greene Publishing Co. NY, (1995).
  • RNA polyA RNA
  • mRNA polyA RNA
  • the total RNA/mRNA can be isolated using a variety of techniques. Numerous techniques are well known (see Sambrook et al., Molecular Cloning: A Laboratory approach, Cold Spring Harbour Press, NY (1987), and Ausubel et al., Current protocols in Molecular Biology, Greene Publishing Co. NY, (1995)). In general, these techniques include a first step of lysing the cells and then a second step of enriching for or purifying RNA.
  • RNA-directed DNA polymerase such as "reverse transcriptase” isolated from such retroviruses as AMV, MoMuLV or recombinantly produced.
  • reverse transcriptase isolated from such retroviruses as AMV, MoMuLV or recombinantly produced.
  • Many commercial sources are available (e.g., Perkin Elmer, New England Biolabs, Stratagene Cloning Systems).
  • the mRNA is reversed transcribed into cDNA and at the same time a label is incorporated for later detection of the hybridised amplified products on the array.
  • the amplification by PCR may be performed according to Example 2.
  • the label may vary dependent on the system to be used for the detection and several labels are well known in the area of molecular biology (e.g. radioactive labels, fluorescent labels, coloring labels, chemical labels etc.)
  • the labelled cDNA is then denaturated and used for hybridisation on the array.
  • the hybridisation conditions vary and are dependent on the aim with the expression profile obtained after the hybridisation.
  • One example is found in Example 6.
  • the specific differential display array is washed to remove the cDNA which have not hybridised to the fragments and the hybridised labelled cDNA are detected by a suitable means and an expression profile obtained.
  • most diseases or conditions might influence the expression profile of the second biological material.
  • in vivo models such as, e.g., a rat model in which at least a first and a second experimental group are used.
  • the first group is non-stressed and the second group stressed in such a way that the expression of one or more of the polynucleotides on the specific differential display array are influenced in such as way that an increase or a decrease of the expression is obtained, when the expression profiles are analysed using array and the method according to the invention.
  • the second group may be either permanently stressed or stressed during a certain period of time and after the period of stress one or more biological materials obtained from the second group and the expression profile determined.
  • the present invention provides a diagnostic method for the determination of the differences of expression profiles between two biological materials, said method comprises:
  • the expression profile from more than two different biological materials are compared, such as biological materials, which are in different stages of a disease.
  • the difference between the first expression profile and the second expression profile is directly or indirectly influenced by a disease.
  • the expression profile from more than two different biological materials are compared, such as biological materials, which are in different stages of a disease.
  • the diagnostic method may be useful in the determination of diseases directly or indirectly caused by different expression profiles and by the use of such a method there will be an enhanced possibility to start the treatment of the disease at an early stage of the disease.
  • the present invention provides a kit for use in a hybridisation assay, said kit comprising a specific differential display array as described above.
  • the kit further comprises reagents for generating a labelled target polynucleotide sample.
  • the kit further comprises a hybridisation buffer.
  • the kit may be used according to the above-mentioned methods for the determination of expression profiles in a biological material as defined above.
  • the present invention provides a database comprising a plurality of gene fragments as identified by the method as described above.
  • the database further comprises additional expressed genes that are substantially relevant to the stressed state of the biological source.
  • the database of the invention contains one or more of the following items of information obtained in gene and protein expression analysis: 1) Genes and proteins identified as differentially expressed (their accession number and sequences)
  • the database of the invention further contains the following items of information obtained in analysis on the Specific Differential Display Arrays:
  • Drug information Drug name, Dose usage, chemical structure, mechanism of action, Drug Interaction, Clinical Trial information, Indications and usage, Contradictions, Drug/Laboratory test interactions, receptor binding studies results, ADME results, PK results, toxicological profile
  • the database of the invention may be used as follows: A gene expression profile is performed on a biological source, e.g. brain tissue, from a treatment of a test animal, e.g. a mouse, with a Novel Chemical Entity (NCE) on a Specific Differential Display Array. This gene expression profile will then be stored in the database and use for a complex datamining against all the gene profiles stored in the database resulting in the possibility to elucidate e.g. potential therapeutic and toxicological effect of a new tested drug and suggesting new modification of this NCE to make it a more efficient NCE towards this therapeutic field.
  • NCE Novel Chemical Entity
  • the disease state may be any disease state giving rise to a differential expression of one or more genes.
  • the disease state is a CNS related disease selected from the group consisting of Asthma, cystic fibrosis, chronic obstructive pulmonary disease and rhinorrhea, convulsions, vascular spasms, coronary artery spasms, renal disorders, polycystic kidney disease, bladder spasms, urinary incontinence, bladder outflow obstruction, irritable bowel syndrome, gastrointestinal dysfunction, secretory diarrhoea, ischaemia, cerebral ischaemia, ischaemic hearth disease, angina pectoris, coronary hearth disease, traumatic brain injury, psychosis, anxiety, depression, dementia, memory and attention deficits, drug addiction and/or abuse, including cocaine or tobacco abuse, Parkinson's disease, Alzheimer's disease, dysmenorrhea, narcolepsy, Reynaud's disease, intermittent claudication, Sjorgren's syndrome, migraine, arrhythmia, hypertension, absence seizures, myotonic muscle dystroph
  • CNS related diseases include a variety of disorders associated with the neural system, for example eating disorders, obsessive compulsive disorders, panic disorders, alcoholism, pain, memory deficits and anxiety. Included among these disorders are disorders such as pseudodementia or Ganser's syndrome, migraine pain, bulimia, obesity, pre-menstrual syndrome or late luteal phase syndrome, post-traumatic syndrome, memory loss, memory dysfunction, social phobia, attention deficit hyperactivity disorder, chronic fatigue syndrome, premature ejaculation, erectile difficulty, anorexia nervosa, disorders of sleep, autism, mutism, trichotillomania or mood syndrone. Also, according to the present invention CNS related diseases include autoimmune diseases, e.g.
  • polynucleotide is intended to mean a single or double stranded polymer composed of nucleotides, e.g. deoxyribonucleotides and/or ribonucleotides from about 10 to about 9,000 nucleotides in length, such as from about 10 to about 6,000, from about 10 to about 3,000, from about 10 to about 1 ,500, from about 10 to about 1 ,000, from about 25 to about 1 ,000, or from about 25 to about 750.
  • nucleotides e.g. deoxyribonucleotides and/or ribonucleotides from about 10 to about 9,000 nucleotides in length, such as from about 10 to about 6,000, from about 10 to about 3,000, from about 10 to about 1 ,500, from about 10 to about 1 ,000, from about 25 to about 1 ,000, or from about 25 to about 750.
  • complementarity is used in relation to the base- pairing rules of nucleotides well known for a person skilled in the art.
  • Polynucleotides may be complete or partial complementary. Partial complementarity means that at least one nucleic acid base is not matched according to the base pairing rules. Complete complementarity means that all nucleotides in a polynucleotide match according to the base pairing rules. The degree of complementary between polynucleotides affects the strength of hybridisation between two polynucleotide strands.
  • the inhibition by hybridisation of the complementary polynucleotide to the target polynucleotide may be analysed by techniques well known for a person skilled in the art, such as Southern blot, Northern blot, and the like under conditions of high stringency.
  • a partially (substantially) homologous polynucleotide will compete for and inhibit the binding of a completely homologous sequence to the target sequence under low stringency.
  • homology is intended to mean the degree of identity of one polynucleotide to another polynucleotide.
  • homology is used in connection with complementarity between polynucleotides within a family or between species. There may be complete homology (i.e. 100% identity) between two or more polynucleotides.
  • degree of homology may be determined by any method well known for a person skilled in the art.
  • polynucleotide composition is intended to mean a composition comprising a polynucleotide together with an excipient.
  • the polynucleotide compositions are applied as spots on the array.
  • the polynucleotide composition comprises a non-conserved region of a polynucleotide family member.
  • polynucleotide composition includes also control or calibrating compositions such as, e.g. compositions comprising polynucleotides corresponding to housekeeping genes.
  • non-conserved region is intended to mean a segment of nucleotides in a polynucleotide that - compared to a segment of nucleotides in another polynucleotide originating from the same species, i.e. intraspecies - has at the most 90% identity.
  • intraspecies gene is intented to mean a gene or polynucleotide arising from the same species, such as such humans, mice or rats.
  • the publicly known genes and polynucleotides may generally be found and downloaded from Genbank or EMBL (www.ncbi.nih.org).
  • the term "intraspecies identity” is intended to mean identity within a group of members belonging to the same species such as such humans, mice or rats (paralogy).
  • interspecies identity is intended to mean identity between a group of different species, such as a group comprising humans, mice and rats (orthology).
  • expression profile is intended to mean the expression of the mRNAs in a biological material. While an expression profile encompasses a representation of the expression level of at least one mRNA, in practice the typical expression profile represents the expression of several mRNAs. For example, an expression profile used according to the present invention represents the expression levels of at least from 1 to 100,000 or more different mRNAs in a biological material. The expression level of the different mRNAs is the same or different. The expression of mRNAs may be up- or down regulated resulting in different expression profiles.
  • biological material include within its meaning organisms, organs, tissues, cells or biological material produced by a cell culture.
  • the biological material may be living or dead.
  • the material may correspond to one or more cells from the organisms, in case the organism is a multicellular organism, the biological material may correspond to one or more cells from one or more tissues creating the multicellular organism.
  • the biological material to be used according to the invention may be derived from particular organs or tissues of the multicellular organism, or from isolated cells obtained from a single or multicellular organism.
  • biological source include within its meaning organisms, organs, tissues, cells or biological material produced by a cell culture.
  • the biological material may be living or dead.
  • the material may correspond to one or more cells from the organisms, in case the organism is a multicellular organism, the biological material may correspond to one or more cells from one or more tissues creating the multicellular organism.
  • the biological material to be used according to the invention may be derived from particular organs or tissues of the multicellular organism, or from isolated cells obtained from a single or multicellular organism.
  • the biological material or biological source may be subject to a number of different processing steps.
  • Such steps might include tissue homogenisation, cell isolation and cytoplasma extraction, nucleic acid extraction and the like and such processing steps are generally well known for a person skilled in the art.
  • Methods of isolating RNA from cells, tissues, organs or whole organisms are known to those skilled in the art and are described in Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbour Press) (1989).
  • the term "organism” is intended to mean any single cell organism such as yeast or multicellular organism, including plants, fungi and animals, preferably mammals, such as humans, rats, pigs, cows, horses, dogs, guinea pigs, ferrets, rabbits, sheep, apes, monkeys and cats.
  • target polynucleotides is intended to mean polynucleotides present in the biological material of interest. If the target polynucleotide has a complementary polynucleotide present on the specific differential display array, it will hybridise thereto and thus give rise to a detectable signal.
  • non-overlapping is intended to mean that when the specific fragments used in the polynucleotide composition spots are obtained from the same polynucleotide, the regions are obtained from different parts of the polynucleotide and the different parts are located in such a manner that the regions not even overlap each other by a single nucleotide.
  • a polynucleotide of e.g. 1 ,000 nucleotides the regions 1 -500 and 501-900 are non-overlapping.
  • the non-overlapping polynucleotide regions may be located with a distance of one or more nucleotides from each other.
  • primer is intented to mean a polymer of 3-50 nucleotides.
  • set of primers is intended to mean one or more primers having the ability to amplify a polynucleotide region under suitable conditions.
  • the length of the primers may be the same or different and dependent on the character of the polynucleotide region to be amplified. Design of such a set of primers is well known for a person skilled in the art.
  • the set of primers having a sufficient length to specifically hybridise to a distinct polynucleotide in the sample and the length of the primers will be from about 3 to 50 nucleotides.
  • stressed state and stressed is intended to mean that the above described “biological material” and “biological source” is influenced compared to the normal condition.
  • the biological material or source may be influenced by some kind of organic/inorganic compound, an environmental agent, a drug substance, pathogen, mutagen, mitogen, receptor mediated signal or the like.
  • the biological material is influenced in such a manner that the expression profile of the polynucleotides in the biological material or source either directly or indirectly is affected resulting in at least one difference between the expression profile of the non- stressed biological material or source compared to the stressed biological material or source.
  • polynucleotides may either belong to the same family or different families and/or being polynucleotides encoding the same polypeptide from the same or different species.
  • Optimal alignment of nucleotides of a polynucleotide for comparison of the homologies may be conducted using the homology algorithm (Smith and Waterman, Adv. Appl. Math. 2: 482 (1981)), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad.
  • mice Male NMRI mice (Age: 12-14 weeks) were treated for 21 days with fluoxetine (10 mg/kg) or clomipramine (15 mg/kg) receiving one intraperitoneal injection per day. Control animals were treated with 0.9% saline in a similar fashion. On day 22, 24 hrs after the last injection the mice were sacrificed by cervical dislocation and the pituitary glands were rapidly isolated and snap-frozen in liquid nitrogen. Total RNA was purified from pituitary glands using TRI reagent according to standard procedure (Chomczynski and Sacchi, 1987).
  • RNA isolated from mice treated as stated in Example 1aand 1 b(pooled from 3 animals) was reverse-transcribed using a poly-dT primer, (T25V from DNA Technology, Arhus, Denmark), and 100 units Reverse Transcriptase in 25 ⁇ l volume. The reaction was incubated at 42°C for 2 hours. For second-strand synthesis, a 50 ⁇ l mix consisting of buffer, dNTPs,
  • restriction fragment differential display PCR template was completed by ligating the digest with a standard adaptor and an EP-adaptor containing an extension protection group (EPG) at 37°C for 3 hours using T4 DNA ligase.
  • EPG extension protection group
  • the incubation was carried out at 37°C and not the standard 16°C to maintain the Taql endonuclease active during the ligation. This prevented religation of cDNA Taql-fragments but not
  • the template was PCR amplified using a 0-extension primer complementary to the EP-adaptor in combination with a 3-extension primer recognizing the standard adaptor and the three nucleotides adjacent to the Taql site.
  • the 0-extension primer was kinase-labelled with [ 33 P]dATP (3000 Ci mmol-1 , equivalent to 1.11 x 1014 Bq mmol-1 , ICN).
  • PCR reactions were carried out in 20 ⁇ l volume using 0.2 ⁇ l template and standard concentration of dNTPs and primers, using the following PCR- amplification profile: precycle 94 °C, 1 min., for the first 10 cycles: 94°C, 30 sec; 60°C with touchdown by 0.5°C for each cycle until 55°C is reached; 72°C, 1 min, for the last 25 cycles: 94°C, 30 sec; 55°C, 30 sec; 72°C, 1 min.
  • RNA from the biological material is obtained according to the procedures of Examples 1A or 1 B
  • RNA was preciptated by centrifugation and the RNA pellet was in 70% ethanol. The RNA was precipitated at 15,000 x g for 15 min. The supernatant was discarded and the pellet air-dried. The RNA was adjusted to a concentration of 1 ⁇ g/ ⁇ l with DEPC-H20. In 2 separate tubes 25 ⁇ l total RNA (1 ⁇ g/ ⁇ l) was placed and 7 ⁇ l DEPC treated H20 added. 4 ⁇ l of oligo-dT (e.g. T25V primer) (1 ⁇ g/ ⁇ l) was added to each tube. The tubes were incubated in a Thermal cycler at 65°C for 3 min. Tube 1 was prepared by adding, 5 ⁇ l 10 x cDNA Buffer (500mM Tris-HCI, pH
  • Tube 2 was prepared by adding, 5 ⁇ l 10 x cDNA Buffer (500mM Tris-HCI, pH 8.3; 800 mM KCI; 100 mM MgCI2; 40mM DTT), 2 ⁇ l Cy5-dUTP (1 mM, Cat. No. PA55022, Amersham Pharmabiotech), 5 ⁇ l 10 x dNTP (5mM dATP; 5 mM dCTP; 5 mM dGTP; 5mM dTTP). The contents were mixed and added 2 ⁇ l reverse transcriptase (100 U/ ⁇ l). Incubate tube 1 and 2 for 42 °C for 60 min and at 65°C for 15 min. The temperature was decreases to 42°C.
  • 5 ⁇ l 10 x cDNA Buffer 500mM Tris-HCI, pH 8.3; 800 mM KCI; 100 mM MgCI2; 40mM DTT
  • 2 ⁇ l Cy5-dUTP (1 mM, Cat. No
  • reverse transcriptase 100 U/ ⁇ l
  • reverse transcriptase 100 U/ ⁇ l
  • the DNA was precipitate with 3M Na-acetate and 96% ethanol.
  • the pellet in each tube was washed in 80% ethanol.
  • Each pellet was resuspended in RNase Mix (10 mM Tris-HCI (pH 7.5), 0.1 mM EDTA (pH 8.0), RNAase A 100 m ⁇ /ml).
  • the tubes were incubated at 37°C for 60 min and then added 30 ⁇ l sterile H20 to each tube.
  • the DNA was precipitated using 3M Na-Acetate (pH 6.0) and ice-cold 96% ethanol.
  • the pellets were washed in 80% ethanol and air-dried. Each pellet was resuspended in 15 ⁇ l hybridization buffer (5 x SSC, 0.1 % SDS, 100 ⁇ g/ml, blocking RNA). The two fluorescents probes were mixed 1 :1 in a PCR tube. This is the Sample-Mix.
  • the Sample-mix was denatured 100°C for 3 min and then transferred directly to 55°C for 30 sec.
  • the Sample-Mix was placed on ice and then added to the microarray slide (Pan Array; MWG Germany).
  • the microarray slide was placed in a box and inside the petri dish with the pre-wetted 3MM paper. The lid was replaced back onto the petri dish. The petri dish was placed in a plastic bag.
  • the microarray and protocol used e.g. Affymetrix, Pan Array from MWG
  • Information about the Biological material use for the analysis e.g. mouse brain.
  • Drug information Drug name, Dose usage, chemical structure,
  • Lysis Buffer 20 50 mM Tris-HCL, pH 7.4, 150 mM NaCI, 0.1 % SDS, Protease Inhibitor Cocktail Protease Inhibitor Cocktail (100x concentration): 20 mM AESBF, 1 mM Leupeptin, 100 ⁇ M Pepstatin
  • Tissue (Examplel) was taken directly from -80°C and kept on ice.
  • Protein preparations from mouse brain tissue were labeled with acidcleavable ICAT- reagent and trypsinized (last step according to Section 7.1.4).
  • Eluent B 10 mM K-P0 4 , pH 3.0, 25 % ACN, 350 mM KCL
  • Linear gradient 0-100 % B in 1 hour at flowrate 0.2 ml/min, room temp.
  • Eluent A 0.1 % TFA in 5 % Acetonitril, 95 % H 2 0 (Milli-Q ® )
  • Eluent B 0.1 % TFA in 95 % Acetonitril, 5 % H 2 0 (Milli-Q ® )
  • Linear gradient 2-30 % B in 2 hours at flowrate 200 nl/min.
  • MS/MS analyses standard MS/MS acquisition methods was used. MS spectra was first recorded for quantitation. Peak pairs representing ICAT reagent labeled peptides was recognized by mass differences (multiplums of 9.03 m/z). Precursor ions for MS/MS analysis were selected. MS/MS analyses were runned in automated mode. The differentialy expressed Proteins were identified using GPS ExplorerTM software from MS/MS spectra of ICAT reagent-labelled peptides.
  • Drug information Drug name, Dose usage, chemical structure, mechanism of action, Drug Interaction, Clinical Trial information, Indications and usage, Contradictions, Drug/Laboratory test interactions, receptor binding studies results, ADME results, PK results, toxicological profile and Results from animal models
  • PCR fragments from example 2a were cloned using a commercially available cloning kit according to the manufacturers instructions.
  • the selected clones were then sequenced using the Beckman CEQ-2000 sequencer. Sequences were converted to FASTA format and any vector sequence was masked using the 'cross_match' program running on a Linux platform.
  • Each sequence was used to search for matches in the GenBank database and the EST subset of Genbank using the BLAST sequence similarity search program. For each sequence the most significant hit was reported as a bit score, length of alignment region, percentage identity, and the GenBank gene name annotation.
  • RNA from Example 1 A was identified from analysis on RNA from Example 1 A using the method desribed in Example 2a. This fragment corresponded to mus musculus corticotropin releasing hormone receptor [CRF-R, ace. no. NM_007762].
  • the cDNA sequence of NM_007762 was downloaded from Genbank.
  • the alignments were performed to identify non-conserved regions of CRF-R regions having less than 75% sequence homology to other sequences within the species.
  • the size of the region is preferably from 25 to 750 bases.
  • the numbering of the nucleotide sequence start at the 5' end of the polynucleotide and correspond to a [atg].
  • the first homology search was done to generate a fragment that is highly specific for CRF-R.
  • interspecies homology-search was performed to ensure the fragment can be used for identification of CRF-R in other species.
  • the cDNA sequence of mouse Gas6 (X59846) was downloaded from Genbank. The alignments were performed to identify non-conserved regions of Gas6 having less than 75 % sequence homology to other sequences within the species. The size of the region is preferably from 25 to 750 bases. The numbering of the nucleotide sequence start at the 5'end of the polynucleotide and correspond to a [atg]. The first homology search was done to generate a fragment that is highly specific for Gas6. Secondly, interspecies homology-search was performed to ensure the fragment can be used for identification of Gas6 in other species. Using clustal alignment a 240 region was identified having 94 % homology (interspecies) to rat Gas6 [Ace. No. D42148] and 87% homology (interspecies) to human Gas6 [Ace. No. M33031]. The result of a few of the analysed gene fragments are listed below
  • Drug information Drug name, Dose usage, chemical structure, mechanism of action, Drug Interaction, Clinical Trial information, Indications and usage, Contradictions, Drug/Laboratory test interactions, receptor binding studies results, ADME results, PK results, toxicological profile and Results from animal models EXAMPLE 5
  • RNA Brain total RNA was purchased from Research Genetics (D6030-01 NTR Brain) The cDNA was synthesized using the Omniscript RT Kit (Qiagen, 205111).
  • the Gas6 region 794-1033 was amplified using conventional PCR well known for a person skilled in the art using following primers:
  • Mouse liver mRNA was purchased from Clontech laboratories (cat # 6616-1) The cDNA was synthesized using the Omniscript RT Kit (Qiagen, 205111 ).
  • PCR generated fragments were separated using on a conventional agarose gel and cloned into a suitable vector according to supplier's instruction and the nucleotide sequence was analysed using CEQ 2000 DNA Analysis System (Beckman Coulter, U.S.A.).
  • the glycerol stocks were prepared in 96 wells-trays (Corning Cat. No. cci3793) from the bacterial tranformations of the cloned PCR fragments described in example 3 on a Biomek. 50 ⁇ l glycerol media was transferred into each well of a plane 96-well tray (Corning Cat. No. cci3793). 50 ⁇ l bacterial culture was transferred into each well of the plane 96-well tray and mixed with 2 x 100 ⁇ l. A Storage Mat-I lid (Corning, Cat. No. 3094) was placed on each tray and the trays stored at -80 °C.
  • Circlegrow medium/ampicillin was added to each well in a 4 x 2 ml 96 deep well tray (Corning Cat.No. cci 3961).
  • the glycerol stocks from example 6 were added to each well.
  • the tray was sealed with sealing sheet (Merck Eurolab A/S, Denmark), and incubated with shaking at 37°C for 16 hours prior to plasmid purification.
  • the plasmid purification was performed using Biomek or a conventional method according to Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbour Press) (1989) well known for a person skilled in the art.
  • Following plasmide purification the specific fragments were generated by standard PCR using specific primer sets. The fragments were made "spot-ready" by precipitation according to standard procedure and dissolving in spot buffer in a suitable concentration.
  • oligonucleotide based Specific Differential Display Arrrays were produced. From the identified differentially expressed genes the bioinformatic analysis as described in Example 4 was carried out with the object of identifying 50bp regions with high interspecies homology and low intraspecies homology. The identified 50bp fragments were produced as long oligonucleotides with a primary amine in the 5'-end using oligonucleotide synthesizer (DNA Technology).
  • the oligonucleotides were made "spot-ready" by diluting the oligonucleotides to 20 ⁇ M according to standard procedure in spotting buffer as in Example 7. After spotting onto 3D-Link Amine-Binding slides (Motorola, USA) the microarray slides were treated as in Example 8.
  • 3D-Link Amine-Binding slide may be obtained from (Motorola USA). To have an orientation on the arrays visible after scanning of the slides the each of the arrays corners are marked by a double labelled Cy3 and Cy5 primer with a 5' end amino group (5'-Cy3/5) in a final concentration of 1 pmol/ ⁇ l to enable the possibility to place a grid on the scanned array. After spotting, seal with sealing tape and store at -20°C until use.
  • 3MM paper was pre-wetted with saturated NaCI solution. All the slides were placed in a slide box without a lid and then placed in a plastic bag containing the NaCI saturated 3MM paper. The plastic bag was closed and incubated at room temperature for 36 hours. After incubation, the slides were removed from the plastic bag. The slides were then placed in pre-warmed blocking solution (0.1% SDS, SurModics Blocking Solution. Incubated for 20 minutes at 50 °C. Washed in redistilled H 2 0. Incubated in 4 x SSC, 0.1% SDS solution (50°C), and washed at room temperate in redistilled H 2 0. The slides were boiled in redistilled H 2 0 for 2 min. Washed in redistilled H 2 0 at room temperature.
  • the slide was incubated in pre-hybridisation buffer prewarmed to 50°C (50 ml 20 x SSC, 10 ml 100 x Denhardt solution, 2 ml 10% SDS, 4 ml Salmon Sperm DNA (10 mg/ml), 134 ml Redistilled H 2 0) at 50°C for 30 min. Washed in redistilled H 2 0. The slides were stored at room temperature; dry and dark until use.
  • RNA from the biological material is obtained according to the procedures of Examples 1A or 1 B.
  • RNA was preciptated by centrifugation and the RNA pellet was in 70% ethanol. The RNA was precipitated at 15,000 x g for 15 min. The supernatant was discarded and the pellet air-dried. The RNA was adjusted to a concentration of 1 ⁇ g/ ⁇ l with DEPC-H20. In 2 separate tubes 25 ⁇ l total RNA (1 ⁇ g/ ⁇ l) was placed and and 7 ⁇ l DEPC treated H20 added. 4 ⁇ l of oligo-dT (e.g. T25V primer) (1 ⁇ g/ ⁇ l) was added to each tube. The tubes were incubated in a Thermal cycler at 65°C for 3 min.
  • oligo-dT e.g. T25V primer
  • Tube 1 was prepared by adding, 5 ⁇ l 10 x cDNA Buffer (500mM Tris-HCI, pH 8.3; 800 mM KCI; 100 mM MgCI2; 40mM DTT), 2 ⁇ l Cy3-dUTP (1 mM, Cat. No. PA53022, Amersham Pharmabiotech), 5 ⁇ l 10 x dNTP (5mM dATP; 5 mM dCTP; 5 mM dGTP; 5mM dTTP). The contents were mixed and added 2 ⁇ l reverse transcriptase (100 U/ ⁇ l).
  • 10 x cDNA Buffer 500mM Tris-HCI, pH 8.3; 800 mM KCI; 100 mM MgCI2; 40mM DTT
  • Cy3-dUTP 1 mM, Cat. No. PA53022, Amersham Pharmabiotech
  • 5 ⁇ l 10 x dNTP 5mM dATP; 5 mM dCTP; 5 m
  • Tube 2 was prepared by adding, 5 ⁇ l 10 x cDNA Buffer (500mM Tris-HCI, pH 8.3; 800 mM KCI; 100 mM MgCI2; 40mM DTT), 2 ⁇ l Cy5-dUTP (1 mM, Cat. No. PA55022, Amersham Pharmabiotech), 5 ⁇ l 10 x dNTP (5mM dATP; 5 mM dCTP; 5 mM dGTP; 5mM dTTP). The contents were mixed and added 2 ⁇ l reverse transcriptase (100 U/ ⁇ l). Incubate tube 1 and 2 for 42 °C for 60 min and at 65°C for 15 min. The temperature was decreases to 42°C.
  • 5 ⁇ l 10 x cDNA Buffer 500mM Tris-HCI, pH 8.3; 800 mM KCI; 100 mM MgCI2; 40mM DTT
  • 2 ⁇ l Cy5-dUTP (1 mM, Cat. No
  • reverse transcriptase 100 U/ ⁇ l
  • reverse transcriptase 100 U/ ⁇ l
  • the DNA was precipitate with 3M Na-acetate and 96% ethanol.
  • the pellet in each tube was washed in 80% ethanol.
  • Each pellet were resuspended in RNase Mix (10 mM Tris-HCI (pH 7.5), 0.1 mM EDTA (pH 8.0), RNAase A 100 m ⁇ /ml).
  • the tubes were incubated at 37°C for 60 min and thenadded 30 ⁇ l sterile H20 to each tube.
  • the DNA was precipitated using 3M Na-Acetate (pH 6.0) and ice-cold 96% ethanol.
  • pellets were washed in 80% ethanol and air-dried. Each pellet were resuspended in 15 ⁇ l hybridization buffer (5 x SSC, 0.1% SDS, 100 ⁇ g/ml, blocking RNA). The two fluorescents probes were mixed 1 :1 in a PCR tube. This is the Sample-Mix.
  • the Sample-mix was denaturedat 100°C for 3 min and thentransferred directly to 55°C for 30 sec.
  • the Sample-Mix was placed on ice and then added to the microarray slide.
  • the microarray slide was placed in a box and inside the petri dish with the pre-wetted 3MM paper. The lid was replaced back onto the petri dish. The petri dish was placed in a plastic bag.
  • the petri dish incubated in a dark incubator at 65°C 12-16 hours. Tlides was washed in Washing Buffer I (2xSSC). The slides were submerged in pre-warmed Washing Buffer II (2 x SSC, 0.1 % SDS). Pre-warmed at 65°C for 1 hour in a volume of least 10 ml/slide to cover the slides. Incubated on an orbital shaker at 65°C for 10 min. The slides were washed in Washing Buffer III (0.2 x SSC) in a volume of least 10 ml/slide to cover the slides. Incubated on an orbital shaker at room temperature for 3 min. The slides weres washed in Washing Buffer IV (0.1 x SSC).
  • Washing Buffer V Washed in Washing Buffer V (0.5 x SSC). The washing with washing buffer V was repeated for additional 3 times. All Washing Buffer V was removed by centrifuging at 800rpm for 3 min. Theslide was scanned and evaluation performed using Affymetrix 418 Scanner, Affymetrix 418 Scanner Software and the software ImaGene 4.0 (BioDiscovery) according to the user manual.
  • Differential Display Arrays are stored in the database together with all the information about the following points.
  • Drug information Information about the treatment of the biological source e.g. mouse treated with the 10mg/kg antidepressant fluoxetine one time/day for 21 days.
  • Drug information Drug name, Dose usage, chemical structure, mechanism of action, Drug Interaction, Clinical Trial information, Indications and usage, Contradictions, Drug/Laboratory test interactions, receptor binding studies results, ADME results, PK results, toxicological profile and Results from animal models
  • a gene expression profile is performed on brain tissue from a treatment of a mouse with a new NCE on a Specific Differential Display Array. This gene expression profile will then be stored in the database and used for a complex datamining against all the gene profiles stored in the database resulting in the possibility to elucidate e.g. the potential therapeutic and toxicological effects of a new tested drug and suggesting new modification of this NCE to make it a more efficient NCE towards this therapeutic field.

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Abstract

L'invention concerne un procédé permettant d'identifier un fragment génique différentiellement exprimé dans un état non stressé et dans un état stressé d'une source biologique, ledit procédé consistant à (i) identifier un gène qui est différentiellement exprimé dans un état non stressé et dans un état stressé, et (ii) identifier un fragment du gène identifié, ledit fragment comprenant une zone non conservée.
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EP2290076B1 (fr) 2004-05-28 2015-03-25 Asuragen, Inc. Procédés et compositions impliquant du microARN
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