Biochemical Techniques and Instrumentation

5.2.2. Blotting Technique

There are eight steps in Northern blotting:

1. Isolation of mRNA: The total RNA is first extracted from a homogenized tissue sample. The mRNA is then isolated through the use of oligo (dT) cellulose chromatography to obtain only those RNAs with a poly(A) tail.
2. Agarose gel electrophoresis: The isolated RNA is separated on agarose gels containing formaldehyde as a denaturing agent based on their size. Formaldehyde ensures linear conformation of RNA. Gels are then stained with ethidium bromide (EtBr) and viewed under UV light to observe the quality and quantity of RNA before blotting. Polyacrylamide gel electrophoresis with urea is commonly used for fragmented or microRNAs. An RNA ladder is often run alongside the samples to observe the size of fragments.
3. Transfer: The RNA separated on the gel is then transferred to a nylon membrane through a capillary or vacuum blotting system. A nylon membrane with a positive charge is most effective since the negatively charged nucleic acids have a high affinity for them. The transfer saline sodium citrate (SSC) buffer used usually contains formamide or formaldehyde as it lowers the annealing temperature of the probe-RNA interaction and thus prevent RNA degradation at high temperatures.
4. Fixation: Once the RNA has been transferred to the membrane, it is immobilized through covalent linkage to the membrane by UV light or heat.
5. RNA probes: RNA probes are also called as riboprobes. They are composed of nucleic acids with a complementary sequence to all or part of the RNA of interest. Commonly cDNA is created with labelled primers for the RNA sequence of interest to act as the probe in the northern blot. Probes can withstand more rigorous washing steps.
6. Probe labeling: Probes are labelled either with radioactive isotopes (32P) or with chemiluminescence in which alkaline phosphatase (AP) or horseradish peroxidase (HRP) breakdown chemiluminescent substrates to produce a detectable emission of light. Chemiluminescent labelling occur in two ways, the probe attached to the enzyme or the probe attached with a ligand (biotin) for whihc the antibody (avidin) is attached to the enzyme. Same membrane can be probed up to five times without a significant loss of the target RNA.
7. Hybridization: After a probe has been labeled, it is hybridized to the RNA on the membrane. The membrane is washed to ensure that the probe has bound specifically.
8. Detection: The hybrid signals are then detected by X-ray film and can be quantified by densitometry. X-ray film can detect both the radioactive and chemiluminescent signals. Chemiluminescent signals are faster, more sensitive and reduce health hazards associated with radioactive labels.