title: Isolation of microarray-quality RNA from primary human cells after intracellular immunostaining and fluorescence-activated cell sorting.
abstract: Microarrays have made it possible to perform high-throughput, genome-wide analyses of RNA expression from an extremely wide range of sources. This technology relies on the ability to obtain RNA of sufficient quantity and quality for this type of application. While there are means to circumvent limitations in the former, recovery of RNA suitable for microarray analysis still represents a major issue when working with some biological samples, particularly those treated with and preserved in nucleic acid-modifying organic reagents. In the present report we describe a procedure for the isolation of RNA suitable for microarray analysis from cells purified by fluorescence-activated cell sorting after fixation, permeabilization and intracellular staining with fluorochrome-conjugated antibodies. We show that - although the RNA isolated from these samples presented some degradation - it performed remarkably well in microarray analysis. The method we describe here makes it available to genome-wide expression profiling a variety of biological samples that so far were confined to single-gene analysis.
authors: Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD, United States.
authors: Maria
authors: M
authors: Iglesias-Ussel
authors: Luigi
authors: L
authors: Marchionni
authors: Fabio
authors: F
authors: Romerio
mesh_terms: D015496:CD4-Positive T-Lymphocytes / Q000737:chemistry* / Q000276:immunology / Q000821:virology; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D005434:Flow Cytometry; D019085:Fluorescent Antibody Technique, Direct; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D015497:HIV-1 / Q000276:immunology; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D061251:Primary Cell Culture; D012313:RNA / Q000302:isolation & purification*; D020871:RNA Stability; D015203:Reproducibility of Results; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000276:immunology
chemical_list: D054301:gag Gene Products, Human Immunodeficiency Virus; D012313:RNA