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 32P 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).
KeywordsSodium Dodecyl Sulfate Hybridization Solution Guanidine Hydrochloride Rare Transcript Guanidine Thiocyanate