For gene expression studies in pancreatic cancer, samples of the tumor are usually compared with those obtained from corresponding non-neoplastic tissue of the same or of a different patient. Purification procedures include the yield of homogeneous samples and isolation of individual cell types from clinical material. “normal” is clearly insufficient, in particular at a timepoint when the close molecular links between cancer and inflammation begin to be highlighted. the International Classification of Diseases of the World Health Organization, or the Tumor-Node-Metastasis Classification of the International Union against Cancer) to permit later comparisons. Furthermore, diagnosis has to be classified and staged according to international standards (e.g. Unfortunately, tissue located next to the biopsy is barely examined. The outcome of tissue biopsy done in the operating room or in the pathology laboratory is usually influenced by the macroscopic aspect of tissue, but diagnosis must be confirmed by a surgical pathologist, using accepted techniques (histopathology, immunohistochemistry, etc.).
However, there are several critical preconditions for the success of such a development. For example, proteins that are overexpressed and essential for growth or cell survival are interesting targets for the development of specific inhibitors or antibodies. The identification of tumor-specific expression patterns at the protein and/or mRNA level may allow us to develop new therapeutic or diagnostic targets. Moreover, a quality control protocol addressing the needs of the industry and the requirements of regulatory agencies is proposed. Compared with other cell purification procedures, this method is characterized by several advantages: a large quantity of cells available for downstream analysis, combined transcriptomics and proteomics studies using the same samples, better reproducibility of proteomics studies, and an acceptable yield (63%) for gene expression arrays studies. This method, which uses epithelial cell surface antibody Ber-Ep4, proteases, and RNAases inhibitors, leads to a significant enrichment (>95% purity) of epithelial cells from fresh human tissue samples and allows for both proteomics (Western Blot, 2D PAGE) and transcriptomics studies (rtPCR, cDNA microarray). We developed a successful method that addresses these different problems. In pancreatic tissue, the high amount of RNAases is a further problem when it comes to obtaining high-quality RNA, and the presence of secreted proteases accelerates protein degradation. In particular, in diseases such as pancreatic cancer and pancreatitis, isolating epithelial cells is an important step preceding such research. Standardized sample preparation procedures constitute a prerequisite for obtaining reliable and reproducible results in gene expression research in humans.
0 kommentar(er)
