Quantification of mRNA Levels Using Northern Blotting

Quantification of mRNA levels can be performed using polymerase chain reaction (PCR)-based techniques (e.g., competitive reverse transcription PCR; RT-PCR), RNase protection, or Northern blotting. Northern blotting is less sensitive than the other techniques; however, the methodology is less complex and quantification, certainly compared with RT-PCR, is more straightforward. Aliquots of total RNA or mRNA are separated according to size in an agarose gel; the separated species are then transferred to a filter where the RNA transcripts are immobilized. The filter is then probed with a labeled single-stranded DNA probe specific for a certain mRNA species. The immobilized RNA species on the filter maintain an ability to anneal to single-stranded DNA to form stable heteroduplexes or hybrids. The resulting hybrids are detected using a technique related to the specific type of label attached to the DNA probe. There are now several approaches available for labeling DNA probes and detecting resulting hybrids. The use of ³² P as a label will be described in this chapter. There are other procedures for labeling of DNA that involve the use of other detectable compounds (e.g., fluorochromes, colorimetric, and antibody-based techniques).