UNIT 10: NORTHERN BLOTTING
Northern blotting is in principle the same as Southern blotting except that RNA instead of DNA is isolated, size fractionated on an agarose gel, transferred and linked to a membrane, and then probed. The specifics of Northern blotting however, differ substantially from Southern blotting for several reasons. The first major consideration is that RNA is both chemically and biologically far more labile than DNA. From a practical viewpoint the extreme sensitivity of RNA to RNases and the wide prevalence and stability of these enzymes means that in order to prepare intact molecules of RNA (particularly large RNA), one must pay careful attention to eliminating RNases. Secondly, only a few percent of the total RNA of a cell is m-RNA, so that blotting against total RNA is not a very sensitive way of detecting rare mRNA. Therefore, although one can often detect a target mRNA in a preparation of total RNA, one can attain greater sensitivity by isolating first the poly A+ mRNA fraction and carrying out Northern analysis. Ten mg of total RNA is sufficient to detect an abundant message, whereas several mg or more of poly A+ RNA (equivalent to more than 100 mg total RNA) is generally required to detect rare messages. Poly A+ RNA can be isolated directly or from total RNA by means of oligo dT cellulose run either in a column or batch mode, by magnetic beads crosslinked to oligo dT, or by strepavidin coated magnetic beads capable of binding biotinylated oligo(dT) which has been hybridized to mRNA.
Another major difference between Northern and Southern analysis is that RNA molecules often form secondary structures that grossly alter their mobility in agarose gels. Therefore to denature the RNA so it will run according to its true size, the RNA is usually separated on a formaldehyde agarose gel. Alternatively one can react RNA with glyoxal (a formaldehyde analog) and run the RNA a standard agarose gel lacking formaldehyde. The advantage of glyoxal is that it does not give off toxic fumes like formaldehyde. The disadvantage is that it becomes essential to control the pH during electrophoresis to prevent dissociation of the glyoxal/RNA complex. Another reagent which has been used as a denaturant in RNA electrophoresis is methymercuric hydroxide. Methymercuric hydroxide’s considerable toxicity however is a serious deterrent to using this procedure. Whichever procedure is used for RNA denaturation, following electrophoresis RNA can be transferred using modifications of the methods used for DNA transfer, i.e. capillary blotting, vacuum blotting or electrophoretic blotting. Each has its own adherents.
No matter which method is used for the transfer step, Northern hybridizations are usually carried out at in the presence of 50% formamide to lower the melting temperature of the hybrids so that the RNA is not exposed to excessive heat for long periods. It is possible that this precaution is not really needed, but it is widely observed. RNA probes appear to be generally superior to DNA probes for Northern hybridizations.
The extent which you must go to eliminate RNases is controversial and subject to much superstition. We have found the following steps are important for running successful Northerns.
1) Make all solutions in clean glassware and autoclave them immediately.
2) Whenever possible, use presterilized plasticware which has been removed from the container using a gloved hand.
3) Treat the solutions used for isolation and electrophoresis of RNA with DEPC (Diethypyrocarbonate) to inactivate RNases. Note that DEPC reacts with amines such as Tris. Hence, Tris and similar buffers should be made from DEPC treated water, but not treated with DEPC directly. (DEPC treated water is made by adding 1 ml DEPC per liter of H2O. Mix and let sit 1/2 h to overnight and autoclave. DEPC is highly mutagenic and should only be handled in a hood. Be careful not to allow water to enter the stock bottle of DEPC because in presence of water DEPC breaks down to ethanol and CO2. The increase in CO2 pressure can lead to explosions. For this reason, try to use up all the DEPC you order at one time. Old bottles are potentially hazardous!)
4) Use a very clean gel box that has been rinsed with DEPC treated water. Ideally do not use a gel box that has been used for DNA minipreps for RNA work, since they may well be contaminated with traces of RNase from previous minipreps. Boxes that may have been contaminated with ribonucleases can be cleaned by soaking them in dilute solutions of NaOH or hydrogen peroxide followed by thorough washing with DEPC-treated water. Note however that some plastics may be attacked by these reagents. Commercially available reagents for RNAse removal such as RNAse-Zap or RNAseAway may well provide a better solution.
5) Keep RNA solutions on ice on much as possible, and if possible store RNA as an ethanol-precipitated pellet at -70 oC or in formamide at -70 oC. The formamide should have a pH > 5.2.
6) Wear gloves while working with RNA and change them frequently.
It is probably not necessary to render the solutions used for Northern transfer and hybridization RNase free. While it is prudent to autoclave solutions whenever possible or to make them up from autoclaved water, the levels of RNase possibly remaining are of little consequence. This is probably because under the high salt or alkaline conditions used for capillary transfer, nucleases are unlikely to be active. Furthermore, while a few endonucleolytic cleavages prior or during electrophoresis are potentially catastrophic since they could completely alter the size of the target RNA, such cleavages after the RNA has been separated will not alter the position of the RNA on the blot. So be very cautious about RNases until your RNA is safely electrophoresed-then your normal level of paranoia should be sufficient to ensure the integrity of your RNA.
Other laboratories are more cautious and in addition to the precautions outlined here bake all glassware at 250oC at least 4 hours prior to use. In addition, it is probably prudent to add inhibitors of RNases such as human placental RNase inhibitor to solutions used for the enzymatic synthesis of RNA such as reverse transcriptase reactions.
Special Considerations With Respect To Using Genius Non-radioactive Detection Methods
The Genius probes are labeled with digoxigenin (DIG). Like Northern blotting with 32P-labeled probes, Northern blotting with DIG labeled probes tends to be more difficult than Southern blotting or colony/plaque hybridizations due largely to the lability of RNA. The chemiluminescent Genius Procedure adds another layer of complication. Since ultimately one is measuring tiny amounts of alkaline phosphatase activity it is important to always be aware of the possibility of contamination of your reagents and apparatus by either RNAse or alkaline phosphatase.
Probe Preparation
As in the case with radioactive probes, DIG-labeled RNA probes demonstrate stronger signals and less non-specific hybridization than DNA probes on Northern and Southern Blots. If a DNA probe must be used, we recommend that you use the High SDS hybridization buffer or DIG Easy Hyb to reduce background.
Optimization of the Probe Concentration
Optimize the probe concentration before all hybridization experiments. This is necessary to avoid background staining, and it can be easily performed with a series of mock hybridizations, where increasing concentrations of DIG-labeled probes are incubated with naked pieces of membrane or hybridized to dots of homologous DNA or RNA.
Avoidance of RNase Contamination
Throughout the Northern blot experiment, be careful to avoid the introduction of RNases, as RNA is susceptible to degradation even after its immobilization on a nylon membrane. Roche Molecular Biochemicals (formally Boehringer Mannheim) recommends use of sterile solutions made with DEPC treated water for all reagents that will come into contact with the RNA or Northern blot. While it is hard to fault this advice, it may not be absolutely essential to use DEPC-treated solutions for the transfer and washing steps as we noted earlier. However autoclaved solutions and scrupulously cleaned containers should be used for all washes to reduce contamination by RNAses and alkaline phosphatase.
Throughout the experiment, use clean forceps whenever possible, handle the membranes only by the edge and wear gloves.
Optimal Blotting Conditions
Salt concentrations between 10X and 20X SSC give equivalent results for the transfer of RNA from a 1% agarose formaldehyde gel to a nylon membrane. The optimal blotting duration is overnight at room temperature for a conventional blot. However, it appears that downward blotting can be achieved in 1 to 2 hours using a low concentration alkaline buffer (Protocol 10.6).
PROTOCOL 10.1: ISOLATION OF TOTAL RNA USING TRIZOL
SOURCE: GIBCO/BRL TRIZOL TECHNICAL MANUAL.
INTRODUCTION:
TRIzol (Gibco/BRL) is an acid phenol extraction reagent containing detergents and guandine thiocyanate to denature RNAses (see Chomczynski, BioTechniques 15:532-537). Similar reagents are available from a number of different vendors which are listed in the BioTechniques article. The technique is quick and gives good yields of high quality RNA. It also has the advantage that proteins and DNA partition into a separate phase from the RNA and can also be easily recovered. This makes this reagent particularly attractive when you have a small amount of tissue from which you wish to obtain proteins, DNA and RNA simultaneously.
Note: Guanadine thiocyanate is very powerful protein denaturant, but it does not irreversibly inactive RNAses. Hence, if residual RNAses contaminate the sample after deprotienization, these enzymes can become active again following the removal of the denaturant. Hence, is important that the RNA containing aqueous phase be cleanly separated from the lower organic phase, and not be recontaminated by protein from dirty containers or gloves.
PROCEDURE:
CAUTION : Guanidine Thiocyanate is VERY TOXIC. Therefore, manipulations with it should be carried out in a fume hood. Contact with skin should be avoided. Liquid and solid waste should be discarded as toxic waste. Do not use polycarbonate tubes with guanidine thiocyanate.
1. Homogenization
Pellet cells by centrifugation and remove supernatant. Lyse cells in TRIzol Reagent by repetitive pipetting. (Note: do not pipette up and down too rapidly or the TRIzol reagent will shoot up and soak the internal plug.) Resuspend the pellet in 1 ml of the Trizol reagent per 5-10 X 106 of animal, plant or yeast cells, or per 1 x 107 bacterial cells. Washing cells before addition of TRIzol Reagent should be avoided as this increases the possibility of mRNA degradation. Disruption of some yeast and bacterial cells may require the use of a homogenizer.
Alternative sources of cells can be used, e.g.:
A. Tissues:
Homogenize tissue samples in 1 ml of TRIzol Reagent per 50-100 mg of tissue using a glass-Teflonâ or power. The sample volume should not exceed 10% of the volume of TRIzol Regent used for homogenization. Do not use polycarbonate tubes with guanidine thiocyanate which is contained in the TRIzol reagent.
B. Cells Grown in Monolayer:
Lyse cells directly in a culture dish by adding 1 ml of TRIzol Reagent to a 3.5 cm diameter dish, and passing the cell lysate several times through a pipette. The amount of TRIzol Reagent added is based on the area of the culture dish (1 ml per 10 cm2) and not on the number of cells present. An insufficient amount of TRIzol Reagent may result in contamination of the isolated RNA with DNA.
Phase separation
2. Incubate the homogenized samples or lysed tissue culture cells for 5 minutes at room temperature to permit the complete dissociation of nucleoprotein complexes.
3. Add 0.2 ml of chloroform per 1 ml of TRIzol Reagent.
4. Cap sample tubes securely. Shake tubes vigorously by hand for 15 seconds
5. Incubate tubes at room temperature for 2 to 3 minutes.
6. Centrifuge the samples at no more than 12,000 x g (14K in microfuge) for 15 minutes at 4°C.
7. Following centrifugation, the mixture separates into a lower, red, phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. The volume of the aqueous phase is about 60% of the volume of TRIzol Reagent used for homogenization.
RNA Precipitation
8. Transfer the aqueous phase to a fresh tube, and save the organic phase if isolation of DNA or protein is desired.
Note: Be especially careful not to pick up the white DNA precipitate in the interphase as it will contaminate your RNA, making it hard to quantify how much RNA is recovered. If your sample is very viscous, this indicates contamination with intact genomic DNA.
9 Precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5 ml of isopropyl alcohol per 1 ml of TRIzol Reagent used for the initial homogenization. Once you add the isopropanol, you may or may not see a cloudy suspension of RNA. Do not panic if you fail to see the cloudy suspension, just keep going. Incubate samples at room temperature for 10 minutes and centrifuge at no more than 12,000 x g (14K in microfuge) for 10 minutes at 4°C. The RNA precipitate, forms a pellet on the side and bottom of the tube.
RNA Wash
10. Remove the supernatant.
11. Wash the RNA pellet once with 75% RNAse-free ethanol, adding at least 1 ml of 75% ethanol per 1 ml of TRIzol Reagent used for the initial homogenization.
12. Mix the sample by vortexing and centrifuge at no more than 7,500 x g (~8 K in microfuge) for 5 minutes at 4°C.
Redissolving the RNA
13. At the end of the procedure, briefly air dry the RNA pellet for 5-10 minutes. Do not dry the RNA by centrifugation under vacuum. It is important not to let the RNA pellet dry completely as this will greatly decrease its solubility. Partially dissolved RNA samples have an A 260/280 ratio < 1.6. Dissolve RNA in RNAse-free water by passing the solution a few times through a pipette tip, and incubating for 10 minutes at 55°C - 60°C.
Note: For long term storage, RNA is best kept in formamide. The details of long-term storage are given in Chomczynski, P. Solubilization in formamide protects RNA from degradation. Nucleic Acid Res. 20:3791-3792.
MATERIALS:
1. Cells (5 X 106 HeLa)
2. DEPC treated water
Add 1 ml of DEPC to 1 L of deionized water, cap, and shake. Let sit several hours and autoclave.
Note: DEPC is highly toxic and should be aliquoted in a hood.
3. Chloroform (ACS grade)
4. Isopropanol (ACS grade)
5. 75% Ethanol (ACS grade EtOH and DEPC treated water)
6. TRIzol reagent
PROTOCOL 10.2: ISOLATION OF TOTAL RNA USING GUANIDINE THIOCYANATE AND ULTRACENTRIFUGATION
REFERENCES: CHIRGWIN, J.M., PRZYBYLA, A.E., MACDONALD, R.J. AND RUTTER, W. M. BIOCHEMISTRY 18:5249, 1979. ULLRICH, A.J., SHINE, J., CHIRGWIN J. M., PICTET, R., TISCHER, E., RUTTER W.M., AND GOODMAN, H.M. SCIENCE 196:1313, 1977. SETZER, D.R., MCGROGON, M., NUNBERG, J.H. AND SCHIMKE, R.T. CELL 22361-370, 1980.
INTRODUCTION:
This method was developed for isolation of RNA from pancreas which contains a high level of RNase. The procedure entails rapid denaturation of nucleases with guanidine thiocyanate and sedimentation of RNA through a CsCl cushion. The dense RNA is pelleted in the bottom of the tube, the DNA bands in the CsCl and proteins band in the top phase. The DNA may be saved and is not denatured during treatment. This is the method of choice when tissue is not limiting and large amounts of RNA are required.
PROCEDURE:
A. ISOLATION FROM TISSUE
1. Remove tissue and immediately drop in liquid nitrogen. Weigh frozen tissue and store on dry ice. The tissue may be stored in liquid nitrogen before homogenization, but should be worked up as soon as possible.
2. Pulverize tissue in liquid nitrogen using mortar and pestle.
3. Store pulverized tissue in 35 ml Oak Ridge tubes on dry ice until ready to homogenize.
4. Add 16 ml guanidine thiocyanate per gram on tissue to one sample at a time; then homogenize immediately.
5. Homogenize with Tissuemizer for one minute on high speed.
6. Wash homogenizer with sterile water and guanidine thiocyanate before each sample.
7. Balance tubes (50 ml) and centrifuge at 10,000 rpm in SS34 rotor for 20 to 30 min. at 10-25oC to pellet the cellular debris. Colder temperatures will cause the guanidine thiocyanate to precipitate.
8. Add 4 ml of 5.7M CsCl to clean SW41 rotor tubes to form a CsCl cushion.
9. Remove the supernatant fluid from the 50 ml tube and layer carefully onto a CsCl cushion using a 10 ml sterile pipette. Tubes must be full to within 4 mm of top.
10. Place tubes in numbered buckets and record order of samples. Balance tubes in the buckets and hook buckets on rotor.
11. Centrifuge at 35,000 rpm for 16-20 hours at 25oC.
12. Proceed to Step C.
B. ISOLATION FROM CELL CULTURES
1. Omit steps 1-6.
2. Remove medium from cells. Immediately add guanidine thiocyanate, (8 ml per 1 to 4 100 mm dishes of confluent cells) swirl, and pipette the viscous extract up and down in a 10 ml pipette.
3. When the cells have been disrupted, transfer the extract to a sterile 15 ml tube. Continue to pipette the mixture up and down until it flows in a continuous smooth stream out of the pipette. This step shears the DNA.
4. Samples may be centrifuged immediately or stored at 4 oC overnight or at -20 oC for at least a week.
5. Continue as described in A-7 Collection of RNA Pellet. DO IN A FUME HOOD
C. PROCESSING OF RNA AFTER CENTRIFUGATION
1. Remove tubes from rotor and aspirate the guanidine thiocyanate with a 10 ml sterile pipette down to the CsCl. Pipette off the CsCl using a one ml Pipetman. Change tip for each ml removed.
2. Now a translucent pellet of total RNA should be discernible. Invert the tube to drain.
3. Cut off the top of each tube with a single-edged razor blade leaving about 2 cm at the bottom. Alternatively, the inside of tube may be wiped with a sterile cotton swab while inverted.
4. Dissolve the pellet in 200 ml sterile deionized water and transfer to a sterile 1.5 ml microfuge tube. Wash out the tube with another 200 ml of sterile water. Transfer to a microfuge tube.
5. Add 40 ml 3M NaAcetate pH 5.5 and 1 ml absolute ethanol.
6. Mix and store overnight at -20 oC, Centrifuge 15 minutes in Microfuge at 4 oC. Wash twice with 70% ethanol, dry briefly in Speed-Vac.
7. Dissolve RNA in 200 ml sterile water and determine concentration. Store at -80 oC.
8. Poly (A)-RNA can be purified with oligo(dT) cellulose.
CAUTION
Guanidine Thiocyanate is VERY TOXIC. Therefore, manipulations with it should be carried out in a fume hood. Contact with skin should be avoided. Liquid and solid waste should be discarded as toxic waste. Do not use polycarbonate tubes with guanidine thiocyanate.
MATERIALS:
Chemicals:
Liquid Nitrogen, mortar and pestle
Dry Ice
Equipment:
Tissuemizer or Polytron
Sterile 50 ml polypropylene Oak Ridge tubes
Polyallomer tubes for the SW41 rotor
SW41 rotor (Beckman) and ultracentrifuge
MATERIALS:
1M Na Citrate, pH 7.0 (Autoclave)
2M Na Acetate, pH 5.0 (Autoclave)
4M Guanidine Thiocyanate
Add to sterile cylinder:
Guanidine thiocyanate (weigh wearing mask and gloves) 50g
1M Na Citrate, pH 7.0 1.67 ml
Na lauryl sarcosine 0.5g
Dissolve in sterile deionized distilled water and adjust to 98 ml
Adjust pH to 7.0 with 1M NaOH
Filter sterilize. (The solution cannot be filtered after Antifoam is added).
Add:
Sigma Antifoam A 0.167 ml
b-mercaptoethanol (add immediately before use) 0.70 ml
Store in aluminum foil covered bottle at 4 oC.
The solution can be used for one month if stored without mercaptoethanol.
5.7M Ultrapure Cesium Chloride (Gallard-Schlesinger biochemical grade)
Add to graduated cylinder:
CsCl 96 g
2M Na Acetate pH 5. 0.83 ml
Add water, dissolve solids and adjust volume to 100 ml
Filter and autoclave
PROTOCOL 10.3: POLY-A RNA ISOLATION FROM TOTAL RNA USING POLYATRACT® SYSTEM III
MODIFIED FROM PROMEGA MANUAL #TM021 REVISION 4/95
INTRODUCTION:
The following Poly-A RNA isolation procedure is designed for small-scale mRNA isolation. Total RNA is denatured and incubated with a biotinylated-oligo(dT) probe in the presence of 0.5X SSC. Under these salt conditions the poly-A RNA binds to the oligo(dT) probe. The biotinylated oligo(dT) -mRNA complexes are then incubated with Streptavidin-MagneSphereâ Paramagnetic Particles (SA-PMP). The biotinylated portion of the oligo(dT)-mRNA complex binds the streptavidin complex. The entire complex is then isolated by placing the tube containing the mixture next to a magnet that gently concentrates all the magnetic particles at the bottom of the tube. The supernatant is removed and the beads are washed in 0.1X SSC to remove unbound contaminants. The concentration/wash steps are then repeated several times to remove remaining contaminants. Finally the mRNA is removed from the magnetic particles by incubating the samples in DEPC treated water. The final volume is 250 ml. If the mRNA in the eluted sample is too dilute for your purposes, the RNA can be concentrated by drying in a Speed-Vac.
PROCEDURE:
Annealing of Probe
1. In a sterile, 1.5 ml RNAse-free microfuge tube, bring 0.1-1.0 mg of total RNA to a volume of 500 ml in DEPC-treated water.
Note: Less total RNA may be used, but the mRNA obtained may not be detectable using a spectrophotometric assay.
2. Heat the tube at 65 oC for 10 minutes.
3. Add 3 ml of the Biotinylated-Oligo(dT) Probe and 13 ml of 20X SSC to the RNA. Mix gently and incubate at room temperature until completely cooled. This should require 10 minutes or less. While this solution is cooling, prepare stock solutions of 0.5X and 0.1X SSC.
Stock Solution Preparation
4. Prepare 1.2 ml of sterile 0.5X SSC by combining 30 ml of 20X SSC with 1.17 ml of DEPC-water in an RNAse-free tube.
5. Prepare 1.4 ml of sterile 0.1X SSC by combining 7 ml of 20X SSC with 1.393 ml of DEPC-water in a sterile RNAse-free tube.
Washing of Streptavidin Paramagnetic Particles
6. Resuspend the SA-PMPs by gently flicking the bottom of the tube until they are completely dispersed and then capture them by placing the tube in the magnetic stand until the SA-PMPs have collected at the side of the tube (approximately 2 min.). Carefully remove the supernatant. Do not centrifuge the particles.
Note: The Promega protocol calls for capturing the beads in 30 sec. However some workers have claimed that a considerable fraction of the particles are nearly invisible and are captured more slowly. Hence we recommend using this longer incubation time in the capture step until one is certain that shorter times do not compromise the yield.
7. Wash the SA-PMPs three times with 0.5X SSC (300 ml per wash), each time capturing them 2 min. with the magnetic stand and then carefully removing the supernatant with a pipette tip.
8. Resuspend the washed SA-PMPs in 0.1 ml of 0.5X SSC.
Note: The SA-PMPs should be used within 30 minutes of washing as they are less stable in the absence of protein.
Capture and Washing of Annealed Oligo(dT)-mRNA Hybrids
9. Add the entire contents of the annealing reaction to the tube containing the washed SA-PMPs.
10. Incubate at room temperature for 10 minutes.
11. Capture the SA-PMPs using the magnetic stand 2 min. and carefully remove the supernatant without disturbing the SA-PMP pellet. Note: Save the supernatant until you are certain that satisfactory binding and elution of mRNA has occurred.
12. Wash the particles four times with 0.1X SSC (300 ml per wash) by gently flicking the bottom of the tube until all of the particles are resuspended. After the final wash, capture the SA-PMPs and remove as much of the aqueous phase as possible without disturbing the SA-PMP particles.
Elution of mRNA
13. To elute the mRNA, resuspend the final SA-PMP pellet in 0.1 ml of RNAse-free DEPC-water supplied with the kit and resuspend by gently flicking the tube.
14. Magnetically capture the SA-PMPs and transfer the eluted mRNA aqueous phase to a sterile, RNAse free tube. Do not throw the particles away.
15. Repeat the elution step by resuspending the SA-PMP pellet in 0.15 ml of DEPC-water. Repeat the capture step, pooling the elute with the RNA from the first elution. The total volume should be 250 ml. This RNA can be concentrated if necessary by vacuum drying, but try not to dry the RNA pellet completely as this can make dissolving the RNA pellet more difficult.
Note: If any of the particles are carried over at this point, they may be removed by centrifuging at 10,000 X g for 5-10 minutes at 4 oC. Carefully transfer the RNA to a fresh RNAse-free tube. Do not spin the 2 ml tubes provided in a bottomless rotor, as this may cause the tubes to fail.
MATERIALS:
1. PolyATract kit and solutions.
2. Sterile, RNAse-free 1.5 ml plastic microfuge tubes.
3. Sterile, RNAse-free pipettes and pipette tips.
PROTOCOL 10.4: DIRECT ISOLATION OF POLY(A) RNA FOR NORTHERNS
REFERENCE: J. BADELY ET AL., BIOTECHNIQUES 6:114 (1988).
INTRODUCTION:
This is a rapid method to directly isolate poly (A) from cells or tissue. This procedure works well for brain and other tissues that are low in RNase activity. Its usefulness in any other tissue should be determined experimentally. It is relatively cost effective and especially useful when numerous samples must be prepared for Northern blot analysis. RNA prepared by this method should not be used for construction of cDNA libraries, because cDNA libraries synthesized from this RNA yield only very short transcripts. However, this RNA can be used for RNA PCR.
PROCEDURE:
1. Pulverize 1 gram frozen tissue with a mortar and pestle chilled either by surrounding with dry ice or filling with liquid nitrogen. Transfer tissue powder to a sterile 50-ml pre-chilled conical tube and store on dry ice until ready to homogenize.
2. Add 100 ml proteinase K (20 mg/ml) to 9.9 ml Lysis Buffer, mix and pour on tissue powder.
3. Immediately homogenize with Polytron (15 to 30 seconds).
4. Incubate at 50 oC for 1 hour.
5. Add 600 ml 5M NaCl. Any visible debris should be removed by centrifugation at 10K x g for 5 minutes in sterile Oak Ridge tubes.
6. Transfer to 15-ml tube containing oligo (dT)-cellulose (0.3 g).
7. Incubate at room temperature for > 30 minutes with slow shaking.
8. Pellet in table-top clinical centrifuge 2000 rpm for 1 minute at room temperature.
9. Wash pellet twice with 15 ml Buffer B.
10. Pour slurry into column and wash with 15 ml Buffer B.
11. Wash with 4 ml Intermediate Buffer.
12. Elute poly (A)+ RNA with 3 ml Elution Buffer. Elution rate should be about 1 drop/10 seconds.
13. Add 300 ml 3 M NaOAc pH 5.5 and 7 ml cold EtOH - Mix.
14. Precipitate overnight at -20 oC.
15. Centrifuge 30 minutes at 10K x g at 4 oC. Wash pellet with 5 ml 95% EtOH. Use 10-ml Oak Ridge centrifugation tubes (autoclaved).
16. Drain and air dry.
17. Resuspend in 200 ml H2O.
YIELD: About 50 mg poly (A)+ RNA per gram of tissue.
SOLUTIONS AND MATERIALS:
Lysis Buffer:
100 ml
200 mM NaCl 4 ml of 5M
200 mM Tris 7.5 20 ml of 1M
1.5 mM MgCl2 150 ml of 1M
2.0% SDS 20 ml of 10%
Sterile dd H2O 56 ml
Proteinase K 20 mg/ml H2O
[Roche Molecular Biochemicals (formally Boehringer Mannheim]
Buffer B:
100 ml
10 mM Tris pH 7.5 1 ml of 1M
500 mM LiCl 10 ml of 5M
1 mM EDTA 200 ml of 500 mM
0.5% SDS 5 ml of 10%
Sterile dd H2O 84 ml
Intermediate Buffer:
100 ml
10 mM Tris 7.5 1 ml of 1M
100 mM LiCl 2 ml of 5M
1 mM EDTA 200 ml of 500 mM
0.05% SDS 500 ml of 10%
Sterile dd H2O 97 ml
Elution Buffer:
100 ml
10 mM Tris 7.5 1 ml of 1M
1 mM EDTA 200 ml of 500 mM
0.05% SDS 500 ml of 10%
Sterile dd H2O 98 ml
Preparation of Oligo (dT)-Cellulose (Type 2, Collaborative Research)-clean and remove fines.
1. Place 0.3 g Oligo (dT)-cellulose in sterile 15 ml conical tube.
2. Wash with each of the following in sequence by adding 10 ml, shaking briefly, allowing to settle by gravity for 10 minutes and removing supernatant.