Use of acridine orange staining for the detection of rotavirus RNA in polyacrylamide gels.

Abstract

Acridine orange is a metachromatic intercalator used extensively in histochemistry to differentiate double- from single-stranded (ds, ss) nucleic acid by the emission of green and red fluorescence, respectively, under ultraviolet light. In the present study we standardised a protocol in order to use acridine orange to detect rotavirus ds RNA in polyacrylamide gels and compared it to silver and ethidium bromide staining. We demonstrated that the simplest and best condition was attained when gels containing rotavirus ds RNA bands, stained in green, were treated with 4.3 microM acridine orange after electrophoresis and destained with distilled water pH 6 at 37 degrees C. Under this protocol, rotavirus RNA concentration was calculated and the mean minimum amounts of nucleic acid detected by acridine orange, ethidium bromide, and silver staining were 26.0 +/- 4.29, 15.6 +/- 1.48 and 1.06 +/- 0.11 ng, respectively. The comparison of acridine orange sensitivity with ethidium bromide and silver staining, for 25 field strains of rotavirus and one cell-adapted strain (SA11), demonstrated concurrent results in 80% of the specimens. Red colour emission resulting from the interaction of acridine orange with ss nucleic acid was also shown by testing denatured 0.5 kb HindIII digest of lambda phage DNA. Furthermore, it was demonstrated that rotavirus ds RNA could be used for reverse transcription activity, followed by PCR amplification, after acridine orange staining. In conclusion, although acridine orange is less sensitive than ethidium bromide and silver staining, its practicality, low cost, metachromatic properties, and its non-interference on RT-PCR should be considered. It is suggested the use of acridine orange as an appropriate stain for various purposes in virology, as well as for the molecular biology of nucleic acid.

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Journal of Virological Methods 114 (2003) 29–35
Use of acridine orange staining for the detection of rotavirus RNA
in polyacrylamide gels
Flávio Laurettia, Fernando Lucas de Meloa, Fabr´ ıcio José Benatia, Eduardo de Mello Volotãob,
Norma Santosb, Rosa Elisa Carvalho Linharesa, Carlos Nozawaa,∗
aDepartamento de Microbiologia, Centro de Ciˆ encias Biológicas, Universidade Estadual de Londrina, CEP 86051-900, Londrina, Paraná, Brazil
bDepartamento de Virologia, IMPPG, Universidade Federal do Rio de Janeiro, CEP 21941-590, Rio de Janeiro, Brazil
Received 15 May 2003; received in revised form 18 August 2003; accepted 20 August 2003
Abstract
Acridine orange is a metachromatic intercalator used extensively in histochemistry to differentiate double- from single-stranded (ds, ss)
nucleicacidbytheemissionofgreenandredfluorescence,respectively,underultravioletlight.Inthepresentstudywestandardisedaprotocol
in order to use acridine orange to detect rotavirus ds RNA in polyacrylamide gels and compared it to silver and ethidium bromide staining.
We demonstrated that the simplest and best condition was attained when gels containing rotavirus ds RNA bands, stained in green, were
treated with 4.3?M acridine orange after electrophoresis and destained with distilled water pH 6 at 37◦C. Under this protocol, rotavirus
RNA concentration was calculated and the mean minimum amounts of nucleic acid detected by acridine orange, ethidium bromide, and
silver staining were 26.0 ± 4.29, 15.6 ± 1.48 and 1.06 ± 0.11ng, respectively. The comparison of acridine orange sensitivity with ethidium
bromide and silver staining, for 25 field strains of rotavirus and one cell-adapted strain (SA11), demonstrated concurrent results in 80% of the
specimens. Red colour emission resulting from the interaction of acridine orange with ss nucleic acid was also shown by testing denatured
0.5kb HindIII digest of lambda phage DNA. Furthermore, it was demonstrated that rotavirus ds RNA could be used for reverse transcription
activity, followed by PCR amplification, after acridine orange staining. In conclusion, although acridine orange is less sensitive than ethidium
bromideandsilverstaining,itspracticality,lowcost,metachromaticproperties,anditsnon-interferenceonRT-PCRshouldbeconsidered.Itis
suggestedtheuseofacridineorangeasanappropriatestainforvariouspurposesinvirology,aswellasforthemolecularbiologyofnucleicacid.
© 2003 Elsevier B.V. All rights reserved.
Keywords: Acridine orange; Nucleic acid; Metachromasia; Rotavirus
1. Introduction
The fluorescence of intercalating agents such as acridine
orange and ethidium bromide (Glazer and Rye, 1992) is in-
creased when bound to nucleic acid in an aqueous mellieu
when observed under ultraviolet light (Le Pecq and Paoletti,
1966; Kapuscinski et al., 1982). Acridine orange has been
used extensively in cytochemistry to monitor flow cytom-
etry, nuclear fragmentation, and pH gradients in biological
membranes, and for various other applications (Palmgren,
1991; Zelenin, 1993; Hitoshi et al., 1998). Acridine orange
is a basic stain whose planar structure is derived from an-
thracene. It shows a high affinity for nucleic acid, and in-
∗Corresponding author. Tel.: +55-43-3371-4617.
E-mail address: cnoz@uel.br (C. Nozawa).
tercalates into ds or binds electrostatically to ss nucleic acid
emitting a green fluorescence at 530nm and red fluorescence
at 640nm, respectively, under UV light (Kapuscinski et al.,
1982; Kapuscinski, 1990; Zelenin, 1993). This characteris-
tic confers metachromatic property on the stain. Ethidium
bromide is a heterocyclic stain and a derivative of phenan-
thracene, which intercalates in ds and ss nucleic acid emit-
ting red fluorescence under UV light. The interaction of
ethidium bromide with nucleic acid is similar to that of acri-
dine orange, except that it does not display metachromatic
properties (Lillie, 1977; Carmichael and McMaster, 1980;
Bruno et al., 1996). Silver staining, on the other hand, is
based on the binding of silver ion in nucleic acid phosphate
groups (Lewin, 1994) followed by its reduction in alkaline
medium.
Rotaviruses are the causal agents of acute diarrhoea in
young mammals, including humans, and birds (Estes, 1996).
0166-0934/$ – see front matter © 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.jviromet.2003.08.005

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F. Lauretti et al./Journal of Virological Methods 114 (2003) 29–35
Fig. 1. Rotavirus strain OWC 7 submitted to electrophoresis in a 7% polyacrylamide gel loaded with 130ng virus RNA for each well, and stained
with acridine orange at indicated concentrations. Lanes 1–8 correspond to the concentrations of 2.1, 4.3, 6.4, 8.6, 10.8, 12.9, 15.1 and 17.2?M acridine
orange, respectively. Orange filter YA3 was used.
Human strains are responsible for approximately 800,000
deaths yearly, in population under 5 years of age, in devel-
oping countries (Parashar et al., 1998). Virions are 70nm
in diameter and their genome is represented by eleven seg-
ments of ds RNA which can be resolved by electrophore-
sis in a well-defined band pattern (electropherogram) (Estes,
1996; Ramos et al., 2000).
In experimental cytopathology, acridine orange was used
in herpes virus-infected cultured cell (Ross and Orlans,
1958), and latter to differentiate ss- from ds-containing
viruses (Gomatos et al., 1962). Rotavirus detection in
cell culture was also monitored by acridine orange (Estes
et al., 1979), as well as, the timely appearance of cyto-
plasmic metachromasy combined with antigen detection,
virus yields, and ultrastructural features (Guimarães and
Nozawa, 1990). Although acridine orange has been used
extensively in cytochemistry and cytopathology, nothing
has been reported about its application in quantitative an-
alytical virology. Therefore, the aim of the present study
was to establish a protocol for an acridine orange staining
method for rotavirus ds RNA, as well as, take advantage of
its metachromatic properties.
2. Materials and methods
2.1. Virus
Most of the rotavirus strains used were derived from
19 bovine and six human faecal specimens supplied by
Laboratorio de Virologia Animal (DMVP/UEL), Labo-
ratorio de Microbiologia (HURNP/UEL), and Labora-
torio Oswaldo Cruz. Clarified faecal homogenates and
the simian strain (SA11) adapted to cell culture were
submitted to NA extraction as described by Herring
et al. (1982). For RT-PCR virus RNA was extracted with
Table 1
Comparative detection of human (OWC, PAT, PSP and T) and bovine
(EBP and Ba) rotavirus field strains, and a cell culture-adapted simian
strain (SA11) by acridine orange, silver, and ethidium bromide staining
Strains Acridine orangeSilverEthidium bromide
EBP 311
EBP 531
EBP 535
EBP 544
EBP 549
EBP 562
EBP 579
EBP 622
EBP 636
EBP 637
EBP 421
EBP 513
EBP 558
Ba 968
Ba 1102
Ba 1103
Ba 1104
Ba 1108
Ba 1111
OWC 7
OWC 41
OWC 42
PAT 10
PSP 16
T 01
SA11
+
−
+
−
+
−
−
−
−
+
+
+
+
+
−
−
+
+
+
+
+
+
+
+
+
+
+
+
+
−
+
+
+
−
+
+
+
+
+
+
−
+
+
+
+
+
+
+
+
+
+
+
+
+
+
−
+
−
−
−
+
+
+
+
+
+
−
+
+
+
+
+
+
+
+
+
+
+

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F. Lauretti et al./Journal of Virological Methods 114 (2003) 29–35
31
Fig. 2. Rotavirus strain OWC 42 submitted to electrophoresis in a 7% polyacrylamide gel stained with (A) silver, (B) ethidium bromide, and (C) acridine
orange. Lanes 1–8 correspond to the concentrations of 172, 43, 21.5, 10.8, 5.4, 2.7, 1.3 and 0.67ng of virus RNA, respectively. A C12 filter was used
for A, and orange filter YA3 for B and C.

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F. Lauretti et al./Journal of Virological Methods 114 (2003) 29–35
Trizol (Invitrogen, USA) according to manufacturer’s
recommendation.
2.2. Virus RNA determination by silver staining
Rotavirus ds RNA was resolved by polyacrylamide
gel electrophoresis (Herring et al., 1982) in 7% slab gel
for 90min at 25mA. Lambda phage DNA HindIII digest
(Gibco BRL, USA), previously heated at 65◦C for 10min
and chilled on an ice bath, according to manufacturer’s rec-
ommendation, was used as a standard at amount of 0.25?g
and 0.75?g. After staining, gels were analysed by quanti-
fying the RNA bands using Pharmacia Image Master VDS
1D Prime software.
2.3. Acridine orange staining
Rotavirus nucleic acid dried extracts (Ramos et al., 2000)
treated with dissociation buffer were incubated for 10min
with acridine orange (Inlab/Brazil), which was prepared at
4.3mM in distilled water and diluted to final concentrations
varying from 2 to 21?M. Electrophoresis was performed
and gels observed under UV light in a transilluminator fit-
ted with midrange 300nm bulbs. Alternatively, gels were
submitted to various test procedures after electrophoresis
and before staining. Gels were submitted to washings ei-
ther with 20% ethanol-2% acetic acid, PBS, McIlvaine’s
buffer pH 3.3 or pH-corrected (HCl/NaOH) distilled water
at varying pH from 3 to 9. After these treatments gels were
stained with acridine orange at the concentrations varying
from 2 to 21?M, and afterward destained either with PBS,
McIlvaine’s buffer or distilled water at varying pH from 3
to 9, either at 4◦C or 37◦C.
2.4. Silver and ethidium bromide staining
Silver nitrate (Merck, Brazil) and ethidium bromide
(Sigma,USA)stainingwerecarriedoutaccordingtoHerring
et al. (1982) and Sambrook et al. (1989), respectively.
2.5. Acridine orange staining metachromasia
A 2.5?g quantity of lambda phage DNA HindIII di-
gest/40mM Tris–HCl (pH 7.5); 20nM MgCl2; 50mM NaCl
was denatured in boiling water for 3min and then immedi-
ately transferred to an ice bath (Simmonds and Chan, 1993).
A 1.5?g quantity of the same standard, in distilled water,
was denatured by heating at 95◦C either for 5 or 10min and
chilled in an ice bath. One percent dimethyl sulphoxide was
added and DNA submitted to electrophoresis in 7% poly-
acrylamide gels containing 2M urea, for 95min at 23mA.
Gels were stained with 4.3?M acridine orange and observed
under UV light. In parallel, non-treated original DNA digest
was co-electrophoresed as a control.
2.6. RT-PCR
Dried extracts of virus RNA were re-suspended in either
40?L 4.3?M acridine orange or 40?L distilled water and
submitted to RT-PCR as described elsewhere (Das et al.,
1994). Copies of group A rotavirus VP7 gene were gener-
ated.
3. Results
In order to test the best protocol for acridine orange stain-
ing, gels were loaded with 130ng of rotavirus ds RNA per
Fig. 3. Acridine orange metachromasia demonstrated with lambda phage
DNA HindIII digest in a 7% polyacrylamide gel containing 2M urea/1%
dimethyl sulphoxide and stained with 4.3?M acridine orange. Wells 1–4
were loaded, respectively, with 1.0, 2.5, 1.5 and 1.5?g lambda phage
DNA HindIII digest, submitted to: Lane 1, without any treatment. Lane
2, denatured by heating at 95◦C for 3min in Tris–HCl, pH 7.5; 20nM
MgCl2; 50mM NaCl and chilled in ice bath. Lane 3, denatured by heating
at 95◦C for 5min in distilled water and chilled in ice bath. Lane 4,
denatured by heating at 95◦C for 10min in distilled water and chilled in
ice bath. Reddish bands bellow 2kb fragments are shown in Lanes 2–4
(arrow), while all other bands stained in green, under UV light. Orange
filter YA3 was used.

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F. Lauretti et al./Journal of Virological Methods 114 (2003) 29–35
33
Fig. 4. RT-PCR product for group A rotavirus VP7 gene. Lanes 2 and 4, RNA extracts of human virus field strain OWC 7 (genotype G9, 110bp) and
strain OWC 42 (genotype G1, 158bp), respectively, resuspended in distilled water. Lanes 3 and 5, the same strains, respectively, OWC 7 and OWC 42,
resuspended in 4.3?M acridine orange. Lane 1, 100bp marker. Lane 6, Blank.
well, throughout the experiments, unless otherwise stated.
When acridine orange staining was performed before elec-
trophoresis at a dye concentration of 21?M, we demon-
strated that all eleven bands of virus ds RNA emitted a
weak green fluorescence. However, at concentrations higher
than 21?M altered band mobility was observed, mainly for
those larger segments of rotavirus electropherogram. On the
other hand, when gels were stained after electrophoretic run,
washings were necessary to remove excess dye that pro-
duced a slight greenish fluorescence of the gel matrix; how-
ever, all the ds RNA bands were clearly visible and had a
sharp green colour. With this procedure, acridine orange was
tested at concentrations varying from 2.1 to 17.2?M with-
out affecting nucleic acid visualisation, as shown in Fig. 1.
We tested either McIlvaine’s buffer pH 3.3 or PBS pH 7.3
or distilled water as the appropriate diluent for acridine or-
ange and found the latter to be more suitable. Distilled wa-
ter at pH 3–9 produced readings [19.9±3.4 (mean±S.D.)]
that did not differ significantly at the different pH levels ex-
amined (ANOVA, P < 0.05). However, the optimum acri-
dine orange concentration, with minimum gel background
staining, was determined to be 4.3?M (Fig. 1, lane 2). The
use of fixative, such as ethanol-acetic acid, was found to
be unnecessary because gels kept at 4◦C in distilled water
maintained RNA bands intact, for several days. The fixa-
tive caused gel overstaining. The use of McIlvaine’s buffer
pH 3.3 as gel washing solution before staining did not help
whatsoever, and let to greater overstaining of the gel matrix,
although, virus RNA bands could still be observed. There-
fore, gels stained in acridine orange at 4.3?M in neutral
pH distilled water for 10min and destained also with dis-
tilled water at pH 6 for 15min at ambient temperature, were
considered satisfactory. Under the conditions established for
acridine orange staining, rotavirus field strains and SA11
strain submitted to electrophoresis were stained and com-
pared to those stained with silver and ethidium bromide.
Considering the observation of complete electropherogram
of virus ds RNA (11 bands), it was found that 80% of the
results were concurrent, in which there were 18 positives
and 3 negatives for the different methods (Table 1). In or-
der to test the minimum amount of ds RNA detectable by
the staining procedures, dilutions of the strain OWC 42 con-
taining varying amount of ds RNA, from 172 to 0.67ng,
were electrophoresed and stained with acridine orange, sil-
ver and ethidium bromide. The results demonstrated that
the minimum amount detected, where the complete electro-
pherogram could be observed, was 1.3, 10.8 and 21.5ng of
ds RNA for silver, ethidium bromide and acridine orange,
respectively (Fig. 2). In order to demonstrate acridine or-
ange metachromasia, denatured 0.5kb fragments of lambda
phage DNA HindIII digest were electrophoresed (Fig. 3)
and shown as reddish bands with intact fragments staining
green. Fig. 4 shows the product of RT-PCR for group A ro-
tavirus VP7 gene after viral RNA intercalation with acridine
orange, for strains OWC 7 and OWC 42 characterised as
genotypes G9 (110bp) and G1 (158bp), respectively.
4. Discussion
Gel electrophoresis is one of the methods used to detect
rotavirus RNA, and has been also employed for the study of

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F. Lauretti et al./Journal of Virological Methods 114 (2003) 29–35
the molecular epidemiology of the disease. This approach
is useful not only in detecting intraspecies strain variation,
but also in exploring interspecies transmission, interspecies
recombination and species differentiation (Hoshino et al.,
1995; Kapikian and Chanock, 1996). Among the gel staining
protocols, silver staining, which is based on silver ion bind-
ing to phosphate backbones of nucleic acid chains (Lewin,
1994) or either to nitrogen 7 of guanine or adenine (Arakawa
et al., 2001), has been used. Although silver staining shows
high sensitivity, capable of detecting 0.3–0.4ng/band of ro-
tavirus ds RNA (Herring et al., 1982) it is time-consuming
and relatively costly. Alternatively, ethidium bromide has
been used as a straightforward staining method with sensi-
tivity of 0.29–1ng of DNA (Sambrook et al., 1989; Tuma
et al., 1999), and has also been used to detect rotavirus ds
RNA. The relative specificity of ethidium bromide for nu-
cleic acid is probably due to the high negative charge density
of phosphate groups, sugars and hydrogen bonding opportu-
nities in DNA and RNA, and this is also applied for acridine
orange (Bruno et al., 1996). Another disadvantage of com-
monly used silver staining is that stained nucleic acid can-
not be recovered for molecular studies (Peats, 1984). On the
contrary, ethidium bromide protocol does allow nucleic acid
recovery (Sambrook et al., 1989). Acridine orange is also
known as supravital dye and is cytotoxic only at high con-
centrations of about 1.0–10mM (Zelenin, 1993). Therefore,
it is also proposed that acridine orange at the concentration
used would not interfere with nucleic acid recovery, allow-
ing further molecular analysis. It was demonstrated that the
luminescence of acridine orange-stained nucleic acid could
be completely removed by washing the gels and that nucleic
acid can be recovered for reverse transcription, demonstrat-
ing the possibility for further molecular studies. In conclu-
sion, it was found that although acridine orange staining
is the least sensitive method, its use is more practical and
cost-efficientwhencomparedtosilverandethidiumbromide
and it allows the distinction of ss from ds nucleic acid, and
nucleic acid can be recovered for further molecular analysis.
Acknowledgements
We thank to Prof. A. Alfieri (DMVP/UEL), Laboratório
de Análises Cl´ ınicas Oswaldo Cruz/PR, and Laboratório de
Microbiologia Cl´ ınica (HURNP/PR) for animal and human
clinical specimens, and Prof. Édio Vizoni (MAP/UEL) for
help with statistical analysis. We are also grateful to CAPES,
CNPqandCPG/UELforfinancialsupport.F.L.wasrecipient
of scholarship also granted by CAPES, and this work is part
of his M.Sc. manuscript.
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