northern blotting
Summary
Northern blotting detects a specific RNA (usually mRNA) in a sample. RNA is separated by gel electrophoresis, transferred to a membrane, and hybridized to a labeled complementary probe. Band presence, size, and intensity show whether a gene is expressed and how much.
Detail
Meaning: Northern blotting is a technique for detecting a particular RNA in a mixture. The steps are: (1) extract RNA from cells or tissue; (2) separate it by size on a gel, where shorter RNAs migrate farther toward the positive electrode because RNA is negatively charged; (3) transfer the RNA onto a nitrocellulose or nylon membrane; (4) add a labeled single-stranded DNA or RNA probe complementary to the target sequence, which hybridizes by base pairing; (5) detect the label (radioactive or fluorescent) as a band.
On the MCAT: Passages use it to compare gene expression between tissues, between treated and untreated cells, or across time points. A darker band means more of that transcript; no band means the transcript is absent or below detection. Band position gives transcript size, which can hint at splice variants. Questions often ask which blot fits a goal, or what a probe must be complementary to.
Common confusion: Students mix up the blots by what is detected. Southern blot detects DNA, northern detects RNA, and western detects protein (using antibodies, not a nucleic acid probe). The names are a joke on Southern's name, not on compass directions. Also, the probe is complementary to the target, not identical to it. Northern blotting shows transcript level, not protein level, so a band does not prove the protein is made.
Sources
- Molecular Biology of the Cell
- Lehninger Principles of Biochemistry
- Molecular Biology of the Gene
Written for the MCAT and checked before it was published. For study reference only. Spotted an error? Let us know.