TY - JOUR
T1 - Isolation of microarray-quality RNA from primary human cells after intracellular immunostaining and fluorescence-activated cell sorting
AU - Iglesias-Ussel, Maria
AU - Marchionni, Luigi
AU - Romerio, Fabio
N1 - Funding Information:
This work was supported by National Institutes of Health grant AI084711 and by Bill & Melinda Gates Foundation Grand Challenges Explorations grant OPP1035926 (F.R.). L.M. was also supported by NIH-NCI grant P30CA006973 . This paper is subject to the NIH Public Access Policy.
PY - 2013/5/31
Y1 - 2013/5/31
N2 - 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.
AB - 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.
KW - FACS
KW - Fixation
KW - Intracellular staining
KW - Microarray
KW - Permeabilization
KW - RNA
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U2 - 10.1016/j.jim.2013.02.003
DO - 10.1016/j.jim.2013.02.003
M3 - Article
C2 - 23434645
AN - SCOPUS:84876134930
SN - 0022-1759
VL - 391
SP - 22
EP - 30
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
IS - 1-2
ER -