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31.
Optimized blood cell profiling method for genomic biomarker discovery using high-density microarray 总被引:1,自引:0,他引:1
J. Shou C. Dotson H. -R. Qian W. Tao C. Lin F. Lawrence M. N'Cho N. H. Kulkarni C. M. Bull L. M. Gelbert J. E. Onyia 《Biomarkers》2005,10(4):310-320
High-quality biomarkers for disease progression, drug efficacy and toxicity liability are essential for improving the efficiency of drug discovery and development. The identification of drug-activity biomarkers is often limited by access to and the quantity of target tissue. Peripheral blood has increasingly become an attractive alternative to tissue samples from organs as source for biomarker discovery, especially during early clinical studies. However, given the heterogeneous blood cell population, possible artifacts from ex vivo activations, and technical difficulties associated with overall performance of the assay, it is challenging to profile peripheral blood cells directly for biomarker discovery. In the present study, Applied BioSystems' blood collection system was evaluated for its ability to isolate RNA suitable for use on the Affymetrix microarray platform. Blood was collected in a TEMPUS tube and RNA extracted using an ABI-6100 semi-automated workstation. Using human and rat whole blood samples, it was demonstrated that the RNA isolated using this approach was stable, of high quality and was suitable for Affymetrix microarray applications. The microarray data were statistically analysed and compared with other blood protocols. Minimal haemoglobin interference with RNA labelling efficiency and chip hybridization was found using the TEMPUS tube and extraction method. The RNA quality, stability and ease of handling requirement make the TEMPUS tube protocol an attractive approach for expression profiling of whole blood to support target and biomarker discovery. 相似文献
32.
Jianyong Shou Christopher M Bull Li Li Hui-Rong Qian Tao Wei Shuang Luo Douglas Perkins Patricia J Solenberg Seng-Lai Tan Xin-Yi Cynthia Chen Neal W Roehm Jeffrey A Wolos Jude E Onyia 《Arthritis research & therapy》2005,8(1):1-14
Rheumatoid arthritis (RA) is a chronic debilitating autoimmune disease that results in joint destruction and subsequent loss of function. To better understand its pathogenesis and to facilitate the search for novel RA therapeutics, we profiled the rat model of collagen-induced arthritis (CIA) to discover and characterize blood biomarkers for RA. Peripheral blood mononuclear cells (PBMCs) were purified using a Ficoll gradient at various time points after type II collagen immunization for RNA preparation. Total RNA was processed for a microarray analysis using Affymetrix GeneChip technology. Statistical comparison analyses identified differentially expressed genes that distinguished CIA from control rats. Clustering analyses indicated that gene expression patterns correlated with laboratory indices of disease progression. A set of 28 probe sets showed significant differences in expression between blood from arthritic rats and that from controls at the earliest time after induction, and the difference persisted for the entire time course. Gene Ontology comparison of the present study with previous published murine microarray studies showed conserved Biological Processes during disease induction between the local joint and PBMC responses. Genes known to be involved in autoimmune response and arthritis, such as those encoding Galectin-3, Versican, and Socs3, were identified and validated by quantitative TaqMan RT-PCR analysis using independent blood samples. Finally, immunoblot analysis confirmed that Galectin-3 was secreted over time in plasma as well as in supernatant of cultured tissue synoviocytes of the arthritic rats, which is consistent with disease progression. Our data indicate that gene expression in PBMCs from the CIA model can be utilized to identify candidate blood biomarkers for RA. 相似文献