TECHNICAL MANUAL

GIPZ™ Lentiviral shRNA

Product description

Design information

The Dharmacon™ GIPZ™ Lentiviral shRNA Library was developed in collaboration with Dr. Greg Hannon of Cold Spring Harbor Laboratory [CSHL] and Dr.
Steve Elledge of Harvard Medical School. This library combines the design
advantages of microRNA-adapted shRNA with the pGIPZ lentiviral vector tocreate a powerful RNA tool capable of producing RNA interference (RNAi) in
most cell types including primary and non-dividing cells.

Unique microRNA-30 based hairpin design
Short hairpin RNA (shRNA) constructs are expressed as human microRNA-30(miR-30) primary transcripts. This design adds a Drosha processing site to thehairpin construct and has been shown to greatly increase gene silencing
efficiency (Boden 2004). The hairpin stem consists of 22 nucleotides (nt) ofdsRNA and a 19 nucleotides (nt) loop from human miR-30. Adding the miR-30
loop and 125 nucleotides (nt) of miR-30 flanking sequence on either side of thehairpin results in greater than 10-fold increase in Drosha and Dicer processingof the expressed hairpins when compared with conventional shRNA designs(Silva 2005). Increased Drosha and Dicer processing translates into greater
shRNA production and greater potency for expressed hairpins.

GIPZ shRNA is available in glycerol stock or viral particle
format. If viral particle format is purchased, begin work with
Protocol IX – Determining Relative Transduction Efficiency.
Important safety note
Please follow the safety guidelines for use and production of vector-based
lentivirus as set by your institution’s biosafety committee.
•••

For glycerol stocks of E. coli containing lentiviral plasmids, BSL1 guidelines
should be followed
For handling and use of lentiviral products to produce lentiviral particles,
BSL2 or BSL2+ guidelines should be followed
For handling and use of lentiviral particle products, BSL2 or BSL2+ guidelines should be followed

Additional information on the safety features incorporated in the pGIPZ
lentiviral vector and the Horizon Trans-Lentiviral Packaging System can befound on page 3.
Dharmacon™ GIPZ™ shRNA vectors are not compatible withthird generation packaging systems, due to the requirementof the expression of tat, which third generation systems do
not contain. We recommend the Trans-Lentiviral Packaging
System for use with our vectors.

Use of the miR-30 design also allowed the use of ‘rules-based’ designs for target
sequence selection. One such rule is the destabilizing of the 5' end of theantisense strand, which results in strand specific incorporation of microRNA/
siRNAs into RISC. The proprietary design algorithm targets sequences in coding
regions and the 3' UTR with the additional requirement that they contain greater than 3 mismatches to any other sequence in the human or mouse genomes.
Each shRNA construct has been bioinformatically verified to match NCBI
sequence data. To assure the highest possibility of modulating the gene
expression level, each gene is represented by multiple shRNA constructs, eachcovering a unique region of the target gene.

Vector information
Versatile vector design
Features of the pGIPZ lentiviral vector (Figures 1 and 2) that make it a versatile
tool for RNAi studies include:
•
•
•
•
•

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Ability to perform transfections or transductions using the replication
incompetent lentivirus (Shimada 1995)
TurboGFP™ (Evrogen, Moscow, Russia) and shRNA are part of a bicistronictranscript allowing the visual marking of shRNA expressing cells.
Amenable to in vitro and in vivo applications.
Puromycin drug resistance marker for selecting stable cell lines.
Molecular barcodes enable multiplexed screening in poolsFrom PowerPoint (R&D)

Revised versions

Antibiotic resistance

hCMV
tGFP

RRE

pGIPZ contains three antibiotic resistance markers (Table 2).

W

PuroR

IRES

tGFP

RRE

WPRE

PuroR

IRES

hCMV

Ψ

3' SIN LTR

5' LTR

pGIPZ
AmpR

SV40 ori

pUC ori

Antibiotic

Concentration

Ampicillin (carbenicillin) 100 μg/mL
Zeocin™

5ʹ LTRΨ

Table 2. Antibiotic resistances conveyed by pGIPZ.

shRNA

25 μg/mL

Puromycin

VariableshRNA

Utility

Bacterial selection marker
pGIPZ(outside LTRs)
Bacterial selection marker(inside LTRs)R
pUCoriAmp
Mammalian
selection marker SV40 ori

Figure 1. pGIPZ lentiviral vector.

Quality control

The GIPZ Lentiviral shRNA Library has TRE
passed through internal QC processes
UBCto
ensure high quality and low recombination (Figures 3 and 4).

RRE

rtTA3 IRES Puro

tRFP

shRNA

Ψ

UBC
TRE
Vector element
Utility
PuroR WPRE
IRES
rtTA3
tRFP
RRE
hCMV
Human cytomegalovirus promoter drives strong transgene
expression
shRNA
tGFP
TurboGFP reporter for visual tracking of transduction andexpression

Ψ

Table 1. Features of the pGIPZ vector.

5' LTR
3' SIN LTR

Ψ

RRE

RRE

3' SIN LTR

3' self-inactivating
long terminal repeat for increased
hCMV
lentivirus safety
R
WPRE
tGFP
IRESallowsPuro
shRNApackaging
Psi packaging
sequenceviral genome
using lentiviral packaging systems
Rev response element enhances titer by increasing
packaging efficiency of full-length viral genomes
Woodchuck hepatitis posttranscriptional regulatory
element enhances transgene expression in the target cells

pSMART 2.0

5' LTR

WPRE

Vector map

AmpR

5ʹ LTR
RRE

IRES

tGFP

cPPT/CTS

PuroR

shRNA

pSMART 2.0

pUCoriAmpR

SV40 ori

hPGK

U6

U6

hPGK

shRNA

PuroR

W

Figure 4. Gel image of a restriction digest of clones from the GIPZ shRNA libraryshRNA
cultured for 10 successive RRE
generations in an attempt
to determine the tendency of thepGIPZ vector to recombine. Each generation was thawed, replicated and incubatedovernight for 16 hours at 30 °C then frozen, thawed and replicated. This process was
repeated for 10 growth cycles. After the 10th growth cycle, plasmid was isolated andnormalized to a standard concentration. Clones were digestedpLKO.1
with SacII and run onan agarose gel. The pGIPZ vector appears stable without showing any recombination.
Expected band sizes: 1259 bp, 2502 bp, and 7927 bp. 10 kb molecular weight ladder(10 kb, 7 kb, 5 kb, 4 kb, 3 kb, 2.5 kb, 2 kb, 1.5 kb, 1 kb).

PuroR

3' SIN LTR

RSV/5' LTR

pLKO.1

pGIPZ
11.8 kb

AmpR

pUC ori

RSV/5ʹ LTR

Ψ

Ψ

RRE

SV40 ori

Figure 3. Representative GIPZ Lentiviral shRNA clones grown for 16 hours at 30 °C.
Plasmid was isolated and normalized to a standard concentration. Clones were thendigested with SacII and run on an agarose gel with uncut plasmid. The expected bandsizes are 1259 bp, 2502 bp, and 7927 bp. No recombinant products are visible. 10 kb
hCMV
molecular weight ladder (10 kb, 7 kb, 5 kb, 4 kb, 3 kb, 2.5 kb, 2 kb, 1.5 kb, 1 kb).

SV40 ori

pUC ori

pUCori

AmpR

Ψ

shRNA

pTRIPZ

5ʹ LTR

5' LTRIRESΨ

3' SIN LTR

pTRIPZ

Puromycin resistance permits antibiotic-selective pressure
and propagation of stable integrants
Internal ribosomal entry site allows expression of TurboGFP
R
SV40
ori
pUC
Amp
and puromycin
resistancegenes
in aorisingle transcript
microRNA-adapted shRNA (based on miR-30) forgene knockdown
5' long terminal repeat

PuroR

AmpR

pUCori

Additional safety information
hCMV
tGFP

IRES

PuroR

microRNA

WPRE

3' SIN LTR

5' LTRpSMART 2.0

R Geneious
Drawing was createdusing(http://www.geneious.com).
SV40 ori
ori
pUC
Amp

Figure 2. Detailed vector map of pGIPZ lentiviral vector.

horizondiscovery.com-2a- Blast
tGFP
IRES
ORF

5ʹ LTRΨΨ

RRE

Historically, the greatest safety risk associated with ahCMV
lentiviral delivery
platform stems from theRRE
potential generation of recombinantvirusesPuro
that R microRNAtGFP IRES
are capable of autonomous replication. The pGIPZ Lentiviral shRNA platform
minimizes these hazards to the greatest degree by combining a disabled viralgenome with the proprietary Trans-Lentiviral packaging process. Starting with
2.0
the HXB2 clone of HIV-1 (GenBank,
Accession#K03455), the lentiviral backbone
pSMARThas been modified to eliminate all but the most essential genetic elements
necessary for packaging and integration (such as 5' LTR, Psi sequences, polypurine tracts, Rev responsive elements and 3' LTR). The resultant self-inactivatingR
SV40 ori
pUCori
Amp(SIN) vector greatly reduces the probabilityof producing
recombinant particles
and limits cellular toxicity often associated with expression of HIV genes.

hCMV

hCMV

nuc

R

WPRE

RRE

ORF

IRES

tGFPnuc -2a- BlastR

Additional safety features can be incorporated by the packaging processitself. Generation of pGIPZ Lentiviral shRNA particles requires a packaging
step during which the expression construct containing the silencing sequenceis enclosed in a viral capsid. Gene functions that facilitate this process (suchas those encoded by the structural genes gag, pol, env, etc.) are distributed
amongst multiple helper plasmids which do not contain significant regionsof homology. This tactic further minimizes the probability of recombination
events that might otherwise generate viruses capable of autonomous
replication. Among commercially available lentiviral vector systems, theTrans-Lentiviral Packaging System offers a superior safety profile as the
packaging components are separated onto five plasmids. Additionally,
expression of gag-pro and tat-rev are under the control of the conditional
tetracycline-responsive promoter element (TRE), limiting expression ofthese viral components strictly to the packaging cell line. A detailed descriptionof the Trans-Lentiviral Packaging System can be found in (Wu 2000).

Table 3. Materials for plate replication.

With these safety measures in place, GIPZ lentiviral shRNA particles can be
employed in standard Biosafety Level 2 tissue culture facilities.

Replication of plates
Prepare target plates by dispensing ~ 160 μL of 2x LB broth (low salt) medium
supplemented with 8% glycerol** and appropriate antibiotic (25 μg/mL Zeocinand 100 μg/mL carbenicillin).
Prepare source plates
Remove foil seals from the source plates while they are still frozen. This
minimizes cross-contamination. Thaw the source plates with the lid on. Wipe any
condensation underneath the lid with a paper wipe soaked in ethanol.

Any investigator who purchases Horizon viral vector products is responsible
for consulting with their institution’s health and biosafety group for specificguidelines on the handling of lentiviral vector particles. Further, each
investigator is fully responsible for obtaining the required permissions for theacceptance of lentiviral particles into their local geography and institution.
•

In the U.S., download the U.S. Department of Health and Human Services
Centers for Disease Control and Prevention and National Institutes of
Health, Biosafety in Microbiological and Biomedical Laboratories (BMBL),
Fifth Edition, Feb 2007 here.

•

See also: NIH Guidelines For Research Involving Recombinant DNA
Molecules (NIH Guidelines), September 2009, downloadable here.

•

For Biosafety Considerations for Research with Lentiviral Vectors, see.

Replication of individual clones
Once the clone has been streak isolated and the identity of the strain has been
confirmed** by Sanger sequencing (See: What is the sequencing primer for
pGIPZ?), we recommend making a stock of the pure culture. Grow the pureculture in LB broth + appropriate antibiotic (See protocol below: Protocol
1 - replication). Vortex the culture to evenly mix the glycerol throughout theculture. The culture can be stored indefinitely at –80 °C.
**Testing of 3-5 colonies is recommended.

Protocol I – replication
For archive replication, grow GIPZ shRNA clones at 30 °C in 2x LB broth(low salt)* medium plus 25 μg/mL Zeocin™ and 100 μg/mL carbenicillin in orderto provide maximum stability of the clones. Prepare medium with 8% glycerol**and the appropriate antibiotics.
2x LB broth (low-salt) medium preparation
LB-Broth-Lennox
20 g/L
Peptone		
10 g/L
Yeast Extract
5 g/L
Appropriate antibiotic(s) at recommended concentration(s). Glycerol 8% for
long-term storage.
*1x LB medium can be used instead of 2x LB broth medium.
**Glycerol can be omitted from the medium if you are culturing for plasmid
preparation. If making copies of the constructs for long-term storage at–80 °C, 8% glycerol is required

Item

VendorCat #2x LB Broth (low salt)

Fisher Scientific

BP1427500

Peptone, granulated, 2 kg – Difco

Fisher ScientificBP9725-2

Yeast Extract, 500 g, granulated

Fisher Scientific

BP1422-500

NaCl

Fisher ScientificBP3581GlycerolFisher ScientificBP2291

Carbenicillin

Fisher Scientific

BP2648-250

ZeocinInvitrogen

ant-zn-5p

Puromycin

Fisher ScientificBP2956-100

96-well microplates

Fisher Scientific12-565-363

Aluminum seals

Fisher Scientific12-565-475

Disposable replicators

Fisher Scientific

NC9584102

Replicate
1. Gently place a disposable replicator in the thawed source plate and lightly
move the replicator around inside the well to mix the culture. Make sure to
scrape the bottom of the plate of the well.
2. Gently remove the replicator from the source plate and gently place in thetarget plate and mix in the same manner to transfer cells.
3. Dispose of the replicator.
4. Place the lids back on the source plates and target plates.
5. Repeat steps 1-4 until all plates have been replicated.
6. Return the source plates to the -80 °C freezer.
7. Place the inoculated target plates in a 30 °C incubator for 18-19 hours.
Freeze at -80 °C for long-term storage. Avoid long periods of storage at room
temperature or higher in order to control background recombination products.
Due to the tendency of viral vectors to recombine, we
recommend keeping the incubation times as short as
possible and avoid subculturing. Return to your glycerol
stock of your pure culture (see Replication of individualclones) for each plasmid preparation.

Protocol II – Plasmid preparation
Culture conditions for individual plasmid preparations
For plasmid preparation, grow all GIPZ shRNA clones at 37 °C in 2x LB broth(low salt) medium plus 100 μg/mL carbenicillin only.
1. Upon receiving your glycerol stock(s) containing the shRNA of interest,
confirm the clone identity (See: Replication of individual clones) and store
immediately at –80 °C until ready to begin.
2. To prepare plasmid DNA, first thaw your glycerol stock culture and pulse
vortex to resuspend any E. coli that may have settled to the bottom of
the tube.
3. Take a 10 μL inoculum from the glycerol stock into 3–5 mL of 2x LB broth(low salt) medium with 100 μg/mL carbenicillin. Return the glycerol stock(s)to –80 °C.
If a large culture volume is desired, incubate the 3-5 mL culture
for 8 hours at 37 ˚C with shaking and use as a starter inoculum.
Dilute the starter culture 1:500–1:1000 into the larger volume.
4. Incubate at 37 °C for 18–19 hours with vigorous shaking.

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5. Pellet the culture and begin preparation of plasmid DNA. Plasmid DNA
can be isolated using Thermo Scientific™ GeneJET™ Plasmid Miniprep Kit(Cat #K0502) or similar.
6. Run 0.2–1 μg of the plasmid DNA on a 1% agarose gel. pGIPZ with shRNA
is 11774 bp.
Due to the tendency of viral vectors to recombine, we
recommend keeping the incubation times as short as
possible and avoid subculturing. Return to your original
glycerol stock of your pure culture (see Replication ofindividual clones) for each plasmid preparation.
Culture conditions for 96-well bio-block plasmid preparation
Inoculate a 96-well bio-block containing 1 mL per well of 2x LB broth (lowsalt) medium with 100 μg/mL carbenicillin with 1 µL of the glycerol stock
culture. Incubate at 37 °C with shaking (~ 170–200 rpm). We have observedthat incubation times between 18–19 hours produce good plasmid yield.
For plasmid preparation, follow the protocols recommended by the plasmid
isolation kit manufacturer.
For the library collection, we use the above 96-well bioblock plasmid preparation protocol in conjunction witha Qiagen™ Turbo™ Kit (Cat #27191). We use 2 bio-blocks
combined. Do not perform the optional wash and elute the
DNA in molecular grade water.

Protocol III – restriction digest

The following is a sample protocol for restriction enzyme digestion using
Thermo Scientific™ FastDigest™ Restriction Enzymes KpnI (Cat #FD0524), SalI (Cat#FD0644), XhoI (Cat #FD0694) and/or NotI (Cat #FD0594) and Thermo Scientific™
Restriction Enzyme™ SalI (Cat #ER0205) for diagnostic quality control of pGIPZ
lentiviral vectors.
1. Add the following components (Table 4), in the order stated, to a sterile
PCR thin-wall tube.
2. Mix gently by pipetting.
3. Incubate in a thermal cycler at 37 °C for 5 minutes for FastDigest enzymes
or as suggested by the manufacturer.
4. Load the gel with 10 µL of each of the digested samples, KpnI, SacII, SalI,
XhoI and/or NotI on a 1% agarose gel. Run uncut sample alongside thedigested samples. (Figure 5).
Table 4. Restriction digest components.

Component

AmountWater, nuclease-free (Cat #R0581)

X μL

10x FastDigest™ buffer

2 μL

DNA sample (up to 1 µg) in water

X μL

FastDigest enzyme

1 μL

Final Volume

20 μL

Protocol IV – Transfection
Quantities and volumes should be scaled-up according to the number ofcells/wells to be transfected (Table 5). This example is for 24-well plate format.
1. In each well, seed ~ 5 × 104 adherent cells or ~ 5 × 105 suspension cells in
0.5 mL of growth medium 24 hours prior to transfection.
The recommended conﬂuency for adherent cells on the
day of transfection is 70-90%. Suspension cells should bein logarithmic growth phase at the time of transfection.

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1
Lane 1 Lane 2 Lane 3 Lane 4 Lane 5 Lane 6 -

2

3

4

5

6

10 kb molecular weight ladder (10 kb, 7 kb, 5 kb, 4 kb, 3 kb, 2.5 kb,
2 kb, 1.5 kb, 1 kb)
Uncut pGIPZ vector
KpnI digested pGIPZ produces two bands at 1917 bp and 9857 bp.
SacII digest produces three bands at 1345 bp, 2502 bp and 7927 bp
SalI produces three bands at 2188 bp, 4465 bp and 5121 bp
XhoI, NotI double digest produces two bands at 1291 bp & 10,483 bp

2. Dilute 1 µg of DNA in 50 µL of DMEM or other serum-free growth medium.
3. Gently mix DharmaFECT kb transfection reagent and add 3 µL to the diluted
DNA. Mix immediately by pipetting.
4. Incubate 10 minutes at room temperature. Remove medium fromwells and replace with 0.45 mL fresh growth medium.
Prepare immediately prior to transfection. We recommend
starting with 1 µg of DNA and 3 µL of DharmaFECT kb
reagent per well in a 24-well plate (see scale-up Table 5).
Subsequent optimization may further increase transfection
efﬁciency depending on the cell line and transgene used.

5. Gently add 50 µL of the DharmaFECT kb reagent/DNA mixture to each well.
The transfection efficiency with DharmaFECT kb
transfection reagent (Horizon, Cat #T-2006-01) is equallyhigh in the presence of serum. This is not the case withother transfection reagents.
6. Gently rock the plate to achieve even distribution of the complexes.
7. Incubate at 37 °C in a CO2 incubator.
8. Analyze transgene expression 24-48 hours later. For stable transfection,
cells should be grown in selective medium for 10-15 days (see Protocol V –Puromycin Selection).

Table 3. Scale-up ratios for transfection of adherent and suspensioncells with DharmaFECT kb transfection reagent
Tissue culture vessel

Growth area,
cm2/wellVolume ofmedium, mL

Adherent (suspension) cells to seed
the day before transfection*

96-well plate

0.3

0.1

48-well plate

0.7

24-well plate
12-well plate

Amount of DNA

Volume of DharmaFECT kb, μL

µg**

µL***

Recommended

Range

0.5-1.2 × 104 (2.0 × 104)

0.2

10

0.6

0.4-1.0

0.25

1.0-3.0 × 10 (5.0 × 10 )

0.5

25

1.5

0.8-2.2

2.0
4.0

6-well plate
60 mm plate

4

4

0.5

5

2.0-6.0 × 10 (1.0 × 10 )

1.0

50

3.0

2.0-5.0

1.0

0.4-1.2 × 105 (2.0 × 105)

2.0

100

6.0

3.9-9.0

9.5
20

4

2.0

5

0.8-2.4 × 10 (4.0 × 10 )

4.0

200

9.0

6.0-12.0

3.0

2.0-6.3 × 105 (1.0 × 106)

6.0

300

18.0

12.0-24.0

5* These numbers were determined using HEK293T and U2OS cells. Actual values depend on the cell type.
** Amount of DNA and DharmaFECT kb transfection reagent used may require optimization.
*** The volume of DNA should be 1/10 of the volume of the culture medium used for dilution of the DNA.

Protocol V – Puromycin selection
Determining antibiotic dose response (Kill curve)
In order to generate stable cell lines expressing the transgene of interest, it
is important to determine the minimum amount of antibiotic required to kill
non-transfected cells. A simple procedure to test this is as follows:
1. Day 1: Using the same cell type and relative cell densities to be used insubsequent transfection or transduction procedures, plate cells
and culture overnight.
2. Day 2: Replace complete growth medium with growth medium
supplemented with a range of puromycin concentrations (0-15 μg/mL),
including untreated control cells with no antibiotic added.
3. Day 4: Refresh medium and assess viability.
4. Replace medium with fresh medium supplemented with the appropriate
concentration of puromycin every 2-3 days depending on the growthof cells.
5. Examine cells daily and identify the minimal concentration of antibiotic
that efficiently kills all non-transfected/transduced cells between
3-6 days following addition of puromycin.
Puromycin selection
If adding antibiotic for selection, use the appropriate concentration as
determined based on the above kill curve.
1. Add medium containing antibiotic 24 or hours post-transfection or
post-transduction, respectively, to begin selection
It is important to wait at least 24 hours after transfection
before beginning selection.
2. Cells can be harvested for transgene expression 24-72 hours after
starting selection.
3. If longer selection is required for cells to be confluent, replace selective
medium approximately every 2-3 days.
4. Monitor the cells daily and observe the percentage of surviving cells.
Cells surviving selection will be expressing the transgene.
5. If generating stable cell lines (optional), select and grow for 10-15 days.
6. Once non-transfected cells are eliminated and/or you have selected forstably transfected cell lines if desired, you can proceed to assay for target
gene expression. RT-qPCR, Western blot analysis or other appropriate
functional assay can be used; compare treated samples to untreated,
reporter alone, non-silencing control, or other controls as appropriate.

1 2 3 4 5 6 7 8 9 10 11 12
Virus stock 1
Virus stock 2
Virus stock 3
Virus stock 4

Figure 6. Five-fold serial dilutions of virus stock.

Dilution Plate

Protocol VII – Titering
Viral titering
Follow the procedure below to determine the titer of your lentiviral stockusing the mammalian cell line of choice IF YOU HAVE PRODUCED VIRALPARTICLES YOURSELF. This protocol uses the HEK293T cell line that isavailable as part of our Trans-Lentiviral shRNA Packaging Kit (Cat #TLP5918).
You can use a standard HEK293T cell line as an alternative.

If you have generated a lentiviral stock of the expression
control (such as GIPZ non-silencing construct), we
recommend titering this stock as well.
1.

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The day before transduction, seed a 24-well tissue culture platewith HEK293T Cells at 5 × 104 cells per well in DMEM (10% FBS,
1% pen-strep).
The following day, each well should be no morethan 40-50% confluent.

2.

Protocol VI – Packaging lentivirus
The pGIPZ vector is tat dependant, so you must use a packaging systemthat expresses the tat gene. For packaging our lentiviral shRNA constructs,
we recommend the Trans-Lentiviral shRNA Packaging Kit (Cat #TLP5912
or TLP5917). The Trans-Lentiviral shRNA Packaging System allows creationof a replication-incompetent, HIV-1-based lentivirus which can be used todeliver and express your shRNA of interest in either dividing or non-dividing
mammalian cells. The Trans-Lentiviral shRNA Packaging System uses a
replication-incompetent lentivirus based on the trans-lentiviral systemdeveloped by Kappes (Kappes 2001). For protocols and information on
packaging pGIPZ with our Trans-Lentiviral shRNA Packaging System, please
see the product manual available on our website.

A
B
C
D
E
F
G
H

3.
4.

Make dilutions of the viral stock in a round bottom 96-well plateusing serum-free media. Utilize the plate as shown in (Figure 6)
using one row for each virus stock to be tested. Use the procedure below(starting at step 4) for dilution of the viral stocks. The goal is to produce aseries of 5-fold dilutions to reach a final dilution of 390,625-fold.
Add 80 µL of serum-free media to each well.
Add 20 µL of thawed virus stock to each corresponding well incolumn 1 (five-fold dilution).
Pipette contents of well up and down 10-15 times. Discard
pipette tip.

5.

With new pipette tips, transfer 20 µL from each well of column 1
to the corresponding well in column 2.
Pipette up and down 10-15 times and discard pipette tip.

6.

Table 6. Example of set up for dilutions.

Well (Row A, B, C, or D)
Originating(96-well plate)

Repeat transfers of 20 µL from columns 3 through 8, pipetting up anddown 10-15 times and changing pipette tips between each dilution.
It is strongly recommended that you use a high quality
multichannel pipettor when performing multiple dilutions.
Pre-incubate the dilutions of the virus stock for 5 minutesat room temperature.

8.

Label 24-well plate as shown in (Figure 7) using one row for each virus
stock to be tested.
9. Remove culture media from the cells in the 24-well plate.
10. Add 225 µL of serum-free media to each well.
11. Transduce cells by adding 25 µL of diluted virus from the original
96-well plate (Figure 6) to a well on the 24-well destination plate(Figure 7) containing the cells. For example, transfer 25 µL from wellA2 of the 96-well plate into well A1 in the 24-well plate (Table 6).
12. Incubate transduced cultures at 37 °C for 4 hours.
13. Remove transduction mix from cultures and add 1 mL of DMEM
(10% FBS, 1% Pen-Strep).
14. Culture cells for 48 hours.
15. Count the TurboGFP expressing cells or colonies of cells (Figure 8).
Count each multi-cell colony as 1 transduced cell, as thecells will be dividing over the 48 hour culture period. Figure
8 illustrates this principle of cell counting.
16. Transducing units per mL (TU/mL) can be determined using thefollowing formula:# of TurboGFP positive colonies counted × dilution factor × 40 = # TU/mL:
Example: 55 TurboGFP positive colonies counted in well A355 (TurboGFP positive colonies) × 625 (dilution factor) × 40 = 1.38 ×
106 TU/mL

1
Virus stock 1

A

Virus stock 2

B

Virus stock 3

CVirus stock 4

D

2

3

4

5

6

Figure 7. Twenty four well tissue culture plate, seeded with HEK293T cells, used
to titer the virus.

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Volume diluted
virus used

Dilution factor

25 μL

5*

A1

With new pipette tips, transfer 20 µL from each well of column 2 to thecorresponding well in column 3.
Pipette up and down 10-15 times and discard pipette tip.

7.

Destination(24-well plate)

A2

A1

25 μL

25

A3

A2

25 μL

125

A4

A3

25 μL

625

A5

A4

25 μL

3125

A6

A5

25 μL

15625

A7
A8

A6

25 μL
25 μL

78125
390625 **Please note that when expecting very high or very low titers, it would be advisable toinclude either well 8 or well 1 respectively.

A

B

Figure 8. Examples of individual colonies.

Once you have generated a lentiviral stock with a suitable titer, you are ready totransduce the lentiviral vector into the mammalian cell line of choice and assay
for gene silencing
Multiplicity of infection (MOI)
To obtain optimal silencing of your shRNA, you will need to transduce thelentiviral vector into your mammalian cell line of choice using a suitable MOI.
MOI is defined as the number of transducing units per cell.
Determining the optimal MOI
A number of factors can influence determination of an optimal MOI including
the nature of your mammalian cell (actively dividing versus non-dividing), its
transduction efficiency, your application of interest, and the nature of your geneof interest. If you are transducing your lentiviral construct into the mammalian cellline of choice for the first time, after you have titered the lentiviral particles, werecommend using a range of MOIs (for example, 0, 0.5, 1, 2, 5, 10, 20) to determine
the MOI required to obtain optimal expression for your particular application.
It should be noted that to achieve single copy knockdown, an MOI of 0.3 isgenerally used, as less than 4% of your cells will have more than one insert.

Protocol VIII – Transduction
Transduction of target cells
The protocol below is optimized for transduction of the lentiviral particles into
HEK293T, OVCAR-8 or MCF7 cells in a 24-well plate using serum-free media. If a
different culture dish is used, adjust the number of cells, volumes, and reagent
quantities in proportion to the change in surface area (Table 7). It is strongly
recommended that you optimize transduction conditions to suit your target cellline to provide for the highest transduction efficiency possible.
It is preferable that transduction be carried out in medium that is serum
free and antibiotic free. A reduction in transduction efficiency occurs in thepresence of serum; however it is possible to carry out successful transductions
with serum present; you will have to optimize the protocol according to yourcells of interest.

1. On day 0, plate 5 × 104 cells per well in a 24-well plate. Incubate
overnight. You will be using full growth medium (with serum) at
this stage.
2. The next day (day 1), remove the medium and add an appropriate
amount of the virus to acheive the MOI you wish to use. Set up
all desired experiments and controls in a similar fashion. Bring
the total volume of liquid up so that it just covers the cells
efficiently with serum-free media (see Table 7 for guidelines).
If you are using concentrated virus you are likely to use a small
volume of virus if you are using unconcentrated virus, you willfind you need more virus volume.

The higher the MOI you have chosen, the more copiesof the shRNA and puromycin resistance gene you will
have per cell. When selecting with puromycin, it is worth
remembering that at higher MOIs, cells containing multiple
copies of the resistance gene can withstand higher
puromycin concentrations than those at lower MOIs. Adjust
the concentration of puromycin to a level that will selectfor the population of transduced cells you require for yourapplication without going below the minimum antibiotic
concentration you have established in your kill curve.

Table 7. Suggested volumes of media per surface area per well of adherent cells. Once your transduction efficiency is at an acceptable level (with or without
puromycin selection performed post-transduction), you can proceed to assay
TissueSurface area
Suggested total serum-free medium
cells for reduction in gene expression or fluorescent reporter activity by reverse
2
culture dishper well (cm )volume per well (mL)transcription quantitative/real-time PCR (RT-qPCR), Western blot analysis or
100 mm56.0
5.0
other appropriate functional assay. Compare target gene to untreated, reporter
60 mm
20.0
2.0
alone (empty vector), non-silencing shRNA, or other negative controls.
35 mm
8.0
1.0
Optimal length of incubation from the start of transfection
6-well9.4
1.0
to analysis is dependent on cell type, gene of interest,
12-well
3.8
0.5
and the stability of the mRNA and/or protein being
24-well
1.9
0.25
analyzed. RT-qPCR generally gives the best indication of
96-well
0.3
0.1mRNA expression and gene silencing. The use of Western
blot analysis to determine knockdown is dependent on
3. Approximately 4-6 hours post-transduction, add an additional 1 mL
quantity and quality of the protein, its half-life, and thesensitivity of the antibody and detection systems used.
of full medium (serum plus Pen-Strep if you are using it) to your cells
and incubate overnight.
We have experienced low toxicity with transduction in thecell lines tested, therefore removal of virus is not required
for many cell lines. In our experience, higher transduction
efficiencies have been achieved if the virus is not removed
after 6 hours. However, if toxicity is a problem, aspirate
the mixture after 4-6 hours and replace with fresh growth
medium. Additionally, fresh growth medium should be
replenished as required for continued cell growth.

At 48 hours post-transduction examine the cells microscopically forthe presence of reporter gene (TurboGFP) expression as this will be
your first indication as to the efficiency of your transduction.
When visualizing TurboGFP expression, if less than
90% of all cells are green, it is recommended in thesecases to utilize puromycin selection in order to reduce
background expression of your gene of interest fromuntransduced cells.

a. If adding puromycin, use the appropriate concentration as
determined based on the kill curve (see Protocol V). Incubate cellswith the selection medium.
b. Approximately every 2-3 days replace with freshly preparedselective medium.
c. Monitor the cells daily and observe the percentage of surviving
cells. Once the non-transduced control cells are dead, the surviving
cells in the transduced wells will be expressing the shRNA
Optimum effectiveness of the puromycin selection should bereached in 4-6 days with puromycin dependent upon theconcentration of puromycin chosen from the kill curve.

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Protocol IX – Determining relativetransduction efficiency
Follow the procedure below to determine the relative transduction efficiencyof purchased GIPZ lentiviral particles (Cat #VGH5518, VGM5520, VGH5526). This
protocol should only be used with purchased GIPZ shRNA individual clones inviral particle format.
Prior to transducing with purchased GIPZ shRNA individual clones in viralparticle format, we recommend determining the relative transduction efficiencyof your cell type. Lentiviral titers provided with purchased GIPZ lentiviralparticles have been calculated by transducing HEK293T cells. Transduction
efficiencies vary significantly by cell type.
The relative transduction efficiency of your cells may be estimated bydetermining the functional titer of a control virus such as GIPZ Non-silencing
control viral particles (Cat #RHS4348) in your cells of interest.
Follow the procedure below to determine the functional titer of the GIPZ
Non-silencing control shRNA viral stock in the mammalian cell line of yourchoice. The following conditions have been optimized for transduction of
HEK293T cells. When determining the relative transduction efficiency of
your cell type, use the transduction conditions that have been optimized
for your cells of interest.
1. The day before transduction (day 0), seed a 24-well tissue culture platewith your cells at 5 × 104 cells per well in their respective medium.
The following day, each well should be no more than
40-50% confluent.
2. Make dilutions of the Non-silencing control shRNA viral stock in a roundbottom 96-well plate using serum-free medium. Utilize the plate as shownin Figure 9 with one row for each replicate (we recommend performingat least two replicates). Use the procedure below for dilution of the viral
stock. The goal is to produce a series of five-fold dilutions to reach a final
dilution of 390, 625-fold.

1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H

Virus stock 1
Virus stock 2
Virus stock 3
Virus stock 4

14. Add 1 mL of your medium (normal serum concentration).
15. Culture cells for 72 hours.
16. Count the TurboGFP expressing cells or colonies of cells (Figure 11). Counteach multi-cell colony as 1 transduced cell, as the cells will be dividing
over the 72 hour culture period. Figure 11 illustrates this principle ofcell counting. Count the number of TurboGFP expressing colonies inwells corresponding to at least two viral dilutions.
17. Transducing units per ml (TU/mL) can be determined using the following
formula: # of TurboGFP positive colonies counted × dilution factor × 40 =# TU/mL
25 μLof diluted virus was added to the cells. This is
1/40th of a mL.
Example: 55 TurboGFP positive colonies counted in well A3 55 (TurboGFP
positive colonies) × 625 (dilution factor) × 40 =1.38 × 106 TU/mL.

Figure 9. Five-fold serial dilutions of virus stock.

Dilution Plate

3. Add 40 uL of serum-free media to each well in column.
4. Add 80 uL of serum-free media to each well of columns 2-8.
If desired, include 8 μg/mL polybrene in the
dilution medium.
5. Add 10 µL of thawed control shRNA virus stock to each well in column 1(five-fold dilution).
Pipette contents of well up and down 10-15 times.
Discard pipette tip.
6. With new pipette tips, transfer 20 µL from each well of column 1 to thecorresponding well in column 2.
Pipette up and down 10-15 times and discard pipette tip.
7. With new pipette tips, transfer 20 µL from each well of column 2 to thecorresponding well in column 3.
Pipette up and down 10-15 times and discard pipette tip.
8. Repeat transfers of 20 µL from columns 3 through 8, pipetting up anddown 10-15 times and changing pipette tips between each dilution.
It is strongly recommended that you use a high-quality
multichannel pipettor when performing multiple dilutions.
Incubate the dilutions of the virus stock for 5 minutes at room
temperature.
9. Label a 24-well plate as shown in Figure 10 using one row for each
Post dox 72 hours
replicate.
10. Remove culture medium from the cells in the 24-well plate.
11. Add 225 µL of serum-free medium to each well.
12. Transduce cells by adding 25 µL of diluted control shRNA lentivirus
from the original 96-well plate (Figure 9) to a well on the 24-welldestination plate (Figure 10) containing the cells. For example,
transfer 25 µL from well A2 of the 96-well plate into well A1in the
24-well plate (Table 8).
13. Incubate transduced cultures at 37 °C for 4-6 hours.

1
Virus stock 1

A

Virus stock 2

B

Virus stock 3

CVirus stock 4

D

2

3

4

5

18. The functional titer calculated for your cell line under your experimental
conditions can be used to determine the relative transduction efficiencyof your cell type by using the following formula: Functional titer of Nonsilencing control shRNA virus stock in your cell line ÷ Titer of Non-silencing
control shRNA virus stock as calculated in HEK293T = Relative transduction
efficiency.
Table 8. Example of set up for dilutions.
Well (row A, B, C, or D)
Originating(96-well plate)Destination(24-well plate)

Volume diluted
virus used

Dilutionfactor

25 μL

5*

A1
A2

A1

25 μL

25

A3

A2

25 μL

125

A4

A3

25 μL

625

A5

A4

25 μL

3125

A6

A5

25 μL

15625

A7

A6

25 μL

78125

25 μL

390625 *A8*Please note that when expecting very high or very low titers, it would be
advisable to include either well 8 or well 1 respectively.

A

B

6
Figure 11. Examples of individual colonies.

Figure 10. Twenty-four well tissue culture plate, seededwith HEK293T cells, used totiter the virus.

horizondiscovery.com

For example, if the titer of the Non-silencing control shRNA virus stock in
HEK293T (as provided on the product specification sheet) is 6.9 × 106 TU/mL
and the functional titer of the control shRNA virus stock in your cell line is 1.38
× 106 TU/mL, the relative transduction efficiency of your cell type is 0.2. To
extrapolate the average functional titer of the provided GIPZ viral particles,
multiply the average titer of each plate as provided on the product specification
sheet by the relative transduction efficiency of your cell type. In our example,
if the titer of the GIPZ viral particles in HEK293T cells is 2 × 106 TU/mL and therelative transduction efficiency of your cell type is 0.2, the extrapolated averagefunctional titer of that plate your cell type is 4 × 105 TU/mL.

Once the relative transduction efficiency of the GIPZ virus has been
established in your cell line, use the optimized transduction conditions
determined in Protocol VIII to transduce your cell line with the purchased
GIPZ shRNA individual clones in viral particle format. If the titer of the nonsilencing control shRNA virus is not satisfactory in your cell line you might
consider choosing a different cell line more permissive to transduction bylentivirus before proceeding.

Protocol X – PCR
QPCR experimental recommendations
One of the biggest challenges of any qPCR experiment is to obtainreproducible and reliable data. Due to the sensitivity of this multi-step
technique, care must be taken to ensure results obtained are accurate andtrustworthy (see Bustin et al., 2009).
1. Experimental samples should be run at least in duplicate. It should
be noted that with duplicate experiments it will not be possible toassign error bars to indicate consistency from experimental sample
to experimental sample. Using triplicate samples or higher will
enable error bars to be assigned indicating the level of experimental
variation.
2. Reverse Transcriptase reactions for cDNA synthesis should always
include a No Template Control (NTC) and No Reverse Transcriptase(no RT) control to check for reagents contamination and thepresence of contaminating DNA, respectively. Use a robust reverse
transcriptase enzyme for cDNA synthesis such as the Thermo
Scientific™ Maxima™ cDNA Synthesis Kit for RT-qPCR (Cat #K1641).
3. We have found that normalizing the RNA concentration prior to
cDNA synthesis will increase consistency downstream.
4. qPCR should be done at least in triplicate. Again, it should be notedthat with duplicate reactions it will not be possible to assign error
bars to indicate the consistency in your qPCR reactions. Using
triplicate samples or higher will enable error bars to be assigned
indicating the level of variation between qPCR reactions. Usevalidated primer sets for SYBR-based assays or primers/probe forprobe-based assays.
5. Make sure the mRNA you are using as your internal reference control
for qPCR is expressed at a level higher than your target gene's
message.
6. Use only high-quality calibrated pipettes, in conjunction with wellfitting barrier tips.
7. When pipetting, take the time to visually inspect the fluid in thepipette tip(s) for accuracy and lack of bubbles, especially when using
a multi-channel pipette.
8. Be sure to spin your qPCR plate prior to loading in the real-time
instrument in order to collect the sample at the bottom of the welland eliminate any bubbles that may have developed.
9. With regard to knockdown experiments using shRNA, it is vitally
important that you greatly reduce if not eliminate entirely thosecells which are not transduced or transfected from the population.
This can be done in several ways: increase the efficiency of yourtransfection, use a higher mulitplicity of infection (MOI) for
your transduction, utilize the puromycin selection marker andselect against those cells that do not contain the shRNA or utilize
fluorescent sorting to select against those cells that do not containthe shRNA.

horizondiscovery.com

10. Always utilize the non-silencing control as a reference for target gene
expression, as opposed to an untreated sample. The non-silencing
treated samples will most accurately reproduce the conditions in yourexperimental samples. The non-silencing best controls for changes in
qPCR internal reference gene expression.
11. You may also use an untreated sample to indicate substantialchanges in target gene expression as seen in the non-silencing control
due to generic consequences of viral transduction and transgene
expression. However, it should be noted that small changes in
expression levels between an untreated sample and the non-silencing
control are to be expected.
12. Cq values greater than 35 should be avoided as they tend to be morevariable. Samples with such high Cq values should be repeated at
higher cDNA concentrations and with a lower expressing qPCR internalreference control (such as TBP).
13. Cq values less than 11 for the qPCR internal reference control should be
avoided as it is difficult to determine a proper background subtraction
using these values. If this occurs, use Cq values from both your internalreference control as well as your experimental target to determine an
optimum cDNA concentration.
14. It may be necessary to change internal reference controls if conditions
in steps 12 and 13 cannot be simultaneously met.

Residual Gene
Activity

140%
120%
100%
80%

113%
100%

93%

88%

60%
40%
20%
0%

Control

GAPDH

EG5

PP1B

Figure 12. Non-silencing lentiviral shRNA control does not knockdown common
endogenous genes. The above data represents the baseline amount of GAPDH, EG5 or
PP1B mRNA set at 100% in the control. The relative amounts of each of these mRNAs arethen represented after treatment with non-silencing shRNA of these genes.

120%
100%

Residual GAPDHExpression
100%

100%

100%

100%

80%
60%
40%
34%

20%

28%
12%

0%
Untransduced
Control

NS#1

NS#1

NS#1

GAPDH GAPDH

GAPDH

Figure 13. HEK293T cells were transduced with lentiviral particles expressing GAPDHor Non-silencing shRNA at variable MOIs ranging from 9-48. The graph depicts theresidual levels of GAPDH relative to Non-silencing control.

Controls and validation

Table 9. Related Reagents.

RNAintro shRNA starter kits
The use of vector-based RNAi for gene silencing is a powerful and versatile tool.
Successful gene silencing in vitro is dependent on several variables including 1)
The target cell line being studied, 2) transfection and transduction efficiency,
3) abundance of the mRNA or protein of interest in the target cell line, 4) halflife of the protein, and 5) robust experimental protocols. For all these reasons,
it is important to run controlled experiments where the transfection andtransduction efficiencies are as high as possible and measurable.

Related Reagents

Dharmacon Cat #GAPDH Verified Positive Control*

RHS4371

EG5 Verified Positive control*

RHS4480

Non-silencing Verified Negative Control*

RHS4346

DharmaFECT kb transfection reagent 1 mL

2006-01

GIPZ shRNA Empty Vector

RHS4349

Trans-Lentiviral shRNA Packaging System

TLP5912Controls are a critical part of a gene silencing experiment. They enableaccurate representation of knockdown data and provide confidence in thespecificity of the response. Changes in the mRNA or protein levels in cells
treated with negative or non-silencing controls reflect non-specific responses
in cells and can be used as a baseline against which specific knockdown
can be measured. Positive controls are useful to demonstrate that yourexperimental system is functional.
Controls
The EG5 and GAPDH GIPZ lentiviral shRNA vectors have been validated as
positive controls for RNAi experiments performed using the GIPZ shRNAcontaining lentiviral vectors. These shRNA have been tested in transduction
based experiments and have shown efficient knockdown at both mRNA andprotein levels. The EG5 control has been validated to knockdown human EG5 by
RT-qPCR (Figure 14 and 15) and in situ hybridization of cells in tissue culture. The
GAPDH control has been validated to knockdown human and mouse GAPDH
by RT-qPCR (Figure 13). The GIPZ Non-silencing lentiviral shRNA vector has beenvalidated as a negative control for RNAi experiments performed using the GIPZshRNA-containing lentiviral vectors (Figure 12).
120%
100%

Residual EG5
Expression
100%

100%

100%

80%
60%
40%
29%

20%

18%

17%

EG5
EG5
MOI 3.5 MOI 8.5

EG5
MOI 17

0%
NS#1
NS#1
MOI 3.5 MOI 8.5

NS#1
MOI 17

Figure 14. HEK293T cells were transduced with lentiviral particles expressing EG5 or
non-silencing shRNA at MOIs of 3.5, 8.5 and 17. The graph depicts the residual levels of
EG5 relative to its non-silencing control.

OVERLAY

ANTI-TUBULIN

Trans-Lentiviral shRNA Packaging System with HEK293T Cells TLP5917*These items also available in the GIPZ lentiviral transfection starter kit (Cat #RHS11851).

Frequently Asked Questions (FAQs)
What clones are part of my library collection?
A USB containing the data for this collection will be shipped with eachcollection. This file contains the location and accession number for eachconstruct in the collection.
Where can I find the sequence of an individual shRNA construct?
If you are looking for the sequence of an individual shRNA construct, you cansearch for the clone on our website (horizondiscovery.com). Enter the catalog
number or clone ID of your construct into the search at the top of the page. You
should see your product in the catalog number section of the results. Click on
the plus sign to expand the details for this clone and select the Sequence tab.
Which antibiotic should I use?
You should grow all GIPZ shRNA constructs in 2x LB broth medium with both
25 μg/mL zeocin and 100 μg/mL carbenicillin for archive replication. For
plasmid preparations, grow the constructs in 2x LB broth medium containing
only 100 ug/mL carbenicillin.
What packaging cell line should I use for making lentivirus?
The GIPZ shRNA vector is tat dependant, so a packaging system that
expresses the tat gene. For packaging our lentiviral shRNA constructs, werecommend the Trans-Lentiviral shRNA Packaging Kit (TLP5912, TLP5917). The
Trans-Lentiviral Packaging Kit allows creation of a replication-incompetent(Shimada, et al. 1995), HIV-1-based lentivirus which can be used to deliverand express your gene or shRNA of interest in either dividing or non-dividing
mammalian cells. The Trans-Lentiviral Packaging Kit uses a replicationincompetent lentivirus based on the trans-lentiviral system developedby Kappes (Kappes and Wu et al. 2001). For protocols and information on
packaging GIPZ shRNA with our Trans-Lentiviral shRNA Packaging Kit, please
see the product manual available at here.
Can I use any 2nd generation packaging system with the pGIPZ vector?
The pGIPZ vector is tat dependant, so you must use a packaging system that
expresses the tat gene.
What does the number 40 refer to in the formula for the calculation of titer?
The titer units are given in transducing units (TU) per mL, so the number 40 isused to convert the 25 µL used in the titration (“volume of diluted virus used”,
Table 6) to one milliliter.

ANTI-EG5

DAPI

Figure 15. The characteristic phenotype observed by the targeting of the EG5 (KIF11)
gene results in the formation of half spindles, mitotic arrest and monoastral microtubular
arrays (green, see the cell on the left). By contrast, normal cells show bipolar spindles
and microtubule networks in mitosis and in interphase (see the cell on the right). The
comparative expression of EG5 (red) between the cell on the left and the right showsthe extensive knockdown of EG5 in the cell displaying the phenotype (left). The cells
were visualized at 100x magnification using aLeica DMIRB fluorescence microscope.
HEK293T cells were stained for tubulin (anti-tubulin, green), DNA (DAPI, blue) and EG5(anti-EG5, red).

What is the sequencing primer for pGIPZ?
The pGIPZ sequencing primer is 5' - GCATTAAAGCAGCGTATC - 3'Note: The binding site lies from base 5820-5842 and runs in the reverse
complement direction. The melting temperature of this 18mer = 52.7 °C.
Where do you purchase puromycin?
We purchase puromycin from Fisher Scientific Cellgro (Cat #BP2956-100).
How many transfections are available in each volume size of
DharmaFECT kb transfection reagent?
The number of transfections that can be performed depends on the size of theculture dish used.

Troubleshooting

References

For help with transfection or transduction of your lentiviral constructs, pleaseemail technical support at technical@horizondiscovery.com with the answers
to the questions below, your sales order or purchase order number and thecatalog number or clone ID of the construct with which you are having trouble.

Cited References and additional suggested reading
1. Bartel, D. P. (2004). microRNAs: genomics, biogenesis, mechanism, andfunction. Cell 116(2): 281-97.
2. Boden, D., O. Pusch, et al. (2004). Enhanced gene silencing of HIV-1 specificsiRNA using microRNA designed hairpins Nucleic Acids Res 32(3): 1154-8.
3. Chendrimada, T. P., R. I. Gregory, et al. (2005). TRBP recruits the Dicer
complex to Ago2 for microRNA processing and gene silencing. Nature
436(7051): 740-4.
4. Cleary, M. A., K. Kilian, et al. (2004). Production of complex nucleic acidlibraries using highly parallel in situ oligonucleotide synthesis.
Nat Methods 1(3): 241-8.
5. Cullen, B. R. (2004). Transcription and processing of human microRNA
precursors. Mol Cell 16(6): 861-5.
6. Cullen, B. R. (2005). RNAi the natural way. Nat Genet 37(11): 1163-5.
7. Dickins, R. A., M. T. Hemann, et al. (2005). Probing tumor phenotypes using
stable and regulated synthetic microRNA precursors. Nat Genet 37(11):1289-95.
8. Editors of Nature Cell Biology (2003). Whither RNAi? Nat Cell Biol 5(6):489-90.
9. Elbashir, S. M., J. Harborth, et al. (2001). Duplexes of 21-nucleotide
RNAs mediate RNA interference in cultured mammalian cells. Nature
411(6836): 494-8.
10. Fire, A., S. Xu, et al. (1998). Potent and specific genetic interference by
double-stranded RNA in Caenorhabditis elegans. Nature 391(6669):
806-11.
11. Gregory, R. I., T. P. Chendrimada, et al. (2005). Human RISC couples
microRNA biogenesis and posttranscriptional gene silencing.
Cell 123(4): 631-40
12. Kappes, J. C. and X. Wu (2001). Safety considerations in vector
development. Somat Cell Mol Genet 26(1-6):147-58.
13. Kappes, J. C., X. Wu, et al. (2003). Production of trans-lentiviral vector
with predictable safety. Methods Mol Med 76: 449-65.
14. Paddison, P. J., J. M. Silva, et al. (2004). A resource for large-scale
RNA-interference-based screens in mammals. Nature 428(6981): 427-31.
15. Shimada, T., et.al. (1995). Development of vectors utilized for genetherapy for AIDS. AIDS 4.
16. Silva, J. M., M. Z. Li, et al. (2005). Second-generation shRNA libraries
covering the mouse and human genomes. Nat Genet 37(11): 1281-8.

1. Are you using direct transfection or transduction into your cell line?
2. What was the 260/280 ratio of DNA? Over 1.8?
3. What was the transfection efficiency if you used direct transfection?
What transfection reagent was used?
4. Were positive and negative knockdown controls used (such as our
GAPDH or EG5 validated positive controls and the validated nonsilencing negative control)?
5. What were the results of the controlled experiments?
6. How was knockdown measured (for example real-time RT-qPCR or
western blot analysis)?
7. What is the abundance and the half-life of the protein? Does the protein
have many isoforms?
8. What packaging cell line was used if you are using transduction rather
than transfection?
9. What was your viral titer?
10. What was your MOI?
11. Did you maintain the cells in puromycin selection media after
transfection or transduction?
12. How much time elapsed from transfection/transduction topuromycin selection?
If transfection into your cell line is unsuccessful, you may need toconsider the following list of factors influencing successful transfection.
1. Concentration and purity of plasmid DNA and nucleic acids—determine
the concentration of your DNA using 260 nm absorbance. Avoid
cytotoxic effects by using pure preparations of nucleic acids.
2. Insufficient mixing of transfection reagent or transfection complexes.
3. Presence of antibiotics in transfection medium—the presence of antibiotics
do not interfere with both DNA/DharmaFECT kb complex formation and celltransfection. This is not the case for other transfection reagents.
4. Cell history, density, and passage number—it is very important to use
healthy cells that are regularly passaged and in growth phase. The highest
transfection efficiencies are achieved if cells are plated the day before;
however, adequate time should be given to allow the cells to recover fromthe passaging (generally > 12 hours). Plate cells at a consistent density tominimize experimental variation. If transfection efficiencies are low or
reduction occurs over time, thawing a new batch of cells or using cells witha lower passage number may improve the results.
If transduction into your cell line is unsuccessful, you may need toconsider the following list of factors influencing successful transduction.
1. Transduction efficiency is integrally related to the quality and thequantity of the virus you have produced. Factors to consider whentransducing include MOI (related to accurate titer in the target cell line),
the presence of serum in the media, the use of polybrene in the media,
length of exposure to virus, and viral toxicity to your particular cells.
2. High quality transfer vector DNA and the appropriate and efficient viral
packaging are required to make high quality virus able to transduce
cells effectively.
3. See also suggestions 3–5 for factors influencing successfultransfection (above).

Lable licenses
The shRNA Products, use and applications, are covered by pending andissued patents. Certain Label licenses govern the use of the products, these canbe found at dharmacon-licensing-statements. It is each Buyer’s responsibility todetermine which intellectual property rights held by third parties may restrict
the use of Products for a particular application. Please review the Label Licenses
governing all use of the shRNA and gene expression products.

To find the contact information in your country for your technology of interest, please visit us at
horizondiscovery.com/contact-us
Horizon Discovery, 8100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom©2020 The Horizon logo and other trademarks are the property of Horizon Discovery Limited, unless otherwise stated. DHARMACON, GIPZ and DHARMAFECT are trademarks of
Dharmacon Inc. TURBOGFP is a trademark of Evrogen JS . HXB2 is a trademark of GenBank. ZEOCIN, FASTDIGEST, GENEJET, and MAXIMA are trademarks of Thermo Fisher Scientific, Inc

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