Considerations when measuring gene expression
Assumptions underlying mRNA analysis
One of the major assumptions underlying analysis of gene expression is that all cells within a given culture express similar levels of the mRNA being measured in response to a biomaterial. The possibility exists, particularly if phenotypic heterogeneity exists within a culture, such as occurs in rat calvarial cultures (Bellows et al., 1986), that the message levels may not correspond to all cells within the population. The level of complexity increases yet further if trying to measure gene expression in vivo, where multiple cell populations are in contact with the biomaterial. It then becomes extremely problematic to assign any mRNA to any particular cell population, certainly without combining gene expression analysis with methods such as immunohistochemistry (protein) and in situ hybridization (mRNA) for further analysis.
The next major assumption often made by researchers is that if a given mRNA transcript is higher in cells cultured on a biomaterial versus control cultures, then the transcript must be important or required in cell response, and further, the effects of the biomaterial cause this transcript to increase directly. Moreover, it is not always correct to conclude that mRNA levels always have to change for a protein level to change as well. It is now well established that mRNAs have different half-lives, and cells can also store mRNA without ever translating the protein (Gilbert, 2000). Because of these regulatory steps, the measured increase in mRNA is not necessarily reflected at the protein level. Measurement of gene expression represents a temporal snapshot only of what is occurring in any given cell culture or tissue, and if expression of the gene is cyclical in nature, this will not be detected unless multiple time points are analyzed. Typically, researchers in the biomaterial field will measure differentiation markers, which as a rule, are known to increase at the mRNA level as the cells adopt their differentiated function. However, this represents only one measure of cell activity and the gene level, and several other steps occur prior to the mRNA resulting in the presence of a functional protein.