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1. WO2004057026 - MATRICE D'ADN COMPLEMENTAIRE MULTIPATIENT EN COMBINAISON AVEC UNE COLLECTE DE DONNEES

Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

[ EN ]

MULTIPATIENT CDNA ARRAY IN COMBINATION WITH A
DATA COLLECTION

The present invention is directed to an array comprising at least one set of cDNA molecules immobilised to a solid surface at an identifiable position, wherein each set of cDNA molecules is generated from a plurality of RNA molecules isolated from a specific biological specimen isolated from a human or non-human being. The invention relates further to a kit comprising a respective array. Moreover, the invention also relates to a method for producing an array as well as to the use of a respective array or a respective kit for research, diagnostic or therapeutic purposes.

In the recent years, high throughput methods such as microarray techniques have been developed for analysis of gene expression or gene activity in tissue extracts. For producing a microarray, DNA sequences or oligonucleotides are printed on a solid support surface in a predetermined order using, for example, computer-controlled, high-speed robotics. Such DNA microarrays typically include representative sequences from genes of interest and can be used to generate an expression profile of a certain type of cells or of a tissue.

For establishing a gene expression profile of a certain tissue, mRNA has to be isolated from a respective tissue sample using standard techniques known by the skilled person. The isolated mRNA can be reverse transcribed into cDNA, for example, in the presence of fluorescence-tagged deoxyribonucleotides. The obtained fluorescent mixture of cDNA is then hybridised to a microarray of selected DNA probes. The fluorescence intensity of the cDNA molecules hybridised to the DNA probes can be quantified, for example, by a laser confocal scanning microscopy and image analysis. These techniques are known, for example, from Schena et al., Science 270, pp. 467-470, 1995; Schena, BioAssays 18, pp. 427-431; Soares, Current Opinion in
Biotechnology 8, pp. 542-546, 1997; Ramsay, Nature Biotechnology 16, pp. 14-44, 1998; Sen/ice, Science 282, 396-399, 1998; US patent no. 5,700,637.

This approach allows to obtain a quantitative gene expression or gene activity profile for a large number of genes in a plurality of samples. using one microarray.

These microarrays are already used for the routine prognostic screening of cancer patients. See, e.g., Nature Medicine 9, page 9, 2003.

A microarray allows to simultaneously screen a plurality of samples. A great advantage of this screening method is that the results of the various samples are directly comparable with each other since these samples have all been treated under identical conditions of preparation and measurement.

However, the significance of the results obtained using microarrays currently commercialised suffer from the fact that the obtained results strongly depend from the work up or preparation of the samples to be screened on a microarray. Phrased differently, although all incubation steps and measurements steps are identical for the samples screened on one microarray, the samples have a different "work up history" before applied to the microarray. Therefore, the results measured by microarray technology are often strongly influenced by the working up of the samples to be measured.

That is to say the obtained results do not reliably reflect, for example, the biochemical or clinical status of a patient examined but to a great extent the conditions under which the samples had been worked up.

Moreover, the pathological status of a patient is often strongly influenced by the medical history of the patient from which the biological specimen is isolated. For example, the development of cancer or metabolic disease states is often caused by external factors such as consumption of drugs, nicotine, alcohol, sugar, etc. or daily working or life conditions and so on. However, this specific information is not available when providing scientists with biological specimens of diseased tissue. Therefore, the results obtained by analysing respective biological specimens cannot be fully or correctly understood.

For these reasons it is rather difficult, if not impossible, to compare res.ults obtained from different tissues of various patients worked up separately under different conditions and wherein, furthermore, no medical information of said patients, from whom the respective tissues had been isolated, is available and to elucidate the molecular defects responsible for the respective pathological conditions or status of said patients.

Consequently, it would be desirable for the research and medical therapy to be able to compare the pathological status of a plurality of biological specimens taken from patients having similar or identical diseases in order to elucidate the molecular mechanisms responsible, for example, for the development of a disease such as cancer or a metabolic disease.

This object is solved by providing an array comprising at least one set of cDNA molecules immobilised to a solid surface at an identifiable position, wherein each set of cDNA molecules is generated from a plurality of RNA molecules isolated from a specific biological specimen isolated from a human or non-human being, each of said specific biological specimens having been worked up under standardised conditions after isolation, wherein a data set relating to each biological specimen and/or said human or non-human being is assigned respectively to each set of cDNA molecules.

Further embodiments are specified in independent claims 2 to 27.

The object underlying the present invention is further solved by a kit comprising
an array comprising at least one set of cDNA molecules immobilised to a solid surface at an identifiable position, wherein each set cDNA molecules is generated from a plurality of RNA molecules isolated from a specific biological specimen isolated from a human or non-human being, each of said specific biological specimens having been worked up under standardised conditions after isolation, and
a data set relating to each biological specimen and/or said human or non-human being assigned respectively to each set of cDNA molecules.

Further embodiments are specified in dependent claims 29 through 54.

Phrased differently, the present invention provides a multipatient array and a kit on the basis of standardised biological specimens in combination with a data collection.

The object of the invention is further solved by a method for producing an array comprising the steps:
a) working up at least one specific biological specimen under standardised conditions, after isolation from a human or non-human being,
b) isolating RNA molecules from said specific biological specimen,
c) generating a set of cDNA molecules from said RNA molecules isolated in step b),
d) immobilising said set of cDNA molecules on a solid support at an identifiable position, and
e) assigning a data set relating to said specific biological specimen and/or to said human or non-human being to said respective set of cDNA molecules.

The data set or data collection assigned to the respective set of cDNA molecules are obtained from medical records, interviews with the patient, medical examinations of the patient, evaluation of the biological specimens taken from the patient, analyses of samples like blood, urine, stool, sputum, etc. taken form the patient, etc.. That is to say, the pertinent information relating the patient and/or biological specimen(s) is collected. After collecting the pertinent information it is assigned to each set of cDNA molecules.

Further embodiments of the method are specified in dependent claims 56 through 81.

Finally, the object is solved by the use of an array pursuant to any one of claims 1 to 27 for research, diagnostic or therapeutic purposes. The array can be used, for example, in clinical or oncology research. Likewise, the object is solved by the use of a kit pursuant to any one of claims 28 to 54 for research, diagnostic or therapeutic purposes, such as clinical or oncology research.

The array or the kit can further be used to develop, to identify or to check novel diagnostics or therapeutic targets or markers.

The specification of the array applies likewise to both the kit and the method of the present invention.

The array according to the present invention comprises preferably a plurality of sets of cDNA molecules which are immobilised to a solid surface support at identifiable positions. Pursuant to the present invention a set of cDNA molecules is generated from RNA molecules isolated from a specific biological specimen. Preferably, a plurality of sets of cDNA molecules are generated, wherein each set of cDNA molecules is generated from RNA molecules, preferably mRNA molecules, isolated from a specific specimen. That is to say, each set of cDNA molecules relates to a specific and identified biological specimen.

The isolation of the biological specimen itself, i.e. the medical treatment of the patient is not part of the invention and, therefore, disclaimed. The standardised working up of the biological specimens starts from the moment the biological specimen has been isolated from the patient.

The term "array" as used in the present invention refers to a grouping or an
arrangement, without being necessarily a regular arrangement. An array comprises preferably at least 10 different sets of cDNA. Preferably, the array of the present invention comprises at least 50 sets of cDNA molecules, further preferred at least 100 sets of cDNA. Pursuant to another embodiment of the invention the array of the present invention comprises at least 500 sets of cDNA. According to a further embodiment, the array comprises at least 1000 sets of cDNA, such as 5000 or 10,000 sets of cDNA.

The term "array" as used in the present invention relates to both macroarrays and microarrays. Macroarrays contain sample spot sizes of about 300 μm or larger and can be easily imaged by conventional gel and blot scanners. The sample spot sizes in microarrays are typically less than 200 μm in diameter and these arrays usually contain thousands of spots. Microarrays require specialised robotics and imaging equipment which are know to the skilled person.

It is particularly preferred that the array is a microarray, such as a DNA chip or a cDNA chip, respectively. However, macroarrays are as well within the scope of this invention.

Therefore, this application is likewise directed to both a macroarray and a microarray. All explanations given in this application with respect to "array" or "arrays" are also valid for macroarray or macroarrays as well as microarray or microarrays.

Contrary to the prior art, the cDNA molecules obtained from RNA - for example by reverse transcription and, if desired, by amplification - are immobilised to a solid surface or support or solid support surface. This array or microarray is then screened by hybridising nucleic acid probes such as DNA probes or oligonucleotides to the array or microarray. In the prior art the DNA probes or oligonucleotides are immobilised to a solid support and cDNA molecules obtained by reverse transcription and amplifcation are subsequently hybridised to the immobilised DNA probes.

That is to say the array or microarray used in the present invention is of reversed type. Therefore, the array or microarray can also be designated as reverse format array such as reverse format macroarray or reverse format microarray.

The array or microarray pursuant to this invention can, however, also be designated as multipatient array since, preferably, said array or microarray comprises a plurality of sets of cDNA generated from various biological specimens isolated from, for example, patients.

The term "patient" as used in the present application covers humans as well as non-human beings such as animals. The animals are preferably selected from the group consisting of mouse, rat, guinea-pig, rabbit, hare, dog and pig.

These animals can be used to specifically induce certain disease states, like cancer or metabolic diseases, for research purposes. The induction of said disease states can, for example, be effected by treatment. of the animals, for example, with radioactive or chemical substances known to induce cancer or another disease state. The disease states can also be induced using viral transfection systems. It is also possible to use genetically modified animals, in which one or more specific gene function(s) has/have been altered, or knock-out animals such as knock-out mice in which a specific gene function has been deleted.

When carrying out reverse transcription of RNA or mRNA, the cDNA molecules may be labelled radioactively or with fluorescence labels. For example nucleotides containing 3H, 14C, 32P, 33P or 35S can be used, for example, in reverse transcription. Alternatively, the nucleotides may be labelled with fluorescent tags such as cyanine 3 (Cy3), cyanine 5 (Cy5) or phycoerythrin. These labelled cDNA molecules can then be spotted on the solid support.

The solid support can be a polymeric material such as nylon or plastic or an inorganic material such as a silicon, for example a silicon wafer, or ceramic. Pursuant to a preferred embodiment, glass (SiO2) is used as solid support material. The glass can be a glass slide or glass chip. Pursuant to another embodiment of the invention the glass substrate has an atomically flat surface.

The RNA molecules can be isolated, for example, from a defined tissue or plurality of cells, for example, from a particular area or subregion of a defined tissue by
conventional extraction methods. A specific biological specimen can also be obtained by micro-dissection or by cutting an isolated tissue with a laser beam. For example, the biological sample may only comprise epithelial cells and no stroma cells and no adipocytes. Therefore, it is within the scope of the present invention to use only a specific subpopulation of cells as biological specimen from which RNA is isolated.

After extraction and purification the mRNA can be reverse transcribed using standard procedures known to the skilled person.

For example, oligo dT-primers such as Oligo(dT)ι2.1s, can be used to effect reverse transcription of mRNAs into cDNAs. Depending on the approach, random primers, for example random hexanucleotides, or sequence specific primers may also be used as primers.

The reverse transcription can subsequently be carried out using, for example, AMV Reverse Transcriptase, M-MuLV Reverse Transcriptase, or C. therm. Polymerase for Reverse Transcription in combination with a DNA polymerase such as Taq DNA polymerase.

There are various approaches which can be used to generate cDNA from RNA. For example, after having generated cDNA by reverse transcription of RNA, the obtained cDNA can then by amplified by various PCR methods. When amplifying the cDNA, obtained by reverse transcription, by PCR a high sensitivity can be obtained. It is of course possible to use any method for generation of cDNA from RNA.

A "set of cDNA molecules" means pursuant to this invention a complex mixture of cDNA molecules obtained, for example, by reverse transcription of RNA molecules, preferably of mRNA molecules, and optionally by amplification of the generated cDNA molecules. The RNA, preferably mRNA, is isolated from a biological specimen taken from a human or non-human being. Preferably, the samples are taken from humans or animals. Pursuant to a preferred embodiment the RNA from which the cDNA is generated is mRNA.

It is of course possible to effect a single cell RT-PCR to provide a set of cDNA molecules representing the mRNA of a single specific cell. This single cell can, for example, be isolated from central tumour tissue, peripheral tumour tissue, intermediate tumour tissue, metastatic tissue, premalignant tissue such as adenomas, fatty tissue, and/or healthy tissue. The respectively obtained set of cDNA from said single cell can then be spotted on a solid support. By spotting a plurality of sets of cDNA obtained each by single cell RT-PCR an array, preferably a microarray highly specific for defined cell types is obtained.

The whole complex cDNA mixture, i.e. a set of cDNA molecules, representing preferably, but not necessarily, the entire mRNA message of a single cell or a defined plurality of cells or a defined subregion of a tissue is then each spotted at an identifiable position on a solid surface support. That means one spot contains cDNA of the preferably whole mRNA isolated from, for example, normal or healthy tissue, neoplastic tissue such as of a benign or malignant tumour.

The complex cDNA mixture or set of cDNA molecules reflects preferably the whole mRNA of a cell, a plurality of cells, or a selected tissue such as peripheral or central tumour tissue or any other desired specific tissue or population of cells.

According to a preferred embodiment of the present invention, the mRNA is isolated of tumours of various tumour stages such as stage I to IV or tumour grades or various radicalities. These various mRNA isolates are then respectively reverse transcribed into sets of cDNA molecules.

In order to build up an array, a plurality of sets of cDNA, each generated from a specific biological specimen isolated from a patient, i.e. a human or a non-human being, are spotted each at identifiable positions on an solid support surface.

Pursuant to the present invention the biological specimens have been taken from a human or non-human being under highly standardised conditions, preferably, absolutely standardised conditions.

The biological specimen, for example, a non-diseased tissue, non-neoplastic tissue, a diseased tissue, a neoplastic tissue, metastatic tissue, a premalignant tissue, a fatty tissue or a mixture thereof is taken from a human or non-human being in an operating room by a standard operating procedure.

After isolation of the biological specimen the time is precisely taken in order to guarantee the observance of the specified schedule. The one or more biological specimen(s) isolated from the.patient who can be a human or a non-human being, are then worked up under standardised conditions.

After isolation of the biological specimen such as a specified tissue, a photo, for example, a digital photo, is taken from the biological specimen. The photo is taken, for example, 2, 3, 4 or 5 minutes after isolation of the biological specimen from the patient. Thereafter, the biological specimen is cut to obtain biological specimens of a preferably defined volume of, for example, normal tissue, peripheral neoplastic tissue and/or central neoplastic tissue.

Each cut sample is then transferred into a container, for example, a 1.5 ml, 5 ml or 10 ml cryo-tube. At a defined point in time, preferably 10 minutes after isolation of the biological specimen from the patient, the sample material is preserved, for example, by cryo-conservation. Preferably, the sample material is shock frozen in a freezing medium such as liquid nitrogen. The sample tubes containing the cut biological specimen are preferably immersed in a freezing medium such as liquid nitrogen.

Since the sample volume of the cut biological specimen is preferably about 1 mm3, 3 mm3, 50 mm3, 200mm3, 0.5 cm3, 1 cm3, 2 cm3 or 4 cm3 the samples are deep frozen within seconds.

It is, however, also possible to directly immerse the obtained biological specimen in a cryo-medium such as liquid nitrogen or dry ice or carbon dioxide snow.

Alternatively, the preservation of the sample material can be effected by adding 3.5 % (v/v) of aqueous formaldehyde or by adding 5.5 % (v/v) of aqueous glutaric dialdehyde.

However, preferably, the preservation of the sample material is effected by shock freezing in liquid nitrogen in order to modify the sample material as little as possible.

Preferably, a plurality of biological specimens from various patients are obtained and stored under standardised conditions at -80°C or in liquid nitrogen.

After having collected a plurality of biological specimens, such as non-neoplastic tissue, neoplastic tissue, fatty tissue, and the like, the RNA especially mRNA is isolated using standard procedures well known to the skilled person.

According to an embodiment of this invention the neoplastic tissue is selected, but not limited to, from the group consisting of colonic carcinoma, rectocolonic carcinoma, pancreatic carcinoma, mammary carcinoma, prostatic carcinoma, broncho carcinoma, gastric carcinoma, ovary carcinoma and cervical carcinoma.

Moreover, a data set relating to the biological specimen and/or the human or non-human being is assigned to each set of cDNA molecules. That is to say a great deal of clinical and medical information is assigned to each set of cDNA molecules.

Preferably, the data sets comprises information of the human or non-human being such as sex, age, weight, ethnic background, occupation, environment, family medical background, complaints, prior diseases, allergies, children's diseases, infectious diseases, tropical diseases, habit of living, eating or sleeping, excretion habits of stool and urine, diagnostic and prognostic test results, symptoms, drug exposure,
therapeutic agent exposure, result of treatment regimens, incompatibilities of
medicaments, history of alcoholism, drug or tobacco use, time of death, cause of death, medication history, intake of anaesthetics for surgery, surgical procedure, and/or medical follow-up information such as survival time or development of metastases.

It is further preferred that the data sets comprise information relating to the biological specimen, such as, histological characterisation such as acute or chronic inflammation, thromboses, tumour type, tumour grade, tumour size, written documentation of tumour, photographic documentation of tumour, electronic documentation of tumour, and/or accrual information or information about the collection of the biological specimens.

The data set is a comprehensive data collection comprising any relevant information about the patient and/or the biological sample.

The medical, biological and/or clinical information supplements significantly the scientific results obtained by array profiling of the biological specimen. This additional information can provide the missing link to understand the pathological condition of the patient. Due to the standardised working up of the biological specimens reliable information are obtained by array or microarray analyses are obtained.

Every set of cDNA molecules is generated from a specific biological specimen handled under nearly identical, preferably identical, conditions. Therefore, any deviation of gene expression or gene activity detected by array or microarray analysis can be linked with the pathological state of the biological sample from which the RNA or mRNA was isolated. Phrased differently, the deviations are not caused by a different handling and working up of the biological specimen after isolation from the patient.

In combination with the additional medical and clinical information assigned to each set of cDNA molecules, similarities and/or differences between various disease states can be better understood. The array system or microarray system of the present invention allows to detect the up or down regulation of gene activity or gene expression of one or more genes or group of genes or gene clusters and combining this gene activity or expression information with the assigned medical or clinical information.

The combination of the data obtained by the array or microarray analysis with the data set assigned to each set of cDNA molecules provides a valuable tool for research, diagnostics and therapy, particularly in the clinical or oncology field. The array or microarray of this invention is also a valuable tool for the investigation of other diseases like heart disease, circulatory disorders, organ diseases and/or metabolic diseases.

This invention provides arrays wherein complex mixtures of cDNA molecules, representing preferably, but not. necessarily, the entire mRNA molecules of a particular cell or tissue type, are spotted each at an identifiable position. The plurality of biological specimens represent preferably various tumours, various tumour stages, various healthy tissues of organs like lung, breast, lymphatic tissue, ovary, cervix, prostate, brain, kidney, liver, colon, bladder, uterus, stomach, rectum, oral cavity, pancreas, bone, skin, and/or epidermis.

The array system or microarray system of the present invention is preferably a multipatient array, since a plurality of sets of a various patients are spotted each at identifiable positions on one solid support surface.

The normalisation between the various biological specimens can be carried out, for example, using one, two, three, four or more house keeping genes, such as beta-actin, ubiquitin, phospholipase A2, glutamase dehydrogenase, 23-kDa highly basic protein, or transferrin receptor.

The normalisation can be carried out for each set of cDNA on the basis of these house keeping genes. These house keeping genes have a nearly identical activity in all cell or tissue types. Therefore, the activity of these genes can be used for normalisation when quantifying the gene activities.

Of course, it is possible to use any other normalisation approach. There are various normalisation methods currently used for microarrays. These approaches can be likewise used in the present invention.

After having normalised the measured results obtained by the various sets of cDNA, a direct comparison of the various biological samples is possible. A quantification of the measured fluorescence allows to quantify the extent of up or down regulation of a certain gene activity, such as p53 gene or any oncogene like ras or myc, etc..

Using radioactive or fluorescence labelled probes for genes, the activity of which is up or down regulated in cancer cells compared to non-neoplastic cells, for hybridisation on an array according to the invention allows to obtain a new and detailed insight into the molecular mechanism of diseases such as cancer or metabolic diseases. The additional information assigned to each sets of cDNA molecules further deepens and facilitates the understanding of the pathological mechanism of the development of diseases such as cancer or metabolic diseases.

Pursuant to a preferred embodiment of the invention the period between isolation of the biological specimen from the human or non-human being up to the preservation of this biological specimen occurs within a defined period. The working up of the various biological specimens occur within a defined maximum deviation from the defined period.

It is particularly preferred that the defined maximum deviation from said defined period is not more than 10 %, preferably not more than 5 % based on the defined period.

According to another embodiment of the invention, it is preferred that the defined maximum deviation of the defined period for working up is at maximum one minute.

Pursuant to an embodiment of this invention the defined period is less than about 25 minutes, further preferred less than about 15 minutes. Furthermore, it is preferred that the defined period is less than about 12 minutes. It is particularly preferred that the defined period is about 10 minutes or less, such as, 8 minutes or 5 minutes.

When stating that the maximum deviation is not more than 10 %, this means that the maximum deviation of a defined period of 10 minutes is ± 1 minute. When further limiting the maximum deviation to 5 %, the maximum deviation is ± 0,5 minutes.

The highly standardised working up procedure of the biological specimen, after isolation from the patient, makes it possible to obtain high quality biological specimen the profile of gene activity of which is directly comparable to each other.

Pursuant to another embodiment of the invention the period of ischemia, i.e. the time between cutting off the tissue from the blood supply and the preservation of the biological specimen is also defined. Likewise the maximum deviation from this defined period of ischemia is also defined. The cutting-off from the blood supply is important since the tissue is cut-off from the oxygen supply which has a strong impact on the biochemical status of the biological specimen isolated.

Pursuant to an embodiment of the invention the defined maximum deviation from the defined period of ischemia is not more than 10 %, preferably not more than 5 %, based on the defined period of ischemia.

According to another embodiment of the invention, it is preferred that the defined maximum deviation of the defined period of ischemia is at maximum one minute.

According to another embodiment of the invention the period of ischemia is less than about 25 minutes, more preferred less than about 15 minutes. It is particular preferred that the defined period of ischemia is less than about 12 minutes. Most preferably the defined period of ischemia is about 10 minutes.

It is particular preferred that the composition of the set of cDNA molecules reflects the relative abundance of mRNA molecules contained in a specific biological specimen from which the cDNA molecules was generated by, for example, reverse transcription of isolated RNA molecules, preferably mRNA molecules, and, optionally, by
amplification of reverse transcribed cDNA.

Phrased differently, it is preferred that the relative ratio of mRNA molecules is maintained when, for example, reverse transcribing into cDNA and optionally amplifying the latter.

When spotting the complex cDNA mixture of a set of cDNA molecules to one identifiable position on a solid support, it is possible to determine an altered gene activity between normal or non-diseased tissue and diseased tissue.

Moreover, it is also possible to compare different disease states with each other. The different regulation of gene activity in different pathological conditions may also help to explain a pathological mechanism of the development of diseases like cancer or metabolic diseases. Moreover, such a microarray allows to identify novel diagnostic and therapeutic markers.

The present invention also relates to a kit comprising said array or microarray as described above and said data set also described above. The data set assigned to each set of cDNA can be provided on a data carrier like a magnetic memory unit such as hard disk, floppy disk, etc., or an optical memory unit such as CD, DVD, etc.. The data sets can also be provided as printed version. The provision of the data sets on a magnetic or optical memory unit allows to easily combine and evaluate these data with the data obtained by measuring the array or microarray.

The inventive kit including said arrays or microarrays and said data set stored on a data carrier can be easily packed and shipped to the customers.

The array or microarray is particularly useful for research or diagnostic purposes like clinical or oncology research. The array or microarray of the present invention represents a particular useful tool for the pharmaceutical industry. The action or influence of a drug or pharmaceutical compound administered to a patient, taking into account the detailed medical information assigned to each set of cDNA molecules, can be analysed and better understood using the results of the gene activity profile provided by the array or microarray of the present invention. Furthermore, the array or the kit of the present invention allows a significantly improved statistical substantiation.

Example

The following example is merely given for illustrative purposes and does not limit the invention as claimed.

An exemplary schedule for preparing a collection of a biological specimen from which mRNA is subsequently isolated is shown below in Table I. The schedule starts immediately after isolation of the biological specimen (colon resection).

Table I: Schedule of working up of isolated colon tissue


For the manufacture of an array or microarray of the present invention, RNA, particularly mRNA, is isolated from each biological specimen according to standard procedures. The set of cDNA can be generated by reverse transcription and
amplification by methods known to the skilled person.

The obtained set of cDNA molecules or amplified cDNA molecules can be diluted in an appropriate liquid and spotted and coupled at a predefined position on a plurality of substrates, for example to 100, 500 or 1 ,000 substrates. Any method usually designated as delivery approach can be used for coupling the cDNA to the solid surface. The substrate is preferably glass (silicon dioxide), silicon, ceramic, or plastic. This procedure is repeated in order to apply the desired number of sets of cDNA on the substrate. The application of the cDNA can be carried out simultaneously or
sequentially using high-speed robotics. By repeating the procedure or simultaneously applying a plurality of sets of cDNA molecules, a microarray is produced containing sets of cDNA molecules from various tissues, cell types, subpopulations of cells, and patients.

The data set obtained from each patient comprising the medical history is assigned to the respective sets of cDNA molecules. Each set of cDNA molecules reflects preferably the relative abundance of mRNA in each specimen of healthy tissue, peripheral tumour tissue and central tumour tissue.

The array or microarray specified before can then be used to screen the gene activity (expression analysis or RNA profiling) of known or novel genes or group of genes or gene clusters which are important or might be recognised to be important in the development of a certain disease such as cancer or metabolic diseases, taking into account the data set reflecting the medical history of each patient. The screening can be effected using respective labelled or unlabelled probes.

The set of cDNA molecules of the arrays or microarrays of the present invention can also be labelled or unlabelled. The interaction of labelled or unlabelled probes with labelled or unlabelled cDNA molecules can then be detected optically, for example, by phospho-imager, scanner, CCD camera or confocal scanning microscopy. It is of course necessary that either the spotted cDNA molecules or the probes are labelled in order to be able to detect an optical signal. Alternatively, both the spotted cDNA molecules and the probes are labelled, for example, with fluorescence tags. In the latter case the fluorescence tags of the cDNA molecules and of the probes are preferably different from each other.