RNase Protection Assay (Beverly Faulkner-Jones)

RNase Protection Assay - Protocol

This method can be used to detect and quantitate mRNA, to map mRNA termini and to determine the position of introns within the corresponding gene.

High specific activity 32P-UTP-labelled single-stranded cRNA is generated and purified, then hybridised in excess in solution to the target mRNA : this ensures that all / most of the target mRNA is hybridised to the cRNA probe. A combination of RNaseA and RNaseT1 are used to digest unhybridised, single-stranded RNA and the digestion products are analysed by denaturing polyacrylamide gel electrophoresis and autoradiography / PhosphorImaging. The undigested cRNA probe will contain a stretch of plasmid sequence and is therefore larger than the mRNA which it "protects" from the action of RNase by duplex formation. It will migrate slower than the protected fragments and is used for their identification

ProtocolReagents - make up all reagents in sterile MilliQ waterReagents for in vitro transcription - see section on In vitro transcription Target RNA Formamide deionise with BioRad mixed bed resin for 1 hour at room temperature. Check the pH - it should be neutral or less than 7.4. If it is greater, then discard that batch of formamide. Store deionised formamide in aliquots at -20oC1 10 x RNase protection buffer 400 mM PIPES (disodium piperazine-N,N'-bis[2-ethansulphonic acid]),10 mM EDTA and 4 M NaCl, pH 6.4. Add PIPES as the free acid (RMM 302, Boehringer 239 496) and adjust pH to 6.4 : the PIPES will not go into solution at lower pH. Autoclave and store in aliquots at -20oC tRNA at 10 mg / ml in sterile water. Dissolve the tRNA in sterile water, extract twice against phenol:chloroform, ethanol precipitate, wash with 70% ethanol and then resuspend in sterile water. Store in aliquots at -20oC RNaseA 2 mg / ml of Sigma R5250 X-A in 10 mM Tris pH 7.5, 15 mM NaCl. Store small aliquots at -20oC RNase T1 100 mg / ml Sigma R1003 in 50% glycerol and 10 mM NaPO4 pH 6.5. Store small aliquots at -20oC. 10 x RNase digestion buffer 100 mM Tris pH 7.5, 50 mM EDTA, 3M NaCl. Autoclave to sterilise and store at room temperature 20% SDS in sterile water Freshly made proteinase K at 10 mg / ml in water2 Water-saturated acid phenol Chloroform Isopropanol Formamide loading buffer 90% deionised formamide, 1 x TBE and bromophenol blue. (Make 5 ml viscous enough to sink to the bottom of the well without diffusing upwards by adjusting the amount of saturated BPB solution added) Reagents and equipment for denaturing polyacrylamide gel electrophoresis of RNAMethods - read the notes at the end of the protocol BEFORE setting up the assayIn advance1 Prepare template DNA for generation of the cRNA probe 2 Prepare all RNA samples, both the test samples and the positive and negative control 'cold' sense cRNAsDay 13 Make high specific activity a32P-UTP-labelled cRNA. Use a 5 - 6% denaturing polyacrylamide preparative gel. It will take ~ 5 - 8 hours to transcribe, purify and elute the probe 4 Assemble the hybridisation reactions Make all the reactions up from a single 'master mix' to reduce variations between samples. Depending on the mount of RNA to be analysed3, and the amount of contaminating DNA in the sample4, use reaction volumes between 30 - and 60 ml. Use the PIPES buffer at a final concentration of 1 x and the formamide at 80%5 5 All reactions should contain the same amount of RNA in total - make up the difference between tubes with tRNA eg 20 or 50 mg per reaction6 6 Include the following controls :- Negative control : tRNA only. This will indicate if the RNAses are not working, if there is still template DNA in the probe preparation and whether the probe self-hybridises significantly Positive control : essential with an uncharacterised probe. Use synthetic sense cRNA7 or a known source of the 'natural' RNA. A plasmid will do if there is nothing else Positive control : a house-keeping gene mRNA to check the integrity of the mRNA eg GAP-DH. This can be performed in the same reaction - see the Notes section at the end 7 Make sure the RNA is completely dissolved8 - heat at 37oC if necessary - before adding the probe 8 Add 1 ml of the cRNA probe which must be in excess for accurate quantitation. The amount has to be determined empirically but as a rough rule of thumb, using a 200 - 400 base probe and a yellow b-counter, for a low abundance mRNA, 100 - 200 cps / ml is sufficient and for a high abundance mRNA 500 - 600 cps / ml is need 9 Vortex tube and then heat denature the reactions at 80oC for 2-5 minutes and transfer to a pre-warmed rack at 45oC9. Incubate at 45oC10 and allow to hybridise for at least 8 hours11Day 211 Take the tubes out of the incubator and chill on ice Steps 11 - 23 will take most of the rest of the day 12 Immediately add 350 ml of ice cold RNase digestion solution, mix with the hybridisation reaction by vortexing and incubate for 30 minutes. A good starting point is 1/5x RNase ie. 40 ml of RNaseA solution and 40 ml of RNaseT1 solution in 10 ml of 1x RNase digestion buffer (ie 8 mg / ml RNaseA and 0.4 mg / ml RNaseT1), at 37oC12 13 Add 20 ml of 10% SDS and 10 ml of 10 mg / ml proteinase K solution and incubate at 37oC for a further 30 minutes13 14 Extract once with an equal volume of acid-phenol14 15 Remove the aqueous phase, add 20 mg of tRNA and precipitate with 600 ml of isopropanol 16 Mix the tube contents by vortexing and precipitate the digestion products on ice for 10 minutes 17 Recover the digestion products by centrifugation at room temperature for 20 minutes 18 Identify the pellet then aspirate all the isopropanol - do not lose the pellet 19 Wash the pellet extensively with 70% ethanol, re vortex, re centrifuge and aspirate as much ethanol as possible 20 Dry the pellet until all traces of ethanol are gone, and resuspend in 5 - 6 ml of formamide loading buffer15 21 Once the pellet is fully resuspended in loading buffer, denature the digestion products at 80oC for 2 minutes16 22 Chill the sample on ice 23 Analyse the digestion products by denaturing polyacrylamide gel electrophoresis and either autoradiography or PhosphorImaging. - Make the analytical gel with ~>2% more polyacrylamide than the preparative gel used for the cRNA probe. - Always run an aliquot of undigested probe - this will run behind the protected fragments and be an indication if the probe is hybridising to mRNA target or contaminating DNA in the test reactions - Allow a lane between the undigested probe sample and the test sample lanes if possible (eg use the tRNA negative control sample) - Calibrate the amount of undigested probe to load onto the gel. Use the hand-held b-counter to gauge the amount of radioactivity in the positive control and test samples. Load one aliquot that is equivalent to ~half the counts in the positive control sample, and one aliquot that is equivalent to ~half the counts in the test samples. Similar exposure times can then be used for undigested probe + positive control, and undigested probe and test sample(s) - The tRNA plus probe lane should be empty. High abundance mRNA will be visible within the hour. Low abundance (0.1 pg) mRNA will need 5 - 7 days with a PhosphorImager - Kodak or Fuji film give good, long exposure autoradiographs