amplificationtech note 5692

Real-Time qPCR as a Tool for Evaluating RNAi-MediatedGene Silencing
Tom Van Maerken,1 Pieter Mestdagh,1 Sarah De Clercq,2 Filip Pattyn,1Nurten Yigit,1 Anne De Paepe,1 Jean-Christophe Marine,2 Frank Speleman,1
and Jo Vandesompele1
1Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium;
2Laboratory for Molecular Cancer Biology, Flanders Interuniversity Institute for

Biotechnology (VIB), Ghent, Belgium
Contact information: Jo Vandesompele, Center for Medical Genetics, Ghent
University Hospital, MRB, De Pintelaan 185, 9000 Ghent, Belgium. Phone:
32-9-332-5187; Fax: 32-9-332-6549; E-mail: Joke.Vandesompele@UGent.be

Introduction
Real-time quantitative PCR (rt-qPCR) is the method of
choice for accurate, sensitive, and specific quantitation of
nucleic acid sequences. Applications of this technology are
numerous in molecular diagnostics and virtually all fields of life
sciences, including gene expression profiling, measurementof DNA copy number alterations, genotyping, mutation
detection, pathogen detection, measurement of viral load,disease monitoring, and assessment of drug response.
Several factors, such as careful primer design and evaluation,
template preparation, implementation of a robust normalization
strategy, and accurate data analysis, are essential for a
successful and reliable rt-qPCR assay. In this report we
describe how rt-qPCR can be implemented as a tool formonitoring silencing efficiency and assessing functional effects
of RNA interference (RNAi)-mediated knockdown of MYCN
and TP53 genes, which are pivotal in neuroblastoma
pathogenesis.
Neuroblastoma is a childhood cancer that is derived
from precursor cells of the adrenosympathetic system,
originating in the adrenal medulla or sympathetic ganglia.
Three clinicogenetic subtypes of neuroblastoma can bediscerned (Vandesompele et al. 2005), the most notorious
being the subtype characterized by amplification of the
MYCN oncogene. Amplification of MYCN is associatedwith advanced stage of disease and poor outcome, but themechanisms by which this transcription factor exerts its
oncogenic activity and confers an unfavorable prognosis
are poorly understood. Another intriguing feature ofneuroblastoma is the remarkably low frequency of TP53
mutations upon diagnosis (Tweddle et al. 2003). We havepreviously shown that reactivation of the p53 pathway by
the selective small-molecule MDM2 antagonist, nutlin-3,
constitutes a promising novel approach for the treatment ofneuroblastoma (Van Maerken et al. 2006).

To gain insight into the mechanism of action of MYCN and TP53
genes in neuroblastoma pathogenesis, and to create a model
system for future exploration of targeted therapeutics, we
decided to exploit RNAi as an experimental tool for suppressing
the expression of MYCN and TP53. The MYCN gene wastransiently suppressed using several 27-mer siLentMer™*
Dicer-substrate small interfering RNA (siRNA) duplexes, which
are generally considered more effective at gene silencing thancorresponding traditional 21-mer siRNAs (Kim et al. 2005).
Stable knockdown of the TP53 gene was achieved using
lentiviral-mediated short hairpin RNA (shRNA) interference.
The CFX96™ real-time PCR detection system was used as areal-time detection platform.
Methods
Cell Lines

Human neuroblastoma cell lines IMR-32 and NGP carrying
wild-type p53 were grown as a monolayer in RPMI-1640
medium supplemented with antibiotics, 15% fetal calf serum,and 2 mM glutamine. Human embryonic kidney 293T cells
were cultured in DMEM medium supplemented with antibiotics,
10% fetal bovine serum, and 2 mM glutamine. All cells wereincubated at 37°C in a humidified atmosphere containing
5% CO2.
siRNA Transfections and Nutlin-3 Treatment

IMR-32 cells were transfected either with 10 nM of anonspecific control siLentMer siRNA or siLentMer siRNAs
targeting MYCN (target sequence of siRNA 1:5'-CGAGCTGATCCTCAAACGATGCCTTCC-3'; target sequenceof siRNA 2: 5'-GACGCTGATACATAACTAAATTTGATA-3') in a
6-well plate format using 2.5 µl of siLentFect™ lipid transfection
reagent per well. Silencing efficiency was assessed by rt-qPCR
48 hr after transfection. Nutlin-3 (Cayman Chemical) was
dissolved in ethanol and stored at –20°C in small aliquots as
a 10 mM stock solution. Cells were exposed to 0–32 µM ofnutlin-3 for 24, 48, and 72 hr, with the final ethanol concentration
kept constant in each experiment.
Lentiviral Vector Construction, Virus Production, and Infection

The sequence of the human TP53 gene that was targeted using
shRNA was 5'-GACTCCAGTGGTAATCTAC-3'. As a negative
control, we targeted the Trp53 gene, the murine form of p53,
using the sequence: 5'-GTACTCTCCTCCCCTCAAT-3'. Pairs ofcomplementary oligonucleotides containing these sequences* These siLentMers were validated in Dr. Vandesompele’s laboratory.

were phosphorylated, annealed, and ligated into the pSIF-H1Puro vector (System Biosciences). The pSIF-H1-Puro vectors
containing the shRNA sequences were cotransfected withtwo packaging plasmids (pFIV-34N and pVSV-G) (System
Biosciences) into 293T cells using calcium phosphate
precipitation. Viral supernatants, designated as LV-h-p53
and LV-m-p53 for lentiviruses carrying the shRNA constructagainst human TP53 and murine Trp53, respectively, werecollected and filtered through a 0.45 µm filter, and used to
infect IMR-32 and NGP cells in the presence of 5 µg/ml
polybrene (Sigma). Transduced cells were selected with
1 µg/ml puromycin (Sigma) for 48 hr.
Quantitation of mRNA Expression by rt-qPCR

Total RNA was extracted from cells using the RNeasy
mini kit (QIAGEN), with an on-column treatment with
RNase-free DNase I. After an additional RQ1 DNase treatment
in solution (Promega), first-strand cDNA was synthesized from
2 µg total RNA using the iScript™ cDNA synthesis kit. Primer
sequences for rt-qPCR analysis are available in the public
RTPrimerDB database (http://medgen.ugent.be/rtprimerdb/)(Pattyn et al. 2006): GAPDH (ID #3), SDHA (ID #7),
UBC (ID #8), MYCN (ID #180, P1; ID #3870, P2; ID #3871,
P3; ID #3869, P4; ID #3868, P5), TP53 (ID #1186, P1;
ID #3872, P2), MDM2 (ID #3499), and BBC3 (PUMA)(ID #3500). rt-qPCR assay validation was performed using
RTPrimerDB’s in silico assay evaluation pipeline to determine
specificity (BLAST), absence of secondary structure (mfold),and nucleotide polymorphisms in the primer annealing region.
In silico assay evaluation was followed by experimental
evaluation of specificity and PCR efficiency using a standard
curve consisting of six points of 4-fold serially diluted(64 ng down to 62.5 pg) reverse-transcribed human qPCR
reference total RNA (Stratagene). (The protocol for generationof a template dilution series is available on http://medgen.
ugent.be/CMGG/protocols/). Relative mRNA expression levels
were determined using an optimized two-step SYBR Green I
rt-qPCR assay with minor modifications (Vandesompele
et al. 2002a). The PCR was run in duplicate on the CFX96
real-time PCR detection system in 15 µl reactions, containing
7.5 µl of iQ™ SYBR® Green supermix, 250 nM of eachprimer, and cDNA equivalent to 20 ng of total RNA. Gene
expression levels and PCR efficiency, along with its standard
error, were calculated using the qBasePlus 1.0 analysis
software (http://www.biogazelle.com) (Hellemans et al. 2007),
which employs a proven delta-CT relative quantitation modelwith PCR efficiency correction and multiple reference genenormalization using GAPDH, UBC, and SDHA (Vandesompele
et al. 2002b).
Cell Viability Assay

Cells were seeded in duplicate in 96-well plates (1 x 104 cells/well)and incubated for 6 hr to permit adherence to the surface.
Cells were then treated with 0–32 µM nutlin-3 for 24, 48, and72 hr. Cell viability was determined using the CellTiter-Glo
luminescent cell viability assay (Promega). Three independent
experiments were performed.
© 2008 Bio-Rad Laboratories, Inc.

Results and Discussion
From Experimental Design to Analysis of an rt-qPCR Assay

Bioinformatics-based quality assessment of newly designed
rt-qPCR primers can considerably improve the likelihood of
obtaining specific and efficient primers. A number of quality
control parameters have been integrated in our in-housedeveloped RTPrimerDB’s in silico assay evaluation pipeline(Pattyn et al. 2006) that allow for a streamlined assessmentof candidate primer pairs, with automated BLAST specificity
search, prediction of putative secondary structures of theamplicon, indication of which transcript variants of the geneof interest will be amplified, and search for known SNPs in theprimer annealing regions. This in silico evaluation, however,does not preclude the need of experimental validation after
synthesis of the primers. Ideally, experimental evaluation
addresses specificity, efficiency, and dynamic range of theassay using a broad dilution series of template (Figure 1).
Purity and integrity of the RNA template are critical factors
to the success of an rt-qPCR assay. Several kits arecommercially available for producing clean RNA samples.
Contaminants should be avoided or removed, since they cangreatly influence the reverse transcription step or the actual
PCR. The presence of PCR inhibitors can be assessed using
a variety of methods, including the simple and fast PCRbased SPUD assay (Nolan et al. 2006a). An oligonucleotide
target sequence with no homology to human DNA is spiked
into human RNA samples and a water control at a known
concentration. rt-qPCR quantitation of the oligonucleotide
template in both the RNA samples and the (negative) water
control is indicative of possible enzymatic inhibitors present
in the RNA extract. For assessment of RNA integrity,electrophoresis and PCR-based methods are available(Fleige and Pfaffl 2006, Nolan et al. 2006b).
To control for inevitable experimental differences due tovariables such as the amount and quality of starting material,
enzymatic efficiencies, and overall cellular transcriptional
activity, use of a reliable normalization strategy in which thesefactors are taken into account is necessary. In principle,
internal reference genes offer the best way to deal with themultiple sources of confounding variables that might exist
between different samples. A truly accurate normalization canbe achieved only when multiple reference genes are utilized,
since use of a single reference gene results in relatively
large errors in a considerable proportion of the sample set(Vandesompele et al. 2002b). Because no universal set ofreference genes exists, care should be taken when selecting
the genes to be used for normalizing the expression levels.
Different sample origins and experimental manipulations
might require another set of genes to be used as reference
genes. Selection and validation of reference genes can bedone using the geNorm algorithm, which determines the moststable genes from a set of tested candidate reference genes
in a given sample panel and calculates a normalization factor(Vandesompele et al. 2002b).

Bulletin 5692

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Primers

		

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PCR efficiency 98.6 ± 0.4%

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Fig. 2. Importance of primer location for rt-qPCR assessment of siRNAsilencing efficiency. A, percentage silencing of MYCN gene expression
measured by five different primer pairs (P1–P5) in IMR-32 cells 48 hr after
transfection with anti-MYCN siLentMer siRNA duplexes (siRNA 1 or siRNA 2),
compared to cells transfected with a nonspecific control siRNA; B, schematic
representation of the MYCN mRNA structure with location of siRNA-targeted
sequences and primer binding sites.

34
32

P1

95

28
26

Assessment of siRNA Silencing Efficiency Using rt-qPCR in
Cells Treated With Anti-MYCN siLentMer siRNA Duplexes

24
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10 log starting quantity

10

100

Fig. 1. Experimental validation of newly designed rt-qPCR primers.
A, PCR efficiency and dynamic range of the rt-qPCR assay was tested
using a 4-fold serial dilution of six points of reverse transcribed humanqPCR reference total RNA (64 ng down to 62.5 pg) and TP53 P2 primers;
B, specificity of the TP53 P2 primers was assessed by generating a melting
curve of the PCR product; C, standard curve and PCR efficiency estimation(including the error) according to the qBasePlus software. Cq, quantitation
cycle value.

Processing and analysis of the raw rt-qPCR data represent
a multistep computational process of averaging replicate
CT values, normalization, and proper error propagation
along the entire calculation track. This process might prove
cumbersome and deserves equal attention as the previous
steps in order to get accurate and reliable results. This
final procedure has been automated and streamlined in
Biogazelle’s qBasePlus software, a dedicated programfor management and analysis of rt-qPCR data (Hellemans
et al. 2007).

© 2008 Bio-Rad Laboratories, Inc.

Human IMR-32 neuroblastoma cells were transfectedwith different anti-MYCN siLentMer siRNA duplexes or anonspecific control siRNA, and the level of MYCN transcript
was determined 48 hr posttransfection by rt-qPCR. Our
results show variable levels of MYCN silencing with different
primers (Figure 2A), which indicates the importance of primer
location for evaluation of siRNA silencing efficiency, consistent
with a previously published study (Shepard et al. 2005).
The target mRNA sequence is cleaved by the RNA-induced
silencing complex (RISC) near the center of the region
complementary to the guiding siRNA (Elbashir et al. 2001).
Complete nucleolytic degradation of the resulting fragments is
not always guaranteed, which might result in underestimationof siRNA silencing efficiency if primers that do not span thesiRNA target sequence are used for rt-qPCR (Figure 2B).
Evaluation of Silencing Efficiency and Functional Effects ofLentiviral-Mediated shRNA Knockdown of TP53 Using rt-qPCR

To generate stable TP53 knockdown variants ofneuroblastoma cell lines that carry wild-type p53, we infected
IMR-32 and NGP cells with a lentiviral vector encoding an
shRNA directed specifically against the human TP53 gene
(LV-h-p53) or against the murine Trp53 gene (LV-m-p53), as

Bulletin 5692

A. BBC3 (PUMA) expression

Relative mRNA expression of TP53

120

TP53 P1 primers
TP53 P2 primers

100
80
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40

12.00
Relative mRNA expression

IMR-32 Cells

20

10.00
8.00
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Nutlin-3, µM

00shRNA construct
Cell type

NGP Cells

0816
LV-m-p53
IMR-320

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NGP

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LV-h-p53 infection

Fig. 3. Assessment of shRNA-mediated TP53 knockdown efficiency by
rt-qPCR. IMR-32 and NGP cells were infected with a lentivirus carrying an
shRNA construct specific for either the human TP53 gene (LV-h-p53) or themurine Trp53 gene (LV-m-p53) as a control. Efficiency of TP53 gene silencing
was evaluated by rt-qPCR using two different primer pairs (TP53 P1 andTP53 P2). Bars indicate mRNA expression levels of TP53, relative to therespective LV-m-p53 infected cells; error bars depict standard error of themean (duplicated PCR reactions for TP53 and 3 reference genes).

a negative control. rt-qPCR analysis with two different primer
pairs demonstrated that expression of TP53 was reducedby 81–87% in IMR-32 cells infected with LV-h-p53 and by
92–94% in NGP cells infected with LV-h-p53, compared to
the respective control cells infected with LV-m-p53 (Figure 3).
Functionality of the TP53 knockdown variants was validated
by rt-qPCR and analysis of cell viability after treatment withnutlin-3. This small-molecule compound selectively disrupts
the interaction between p53 and its negative regulator MDM2,
resulting in stabilization and accumulation of the p53 protein
and activation of the p53 pathway (Vassilev et al. 2004).
Lentiviral-mediated expression of shRNA against human TP53
prevented transactivation of p53 target genes, such as BBC3
(PUMA) and MDM2, by nutlin-3 (Figure 4A, B). In addition,
downregulation of TP53 transcript level by nutlin-3, a resultof the ability of the p53 protein to negatively autoregulate its
transcriptional expression after accumulation (Hudson et al.
1995), was abrogated in cells infected with LV-h-p53 (Figure
4C, D). At the cellular level, silencing of human TP53 severely
attenuated the cell viability response to nutlin-3 (Figure 5A,
B, D, E), in contrast to control infection with LV-m-p53(Figure 5A, C, D, F), while viability of nontransduced parental
cells (Figure 5A, D) and cells infected with LV-m-p53 (Figure
5C, F) was significantly reduced when treated with increasing© 2008 Bio-Rad Laboratories, Inc.

0shRNA construct
Cell type816
LV-h-p53
IMR-32

C. TP53 expression (using TP53 P1 primers)
1.20
Relative mRNA expression

LV-m-p53 infection

1.00
0.80
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Nutlin-3, µM

0shRNA construct
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D. TP53 expression (using TP53 P2 primers)
1.20
Relative mRNA expression

Relative mRNA expression of TP53

8
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LV-h-p53
IMR-32

1.00
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Nutlin-3, µM
shRNA construct
Cell type0816
LV-h-p53
IMR-320

8
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LV-m-p53
IMR-320

Fig. 4. Functional validation of shRNA-mediated TP53 knockdown throughrt-qPCR analysis of transcript levels of p53-regulated genes after
nutlin-3 treatment. IMR-32 and NGP cells were infected with a lentiviral vector
encoding an shRNA directed specifically against either the human TP53 gene
(LV-h-p53) or the murine Trp53 gene (LV-m-p53). Cells were treated with 0, 8,
or 16 µM nutlin-3 for 24 hr, and expression of BBC3 (PUMA) (A), and MDM2 (B),p53-regulated genes, and TP53 was determined by rt-qPCR. Two different
primer pairs (TP53 P1 and TP53 P2) were used for quantification of TP53transcript levels (C, D). Bars indicate mRNA expression levels relative to therespective vehicle-treated (0 µM nutlin-3) LV-m-p53 infected cells, mean of two
different rt-qPCR measurements; error bars show standard error of the mean.

Bulletin 5692

A. Uninfected IMR-32 cells

B. IMR-32 cells infected with LV-h-p53

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100Fig. 5. Functional validation of shRNA-mediated TP53 knockdown through cell viability analysis after treatment of IMR-32 and NGP cells with nutlin-3.
Effect of nutlin-3 on viability of uninfected cells (A, D), LV-h-p53 infected cells (B, E), and LV-m-p53 infected cells (C, F). Exponentially growing cells were exposed to
0–32 µM of nutlin-3 for 24, 48, and 72 hr, and the percentage cell viability, with respect to vehicle-treated (0 µM nutlin-3) cells, was determined. Error bars indicate
standard deviation of mean cell viability values of three independent experiments.

concentrations of nutlin-3. These results firmly demonstratepotent and selective impairment of p53 function in IMR-32
and NGP cells infected with a lentivirus carrying an shRNA
construct against human TP53.

© 2008 Bio-Rad Laboratories, Inc.

Conclusions
rt-qPCR analysis provides a convenient and reliable method
for evaluating knockdown efficiency and functional effects ofRNAi-mediated gene silencing, as demonstrated by the useof this tool for monitoring knockdown of MYCN and TP53
genes, which are pivotal in neuroblastoma pathogenesis.
Successful application of rt-qPCR requires careful attention to
all of the steps in the assay workflow, including primer designand evaluation, template preparation, normalization strategy,
and data analysis.

Bulletin 5692

Grant Support
The Fund for Scientific Research – Flanders (FWO) grants 011F4004(T. Van Maerken, Research Assistant), G.1.5.243.05 (J. Vandesompele) andG.0185.04; GOA grant 12051203; grants from the Belgian Foundation against
Cancer (S. De Clercq), the Ghent Childhood Cancer Fund, and the Ghent
University Research grant (B.O.F.) 01D31406 (P. Mestdagh, F. Pattyn).

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BLAST is a trademark of the National Library of Medicine.
CellTiter-Glo is a registered trademark of Promega Corporation.
RNeasy is a trademark of QIAGEN Corporation.
SYBR is a trademark of Invitrogen Corporation. Bio-Rad Laboratories, Inc. is licensed by
Invitrogen Corporation to sell reagents containing SYBR Green I for use in real-time PCR,
for research purposes only.
The siLentMer products are manufactured by Integrated DNA Technologies, Inc. (IDT)
and are for research use only. For custom siRNA synthesis, contact IDT.
Bio-Rad’s CFX96 real-time thermal cycler is a licensed real-time thermal cycler underApplera’s United States Patent No. 6,814,934 B1 for use in research and for all otherfields except the fields of human diagnostics and veterinary diagnostics.

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