The use of the PCR to quantitate gene expression.

Several techniques are currently available to measure changes in gene expression. These include the Northern blot, the RNase protection assay, in situ hybridization, and the reverse transcriptase-polymerase chain reaction (RT-PCR). For many purposes, the Northern blot or the more sensitive RNase protection assay is sufficient to detect quantitative differences between samples. However, if the sample quantity is low or the target message is rare such that these techniques are no longer practical, the more sensitive quantitative RT-PCR can be used. In cases where comparisons have been possible, results from the RT-PCR assay are quite comparable to results from Northern blot analysis, (1) slot-blot analysis, (z) and in situ hybridization. (3) In the RT-PCR method, RNA is initially reverse-transcribed to cDNA and the desired target cDNA species is amplified using specific primers. Less than 10 copies of target RNA are required for this procedure, and it has been successful when the RNA is isolated from a single cell. (4) Because of this high sensitivity, the RT-PCR is being increasingly used to quantitate small but physiologically relevant changes in gene expression that would otherwise be undetectable. For example, in a recent study involving analysis of IL-4 gene expression after immunization with antigen, studies with blocking anti-IL-4 antibodies had demonstrated previously the importance of elevated IL-4 in the response, but neither Northern blot analysis nor in situ hybridization could detect corresponding elevations in IL-4 gene expression. A quantitative RT-PCR was developed to analyze IL-4 cytokine gene expression. The quantitative RT-PCR showed that there was a 150x increase in IL-4 message in spleens from immunized mice. (s) Later studies using an ELISPOT assay, which measures protein secretion by individual cells, confirmed that the marked increase in IL-4 message was correlated with marked increases in IL-4 secretion. ( In contrast, protein assays usually require in vitro cell culture, in some cases with mitogens, often resulting in the production of cytokines not originally produced by the cells in vivo.