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 great ly enhance understanding of the molecular basis of biological responses to positive and negative growth regulators. To this end, we compared expressio n of RNA encoding the receptors for interleukin-3 (IL-3), granulocyte-macro phage 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-PC R 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 po ssibility that only a proportion of cells expressed RNA. Therefore, we used in situ RT-PCR to examine growth factor receptor mRNA expression in indivi dual cells. In contrast to RT-PCR, we observed that only 40-80% of Lin(-) c ells and 75-100% of Lin(-) c-Kit(+) cells were positive for expression of t he growth factor receptor subunits, demonstrating that not all cells were r eceptor positive. We found that in situ RT-PCR could also be used to measur e induction or repression of receptor RNA expression in these cell populati ons. Specifically, the percentage of cells expressing IL-6 alpha receptor R NA 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 t o detect and quantify the number of individual cells that express growth fa ctor receptor mRNA, and may also be useful to measure changes in expression of other endogenous genes or genes introduced by transfection and gene the rapy vectors. (C) 2001 Elsevier Science B.V. All rights reserved.