Detection of growth factor receptor RNA in individual hematopoietic cells by in situ RT-PCR; comparison with RT-PCR.
The ability to detect changes in RNA expression in single cells would greatly enhance understanding of the molecular basis of biological responses to positive and negative growth regulators. To this end, we compared expression of RNA encoding the receptors for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, leukemia inhibitory factor (LIF) and stem cell factor (SCF) in populations of primitive hematopoietic progenitors (lineage marker negative, Lin(-), and Lin(-) c-Kit(+)) by RT-PCR and in situ RT-PCR. Both Lin(-) and Lin(-) c-Kit(+) progenitors expressed all receptors by RT-PCR. However, RT-PCR could not distinguish between the possibility that all cells expressed growth factor receptor RNA, or the possibility that only a proportion of cells expressed RNA. Therefore, we used in situ RT-PCR to examine growth factor receptor mRNA expression in individual cells. In contrast to RT-PCR, we observed that only 40-80% of Lin(-) cells and 75-100% of Lin(-) c-Kit(+) cells were positive for expression of the growth factor receptor subunits, demonstrating that not all cells were receptor positive. We found that in situ RT-PCR could also be used to measure induction or repression of receptor RNA expression in these cell populations. Specifically, the percentage of cells expressing IL-6alpha receptor RNA decreased from 88% positive in freshly harvested cells to 9% in Lin(-) c-Kit(+) cells cultured in IL-3 for 18 h. Thus, in situ RT-PCR can be used to detect and quantify the number of individual cells that express growth factor receptor mRNA, and may also be useful to measure changes in expression of other endogenous genes or genes introduced by transfection and gene therapy vectors.
Spence
S E
SE
Intramural Research Support Program, SAIC Frederick, Division of Basic Science, National Cancer Institute-Frederick, P.O. Box B, Frederick, MD 21702, USA. spence@mail.ncifcrf.gov
Lohrey
N C
NC
Ortiz
M
M
Gooya
J
J
Keller
J R
JR
D000199:Actins / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit; D051379:Mice; D008807:Mice, Inbred BALB C; D019009:Proto-Oncogene Proteins c-kit / Q000235:genetics; D012313:RNA / Q000032:analysis* / Q000235:genetics*; D018121:Receptors, Cytokine / Q000235:genetics; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics; D017978:Receptors, Growth Factor / Q000235:genetics*; D016185:Receptors, Interleukin-3 / Q000235:genetics; D019947:Receptors, Interleukin-6 / Q000235:genetics; D053606:Receptors, OSM-LIF; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000199:Actins; D017931:DNA Primers; D053680:Leukemia Inhibitory Factor Receptor alpha Subunit; C507841:Lifr protein, mouse; D018121:Receptors, Cytokine; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D017978:Receptors, Growth Factor; D016185:Receptors, Interleukin-3; D019947:Receptors, Interleukin-6; D053606:Receptors, OSM-LIF; D012313:RNA; D019009:Proto-Oncogene Proteins c-kit