MicroRNA fate upon targeting with anti-miRNA oligonucleotides as revealed by an improved Northern-blot-based method for miRNA detection.
ABSTRACT MicroRNAs (miRNAs) are small non-coding RNAs involved in fine-tuning of gene regulation. Antisense oligonucleotides (ONs) are promising tools as anti-miRNA (anti-miR) agents toward therapeutic applications and to uncover miRNA function. Such anti-miR ONs include 2'-O-methyl (OMe), cationic peptide nucleic acids like K-PNA-K3, and locked nucleic acid (LNA)-based anti-miRs such as LNA/DNA or LNA/OMe. Northern blotting is a widely used and robust technique to detect miRNAs. However, miRNA quantification in the presence of anti-miR ONs has proved to be challenging, due to detection artifacts, which has led to poor understanding of miRNA fate upon anti-miR binding. Here we show that anti-miR ON bound to miR-122 can prevent the miRNA from being properly precipitated into the purified RNA fraction using the standard RNA extraction protocol (TRI-Reagent), yielding an RNA extract that does not reflect the real cellular levels of the miRNA. An increase in the numbers of equivalents of isopropanol during the precipitation step leads to full recovery of the targeted miRNA back into the purified RNA extract. Following our improved protocol, we demonstrate by Northern blotting, in conjunction with a PNA decoy strategy and use of high denaturing PAGE, that high-affinity anti-miRs (K-PNA-K3, LNA/DNA, and LNA/OMe) sequester miR-122 without causing miRNA degradation, while miR-122 targeting with a lower-affinity anti-miR (OMe) seems to promote degradation of the miRNA. The technical issues explored in this work will have relevance for other hybridization-based techniques for miRNA quantification in the presence of anti-miR ONs.
- SourceAvailable from: Marcel Scheideler[Show abstract] [Hide abstract]
ABSTRACT: MicroRNAs (miRNAs) are endogenous small non-coding RNAs of ~23 nucleotides in length that form up a novel class of regulatory determinants, with a large set of target mRNAs postulated for every single miRNA. Thousands of miRNAs have been discovered so far, with hundreds of them shown to govern biological processes with impact on disease. However, very little is known about how they specifically interfere with biological pathways and disease mechanisms. To investigate this interaction, the hunt for direct miRNA targets that mediate the miRNA effects-the "needle in the haystack"-is an essential step. In this review we provide a comprehensive workflow of successfully applied methods starting from the identification of putative miRNA-target pairs, followed by validation of direct miRNA-mRNA interactions, and finally presenting methods that dissect the impact of particular miRNA-target pairs on a biological process or disease. This guide allows the way to be paved for obtaining biologically meaningful miRNA targets.International Journal of Molecular Sciences 10/2014; 15(11):20266-20289. · 2.34 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: MicroRNAs (miRNAs) are important regulators of gene expression, and many pathological conditions, including cancer, are characterized by altered miRNA expression levels. Therefore, accurate and sensitive quantification of miRNAs may result in correct disease diagnosis establishing these small noncoding RNA transcripts as valuable biomarkers. Aiming at overcoming some limitations of conventional quantification strategies, nanotechnology is currently providing numerous significant alternatives to miRNA sensing. In this review an up-to-date account of nanotechnology-based strategies for miRNA detection and quantification is given. The topics covered are: nanoparticle-based approaches in solution, sensing based on nanostructured surfaces, combined nanoparticle/surface sensing approaches, and single-molecule approaches.Chemistry - A European Journal 07/2014; · 5.93 Impact Factor
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ABSTRACT: ncRNAs are emerging as key regulators of physiological and pathological processes and therefore have been identified as pharmacological targets and as markers for some diseases. Oligonucleotide analogues represent so far the most widely employed tool for the modulation of the expression of ncRNAs. In this perspective we briefly describe most of the known classes of ncRNAs and then we discuss the design and the applications of oligonucleotide analogues for their targeting. The effect of modifications of the chemical structure of the oligonucleotides on properties such as the binding affinity toward targets and off targets, stability to degradation and on their biological effect (when known) are discussed. Examples of molecules currently used in clinical trials are also reported.Journal of Medicinal Chemistry 10/2014; · 5.48 Impact Factor
MicroRNA fate upon targeting with anti-miRNA
oligonucleotides as revealed by an improved
Northern-blot-based method for miRNA detection
ADRIAN G. TORRES,1MARTIN M. FABANI,1ELENA VIGORITO,2and MICHAEL J. GAIT1
1Medical Research Council, Laboratory of Molecular Biology, Cambridge, CB2 0QH, United Kingdom
2Laboratory of Lymphocyte Signalling and Development, The Babraham Institute, Babraham Research Campus, Cambridge, CB22 3AT,
MicroRNAs (miRNAs) are small non-coding RNAs involved in fine-tuning of gene regulation. Antisense oligonucleotides (ONs)
are promising tools as anti-miRNA (anti-miR) agents toward therapeutic applications and to uncover miRNA function. Such
anti-miR ONs include 29-O-methyl (OMe), cationic peptide nucleic acids like K-PNA-K3, and locked nucleic acid (LNA)-based
anti-miRs such as LNA/DNA or LNA/OMe. Northern blotting is a widely used and robust technique to detect miRNAs.
However, miRNA quantification in the presence of anti-miR ONs has proved to be challenging, due to detection artifacts, which
has led to poor understanding of miRNA fate upon anti-miR binding. Here we show that anti-miR ON bound to miR-122 can
prevent the miRNA from being properly precipitated into the purified RNA fraction using the standard RNA extraction protocol
(TRI-Reagent), yielding an RNA extract that does not reflect the real cellular levels of the miRNA. An increase in the numbers of
equivalents of isopropanol during the precipitation step leads to full recovery of the targeted miRNA back into the purified RNA
extract. Following our improved protocol, we demonstrate by Northern blotting, in conjunction with a PNA decoy strategy and
use of high denaturing PAGE, that high-affinity anti-miRs (K-PNA-K3, LNA/DNA, and LNA/OMe) sequester miR-122 without
causing miRNA degradation, while miR-122 targeting with a lower-affinity anti-miR (OMe) seems to promote degradation of the
miRNA. The technical issues explored in this work will have relevance for other hybridization-based techniques for miRNA
quantification in the presence of anti-miR ONs.
Keywords: microRNA; miR-122; oligonucleotide; Northern blot; RNA extraction
MicroRNAs (miRNAs) are an important class of small non-
coding RNAs that regulate gene expression post-transcrip-
tionally. They are transcribed as stem–loop-containing pre-
cursors and are processed into shorter duplex RNAs by a
series of enzyme complexes, first in the nucleus and then
following transport into the cytosol, in order to become
fully active (Siomi and Siomi 2010). The final complex,
miRISC, directs one of the two RNA strands (the guide
strand) to bind to the 39-UTR of target mRNAs resulting in
repression of gene expression. Most miRNAs are able to
target a number of mRNAs (Lim et al. 2005; Friedman et al.
2009), and their expression and activity are often associated
with complex cellular pathways, for example, cell growth
and apoptosis (Cheng et al. 2005), metabolism (Esau et al.
2006), viral infection (Skalsky and Cullen 2010), and cancer
(Lu et al. 2005).
Anti-miRNA oligonucleotides (ONs) capable of forming
complementary base pairs with the guide strand of miRNAs
have proved to be of great value in recent years as tools to
understand miRNA action and as potential therapeutics
(Kru ¨tzfeldt et al. 2005; Esau et al. 2006; Kloosterman et al.
2007; Vermeulen et al. 2007; Elme ´n et al. 2008a; Lu et al.
2009; Fabani et al. 2010; Lanford et al. 2010; Robertson
et al. 2010). Several types of ON analogs have been pro-
posed as anti-miRNA (anti-miR) agents in order to en-
hance steric-block potency, biostability, and binding affin-
ity for their RNA targets. Some of these include charge
neutral ONs, such as peptide nucleic acids (PNA) (Fabani
and Gait 2008; Oh et al. 2009; Fabani et al. 2010) and
phosphorodiamidate morpholino oligonucleotides (PMO)
Reprint requests to: Michael J. Gait, Medical Research Council,
Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH,
UK; e-mail: firstname.lastname@example.org; fax: 44-1223-402070.
Article published online ahead of print. Article and publication date are
RNA (2011), 17:933–943. Published by Cold Spring Harbor Laboratory Press. Copyright ? 2011 RNA Society.
(Flynt et al. 2007; Kloosterman et al. 2007; Eberhart et al.
2008); 29-modified ONs such as 29-O-methyl (OMe)
(Hutva ´gner et al. 2004; Meister et al. 2004; Cheng et al.
2005), which also form the basis of antagomiRs (Kru ¨tzfeldt
et al. 2005, 2007) 29-O-methoxyethyl (MOE) (Davis et al.
2006; Esau et al. 2006) and 29-fluoro/29-methoxyethyl
mixmers (29F/MOE) (Davis et al. 2009); and the particu-
larly strong RNA binding analog locked nucleic acid (LNA)
in mixmers with DNA (Ørom et al. 2006; Elme ´n et al.
2008a; Lanford et al. 2010), OMe (Fabani and Gait 2008),
or MOE (Davis et al. 2006, 2009).
Anti-miR ONs inhibit their miRNA target by a steric
blocking mechanism. However, the fate of the miRNA
following anti-miR ON binding is still unclear (Horwich
and Zamore 2008). Some evidence suggests that upon
binding of an LNA/DNA anti-miR, the miRNA becomes
complexed and sequestered in cells, but not degraded
(Elme ´n et al. 2008a,b). In contrast, there are also data
suggesting anti-miR-mediated miRNA degradation for
antagomiRs, MOE, and PNA anti-miRs (Kru ¨tzfeldt et al.
2005; Esau et al. 2006; Fabani and Gait 2008; Ameres et al.
2010). More recently, a report suggested that the miRNA
fate upon anti-miR binding might be chemistry-dependent.
While miRNA inhibition by a 29F/MOE anti-miR ON would
not degrade the miRNA, treatment with MOE anti-miR
ONs would induce miRNA degradation (Davis et al. 2009).
Many techniques have been proposed for detection of
miRNAs. Most of these techniques are dependent on the
hybridization properties between the target miRNA and
a complementary nucleic acid strand probe (Cissell and
Deo 2009). Among these methods, Northern blotting has
proved to be a robust and widely used technique that also
allows detection of not only mature miRNAs but also
their precursors (Lee and Ambros 2001; Lee et al. 2002;
Obernosterer et al. 2006) and that does not require spe-
cialized equipment. It is useful to quantify miRNA levels, to
determine their size and to validate predicted miRNAs
(Va ´rallyay et al. 2007). The sensitivity, safety, and speed of
this assay have been improved significantly by use of LNA-
modified detection probes (Va ´rallyay et al. 2008), by non-
radioactive labeling of the detection probe (Kim et al.
2010), and by EDC-mediated cross-linking of the RNA to
appropriate membranes (Pall and Hamilton 2008). Thus, it
is not surprising that Northern blotting is also the most
frequently used method to monitor miRNA abundance
after treatment with anti-miR ONs (Chan et al. 2005; Davis
et al. 2006; Esau et al. 2006; Kru ¨tzfeldt et al. 2007; Elme ´n
et al. 2008b; Fabani and Gait 2008; Fabani et al. 2010;
Lanford et al. 2010).
Despite the advantages of Northern blotting over other
techniques to detect miRNAs, miRNA detection by North-
ern blot in the presence of anti-miR ONs can give mis-
leading results, which partly explains the lack of consensus
on the mode of action of anti-miR ONs. It has been shown
that anti-miR ONs can interfere with miRNA detection at
two different steps. First, during the Northern blot hybrid-
ization step, the probe for miRNA detection may not be
able to displace the anti-miR ON already bound to the
miRNA of interest, even in samples analyzed in denaturing
PAGE conditions (Chan et al. 2005; Davis et al. 2009). In
this case, uncomplexed miRNA would be detected, while an
miRNA:anti-miR duplex would not be detected, thus lead-
ing to the interpretation that there is a (partial) knockdown
of the miRNA. Second, the miRNA:anti-miR duplexes
might not be recovered in the purified RNA fraction
during the commonly used guanidinium thiocyanate-phe-
nol-choloroform RNA extraction protocol (Chomczynski
and Sacchi 2006) (commercially known as TRI-Reagent or
TRIzol), yielding an RNA extract that does not represent
the physiological levels of the particular miRNA (Fabani
and Gait 2008; Lu et al. 2009; Davis et al. 2009). Therefore,
in light of these technical issues, the inability to detect
an miRNA by Northern blot in the presence of anti-miRs
is not sufficient evidence for anti-miR-mediated miRNA
There have been suggestions on how to overcome some
of these problems. It has been shown that miRNA masking
by the anti-miR ON can be relieved, at least for lower-
affinity analogs, by increasing the denaturing conditions of
the electrophoresis separation (Kru ¨tzfeldt et al. 2005). Al-
ternatively, a competitor PNA ON, having the same se-
quence as the miRNA of interest and that binds to the anti-
miR ON, can be introduced in the RNA samples prior to
PAGE separation, allowing for miRNA release and its de-
tection by Northern blot (Davis et al. 2009). However, there
are currently no reports on how to recover miRNA:anti-
miR duplexes that have been lost during the RNA extrac-
MicroRNA-122 (miR-122), a liver-specific miRNA asso-
ciated with lipid metabolism (Esau et al. 2006) and virus
infection (Triboulet et al. 2007; Chang et al. 2008), has be-
come one of the main miRNA models for anti-miR-me-
diated inhibition of miRNAs, mainly due to the huge in-
terest in it as a target for therapeutics (Jopling et al. 2005;
Kru ¨tzfeldt et al. 2005, 2007; Esau et al. 2006; Elme ´n et al.
2008b; Fabani and Gait 2008; Davis et al. 2009; Lanford
et al. 2010).
Here we show in detail how anti-miRs of four different
chemistries—29-O-methyl (OMe), peptide nucleic acid
(PNA), and LNA/OMe and LNA/DNA mixmers targeting
miR-122—interfere at the two aforementioned steps of the
miRNA detection process by Northern blotting. Further-
more, we demonstrate complete recovery of miRNA:anti-
miR duplexes back into the RNA extract, even for high-
affinity anti-miR ONs, by modification of the widespread
TRI-Reagent-based RNA extraction procedure. Finally, by
use of the new RNA extraction procedure together with a
decoy PNA ON strategy (Davis et al. 2009) and poly-
acrylamide gel electrophoresis (PAGE) separation of RNA
samples under highly denaturing conditions (Kru ¨tzfeldt
Torres et al.
RNA, Vol. 17, No. 5
et al. 2005), we were able to shed light on the cellular
outcome for miRNAs upon anti-miR targeting of different
OMe, LNA/OMe, and LNA/DNA anti-miR interfere
with miR-122 detection by Northern blotting
We first wished to verify whether anti-miR ONs interfere
with miRNA detection. We addressed this issue by de-
tection of miR-122 by Northern blotting in the presence of
anti-miR-122 ONs of four different chemistries: K-PNA-K3
(23-mer + 4 Lys) (Fabani and Gait 2008), OMe (31-mer)
(Jopling et al. 2005), LNA/OMe (23-mer) (Fabani and Gait
2008), and a commercial 23-mer LNA/DNA knockdown
probe (Exiqon). PNA, OMe, and LNA/OMe anti-miR ONs
have been validated previously as effective miR-122 inhib-
itors through additional assays (Jopling et al. 2005; Fabani
and Gait 2008). For miR-122 detection, RNA was extracted
using TRI-Reagent (Sigma), which is also available (TRIzol)
from multiple suppliers, following the manufacturer’s pro-
tocol that involves the use of the standard 0.5 volumes
of isopropanol for each volume of TRI-Reagent for RNA
precipitation. RNA separations were carried out by use of
very highly denaturing PAGE in the presence of 8 M urea
and 20% formamide (Kru ¨tzfeldt et al. 2005). Additionally,
8 M urea and 20% formamide were included in the loading
buffer, and the sample was preheated before loading. These
highly stringent PAGE conditions were used to maximize
the likelihood of the miRNA being single-stranded and thus
most easily detectable. Finally, LNA probes were used for
miRNA detection by Northern blotting because of their
superior sensitivity compared to standard DNA probes
(Va ´rallyay et al. 2008) and their ability to detect miRNAs in
the presence of anti-miRs (Horwich and Zamore 2008).
We treated Huh7 cells that express endogenous miR-122
with the anti-miRs and measured miR-122 levels by North-
ern blot analysis. K-PNA-K3 anti-miR was added to the
cells without the use of a transfection agent as described
previously (Fabani and Gait 2008), while the other three
anti-miRs were lipofected at the indicated concentrations.
As expected from previous studies, treatment with any
of the anti-miRs led to a reduction in miR-122 signal
(Fig. 1A). OMe anti-miR treatment resulted in partial miR-
122 loss of signal, while for K-PNA-K3 and LNA/OMe
anti-miRs, the loss was much stronger. MiR-122 detection
following LNA/DNA anti-miR treatment resulted in a
slight band shift consistent with miR122:LNA/DNA anti-
miR duplex formation as previously reported (Elme ´n et al.
2008a,b). However, the strength of the miR-122:LNA/
DNA anti-miR duplex signal was much weaker as com-
pared to untreated cells (negative control) (Fig. 1A). Note
that in our conditions, an approximate loss of 95% of miR-
122 signal was achieved in the low nanomolar range for
FIGURE 1. Anti-miRs can interfere with miR-122 detection by
Northern blot. (A) Northern blot of total purified RNA from Huh7
cells treated with miR-122 anti-miRs K-PNA-K3, OMe, LNA/OMe, or
LNA/DNA. Total purified RNA from untreated cells was used as
negative controls. (B) Northern blot of total purified RNA after ad-
dition of miR-122 anti-miRs at equivalent amounts to mimic trans-
fections in A to Huh7 cell lysates. Total purified RNA from Huh7 cells
lysates without addition of anti-miR was used as negative control. (C)
Northern blot of total RNA from Huh7 cell lysates treated as in B with
OMe, LNA/OMe, or LNA/DNA anti-miR and further addition of 100
pmol of decoy PNA ON to the purified RNA. RNA from untreated
lysates but with 100 pmol of decoy PNA ON addition was used as
negative control. U6 RNA was used as loading control for all gels.
miRNA fate upon anti-miR targeting
lipofected LNA/DNA and LNA/OMe anti-miRs, in the low
hundreds nanomolar range for lipofected OMe anti-miR
and in the low micromolar range for free delivered K-PNA-
K3 anti-miR. Also note that pre-miR122 bands could be
detected and they remained unchanged in the presence of
any of the anti-miRs tested (data not shown).
To explore this loss of signal, we added to untreated
Huh7 cell lysates in TRI-Reagent equivalent amounts of
each anti-miR to mimic the cell treatments carried out in
the previous experiment prior to RNA extraction and
Northern blotting, an approach that has been commonly
used previously (Kru ¨tzfeldt et al. 2005, 2007; Esau et al.
2006; Davis et al. 2006, 2009; Fabani and Gait 2008; Lu
et al. 2009). As shown in Figure 1B, the presence of a band
corresponding to miR-122 in the sample to which K-PNA-
K3 anti-miR had been added suggests that this anti-miR
does not interfere with miR-122 detection by Northern
blotting under these conditions. In contrast, partial inter-
ference was seen with OMe anti-miR, and a complete loss
of miR-122 signal was seen in cell lysates containing LNA/
OMe or LNA/DNA anti-miR, showing in these cases major
interference with miR-122 detection. To test whether the
lack of miR-122 signal was due to complex formation with
the corresponding anti-miR, hindering the miRNA from
the labeled miRNA detection probe, we repeated the pre-
vious experiment, but before PAGE separation we added an
excess of a competitor (decoy) PNA ON having the same
sequence as miR-122, as previously described by Davis et al.
(2009) (Fig. 1C). No further recovery of miR-122 signal
was achieved under these conditions, suggesting that even
in the presence of excess decoy, miR-122 is not fully detected
in the presence of these anti-miRs. These results show that
OMe, LNA/OMe, and LNA/DNA anti-miR ONs interfere
with the Northern blot detection of the targeted miRNA
and, further, that it is not possible to conclude using pub-
lished protocols for this assay whether or not miR-122 is
degraded or sequestered upon anti-miR treatment.
K-PNA-K3 anti-miR sequesters miR-122 without
causing miR-122 degradation
Since K-PNA-K3 anti-miR was the only backbone chem-
istry that did not seem to interfere with miR-122 detection
when Huh7 cell lysates were supplemented with this anti-
miR, it appeared likely that the reduction in the miR-122
signal strength seen in Huh7 cells treated with this anti-miR
represented a genuine miRNA knockdown (Fig. 1A,B), as
we suggested previously (Fabani and Gait 2008). To further
examine this observation, we treated Huh7 cells with
K-PNA-K3 anti-miR at two different concentrations (1 and
10 mM). As expected, a dose-dependent loss of miR-122
signal was seen (Fig. 2A). We then investigated whether the
observed decrease in miR-122 signal was due to target deg-
radation by supplementing the RNA samples with decoy
PNA ON prior to high denaturing PAGE. To our surprise,
the recovery of miR-122 signal was complete, demonstrat-
ing that K-PNA-K3 anti-miR sequesters miR-122 in Huh7
cells and does not cause its degradation, even at high anti-
miR concentrations (Fig. 2B). Thus, the loss of signal in
Figure 2A cannot be attributed to degradation and must
instead be due to undetected K-PNA-K3:miR-122 complex
formation, resulting because bound K-PNA-K3 anti-miR is
not displaced by the miRNA detection probe under these
conditions. Note that when K-PNA-K3 anti-miR was added
to untreated Huh7 cell lysates in TRI-Reagent at equivalent
amounts to mimic the concentrations used in transfections
in Figure 2A, we found it to interfere with miR-122 de-
tection only at the highest concentration tested, 10 mM
(Supplemental Fig. 1). This result and the dose-dependent
effect seen in Figure 2A suggest that the level of interference
FIGURE 2. miR-122 fate upon targeting with K-PNA-K3 anti-miR.
(A) Northern blot of total purified RNA from Huh7 cells treated with
K-PNA-K3 anti-miR at 1 mM and 10 mM. RNA from untreated cells
was used as negative control. (B) Northern blot of total purified RNA
from Huh7 cells treated with K-PNA-K3 anti-miR as in A and further
addition of 100 pmol of decoy PNA ON to the purified RNA. RNA
from untreated cells but with 100 pmol of decoy PNA ON addition
was used as negative control. U6 RNA was used as loading control for
Torres et al.
RNA, Vol. 17, No. 5
by anti-miR ONs in detection of targeted
miRNAs by Northern blotting is also
dependent on the concentration of anti-
miR used. A similar anti-miR dose-de-
pendent effect on miR-122 detection by
Northern blotting was seen for the other
anti-miRs tested in cell lysates that re-
ceived addition of each anti-miR to
mimic transfections in the nanomolar
range (data not shown).
miR-122:anti-miR duplex loss and
recovery during RNA extraction
The experiments so far indicated that
all anti-miR ONs tested interfere with
miRNA detection by Northern blotting.
However, in contrast to our observation
for K-PNA-K3 anti-miR, miR-122 signal
could not be fully recovered when
OMe, LNA/OMe, or LNA/DNA anti-
miRs were added to untreated Huh7 cell
lysates, even in the presence of decoy
PNA ON (Fig. 1B,C). This might in-
dicate that either the decoy PNA ON is
not able to displace the anti-miR from
the miRNA in the conditions tested,
or that miRNA:anti-miR
are lost during RNA purification, prior
to Northern blotting, as previously sug-
gested (Fabani and Gait 2008; Davis
et al. 2009; Lu et al. 2009). To explore
the second possibility, we added anti-
miRs to untreated Huh7 cell lysates and
supplemented the lysates with small
miR-122 mimic (32P-miR122). Use of
32P-miR122 allows for easy identifica-
tion of miR-122 throughout the RNA
extraction procedure and eliminates the
need for a radiolabeled anti-miR-122
detection probe for Northern blotting.
plexes should be detectable as well as
panel) confirms that, under our condi-
pletely lost from the purified RNA
fraction when RNA was extracted in
the presence of OMe, LNA/OMe, or
LNA/DNA anti-miRs. As expected,
32P-miR122 is totally recovered in the
purified RNA fraction in the presence of
equivalent amounts of K-PNA-K3 anti-
miR to mimic 1 mM transfections, a
32P-miR122 is partially or com-
FIGURE 3. miR-122 loss and recovery during RNA extraction. Denaturing PAGE of cell
lysates supplemented with32P-miR122 and further addition of K-PNA-K3, OMe, LNA/OMe,
or LNA/DNA anti-miRs before RNA extraction, at equivalent amounts to mimic transfections
in Figure 1A. Negative controls correspond to cell lysates spiked with32P-miR122 only. (A)
Purified RNA (upper panel) and the remaining isopropanol supernatant (lower panel) of
samples after RNA extraction with 1 equivalent (v/v) isopropanol. (B) Purified RNA (upper
panel) and the remaining isopropanol supernatant (lower panel) from cell lysates treated as in
A with LNA/OMe anti-miR after RNA extraction with 1, 2, or 3 equivalents (v/v) isopropanol.
(C) Purified RNA from samples treated as in A after RNA extraction with 3 equivalents (v/v)
miRNA fate upon anti-miR targeting
concentration at which K-PNA-K3 did not interfere with
miR-122 detection in lysates of untreated cells (Fig. 1B).
Careful analysis of the individual fractions obtained during
the RNA extraction procedure showed that most of the
RNA precipitation step with isopropanol (Fig. 3A, lower
panel). Note that under our conditions32P-miR122 was not
differentially distributed during the initial phase separation
of the RNA extraction protocol in the presence or absence of
an LNA-based anti-miR (Supplemental Fig. 2), in contrast
to a previous suggestion (Davis et al. 2009).
All available kits based on the guanidinium thiocyanate–
phenol–chloroform RNA-extraction method (Chomczynski
and Sacchi 2006) (TRIzol, Tri-Reagent, etc.) stipulate the
use of 0.5 volumes of isopropanol for each volume of
TRI-Reagent for RNA precipitation. This corresponds ap-
proximately to 1 equivalent (v/v) of isopropanol relative to
the volume of the aqueous RNA-containing phase after the
first phase separation step of the protocol. We thought that
RNA precipitation could be improved by increasing the
amount of isopropanol at the RNA precipitation step. In-
deed, by adjusting the volume of isopropanol from 1 to 3
equivalents (v/v) for RNA precipitation, we observed in-
creasing recovery of32P-miR122 in the purified RNA phase
(Fig. 3B, upper panel) and decreasing abundance of32P-
miR122 in the remaining isopropanol supernatant (Fig. 3B,
lower panel; Supplemental Fig. 3) in the presence of LNA/
OMe anti-miR, as analyzed by denaturing PAGE. Similarly,
32P-miR122 could also be completely recovered by pre-
cipitating the RNA with 3 equivalents (v/v) of isopropanol
in the presence of OMe and LNA/DNA anti-miRs (Fig.
3C). The superior binding strength of LNA-based anti-
miRs is evidenced by the presence of lower-mobility com-
plexes formed between LNA/DNA or LNA/OMe anti-miRs
conditions (8 M urea/20% formamide). The same condi-
tions seem to be sufficient for complex dissociation be-
tween OMe anti-miR and32P-miR122. In the absence of
20% formamide, the OMe anti-miR showed complex for-
mation (Supplemental Fig. 4), as previously shown for
antagomiRs (Kru ¨tzfeldt et al. 2005). Interestingly, under
denaturing conditions, duplexes did not migrate in the gels
at the expected molecular size. However, in vitro binding
assays between32P-miR122 and each anti-miR under these
highly denaturing conditions confirmed complex forma-
tion (Supplemental Fig. 5). Note that in Figure 3B, partial
melting of the32P-miR122:LNA/OMe anti-miR duplex is
seen in the isopropnaol supernatant phase; this is due to the
NAP-10 purification (desalting) procedure.
32P-miR122 remained in the supernatant of the
32P-miR122, even under highly denaturing PAGE
The fate of miR-122 upon anti-miR targeting
is dependent on the anti-miR chemistry
Having optimized the RNA extraction procedure in order
to recover miRNA:anti-miR duplexes back into the RNA
extract (Fig. 3), we set out to determine the cellular out-
come for miR-122 upon targeting with LNA/DNA, LNA/
OMe, or OMe anti-miRs. First, total RNA was obtained
from Huh7 cells transfected with each of these anti-miR
oligonucleotides by standard purification (1 equivalent of
isopropanol compared to the aqueous phase for the RNA
precipitation step) that had been supplemented with PNA
decoy and analyzed by Northern blotting. Figure 4A shows
that under these standard purification conditions, miR-122
signal was only recovered through decoy use in the pres-
ence of K-PNA-K3 anti-miR, but not in the presence of any
of the other three anti-miRs of different chemistry, and
confirms the lack of degradation of the miR-122 target in
the presence of K-PNA-K3 seen in Figure 2B.
As suggested in Figure 3, OMe, LNA/OMe, and LNA/
DNA anti-miR chemistries prevent the miR-122:anti-miR
duplexes from being properly recovered in the RNA extract
following the standard RNA extraction procedure. There-
fore, we decided to test our improved RNA extraction
protocol (3 equivalents [v/v] isopropanol for RNA pre-
cipitation step) in Huh7 cells treated with these three anti-
miR chemistries and by analyzing by Northern blotting as
before. Figure 4B shows that for cells treated with LNA/
DNA anti-miR, the procedure allows for partial detection
of miR-122 in the form of a low-mobility complex. MiR-
122 is released by treatment of this complex with decoy
PNA ON, showing that this high-affinity anti-miR seques-
ters miR-122 without inducing target degradation in cells,
just as for K-PNA-K3 anti-miR. Furthermore, when LNA/
DNA anti-miR was used in mice, the miR-122 signal was
recovered in Northern blotting under the improved RNA
extraction conditions, and once again decoy use resulted in
recovery of the complete miR-122 signal in the case of
K-PNA-K3 (Supplemental Fig. 6).
Treatment of Huh7 cells with the very-high-affinity
LNA/OMe anti-miR showed a similar result, but the
miR-122:anti-miR duplex was not detected, and the re-
covery of the miR-122 signal upon addition of decoy PNA
ON was not complete (Fig. 4C). The most likely explana-
tion is that the less strongly binding decoy PNA ON is not
able to completely displace the highly stable miR-122:LNA/
OMe anti-miR duplex. Addition of a greater excess of de-
coy PNA ON could not be used to enhance the recovery of
the miR-122 signal because the PNA decoy detection com-
petes too efficiently under these conditions for the radio-
active detection probe and masks the ability to detect the
miR-122 (data not shown). Also, cell treatment with a higher
amount (150 nM) of LNA/OMe anti-miR did not result in
a lower miR-122 signal than in the case of use of 50 nM anti-
miR (data not shown), thus ruling out dose dependence of
the signal detection. Our results are best consistent with the
view that this high-affinity anti-miR sequesters miR-122
without causing significant miRNA degradation.
In contrast, for the OMe anti-miR, miR-122 signal was
not recovered following use of the improved method for
Torres et al.
RNA, Vol. 17, No. 5
miRNA recovery and detection by Northern blotting in the
presence of decoy PNA ON (Fig. 4D). The lack of miR-122
signal in the presence of OMe anti-miR under these con-
ditions is very unlikely to be due to an artifact. We have
shown that in the presence of these amounts of OMe anti-
miR, miR-122 would have been detected if present in the
RNA extract when RNA precipitation is carried out with 3
equivalents (v/v) of isopropanol (Fig. 3C). Furthermore,
the miR-122:OMe anti-miR duplex is clearly fully dis-
rupted under our highly denaturing conditions (Supple-
mental Figs. 4, 5), and this is independent of whether
the decoy PNA ON is present or not, ruling out the pos-
sibility that decoy PNA ON is unable to displace the OMe
anti-miR bound to miR-122. Thus, uniquely among the
four anti-miR chemistries tested, OMe anti-miR usage in
Huh7 cells is consistent with degradation of the miR-122
Northern blotting has become a primary method for de-
tection, identification, and validation of novel miRNAs,
and it has been extensively used to demonstrate the effect of
anti-miR ONs on miRNA expression (Chan et al. 2005;
Davis et al. 2006; Esau et al. 2006; Kru ¨tzfeldt et al. 2007;
Elme ´n et al. 2008b; Fabani and Gait 2008; Fabani et al.
2010; Lanford et al. 2010). Of the four chemistries we in-
vestigated, only LNA/DNA has advanced to clinical trials as
reported by Santaris Pharma in 2010. OMe has been the
most widely used anti-miR chemistry (Chang et al. 2004;
Hutva ´gner et al. 2004; Meister et al. 2004; Jopling et al. 2005;
Horwich and Zamore 2008), which also forms the basis of
antagomiRs (Kru ¨tzfeldt et al. 2005, 2007). PNAs containing
cationic amino acids have been shown to inhibit miRNAs in
cells (Fabani and Gait 2008; Oh et al. 2009, 2010; Fabani et al.
2010) and in vivo (Fabani et al. 2010). We reported high-
affinity LNA/OMe anti-miRs for miR-122 inhibition in cell
culture, which proved to be more potent than the OMe anti-
miR, (Fabani and Gait 2008) and for other steric-blocking
applications (Arzumanov et al. 2001; Brown et al. 2005).
Problems with the Northern blotting technique have
hitherto served to obscure the fate of miRNAs targeted by
anti-miRs. We have now demonstrated, through use of a
PNA decoy approach (Davis et al. 2009), that K-PNA-K3
anti-miR acts by sequestering the miRNA without causing
its degradation in cell culture (Fig. 2) or in vivo (Supple-
mental Fig. 6). For OMe, LNA/OMe, and LNA/DNA anti-
miRs, neither analysis under highly denaturing conditions
nor addition of a PNA decoy was sufficient to reveal the
effect of these anti-miRs on miR-122 levels (Figs. 1C, 4A).
Loss of miR-122 in the purified RNA samples treated with
these anti-miRs (Fig. 3A, upper panel) has been reported
previously for high-affinity LNA-containing anti-miRs by
us and others (Fabani and Gait 2008; Davis et al. 2009;
Lu et al. 2009). We did not find anti-miRs to partition
FIGURE 4. miRNA fate upon targeting with LNA/DNA, LNA/OMe,
or OMe anti-miR. (A) Northern blot of total purified RNA extracted
with 1 equivalent (v/v) isopropanol from Huh7 cells treated as in
Figure 1A and further addition of decoy PNA ON to the RNA samples
before PAGE separation. RNA from untreated Huh7 cells but in the
presence of decoy PNA ON was used as negative control. (B–D)
Northern blots of total purified RNA extracted with 3 equivalents
(v/v) isopropanol from HuH7 cells treated as in A with either LNA/
DNA anti-miR (B), LNA/OMe anti-miR (C), or OMe anti-miR (D)
in the presence or absence of decoy PNA ON. RNA from untreated
Huh7 cells was used as negative control. U6 RNA was used as loading
control for all gels.
miRNA fate upon anti-miR targeting
radiolabeled miR-122 (32P-miR-122) into the organic
phase during the RNA extraction (TRI-Reagent) protocol
as measured by scintillation counting (Supplemental Fig.
2), in contrast to similar experiments reported for LNA/
MOE anti-miRs (Davis et al. 2009). Instead, the LNA-con-
taining anti-miRs, and OMe anti-miR (partially), caused
retention of targeted32P-miR122 in the isopropanol super-
natant during precipitation when the standard RNA ex-
traction procedure was followed (Fig. 3A). However, miR-
122 could be recovered fully by use of 3 equivalents (v/v) of
isopropanol instead of the standard single equivalent (Fig.
3B,C; Supplemental Fig. 3).
By use of these improved techniques, it now becomes
clear that LNA/DNA and LNA/OMe anti-miRs, just like
K-PNA-K3, also inhibit miR-122 in cells by complex for-
mation without causing significant miRNA degradation
(Fig. 4B,C), and this applies also for LNA/DNA anti-miRs
in vivo (Supplemental Fig. 6). Our results are consistent
with the conclusions of Elme ´n et al. (2008a,b) that LNA/
DNA anti-miRs act to complex miR-122 without degrada-
tion, but note that we and others (Chan et al. 2005;
Naguibneva et al. 2006; Laneve et al. 2007; Inomata et al.
2009) have been unable to clearly visualize such complexes
by denaturing PAGE under standard RNA extraction con-
ditions. With our higher isopropanol precipitation con-
ditions during extraction and under denaturing PAGE,
miR-122 can be detected by Northern blotting partially as
a complex with the LNA/DNA anti-miR (Figs. 3C, 4B;
Supplemental Fig. 6b). Addition of decoy PNA ON resulted
in full recovery of the miR-122 signal as single-stranded
miRNA, thus confirming the miR-122 sequestration during
cell treatment rather than degradation (Fig. 4B). Our study
is the first to report that high-affinity LNA/OMe anti-miR
also does not give rise to significant miRNA degradation
(Fig. 4C). Davis et al. (2009) reported that the high-affinity
29F/MOE anti-miR also sequesters miR-122 without caus-
ing miRNA degradation, but we have been unable to in-
vestigate MOE-containing anti-miR ONs, because such
ONs are not commercially available. We therefore conclude
that miRNA sequestration without significant miRNA deg-
radation is a general feature of high-affinity anti-miRs.
The OMe anti-miR was the only one of the four tested
that appears to induce miR-122 degradation upon targeting
(Fig. 4D). This 31-mer OMe ON is the same as that shown
to be functionally active as an anti-miR122 in Huh7 cells
(Jopling et al. 2005). Several other studies that showed
functional inhibition of miRNA activities in a variety of cell
types by OMe ONs also showed loss of miRNA signal on
Northern blotting, suggesting that these miRNAs were
degraded by the OMe anti-miR in cells (Hutva ´gner et al.
2004; Chan et al. 2005; Krichevsky et al. 2006; Yang et al.
2008). However, in all these cases, the standard RNA
extraction conditions were used, and in some cases, only
lower-affinity DNA probes were used for miRNA detection,
which may have compromised the Northern blotting as-
says. Horwich and Zamore (2008) reported that in Dro-
sophila cells OMe anti-miRs had no significant effect on
endogenous miR-277 levels in Northern blotting, which
contrasts with all other studies in mammalian cells.
Kru ¨tzfeldt et al. (2005, 2007) reported that in mice, inhib-
ition of miR-122 by antagomiRs (cholesterol-conjugated
OMe ONs partially modified with a phosphorothioate
backbone) led to target miRNA degradation, but that the
equivalent OMe anti-miR (partially or fully modified with
phosphorothioate backbone but not cholesterol-conju-
gated) resulted in detection of the miR-122 RNA in North-
ern blotting in denaturing PAGE, suggesting miR-122
complex formation (Kru ¨tzfeldt et al. 2005). Cholesterol-
unconjugated all PO OMe ONs were not reported in this
study. Very recently, Ameres et al. (2010) showed that in
HeLa cells, antagomiRs targeted to miR-16 or miR-21 lead
to miRNA degradation through tailing (addition of pre-
dominately adenosines and uridines at the 39 end of the
miRNA) and trimming (usually 39 to 59) of the targeted
miRNA. Ameres’s work suggests an interesting mechanism
for anti-miR-mediated degradation of miRNAs; however,
under our conditions, we failed to detect trimming or
tailing of miR-122 by Northern blotting after cell treatment
Under our improved isopropanol precipitation condi-
tions of RNA and use of a decoy PNA ON, we observed
clear loss of the miRNA target in Northern blotting and
therefore, in agreement with the large majority of studies
using OMe ONs as anti-miRs, we conclude that degrada-
tion remains the most probable outcome of OMe targeting.
Note also that Esau et al. (2006) reported miR-122 deg-
radation upon MOE/PS anti-miR targeting in mice under
standard RNA extraction conditions, and Davis et al.
(2009), using the PNA decoy approach, recently showed
clear evidence for miR-122 degradation in mice treated
with MOE/PS anti-miRs, even though MOE/PS ONs were,
in principle, able to be complexed fully by the PNA decoy.
While high-affinity anti-miR chemistries (K-PNA-K3,
LNA/DNA, LNA/OMe) in all cases maintain complex
formation with their target miRNAs without evidence for
significant miRNA degradation, the overwhelming balance
of experimentation across the available literature is consis-
tent with miRNA degradation being observed only when
lower-affinity anti-miRs are used. It seems unlikely that this
degradation is because of cellular instability of the anti-
miR, because miRNA degradation is clearly seen when, for
example, highly stable MOE/PS ONs are used (Esau et al.
2006; Davis et al. 2009). Instead, it seems likely that miRNA
duplexes formed with lower-affinity anti-miRs are more
prone to being broken in cells during inhibition experi-
ments, which are often carried out over extended periods
and subsequently become vulnerable to degradation by
nucleases. This would imply possible displacement of the
miRNA from miRISC. In contrast, stronger binding PNA
and LNA anti-miRs are highly stable inside cells and form
Torres et al.
RNA, Vol. 17, No. 5
very strong complexes with miRNAs. Irrespective of
whether they are displaced from miRISC, such strong com-
plexes would protect the miRNA from nuclease degrada-
tion. Verification of this hypothesis would ideally require a
fully independent method of miRNA level measurement
within cells, perhaps through use of exogenously intro-
duced and quantifiable miRNA mimics as well as anti-miR
complements in otherwise miRNA-depleted cells, experi-
ments beyond the scope of this study.
Our improvements to the technical aspects of Northern
blotting in the presence of anti-miRs will have relevance
also to other hybridization-based miRNA detection proce-
dures such as in situ hybridization (Kloosterman et al. 2006,
2007; Elme ´n et al. 2008b; Jørgensen et al. 2010), microarrays,
BRET-based, bioluminescence and electrochemical assays,
and real-time quantitative PCR (RT-qPCR) (for an overview
on these techniques, see Cissell and Deo 2009), which are
subject to misleading interpretation due to anti-miR inter-
ference, for example, shown in RT-qPCR (Davis et al. 2009;
Lu et al. 2009). However, for each technique, further par-
ticular optimization will need to be addressed to develop a
reliable method for miRNA quantification and detection in
the presence of anti-miRs. We believe that our results will be
of wide interest to those using such miRNA quantification
and detection methods and serve also as an additional re-
minder that evaluation of anti-miR efficiency as potential
therapeutics in cells and in vivo requires additional criteria
beyond Northern blotting, such as measurement of the levels
of mRNA targets of the specific miRNA inhibited by the
anti-miR, as we and others have advocated previously (Esau
2008; Fabani and Gait 2008; Davis et al. 2009).
MATERIALS AND METHODS
Synthetic miR-122 RNA ON corresponding to the human miR-
122 sense strand was purchased from Dharmacon, and the se-
quence was obtained from the miRBase Sequence Database
(Release 9.2): 59-UGGAGUGUGACAAUGGUGUUUGU-39.
LNA/OMe ON was synthesized as previously described (Turner
et al. 2006), and the ON sequence corresponded to that previously
reported (Fabani and Gait 2008): 59-aCaAaCaCcAuuGuCaCaCu
PNA ONs were synthesized on a Liberty (CEM Corporation)
microwave-assisted peptide synthesizer as described previously
(Fabani et al. 2010). For in vitro studies and cell work, a 23-mer,
fully complementary to mature wild-type miR-122, PNA sequence
was synthesized containing an N-terminal Cys and four (L)-Lys
residues K-PNA-K3: Cys-(L)K-59-ACAAACACCATTGTCACAC
TCCA-39–(L)K(L)K(L)K (Fabani and Gait 2008).
The 31-mer 29-OMe fully modified ON (Jopling et al. 2005)
was purchased from Dharmacon: 59-AGACACAAACACCAUUG
For in vitro and cell assays, an miRCURY LNA/DNA
knockdown probe was used and was purchased from EXIQON
(59-ACAAACACCATTGTCACACTCCA-39; Cat No: 118019-00
Decoy PNA was obtained from Panagene (Davis et al. 2009):
Cell culture, transfections, and RNA extractions
Huh7 cells were plated in a 6-well plate format and maintained in
DMEM/10% FBS with antibiotics (Full Media) for z20 h before
transfection at 37°C/5% CO2. For PNA ON treatment, cells were
carefully washed once with PBS and media was replaced by 1 mL
of opti-MEM (Invitrogen) containing PNA ON at the desire con-
centration. Four hours later, media was replaced by Full Media.
All other ONs were lipofected using Lipofectamine 2000 (Invi-
trogen) in serum-free media following the manufacturer’s pro-
tocol. Four hours after lipofection, the media was replaced by Full
Media. Cells were incubated for 20 h at 37°C/5% CO2after trans-
fection, washed once with PBS, and lysed using 1 mL of TRI-
Reagent (Sigma). RNA was extracted following the TRI-Reagent
manufacturer’s protocol unless stated otherwise. For 2-equivalent
and 3-equivalent isopropanol (v/v) samples, the TRI-Reagent
protocol was slightly modified as follows: z500 mL of aqueous
(RNA) phase was extracted and precipitated with 1 mL or 1.5 mL
of isopropanol, respectively. The RNA pellet was then washed
twice with 2 mL of 75% ethanol and dissolved in 100 mL of water.
RNA quantification and quality control were measured using a
NanoDrop ND-1000 spectrophotometer (only for non-radioac-
tive RNA). Addition of more than recommended isopropanol
during the RNA precipitation step induces precipitation of other
contaminants to the RNA fraction; hence the need for more RNA
washing steps. For samples extracted with 3 equivalents (v/v) iso-
propanol, RNA was re-precipitated after the extraction protocol
using 2.5 volumes of ethanol 96% and 0.1 volume of 3 M NaOAc,
samples were incubated for 2 h at ?80°C, and the RNA was
recovered by centrifugation for 30 min at 4°C at 13,200 rpm. Re-
precipitated RNA was washed once with 1.5 mL of 75% ethanol,
dried at room temperature, and dissolved in 100 mL of water.
MiR-122 recovery in the presence of any of the anti-miRs used in
this study was not compromised during the RNA re-precipitation
Isopropanol supernatants of radioactive samples were concen-
trated to z200 mL (using a Savant SpeedVac DNA 110 concen-
trator) where a precipitate was detected. The precipitate was
dissolved in 500 mL of water and was NAP-10-purified (GE
Healthcare) following the manufacturer’s protocol.
Cell lysate experiments
Huh7 cells were plated in 6-well plate format and kept in Full
Media for 48 h as described above. Cells were then lysed using 1
mL of TRI-Reagent (Sigma). Equivalent amounts of anti-miR
ONs were added to the cell lysates to mimic transfections at the
desired concentrations, and RNA was extracted.
For experiments containing
synthetic miR-122 sense strand ON was 59-end-radiolabeled
using [g-32P]ATP. The reaction product was NAP-10-purified
(GE Healthcare), and the eluate was concentrated to half of its
volume, z750 mL, using a Savant SpeedVac DNA 110 concen-
trator. Twenty microliters of this solution was added to each cell
32P-miR122 ON, 250 pmol of
miRNA fate upon anti-miR targeting
Polyacrylamide gel electrophoresis (PAGE)
and Northern blots
PAGE and Northern blotting were carried out as described
previously (Fabani and Gait 2008) with minor modifications: 4
to 10 mg of total RNA from each experimental condition was
dissolved in loading buffer (8 M urea; 50 mM EDTA; 20%
formamide; Bromophenol Blue; Xylene Cyanol F), loaded onto
the gels, and run for 2 h at 10 W at room temperature. A32P-
labeled RNA ladder (Decade-Markers; Applied Biosystems) was
used to estimate band sizes. Blotted membranes were cross-linked
but not baked. After overnight membrane hybridization with
specific radiolabeled probes, membranes were sequentially washed
with 23 SSC/0.1% SDS (30 min), 13 SSC/0.1% SDS (30 min),
and 0.53 SSC/0.1% SDS (1 min) at 42°C.
For experiments containing32P-miR122, 5 mL of extracted total
RNA or purified isopropanol supernatant was mixed with 10 mL
of loading buffer and loaded in a gel and run as described above.
After the electrophoresis, the gels were exposed to a radiographic
film for 30 min at ?80°C.
PNA decoy experiments
PNA decoy experiments were carried out as described by Davis
et al. (2009) with minor modifications: Gel composition and RNA
loading buffer contained 20% formamide as described above. The
whole gel was transferred to a membrane as described previously
(Fabani and Gait 2008), and the membrane was cut prior to
hybridization with miR-122 probe. The pre-hybridization buffer
was not exchanged after the membrane pre-hybridization step.
Hybridization of the miR-122 probe was carried out overnight,
and the membrane was then washed as described above.
Supplemental material is available for this article.
We thank Donna Williams for PNA synthesis. We also thank
David Loakes and Andrey Arzumanov for technical advice. We
thank Andrew Newman for reading and commenting on the
manuscript. A.G.T. is funded by a Cesar Milstein Scholarship
from the Darwin Trust of Edinburgh, Scotland. The work was
supported by the Medical Research Council (MRC Unit pro-
Received November 9, 2010; accepted February 15, 2011.
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