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

Successful RNA
interferenceexperiments indifficult-to-transfectcell lines.

Successful gene silencing experiments that help clarify abiological pathway rely on good experimental planning anddesign. The main components of a well-planned experiment
include optimal delivery of the functional silencing reagents
into a cell type and accurate confirmation of gene knockdown.
RNA interference (RNAi) silencing methods borrow an elegant
endogenous biological pathway that can be applied tomodulate gene expression thus permitting the detailed studyof a particular gene target, a defined biological pathway or
a high-throughput screen of an entire genome. By this one
method, a researcher can characterize a specific phenotype
or disease state. Regardless of the experimental approach(targeted knockdown or high-throughput screen), RNAi
experiments can be described with a general workflow thatAuthor

includes1 identification of functional siRNAs,2 optimization ofAnnaleen Vermeulenand finally,4 investigation of the biological phenotype (Figure 1).

siRNA delivery, followed by3 detection of gene down-regulationRevvity, Inc.

Delivery solutions
Early hurdles of finding potent siRNAs have essentiallybeen eliminated with the availability of rational design
methods for functional siRNAs as with the Dharmacon™
SMARTselection™‑designed siRNAs. However, delivery of thesefunctional siRNAs remains a significant challenge in certain
cell types. Lipid‑based transfection and electroporation are
widely utilized, well-validated methods for many standard celllines. Unfortunately, these methods are often inefficient for
more biologically relevant model systems represented by celltypes that are refractory to traditional transfection strategies.

Successful RNA interference experiments in difficult-to-transfect cell lines.

For example, human suspension (hemapoetic cell lines) andenhanced for delivery in the absence of lipid. Accell siRNAneural cell types, of interest in immune and neurologicalrepresents a new delivery technology to empower RNAi

studies, are typically intractable to standard lipid-mediatedexperiments in many difficult-to-transfect cell types, withoutsiRNA delivery. A variety of alternative vehicles or delivery

transfection reagents, viral vectors or instrumentation.

modes have been developed specifically for these difficult

Accell siRNAs are shown to be effective in a wide range of

to-transfect cell types. Revvity’s Dharmaconl™ Accell™ siRNA

human, mouse and rat cell types that have been traditionallytechnology represents one such innovation that successfully

identified as difficult-to-transfect by conventional lipid

combines bioinformatics and chemical modificationdelivery methods.

strategies to provide functional and specific siRNAs that areFigure 1: Workflow for successful RNAi gene silencing experiments

Detection solutions

Workflow and result

Detection of the level of gene knockdown represents

Accell application in neuroblastoma cells and T-cells

yet another challenge during the optimization of RNAi
experiments. Reverse transcription quantitative real-time
PCR (RT-qPCR) represents the most commonly
recommended method for mRNA detection. Whileoften perceived as an easy, quick and reliable process,
RT-qPCR methods often suffer from variability and poor
reproducibility. The Thermo Scientific™ Verso™ QRT-PCRSYBR Green kits incorporate a unique blend of highperformance reaction components optimized to eliminate
variability in detection—giving reliable and consistent mRNAlevel assessments.

Delivery optimization is a balance between potent
knockdown and the maintenance of cell viability. Applicationof Accell siRNA to your cell line of interest is relatively
straightforward (Figure 2) and is described in more detail in
the Accell siRNA Protocol (horizondiscovery.com/en/genemodulation/knockdown/sirna/accell-sirna). Briefly, cells
were either pre-plated in their specific growth medium theday before (SH-SY5Y) or just prior to Accell transfectionin Accell Delivery Media (Jurkat) in 96-well formats atdensities optimized for growth conditions, viability, and geneknockdown assays (see Materials and Methods for details).

Here we provide a step by step description of an

Target-specific, positive, and negative control Accell siRNAs

experimental workflow that combines the use of functional

were each reconstituted to 100 µM stocks in 1x siRNA buffer,siRNAs enhanced for delivery and potent target knockdown(diluted from 5x siRNA buffer; Dharmacon, Cat #B-002000-

and a robust method for determining gene expression

UB-100). For delivery, the Accell siRNA stocks were mixed

levels (Figure 2). Specifically, we highlight: (1) Delivery ofwith Accell Delivery Media (Dharmacon, Cat #B-005000-100)

Accell control siRNAs into two difficult-to-transfect celland added to cells at a final concentration of 1 µM in 100 µL

lines: SH-SY5Y (adherent neuroblastoma cells) and Jurkat

volume per 96-well. Delivery, viability and gene knockdown(suspension T-cells), and. (2) Detection of target gene

was determined after a 72 hours incubation period.

knockdown using a Verso qRT-PCR SYBR Green kit.

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2

Successful RNA interference experiments in difficult-to-transfect cell lines.

SH-SY5YA

BJurkat
CFigure 2: Experimental workflow for Accell siRNA-mediated genesilencing and detection of mRNA knockdown.

DFigure 3: Cellular Uptake of Accell Red Non-targeting siRNAin SH-SY5Y and Jurkat Cells. Accell Red Non-targeting siRNAwas delivered to SH-SY5Y (A, B) and Jurkat (C, D) cells. Cellularuptake (red) in live cells was visualized using epifluorescencemicroscopy. Nuclei were stained with Hoechst 33342 (blue). Accell
Red Non-targeting siRNA localizes in the cytoplasm of both celltypes. Images (A) and (C) are merged epifluorescence micrographsof Accell Red Non-targeting siRNA and Hoechst 33342. Images(B) and (D) are epifluorescence micrographs merged with phase
contrast micrographs.

Qualitative visualization of Accell siRNA cellular uptake
A qualitative assessment of delivery can be made throughout

Workflow and results

the 72 hours incubation period. Uptake of dye-labeled

Relative quantification of mRNA with Verso™ SYBR GreenAccell Red Non-targeting siRNA into cells was visualized by

qRT-PCR Kits

fluorescent microscopy. Accell siRNA accumulates mainlyin the cytoplasm of the cells and is observed as early as

While the ultimate goal for RNAi experiments is to assess

24 hours* but increases and persists by 72 hours (Figure 3).

affects on phenotype, the level of gene silencing must be*Note: While uptake of dye-labeled siRNA is observed 24 hours postdelivery, the optimal time point for detecting mRNA knockdown isusually 72 hours post delivery.

Assessment of cell viability
It is important to determine whether the cell line wasadversely affected by Accell application using a cell viability
assay. Although Accell siRNAs do not induce the gene
responses that are common with lipid-mediated delivery,
it is always necessary to see if a delivery method or medium
condition is compatible with your biological system. In thisreport, cell viability was assessed using a method thatmeasures the reduction of resazurin (non-fluorescent blue torusorufin (fluorescent pink) based on the metabolic capacityof the cells. Accell siRNA delivery had little to no effect on
viability as compared to untreated cells for both SH-SY5Y andJurkat cells (data not shown). All experiments described here
showed cell viability of > 95% compared to untreated cells.
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determined to demonstrate working experimental design.
Gene knockdown can be determined by both mRNA andprotein detection, although the standard for detection ofgene silencing is RT-qPCR.
Detection results of target mRNA knockdown by RT-qPCR
are shown in Figures 4 and 5. Amplification curves forboth target (PPIB or GAPD and housekeeping (ACTB) genes
are presented. Successful delivery of Accell GAPD siRNA
resulted in robust silencing of GAPD expression. GAPD
expression was silenced 95% in SH-SY5Y and 93% inJurkat cells. Furthermore, delivery of Accell PPIB siRNA
into either cell line also resulted in knockdown of PPIBmRNA expression. PPIB expression was reduced by 75% inSH-SY5Y and 60% in Jurkats. In both SH-SY5Y and Jurkat
cells there was no significant effect on the expression of thenon-targeted housekeeping gene, ACTB, in either control ortarget wells.
3Successful RNA interference experiments in difficult-to-transfect cell lines.

Summary
The ultimate goal of RNAi experiments is to investigate thebiological impact of target gene knockdown in a particularbiological system. However, significant and relevant results
depend on a number of factors that we have addressedin this basic workflow including delivery, detection andanalysis. Of these, optimal delivery of functional siRNAsand accurate confirmation of gene knockdown remainparamount for correctly interpreting and assigning functionalroles to specific genes. After optimizing siRNA delivery andthe parameters to assess mRNA knockdown in a given cell
type, researchers are able to confidently proceed to thestudy of the resulting biological phenotype through highcontent or endpoint assays.

Figure 4: RT-qPCR quantification of Accell siRNA Geneknockdown in SH-SY5Y Cells. Experiments were performed
in biological triplicate for all samples. (A) qPCR amplification
curves detecting GAPD in all samples. (B) qPCR amplification
curves detecting Cyclophilin B (PPIB) in all samples. (C) qPCR
amplification curves detecting ACTB in all samples. (D) Calculated
results of GAPD and PPIB knockdown in SH-SY5Y cells normalized
to housekeeping gene (ACTB) and Non-targeting control #1.

Materials and methods
Accell siRNA delivery
SH-SY5Y cells were plated at 10,000 cells per well in a
96-well plate and allowed to adhere overnight. Growth
medium for SH-SY5Y cells was removed by aspiration.
Prior to transfection, SH-SY5Y cells were washed in Accell
Delivery Media as serum has been identified as a growth
medium component that interferes with efficient AccellsiRNA uptake. Accell siRNA was added to Accell Delivery
Media resulting in a final concentration of 1 μM. 100 μL ofthe Accell siRNA and Delivery Media mixture per well wasthen added to SH-SY5Y cells in a 96-well plate. Jurkat cells
were plated at 30,000 cells in 50 μL of Accell Delivery Mediaper well in a 96-well plate on the day of delivery. Accell
siRNA was added to Accell Delivery Media resulting in a
final concentration of 2 μM. 50 μL of the Accell siRNA andDelivery Media mixture per well was then added to Jurkat
cells in a 96-well plate. Cells were incubated for 72 hours
at 37 ºC and 5% CO2. A sample 96-well plate map is shownin (Figure 2). It is highly recommended to have biologicaltriplicates for each Accell siRNA and controls. Optimaldelivery and siRNA knockdown is observed 72 hours after
the original application of Accell siRNAs.

Figure 5: RT-qPCR Quantification of Accell siRNA Gene
Knockdown in Jurkat Cells. Experiments were performed
in biological triplicate for all samples. (A) qPCR amplification
curves detecting GAPD in all samples. (B) qPCR amplification
curves detecting Cyclophilin B (PPIB) in all samples. (C) qPCR
amplification curves detecting ACTB in all samples. (D) Calculated
results of GAPD and PPIB knockdown in Jurkat cells normalized
to housekeeping gene (ACTB) and Non-targeting control #1.
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4Successful RNA interference experiments in difficult-to-transfect cell lines.

Table 1: Dharmacon Accell siRNA reagentsAccell control siRNA kits

Cat #H

K-005000-R1-01

M

K-005000-R1-02

R

K-005000-R1-03

SpeciesCat #H

D-001920-01

MD-001920-02

RD-001920-03

HD-001930-01

M

D-001930-02

auto-fluorescence of the medium. It is recommended toRD-001930-03

replace medium with pre-warmed 1x PBS with or without aH

D-001975-01

nuclear counterstain dye (Hoechst 33342) to clearly visualizeM

D-001975-02

RD-001975-03

SpeciesCat #—

D-001960-01

maxima for Hoechst 33342 is 350/461 nM and for Accell

SpeciesCat #Red is 557/574 nM. Photos were taken with SPOT Insight™

H

K-005000-R1-01

M

K-005000-R1-02

R

K-005000-R1-03

Accell control siRNA kits (Red)
Accell positive control
reagents
Accell cyclophilin B
control siRNA

Accell GAPD control siRNA

Accell red cyclophilin Bcontrol siRNA
Accell negative control
reagents
Accell red non-targeting siRNA
Accell control siRNA kits

Viability assay

Species

Accell control siRNA kits (Red)Resazurin was added to cells at a final concentration of25 μg/mL 72 hours post-delivery. Cells were returned to theincubator for 1 to 3 hours. Plates were read on a Wallac™
VICTOR 2 (Revvity Life Sciences) plate reader (Excitation
530 nM, Emission 590 nM and 1 second exposure).

Microscopy
Although it is possible to observe Accell Red siRNAfluorescence directly in experimental wells there isa large amount of background fluorescence due touptake. Briefly, 72 hours post-delivery cells were washedonce with 1x PBS and then incubated for 5-10 minuteswith 1x PBS plus Hoechst 33342 (1 µg/mL) for staining ofthe nuclei. Epifluoresence was visualized with a Leica™ DM
IL Microscope (Meyer Instruments™). Excitation/Emission

camera and software (Diagnostic Instruments Inc.).

mRNA detection by RT-qPCR
Primers were designed using Primer3 software (frodo.wi.mit.edu)

Verso™ SYBR Green 1- and 2- Step qRT-PCR Masterand synthesized by Operon. The sequences are listed inMixes were used in a final reaction volume of 25 μL.

Table 2. All primers were tested for technical reproducibility

Gene-specific primers with a final concentration of(standard deviation +/- 0.3 Cq), dynamic range (50 ng to87.5 nM and ~ 15 ng of template were added to each80 pg total RNA input) and amplification efficiency (> 90%reaction. 1- Step RT-qPCR cycling was performed inand 110%; data not shown). It is recommended for

a Stratagene™ Mx3005P thermocycler. The thermalresearchers to optimize all primers before assessing percent

cycling program consisted of: 50 ºC for 30 minutes

gene silencing. Cells were harvested for RNA purification(1 cycle); 95 ºC for 15 minutes (1 cycle); 95 ºC for(Qiagen™ RNeasy™ Plus) 72 hours post-delivery. Average

15 seconds/60 ºC for 30 seconds/72 ºC for 30 seconds

amount of RNA purified from each well was ~ 50 ng total.

(40 cycles); plus a melt curve profile.

Table 2: Primer pairs for each target

Target (human)

Accn No.

beta-Actin (ACTB)
Cyclophilin B (PPIB)
Glyceraldhyde-3-phosphate dehydrogenase (GAPD)

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Forward (5'→3')

Reverse (5’→3’)

NM_001101

tggacatccgcaaagacctg

ccgatccacacggagtactt

NM_00942

gatggcacaggaggaaagag

agccaggctgtcttgactgt

NM_002046

tgcaccaccaactgcttag

ggatgcagggatgatgtt

5Successful RNA interference experiments in difficult-to-transfect cell lines.

For 2-Step RT-qPCR, the RT thermal cycling program
consisted of 42ºC for 30 minutes (1 cycle); 95 ºC for
2 minutes (1 cycle); 4 ºC hold, and the thermal cycling
qPCR Program was 95 ºC for 15 minutes (1 cycle); 95 ºC

References
1. Bustin, Stephen A. A-Z of Quantitative PCR. LaJolla:International University Line, 2004.

for 15 seconds/60 ºC for 30 seconds/72 ºC for 30 seconds(40 cycles); plus a melt curve profile.
The ΔΔCt method1 was used to calculate relative
expression. The housekeeping gene, beta-Actin, was used
to normalize gene expression among wells. Expression
levels were further normalized to non-targeting control andare reported as a percentage of the non-targeting control
expression level.

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