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.

 

a.  Sterile water

b.  0.1N NaOH/5 mM EDTA

c.  Sterile water

d.  Buffer B (leave 1 ml in the tube)

 

Preparation of Column

 

Wash 2-ml disposable column (Bio-Rad) and stopcock (Kontes)

 

a.  0.1 N NaOH

b.  Sterile distilled water

c.  Buffer B (leave 1 ml in the column)

 

Regeneration of Oligo (dT)-cellulose

 

Used Oligo (dT)-cellulose can be regenerated and reused many times (~10).  Wash cellulose in column with:

 

a.  10 ml NaOH

b.  20 ml H2O

c.  10 ml 100% EtOH

 

Vacuum dry and store desiccated at -20 oC.

PROTOCOL 10.5:  FORMALDEHYDE AGAROSE GEL ELECTROPHORESIS OF RNA

 

MODIFIED FROM R. KROCZEK AND E. SIEBERT ANALYT. BIOCHEM:184, 90-95 (1990) AND THE ROCHE MOLECULAR BIOCHEMICALS (FORMALLY BOEHRINGER MANNHEIM)  GENIUS PROTOCOL BOOK (SECOND EDITION)

 

INTRODUCTION:

 

This procedure for running ethidium bromide stained RNA on Northern gels  is not quite as sensitive as techniques in which the RNA is not stained with ethidium bromide.  However, staining the RNA with ethidium bromide prior to electrophoresis offers several advantages which for most experiments probably outweigh the disadvantages.  To begin with, immediately following electrophoresis one can check the gel to find out if intact RNA was isolated (by examination of the sharpness of the 28S (~ 4700 bp) and 18S rRNA (~1950 bp) bands.  Furthermore, using stained RNA allows for easy checking of the efficiency of transfer.  This procedure also uses a much lower concentration of formaldehyde than the "classic" Northern protocols which allows gels to be run faster at a higher voltage.  Finally by using downward alkaline blotting techniques we can reduce the transfer time from 16 hours to 1-2 hours.

 

Notes:  More recent discussions of the pros and cons of ethidium bromide staining of RNA, optimum concentrations, and the effect of staining on Northern blots can be found in Z Gong , Biotechniques 12:74-76 (1992) and in B. Ogretman et al. Biotechniques 14: 932-935 (1993).  The use of UV illumination to visualize RNA on a gel is discussed by T. Mukhopadhyay and J. Roth Biotechniques 17:619-620 (1994).  While this technique can’t be used to follow transfer and is only sensitive to about 2 mg of total RNA in a lane, it presumably would have no effect on Northern blotting.

 

PROCEDURE

 

1.         Prepare a 1.2% agarose midigel containing 1% formaldehyde.  For 100 mls:

 

            a.         This should be done in the hood.  In a sterile flask melt 1.2 g agarose in either 87.3 ml autoclaved dI H2O or preferably DEPC H2O.

 

            b.         Bring this solution to 55oC in a water bath.

 

            c.         Add:

                                    10 mls 10x MOPs buffer

                                    2.7 mls 37% formaldehyde (pH > 4.0 - 1% final)

 

                        Note: The pH of the MOPs has to be 7.0 or you run the risk of having the RNA migrate toward the cathode, i.e. out the top of the gel.

 

            d.         Mix gently.

            e.         Pour IN HOOD to a depth of approximately 0.75 cm. (Volume = 100 ml)

            f.          Allow to solidify at room temperature (about 45 minutes).  Because of the low formaldehyde concentration in this gel, it should be used soon after casting to minimize evaporation of the formaldehyde.

 

2.                  Mix your vacuum-dried RNA (0.15-10 mg) with Sigma Loading Buffer in a ratio of between 2:1 to 5:1 RNA:Loading Buffer.  This loading buffer contains ethidium bromide and loading dye.

 

If Sigma RNA loading buffer is not available you can dilute you RNA with 20 ml of Sample Buffer and then add 5 ml loading dye.  At this point you have the option of adding 1 ml of 0.5 mg/ml ethidium bromide before proceeding to step 3.  Mix all components gently.  Adding the ethidium at this point will slightly increase the sensitivity of the staining, but will somewhat decrease the ability of the RNA to subsequently hybridize.

 

3.         Heat the RNA to 55oC for 15 min or 65oC for 10 min. - Do not let the heating go longer than specified, briefly quench on ice.  If you have not already added the ethidium bromide in step 2, then add 1 ml of 0.5 mg/ml ethidium bromide solution, and load on the gel.

 

4.         Electrophorese for 31/2 h at 70 V (gels 15 cm wide, 10 cm long) using 1 X MOPs electrophoresis buffer, pH 7.0.  Slightly higher voltages for shorter times may be acceptable.  Ideally the buffer should be recirculated, but if this is impractical, mix the buffers in the anode and cathode chamber every hour.  Do not let the buffer go longer than an hour without mixing or checking the pH.

 

5.         At the completion of the electrophoresis examine the gel on a UV light box and take a picture with a fluorescent ruler set beside the gel to both check the quality of the preparation (ideally the intensity of the 28S r-RNA band should be about double that of the 18S r-RNA band) and to allow you to estimate the molecular weight of any bands you detect (Do not leave the gel on the light box for more than a few seconds to prevent UV damage).  Then carry out the downward blotting procedure in Protocol 10.6.

 

 

MATERIALS:

 

1.  DEPC-treated H20

 

        Add diethylpyrocarbonate to 0.1%, shake briefly, incubate for 30 minutes at room temperature, and autoclave.  Diethylpyrocarbonate should be handled in a fume hood.

 

2.  Formaldehyde 37%

 

The formaldehyde should have a pH > 4.0.  Do not use if it is less than 4.0.  Adjust with NaOH if necessary.

 

3.  Formamide

 

        Formamide can be used to store RNA and reduce chances of degradation.  It is best to buy small bottles and use them up relatively quickly or to deionize the formamide with a Dowex resin and store frozen under N2.  In either case the pH should be checked to make sure it is greater than about 5.2.

 

4.  0.5 M EDTA

 

        Add 186.1 g of disodium ethylenediaminetetra-acetate.2H2O to 800 ml of H2O.  Stir vigorously on a magnetic stirrer.  Adjust the pH to 8.0 with NaOH (~20 g of NaOH pellets).  Dispense in aliquots and sterilize by autoclaving.

 

5.  1 M Sodium Acetate (82.03 g/l) or Sodium Acetate Trihydrate (136.08 g/l)

 

                Make with DEPC water and autoclave or filter sterilize

 

6.  Loading Buffer (use DEPC water)

        Final Concentration                         Stock Solution                      Amount/10 ml

        1 mM EDTA, pH 8.0                        500 mM                                      20 ml

        DEPC-Treated water pH 7.0                                                                5 ml

        0.25% bromophenol blue                                                                    25 mg

        0.25% xylene cyanol                                                                            25 mg

        50% glycerol                                                                                         5 ml

 

7.  10 X MOPS Running Buffer.   This solution must have the correct pH or the RNA may migrate backwards out of the gel!

 

                                                                                                          per L

                0.4 M MOPS, pH 7.0                                            83.71 g MOPs Free acid

                0.1 M Sodium Acetate                                         8.20 g NaOAc, anhydrous

                0.01 M EDTA                                                        3.72 g Disodium EDTA

                Make with DEPC treated water                          to 1 L, pH 5.5-7.0

 

                pH to 7.0 with NaOH

 

8.                    10X MOPS Formaldehyde Running Buffer From Five Prime Three Prime

 

                This buffer is essentially the same as in solution 7.  It should be diluted 1:10 with DEPC-treated water.

 

 

9.  1 M MOPS, pH 7.0)

 

                209.27 g/L MOPS Free acid and 231.27g/l of the MOPS Monosodium Salt

                Make in DEPC treated water and autoclave

 

10.                 Sigma RNA Loading Buffer

 

Deionized Formamide, 62.5% (v/v)

Formaldehyde, 1.14 M

Bromophenol blue, 200 mg/ml

Xylene cyanol 200 mg/ml

MOPS-EDTA-Sodium acetate solution at 1.25X working concentration (see note below)

Ethidium bromide 50 mg/ml

 

Note: 10X Solution Of MOPS-EDTA-Sodium Acetate solution is:

                0.4 M MOPS, pH 7.0

0.1 M Sodium Acetate

10 mM EDTA

 

11.           Sample Buffer

 

                Final Concentration                                             Stock                                      Amt. Stock Per 50 ml

 

0.2 M MOPS                                                         1 M MOPS                            10.0 ml

                0.005 M Sodium Acetate                                     1 M NaOAc                             0.25 ml

                0.001 EDTA                                                           0.5 M EDTA                            0.10 ml

6.54% formaldehyde                                            37%                                          8.84 ml

50% deionized formamide                                   100%                                      25.0 ml

                H2O                                                                                                                           5.81 ml

 

10.           0.5 mg/ml EtBr Stock

 

                Dissolve 0.5 mg Ethidium bromide/ ml DEPC treated water.

 

PROTOCOL 10.6: DOWNWARD CAPILLARY TRANSFER OF RNA TO NYLON MEMBRANES USING AN ALKALINE TRANSFER BUFFER

 

SOURCE:  MODIFIED FROM S. MÜNCH, BIOCHEMICA 11: 29 (1994).  (BIOCHEMICA IS PUBLISHED BY ROCHE MOLECULAR BIOCHEMICALS - FORMALLY BOEHRINGER MANNHEIM)

 

INTRODUCTION:

 

Capillary blotting for RNA differs in only a few details from the procedures used for capillary blotting DNA.  Because RNA molecules are already relatively small there is no need to carry out an acid depurination step as for DNA.  Also, the relative alkali lability of RNA limits the extent to which one can use alkali buffers during blotting, although blotting with buffers containing up to 10 mM NaOH is quite feasible in rapid downward blotting protocols.  The main advantages of the present protocol are that it is rapid, does not use expensive equipment, and provides high sensitivity without the use of radioactivity.

 

PROCEDURE:

 

1.         The gel is blotted with no pretreatment.  Place the gel in the Alkaline Transfer Buffer while you assemble the blotting stack.

 

2.         Set up your Northern Blot as follows:

 

 

a.         Measure dimensions of gel.

 

b.         WITH GLOVES, and using the gel measurements, cut a piece of nylon filter that will fit exactly on top of the gel.

 

c.         Wet the filter by gently laying it on top of a pan of RNA Alkaline Transfer Buffer and letting the membrane wet from beneath.  Once it is completely wet, submerge the membrane.

 

d.         Cut 2 sheets of Whatman 3MM paper that are the size of the gel in both dimensions.  Wet both in the transfer buffer. 

e.         Stack paper towels, which have been cut to approximately the same size as the gel, on top of a piece of Saran Wrap.  Make a column of towels approximately 4 to 6 inches high.  Put a piece of thick blotting paper on top.  This constitutes the gel platform.

 

f.          CAREFULLY, remove the gel and place on a piece of Saran Wrap. Be careful how you handle the gel because it is very slippery when trying to pick it up with gloved hands.  It is easy to drop and break the gel at this point unless you are very careful.  Next, while gently lifting up one side of the Saran Wrap with one hand, and supporting the gel with the other hand, gently flip the agarose gel over so that the bottom of the gel when the gel was cast is now facing up.  This bottom surface is more uniform than the top surface and hence makes it easier to place the membrane without distortion or bubbles forming.

 

g.         Carefully place the WET membrane squarely on top of the inverted gel.  It is easiest to "bow" the filter down in the middle to the gel and lower the filter onto the gel at the edges.  This will prevent trapping air bubbles between the filter and the gel.

 

h.         Place one piece of wetted Whatman paper on the thick blotting paper at the top of the gel platform.

 

i.          Now carefully invert the gel plus membrane and CAREFULLY place the gel plus membrane onto the wetted Whatman paper on top of the gel platform.  You can sandwich the gel between 2 glass plates or Saran Wrap and then quickly flip the "sandwich" (being careful not to let the gel slide out of the sandwich).

 

j.          Next take a sharp pencil and go down through the bottom of the wells and mark where the lanes should be.  Pencil survives the hybridization better than many ink pens.  Then  cut off the wells with a scalpel.  Alternatively, after marking the position of the wells, fill the wells with molten agarose to prevent the transfer buffer short circuiting through the wells.

 

            Note:  Even though the gel will ultimately reside on top of the membrane, it is probably easier to build the stack upside down as we do here since in this way the membrane can be carefully lowered onto the gel.  If you choose you may wish to build the entire transfer stack from the bottom up, but this entails lowering the gel carefully onto the membrane which can be a bit tricky.

 

k.         Remove any air bubbles that may be trapped between the gel and the wick by gently rolling a 5 ml sterile pipette over the gel and rolling the bubbles out to the sides of the gel.

 

l.          Add RNA Alkaline Transfer Buffer to a clean baking dish and saturate a clean sponge.  Squeeze lightly (so it is not dripping).  Place the wetted Whatman paper which has been cut to exactly to the same size on top of the gel and then lay the sponge on top of that.  Be sure that the Whatman paper and the sponge do not overhang the gel and touch the paper towels or blotting pads below.  If contact is allowed to happen, the buffer used for blotting will bypass the gel, thereby lowering transfer efficiency.  The result of this short circuit is usually no bands on the blot.  An easy way to avoid this problem is by placing parafilm or Saran Wrap around the side of the blot to prevent the sponge from making contact with the blotting pads.

 

m.        Allow to blot  1.5 to 2 hours at room temperature

 

 

3.  Tear down Northern Blot:

 

a.         Remove the sponge from the top of the gel.

 

b.         Remove gel/filter/paper sandwich (in one piece) and peel the Whatman paper off of the gel.

 

c.         Place gel side of sandwich down on bench top.

 

d.         Peel the piece of Whatman paper off of the membrane.

 

e.         Peel membrane off of the gel and place on a clean (wetted) piece of Whatman paper.  Mark the RNA side of the filter (the side that was in contact with the gel).

 

f.          Neutralize the blot by soaking it for 15 minutes in 200 mM sodium phosphate buffer pH 6.8.  Do NOT UV irradiate the blot as this lowers the detection efficiency.  The RNA will bind irreversibly to the nylon filter in the presence of alkali even without irradiation or baking.

 

g.         Check the blot briefly on the long wavelength transilluminator.  You should easily see the bands corresponding to r-RNA if one or more of your lanes contains total RNA.  Note that some of the rRNA may remain in the gel after these short transfer times, but that the mRNA which is in far lower concentration seems to completely transfer more rapidly.  Attempting to increase the sensitivity by prolonging the transfer time may be counter productive. The membrane can now be prehybridized, or stored dry at +4oC in a desiccation chamber for future detection.

 

 

h.         Place the blot in a hybridization bottle and prehybridize in RNA Hybridization Solution for 1 to 3 hours at 680C.  Longer times may be preferable.

 

i.          Remove the prehybridization solution and add fresh hybridization solution containing freshly denatured RNA probe.  (The probe is initially denatured by placing the probe in a screw cap tube and incubating in a boiling water bath for 10 minutes, then snap cooled in a ice water bath for 3 to 5 minutes.  For subsequent use, hybridization buffer containing the probe can be heated to 680C for 15 minutes to denature.).  Hybridize overnight at 680C.  Longer hybridization times may lead to increased sensitivity since RNA probes unlike their DNA counterparts can not self-anneal.

 

j.          At the end of the hybridization period pour off the hybridization solution containing the probe and store it at -700C.  Rinse the membrane briefly in 2X SSC, 0.1% SDS and discard to remove the most of the remaining hybridization solution.  Next, wash the blot two times in 2X SSC, 0.1% SDS for 5 minutes at room temperature, then do two 15 minute washes in 0.1X SSC, 0.1% SDS at 68oC if using a fully homologous probe. 

 

k.         The subsequent development of the blot is exactly as described for a Southern blot in Protocol 9.4.  If the background is too high you can try rewashing the blot in 0.1X SSC at 68oC or treating it with RNAse as described in the appendix of the Genius protocol manual.

 

MATERIALS:

 

(Note: We have specified DEPC treated water throughout this procedure.  This may be an unnecessary precaution since after the gel has been run a nick or two in the RNA will not alter the results of the experiment.  However stringent attention to cleanliness is still a good idea to reduce the chance of a high background on the blots).

 

1.             Baking dish

 

2.             Paper towels

 

3.             Positively charged nylon filters e.g. Hybond N

 

4.             Whatman 3MM paper

 

5.             RNA Alkaline Transfer Buffer

 

                3.0 M NaCl  (175 g/l) made in sterile dIH2O DEPC-treated water.  After autoclaving add NaOH to 8 mM and sodium lauryl sarcosine (=N-lauryl sarcosine sodium salt FW=293.4) to 2 mM.  Final pH is about 12.

 

 

6.             Neutralization Buffer

 

                200 mM Na Phosphate pH 6.8 in DEPC-treated water

 

7.             RNA Hybridization Solution in DEPC treated water

 

                50% Formamide

                5X SSC

                2% Genius Blocking reagent

                0.1% sodium lauryl sarcosine

                0.02% sodium dodecyl sulfate

 

8.             Saran wrap

 

9.             2X wash solution

 

                2X SSC containing 0.1% SDS

 

10.           0.5X wash solution

 

                0.5X SSC containing 0.1% SDS

 

11.  High SDS Buffer

 

                5X SSC

                2.0% (w/v) blocking reagent for nucleic acid hybridization (Roche Molecular Biochemicals)

                50 mM sodium phosphate (pH 7.0)

                0.1% N-lauroylsarcosine

                7% SDS

                50% Formamide

 

12.  0.5X Wash Solution

 

                0.5X SSC

                0.1% SDS

 

13.  20X SSC

 

                3M NaCl

                300 mM sodium citrate

                pH 7.0 made with DEPC treated water