Northern Blot of Large mRNAs 305
305
25
Northern Blot Analysis of Large mRNAs
Nicole Porchet and Jean-Pierre Aubert
1. Introduction
Northern blot analysis has historically been one of the most common methods used
to provide information on the number, length, and relative abundance of mRNAs
expressed by a single gene. This technique also generates a record of the total mRNA
content expressed by a cell culture or by a tissue, which can be analyzed and compared
on the same specimens by successive hybridizations with specific probes.
There are two main difficulties often associated with this technique. The fisrt is that
Northern blotting is generally considered to be rather insensitive, requiring large
amounts 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 high
purity 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 less
polydisperse 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 from
two 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 efficiency
of the transfer of large RNAs onto membranes, and thus the poor detection of the large
intact mRNA species.
Moreover, efficiency of large poly(A
+
)RNA selection is very poor with preferential
loss of the largest transcripts. Hence, there is a risk of misinterpretation of the data in
the 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 that
have successfully been used in our laboratory to examine mucin gene expression both
in 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 Mucins
Edited by: A. Corfield © Humana Press Inc., Totowa, NJ
306 Porchet and Aubert
stomach, colon, small intestine). These protocols can also be adapted to analyze other
large mRNAs such as ApoB transcripts (1,2).
2. Materials
2.1. 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 dissolving
23.6 g of guanidinium isothiocyanate, 73.5 mg of sodium citrate, and 250 mg of sodium
N-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 M
EDTA, 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. Adjust
to 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 3H
2
O in 800 mL of
water. Adjust pH to 5.5 with glacial acetic acid. Adjust volume to 1 L. Dispense in aliquots
and sterilize by autoclaving.

7. Ethanol: 100 and 95%.
2.2. Electrophoresis
1. Gel box, castinng tray, and combs: carefully clean with 0.1 N NaOH overnight or for at
least 3 h, rinse with distilled water (verify the pH), rinse with ethanol, and give a final
rinse with sterile distilled water just before use. Use gloves in all steps. Set up the casting
tray and comb in a fume hood because of toxic vapors given off during the pouring and
setting of the gel (hot formaldehyde).
2. DEPC-treated water.
3. 10X Morpholino-propane-sulfonic acid (MOPS) stock solution (0.2M MOPS): Dissolve
MOPS (10 g) in DEPC water (200 mL) containing 3 M sodium acetate (4.2 mL), and 0.5 M
EDTA, pH 8.0 (5 mL). Adjust the pH to 7.0 with 3 M NaOH and the final volume to 250 mL
with 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 M
MOPS, pH 7.0.
6. Denaturing gel: 0.9% agarose gel (13 × 18 × 0.3 cm) containing 18% formaldehyde and
MOPS 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 Crosslinking
1. 20X Sodium chloride sodium citrate (SSC) buffer: Dissolve 175 g of NaCl and 88.2 g of
trisodium 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 307
2.4. Filter Hybridization
1. 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 bovine
serum albumin are dissolved in 100 mL of H
2
O. 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. Methods
3.1. Preparation of mRNA (
see
Notes 1–3)
The original guanidinium isothiocyanate method is recognized for the purity and
quality of the RNA obtained. Guanidinium isothiocyanate combines the strong dena-
turing characteristic of guanidine with the chaotropic action of isothiocyanate and
efficiently solubilizes tissue homogenates. Effective disruption of cells can be obtained
without the use of a homogenizer, which has otherwise been used routinely, especially
when 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 guanidinium
isothiocyanate-phenol/chloroform (3), lithium chloride-urea (4), or different optimized
commercial total RNA preparation kits from Bioprobe (Montreuilsous Bois, France)
or Clontech Inc. (Palo Alto, CA). The best protocol to prepare intact large RNAs was
the following improved method that we developed, derived from the guanidinium
isothiocyanate protocol (5):
1. Grind cells (1.5 × 10
6
) or tissues (optimal weight of 1 g) to a fine powder in a mortar and
pestle in liquid nitrogen and mix with 10 mL of homogenization buffer (Subheading

2.1., item 2) still in liquid nitrogen.
2. Then allow the homogenate to thaw gradually at room temperature, during which time the
guanidinium 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 in
a Beckman SW41 rotor. Remove the supernatant, cut off the bottom of the tube, and
carefully resuspend the white pellet of total RNA in 2 × 1 mL TE buffer, pH 8.0 (see
Subheading 2.1., item 4), 0.1% SDS by using wide-mouth pipets, carefully avoiding all
shear 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 to
dissolve. During the washing steps, the tubes must be mixed only by gentle inversion, and
vortexing is strictly avoided.
5. Carefully remove the top aqueous phase, which contains the RNA, with wide mouth
pipettes and transfer it to a fresh tube. Precipitated the RNA by adding 0.1 vol of 3 M
sodium 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 then
100% ethanol, centrifuge it again at 10,000g for 30 min at 4°C, and let it air-dry.
308 Porchet and Aubert
6. Redissolve the RNA pellet carefully in DEPC-treated water and quantify by measuring
the A
260nm
of an aliquot.
7. Store the final preparation at –80°C until it is needed.
3.2. Electrophoresis
Studying the isolation of poly(A+)RNA by using standard protocols or more recent
systems (Poly [A] tract
®
RNA Isolation System from Promega, Charbonnieres, France),
we concluded that selection of poly(A+) is not recommended in the case of large

mRNAs because of a very poor yield and additional risks of mechanical damage (1).
Thus, electrophoresis must be performed starting from total RNA. Moreover, in the
case of large mRNAs, no risk of misinterpretation of the data can be expected from the
presence of ribosomal bands.
The methods for electrophoresis of RNA have been described in many books. The
protocol 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 final
volume 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 on
ice. 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 put
onto a silica gel plate containing a fluorescent indicator F 254 (Merck) when exposed to
UV 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 optimal
signal 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 and
then expose to 254 nm of UV light for 4 min. The filter is now ready for hybridization.
3.4. Filter Hybridization

A large variety of hybridization buffers are available and can be used with equal
success 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 the
apoB-100 probes (15–17) are labeled with [
32
P] 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 × 10
6
cpm/mL and 10
6
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 309
2. Remove the filter and rinse with 50 mL of 2X SSPE (see Subheading 2.4., item 2) at
room 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 film
while moist. Expose to autoradiography at –80°C with an intensifying screen and Kodak
X-Omat film (Fig. 1).
5. After analysis of the results, strip the filters by two washings in a 0.1% SDS boiling
solution for 15 min (this can be repeated if necessary, testing for remaining label by
autoradiography).
3.5. Estimation of the Sizes of Large Mucin mRNAs
The use of standard RNA molecular weight markers or total RNA controls (28S
and 18S ribosomal bands) is useful to evaluate the quality of electrophoretic migration
Fig. 1. Efficiency of this improved protocol for large RNA isolation. Total RNA from the
same 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 smear

from up to 20 kb to about 0.5 kb is detected by MUC2 probe while in (B) and (C) a discrete
unique band is obtained. In (C) (compared to B) the efficiency of the transfer was increased at
least ten fold by using a pre-treatment of the gel with 0.05 N NaOH.