Northern blotting; definition, technique, applications and disadvantages:
Blotting is a technique that allows the detection and quantification of specific RNA species from a particular cell type. This technique was developed by James Alvin and George Stark in 1975.
Generally, this technique involves isolation of the RNA molecules from source of interest and the separation of the isolated RNA by agarose or polyacrylamide gel electrophoresis. The separate RNA in the gel is then transferred to a solid matrix such as filter paper through the blotting process. The isolated RNA is electrophoresd through an agarose gel which separates the RNA species by size (such that the smaller RNA migrating faster). The isolated RNA is stained with ethylene bromide and is visualised using UV light. To probe for a specific messenger RNA, transfer the RNA from the agarose gel to a nylon membrane. This RNA is detected by hybridization using a chemically or radioactivity labelled RNA probe.
- RNase free water,
- Running buffer,
- Loading dye,
- Ethedium bromide,
- UV trans-illuminator,
- Nylon membrane,
- 20 x SSC (Na3citrate, NaCl)
- Saran wrap,
- Glass plate,
- Whatman paper,
- Glass as weight for blot,
- Sponge soaked in 20 x SSC.
- Preparing RNA sample for loading:
Add RNA to a sterile eppendorf tube. Add formamide- it is for lowering the annealing temperature and to prevent degradation by high temperature. Now add formaldehyde (which is the designation agent for RNA), buffer and loading dye.
- Loading and running of samples into the wells at 100 volts for 2 hours:
The RNA samples are most commonly separated on agarose gels which contain formaldehyde as a de-naturing agent. This is done for the RNA to limit secondary structure.
- Visualisation of RNA:
The gel is stained by ethedium bromide and viewed under UV light to observe the quality and quantity of RNA before blotting.
- Transfer to nylon membrane:
Set up the transfer. First place whatman filter paper, add transfer buffer and place filter paper. Remove the air bubbles and place gel and a nylon membrane over it. Now flood the membrane with transfer buffer. Again remove the air bubbles. Cover it with whatman filter paper, then with a plastic wrap and then stack paper towels above the gel. Allow transfer to go overnight.
- Disassemble the transfer system:
To disassemble the transfer system, remove the paper towel and filter papers. Fix the RNA to nylon membranes using UV cross-linker.
Pre-hybridization membrane in pre-hybridize buffer. Incubate dor 2 to 4 hours. Now discard the pre-hybridization buffer and add hybridization buffer.
- Addition of the probe.
Incubate the probe in a hybridization chamber, overnight and after incubation, discard the solution. Add wash solution and again incubate at 52 degree C for 30 minutes. Repeat this step 3 times and then remove the membrane.
Membrane is now ready to be exposed to film.
Following are the applications of northern blotting technique.
- It is a standard for direct study of the gene expression at the level of mRNA.
- Detection of mRNA transcript size.
- Study of RNA splicing- can detect alternatively spliced transcripts.
- Study of RNA half life.
- It is a time consuming process.
- RNA samples can be degraded by the RNase.
- Use of radioactive probes.
- Detection with multiple probes is a problem.
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