Transcriptional analysis of intracytoplasmically stained, FACS-purified cells by high-throughput, quantitative nuclease protection.
Analyzing specialized cells in heterogeneous tissues is crucial for understanding organ function in health and disease. Thus far, however, there has been no convenient method for studying gene expression in cells purified by fluorescence-activated cell sorting (FACS) using intracellular markers. Here we show that the quantitative nuclease protection assay (qNPA) enables transcriptional analysis of intracytoplasmically stained cells sorted by FACS. Applying the method to mouse pancreatic islet-cell subsets, we detected both expected and unknown lineage-specific gene expression patterns. Some beta cells from pregnant animals were found to express Mafb, previously observed only in immature beta cells during embryonic development. The four 'housekeeping' genes tested were expressed in purified islet-cell subpopulations with a notable variability, dependent on both cell lineage and developmental stage. Application of qNPA to intracellularly stained, FACS-sorted cells should be broadly applicable to the analysis of gene expression in subpopulations of any heterogeneous tissue, including tumors.
Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Susanne
S
Pechhold
Melissa
M
Stouffer
Gregory
G
Walker
Ralph
R
Martel
Bruce
B
Seligmann
Yan
Y
Hang
Roland
R
Stein
David M
DM
Harlan
Klaus
K
Pechhold
D000375:Aging / Q000235:genetics; D000818:Animals; D002469:Cell Separation; D003593:Cytoplasm / Q000235:genetics*; D004210:Dissection; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D057166:High-Throughput Screening Assays / Q000379:methods*; D007515:Islets of Langerhans / Q000166:cytology / Q000378:metabolism; D051258:MafB Transcription Factor / Q000235:genetics / Q000378:metabolism; D051379:Mice; D023282:Nuclease Protection Assays / Q000379:methods*; D013194:Staining and Labeling; D014158:Transcription, Genetic
D051258:MafB Transcription Factor; C490406:Mafb protein, mouse