I) Isolation *
Isolation Of Eukaryotic RNA *
Ii) hybridisation *
Northern Blots *
S1-Mapping Of Oocyte RNA *
RNA Dot Blots *
Hybrid Selection *
III RNA Sequencing *
_________________________________
RNA analysis
A. Bannister
1. Grind tissues to a fine powder under liquid nitrogen using a pestle and mortar.
2. Homogenise the frozen tissue (or cultured cells) in 7 vols. of homogenisation buffer;
4M Guanidine thiocyanate,
10mM EDTA,
50mM Tris.HCl pH 7.5,
8% (v/v) 2-mercaptoethanol.
3. Precipitate the RNA by adding 7 vols. of 4M LiCl and incubate at 4 oC for 12-18 hours. RNA may be stored in this state for many days without detectable degradation.
4. Pellet RNA by centrifuging at 11,000g for 90 min. at 4 oC. Eg. Sorvall SS34 rotor, Corex tubes.
5. Resuspend pellet into 5ml 2M LiCl (or 5ml of 4M urea, 2M LiCl if pellet is 'heavy'). 5ml is the correct volume for an expected total RNA yield of 1-15mg.
6. Centrifuge as above for 60 min..
7. Resuspend pellet into 5ml of; 0.1% (w/v) SDS,
1mM EDTA,
10mM Tris.HCl pH 7.5,
and incubate on ice for 45 min..
8. Freeze sample in liquid nitrogen (or at -70 oC) and vortex whilst thawing.
9. Extract with an equal volume of phenol-chloroform (saturated in TNE) and then with an equal volume of 24:1 (v/v) chloroform: iso-amyl alcohol.
10. Precipitate RNA by adding 0.1 volume of 3M NaOAc pH 5.2 and 2 vols. of EtOH. (-70 oC for 1hr. or -20 oC O/N).
11. Pellet RNA by centrifuging as above for 30 min., wash in 70% (v/v) EtOH prior to resuspending in sterile distilled water. Store aliquoted under liquid nitrogen.
RNA prepared by this method is essentially free of contaminating DNA. The RNA has been used to construct a cDNA library from parotid RNA (a gland with very high endogenous levels of RNase). The same RNA has been sequenced with rev. transcr., used in Northerns to reveal discrete bands, and has been in vitro translated.
A. Bannister
Denaturation of RNA using glyoxal may require deionised glyoxal.
Deionisation Of Glyoxal.
1. Prepare 500ml of a 40% (w/v) glyoxal solution. Add 250g of a mixed bed ion-exchange resin and stir until the resin is exhausted (BDH add a dye to their 'Duolite' resin which changes colour when exhausted).
2. Filter the solution under vacuum and repeat until no colour change is observed (usually 3-5 resin changes). The pH should be between 5.5 and 6.0.
3. Freeze the glyoxal in 100 aliquots at -20 oC.
Denaturation Of RNA, Gel Electrophoresis and Northern Transfer to Nitrocellulose.
1. To the RNA (<20 g) in 5 l of water add;
|
Final concentration |
|
|
4 ul deionised glyoxal |
1M |
|
3 ul 80 mM NaPO4 buffer, ph 6.5 |
10 mM |
|
12 ul DMSO |
50% |
2. Heat at 50 oC for 1 hr.
3. Chill on ice and add 5 ul sample buffer (0.05% BPB, 50% sucrose, 10mM NaPO4 pH 6.5).
4. Prepare an agarose gel gel of appropriate percentage. Use 10mM NaPO4 buffer ph 6.5 to prepare the gel. Once set place the gel in an RNase free gel tank containing 10 mM NaPO4 pH 6.5 as running buffer. Load the samples - DNA size markers which should be radiolabelled and they will have been glyoxylated exactly as for RNA.
5. For a 20 cm long gel, electrophorese at 100 V for 3-4 hr. During electrophoresis recirculation of buffer is essential but only after samples have entered the gel. Also, the gel may need to be weighted down during the run to avoid it floating. To do this place two clean glass rods down each side.
6. Blotting of the gel after electrophoresis is essentially identical to Southern transfer of DNA except that there is no pre-treatment of the gel. The blotting solution is 20 X SSC and the nitrocellulose is pre-wetted in 4 X SSC. N.B. Treatment of the gel with alkali or Ethidium Bromide solution prior to transfer greatly reduces transfer.
7. Transfer at least 12 hr and then air dry and finally bake the filter at 80 oC for 2 hr.
8. Place the filter in 200 ml of 20 mM Tris-HCl pH 8.0 at 100 oC and allow to cool to room temp.. This removes residual glyoxal.
9. Detect RNA via hybridisation.
(T. Moss and R.F. de Winter)
10x hybridisation buffer:
|
Final Concentration |
||
|
NaCl |
4.7 g |
4 M NaCl |
|
1.0 M PIPES, pH6.4 |
8.0 ml |
400 mM PIPES |
|
0.5 M EDTA, pH near neutral |
0.4 ml |
10 mM EDTA |
|
_______ |
||
|
final volume |
20 ml |
Make up solution, then adjust pH to 6.4. and sterilise by filtration.
10x S1 Nuclease Buffer:
|
Final Concentration |
||
|
NaAc (.0H2O) |
166 mg |
300 mM NaAc |
|
ZnSO4.7H2O |
57 mg |
20 mM ZnSO4 |
|
NaCl |
1.16 g |
2 M NaCl |
|
Glacial acetic acid |
0.17 ml |
pH 4.5 |
|
______ |
||
|
final volume |
10 ml |
Sterilise by filtration.
1. RNA is dissolved in formamide at a concentration of 4 ul per oocyte (4.5-5 ug of RNA).
2. Per S1 reaction, 24 ul of RNA (27-30 ug) is mixed with 3 ul 10x hybridisation buffer and 3 ul of 5' end-labelled DNA probe (0.05-0.005 pmole), which is dissolved in water.
3. After denaturation in boiling water for approximately 1 min, samples are incubated at 55-57oC overnight.
4. 270 ul ice cold 1x S1 buffer containing 25 units of S1 (Pharmacia) is added, samples vortexed and kept on ice. After a short spin samples are then incubated at 45oC for 1 hr.
5. Extract once with phenol/chloroform, precipitate with 2.5 vol EtOH -20oC >1hr, spin 10,000xg 5 min, remove S/N and wash pellet with 80% ethanol. Dry and redissolve in 4 to 6ul 1x urea loading buffer.
6. Samples are run hot on a 25 cm x25 cm 8% polyacrylamide, 7 M urea gel at ~30 watts.
Reference:
Berk A.J. and Sharp P.A. (1977) Cell 12, 721-732. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease digested hybrids.
A. Bannister
1. Place up to 10 ug of RNA in 2 ul of water and add 2 ul of denaturation solution;
For 100 ul Denaturation Soln.,
|
20 ul |
0.1 M NaxPO4 pH 6.5, |
|
34 ul |
de-ionised glyoxal, |
|
46 ul |
water. |
2. Incubate at 50 oC for 1 hr.
3. Prepare suitable dilutions of the samples in 0.1 % SDS, each dilution to be 4 ul final volume.
4. Prepare nitrocellulose by soaking in water then in 20 X SSC for 5 min. at room temp..
5. Dry the filter under heat lamp.
6. Apply the sample in one 4 ul application.
7. Air dry the filter then bake at 80 oC for 2 hr..
8. Place the filter in 200 ml 20 mM Tris-HCl pH 8.0 at 100oC and allow to cool to room temp.. Then hybridise.
A. Bannister
1. Wet a piece of nitrocellulose in 2 X SSC and place in position on a manifold suction device
2. Boil 10 ug of the cloned DNA in 50 ul of 1 mM EDTA, 20 mM Tris(pH 7.6) for 10 min.
3. Add 50 ul of 1 M NaOH and incubate at 20 oC for 20 min..
4. Place on ice and add 400 ul of neutralisation buffer (1.4 M NaCl, 0.15 M sodium citrate, 0.25 M Tris.HCl pH 8.0, 0.25 M HCl).
5. Pipette the DNA sample into the manifold well under gentle suction (>5 min. total passage time).
6. Air dry the filter then bake at 80 oC for 2 hr..
7. Wet the filter by placing on 3MM paper saturated with water and cut the DNA dots out of the wet filter.
To remove loosely bound DNA before first use.
1. Place each filter in 1 ml of water and incubate for 1 min. in boiling water bath.
2. Chill on ice and remove water by aspiration.
3. Add 1 ml water, vortex, and remove the water.
4. Blot the filters dry on 3MM paper.
1. Place the filters in 100 ul of hybridisation buffer (50% deionised formamide, 0.9 M NaCl, 0.2% SDS, 1 mM EDTA, 20 mM Pipes pH 6.4).
2. Incubate at room temp. for 10 min. then remove buffer.
3. Add fresh 100 ul containing 250 ug of the total RNA of interest and incubate at 37 oC for 6 hr.
4. Wash with 100 ml washing buffer (50 % deionised formamide, 20 mM NaCl, 8 mM sodium citrate, 1mM EDTA, 0.5% SDS - BUFFER MUST BE MADE FRESH) for 15 min. at 37 oC.
5. Change the washing buffer and repeat step 4 for a total of five washes. Blot the filters dry on 3MM paper.
1. Add 1 ml water to each filter and vortex 5 sec.. Remove the water and repeat the wash.
2. Add 300 ul 1 mM EDTA to the filter and place in a boiling water bath for 1 min..
3. Snap freeze by plunging into dry ice and thaw on ice to remove filter.
4. To the solution add 10 ul of 1 mg/ml of phenolised calf liver tRNA and sodium acetate to 0.2 M. Extract with phenol: chloroform: iso-amyl alcohol (1:1:0.04).
5. Add 2.5 vols EtOH and place at -70 oC for 30 min. Centrifuge to recover the RNA and wash the pellet once with 70% EtOH and once with 100%.
6. Dry the RNA under vacuum and prepare for in vitro translation.
Removing residual RNA from filters before reuse.
1. To each filter add 1 ml of 2 X SSC, 0.1 M NaOH. Leave at room temp. for 30 min. and then remove liquid.
2. Add 1 ml of 2 X SSC to the filter, vortex and remove. Repeat this wash four more times
3. Wash once only as in step 2 using 1 ml water and then air dry the filters. Store filters in sealed plastic bags at 4 oC.
Immunoprecipitation Of In Vitro Translation Products
A. Bannister
1. In vitro translation products (in a translation reaction volume of ~10-50 ul) are diluted to 800 ul with immunobuffer (50 mM Tris.HCl pH 7.5, 0.15 M NaCl, 0.1% [w/v] NaN3, 1% [v/v] Triton-X-100, 1% [v/v] A-Protinin).
2. 200 ul of antibody is added to the reaction and incubated at room temp. for 4 hr.. The antibody should be diluted in immunobuffer if required and for quantitative results should be prior titrated with a constant amount of in vitro products.
3. 8 mg of protein A-sepharose in 100 ul of immunobuffer containing 0.1% [w/v] SDS is added to the reaction and the mixture roller mixed overnight at 4 oC. The amount of Protein A added should also be titrated to ensure that it is in excess. Allow at least 1 hr. for the sepharose to swell before adding to the immunoprecipitation.
4. Pellet by centrifugation (10,000g for 10 min. at room temp.) and resuspend the pellet in 100 ul of immunobuffer containing 0.1% [w/v] SDS.
5. Transfer the suspension onto a 1 ml cushion of 1 M sucrose, 0.1% [w/v] SDS, 1% [v/v] Triton-X-100 and centrifuge as above.
6. Wash the pellet three times with immunobuffer containing 0.1% [w/v] SDS and then resuspend in 50 ul of 50 mM Tris.HCl pH 6.8, 2% [w/v] SDS, 10% [v/v] b-mercaptoethanol.
7. Heat the suspension at 95 oC for 5 min. to dissociate antibody-antigen complexes. Prepare for SDS-PAGE.
III RNA Sequencing
A. Bannister
1. Mix 2 ug of poly (A) RNA with 50 ng end-labelled oligonucleotide such that the solution is 0.2 M NaCl and 5 ul final volume.
2. Incubate at 65 oC for 10 min., and allow to cool slowly to room temp..
3. Add 2.5 ul 10 X sequencing buffer ( 0.5 M Tris.HCl pH 7.5, 0.1 M MgCl2, 50 mM DTT, 500 ug/ml actinomycin D), 10 uCi [ a-32P]dATP and 30 units AMV reverse transcriptase to a final volume of 25 ul.
4. Incubate 37 oC for 1 min..
5. Aliquot 2 ul of this solution into a tube containing 18ul of 1 X sequencing buffer containing 500 uM of each of the four dNTPs and 150 uM of the relevant ddNTP.
6. Incubate at 42 oC for 1 hr. prior to EtOH precipitation of the samples. Resolve products by gel electrophoresis.