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In this article we will discuss about the principle, requirements and procedure for northern blotting and hybridisation.
By this method specific mRNA sequences in RNA preparations can be detected. Northern blots are carried out in order to detect as to which of the several RNA molecules on agarose gel hybridize to a particular probe. It also provides information regarding the size and amount of specific mRNA and any precursors.
They are useful to determine whether a cDNA clone used as a probe is full length or whether it is one of a family of related transcripts. By this process it is possible to identify as to whether a genomic clone has regions that are transcribed and also, if the RNA on the blot is made from different tissues, where these transcripts are made.
Northern blotting can be made by capillary action, electro transfer, centrifugation or vacuum transfer.
Principle:
By electrophoretic separation, total RNA is transferred to a filter and is hybridised to a complementary probe.
Requirements:
Reagents:
All solutions should be prepared using sterilised deionised water which has been treated with Di-Ethyl Pyrocarbonate (DEPC) to prevent RNAse activity:
1. NaOH IN
2. SSC 20x
3. Formamide prehybridisation Buffer:
SSC 2.0x / 0.1% W/V SDS.
SSC 0.2x / 0.1% W/V SDS at room temperature and at 42°C.
SSC 0.1x / 0.1% W/V SDS at 68°C.
4. Waterbath 60°C.
5. Nylon membrane.
6. Whatman 3 mm filter paper.
7. U.V. transparent plastic wrap.
8. U.V. transilluminator.
9. Hybridisation oven.
Procedure:
1. Rinse the gel with RNA in deionized water several times to remove formaldehyde.
2. Soak the gel in ten gel volumes of 0.05M NaOH for 30 minutes, rinse with deionised water and soak in 10 gel volumes of 10x SSC by placing it over a 3 mm Whatman filter paper.
3. Cut a nylon membrane to the same size as that of the gel, wet it in deionised water for 10-15 minutes and place it over the gel and then a layer of Whatman 3 mm filter paper previously soaked in 2x SSC taking care to see that no air bubbles are caught in between.
4. Place plastic sheets on the sides and place more Whatman 3 mm filter paper. Keep a weight of 400-500 g and incubate over-night.
5. Dismantle it the next day. Remove nylon membrane and gel. Mark the positions of the well of gel on the nylon membrane with pencil.
6. Rinse the membrane in 2xSSC. Keep it on Whatman 3 mm filter paper and allow it to dry.
7. This dry membrane is wrapped in UV transparent plastic wrap and place with RNA-side down on a UV cross linker of 254 nm wavelength and irradiate. Blot is ready for hybridisation.
8. Pre-hybridisation is the same as that of DNA gel blot.
9. Prepare probe labelled to a specific activity of >108 dpm/µg and with unicorporated nucleotides removed.
10. Wet the membrane with immobilized RNA with 6xSSC and place it in the hybridisation tube with RNA side up.
11. Add approximately one ml of formamide prehybridisation solution per 10 cm2 of membrane and keep the tube in hybridisation oven and incubate for three hours at 60°C with rotation.
12. In case of a double stranded probe, it is denatured by heating in a water bath or incubator for ten minutes at 100°C. Transfer it to ice.
13. Pipette out desired volume of probe (to give 10 ng/ml if specific activity is 109 dpm/p.g) into hybridisation tube.
14. Incubate again at 60°C overnight with rotation. Then pour out the hybridisation solution. Wash twice with SSC 2.0x / 0.1% W/V SDS for five minutes with rotation at room temperature-low stringency wash.
15. Wash twice with pre-warmed SSC 0.2x / 0.1% W/V SDS for fifteen minutes at 42°C (moderate stringency wash).
16. Wash twice with pre-warmed SSC 0.1x / 0.1% W/V SDS for fifteen minutes at 68°C (high stringency wash).
17. Rinse the membrane with 2xSSC at room temperature. Remove excess water. Cover in U.V. transparent sheet (plastic) and autoradiograph.
N.B: DEPC is carcinogenic and should be handled carefully. (DEPC reacts with ammonium ion to produce ethyl carbonate which is a potent carcinogen).