Profiling of gene expression in individual hematopoietic cells by global mRNA amplification and slot blot analysis.
The pattern of expressed genes defines the structure and functional status of cells. Currently, most methods used in gene expression studies depend on large numbers of cells. Thus, their application may be hampered by the heterogeneity of cell populations, and by the low numbers of cells obtainable from in vivo sources. Such drawbacks may be overcome by methods suitable for the profiling of gene expression at the single cell level. We studied whether polymerase chain reaction (PCR) products synthesized from individual cells by global amplification of messenger RNA (mRNA) were suitable as probes for gene expression analysis. For this purpose, cells were subjected to reverse transcription and PCR using sequence independent primers (SIP RT-PCR). The resultant cDNA products were radiolabeled and hybridized to cDNA clones arrayed on a nylon membrane by vacuum slot blotting (a method referred to as slot blot analysis). The SIP RT-PCR procedure was reproducible and allowed the detection of twofold changes in input RNA copies per cell (range: 80-10.000 copies of an in vitro transcribed poly(A)-tailed RNA/cell). Analysis of total RNA and amplified cDNA, obtained from neutrophil granulocytes and the promyelocytic HL-60 cell line, demonstrated comparable gene expression profiles as measured by Northern blot and slot blot analysis. Slot blot analysis of HL-60 cells indicated that individual cells from an apparently homogenous population have varying expression of specific transcripts, which all contribute to the mRNA phenotype of their population. Interestingly, the genes that were detected in some but not all individual HL-60 cells were those found to peak within 2 days of retinoic acid-induced granulocytic differentiation. This study demonstrates the potential of cDNA, synthesized from individual cells by global amplification of mRNA, as probes for cDNA arrays.
Theilgaard-Mönch
K
K
The Granulocyte Research Laboratory, Department of Hematology-9322, The Finsen Center, Rigshospitalet, University of Copenhagen, Blegdamsvej 9, 2100, Copenhagen, Denmark. kimthei@rh.dk
Cowland
J
J
Borregaard
N
N
D015152:Blotting, Northern / Q000379:methods; D002454:Cell Differentiation; D017931:DNA Primers; D018076:DNA, Complementary / Q000096:biosynthesis; D005784:Gene Amplification; D020869:Gene Expression Profiling / Q000379:methods*; D006098:Granulocytes; D018922:HL-60 Cells; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D012333:RNA, Messenger; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017423:Sequence Analysis, RNA
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger