Compositions and methods for detecting and quantifying gene expression

#This invention relates to compositions and methods for improved analysis of gene expression, genetic polymorphism or gene mutation using nucleic acid microarrays for genetic research and diagnostic applications.

#Nucleic acid microarrays, often containing thousands of gene sequences, are useful for identifying differential gene expression in diseased tissue relative to normal tissue of the same type, for example. Using nucleic acid microarrays, test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to generate cDNA probes. The probes are then hybridized to an array of nucleic acids immobilized on a solid support. The array is configured such that the sequence and position of each member of the array is known. For example, a selection of genes that have potential to be expressed in certain disease states may be arrayed on a solid support. Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe was derived expresses that gene. Differential gene expression analysis of disease tissue can provide valuable information. For example, if hybridization of a probe from a test (disease tissue) sample is greater than hybridization of a probe from a control (normal tissue) sample, the gene or genes expressed in the diseased tissue may be a significant diagnostic indicator of a potential drug target.

#Detection sensitivity is a limiting factor for effectively analyzing test versus control samples such that gene expression, a genetic polymorphism, or a gene mutation associated with the disease may be recognized. For the study of human genes using DNA microarrays, successful analysis of many disease states requires sensitive detection to work with limiting sample quantities.

#FIG. 1 is a photograph of microarray images generated using fluoroprobes synthesized by the method of the invention from 1-5 ng total RNA from microdissected colon tumor cells.

#FIG. 2A is a photograph of microarray images generated using fluoroprobes synthesized by the method of the invention from 5 μg total RNA isolated from formalin-fixed paraffin-embedded liver tissue. FIG. 2B is a photograph of microarray images generated using fluoroprobes synthesized by the method of the invention from 5 μg total RNA isolated from fresh frozen adult liver. Probes generated from paraffin-embedded starting material were comparable in detection sensitivity to probes generated from fresh frozen tissue (compare FIG. 2A and FIG. 2B). FIG. 2C is a photographic image of a microarray analysis from a formalin-fixed paraffin-embedded colon tumor, 4 μg total cellular RNA starting material. FIG. 2D is a scatter plot of the fluorescence intensities from microaray analysis of colon tumor RNA isolated from the same patient, a fresh-frozen sample (X axis) versus a formalin-fixed paraffin-embedded sample (Y axis).

#FIG. 3A is a photograph of microarrays showing hybridization of probes synthesized from breast tumor RNA. FIG. 3B shows hybridization of probes synthesized from epithelial tissue RNA pool reference sample. In general, gene expression is quantified by comparison of the intensity and wavelength emitted from each spot for test versus control samples.

#The following examples are offered by way of illustration and not by way of limitation. The examples are provided so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the compounds, compositions, and methods of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to insure accuracy with respect to numbers used (e.g. amounts, temperature, etc.), but some experimental errors and deviation should be accounted for. Unless indicated otherwise, temperature is in degrees C. (° C.). The disclosures of all citations in the specification are expressly incorporated herein by reference.

#The invention is useful for detecting the presence of nucleic acids in any mixture of nucleic acids. The present invention finds its preferred use, however, in the detection and quantification of gene expression in tissue samples, a medium in which detection of gene expression has heretofore posed distinct challenges. The present invention solves this problem by providing a method of improving the detection limit for gene expression in tissue samples.

#Collecting Cells for Control or Test Samples: Microarray analysis allows the direct comparison of cellular states between test and control samples of cells, tissue, body fluids, and the like. Such comparisons are optimized when the test or control sample comprises exclusively or substantially only the cells of interest. For example, a diseased tissue, such as cancer tissue, frequently comprises cancerous cells that have infiltrated an area of normal cells. Thus, a sample of cancerous tissue will often contain a mixture of normal and diseased cells and may also include several cell types found in the tissue or associated with the cancer, such as cells associated with the inflammatory and immune responses to cancer. Preferably, a sample comprises only those cells important to the analysis. According to the present invention, it is preferred that the test sample comprises a collection of cells collected specifically by cell type or other desired state such that contamination of the sample by cells of a different type or state are excluded.