Size Separation and Quantification of mRNA by Northern Analysis

Most acute changes in cell activity often involve changes in activity of existing metabolic pathways either as a consequence of cell signaling changing the phosphorylation of rate-limiting proteins, or allosteric control as a consequence of changes in the environment. Long-term changes in cell activity, however, may also relate to changes in expression of key proteins. This is particularly true in changes in gestation/development or in removal of cells from an in vivo to an extended in vitro culture system. Changes in protein levels may occur through mechanisms that are transcriptional (i.e., altered rates of transcription) or posttranscriptional (changes in mRNA turnover), translational (altered synthesis of protein from existing mRNA) or posttranslational (altered protein stability). Northern analysis is a standard procedure by which abundant levels of mRNA can be characterized by size and quantified so that effects of treatment on message level can be determined. Whereas a change in message level with treatment is an indication that regulation may be occurring at the level of mRNA it is not, however, sufficient alone to demonstrate whether this is transcriptional or posttranscriptional. Results from Northern analysis should always be normalized for loading using an appropriate housekeeping gene product (see Note 1 ) and should also be compared to the results of Western analysis for quantification of the corresponding protein wherever possible.