1) The document describes a cell-based multi-pathway profiling array that was developed to rapidly and sensitively interrogate the functional and biological effects of siRNAs on key cell signaling pathways in vivo. 2) The array uses luciferase reporter technology to monitor the activity of 13 cell signaling pathways, including NFkB, PKC/Ca2+, Notch, Wnt/β-Catenin, cAMP/PKA, p53, E2F, TGFβ, MAPK, and JAK/STAT pathways. 3) Testing of 5 siRNAs on the array found it could provide robust data on both the specific on-pathway and off-pathway effects of siRNAs, as well as highlight potential

1. Cell-based Multi-Pathway Profiling Array: An approach for siRNA phenotype analysis
Qiong Jiang, Abigail Harris, Yexun Wang, Song Tian, Siu Lan Lee, Xiao Zeng, Li Shen, Vikram Devgan
SuperArray Bioscience Corporation, 7320 Executive way, Suite 101, Frederick, MD 21704, USA
Abstract
Recent augmented use of siRNA technology highlighted the importance of comprehensive phenotypic
analysis of siRNA. We have developed a cell-based multi-pathway profiling array for rapid and
sensitive interrogation of functional and biological effect(s) of a siRNA on key signal trans-duction
pathways in vivo. The cellular assay of the array relies on luciferase reporter technology, and covers a
wide range of cell signaling pathways. In the present study, phenotypic effects of 5 siRNA molecules
were screened against 13 cell signaling pathways such as NFkB, PKC/Ca2+, Notch, Wnt/β-Catenin,
cAMP/PKA, p53, E2F, TGFβ, MAPK, interferon including JAK/STAT pathways. Taken together, the
results emphasize the efficacy of cell-based multi-pathway profiling array to advance the screening of
siRNA phenotype by providing robust data for specific on-pathway and off-pathway effects, and
highlight the utility of the array to study cross-talk between cell signaling pathways. Our results also
indicate that cell-based multi-pathway profiling array can also be used to study the physiological
effects of any given stimulus, drug candidate, or gene of interest on cell signaling network.
EXCELLENT REPRODUCIBILITY
FUNCTIONAL GENOMICS - RNAi
Functionally validated dual reporter formulation minimizes variability,
increasing biological relevance of each experiment
Establish mechanisms of action using gene expression knockdown tools
Figure 3. Effects of p53 siRNA, Dicer siRNA and MAPK1 siRNA on the activity of thirteen (13) key signaling pathways
under basal conditions
A
C
Can monitor both up and down-regulation
Analyze the pathway-specific biological effects of gene of interest
expressed from plasmid- or viral-based expression vector
HIGH SENSITIVITY
FUNCTIONAL PROTEOMICS
Use of destabilized luciferase increases signal to noise ratio,
maximizing assay sensitivity
Examine the pathway-specific functional effects of recombinant
proteins or peptides
ENHANCED CONVENIENCE
CHEMICAL GENETICS:
Transfection ready constructs, including positive and negative controls
enable rapid analysis of 13 signaling pathways simultaneously
Study the effect of small chemical molecules or organic products on the
specific signaling pathways
Performance of CignalTM Multi-pathway Profiling Array
MultiFigure 1. CignalTM Multi-pathway Profiling Array can be used for the study of siRNA/shRNA effects
Principle
The Cignal Multi-Pathway profiling Array is based on dual-luciferase reporter assays and reverse transfection
technology for monitoring the activity of thirteen (13) signal pathways simultaneously in cultured cells. The
system uses transcription factor (TF) responsive elements to report corresponding signaling pathway activity.
By determining the activity of thirteen (13) TF using firefly luciferase and normalizing their activity to the
internal control Renilla luciferase activity, the array allows more reliable analysis of the effects of siRNA,
chemical compound, or other treatments on multi-pathways activity in any cell line .
A
HEK-293H cells
B
HeLa cells
HEK-293H cells were reverse transfected with either chemical synthesized negative control siRNA or p53 siRNA (A), Dicer siRNA (B) and MAPK1 siRNA (C) using Cignal
Multi-pathway Profiling Array. After 48 hours of transfection, dual luciferase assay (Promega) was performed. Promoter activity values are normalized using a Renilla reporter
activity and expressed as fold change compared to negative control siRNA. Cignal Multi-pathway Profiling Array reported 3 fold decrease in p53 signaling by p53siRNA.
Moreover, p53siRNA induce Notch, Wnt, MAPK (ERK) and C/EBP signaling. This is consistent with previous reports that p53 inhibit Notch, Wnt, ERK and C/EBP signaling.
Likewise, this array showed the effects of Dicer siRNA and MAPK1siRNA on 13 crucial signal transduction pathways. This data indicate the effectiveness of the Cignal Multipathway Profiling Array for sensitive, specific and rapid screening siRNA effects even in a basal (non-stimulated) condition.
Figure 4. Effects of MAPK1 siRNSA, RelA shRNA and GSK3β siRNA on the activity of thirteen (13) key signaling
pathways under stimulated conditions
Schematic representation of constructs of CignalTM Multi-pathway Profiling Array
Transcription factor
A
TATA
box
Luciferase
B
FUNCTIONAL GENOMICS - OVEREXPRESSION
VERSATILITY
What is the CignalTM Multi-pathway Profiling Array?
Multi-
A.
siRNA/shRNA phenotype analysis using CignalTM Multi-pathway profiling array
Applications
Benefits
B
C
The inducible transcription factor responsive construct (TREs-luc)
Tandem repeats
of TREs
B.
C.
CMV immediate early
enhancer/promoter
Renilla
TATA
box
The constitutively expressing Renilla construct (CMV-Ren)
Luciferase
D.
E.
The non-inducible reporter construct (Pmin-luc)
CMV immediate early
enhancer/promoter
Luciferase
The constitutively expressing luciferase construct (CMV-luc)
CMV immediate early
enhancer/promoter
MGFP
The constitutively expressing GFP construct (CMV-GFP)
Layout of CignalTM Multi-pathway
Profiling Array
How It Works
Prepare siRNA/shRNA stock.
Experimental cells
1
Control cells
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5
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8
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9
10
12
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12
13
-ve
13
-ve
+ve
20 minutes
5 minutes
Aliquot 100 ul of cell suspension into each well of
Cignal multi-pathways Profiling Array. Mix well and
incubate it at 37°C in a CO2 incubator.
5 minutes
Prepare cell suspension of 2X105 cells/ml in OptiMEM medium containing 5% FBS.
5 minutes
Aliquot 25 ul of dilute SureFECT (or any other
transfection reagent) into each well of the array. Mix
well and incubate it at room temperature for 20 min.
2 days
30 minutes
Perform dual luciferase assay (Promega)
2
A
HEK-293H cells
B
HeLa cells
HEK-293H cells were reverse transfected with shRNA control or Rel A shRNA (A); or with chemical synthesized negative control siRNA or MAPK1 siRNA (B) or GSK3β
siRNA (C) using Cignal Multi-pathway Profiling Array. After 48 hours of transfection, cells were treated with different stimuli as described above. Dual luciferase assay was
performed 66 hours after transfection. Promoter activity values are normalized using a Renilla reporter and then expressed as fold change compared to control shRNA (A) or
control siRNA (B and C). The Cignal Multi-pathway Profiling Array showed that RelA shRNA specifically modulate the NFkB signaling (4 fold decrease). Thereby, potentiate the
specificity of the array even after stimulation. Importantly, our data indicate that MAPK siRNA downregulate activity of same signaling pathways in both basal and stimulated
conditions except the C/EBP signaling. Besides, the array showed that knock-down of GSK3β has suppressive effect not only on Wnt signaling but also on Notch, NFkB, PKC/Ca2+,
cAMP/PKA, MAPK (AP-1), C/EBP and TGF β signaling. Thereby, highlights the utility of this array for studying crosstalk between cell signaling pathways.
+ve
Untransfected
control
Dilute 31.5 ul of SureFECTTM (or any other transfection
reagent) in 2.625 ml of Opti-MEM serum free medium.
Mix well and incubate it at room temperature for 5 min.
Figure 2. CignalTM Multi-pathway Profiling Array can be used for the study of biological effects of gene of
interest, chemical compounds and recombinant proteins
4
6
11
5 minutes
4
5
5 minutes
3
9
Aliquot 4-10 pmol of siRNA or 200ng of shRNA plasmid
into each well of CignalTM multi-pathways Profiling Array.
1
7
Add 25ul of Opti-MEM serum free medium into each
well of CignalTM multi-pathways Profiling Array.
2
Cells were reverse transfected using Cignal Multi-pathway Profiling Array. After 48 hours of transfection, dual luciferase assay (Promega) was performed.
Promoter activity values are normalized using a Renilla reporter activity and expressed as fold change compared to negative control (more than two fold change
was considered significant). The inducible transcription factor responsive elements (TREs-luc) showed distinct basal activity of thirteen signaling pathways in
different cell type indicating the potential use of Cignal Multi-pathway Profiling Array for studying repression in the cell signaling pathways activity.
Untransfected
control
Each well has 100 ng of specific reporter element (TREsluc) + 2.5 ng of renilla element (CMV-Ren) distributed in
triplicate.
: Notch signaling pathway (RBP-Jk reporter assay)
: Wnt signaling pathway (TCF/LEF reporter assay)
: DNA Damage (p53) pathway (p53 reporter assay)
: Cell cycle pRB-E2F pathway (E2F reporter assay)
: NFkB signaling pathway (NFkB reporter assay)
: PKC/Ca2+ signaling (NFAT reporter assay)
: cAMP/PKA pathway (CRE reporter assay)
: MAPK signaling pathway (AP1 reporter assay)
: MAPK signaling pathway (SRE reporter assay)
: C/EBP pathway (C/EBP reporter assay)
: TGFβ signaling pathway (SMAD reporter assay)
: Interferon signaling (ISRE reporter assay)
: IFNγ signaling pathways (JAK/STAT1 pathway)
(GAS reporter assay)
-ve: The non-inducible reporter element (Pmin-luc)
+ve: The constitutively expressing luciferase element
(CMV-luc) and GFP construct (CMV-GFP)
Conclusions
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SuperArray’s functionally validated panel of Cignal™ Pathway Reporter Assays can be used for measuring the change (both
increase and decrease) in the intracellular activity of 18 cell signaling pathways (data of 5 reporter assays were not shown).
Cignal™ Pathway Reporter Assays cover wide range of biology; cancer biology: Notch, Wnt/β-Catenin, TGFβ, p53, c-Myc,
NFkB signaling; Cell cycle control: E2F, p53, c-Myc pathways; immunology: NFkB, IFN, INFγ signaling; cell proliferation:
MAPK (AP-1), MAPK (SRE), C/EBP; GPCR assay: NFAT (PKC/Ca2+), CRE (cAMP/PKA), AP-1 (MAPK-AP1), nuclear
hormone receptors: glucocorticoid receptor, retinoic acid receptor, estrogen receptor and toxicology: Hypoxia, p53 pathways.
Cells were reverse transfected using Cignal Multi-pathway profiling array. After 24 hours of transfection, cells were treated with different stimuli as described
above. After 42 hours of transfection, dual luciferase assay (Promega) was performed. Promoter activity values are normalized using a Renilla reporter activity and
expressed as fold change compared to non-stimulated control. The inducible transcription factor responsive elements (TREs-luc) reported induction in the activity of
corresponding cell signaling pathways in response to specific stimulus in different cell types. The induction of TREs-luc activity, in turn of corresponding signal
transduction pathway, is specific and dose dependent (data not shown).
CignalTM Multi-pathway Profiling Array is an innovative tool for measuring the intracellular activity of 13 different signal
transduction pathways simultaneously.
The array provides accurate, specific, sensitive and rapid evaluation of siRNA/shRNA phenotype on the activity of 13
different signaling pathways.