In Situ Hybridization Applicable to
mRNA Species in Cultured Cells
RNA in situ
hybridization (ISH) techniques allow
the study of gene expression at the individual cell level
in a histocytomorphological context. These techniques
often involved the use of radioactive-labeled probes.
However, because of resolution drawbacks limiting
their applicability in the study of RNA distribution at
(sub)cellular sites, these probes are replaced more
frequently by hapten- or fluorophore-labeled ones.
Nevertheless, radioactive ISH is still useful for detecting
relatively low abundant RNA transcripts in tissue
From the early 1980s, several nonisotopic-labeling
methods have been developed that are most often
based on the introduction of haptens (e.g., biotin and
digoxigenin) or fluorochromes (e.g., fluorescein, rhodamine,
coumarin, Cy3, and Cy5) that are conjugated
to allyl or alkylamine-dUTP in DNA or RNA probes.
Such probes provide a high spatial resolution and
allow simultaneous detection of multiple RNA
sequences (Dirks et al.
, 1991; Levsky et al.
, 2002) or
RNA sequences together with proteins (Dirks, 1998;
Snaar et al.
Direct ISH techniques, using fluorochrome-labeled
probes, are used to detect relatively abundant mRNA
species by conventional fluorescence microscopy.
Using more advanced digital imaging microscopy,
visualization of single RNA transcripts proved even
possible when oligodeoxynucleotide probes were
labeled with five fluorochromes per molecule and 10
oligonucleotide probes were hybridized to adjacent
sequences on an RNA target (Femino et al.
Indirect ISH techniques, using haptenized probes,
are often the method of choice because of their better
sensitivity compared to direct fluorescence ISH
approaches (Dirks et al.
, 1993). Furthermore, enhancement
of ISH signals can be accomplished by applying
multiple antibody layers or by the use of a tyramidebased
detection method (Adams, 1992; Raap et al.
1995; van de Corput et al.
, 1998). Tyramide-based
detection methods involve the use of an antihapten
peroxidase-labeled antibody as a first antibody layer
and a biotin, DNP, or fluorochrome tyramide that
serves as a peroxidase substrate to generate and
deposit many reporter molecules close to the
This article describes a protocol that allows sensitive
detection of RNAs at cytoplasmic as well as at
nuclear sites of cells that are grown or attached to
II. MATERIALS AND
Dulbecco's modified Eagle medium without phenol
red (DMEM, Cat. No. 1188-36), fetal bovine serum
(FBS, Cat. No. 1050-64), l-glutamine (Cat. No. 2503-24),
penicillin-streptomycin (Cat. No. 1514-22), and
trypsin (Cat. No. 2509-28) are from Invitrogen Life
Technologies. Salmon testes DNA (Cat. No. D-7656),
dithiothreitol (DTT, Cat. No. D-0632), thimerosal (Cat.
No. T-5125), polyvinylpyrrolidone (PVP, Cat. No. PVP-
40), Tween 20 (Cat. No. P-1379), mouse monoclonal
antidigoxin (Cat. No. D-8156), rabbit antimouse fluorescein
isothiocyanate (FITC) (Cat. No. F-7506), Streptavidin-
FITC (Cat. No. S-3762), and goat antirabbit
FITC (Cat. No. F-9262) are from Sigma. Ficoll PM 400
(Cat. No. 1-30-0) is from Amersham Biosciences. Bovine serum albumin fraction V (BSA, Cat. No.
44155) is from BDH. Formamide (Cat. No. 7042) and
acetic acid (Cat. No. 6052) are from J. T. Baker. Amberlite
MB1 ion exchanger (Cat. No. 40701) and 4,6,-
diamidino-2-phenylindol 2HCl (DAPI, Cat. No. 18860)
are from Serva. Acid-free formaldehyde (Cat. No.
3999) is from Merck. dATP (Cat. No. 1051 440), dCTP
(Cat. No. 1051 458), dGTP (Cat. No. 1051 466), dTTP
(Cat. No. 1051 482), DNase I (Cat. No. 104 158),
digoxigenin-11-dUTP (Cat. No. 1093 088), sheep
antidigoxigenin HRP (Cat. No. 1207 733), and blocking
reagent (Cat. No. 1096 176) are from Roche. DNA
polymerase I (Cat. No. M2051) is from Promega. Vectashield
mounting medium is from Vector. Staining
jars (100ml) for object slides (Cat. No. L4110) are from
Agar Aids Ltd. TSA fluorescein system (Cat. No.
NEL701), TSA tetramethylrhodamine NEL702), TSA
coumarin system (Cat. No. NEL703), and TSA biotin
system (Cat. No. NEL700) are from NEN.
Fluorescence ISH results were examined with an
epifluorescence microscope (DM, Leica) equipped
with a 100-W mercury arc lamp and a triple excitation
filter for red, green, and blue excitation (Omega).
Digital images were captured with a cooled CCD
A. Labeling of DNA by Nick Translation
- Nick translation buffer (10x): 0.5M Tris-HCl, pH
7.8, 50mM MgCl2, and 0.5mg/ml BSA. To make 10ml
of the solution, add 5ml of an autoclaved 1M stock
solution of Tris-HCl, pH 7.8, 0.5 ml of an autoclaved
1M solution MgCl2, and 5 mg of nuclease-free BSA and
complete to 10ml with autoclaved distilled water.
Aliquot in 100-µl portions and store at -20°C.
- 0.1M DTT: To make 10ml, dissolve 150mg of
DTT in 10ml of 0.01M sodium acetate (pH 5.2). Filter
the solution through a 0.2-µm filter, aliquot in 1-ml
portions, and store at -20°C.
- Nucleotide mix: 0.5mM dATP, 0.5mM dCTP,
0.5mM dGTP, and 0.1mM dTTP. To make 1ml of the
solution, add 5µl each of 100mM stock solutions dATP,
dCTP, and dGTP and 1µl of 100mM stock solution
dTTP. Complete to 1ml with autoclaved distilled water
and store at -20°C.
- 1mg/ml DNase I: To make 1ml, add 1mg of
DNase, 20µl of a 1M stock solution of Tris-HCl, pH
7.6, 50µl of a stock solution of NaCl, 10µl of a 100mM stock solution of DTT, 0.1mg of BSA, and 0.5ml of
glycerol. Complete to 1ml with autoclaved distilled
water and store at -20°C.
- 10mg/ml salmon testes DNA: To make 10ml, dissolve
100mg DNAin 100ml 0.3M NaOH in TE, pH 7.8
(10mM Tris-HCl, pH 7.8, 1mM EDTA). Boil for 20 min
at 100°C. Neutralize the solution by adding 5ml of
2M Tris-HCl, pH 7.5, 7.5ml of 4M HCl, and 12ml
of 2M sodium acetate. Precipitate the DNA by adding
2 volumes of 100% -20°C ethanol and incubate for 1h
on ice. Centrifuge for 10min at 2600g, remove the
ethanol, and dissolve the DNA pellet in 10 ml TE buffer.
- Deionized formamide: To make 100ml, add 5g of
ion exchanger to 100ml formamide. Stir for 2h and
filter twice through Whatmann No. 1 filter paper.
Aliquot in 1-ml portions and store at -20°C.
- 20x SSC: To make 1 liter, add 175.3g NaCl and
88.24 g sodium citrate to distilled water. Adjust to pH
7.0 and complete to 1 liter. Autoclave the solution and
store at room temperature.
- 50X Denhardt's solution: 1% PVP, 1% Ficoll (type
400), and 1% BSA. To make 500ml of the solution, add
5g Ficoll, 5g PVP, 5g BSA (fraction V), and distilled
water to 500ml. Sterilize the solution by filtration and
store at -20°C.
B. Culturing and Fixation of Cells
10x PBS (pH 7.2)
- Thaw the required stock solutions and keep them
on ice. Prepare the labeling solution on ice and mix
well. To make the labeling mixture, combine 26µl
autoclaved distilled water, 5µl 10x nick translation
buffer, 5 µl 100mM DTT, 4 µl nucleotide mix, 2 µl digoxigenin-
11-dUTP (0.25mM stock solution), 1µl probe
DNA (1µg, e.g., cDNA), 2µl DNA polymerase I, and
5µl of a 1:1000 DNase I dilution from a 1-mg/ml stock
- Place the labeling mixture for 2 h in a 16°C water
- Add 250µl distilled water and precipitate the
labeled probe by adding 30µl of 3M sodium acetate,
pH 4.8, 5 µl salmon testes DNA, and 750µl -20°C 100%
ethanol. Mix well and place the solution on ice for
- Centrifuge for 30 min at 4°C in an Eppendorf centrifuge
at maximum speed. Remove the ethanol completely
and resuspend the pellet in 200µl hybridization
mixture to reach a probe concentration of 5ng/µl.
To make 10ml hybridization mixture, combine
5ml deionized formamide, 1ml 20X SSC, 1ml of
0.5M sodium phosphate buffer, pH 7.0, 1ml 50x
Denhardt's solution, and 2ml autoclaved distilled
water. The probe mixture can be stored at 4°C for
at least 1 year.
: Add 80g NaCl, 2g KCl, 15g
O, and 1.2 g KH2
and adjust to 1 liter.
Autoclave the solution and store at room temperature.
C. Pretreatment and Hybridization
- Trypsinize subconfluent cells that are grown in
tissue culture flasks as a monolayer in DMEM supplemented
with 10% FCS, 100 U of penicillin/ml, and
100µg of streptomycin/ml and seed into petri dishes
containing five microscopic object slides and 20ml
medium. Grow cells to subconfluency at 37°C in a
humidified 5% CO2 atmosphere.
- Wash the object slides containing cells in PBS for
I min at room temperature.
- Fix the cells in PBS containing 3.7% formaldehyde,
5% acetic acid for 20min at room temperature.
To make 100ml fixative, combine 75 ml distilled water,
10 ml 10x PBS, 10 ml formaldehyde (37% stock), and
5 ml acetic acid.
- Wash the cells in PBS for 5 min at room temperature
and transfer the object slides to a 100-ml staining
- Wash the cells in 70% ethanol and store the object
slides in 70% ethanol at 4°C until use.
: To make 100ml, dissolve 0.1 g pepsin in
distilled water, adjust pH to 2.0, and bring to 100ml.
Make this solution about 15 min before use and place
in a 37°C water bath.
D. Posthybridization Washes Solutions
- Wash the cells in PBS for 3 min at room temperature.
- Incubate the cells in 0.1% pepsin solution for 2min
- Wash the cells in 70% ethanol for 30s.
- Wash twice in PBS for 30s each.
- Incubate in 1% formaldehyde in PBS for 5 min at
- Wash in PBS for 5 min and dehydrate the cells successively
in 70, 90, and 100% ethanol for 3 min each.
- Denature the probe dissolved in hybridization
mixture for 5 min in an 80°C water bath.
- Place the probe for I min on ice and spin down in a
- Apply 10 gl of the denatured probe mixture on the
object slide and hybridize overnight at 37°C in a
moist chamber, which consists of a l-liter beaker covered with aluminium foil containing tissues
moistened with 50% formamide/2x SSC, pH 7.0.
- 50% formamide/2x SSC wash solution: To make
400ml, add 200ml formamide, 40ml 20x SSC, and
160ml distilled water and adjust the pH to 7.0.
- 10x TBS: To make 1 liter, dissolve 121.4g Tris and
87.4 g NaCl in 800ml distilled water. Adjust the pH
to 7.4 with 6 N HCl and bring to a total volume of
E. Conventional Immunocytochemical
- Wash the slides in the 50% formamide/2x SSC solution
at room temperature until the coverslips are
- Transfer the slides to a new jar filled with 100ml
50% formamide/2x SCC and wash for 10min.
- Wash the slides for 10min each in 50% formamide/
2x SSC at 42°C and in the same solution at
- Wash the slides in 2x SSC for 5min at room
- Wash the slides in 1× TBS for 5min at room
: To make 100ml, add 0.5 g blocking
reagent and 100µl thimerosal (to prevent bacterial
growth). Complete to 100ml with 1× TBS. Heat the
mixture for 1h at 60°C to dissolve the blocking reagent
and aliquot in 10-ml portions. Store at -20°C.
F. Tyramide Signal Amplification
- Take a slide from the TBS solution and apply 100µl blocking solution. Cover it with a 24 × 50-mm2 coverslip and incubate for 30 min at 37°C in a moist
chamber (l-liter beaker covered with aluminium
foil containing tissue moistened with water).
- Wash briefly with TBS to remove the coverslips.
- Drain of as much of the TBS solution as possible
and incubate the slides with mouse antidigoxigenin,
diluted 1:500 in blocking solution under
a coverslip for 45min at 37°C in a moist
- Remove the coverslips by a brief wash in TBS
and wash the slides 3 × 5min with TBS at room
- Drain of as much of the washing solution as
possible and incubate the slides with rabbit antimouse
FITC diluted 1:500 in blocking solution as
described in step 3.
- Wash the slides as described in step 4.
- Drain of as much of the washing solution as possible
and incubate the slides with goat anti-rabbit
FITC, diluted 1:500 in blocking solution, as
described in step 3.
- Wash the slides as described in step 4.
- Dehydrate the slides for 3 min each in 70, 90, and
100% ethanol and air dry.
- Apply 30µl Vectashield containing 10ng/µl DAPI
(blue fluorescent DNA counterstain) on a slide and
cover with a 24 × 50-mm2 coverslip.
- Examine the slides with a fluorescence microscope
equipped with appropriate excitation and emission
filters for FITC and DAPI fluorescence.
: To make 1 liter, dissolve 121.4g Tris and
87.4 g NaCl in 800ml distilled water. Ad 0.5 ml Tween
20. Mix thoroughly, adjust the pH to 7.4, and bring to
a total volume of 1 liter.
- Take slides from the TBS solution and apply 100µl
blocking solution. Cover with a coverslip and incubate
for 30min at 37°C in a moist chamber.
- Wash briefly with TBS to remove coverslips.
- Drain of as much of the washing solution as possible
and incubate the slides with antidigoxigenin-
HRP, diluted 1:500 in blocking solution, under a
coverslip for 45 min at 37°C in a moist chamber.
- Remove the coverslips by a brief wash in TNT and
wash the slides 3 × 5min with TNT at room
- Drain of as much of the TNT solution as possible
and apply 300µl of a 1:50 dilution of a fluorescent
(red, green, or blue) or biotin tyramide in 1× amplification
diluent on each slide. Incubate the slides for
approximately 10-30min at room temperature
without a coverslip.
- Wash the slides 3 × 5min with TNT at room
- For detection of biotin tyramide, incubate the slides
with steptavidin FITC diluted 1:1000 in blocking
solution as described in step 3.
- Wash the slides as described in step 4.
- Dehydrate the slides in ethanol, air dry, and mount
The protocol just described allows sensitive detection
of specific mRNAs in a variety of cell types,
including cultured cells, trypanosomes (Chaves et al.
1998), malaria parasites (Vervenne et al.
, 1994), and
blood cells. The various steps in this protocol have
been optimized for detecting RNA molecules in cultured
mammalian cells by trial and error (Dirks et al.
1993) and may need some adjustments for specific
applications. For example, if RNA FISH is applied at
the electron microscopic level of resolution, sample
preparation, fixation, and pretreatment conditions
|FIGURE 1 (A) Detection of HEF mRNA in
HeLa cells using a
plasmid probe. The hybridized probe was
visualized using a fluorescein-conjugated
secondary antibody. (B) A
hybridized HEF probe
was visualized using a peroxidase-labeled
antidigoxigenin antibody and fluorescein-tyramide.
(C) Detection of
IL-6 mRNA in 5637 bladder
carcinoma cells using a cocktail of three
HRP-labeled oligodeoxynucleotides and the TSA
(D) Detection of G-CSF mRNA in
cells using a cocktail of three
oligodeoxynucleotides and the TSA biotin system
details, see van de Corput et al., 1998).
need to be adjusted (Macville et al.
Compared to conventional immunocytochemical
detection systems, the use of tyramide-based detection
systems results in an at least a 10-fold increase in signal
intensity (Raap et al.
, 1995). This is illustrated in Fig. 1,
showing hybridization signals of human elongation
factor (HEF) mRNA in HeLa cells and of IL-6 and
G-CSF mRNA in human bladder carcinoma cells
grown on microscope slides. If required, the localization
properties of tyramides can be improved by the
addition of dextran sulfate to the amplification diluent
(Van Gijlswijk et al.
This hybridization protocol also allows bright-field
microscopic visualization of hybridization signals
when conjugates with peroxidase or alkaline phosphatase are used instead of fluorochrome antibody
conjugates (Dirks et al.
As a positive control on the procedure, probes specific
for housekeeping gene transcripts, like human
elengation factor and actin mRNA, or for rRNA, such
as 28S rRNA, can be used.
Finally, in order to study dynamic aspects of RNA
localization, methods have been developed that allow
hybridization of probes to RNAs and its subsequent
visualization in living cells (Molenaar et al.
- Probe labeling can be checked by performing a
filter spot test. Spot 1µl of a dilution series of the
labeled probe on a nitrocellulose filter and incubate
with sheep antidigoxigenin-alkaline phosphatase.
After performing the NBT/BCIP reaction, a probe concentration
of 5 to 1 pg/µl should be visible.
- We noticed that it is important to use for the
fixation of cells a good source of formaldehyde.
Formaldehyde solutions of poor quality may lead to
poor cell morphology, high levels of autofluorescence,
or weak hybridization signals.
- It is important to titrate the pepsin concentration
and/or time of incubation in order to find a balance
between morphology and signal intensity.
- For optimal results it is sometimes necessary to
denature the target mRNA. This is done just before
hybridization, after applying the probe solution and
coverslip on the slide, by placing the slide on an 80°C hot plate for 2 to 3 min.
- The dilution and reaction time of labeled tyramides
need to be optimized for each application. To
diminish nonspecific background staining, the first
antibody may be diluted further.
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