Analytical Biochemistry 434 (2013) 6066

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Analytical Biochemistry
journal homepage: www.elsevier.com/locate/yabio

Whole-mount in situ detection of microRNAs on Arabidopsis tissues

using Zip Nucleic Acid probes
M. Begheldo a,,1, F.A. Ditengou b,1, G. Cimoli c, S. Trevisan a, S. Quaggiotti a, A. Nonis a,
K. Palme b,d,e,f,g,, B. Ruperti a
a
Department of Agronomy, Food, Natural Resources, Animals and Environment, University of Padova, 35020 Legnaro (Padova), Italy
b
Institute of Biology II/Botany, Faculty of Biology, Albert-Ludwigs-University of Freiburg, D-79104 Freiburg, Germany
c
Tecan Italia S.r.l., Cernusco Sul Naviglio, 20063 Milano, Italy
d
Centre of Biological Systems Analysis, Albert-Ludwigs-University of Freiburg, D-79104 Freiburg, Germany
e
Freiburg Institute of Advanced Sciences, Albert-Ludwigs-University of Freiburg, D-79104 Freiburg, Germany
f
Centre for Biological Signalling Studies, Albert-Ludwigs-University of Freiburg, D-79104 Freiburg, Germany
g
Freiburg Initiative for Systems Biology, Albert-Ludwigs-University of Freiburg, D-79104 Freiburg, Germany

a r t i c l e i n f o a b s t r a c t

Article history: MicroRNAs (miRNAs) affect fundamental processes of development. In plants miRNAs regulate organ
Received 20 August 2012 development, transition to owering, and responses to abiotic/biotic stresses. To understand the biolog-
Received in revised form 26 October 2012 ical role of miRNAs, in addition to identifying their targeted transcripts, it is necessary to characterize the
Accepted 31 October 2012
spatiotemporal regulation of their expression. Many methods have been used to dene the set of organ-
Available online 10 November 2012
specic miRNAs by tissue dissection and miRNA proling but none of them can describe their tissue and
cellular distribution at the high resolution provided by in situ hybridization (ISH). This article describes
Keywords:
the setup and optimization of a whole-mount ISH protocol to target endogenous miRNAs on intact Ara-
ISH
Whole mount ISH
bidopsis seedlings using DIG-labeled Zip Nucleic Acid (ZNA) oligonucleotide probes. Automation of the
miRNA main steps of the procedure by robotized liquid handling has also been implemented in the protocol
ZNA for best reproducibility of results, enabling running of ISH experiments at high throughput.
Robotized liquid handling 2012 Elsevier Inc. All rights reserved.

In the past two decades thousands of microRNAs (miRNAs)2 have
been identied among animals, plants, and viruses, thus raising them
to the most abundant class of small (2025 nt) non-protein-coding
RNAs (miRBase [1]). Although there is no evidence of similarities be-
tween miRNAs of animals and plants, they are conserved in distantly
related species ranging from mosses to higher owering plants and
from worms to animals [2]. For this reason it has been suggested that
they might play a key role in all kingdoms in regulating essential bio-
logical processes such as stem cell differentiation, signal transduction,
disease, and responses to environmental stresses [35].
As regulatory molecules miRNAs control gene expression at the
post-transcriptional level by three major mechanisms, namely
translational repression, exonucleolytic decay of mRNA, or direct
mRNA degradation (slicing). This last case often occurs in plants
(even though not exclusively) [6] and it is due to the high similarity
Corresponding authors. Fax: +39 049 827 2850 (M. Begheldo), +49 761 203
2629 (K. Palme).
E-mail addresses: maura.begheldo@unipd.it (M. Begheldo), klaus.palme@biolo-
gie.uni-freiburg.de (K. Palme).
1
These authors contributed equally to this work.
2
Abbreviations used: miRNA, microRNA; ISH, in situ hybridization; qPCR, quanti-
tative real-time PCR; ZNA, Zip nucleic acid; DIG, digoxigenin; GUS, b-glucuronidase;
GFP, green uorescent protein; QC, quiescent center.
between plant miRNAs and their corresponding targeted mRNAs.
Because of this specic interplay between plant miRNAs and their
targets, there are only a few targets for a majority of miRNAs in
plants, while, for instance, there can be many for each animal miR-
NA [7].
Traditional genetic approaches based on microarray technology,
analysis of target expression using miRNA loss-of-function mu-
tants [8,9], and computational prediction [10,11] have been suc-
cessfully employed to identify new small RNA molecules and
their target sequences.
qPCR and microarray technology have been used to study miR-
NA expression patterns and thus infer their presumptive function.
Nevertheless, even though these techniques ensure nearly quanti-
tative analysis of expression in a large-scale approach, only in situ
hybridization (ISH) allows one to determine directly the tissue dis-
tribution of specic small non-coding RNA molecules and their tar-
geted sequences. ISH, if compared with other expression proling
methods, gives a far better spatiotemporal denition of RNA regu-
lation at high resolution. In the case of miRNAs, this may be further
facilitated by the fact that they are present at high copy numbers
(mammalians) [12] or usually highly expressed (plants) [13], thus
making easier their cellular and subcellular localization by ISH
with sufcient sensitivity. Conversely, a major drawback of ISH

0003-2697/$ - see front matter 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ab.2012.10.039
 Whole mount Arabidopsis miRNAs detection by ZNA probes/M. Begheldo et al. / Anal. Biochem. 434 (2013) 6066
was considered to be its low throughput in comparison to qPCR
and microarray techniques, because of its requirement for labori-
ous procedures. However, this limitation has been partially over-
come through the development of robotized platforms through
which ISH procedures can be automated and standardized, thus
raising ISH throughput to relatively high levels ([12]; Gene Paint
Project http://www.genepaint.org).
Because of the chemical and structural nature of miRNA mole-
cules (short and often GC-rich), the localization of miRNAs with
satisfactory reliability and specicity requires carefully developed
dedicated protocols.
A decisive improvement in the in situ detection of specic miR-
NAs has been provided by the development of locked nucleic acid
(LNA) probes, which overcame the low capability to enhance the
signal-to-noise ratio owing to the use of 20-nt-long unmodied
probes [14]. In fact, the LNA modication (a methylene bridge con-
necting the 20 oxygen atom with the 40 carbon atom of the ribose
backbone) locks the hybridized duplex so that the acquired rigid-
ity leads to increased melting temperature of the complex made by
the LNA probe and its complementary miRNA. This leads to a sig-
nicant reduction in the background, arising from the non-specic
binding of probes in ISH experiments, by enabling highly stringent
hybridization/washing conditions. Since LNA probes were rst
introduced into the ISH protocol a number of technical applications
have been published [1518]. Recently Trevisan et al. [19] showed
that another type of modied nucleotides, namely ZNA (Zip Nu-
cleic Acid) oligonucleotides, already adopted for real-time PCR
[20], can be used on plant sections to detect in situ expression of
miRNAs. The stability of the ZNAprobemiRNA duplex, different
from LNA, is due to the presence of cationic spermine units (Z)
linked at the 50 end of the oligonucleotide, so that increased afnity
for the target is achieved by decreasing electrostatic repulsion be-
tween the negatively charged anionic single-strand nucleic acid
target and the probe [20].
Here we describe the use of the ZNA oligonucleotides for the
optimization of a whole-mount protocol for localization of miRNAs
in Arabidopsis thaliana seedlings. We provide evidence that ZNA
oligonucleotides can be used for ISH on both whole mounts and
sections of Arabidopsis seedlings, thus offering a valid and more
economic alternative to the use of LNA probes. We also show that
automation of the whole-mount protocol described in this paper
supports the use of ZNA oligonucleotides and miRNA localization
by ISH at high-throughput levels.
Materials and methods
Plant material and xation
Three- to ve-day-old seedlings of A. thaliana ecotype Columbia
(Col-0) were grown on half-strength Murashige & Skoog medium
at 22 C under short-day conditions (8 h light/16 h dark). For the
whole-mount ISH protocol, seedlings were xed in a 1:1 (v/v) mix-
ture of xation solution (5% paraformaldehyde (PFA), 15% dimethyl
sulfoxide, 0.08 M EGTA, 0.1% Tween in phosphate-buffered saline
(PBS), pH 7.4) and heptane for 30 min [21]. For experiments on sec-
tions, plants were xed in 4% paraformaldehyde in PBS for 2 h at
room temperature, vacuum inltrated for 5 min three or four
times, and left overnight in xation solution at +4 C. After xation,
the plants were embedded in Paraplast [22] for later sectioning and
ISH [19].
Whole-mount in situ hybridization
Five 50 -DIG-labeled ZNA-4 modied oligonucleotides containing
four spermine units (4Z; Metabion International AG, Martinsried,
61
Germany) [23] were chosen for selected known mature miRNA
sequences (listed in Table 1, miRbase registry http://microrna.
sanger.ac.uk/sequences). These probes were used to perform
whole-mount experiments on Arabidopsis seedlings by testing
serial concentrations at 15, 30, 50, and 70 nM. A ZNA-modied
oligonucleotide designed to target the mouse mmu-miR124a was
used as negative control [39].
After xation, seedlings were dehydrated through a graded eth-
anol series (30%, 50%, 70%, and 100% in 1 PBS Triton 0.01% (PBST),
30 min each) and stored in 100% methanol at 20 C until hybrid-
ization. Before use xed plants were rinsed in 100% ethanol for
30 min at room temperature. Sample permeabilization steps were
carried out following the protocol described by Hejtko et al. [40]
with some modications. After Histo-Clear treatment and rehydra-
tion through a graded ethanol series, plants were washed twice in
0.85% NaCl solution for 5 min at room temperature and immersed
in 0.2 mM HCl for 20 min and again washed in 0.85% NaCl for
10 min. Proteinase K digestion (70 lg/ml in 0.02 mM TrisHCl,
pH 7.5, 2 mM CaCl2 buffer) was run for 40 min at 37 C and stopped
by 0.2% glycine in PBST for 5 min at room temperature (RT). Plants
were washed with PBS twice for 5 min and subsequently xed
again in 4% PFA with PBST for 30 min at RT and rinsed overnight
in 0.1 M triethanolamine, pH 8.0, supplemented with 0.1% (v/v)
acetic anhydride. Plants were subsequently exposed for 20 min
to vapors of TrisEDTA, pH 9, buffer (DAKO, Glostrup, Denmark)
heated at 90 C in a water bath and prehybridized for 2 h at
50 C in 50% formamide, 2 standard saline citrate (SSC), and
0.1 mg/ml heparin. Samples were hybridized for 16 h at 50 C in
a solution made up of 50% formamide, 2 SSC, 2.5 Denhardts
solution, 0.5% dextran sulfate , 500 lg/ml tRNA, and a 5070 nM
concentration of denatured miRNA probe (70 C for 5 min). After
hybridization, seedlings were consecutively washed once with TN
buffer (0.1 M TrisHCl, pH 7.5, 150 mM NaCl) for 15 min at 37 C
and with a range of SSC solutions (1.5 SSC at 45 C, 1.0 at
50 C, and 0.5 at 50 C) 5 times and for 10 min each. A nal 5-
min rinsing step with PBST at 50 C was included and repeated 3
times. Blocking was carried out with 2% bovine serum albumin in
PBST solution at RT for 60 min, followed by incubation at +4 C
(or alternatively 2 h at room temperature) with fresh blocking buf-
fer supplemented with anti-digoxigeninAP Fab fragments diluted
1:2000. After 16 h, the plants were rinsed 8 times in TN buffer for
10 min and 10 times in TNM buffer (0.1 M TrisHCl, pH 9.5, 0.1 M
NaCl, 50 mM MgCl2) for 10 min.
The procedure was terminated by incubation in TNM detection
buffer supplemented with 2 mM levamisole and NBT/BCIP (Roche,
Basel, Switzerland). Staining was continued until a clearly detect-
able signal was visible under a light microscope. Plants were
mounted in 50% (v/v) glycerol and observed with an Olympus
BX50 microscope (Olympus Corp., Tokyo, Japan) equipped with
DIC optics. Images were captured with an MRc5 Axiocam color
camera (Carl Zeiss, Oberkochen, Germany) and processed with
Adobe Photoshop CS4 (Adobe, San Jose, CA, USA).
Embedding of seedlings and in situ hybridization on sections
Fixed plants were rinsed two times in PBS and subsequently
preembedded in 1% agarose in PBS (as described in [22]). The xed
tissue was dehydrated in ethanol, cleared in Hemo-De (Thermo
Fisher Scientic, Waltham, MA, USA), and embedded in Paraplast-
Plus (Thermo Fisher Scientic). Tissue sections (7 lm thick) were
cut with a 2035 Leica microtome (Leica Microsystems GmbH,
Wetzlar, Germany), oated on water, and mounted on Superfrost-
Plus slides (Thermo Fisher Scientic).
The slides were air-dried at 37 C and used for ISH as described
by Trevisan et al. [19]. The same ZNA oligo probes used for the
62 Whole mount Arabidopsis miRNAs detection by ZNA probes/M. BegheldoF.A. Ditengou et al. / Anal. Biochem. 434 (2013) 6066

Table 1
Arabidopsis thaliana microRNA probes adopted in the experiments.

miRNA Targets Biological functions Probe sequence Ref.

0 0
ath-miR160 ARF10, ARF16, ARF17 Stem cell differentiationroot development; auxin signal 4Z5 -TGGCATACAGGGAGCCAGGCA-3 [2426]
transduction
0 0
ath-miR164a/b CUC1, CUC2 Stem cell maintenance and signal transductionestablishment 4Z5 -TGCACGTGCCCTGCTTCTCCA-3 [2731]
of axillary meristems and boundary domain, lateral root
development
ath-miR167a/b ARF6, ARF8 Phase change ovule and anther fertility control; organ 4Z50 -TAGATCATGCTGGCAGCTTCA-30 [32,33]
developmentlateral root emergence; auxin signal transduction
ath-miR390 TAS3-ARF2|ARF3|ARF4 Signal transduction and organ developmentauxin-mediated 4Z50 -GGCGCTATCCCTCCTGAGCTT-30 [3436]
polarity establishment in root, leaf, and ower development
0 0
zma-miR166j/k/n HD-ZIP-III Stem cell maintenance and developmentcell organization in 4Z5 -AGGGATTGAAGCCTGGTCCGA-3 [9,37,38]
root and shoot

Investigated miRNAs, their targets, and the corresponding oligonucleotide sequences of probes are listed along with references reporting their characterization and biological
roles. For each miRNA the probe sequence refers to oligos modied with four ZNA spermine (Z) building blocks.

whole-mount protocol (listed in Table 1) were used at a nal con-
centration of 20 nM.
Automation of liquid handling for ISH standardization
All ISH steps, with the exception of xation and staining, were
carried out using an adaptation of a universal liquid handling ro-
bot, Freedom EVO 100, from Tecan (http://www.tecan.com). For
ISH on sections, a minimum number of six slides (each including
at least ve sections) per probe concentration were tested using
the Gene Paint suite accessories as described by Trevisan et al.
[19]. For ISH on whole-mount specimens xed plants were placed
in a custom-developed 48-well plate positioned on a thermostat-
controlled carrier. Three independent biological replicates per miR-
NA probe per concentration were performed, in three independent
wells each containing ve or six seedlings.
Results and discussion
Optimization and automation of whole-mount in situ hybridization
protocol on Arabidopsis seedlings
Changes were applied to some steps of the whole-mount ISH
protocol described by Hejtko et al. [40], to improve reproducibil-
ity of probe penetration and signal-to-noise ratio. For this dual pur-
pose, specic crucial steps were modied to improve penetration
of reagents within the tissues and to reduce background, making
a compromise between increased accessibility of targets and loss
of tissue structure organization.
To this end, an HCl step was added in the permeabilization
phase before protein digestion with proteinase K, while the pro-
teinase K working concentration was optimized to a relatively
low concentration (70 lg/ml versus the 125 lg/ml used by Hejtko
et al. [40]) with a digestion time of 40 min. Before the hybridiza-
tion step, and after postxation, samples were rinsed in triethanol-
amine supplemented with acetic anhydride, a treatment reported
to inactivate RNases and to improve signal-to-noise ratio [41].
When oligonucleotide probes are used, RNases should not repre-
sent a problem; nevertheless the preservation of RNA species in
the processed tissues is also fundamental to enhance sensitivity.
This RNA protection is usually carried out using a crosslinking x-
ative such as formaldehyde. If on one hand this step is essential, on
the other crosslinking itself prevents the probes from reaching
their target molecules [42]. To solve this problem, an mRNA retrie-
val treatment is normally carried out. mRNA retrieval can be per-
formed enzymatically, by digestion with proteinase K, which
should not be prolonged too much to avoid disruption of tissues,
or by heat treatment of the specimen. Heat retrieval techniques,
borrowed from immunocytochemistry, are commonly applied to
enhance the extraction of nucleic acids in ISH embedded samples
and to allow greater penetration of the probes [43]. Thus, we have
adopted a combination of a heat-retrieval method, based on the
use of a warmed ionic solution of TrisEDTA, with a proteinase K
digestion step at the lowest effective concentration (70 lg/ml) to
keep a better tissue integrity in comparison to short-term and
high-concentration proteinase K treatment alone [44,45]. In the
hybridization solution no herring sperm was used, but tRNA and
Denhardts solution were added to maintain a clear background,
along with dextran sulfate, to increase the rate of hybridization
[46]. Washing steps were carried out with an initial 1.5 SSC solu-
tion at 45 C, followed by a decreasing concentration series of SSC
solutions at 50 C, to maintain stringency by acting on temperature
instead of using formamide, thus minimizing the use of hazardous
chemicals. After anti-digoxigeninAP incubation the antibody ex-
cess was rinsed off in TN buffers instead of PBS to equilibrate the
plants in detection buffer.
Overall changes applied to the protocol resulted in an improve-
ment in terms of reproducibility of signals from the starting 30%,
obtained with the unimproved protocol, to ca. 70% of plants show-
ing a well-preserved structure and detectable signals (data not
shown). In addition, no tissue clearing steps were necessary, such
as mounting of specimens with chloral hydrate solution.
Since a major drawback of ISH procedures is their time-con-
suming low throughput, we implemented the protocol in an auto-
mated robotized platform (Tecan Freedom EVO 100) for liquid
handling of all steps except xation and nal staining.
An upgraded Gene Paint suite (http://www.genepaint.org, [47])
was set up for this purpose and a custom-designed thermostat-
controlled 48-well plate was developed to process 48 independent
samples at the same time.
Special care was taken to achieve effective liquid handling with-
out damaging the fragile biological structure of samples that were
repeatedly exposed to several different solutions. The automation
brought the throughput of the entire procedure to a high level,
by signicantly increasing the technical reproducibility of results,
by ensuring highly standardized conditions and running several
technical replicas, and, most of all, by enabling the evaluation of
high numbers of biological replicates, thus overall enormously
improving condence in the results.
Whole-mount detection of miRNAs with ZNA probes
The performance of the optimized automated whole-mount
miRNA ISH protocol was evaluated by determining the localization
of ve different selected miRNAs on Arabidopsis seedlings. These
miRNAs were chosen based on the facts that: (a) for some of them
a partial characterization of tissue-specic expression (e.g., by pro-
moter::GUS fusion studies) was available [24,36], thus providing a
 Whole mount Arabidopsis miRNAs detection by ZNA probes/M. Begheldo et al. / Anal. Biochem. 434 (2013) 6066 63

control for the reliability of the results obtained, and (b) their
expression in distinct types of tissue (hypocotyl and root)
[24,26,36] made them good candidates to test the degree of oligo
probe penetration within various cell structures and thus tissue
accessibility.
The oligonucleotides used, their corresponding miRNA targets,
and their sequences are listed in Table 1 along with references
reporting their characterization and biological roles. Four of the
chosen miRNAs (ath-miR160a/b, -miR164a/b, -miR167a/b, and -
miR390a/b) referred exclusively to A. thaliana, while the fth
(zma-miRNA166j/k/n) belonged to the Zea mays miRNA database
and had two mismatching nucleotides in comparison with its cor-
responding ath-miRNA166 from Arabidopsis. This miRNA was se-
lected to conrm also in Arabidopsis the results that were
previously obtained on maize root sections [19] and to verify the
ability of ZNA probes to potentially discriminate miRNAs belonging
to the same family across species and displaying minimal nucleo-
tidic mismatches. 50 -DIG-labeled ZNA (4Z) oligonucleotides were
used instead of the most commonly used LNA [19,48]. For each
ZNA probe various probe concentrations (15, 30, 50, and 70 nM -
nal concentration) were tested to identify the optimal range yield-
ing the best signal-to-noise ratio. At 15 nM no signal could be
detected (Fig. 1A), while the presence of detectable signals with in-
creased intensity could be appreciated within the 3050 nM range
for the majority of probes (two representative examples are shown
in Fig. 1B). A dened signal with higher signal-to-noise ratio was
detectable for all probes at 70 nM. Hybridizations with mouse miR-
NA mmu-miR124a [39] gave no signals up to 70 nM probe concen-
tration, while at higher molarities the general background
increased because of non-specic binding (data not shown). Based
on these results, a 5070 nM (depending on target) nal probe con-
centration was used for all subsequent experiments.
All the results obtained from the hybridizations of the ve se-
lected probes are shown in Fig. 2 and appeared in agreement with
data obtained previously from promoter::GUS reporter lines and
from ISH [24,26,36,38]. The signal of the miRNA160 probe appeared
localized specically at the shoot apical meristem (SAM), in leaf
primordia, and in the root provascular tissue (from the stem cell
niche) and lateral root primordia (LRP) (Fig. 2AC). This result is
consistent with previous data suggesting that this family of miR-
NAs is probably involved in promoting lateral root development
and in the regulation of primary root length by acting in the quies-
cent center [2426]. The specic signal in SAM was compatible
with embryo GUS reporter line observations and with a hypothet-
ical role for miRNA160 in stem cell differentiation [26]. miRNA164a/
b also appeared localized in SAM, in leaf primordia, and in root pro-
vascular tissues, even if with a slightly weaker signal (Fig. 2DF),
conrming its putative involvement in the regulation of the cell
proliferation in these tissues [29]. Concerning miRNA166 and miR-
NA167a/b (and to a lesser extent miRNA390), a more intense and
diffused signal could be detected in the SAM compared to those ob-
tained with the rest of probes. A slight signal in the root provascu-
lar tissues and LRP was visible for miRNA166 and miRNA167a/b,
while a much stronger signal was evident in the root provascular
tissue only for miRNA390, which in contrast did not show any sig-
nal in LRP.
These results overlap with previous expression data reported in
the literature and support the hypothesis that both miRNA167a/b
and miRNA390 could be involved in regulatory mechanisms
linked to auxin signaling and metabolism in driving lateral root

Fig.1. Whole-mount in situ hybridization on Arabidopsis 3- to 5-day-old seedlings at various oligo ZNA (4Z) probe concentrations. (A) Root tissue hybridizations conducted
with various probes (ath-miR160a/b, ath-miR164a/b, ath-miR167a/b, ath-miR390a/b and zma-miRNA166n/j/k) at 15 nM are presented, and (B) examples of hybridization at
30 and 50 nM are displayed for two oligo probes (ath-miR160a/b, ath-miR390a/b) on shoot apical meristem (SAM) and root. Scale bars, 50 lm.
64 Whole mount Arabidopsis miRNAs detection by ZNA probes/M. BegheldoF.A. Ditengou et al. / Anal. Biochem. 434 (2013) 6066

study. Carlsbecker et al. [38] demonstrated that LNA-modied oli-

gonucleotides specic for miRNA165 and 166, differing only by one
nucleotide substitution, gave overlapping hybridization patterns
and thus highlighted the impossibility of differentiating between
the signals coming from either probe. Since the results obtained
by Carlsbecker et al. are comparable to those presented here we
could assume that also in the case of ZNA oligonucleotides it is
probably not possible to discern miRNA members of the same
family by using a 1-base substitution. Moreover, because the

Fig.2. Whole-mount in situ hybridization on Arabidopsis 3- to 5-day-old seedlings

with oligo ZNA (4Z) probes at 70 nM nal concentration. (AC) ath-miR160a/b,
(DF) ath-miR164a/b, (GI) ath-miR167a/b, (JL) ath-miR390a/b, (MO) zma-
miRNA166n/j/k oligo hybridizations are shown for three main tissues: shoot apical
meristem (SAM), root, and lateral root primordia (LRP). (PR) Negative control is
represented by mmu-miR124a. Scale bars, 50 lm.

emergence (miRNA167a/b) and in the development of aerial organs

and vascular tissues (miRNA390) [24,36]. Moreover, the miRNA166
expression prole obtained in this study by using a specic maize
miRNA166 probe is in agreement with observations that localized
mature miRNA165/6, particularly in the QC and surrounding cells,
becoming progressively diffused in the neighboring root layers
[38]. A similar ISH prole along the provascular tissues was also
obtained with maize root sections [19].
A specic comment must be made on the recognition of miR-
NA166 family members by the zma-miR166j/k/n probe used in this
Fig.3. ISH hybridization on Arabidopsis sections of plants 56 days of age with oligo
ZNA (4Z) probes at 20 nM nal concentration. (AC) ath-miR160a/b, (DF) ath-
miR164a/b, (GI) ath-miR167a/b, (JL) ath-miR390a/b, (MO) zma-miRNA166n/j/k
oligo hybridizations are shown for three main tissues: shoot apical meristem (SAM),
root, and lateral root primordia (LRP). (PR) Negative control is represented by
mmu-miR124a. Scale bars, 50 lm.
 Whole mount Arabidopsis miRNAs detection by ZNA probes/M. Begheldo et al. / Anal. Biochem. 434 (2013) 6066
zma-miR166j/k/n oligo differs from ath-miR166 by 2 nucleotides
(miRBase [1]) we could infer that it is probably not possible to dis-
tinguish between miRNAs differing in two base substitutions
across species, as previous results we obtained with maize root
sections evidenced a comparable ISH prole [19].
No signal was detected for ISH conducted with the negative
control probe miR124a [39] (Fig. 2).
To test the reliability of the whole-mount results, ISH was car-
ried out also on sections. The expression patterns observed on sec-
tions gave support to the whole-mount data, conrming the
expression of the selected miRNAs in the root apical meristem, in
leaf primordia, in primary root meristem, and in lateral root pri-
mordia (Fig. 3). Slight differences between ISH data from whole
mounts and sections, which were more evident in root tissues,
are probably due to the fact that tissues from sections were ob-
tained from seedlings that were 1 or 2 days older than those used
for whole mount. This was dictated by the technical requirement of
sufciently developed plants to be handled for embedding and sec-
tioning. Remarkably, different optimal probe concentrations were
required in the two protocols: higher concentrations (between 50
and 70 nM) were necessary to obtain detectable signals with good
signal-to-noise ratios in whole-mount experiments, while a signif-
icantly lower concentration (20 nM) enabled retrieval of strong
signals in sections (7 lm). This low concentration was probably
not sufcient to allow a good penetration of the probe in all tissue
layers in whole-mount experiments, while sections were readily
penetrated. The ISH data presented in this work support the reli-
ability of the whole-mount miRNA procedure described, which en-
ables one to discriminate the expression domain at high resolution
with low background and high signal/noise ratios. Only few whole-
mount data are available from other authors for miRNA localiza-
tion [36], providing a partially clear whole-mount perspective.
The construction of promoter::GUS/GFP reporter fusions to study
tissue-specic expression of miRNAs requires longer and tedious
procedures of cloning, transformation, and selection of transgenic
lines [24,36,38]. Although these approaches give stable transcrip-
tional reporter lines, the availability of a reliable ISH procedure
represents a faster tool to dene the spatiotemporal localization
of specic miRNAs.
In this paper we describe a protocol for the whole-mount
hybridization of miRNAs, using ZNA (4Z)-modied oligonucleo-
tides. Overall, our results show that ZNA oligos perform well in
whole-mount ISH of miRNAs on Arabidopsis seedlings and conrm
the value of these modied probes as a real alternative for ISH on
sections in different plant species (i.e., Arabidopsis in addition to
maize). Furthermore, our data indicate that also for ZNA oligos,
as for LNA, a 2-nucleotide mismatch does not allow discrimination
between miRNAs of the same family. Further investigations will be
necessary to dene the minimum number of mismatching nucleo-
tides required for discrimination. The relatively low cost of ZNA-
modied oligos makes them certainly an interesting alternative
tool to LNA-modied oligos for in situ localization of miRNA
expression.
Conclusion and perspectives
miRNA exploration is becoming a more and more attractive
eld for scientists. Uncovering the biological roles of miRNAs, in
the modulation of developmental events or in the regulation of
stress responsiveness, is an important and ambitious challenge.
Multiple approaches should be used to address the numerous
questions about miRNAs involvement in biological and develop-
mental processes. In this scenario reliable and high-throughput
ISH methods are indispensable in studies for spatiotemporal reso-
lution of miRNA expression, considering the ever-increasing num-
ber of miRNAs being uncovered.
65
Automation of protocols gives important support in this
direction in terms of reproducibility and reliability of results and
enables us to expand the capability of determining the tissue-
specic expression of miRNAs at high throughput and, most of
all, under several experimental conditions. Furthermore, lower
cost chemicals are important as well for large-scale experiments
and the ZNA (4Z) chemistry seems to represent an interesting
option for this aim.
Acknowledgment
This project was funded by the European Project AUTO-
SCREEN (LSHG-CT-2007-037897).
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