One-step RT-PCR for screening microdissected tissue samples.

The technology of laser-capture microdissection (LCM) enables the easy extraction of single cells or defined groups of cells from a tissue section (1,2). This technique uses a laser pulse to activate a plastic polymer, which binds to and captures cells from a frozen or paraffin-embedded tissue section. Captured cells present on this solid support can then be used for nucleic acid and/or protein analysis. This technology is becoming increasingly important for the characterization of molecular profiles of cell subpopulations within a heterogeneous tissue (4). Despite the enormous power of LCM technology, a constant challenge for investigators has been the consistent manipulation of small quantities of material. Most protocols require an amplification step, preceded by multistep procedures to isolate and purify the molecular components (3). For example, the steps involved in gene expression studies using RT-PCR include microdissection, denaturation of RNA using guanidinium isothiocyanatebased reagents, phenol-chloroform treatment to remove proteins, precipitation of nucleic acids, washing, resuspension of RNA, re-extraction of RNA when required for increased purity, reverse transcription to produce cDNA, and PCR using gene-specific primers. Although this standard protocol is effective, RNA can potentially be lost at each step. In this report, we describe a one-step protocol for screening gene expression in microdissected cells using RT-PCR. Significantly, this procedure does not require an initial isolation of RNA. Rather, the reverse transcription and subsequent PCR amplification of specific genes are conducted directly from the microdissected cells. This procedure is a rapid, specific, and cost-effective screening technique. When we started this project, we hypothesized that, because of the small Benchmarks