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Northern Blot of Large mRNAs 30530525Northern Blot Analysis of Large mRNAsNicole Porchet and Jean-Pierre Aubert1. IntroductionNorthern blot analysis has historically been one of the most common methods usedto provide information on the number, length, and relative abundance of mRNAsexpressed by a single gene. This technique also generates a record of the total mRNAcontent expressed by a cell culture or by a tissue, which can be analyzed and comparedon the same specimens by successive hybridizations with specific probes.There are two main difficulties often associated with this technique. The fisrt is thatNorthern blotting is generally considered to be rather insensitive, requiring largeamounts of starting material and consuming large amounts of tissue. The second prob-lem stems from the fact that the RNA isolated from cells or tissues must be of highpurity and high quality, and nondegraded; maintaining these qualities can be difficult,specifically in the case of large mRNAs, even for experienced workers.Messenger RNAs, larger than 10 kb, encoding human titin (23 kb), nebulin (20.7 kb),apolipoprotein B-100 (14.1 kb), dystrophin (14 kb), and secreted mucins MUC2,MUC3, MUC4, MUC5B, MUC5AC, MUC6 (14–24 kb) usually show more or lesspolydisperse patterns on Northern blots, which are attributable to artifactual causes.These patterns are in the form of a smear or very wide bands and result mainly fromtwo technical problems: (1) the high sensitivity of large mRNAs to mechanical dam-age that occurs during extraction and purification steps, and (2) the lack of efficiencyof the transfer of large RNAs onto membranes, and thus the poor detection of the largeintact mRNA species.Moreover, efficiency of large poly(A+)RNA selection is very poor with preferentialloss of the largest transcripts. Hence, there is a risk of misinterpretation of the data inthe case of mucin genes that express allelic transcripts of different size owing to vari-able numbers of tandem repeat polymorphism.This chapter describes in detail the protocols for carrying out Northern blots thathave successfully been used in our laboratory to examine mucin gene expression bothin cell cultures (HT-29 MTX) and in tissues from various mucosae (trachea, bronchus,From:Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield © Humana Press Inc., Totowa, NJ 306 Porchet and Aubertstomach, colon, small intestine). These protocols can also be adapted to analyze otherlarge mRNAs such as ApoB transcripts (1,2).2. Materials2.1. Preparation of mRNA (see Note 1)1. Cultured cells or tissues: snap-freeze in liquid nitrogen and store in liquid nitrogen until used.2. Homogenization buffer: 4 M guanidinium isothiocyanate buffer is prepared by dissolving23.6 g of guanidinium isothiocyanate, 73.5 mg of sodium citrate, and 250 mg of sodiumN-lauroylsarcosine in 50 mL of water, treated with 0.1% diethylpyrocarbonate (DEPC),and then autoclave. Add 2-mercaptoethanol to 100 mM just before use.3. Cesium chloride 5.7 M EDTA cushion: Dissolve 19.2 g of cesium chloride at 60°C in 0.1 MEDTA, pH 7.5, treated with 0.1% DEPC to a final volume of 20 mL and then autoclaved.Keep at 4°C in dark bottles.4. TE buffer: Dissolve 1.21g of Tris base and 0.37g of EDTA-disodium salt per liter. Adjustto pH 8.0 with HCl. Sterilize by autoclaving.5. Chloroform:n-butyl alcohol (4:1).6. 3 M Sodium acetate, pH 5.5: Dissolve 408.1 g of sodium acetate 3H2O in 800 mL ofwater. Adjust pH to 5.5 with glacial acetic acid. Adjust volume to 1 L. Dispense in aliquotsand sterilize by autoclaving.7. Ethanol: 100 and 95%.2.2. Electrophoresis1. Gel box, castinng tray, and combs: carefully clean with 0.1 N NaOH overnight or for atleast 3 h, rinse with distilled water (verify the pH), rinse with ethanol, and give a finalrinse with sterile distilled water just before use. Use gloves in all steps. Set up the castingtray and comb in a fume hood because of toxic vapors given off during the pouring andsetting of the gel (hot formaldehyde).2. DEPC-treated water.3. 10X Morpholino-propane-sulfonic acid (MOPS) stock solution (0.2M MOPS): DissolveMOPS (10 g) in DEPC water (200 mL) containing 3 M sodium acetate (4.2 mL), and 0.5 MEDTA, pH 8.0 (5 mL). Adjust the pH to 7.0 with 3 M NaOH and the final volume to 250 mLwith DEPC-treated water.4. Gel running buffer: 0.02 M MOPS, pH 7.0 (1X MOPS stock solution).5. Denaturing buffer: 50% deionized formamide, 18% deionized formaldehyde, 0.02 MMOPS, pH 7.0.6. Denaturing gel: 0.9% agarose gel (13 × 18 × 0.3 cm) containing 18% formaldehyde andMOPS stock solution, pH 7.0 (final concentration : 1X).7. Loading buffer: 0.1% xylene cyanol, 0.1% bromophenol blue, 1X MOPS, pH 7.0, solu-tion, 50% glycerol. Make with DEPC-treated water and autoclaved glycerol.8. Molecular weight markers (Roche Diagnostics, Meylan, France).9. Fluorescent indicator F 254 (Merck, Darmstadt, Germany).2.3. Transfer and Crosslinking1. 20X Sodium chloride sodium citrate (SSC) buffer: Dissolve 175 g of NaCl and 88.2 g oftrisodium citrate dihydrate per liter. Adjust to pH 7.0.2. Hybond™ N+ membrane (Amersham).3. Ultraviolet (UV) light source, 254 nm. Northern Blot of Large mRNAs 3072.4. Filter Hybridization1. Random-primed labeling kit (Boehringer Mannheim).2. 20X SSPE buffer: Dissolve 174 g of NaCl, 27.6 g of sodium dihydrogen phosphate mono-hydrate, and 7.4 g of EDTA per liter. Adjust to pH 7.4 with 10 N NaOH.3. 50X Denhardt’s solution: 1 g of Ficoll 400, 1 g of polyvinylpyrrolidone, and 1 g of bovineserum albumin are dissolved in 100 mL of H2O. Sterilize by filtration.4. Salmon sperm DNA (Boehringer Mannheim).5. Hybridization solution: 50% formamide, 5X SSPE, 10X Denhardt’s solution, 2% (w/v)sodium dodecyl sulfate (SDS), and 100 mg/mL of sheared salmon sperm DNA.6. Kodak X-Omat film (Kodak, Rochester, NY).3. Methods3.1. Preparation of mRNA (see Notes 1–3)The original guanidinium isothiocyanate method is recognized for the purity andquality of the RNA obtained. Guanidinium isothiocyanate combines the strong dena-turing characteristic of guanidine with the chaotropic action of isothiocyanate andefficiently solubilizes tissue homogenates. Effective disruption of cells can be obtainedwithout the use of a homogenizer, which has otherwise been used routinely, especiallywhen the starting material comes from tissues. In the specific case of large mRNAs,great care must be taken to prevent all risks of mechanical degradation. For compari-son, RNA was also isolated in our laboratory by other methods using guanidiniumisothiocyanate-phenol/chloroform (3), lithium chloride-urea (4), or different optimizedcommercial total RNA preparation kits from Bioprobe (Montreuilsous Bois, France)or Clontech Inc. (Palo Alto, CA). The best protocol to prepare intact large RNAs wasthe following improved method that we developed, derived from the guanidiniumisothiocyanate protocol (5):1. Grind cells (1.5 × 106) or tissues (optimal weight of 1 g) to a fine powder in a mortar andpestle in liquid nitrogen and mix with 10 mL of homogenization buffer (Subheading2.1., item 2) still in liquid nitrogen.2. Then allow the homogenate to thaw gradually at room temperature, during which time theguanidinium isothiocyanate and 2-mercaptoethanol efficiently solubilize the cell or tissue mixture.3. Gently transfer the homogenate obtained onto 3.2 mL of 5.7 M cesium chloride cushion(see Subheading 2.1., item 3). Ultracentrifugation is performed for 16 h at 29,500 rpm ina Beckman SW41 rotor. Remove the supernatant, cut off the bottom of the tube, andcarefully resuspend the white pellet of total RNA in 2 × 1 mL TE buffer, pH 8.0 (seeSubheading 2.1., item 4), 0.1% SDS by using wide-mouth pipets, carefully avoiding allshear forces.4. Remove chloride cesium salts from the pellet by two washings with 2 vol of chloroform/n-butyl alcohol (4:1) mixture. This purification of the pellet of RNA makes it easier todissolve. During the washing steps, the tubes must be mixed only by gentle inversion, andvortexing is strictly avoided.5. Carefully remove the top aqueous phase, which contains the RNA, with wide mouthpipettes and transfer it to a fresh tube. Precipitated the RNA by adding 0.1 vol of 3 Msodium acetate, pH 5.5, and 2.5 vol of ethanol, at –80°C for 15 h. Centrifuge the precipi-tate of RNA at 10,000g for 30 min at 4°C, wash it with ice-cold 95% ethanol, and then100% ethanol, centrifuge it again at 10,000g for 30 min at 4°C, and let it air-dry. 308 Porchet and Aubert6. Redissolve the RNA pellet carefully in DEPC-treated water and quantify by measuringthe A260nm of an aliquot.7. Store the final preparation at –80°C until it is needed.3.2. ElectrophoresisStudying the isolation of poly(A+)RNA by using standard protocols or more recentsystems (Poly [A] tract®RNA Isolation System from Promega, Charbonnieres, France),we concluded that selection of poly(A+) is not recommended in the case of largemRNAs because of a very poor yield and additional risks of mechanical damage (1).Thus, electrophoresis must be performed starting from total RNA. Moreover, in thecase of large mRNAs, no risk of misinterpretation of the data can be expected from thepresence of ribosomal bands.The methods for electrophoresis of RNA have been described in many books. Theprotocol below represents a modified version of the standard technique described in ref. 6,and only the modifications introduced to fractionate mucin RNAs are described in detail.1. Prepare the RNA samples: The optimal quantity is 10 µg (2 µL or less). Adjust to a finalvolume of 10 µL with deionized formamide (5 µL), deionized formaldehyde (1.78 µL),10x MOPS stock solution pH 7.0 (1 µL), and DEPC-treated water.2. Heat shock the samples to denature the RNA at 68°C for 10 min in a water bath and cool onice. Add 3 µL of loading buffer (see Subheading 2.2., item 7) and load the gel immediately.3. Run the gel (see Subheading 2.2., item 6) for 16 h at 30 V.4. Stop electrophoresis and cut off the molecular weight markers, and RNA control lanes.The different bands (markers) or ribosomal bands (control) appear as shadows when putonto a silica gel plate containing a fluorescent indicator F 254 (Merck) when exposed toUV illumination.3.3. Transfer and Crosslinking (see Note 4)1. Prior to transfer, soak the gel in 0.05 N NaOH with gentle shaking. Obtain the optimalsignal after treatment for 20 min (for a 3-mm thick gel).2. Then rinse the gel in DEPC-treated water and soak for 45 min in 20X SSC (see Subhead-ing 2.3., item 1).3. Use capillary transfer in 20X SSC to transfer the RNA from the gel in a standard manner(7) or via vacuum blotting for 1 h.4. The UV-crosslinking method proposed is based on tests designed to optimize the perma-nent binding of RNA to Hybond N+ membrane: bake at 80°C in a vacuum for 30 min andthen expose to 254 nm of UV light for 4 min. The filter is now ready for hybridization.3.4. Filter HybridizationA large variety of hybridization buffers are available and can be used with equalsuccess in the filter hybridization. In this method, all the probes used MUC1 (8), MUC2(9), MUC3 (10), MUC4 (11), MUC5AC (12), MUC5B (13), and MUC6 (14), and theapoB-100 probes (15–17) are labeled with [32P] dCTP using a commercial random-primed labeling kit according to the manufacturer’s protocol (see Subheading 2.4.,item 1). These probes are used at 1 × 106 cpm/mL and 106 cpm per lane.1. Preform prehybridization and hybridization in 10 mL of hybridization solution (see Sub-heading 2.4., item 5) for 2 and 16 h, respectively, at 42°C in a hybridization oven. Northern Blot of Large mRNAs 3092. Remove the filter and rinse with 50 mL of 2X SSPE (see Subheading 2.4., item 2) atroom temperature to remove most of the nonhybridized probe.3. Wash the membranes twice in 0.1X SSPE and 0.1% SDS buffer for 15 min at 65°C.4. After a final wash with 6X SSPE, at room temperature, wrap the membrane in plastic filmwhile moist. Expose to autoradiography at –80°C with an intensifying screen and KodakX-Omat film (Fig. 1).5. After analysis of the results, strip the filters by two washings in a 0.1% SDS boilingsolution for 15 min (this can be repeated if necessary, testing for remaining label byautoradiography).3.5. Estimation of the Sizes of Large Mucin mRNAsThe use of standard RNA molecular weight markers or total RNA controls (28Sand 18S ribosomal bands) is useful to evaluate the quality of electrophoretic migrationFig. 1. Efficiency of this improved protocol for large RNA isolation. Total RNA from thesame human colon mucosa specimen was isolated by the original guanidinium isothiocyanate-ultracentrifugation protocol (A) or by this improved method (B and C). In (A) a large smearfrom up to 20 kb to about 0.5 kb is detected by MUC2 probe while in (B) and (C) a discreteunique band is obtained. In (C) (compared to B) the efficiency of the transfer was increased atleast ten fold by using a pre-treatment of the gel with 0.05 N NaOH. 310 Porchet and Aubertbut is not adapted to determine large sizes (nonlinear curves). Moreover the demon-stration of the integrity of the RNA preparation using a probe such as β-actin, GAPDH,or 28S rRNA is misleading in the case of large RNAs because these probes hybridizeto short messages, for which the degradation problem is not encountered using usualprotocols of RNA preparation. Using the improved protocols presented here, we foundthat each of the MUC2-6 genes expresses mRNAs of much larger size than usuallyfound in eukaryotes (14–24 kb). Moreover, allelic variations in length of these mucintranscripts were observed, directly related to the variable number of tandem repeatpolymorphisms seen at the DNA level (1). So it is of great interest, since mucin geneexpression and polymorphism is implicated in the increase of susceptibility to anypathology, to estimate the size of the mucin transcripts. Because the largest transcriptsfound in standard RNA molecular size markers are not larger than 10 kb, we use apoB-100 (15–17) and MUC5B (Laine, A., unpublished results) probes, which detect largeunique transcripts of 14.1 kb (small intestine-colon) and 17.6 kb (bronchus, trachea,gallbladder, submaxillary glands), respectively (1,18).The standard curve is derived by using β-actin (see Fig. 2) (2 kb, point A), 28SrRNA (5 kb, point B) apoB-100 (14.1 kb, point C) ,and MUC5B (17.6 kb, point D) asstandards. The curve between points A and C is approximately a straight line. Thecurve between points A and D is a nonlinear curve of the following formula: Y = 31.05exp(–0.273 X), where Y represents size in kb and X represents distance migration incentimeters. A simple method of drawing this nonlinear curve consists in joining pointsA, B, and C (first straight line) and points C and D (second straight line). Using thismethod, we found that the largest transcrpts of human mucin were from MUC4 (24kb). Their size is deduced from the second straight line (point E). In the future, once ithas been precisely sized after complete sequencing, it will also be possible to use thelargest MUC4 allele, which is common, as an additional size standard for a better sizeestimation of mRNAs encoding mucins and other large RNAs.4. Notes1. Methods for isolation and analysis of large RNAs require the same precautions as for allother RNAs and involve using of pure high-grade analytical reagents and taking care toavoid accidental introduction of RNAses. Standard precautions can be used to avoid prob-lems with RNAses such as wearing gloves at all times, autoclaving buffers and deionizedor distilled water and decontaminating general laboratory glassware or plasticware with0,1% DEPC (6,7).2. Extreme care must be taken at each step during the experiments to prevent any risk ofmechanical degradation. Homogenization, the use of syringes, and vortexing are strictly avoided.3. After centrifugation, if the RNA pellet is difficult to dissolve, warm it to 45°C for 20 min.4. Soaking the gel in NaOH before transfer is an important step that must not be omitted andmust be optimized according to the gel thickness. This treatment partially hydrolyzes theRNA and improves (at least 10-fold) the efficiency of transfer of very large RNA species.AcknowledgmentsSpecial thanks to Dr. Virginie Debailleul for her contribution to the development ofthe method presented herein. The authors also thank Dr. Dallas Swallow for her con- Northern Blot of Large mRNAs 311stant encouragement and assistance in preparing the manuscript. Support was receivedfrom l’Association de Recherche sur le Cancer, the Comité du Nord de la LigueNationale sur le Cancer and the CH et U de Lille (n°96/29/9595).References1. Debailleul, V., Laine, A., Huet, G., Mathon, P., Collyn d’Hooghe, M., Aubert, J.P. andPorchet, N. (1998) Human mucin genes MUC2, MUC3, MUC4, MUC5AC, MUC5B andMUC6 express stable and extremely large mRNAs and exhibit a variable length polymor-phism: an improved method to analyse large mRNAs. J. Biol. Chem. 273, 881–890.2. Debailleul, V. (1997) Expression des gènes de mucines humaines: étude de la stabilité, del’hétérogénéité et du polymorphisme des ARNm. Thèse de Doctorat d’Université des Sci-ences de la Vie et de la Santé, Lille, France.3. Chomczynski, P. and Sacchi, N. (1987) Single-step method of RNA isolation by acidguanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159.4 Auffray, C. and Rougeon, F. (1980) Purification of mouse immunoglobulin heavy chainRNAs from myeloma tumor RNA. Eur. J. Biochem. 107, 303–314.Fig. 2. Estimation of the size of large mRNA species. The use of standard RNA molecularsize markers is not adapted to determine large sizes. We therefore recommend use of apoB-100and MUC5B for analysis of mucin messages. The standard curve is derived by using 4 points:β-actin (2 kb point A), 285 rRNA (5 kb, point B), apo-100 (14.1 kb, point C) and MUC5B(17.6 kb, point D). The best formula corresponding to the nonlinear curve joining these fourpoints is: Y = 31.05 exp (–0.273x) where Y represents size in kb and X represents distancemigration in centimeter. A simple method of drawing this curve is shown in this graph: a firststraight lane is obtained joining points A, B, and C. A second straight is obtained by joiningpoints C and D. An additional point E represents the size of the largest MUC4 mRNA messagewhich is deduced from this second straight lane. 312 Porchet and Aubert5. Chirgwin, J. M., Przybyla, A. E., McDonald, R. J., and Rutter, W. J. (1979) Isolation ofbiologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry18, 5294–5299.6. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular cloning: A LaboratoryManual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) pp. 7.3–7.52.7. Krumlauf, R. (1991) Northern blot analysis of gene expression, in Methods in MolecularBiology, vol. 7: Gene Transfer and Expression Protocols (Murray, E. J., ed.), Humana,Clifton, NJ.8. Gendler, S. J., Lancaster, C. A., Taylor-Papadimitriou, J., Duhig, T., Peat, N., Burchell, J.,Pemberton, L., Lalani, E., and Wilson, D. (1990) Molecular cloning and expression of ahuman tumor-associated polymorphic epithelial mucin. J. 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Degand, P., Aubert, J. P., and Porchet, N. (1995) Characterizationof the human mucin gene MUC5AC: a consensus cysteine-rich domain for 11p15 mucingenes? Biochem. J. 305, 211–219.13. Dufossé, J., Porchet, N., Audié, J. P., Guyonnet-Dupérat, V., Laine, A., Van Seuningen, I.,Marrakchi, S., Degand, P., and Aubert, J. P. (1993) Degenerate 87 base pair tandem re-peats create hydrophilic/hydrophobic alternating domains in human mucin peptidesmapped to 11p15. Biochem. J. 293, 329–337.14. Toribara, N. W., Robertson, A. M., Ho, S. B., Kuo, W. L., Gum, E., Hicks, J. W., Gum, J.R., Byrd, J. C., Siddiki, B., and Kim, Y. S. (1993) Human gastric mucin. Identification ofa unique species by expression cloning J. Biol. Chem. 268, 5879–5885.15. Chen, S. H., Yang, C. Y., Chen, P. F., Setzer, D., Tanimura, M., Li, W. H., Gotto, A. M.,and Chan, L. (1986) The complete cDNA and amino acid sequence of human apoliproteinB-100. J. Biol. Chem. 261, 12,918–12,921.16. Law, S. W., Grant, S. M., Higuhi, K., Hospattankar, A., Lackner, K., Lee, N., and BrewerH. B., Jr (1986) Human liver apolipoprotein B-100 cDNA complete nucleic acid and de-rived amino acid sequence. Proc. Natl. Acad. Sci. USA 83, 8142–8146.17. Cladaras, C., Hadzopuolou-Cladaras, M., Nolte, R. T., Atkinson, D., and Zannis V.J.(1986) The complete sequence and structural analysis of human apolipoprotein B-100relationship between apo B-100 and apo B-48 forms. EMBO J. 5, 3495–3507.18. Vinall, L. E., Hill, A. S., Pigny, P., Gum, J. R., Kim, Y. S., Porchet, N., Aubert, J. P., andSwallow, D. M. (1998) Variable number tandem repeat polymorphism of the mucin geneslocated in the complex on 11p15.5. Hum. Genet. 102, 357–366. . cultures (HT-29 MTX) and in tissues from various mucosae (trachea, bronchus,From :Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: . 10,000g for 30 min at 4°C, and let it air-dry. 308 Porchet and Aubert6. Redissolve the RNA pellet carefully in DEPC-treated water and quantify by measuringthe