Utilization of Polymerase Chain Reaction for Clonal Analysis of Gene Expression

Abstract

The reverse transcription-polymerase chain reaction (RT-PCR) is a powerful tool when studying gene expression in a limited number of cells. RT-PCR was first described by Veres et al. (1) and numerous accounts have since followed. Classic studies of gene expression have utilized Northern blot analysis to monitor expression of transcripts in response to developmental and activational signals. Problems associated with using the Northern blot technique include the necessity for an abundant number of cells, the limited numbers of genes that can be analyzed on a single blot, and that, usually, only one gene can be monitored at a time. Finally, little information on which cells within the cell population being analyzed are expressing the gene(s) is provided by Northern blot analyses. For example, on a Northern blot it is virtually impossible to determine whether all cells contain a transcript for a specific gene(s) or whether all cells exclusively express the particular gene of interest. In situ hybridization has been used in conjunction with Northern blot analyses to determine which cells in a population are expressing a particular gene. However, this technique is limited by low sensitivity and by the fact that usually only one gene can be monitored at a time. RT-PCR alleviates these problems because specific transcripts can routinely be detected in RNA quickly isolated (2) from 5–10 cells (3-5). The principle of clonal analysis by RT-PCR is to use 50–100 cells that are clonally derived from a single progenitor cell. RNA is then isolated from individual clones, reverse-transcribed into cDNA, and amplified with transcript-specific primers. Two steps are involved in RT-PCR:

• 1. Isolation and reverse-transcription of the specific transcripts or total mRNA into cDNA.

• 2. Amplification of specific sequences of transcripts by PCR.

During the PCR step, primers used for amplification of specific sequences are designed in order to amplify sequences of predetermined size. In addition, it is also useful to design primers that span across introns to assure that contaminating DNA is not being amplified. There can be numerous technical difficulties associated with amplifying cDNA sequences from limited transcripts obtained from low cell numbers. We have used an internal nested primer that results in increased specific amplification with decreased background amplification to overcome this problem (4). In demonstrating the RT-PCR technique, we describe in this chapter the approach we have developed to molecularly phenotype colonies of bone marrow-derived macrophage obtained using hematopoietic growth factors (5).