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
Reagents - make up all reagents in sterile MilliQ water Reagents 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 RNA
Methods - read the notes at the end of the protocol
BEFORE setting up the assay
In advance 1 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 cRNAs
Day 1 3 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 hours11
Day 2 11 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
Notes 1 The standard cRNA probe preparation is 11 ml volume containing 40 mM TrisHCl (pH 8.0), 6 mM MgCl2, 2 mM spermidine HCl, 10 mM NaCl, 10 mM DTT, 100 mg/ml BSA, 20 nM linearised phagemid template DNA, 250 mM ATP, 250 mM CTP, 250 mM GTP, 1 unit ml of RNasin, 500 - 1000 units / ml of T7 RNA polymerase, 5 ml of a32P-UTP (3000 Ci / mmol, 10 mCi/ ml) at a final concentration of 1.5 mM and unlabelled UTP at a final concentration of 10 mM 2 Hotter probes do not necessarily make more sensitive RPAs, and due to the greater radiolysis of a higher specific activity probe, they may be less sensitive, less aesthetic or both. A good compromise for specific activity of cRNA probes is 11.5 mM UTP - 1.5 mM hot and 10 mM cold - reasonable proportion of full length transcripts and a hot probe. The standard protocol above generates a theoretical maximum of 167 ng of cRNA with an SA. of 0.72x109 dpm/mg, which is 14% of the theoretical maximum SA. of 5.1x109 dpm/mg. 3 DNaseI treatment of transcription reactions prior to gel purification to remove the DNA template : template contamination will be a problem if you don't. Especially noticeable on longer exposures ® unaesthetic / uninterpretable results 4 Use the probes as soon as possible after gel purification and definitely within 24 hours. All the radiolysed fragments are complementary to the target and will protect partial fragments. The resulting RPA will be less aesthetic and less sensitive 5 Poly A+ RNA gives cleaner results than total RNA. Although you can use a large amount of RNA in a single reaction, it generates more sub bands and a less aesthetic RPA. Any mistakes during the course of the assay will be compounded and be even more apparent than usual! 6 I have used both 'dried down' and precipitated RNA - I don't really think that there is a difference between the two methods as long as the RNA is fully resuspended before the experiment starts. If drying down RNA, don't completely desiccate it - it will be very difficult to get into solution. If the RNA has been precipitated, wash out the salt with 70% ethanol before resuspension 7 To determine the amount of probe needed to produce acceptable results, do a series of reactions with varying ratios of probe to (synthetic) target mRNA to find the amount of probe giving the best signal : noise ratio. 8 Determine the amount of target mRNA needed to produce acceptable results by doing a series with the appropriate amount of probe. 9 If using a house-keeping gene as a positive control, it can be done in the same reaction tube as the test probe as long as the protected fragment of the house-keeping gene is smaller than the gene of interest. Check that the two probes do not hybridise together 10 For a new probe and / or new target mRNA, the RNase conditions have to be determined empirically. The standard starting point for a fully homologous target is overnight hybridisation at 45oC with digestion at 1/5x RNase for 30 minutes at 37oC. It is easier to vary one parameter : I usually vary the RNase conditions before altering the temperature. A partly homologous target will need much less stringent conditions to allow full-length protection by the cRNA probe. Try 1/20x, 1/10x RNase at 37oC, 22oC, 16oC or even 4oC for 30 minutes or less and compare with the synthetic sense = fully homologous target mRNA. To demonstrate differences between two or more distinct although highly homologous targets, you will need very stringent RNase digestion conditions eg. 1x or 2x RNase for up to 60 minutes etc. It is easier to demonstrate differences between target mRNAs if there are two or three consecutive nucleotide differences rather than scattered base changes 11 Found that xylene cyanol (particularly at the gel purification stage) interferes with the electrophoresis of the cRNAs as they often run at the same level. Bands ugly and diffuse. 12 What can go wrong - everything! No cRNA - see section 'In vitro transcription' RNA degraded - a low molecular weight smear on gel with no large molecular weight bands. Usually operator error - not careful enough or poor reagents Multiple background bands - increase hybridisation temperature, decrease probe concentration, increase RNase digestion stringency, use PolyA+ RNA, use a different subclone for cRNA synthesis No protected bands - nothing there anyway, lost the pellet, didn't resuspend the final pellet in formamide loading buffer well enough, RNA degraded Ugly gel - acrylamide / urea degrading samples due to decomposition or they are contaminated, samples not dry before resuspending, salt left in pellets, gel run too hot >50 mA, urea left in wells, poor loading technique, gel knocked after loading and before running, wrong buffer (TAE instead of TBE), comb and spacers do not match, acrylamide in wells etc Streaking - poor resuspension in loading buffer, too large a volume used, crap in wells, wells allowed to dry out before loading etc
Reference #116 B. E. Faulkner-Jones, D. S. Cram, J. Kun and L. C. Harrison (1993) Localisation and quantitation of expression of two glutamate decarboxylase genes in pancreatic b-cells and other peripheral tissues of mouse and rat Endocrinol 133:2962 - 2972
Reference #6 D. A. Melton, P. A. Krieg, M. R. Rebagliati, T. Maniatis, K. Zinn and M. R. Green (1984) Efficient in vitro synthesis of biologically active RNA and RNA hybridisation probes from plasmids containing a bacteriophage SP6 promoter Nucleic Acids Res 12:7035-7056
Reference #7 P. A. Krieg and D. A. Melton (1987) In vitro RNA synthesis with SP6 RNA polymerase Methods Enzymol 155:397-415
Reference #489 D. E. Titus (1991) Promega Protocols and Applications Guide, Second Edition.
Reference #12 K. Zinn, D. DiMaio and T. Maniatis (1983) Identification of two distinct regulatory regions adjacent to the human b-interferon gene Cell 34:865-879 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.
BFJ - Thesis, R. Harvey - notes from Boston including original protocol from Zinn, C. Martens, DNAX 1 All reagents are made up in sterile MilliQ water unless otherwise stated 1 The solution turns yellow but this does not seem to interfere with its performance 1 Use saturated formaldehyde solution - Analar or equivalent. The concentration is ~37% in water and the pH should be <4.0. If the pH is >4.0 or if there is a lot of yellow 'sludge' on the bottom of the container, get fresh stocks 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 1 Make up saturated BPB solution in sterile MilliQ water 1 This is ~7.4% formaldehyde or ~2.4M. This can be reduced to ~4% / 1.2M unless the RNA of interest is particularly large 1 The gel will be ~60oC after addition of ~50 ml of reagents at room temperature 1 A paper tissue held over the mouth of the flask will catch bubbles / particle of partially dissolved agarose 1 Setting of the gel can be accelerated by placing the former in a cold room 1 This helps to free the teeth of the gel comb and prevents them tearing the gel when the comb is removed. This should always be done for low percentage and low melting point agarose gels 1 There are four common gel loading buffers which are all 6 x concentrates : I = 0.25% BPB, 0.25% XC in 40% w/v sucrose; II = 0.25% BPB, 0.25% XC in 15% w/v Ficoll 400; III = 0.25% BPB, 0.25% XC in 30% v/v glycerol and IV = 0.25% BPB in 40% w/v sucrose 1 Using too large a sample volume in the slot can result in contamination of adjacent lanes. 1 Marker RNAs may be used 1 If the RNA sample contains any alcohol, the RNA will 'creep' out of the well after loading - heat to 70oC for 10 minutes in an open tube before adding the loading buffer 1 The gel may be stained in ethidium bromide solution after electrophoresis is complete. Immerse in 0.5 mg / ml ethidium solution for ~30 - 45 minutes at room temperature 1 Formaldehyde should be difficult to smell in an adequately washed gel 1 Acrylamide is a potent, skin absorbed neurotoxin 1 Acrylamide and bis-acrylamide are slowly deaminated to acrylic acid ; the reaction is catalysed by light and alkali. Check the pH of the solution (neutral) and keep it dark and cool. Re-make solutions every few months 1 Cheaper grades of acrylamide often contain contaminants so always use sequencing grade reagents 1 Discard the solution when a precipitate forms 1 Use sequencing grade reagents. 1 Will keep for a few weeks at 4oC 1 Keep at 4oC 1 Deionise the formamide with a mixed bed ion-exchange resin (AG 501-X8 Resin, BioRad, Hercules, CA). If the pH of the formamide after deionisation is >7.4, discard it 1 Thin gels (0.3 mm - 0.5 mm) give better resolution, do not heat up as much and are easier to fix and dry than thick (1 mm) gels. However, they are more fragile, more difficult to cast and don't allow such large amounts of sample to be loaded 1 Conventional well-forming combs can be used but, especially at low acrylamide concentrations, there are frequently problems with tearing and deformation of wells. They give good results when analysing RNase protection assays or purifying probes or nucleic acids 1 Alternatively, sharks teeth combs can be used. These give a flatter, more uniform loading-surface than the well-forming combs and reduce the risk of tearing/damage to the gel. They give good results when analysing sequencing reactions : the close proximity of the lanes allows easier reading of the sequence, but they are prone to allowing leakage of samples between wells 1 The thinner and longer the gel, the more important it is to thoroughly clean and siliconise the plates. Dirty plates will not allow even casting of the gel (air bubbles) and the gel is liable to tear when the plates are separated after electrophoresis 1 If pouring a low-percentage acrylamide gel, casting is easier if the back-plate is also siliconised 1 If a well-forming comb is to be used, do not siliconise the very top of the back-plate : the teeth of the well will collapse if the glass surface is too slippery 1 Plates washed with distilled water after previous electrophoresis just need a methanol wash and re-siliconising 1 Siliconising agents are toxic : use in a fume hood 1 This reduces the likelihood of injecting air bubbles trapped within the syringe 1 Only necessary if the full width of the gel is to be used or when desperate! 1 Gels cast the day before tend to produce smeary RNA bands - ? urea / acrylamide decomposition ? 1 When preparing low-percentage gels with well-forming combs, the wells tend to collapse. Accelerating polymerisation helps - use warmed acrylamide solutions BUT there is no leeway to make mistakes when pouring : the gel sets fast! 1 High percentage gels will polymerise well with half the amount of AP and TEMED and give more time to cast them and make mistakes 1 Allow all unused acrylamide to polymerise - essentially non-toxic - before disposal 1 Not all makes of vertical gel apparatus needs this : many have an integral metal plate positioned against the back (notched) glass plate. Also, it is usually unnecessary for small 15 x 15 cm gels and may not be a problem with a large RNase protection assay gel which will only be run ~15 cm (BPB dye front) 1 There are a variety of methods available. I use tips that are flattened front to back and slip between the two plates if a 0.4 mm spacer is used. A single tip can be used for each gel by washing it out in the anode chamber with 1 x TBE between loadings 1 Wet evenly with distilled water and blot off the excess with paper towels 1 Handle with gloves - EtBr is carcinogenic 1 This is the method to use when radiolabelled-probes are to be recovered from gels. If 32P is used as the radio-label, the gel does not have to be fixed and dried prior to autoradiography, but the resolution is poorer 1 Fixing RNase protection assay gels in methanol / acetic acid reduces the sensitivity of the assay as some of the sample is lost into the fixative 1 Wet evenly with distilled water and blot off the excess with paper towels 1 Handle with gloves - EtBr is carcinogenic 1 Radiolysis of 32P-labelled cRNA is rapid : keep for RNase protection assays for no more than 1 day after purification 1 Purified cRNA for northern hybridisations will keep a few days at -70oC 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 1 Make up saturated BPB solution in sterile MilliQ water 1 An overnight immersion in 0.1% formaldehyde may be required - depends what has been done with it! 1 Formamide decomposes to ammonia - not good for RNA integrity! 1 Anecdotally, stored Proteinase K solutions adversely affect the assay 1 I have used up to 60 mg of total RNA and 200 mg of poly A+ RNA in a single 60 ml reaction 1 DNA contaminated RNA preparations are very difficult to get into solution, and will often precipitate during the hybridisation and processing steps - these will not make an aesthetic analytical gel 1 I have had to use up to 90% for very CG rich probes (eg hIGAD 65N = 80% C+G) 1 This standardises the substrate concentration for the subsequent RNase digestion step 1 The sense cRNA from the same template stock as the probe will be perfectly homologous to the antisense cRNA probe. This shows what pattern is produced between the probe and a perfectly homologous target using the chosen RNase digestion conditions. Natural RNAs encoding the same gene may have up to 3% difference at the nucleotide level between different strains 1 DNA contaminated RNA preparations are very difficult to get into solution, and will often precipitate during the hybridisation and processing steps - these will not make an aesthetic analytical gel 1 After heat denaturing the reaction prior to hybridisation, do not let the temperature fall below 45oC 1 Some probes will give better results at 50oC - trial and error 1 Increase the minimum time to 10 hours if the formamide concentration is increased to 90% 1 The accurate timing of this stage is important - limit the number of samples to ensure that they are all digested for a similar length of time 1 The timing of this stage is not as crucial as for step 12, but RNaseA will still be partially active until the sample has been phenol-extracted 1 Any residual DNA will tend to partition into the organic phase when phenol is < pH 8.0. Chloroform can be added if the interface between the two phases is not very sharp 1 When loading gel, try to keep the sample as small a volume as possible (< 7 ml) - the results from the analytical gel will look better 1 Do not use un deionised / old formamide in the loading buffer. Formamide decomposes to ammonia and urea and will degrade the RNA. Do not heat denature for too long in the loading buffer - the formamide will start to decompose
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