In Situ Hybridization
Interpretation of ISH results
Changes in area vs. density of autoradiographic in situ hybridization signal
In autoradiographic ISH, hybridization is measured as the optical density of film exposed to tissue hybridized with a radioactive probe. Both area and density (i.e. darkness of the exposed film) of the hybridization signal are presumed to reflect mRNA levels in the tissue.
Autoradiographic ISH assumes that there is a threshold of detection for measuring a hybridization signal above background. (Typically, we chose 2 standard deviations above the average background optical density of the film and nonhybridizated tissue as an objective threshold.). Thus, a tissue must express a certain minimum amount of mRNA in order for a hybridization signal to be visible on the film. Furthermore, if above the detection threshold, the signal is assumed to be roughly linear with increasing amounts of mRNA above threshold.
Given these assumptions, we interpret the variables as follows: "Area" represents the tissue area (i.e. number of cells) that express mRNA above the threshold of detection, within the specificed region. “Density” represents the amount of mRNA per unit area (i.e. mRNA per cell) within the specificed region. The total amount of RNA expressed within the specified region is the product of the two values (area X density).
Changes in either area or density represent changes in mRNA levels. Differential results for area and density are a consequence of ISH methodology. An increase in signal area after treatment would imply that some cells in the specified area express the mRNA below the threshold for detection in the control condition, and that the treatment increased mRNA levels in those cells above the detection threshold. A concomittant increase in signal density would imply a higher level of mRNA expression throughout the region, including those cells that were already above threshold in the control condition. If the signal area is increased but signal density is not increased, this would imply that the cells which expressed detectable levels of mRNA in the control condition did not increase their expression after treatment. The failure to increase mRNA levels in cells that already express the gene could be due either to insensitivity to the treatment or to a “ceiling” of maximal possible expression.
Note that the ceiling or maximal level of expression may vary across regions, even for the same gene product. For example, the MR has a lower and sparser expression of 5HTT and Pet-1 mRNA than the DR under baseline conditions. Thus the dynamic range of MR cells may be lower than cells in the DR, and an increase in expression within the MR may be detected as an increase in area rather than in density. The physiological bases for the heterogenity in baseline and stimulated levels of gene expression within the raphe nuclei is a profound topic, to which this paper makes a small contribution with respect to estrogen effects.