# US 6,720,138 B2 · Method of preparing a standard diagnostic gene transcript pattern > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 6,720,138 B2 - Document ID: US6720138B2 - Status: Granted - Kind: B2 - Application number: 09/429,003 - Filed: 1999-10-29 - Published: 2002-02-21 - Granted: 2004-04-13 - Earliest claimed: 1997-04-30 - Assignees: Diagenic AS - Inventors: Praveen Sharma; Anders Lönneborg - Web page: https://patentgrep.com/patent/US6720138B2 ## CPC - C12Q 1/6883: for diseases caused by alterations of genetic material - C12Q 1/6809: Methods for determination or identification of nucleic acids involving differential detection - C12Q 1/6816: characterised by the detection means (C12Q1/6804 takes precedence) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract A method for preparing a gene transcript pattern probe kit characteristic of a disease or condition or a stage thereof in a prokaryotic or eukaryotic organism using mRNA which is differentially expressed in the disease or condition or stage as probes, methods of diagnosis using the method and kits for performing the same are disclosed. ## Claims ### Claim 1 A method of obtaining isolated selected mRNA species or isolated selected cDNA species useful for diagnosing or identifying Alzheimer's disease or stage thereof in a human comprising the steps of: (a) isolating mRNA from cells of one or more humans which are known to have said disease or a stage thereof (diseased sample), wherein said cells are obtained from blood, wherein the resulting isolated mRNA is optionally subjected to reverse transcription to obtain isolated cDNA; (b) isolating mRNA from corresponding cells of one or more normal humans (normal sample), wherein the resulting isolated mRNA is optionally subjected to reverse transcription to obtain isolated cDNA; (c) separating, by a non-sequence based separation technique, mRNA species or cDNA species present within each of the resulting isolated mRNA or isolated cDNA of step (a) and step (b), wherein the resulting separated mRNA species are optionally subject to reverse transcription to obtain separated cDNA species; (d) selecting two or more mRNA species or two or more cDNA species from the resulting separated mRNA species or resulting separated cDNA species obtained in step (c), respectively, which are present at a different level in the normal sample than in the diseased sample by identifying a signal corresponding to each mRNA species or cDNA species, wherein the resulting selected two or more mRNA species are optionally subjected to reverse transcription to obtain two or more selected cDNA species; and (e) isolating the resulting two or more selected mRNA species or resulting two or more selected cDNA species obtained in step (d) to obtain isolated selected mRNA species or isolated selected cDNA species, wherein the resulting isolated selected mRNA species are optionally subjected to reverse transcription to obtain isolated selected cDNA species. ### Claim 2 (depends on 1) The method as claimed in claim 1, wherein in steps (a) and (b), the resulting isolated mRNA is subjected to reverse transcription to obtain isolated cDNA. ### Claim 3 (depends on 2) The method as claimed in claim 2, wherein said isolated cDNA is amplified. ### Claim 4 (depends on 1) The method as claimed in claim 1, wherein said isolated cDNA is labelled. ### Claim 5 (depends on 1) The method as claimed in claim 1, wherein, in step (e), between 50 and 100 mRNA species or cDNA species are isolated and selected. ### Claim 6 (depends on 1) The method as claimed in claim 1, wherein, in step (e), between 10 and 500 mRNA species or cDNA species are isolated and selected. ### Claim 7 (depends on 1) The method as claimed in claim 1, wherein, in step (c), said separation technique is gel electrophoresis. ### Claim 8 A method of preparing a gene transcript pattern probe kit comprising the steps of: (a) isolating mRNA from cells of one or more humans which are known to have Alzheimer's disease or a stage thereof (diseased sample), wherein said cells are obtained from blood, wherein the resulting isolated mRNA is optionally subjected to reverse transcription to obtain isolated cDNA; (b) isolating mRNA from corresponding cells of one or more normal humans (normal sample), wherein the resulting isolated mRNA is optionally subjected to reverse transcription to obtain isolated cDNA; (c) separating, by a non-sequence based separation technique, mRNA species or cDNA species present within each of the resulting isolated mRNA or isolated cDNA of step (a) and step (b), wherein the resulting separated mRNA species are optionally subject to reverse transcription to obtain separated cDNA species; (d) selecting two or more mRNA species or two or more cDNA species from the resulting separated mRNA species or resulting separated cDNA species obtained in step (c), respectively, which are present at a different level in the normal sample than in the diseased sample by identifying a signal corresponding to each mRNA species or cDNA species, wherein the resulting selected two or more mRNA species are optionally subjected to reverse transcription to obtain two or more selected cDNA species; (e) isolating the resulting two or more selected mRNA species or resulting two or more selected cDNA species obtained in step (d) to obtain isolated selected mRNA species or isolated selected cDNA species, wherein the resulting isolated selected mRNA species are optionally subjected to reverse transcription to obtain isolated selected cDNA species; and (f) immobilizing the resulting isolated selected mRNA species or isolated selected cDNA species of step (e) on at least one solid support so as to form a gene transcript pattern probe kit. ### Claim 9 (depends on 8) The method as claimed in claim 8, wherein, prior to immobilizing in step (f), the resulting isolated selected mRNA species or isolated selected cDNA species of step (e) are amplified. ### Claim 10 (depends on 8) The method as claimed in claim 8, wherein said solid support is a filter. ### Claim 11 A method of preparing a standard gene transcript pattern characteristic of Alzheimer's disease or stage thereof comprising the steps of: (a) isolating mRNA from cells of one or more humans known to have said disease or a stage thereof, wherein said cells are obtained from blood, wherein the resulting isolated mRNA is optionally subjected to reverse transcription to obtain isolated cDNA; (b) hybridizing the resulting isolated mRNA or isolated cDNA of step (a) to two or more mRNA species which are present at a different level in cells in a normal sample than corresponding cells in an Alzheimer's diseased sample, wherein the two or more mRNA species or cDNA species are specific for said disease or stage thereof and wherein said cells are obtained from human blood, or to two or more cDNA species transcribed from said mRNA species, wherein said mRNA or cDNA species are immobilized on a solid support; and (c) assessing the amount of hybridisation so as to obtain said standard gene transcript pattern. ### Claim 12 A method of preparing a test gene transcript pattern for Alzheimer's disease or stage thereof comprising the steps of: (a) isolating mRNA from cells of a test human, wherein said cells are obtained from blood, wherein the resulting isolated mRNA is optionally subjected to reverse transcription to obtain isolated cDNA; (b) hybridizing the resulting isolated mRNA or isolated cDNA of step (a) to two or more mRNA species which are present at a different level in cells in a normal sample than corresponding cells in an Alzheimer's diseased sample, wherein the two or more mRNA species or cDNA species are specific for said disease or stage thereof and wherein said cells are obtained from human blood, or to two or more cDNA species transcribed from said mRNA species, wherein said mRNA or cDNA species are immobilized on a solid support; and (c) assessing the amount of hybridization so as to obtain said test gene transcript pattern. ### Claim 13 A method of diagnosing or identifying Alzheimer's disease or stage thereof in a test human comprising the steps of: (a) isolating mRNA from cells of a test human, wherein said cells are obtained from blood, wherein the resulting isolated mRNA is optionally subjected to reverse transcription to obtain isolated cDNA; (b) hybridizing the resulting isolated mRNA or isolated cDNA of step (a) to two or more mRNA species which are present at a different level in cells in a normal sample than corresponding cells in an Alzheimer's diseased sample, wherein the two or more mRNA species or cDNA species are specific for said disease or stage thereof and are obtained from human blood, or to two or more cDNA species transcribed from said mRNA species, wherein said mRNA or cDNA species are immobilized on a solid support; (c) assessing the amount of hybridization so as to obtain a hybridization pattern; (d) comparing the resulting hybridization pattern obtained in step (c) with a hybridization pattern obtained by hybridizing isolated mRNA or isolated cDNA prepared from corresponding cells from one or more humans known to have said disease or stage thereof to the two or more mRNA species or two or more cDNA species transcribed from said RNA species, wherein said mRNA species or cDNA species are immobilized on a solid support; and (e) assessing the amount of hybridization, so as to determine the degree of correlation indicative of the presence of said disease or stage thereof, and so as to diagnose or identify said disease or a stage thereof in said test human.