RNAi microarray by reverse transfection of siRNA and shRNAfor functional genomics
1

Zheng-Fu Piao1, 2, Fumitaka Takahashi1, Kaoru Saigo1, Kumiko Ui-Tei1

Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan, and 2Present address: Institute of Hepatitis, Beijing You-an Hospital, Affiliated to Capital Universityof Medical Sciences, Beijing 100069, China
Abstract:
RNA interference (RNAi) is a widely used method for genefunction analyses in a variety of organisms. Establishment of alarge-scale, high-throughput platform for analysing genefunction may require development of an RNAi microarraysystem using reverse transfection. Here, we show that shRNA ismore effective in microarray gene silencing compared to siRNA.
Endogenous mammalian genes are suppressed efficiently ifhighly functional class I sequences were used for siRNAs
produced via shRNAs. Furthermore, the method appears
applicable for functional genomics. Indeed, apoptotic cell death
induced by staurosporine and actinomycin D was found to be
blocked by shRNA against apoptotic protease-activating
factor-1 and caspase-3, both of which are required in thecytochrome c-dependent apoptosis cascade.

1. INTRODUCTION

2. METHODS

Double-stranded
RNA(dsRNA)
directs
thesequence-specific degradation of mRNA through a genesilencing process known as RNA interference (RNAi) [1,2].
In mammalian cells, RNAi is triggered by the transfection ofshort-interfering RNA (siRNA), a dsRNA 19-bp in lengthwith 2-nt 3′ overhangs [3], or by short hairpin RNA(shRNA)producedwithincells[4]throughsequence-specific cleavage of a cognate transcript. Thus,
siRNA is usually chemically synthesized, whereas shRNA istranscribed from a DNA plasmid containing an appropriate
promoter such as the H1 or U6 RNA polymerase III
promoter. In the latter case, RNA produced within cells
possesses a hairpin structure containing a stem sequence ofapproximately 20-bp that matches the target RNA sequence
precisely. This hairpin RNA is thought to be processed by
Dicer and other enzymes to generate active siRNA in vivo[5]. RNAi has been rapidly adopted as a technology for the
analysis of gene function, together with ongoing efforts forconstructing large-scale human siRNA libraries.
Cell microarray is a tool that allows the functional
analysis of many genes in a systematic and high-throughput
fashion [6−9]. A gelatin solution containing plasmids andtransfection reagents is spotted onto a glass slide using arobotic microarray system, and mammalian cells are thenapplied across the surface of the entire slide. Cells growing
on gelatin spots, each of which may have a different set ofplasmids, undergo reverse transfection by incorporating that
locally applied plasmids, whereas cells growing outside thegelatin spots are non-transfectants. Thus, the cell microarraysystem makes it possible to introduce many different

978-1-4244-2919-6/08/$25.00 ©2008 IEEE

plasmids separately into mammalian cells. Several groups
have already attempted to establish an RNAi microarraysystem but initial attempts have not been very successful,particularly with the regard to endogenous gene silencing.
Here, we report improvements in the cell microarraysystem combined with RNAi technology and reverse
transfection that can be used for large-scale analysis of genefunction by high-throughput RNAi. Our results show thatthe efficiency of gene silencing by shRNA is much higher
than that of siRNA when functional class I sequences [10]are used. Endogenous genes were inactivated by our reverse
transfection system depending on shRNA, indicating thatshRNA-mediated RNAi microarray may be applicable to thefunctional RNAi.

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2.1 siRNA preparation and construction of the shRNA
expression plasmid
RNA oligonucleotides were purchased from Proligo andsiRNA was prepared as described previously [10]. The
passenger and guide strand sequences of siRNAs are as
follows:
5’-GCCACAACGUCUAUAUCAUGG-3’/5’-AUGAUAUAGACGUUGUGGCUG-3’(siEGFP),
5’-CCGACAUCCCCGACUACAAGA-3’/5’-UUGUAGUCGGGGAUGUCGGUG-3’(siDsRed),
and5’-UAAAGAAAGGCCCGGCGCCAU-3’/5’-GGCGCCGG
GCCUUUCUUUAUG-3’(siLuc). The hairpin shRNA
expression vectors were constructed by inserting theannealed synthetic oligonucleotides into BamHI/HindIIIsites in the pSilencer3.1-H1 puro (Ambion) or
pSUPER.retro.puro (OligoEngine) vector. The inserted
oligonucleotide sequences are as follows: pEGFP441:
5’-GATCCCGCCACAACGTCTATATCATGGGCTTCCTGTCACCCATGATATAGACGTTGTGGCTTTTTTGGAA
A-3’
and5’-AGCTTTTTCCAAAAAAGCCACAACGTCTATATCATGGGTGACAGGAAGCCCATGATATAGACGTTGTGG
GG-3’.
pYes270:
5’-GATCCCAGGTGGTGTTACTATATTTGTGCTTCCTGTCACACAAATATAGTAACACCACCTTTTTTTGGAA
A-3’
and5’-AGCTTTTTCCAAAAAAAGGTGGTGTTACTATATTTGTGTGACAGGAAGCACAAATATAGTAACACCACC
TGG-3’.
pYes430:
5’-GATCCCAGCAATTATGTAGCGCCTGCAGCTTCCTGTCACTGCAGGCGCTACATAATTGCTTTTTTTGGAA
A-3’
and5’-AGCTTTTTCCAAAAAAAGCAATTATGTAGCGCCTGCAGTGACAGGAAGCTGCAGGCGCTACATAATTGC
TGG-3’.
pVim812:5’-GATCCCGTACGTCAGCAATATGAAAGTGCTTCCTGTCACACTTTCATATTGCTGACGTACTTTTTTGGAA
A-3’
and5’-AGCTTTTTCCAAAAAAGTACGTCAGCAATATGAA
AGTGTGACAGGAAGCACTTTCATATTGCTGACGTA
CGG-3’.
pVim33:5’-GATCCCGCAGGATGTTCGGCGGCCCGGGCTTCCTGTCACCCGGGCCGCCGAACATCCTGCTTTTTTGGAA
A-3’
and5’-AGCTTTTTCCAAAAAAGCAGGATGTTCGGCGGCC
CGGGTGACAGGAAGCCCGGGCCGCCGAACATCCTG
CGG-3’.
pFyn444:
5’-GATCCCGTGGTACTTTGGAAAACTTGGGCTTCCTGTCACCCAAGTTTTCCAAAGTACCACTTTTTTGGAA
A-3’
and5’-AGCTTTTTCCAAAAAAGTGGTACTTTGGAAAACTTGGGTGACAGGAAGCCCAAGTTTTCCAAAGTACCA
CGG-3’.
pFyn89:5’-GATCCCATGGCACAGACCCCACCCCTGCTTCCTG
TCACGAGGGGTGGGGTCTGTGCCATTTTTTTGGAAA-3’and5’-AGCTTTTTCCAAAAAAATGGCACAGACCCCACCC
CTGGTGACAGGAAGCAGGGGTGGGGTCTGTGCCAT
GG-3’.
pCaspase-3:
5’-GATCCCGACATGGCGTGTCATAAAATAGCTTCCTGTCACTATTTTATGACACGCCATGTCTTTTTTGGAA
A-3’
and5’-AGCTTTTTCCAAAAAAGACATGGCGTGTCATAAAATAGTGACAGGAAGCTATTTTATGACACGCCATGT
CGG-3’.
pApaf-1:5’-GATCCCGAAAGTCGTTTCGTTATTATTGCTTCCTG
TCACAATAATAACGAAACGACTTTCTTTTTTGGAAA-3’and5’-AGCTTTTTCCAAAAAAGAAAGTCGTTTCGTTATT
ATTGTGACAGGAAGCAATAATAACGAAACGACTTT
CGG-3’. Two expression constructs, pCx-DsRed encoding
DsRed and pCx-EGFP encoding EGFP [11], were used asreporters.
2.2 Microarray printing and reverse transfection
Microarray printing was performed using a modified
procedure of lipid-DNA method developed originally byZiauddin and Sabatini [5]. The siRNA or shRNA expression
plasmid with or without target plasmid DNA was suspended
in 15 µl of DNA-condensation buffer (EC buffer) containing
0.35 M sucrose. After incubation with 1.8 µl of Enhancer
solution and 6 µl of Effectene transfection reagent, one
volume of 0.2 % gelatin was added and re-mixed. A
resultant solution (20 µl) was collected into a well of
96-well plate for subsequent printing. A robotic arrayer
spotting system (SPBIOTM, Hitachi Soft) equipped with

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Fig. 1. Outline of the protocol used for RNAi microarray. The mixtureof transfection reagent and reporter plasmids are spotted onto GAPSII
glass slides using an SPBIO robotic microarray system. The glass slide
was then placed inside of a culture dish and cells were poured into thedish. After incubation, the expression of reporter plasmids expressing
DsRed or EGFP were detected via fluorescent microscopy.

stealth pins was used to print siRNA or shRNA plasmid
solution onto CMT GAPS glass slides (2549, Corning). For
RT-PCR, Western blotting and immunohistochemistry, asiRNA-DNA-lipid mixture in gelatin solution was directly
printed onto a glass slide and stored at room temperature for
16–24 h in a vacuum desiccator with anhydrous calcium
sulphate pellets.
2.3 Reverse transfection
HEK293 or HeLa cells were cultured at 37 ºC under 5 %CO2 in Dulbecco’s modified Eagle’s medium (DMEM;

Gibco-BRL) supplemented with 10 % heat-inactivated fetal
bovine serum (FBS; Mitsubishi Kagaku) and antibiotics (10U/ml penicillin and 50 µg/ml streptomycin; Meiji). Reverse
transfection was carried out using an Effecte™ transfection
kit (301425, Qiagen). Enhancer solution (16 µl) was added
to 150 µl of EC buffer, mixed, and allowed to sit at room
temperature for about 5 min followed by the addition of 25µl of Effectene transfection reagent (25 µl). The total
volume of the mixture was used to coat the surface of a
printed glass slide, sealed using the Hybriwell sealing
system (Molecular Probes), and then inbubated at room
temperature for 20 min. After removing the Hybriwell slip,
the printed slide was placed printed side up in a 100×100×10
mm square culture dish (Corning). The cell suspension(1×107 cells in 25 ml of medium) was gently poured into thedish, after which the dish was placed in a humidified
incubator under 5 % CO2 at 37 ºC for 3 days.
2.4 Laser scanning
The microarrays were imaged using a laser fluorescencescanner (GenePix4000B; Axon Instruments) at a 5 µm
resolution. EGFP and Cy3 emissions were measured
separately after sequential excitation of the FITC and Cy3
fluorophores, respectively.
2.5 RT-PCR
Puromycin (3 µg/ml) was added to the medium 1 day after
reverse transfection with shRNA expression vectors and
pCAGIPuro-EGFP [10], which encodes the puromycin
resistant gene and EGFP, and cells were incubated for 2
additional days to select the pCAGIPuro-EGFP-transfected
HeLa cells. Total RNA was extracted from cells using
RNeasy 96 (Qiagen) according to the manufacturer’s
protocol. After cDNA was synthesized with Oligo(dT)primers, the following primers were used for amplificationof human genes. yes; 5’-CACCATTTGGAGGATCCTCAG
and5’-CCTCGTTGATTTCCAGGATTC,
Fyn;5’-CTCAGCACTACCCCAGCTTC
and5’-CATCTTCTGTCCGTGCTTCA,
vimentin;
5’-GATGTTGACAATGCGTCTCTG
and5’-TTAAGGGCATCCACTTCACAG,
β-actin;5’-CACACTGTGCCCATCTACGA
and5’-GCCATCTCTTGCTCGAAGTC.
2.6 Western blotting
Cells were lysed in 1 % NP-40 lysis buffer (20 mM HEPES,[pH 7.3], 1mM CaCl2, 1 mM Mg(OAc)2, 125 mM KOAc, 5mM EGTA, 1 mM dithiothreitol, and complete proteaseinhibitor [Roche]. Cell extracts (20 µg) were fractionated by
SDS-PAGE and transferred onto a polyvinylidene difluoride
membrane (Immunobilon-P; Millipore). The blots weretreated with rabbit anti-human Fyn antibody (Upstate
Biotechnology), rabbit anti-human Yes antibody (Upstate
Biotechnology) or mouse anti-porcine vimentin antibody(Oncogene Research Products). The same membrane was-348-

also treated with mouse anti-β-actin antibody (Sigma). The
resultantmembranewastreatedwithalkaline
phosphatase-conjugated anti-rabbit or anti-mouse secondary
antibody (Amersham), and visualised by chemiluminescence
CDP-Star detection reagent (Amersham).
2.7
Immunostaining
and
immunofluorescencemicroscopy
Three days after reverse transfection, cells were washed inphosphate-buffered saline (PBS) and fixed in 3.7 %formaldehyde in PBS for subsequent immunofluorescencestaining. After permeabilisation in 0.1 % Triton X-100 in
PBS for 30 min and blocking with 5 % goat whole serum for
1 h, cells were incubated with primary antibodies for 12 h at
4 ºC. Rabbit anti-human Fyn antibody (Upstate
Biotechnology) and rabbit anti-human Yes antibody (Upstate
Biotechnology) were diluted to 1/150 in 1 % whole goat
serum in PBS and used as primary antibodies. The cells were
washed three times with PBS and incubated with thesecondary antibody, Cy3-conjugated anti-rabbit IgG(Jackson Immuno Research), at a dilution of 1/300 for 30
min at room temperature. After washing, cells were observed
under a fluorescence/phase-contrast inverted microscope(Axiovert 200, Carl Zeiss) equipped with an AxioCam HRC
digital camera.
2.8 Hoechst33342 staining
Apoptotic cell death was detected by staining with the
DNA-binding fluorescent dye, Hoechst 33342 (Molecular
Probes). After 24 h treatment with staurosporine (0.3 µM) or
actinomycin D (0.5 µg/ml), cells were washed twice in PBS
and incubated with 10 µg/ml Hoechst 33342 (Molecular
Probes) at room temperature for 30 min. Apoptotic cell death
was determined by the presence of brightly staining
condensed chromatin or nuclear fragmentation.
3. RESULTS AND DISCUSSION
3.1 RNAi microarray using reporter genes
As shown in Fig. 1, siRNA and/or DNA-lipid mixtures ingelatin solution were spotted onto a GAPS glass slide using
the SPBIOTM Robotic arrayer. After drying, the glass slides
were treated with transfection reagents and cells suspendedin culture medium were applied to a printed glass slide in asquare culture dish. After an appropriate culture period, genesilencing effects were examined.
Using visible EGFP and DsRed genes as reporters, genesilencing efficiency by reverse transfection was examined in
HEK293 cells 3 days after reverse transfection. As shown in
the first and seventh rows, labelled with siRNAconcentration = 0, in Fig. 2A, clear signals indicative of
DsRed and EGFP transfection were observed in cells
situated within six spotted areas of about 500–600 µm in
diameter, in which pCx-EGFP or pCx-DsRed plasmids (32ng/µl) had been printed. Transfected gene activity was
detected using a laser fluorescence scanner. When siRNAs

by non-cognate siRNA or control shRNA-encoding DNA
treatment (siLuc). However, a difference in target
knockdown efficiency between cognate siRNA andshRNA-encoding DNA treatment was evident. Although>12 ng/µl (>1000 nM) of siRNA was necessary to almost
completely knockdown the EGFP signal, >2 ng/µl (>1nM)
of shRNA-encoding plasmid was sufficient to suppress
EGFP expression, indicating that shRNA-encoding DNA
treatment is much more effective than siRNA treatment atleast in human cells.

Fig. 2. Dose-dependent silencing effects of siRNA and shRNA for reporter
genes. (A) Effects of siRNAs against EGFP and DsRed. The reporter
plasmid, pCx-DsRed or pCx-EGFP (32 ng/µl), was introduced into
HEK293 cells with siEGFP or siDsRed (0-48 ng/µl), and the silencing
effects were observed via fluorescent microscopy after 3 days. siLuc wasused as a control. (B) Comparison of siRNA versus shRNA expression
plasmid on silencing of EGFP. pCx-EGFP (2 ng/µl) was introduced into
HEK293 cells with siEGFP (0-48 ng/µl) or pEGFP441 (0-32 ng/µl) and
EGFP silencing effects were observed via fluorescent microscopy after 3
days. siLuc and pSilencer vector were used as siRNA and plasmid controls,
respectively.

against EGFP or DsRed were co-transfected, EGFP or
DsRed signals, respectively, decreased with increasingsiRNA concentration (Fig. 2A, rows 2–6). In contrast, nochange was detected when a control siRNA, siLuc, was used(rows 8–12). Next, we compared the silencing efficiencies ofsiRNA and shRNA treatment using EGFP as a target (Fig.
2B, rows 1–6). The pEGFP441 vector expresses a shRNA,
whose target sequence is very similar, if not identical, to thatof the siRNA used. Both siRNA and shRNA inhibited EGFP
expression, and no change in EGFP expression was induced-349-

3.2 Identification of shRNA sequence for highly effective
reversetransfection-dependent
endogenous
gene
silencing
Although efficient silencing of reporter genes has been
demonstrated by other groups [6,7], successful studies
targeting endogenous genes are limited [8]. Our previous
experiment [10] indicated that the efficiency ofsiRNA-dependent gene silencing varies considerably
depending on the siRNA sequence used. Thus, the siRNAsequence dependency of reverse transfection-mediated genesilencing in HeLa cells was examined.
SiRNAs may be classified into three groups, class I, II
and III [10]. Class I siRNAs are highly functional in typicalgene silencing and satisfy the following conditions
simultaneously: A/U residues at the 5′ end of the guidestrand; G/C at the 5′ end of the passenger strand; at least
four A/U residues in the 5′ terminal 7 bp of the guide strand.
In contrast, siRNAs opposite in these features give rise tolittle gene silencing in mammalian cells, and are grouped asclass III. In addition, a G/C stretch, more than 9 bp in lengthis absent in class I siRNAs. The siRNAs other than those ofclass I and III are defined as class II. To confirm whether
our algorithm for siRNA classification is applicable forreverse transfection, the effects of the plasmids encoding
either class I or class III siRNA as shRNA were examined.
Three endogenous genes (the human src family kinase genes,
Yes and Fyn, and the intermediate filament protein gene,vimentin) were used as targets. pYes270, pVim812 andpFyn444 are shRNA plasmids capable of producing class I
siRNA, whereas pYes430, pVim33 and pFyn89 are shRNA
plasmids producing class III siRNA. These shRNA plasmids
were printed separately on a glass slide with
pCAGIPuro-EGFP, and reverse transfection was carried out.
After selecting pCAGIPuro-EGFP transfectants after a 2-day
treatment with puromycin (3 µg/ml), cells were recoveredfrom the glass slide and gene silencing effects were
examinedby
RT-PCR,
Westernblotting
and
immunohistochemical analyses (Fig. 3). pSilencer was used
as a negative control. In all cases examined, shRNA
plasmids capable of producing class I siRNAs markedly
suppressed target gene expression, whereas little target
silencing was observed in cells treated with shRNA plasmids
producing class III siRNA. We conclude that our algorithm
for classifying siRNAs for standard RNAi is also applicablefor a selection of shRNA sequences for effective reverse

Fig. 3. The silencing effects of shRNA expression vectors for yes,vimentin and Fyn. The highly effective class I (pYes270, pVim812,
pFyn444) and ineffective class III (pYes430, pVim33, pFyn89)
siRNA sequences were used for reverse transfection. Each shRNA
expression plasmid was introduced into HeLa cells. After 24 hr, thecells were selected with puromycin (3 µg/ml) for 2 days. Effects ofshRNA on the expression of endogenous genes, Yes (A-C),
vimentin (D, E) and Fyn (F-H) were analyzed by RT-PCR (A, D,
F), Western blotting (B, E, H) and immunostaining (C, H). For RTPCR, 0.2 µg (A) or 0.2, 0.4 and 0.8 µg (D, F) of shRNA plasmids,
and for immunohistochemistry, 0.8 µg of shRNA with 0.2 µg
pCAGIPuro-EGFP was introduced into HeLa cells by reverse
transfection. β-actin was detected by RT-PCR and western blot asinternal control. The plasmids containing the highly effective class I
shRNA sequences eff iciently suppressed the expression of eachtarget gene. However, the plasmids containing ineffective class III
shRNA sequences showed almost no effects. pSilencer, a shRNA
expression plasmid with no insert, was used as a control.

transfection.
3.3 Functional analysis of drug-induced apoptosis byreverse transfection
To demonstrate the efficacy of reverse transfection forfunctional genomics, we used a model system involving
drug-induced apoptosis. Staurosporine and actinomycin D
are known to trigger cytochrome c release frommitochondria[13].
Cytochrome
c/Apaf-1/procaspase-9-containing apoptosomes formed in-350-

the cytosol activate downstream caspases, caspase-3, -6 and-7, leading to cell death [14,15]. If reverse transfection iseffective in endogenous gene phenotype analysis, shRNA
plasmids expressing human caspase-3 and Apaf-1 would be
expected to suppress drug-induced apoptosis.
Highlyfunctional class I shRNA expression plasmids for caspase-3
and Apaf-1 were introduced into HeLa cells by reverse
transfection, and the cells were treated with staurosporine
and actinomycin-D. As shown in Fig. 4, apoptosis was
significantly inhibited in cells co-transfected with shRNAtriggered reverse transfection-mediated gene silencing mosteffectively, which may facilitate genome-wide scale
analyses of gene function.
ACKNOWLEDGEMENTS
This work was supported in part by Special Coordination Fund for
promoting Science and Technology to K.S., and grants from the
Ministry of Education, Culture, Sports, Science and Technology ofJapan to K.U-T.

REFERENCES

Fig. 4. Functional analysis using reverse transfection. Highly
effective class I shRNA expression plasmids against human
caspase-3 (C, G) and Apaf-1 (D, H) were introduced into HeLa
cells, respectively, using reverse transfection with pCAGIPuroEGFP. After 24 h, puromycin (3 µg/ml) was added to the culture
and incubated for 2 days. At 3 days after transfection, the cells
were treated with staurosporine (0.3 µM) for 6 hr (B-D) andactinomycin D (0.5 µg/ml) for 12 hr (F-H), and stained with
Hoechst 33342. Arrowheads in (B) and (F) indicate the condensed
or fragmented nuclei in the cells undergoing apoptosis. Control
cells were no treated cells (A, E). pSUPER, a shRNA expression
plasmid with no insert, was used as a control vector (B, F).

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4. CONCLUSION
In summary, we established an efficient shRNA-mediatedRNAi microarray system. The shRNA appeared to induce
reverse transfection much more effectively than did siRNA,
at least in HEK293 cells. As with standard siRNA-mediatedgene silencing, class I siRNA generated via shRNA-351-[1] Hannon, G.J. (2002) RNA interference. Nature, 418, 244-251.
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