A P P L I C AT I O N N O T E

DharmaconAccell siRNA
reagents: achieving
long-term genesilencing.

Abstract
Developing a thorough understanding of a gene’s contribution
to a particular phenotype is problematic when the protein has
an extended half life or the phenotype under study requires
days to materialize. Under these circumstances, reagents thatinduce long-term gene silencing are needed to sufficiently
deplete internal cellular stores for extended periods of time.
To address this need, we have developed Dharmacon™ Accell™
siRNA reagents, with novel modifications enabling uptake
by cells without the aid of lipid transfection reagents. This
innovative delivery technology allows repeated application ofDharmacon Accell siRNA reagents to provide extended duration

Authors
Zaklina Strezoska
Christina Yamada

gene knockdown with only minimal effects on cell viability andthe innate immune response. These attributes greatly broaden
the range of biological questions and cell types that can beinvestigated by researchers using RNA interference (RNAi).

RevvityIntroduction
Over the years, gene silencing by RNAi has relied heavily on theuse of cationic lipid-based delivery reagents for transfecting
cells with small interfering RNAs (siRNAs). In many cases,
lipid-based transfection is sufficient and provides efficientlevels of gene knockdown. Yet in a significant fraction of cases,
cells are either refractory to lipid-mediated delivery of siRNAs
or aversely sensitive to the presence of lipids.

Dharmacon Accell siRNA reagents: achieving long-term gene silencing.

The Dharmacon Accell siRNA product line represents a
recently developed technology that promotes deliveryof siRNA independent of viral vectors or lipid-basedtransfection reagents (also referred to as “passive delivery”)
to virtually any cell type. Through the incorporation ofa unique combination of chemical and bioinformaticenhancements that increase functionality, stability, andlipid-independent delivery, Dharmacon Accell siRNAs canbe used in a wide range of human, mouse, and rat cell linesthat have been traditionally identified as difficult to transfect.
(Appendix I, see back page for a list of cells into which
Dharmacon Accell siRNAs have been successfully tested.)
This novel delivery technology permits gene silencing withminimal off-target events (as assessed by genome-wideprofiling) has limited repercussions on cell viability, andtriggers a negligible innate immune response as assessedby quantitation of nine inflammatory response proteinsusing the SearchLight™ Array platform (Human Inflammatory
Cytokine Array 1; Thermo Scientific Cat. #84619). Thus,
unlike electroporation, which is historically the firstalternative delivery method for cells found to be averse toFigure 1: Repeated application workflow for inducing long-term
gene silencing in cultured cell lines. Following the initial
plating in growth medium (medium plus serum, supplements
and antibiotics), cells are treated with Dharmacon Accell siRNA
resuspended in the Dharmacon Accell Delivery Media. After
24-48 hours, the delivery mix is replaced with complete medium
until cells are confluent, upon which the cultures are split andDharmacon Accell delivery mix is re-applied.

lipid-based transfection reagents, Dharmacon Accell siRNA
reagents can be employed without significant effects on cell
physiology.
Over the course of investigations, it is often necessary
to knock down gene expression for extended periods oftime to accurately assess the contribution of a particular
protein to a given biological event. Long-term reductionof gene expression can be problematic, particularly incases where there are large intracellular reservoirs of
the target or the half-life of the protein is drawn out.
In some instances, researchers have attempted multiple,
consecutive lipid-mediated transfections of siRNAs into
cultured cells. Unfortunately, this approach is frequently
cytotoxic and can induce brief (24-72 hours) changes inthe state of the innate immune response. To overcome
these barriers, DNA-mediated RNAi, commonly exploiting
viral delivery platforms, has been adopted. This approach
can be successful, but is costly and requires significant
development time. For a solution that requires neither lipid
transfection nor viral delivery, this Application Note provides
details of how Dharmacon Accell siRNA reagents may be
applied to provide long-term silencing of gene expression.

Workflow for long-term gene knockdown
using Dharmacon Accell siRNA
Figure 1 provides an overview of how Dharmacon Accell
siRNA can be used to induce gene knockdown for extended
periods of time. The day after cells are trypsinized, counted
and plated, the growth medium used to culture cells(containing serum, supplements and antibiotics) is replacedwith Dharmacon Accell Delivery Media (Cat. #B-005000100, B-005000-500) containing Dharmacon Accell siRNAtargeting the gene of interest (referred to as the Dharmacon
Accell delivery mix). Passive transfection then proceeds for
24-48 hours, whereupon the Dharmacon Accell delivery
mix is exchanged for growth medium, and cells are cultured
for an additional period (generally 24-48 hours). Prior toreaching confluency, cultures are split, re-plated, and treatedagain using the same protocol described in the previous
round. If cells require additional time prior to splitting, they
may be treated again with Dharmacon Accell delivery mix
without splitting. This may vary depending upon the celltype; however, the cycling between growth medium andDharmacon Accell delivery mix can be performed with little
affect on cell viability to provide long-term gene knockdown
in your cell type of choice.

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PPIB
PPIB

NTC
NTC

GAPD
GAPD

PPIB
PPIB

NTC
NTC

1.0 μM

1.0 μM

0.2
0.200

0.5 μM

0.6
0.6
0.4
0.4
0.1 μM

1.0 μM

1.0 μM

0.5 μM

0.1 μM

1.0 μM00

0.5 μM

0.25
0.25

11
0.8
0.8

1.0 μM

0.50
0.50

1.4
1.4
1.2
1.2

0.5 μM

1.00
1.00
0.75
0.75

day
day 33day
day 77day
day 11
11day
day 1515day
day 19191.6
1.6

0.1 μM

1.25
1.25

Cell viability (UT=1)

day
day 33day
day 77day
day 11
11day 15
15day
day
day 19
19

1.50
1.50

0.1 μM

Relative target expression

Dharmacon Accell siRNA reagents: achieving long-term gene silencing.

GAPD
GAPD

UT
UT

UT
UT

Figure 2: Demonstration of long-term gene knockdown using Dharmacon Accell siRNA technology. SHSY5Y neuroblastoma cells wereplated at a density of 5,000 cells per well (96-well plate) and cultured overnight. The following day, the complete medium was exchanged withthe Dharmacon Accell delivery mix containing Dharmacon Accell siRNA targeting housekeeping gene PPIB at 0.1, 0.5 and 1 μM or GAPDH at
1 μM. Dharmacon Accell Non-targeting siRNA #1 (NTC) was also used at 0.1, 0.5 and 1 μM concentrations at every passage. After 48 hours,
the overlying delivery mix was replaced with complete medium. Target knockdown (A.) and cell viability (B.) was assessed on days 3, 7, 11, 15,
and 19 using bDNA and Thermo Scientific™ alamarBlue™ (BioSource, Intl), respectively. (UT – Untreated control cells that have gone throughthe same procedure of medium changes as Dharmacon Accell-treated cells).

(Cyclophilin B, NM_000942) in SH-SY5Y neuroblastoma cells.
Dharmacon Accell siRNA targeting PPIB was delivered at
one of three concentrations (100 nM, 500 nM, or 1 µM)
and the levels of mRNA knockdown were assessed using
branched DNA (bDNA; Panomics) on days 3, 7, 11, 15, and 19.
Dharmacon Accell Non-targeting siRNA #1 (Cat. #D-00191001) was included to assess the specificity and overall effectsof the treatment on cell viability. Finally, at each passage,
untreated cells subjected to medium changes and splitting
were treated with Dharmacon Accell siRNA targeting GAPDH
as a control for transfectability of the cells.
Figure 3: Demonstration of long-term gene knockdown using
Dharmacon Accell siRNA technology. HeLa cells were plated
at a density of 10,000 cells per well (96-well plate) and cultured
overnight. The following day, the complete medium wasexchanged with the Dharmacon Accell delivery mix containing
1 μM Dharmacon Accell siRNA targeting the housekeeping gene
PPIB. Dharmacon Accell Non-targeting siRNA #1 was also used
at 1 μM concentrations at every passage. Following 24 hours oftreatment, the delivery mix was replaced with complete medium.
This workflow (Figure 1) was repeated, and target knockdown andcell viability were assessed on days 2, 6, 10, 14, 18, 22, 26, and
30 using bDNA and alamarBlue™, respectively.

Demonstration of long-term gene knockdown
using Dharmacon Accell siRNA targeting PPIBTo demonstrate the feasibility of providing

The results of these experiments demonstrate the efficacyof Dharmacon Accell technology in providing long-term geneknockdown (Figure 2). Dharmacon Accell siRNA-mediatedgene knockdown of PPIB exhibited a dose-dependent
response, with 1 µM concentrations providing greater than
70% reduction in gene expression in SH-SY5Y cells. Equallyimportant, knockdown was specific while cell viability
generally remained at 80% over the course of the 5 passages(19 days). The repeated application workflow was also
optimized for HeLa cells, resulting in a 30-day duration ofsilencing (Figure 3).

Compatibility of Dharmacon Accell
technology with lipid-mediated delivery

long-term gene knockdown using Dharmacon Accell

To test whether long-term gene silencing using Dharmacon

technology, the workflow described above wasAccell siRNA was compatible with subsequent lipid-mediatedemployed to target the housekeeping gene PPIB

siRNA transfections, HeLa cells treated over the course of
10 days with an Dharmacon Accell PPIB targeting siRNA

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Dharmacon Accell siRNA reagents: achieving long-term gene silencing.

were thereafter transfected with Dharmacon™ siGENOME™

the effects of passive delivery on cell viability and overall(unmodified) siRNA targeting GAPDH (Cat. #D-001140-

physiology are considered prudent to ensure that repeated01-05) and delivered using Dharmacon™ DharmaFECT™

exposure to Dharmacon Accell delivery mix does not

1 Transfection Reagent (Cat. #T-2001-01). The results ofintroduce unwanted changes in the cell’s phenotypic state.

these experiments presented within Figure 4, show that
passive delivery using Dharmacon Accell siRNA technology
is compatible with standard lipid transfection techniques.
In cases where cells were treated with both the Dharmacon
Accell PPIB siRNA and the siGENOME GAPDH siRNAdelivered by DharmaFECT 1, overall knockdown for eachgene was 80% or greater (day 13). These studies attest to
the flexibility of experimental design when using Dharmacon
Accell siRNA reagents and demonstrate the ability to achieve
multi-gene knockdown when using Dharmacon Accell siRNAin conjunction with lipid-mediated delivery techniques.

Long-term gene knockdown is essential when protein
stability and/or assay design requires sustained silencing.
As demonstrated above, repeated application with theDharmacon Accell delivery mix resulted in continuous
silencing for up to 30 days. The described workflow isrelatively non-toxic and compatible with lipid-mediatedtransfection (thus allowing multi-gene or combinatorialknockdown). This novel passive delivery technology
significantly expands the experimental design optionsof researchers by permitting the use of cells that are
historically refractory to lipid-mediated transfection.
Human adherent cell lines (24)Normalized expression

1.21PPIB mRNAGAPDH mRNA

0.80.60.4

Lipid: siRNA(single application)

LAN-5

HEK293T

A549

U-87 MG

A-375

HeLa

HeLa S3

MIA PaCa-2

HEK293

MCF7

MCF 10A SK-BR3

OVCAR-3

NCI/ADR-RES

U2OS

Huh7

GTM-3

HT-1080

HepG2

DLD-1

3T3-L1

H9c2

C2C12

NIH/3T30(repeated application)

IMR-32LNCaP

DU 145

Mouse and rat cell lines (7)0.2

DharmaconAccell siRNASH-SY5Y+++--+

Untreated-PC-12

Human suspension cell lines (4)JurkatFigure 4: Compatibility of Dharmacon Accell siRNA technology
with alternative delivery methods. HeLa cells were treated with
1 μM Dharmacon Accell PPIB siRNA over the course of threepassages (10 days) as described. On day 10, cells were split,
re-plated and transfected with GAPDH-targeting siRNA (100 nM)
using Dharmacon DharmaFECT 1 transfection reagent (DF1).
Twenty-four hours after lipid-mediated transfection, the expression
levels of PPIB and GAPDH were assessed using technologies.

ES-D3

Rat2
IM-9

THP-1

PBMC

Human differentiated stem cells (2)Osteoblasts and adipocytes derived from hMSC
Human, mouse, and rat primary cells (9)HUVEC

HUASMCPrimary mouse
hepatocytes

hMSC

Primary
primary
primaryhumanhuman
human
astrocytes keratinocytes T-cells

Primary rat corticalneurons

primary rat striatum
neurons

Cell types that failed Dharmacon Accell sirna application (0)Summary
While application of Dharmacon Accell siRNA reagentsfor short-term gene silencing requires little optimization,
variations should be tested to identify the best
protocol for long-term gene silencing in your cell type.
Incorporation of controls that allow for assessment ofRevvity, Inc.
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