Clonal analysis of gene expression by PCR

Abstract

A method for determining the heterogeneity of gene expression from isolated macrophage colonies is described. Clones derived using specific growth factors were isolated by soft agar cloning and used as sources of mRNA for analysis. Gene expression was monitored by the reverse transcription polymerase chain reaction method to permit a number of specific genes to be simultaneously amplified. The technique described here involves the use of internal primers in addition to the usual two external primers typically used in gene amplification. Such internal primers greatly increased the accuracy and precision of amplification by adding an extra constraint such that the gene of interest was reliably amplified while decreasing background noise. This method permits gene expression to be monitored in cell colonies, thereby allowing the extent of phenotypic heterogeneity in developing macrophages (or other cell types) to be determined. This technique may also be useful in gene expression studies involving other cell types under the influence of specific growth factors.