Quantitation of In Situ Hybridization Using Image Analysis of Radioactively Labeled RNA Probes

Quantitative analysis of messenger RNA (mRNA) from tissue homogenates, cell extracts, or fixed tissue sections is vital for studies involving gene regulation and expression. Quantitative analysis of mRNA allows the investigator to establish the transcription level of particular genes either in relative or absolute terms. Traditionally Northern blot analysis is a comparative technique that detects the amount of mRNA of the gene of interest normalized to the amount of a housekeeping gene (1 ,2 ). This is generally performed by densitometry of band intensities from an autoradiograph. The method has been employed for more than two decades, and its relative simplicity has made it the first port of call when examining RNA expression. It is particularly useful for examining mRNA transcription in tissue/cells exposed to various treatments. While Northern analysis will inform the investigator of the size and relative abundance of the mRNA of interest, it is a limited technique. Some of the limitations include: inefficient transfer of the RNA to the filter, its relative insensitivity for examining smaller quantities of mRNA and the saturable nature of autoradiography when film is used (3 ). The last limitation has been overcome by phos-phorimaging, which allows measurement of band intensity in a linear fashion. Therefore, Northern blotting is best used as a semiquantitative method of examining relatively abundant mRNA.