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This methods allows the rapid analysis of numerous small samples for the sequence of interest and is less time consuming than the gel electrophoresis methods. 'Dots' or 'slots' of RNA are made onto a filter using a manifold and the filter is then hybridised with a labelled probe. Partially degraded RNA can also be used with good semi-quantitative results. However, high false positive rates need careful use of appropriate controls
Protocol
Reagents
20 x SSC Standard saline citrate : 3 M NaCl, 300 mM tri-sodium citrate, pH 7.0. Add 175.3 g of NaCl and 88.2g of tri-sodium citrate to 800 ml of water. Adjust the pH to 7.0 with 1M HCl and then add water to 1 litre. Autoclave to sterilise Nitrocellulose / reinforced nitrocellulose or nylon membrane RNA loading buffer 1 x MOPS buffer (ie, 20 mM MOPS, 8 mM sodium acetate and 1 mM EDTA, pH 7.0), 7% (w/v) formaldehyde pH 4.0, 5% (v/v) sterile glycerol, 50% (v/v) deionised1 formamide and 0.025% (v/v) of saturated aqueous bromophenol blue solution 2
Equipment
BioDot or BioSlot apparatus (BioRad) Vacuum source
Methods
In advance
1 Prepare RNA samples for analysis. Have up to 20 mg in 10 ml of sterile water or 2 mm DTT with RNasin 2 Clean the dot / slot blot manifold thoroughly. Immerse in dilute detergent, rinse in running tap water and then rinse in distilled water3 3 Cut a piece of membrane to fit the dot / slot blot apparatus and pre-wet in sterile water
Method
4 Assemble the apparatus as per the manufacturers instructions - be particularly careful that no leakage is occurring between wells before loading the RNA samples - use BPB in sterile water as a marker dye 5 Rinse the wells twice with 10 x SSC and suck through the manifold under vacuum. Turn off the vacuum and add a further 50 ml of 10 x SSC to each well 6 Add two volumes of RNA loading buffer to each sample and heat denature at 70oC for 10 minutes. Chill on ice before loading 7 Load the RNA samples into the wells already containing 10 x SSC and aspirate through under vacuum 8 Rinse the wells twice with 200 ml of 10 x SSC and suck through the manifold under vacuum 9 Rinse the resulting dot blot in distilled water and briefly air-dry 10 For nitrocellulose filters - bake under vacuum for 2 hours at 80oC For nylon filters - UV cross-link the RNA in place and then bake for 15 minutes under vacuum at 80oC 11 Store the resulting dot blot at room temperature until required for hybridisation.
Notes
1 Known standards can be dotted on to the membrane to calibrate the system
2 If a manifold is not available, samples can be dotted on by hand :- care must be taken to keep the spots of uniform size, especially if semi-quantitation is required
References
Reference #143 Tan Lab Library 07-94> Sambrook J, Fritsch EF, Maniatis T. 1989 Molecular Cloning, A Laboratory Manual, Second Edition. Cold Spring Harbour Laboratory Press. Reference #151 Tan Lab Library 07-94> Davis LG, Dibner MD, Battey JF. 1986 Basic Methods in Molecular Biology. Elsevier. New York
This page is maintained by Beverly Faulkner-Jones (b.jones@anatomy.unimelb.edu.au) using HTML Author. Last modified on 10/25/95. 1 Formamide was routinely deionised using a mixed bed ion-exchange resin (AG 501-X8 Resin, BioRad, Hercules, CA). If the pH of the formamide after deionisation was>7.4, it was discarded 2 Make up saturated BPB solution in sterile MilliQ water 3 An overnight immersion in 0.1% formaldehyde may be required - depends what has been done with it!