TECHNICAL MANUAL

Inducible Dharmacon™ TRIPZ™
Lentiviral shRNA

Product description

The TRIPZ Inducible Lentiviral shRNA Collection was developed incollaboration with Dr. Greg Hannon of Cold Spring Harbor Laboratory (CSHL)
and Dr. Steve Elledge of Harvard Medical School. This collection combines thedesign advantages of microRNA-adapted short hairpin RNA (shRNA) with thepTRIPZ lentiviral inducible vector to create a powerful RNAi trigger capableof producing gene silencing. The vector is engineered to be Tet-On andproduces tightly regulated induction of shRNA expression in the presenceof doxycycline.

Important safety note

Please follow the safety guidelines for use and production of lentiviralparticles 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 pTRIPZ
lentiviral vector and the Trans-Lentiviral Packaging System can be foundon page 3.
pTRIPZ Inducible shRNA vectors are not compatible
with third generation packaging systems, due to therequirement of the expression of tat, which third
generation systems do not contain. We recommend
the Dharmacon™ Trans-Lentiviral™ Packaging System foruse with our vectors.

Design information
Unique microRNA-30 Based Hairpin Design
Dharmacon shRNA constructs are expressed as human microRNA-30 (miR-30)
primary transcripts. This design adds a Drosha processing site to the hairpin
construct and has been shown to greatly increase knockdown efficiency(Boden 2004).

dharmacon.horizondiscovery.com

The hairpin stem consists of 22 nucleotides of dsRNA and a 19-nucleotide
loop from human miR-30. The addition of the miR-30 loop, along with the
125-nucleotide miR-30 flanking sequences, resulted in a 10-fold increase in
Drosha and Dicer processing of the expressed hairpins when compared withconventional shRNA designs without microRNA (Silva 2005).
The proprietary design algorithm targets sequences in coding regions andthe 3’ UTR with the additional requirement that they contain greater than
3 mismatches to any other sequence in the human or mouse genomes.
Use of the miR-30 design also allows the use of ‘rules-based’ target sequence
selection. One such rule is the destabilizing of the 5' end of the antisense strand
which results in strand specific incorporation of miRNAs into RISC.
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,
each covering a unique region of the target gene.

Tet-on system design of the pTRIPZ Inducible
shRNA vector
The pTRIPZ vector is engineered to be Tet-On. This equips the pTRIPZ vector
to provide induced expression of an shRNA in the presence of doxycycline.
There are two main components on the pTRIPZ vector enabling induction:1) the tetracycline response element (TRE), and 2) the transactivator. The TRE,
modified from its natural state to consist of a string of operators fused to the
CMV minimal promoter, exhibits reduced basal expression and tighter binding
to the second component, the transactivator. The transactivator, known as thereverse tetracycline transactivator 3) (rtTA3) binds to and activates expression
from TRE promoters in the presence of doxycycline.
The rtTA3 transactivator is a modified version of the wildtype in two ways. First,
unlike the original tetracycline transactivator, the rtTA3 is modified to bind to
the TRE in the presence of doxycycline rather than in its absence. Secondly,
there are three mutations within the transactivator that increase its sensitivity
to doxycycline by 25-fold over the initial rtTA without increasing background
activity (Das 2004).

TurboRFP in the pTRIPZ inducible shRNA vector

Vector map

In addition to driving expression of the shRNA, the TRE also drives theexpression of a TurboRFP reporter. This induced expression of TurboRFP
enables the user to easily observe expression from the TRE promoter, allowing
quick assessment of factors such as delivery efficiency and promoter activity.

Tet-on or Tet-off configuration is possible
The pTRIPZ vector is versatile in that it can be easily converted to a Tet-Off
capable vector using Cre/loxP technology or classical restriction digest.
The rtTA3 is flanked by loxP sites allowing in vitro or in vivo excision of the
rtTA3 by exposure to Cre recombinase. The rtTA3 is also flanked by a pair of
BamHI restriction sites allowing for cleavage and ligation of the vector to
remove the rtTA3. Without the rtTA3 present on the vector, a tetracyclinetransactivator (tTA) can be added to the system in trans; thus creating a Tet-Offsystem where expression of shRNA and TurboRFP are induced in theabsence of doxycycline.

pTRIPZ
13.4 kb

Revised versions

From PowerPoint (R&D)

Vector information

5' LTR

IRES

PuroR

WPRE

Drawing was created usingshRNA
Geneious (http://www.geneious.com).

3ʹ SIN LTR

3' SIN LTR

pGIPZ

Figure 2. Detailed Vector Map of the pTRIPZ lentiviral vector.
he empty vector is 13,362 bp in size.

AmpR

pUC ori

SV40 ori

Antibiotic resistanceTRE
pTRIPZ contains 3 antibiotic
resistance markers UBC(Table 1).

TRE

UBC

tRFP

rtTA3

IRES

PuroR

RRE

WPRE

tRFP

rtTA3

Table 1. Antibiotic resistances conveyed by pTRIPZ

IRES PuroR

WPRE

Ψ

RRE

tGFP

Ψ

shRNA

• Ability to use the vector in either a Tet-On or Tet-Off configuration• TurboRFP and shRNA are partpGIPZ
of a single transcript allowing the visual
marking of shRNA-expressing cells• Amenable to in vitro and in vivo applications
AmpR
pUC ori
SV40 ori• Inducible RNAi in both dividing and non-dividing cell lines• Puromycin drug resistance marker for selecting stable cell lines

hCMV
RRE

5ʹ LTRΨ

Versatile vector design
Features of the pTRIPZhCMV
inducible lentiviral shRNA vector (Figures 1-2) that
IRES
PuroR
WPRE
RRE
make it a versatile
tooltGFP
for RNAi
studies
include:pUC ori

TRE

Tetracycline-inducible
promoter
hCMV

tRFP

tGFP
IRES
WPRE
shRNA
TurboRFP reporterfor visual trackingof transduction
andshRNA expression
microRNA-adapted shRNA (based on miR-30) for genepSMART 2.0
knockdown
Human ubiquitin C promoter for constitutive expression ofR
rtTA3
puromycin
resistancepUC ori
SV40 origenes
AmpandReverse tetracycline-transactivator 3 for tetracyclinedependent induction of the TRE promoter
Puromycin
resistance permits antibiotic-selective
pressure
hPGK
U6
and propagation
shRNA of stable integrantsPuroR
Internal ribosomal entry site allows expression of rtTA3 andpuromycin resistance genes in a single transcript

RSV/5' LTR

3' SIN LTRΨRRE

Figure 1. Schematic and features of the pTRIPZ Inducible Lentiviral shRNA vector.
AmpR

pUC ori

SV40 oriΨ

5ʹ LTR

hPGK

shRNA

PuroR

pLKO.1pUC ori

AmpR

hCMV
RRE

tGFP

IRES

PuroR

microRNA

WPRE

3ʹ SIN LTR

pSMART 2.0

3' SIN LTR

3' self-inactivating long terminal repeat for increasedR
lentivirusAmp
safetypUC oriPsi packaging sequence allows viral genome packaging
using lentiviral packaging systems
hCMV
Rev response
element enhances titer by increasing packaging
microRNA WPRE
RRE
tGFP
IRES
Puro
efficiency
of full-lengthviralR genomes
Woodchuck hepatitis posttranscriptional regulatory
element enhances transgene expression in the target cells

5' LTRΨWPRE

pLKO.1
5' long terminal
repeat3' SIN LTR

5' LTR

U6
RRE

3ʹ SIN LTR

IRES

Ψ

RRE

Mammalian selection marker

The pTRIPZ Inducible Lentiviral shRNA library has passed through internal QC
RRE
cPPT/CTStGFPandIRES
PuroR shRNA(Figure
WPRE
processes
to ensurehigh quality
low recombination3). Individualclones can be digested with SalI to assess the integrity of the plasmid and ruleout recombination. For best results, digest 400 ng of purified plasmid andpSMART 2.0
run the complete digest on a 1% agarose gel alongside undigested plasmid.
Expected band sizes for the empty vector are: 7104 bp, 4028 bp, 2188 bp. For
a pTRIPZ vector with Ampa hairpin
thepUC
7104
willoribe slightly larger. YouR
oribp band
SV40
should also be aware of the possibility that any particular shRNA may containa SalI site of its own, generating a fourth band.

RSV/5ʹ LTR ΨΨ
5' LTR

PuroR

Variable3ʹ SIN LTR

rtTA3(outside LTRs)
Bacterial selection marker
R μg/mL
Amp25
pUC ori
SV40 ori(inside LTRs)

hCMV

PuroR

3' SIN LTR

UBC

100 μg/mL

Puromycin

UtilityshRNA

5ʹ LTR

Zeocin™

SV40 ori

Vector element
RRE

Ampicillin(carbenicillin)

UtilitypTRIPZ Bacterial selection marker

Ψ

5' LTR

AmpRshRNA
Concentration

Antibiotic

3ʹ SIN LTR

3' SIN LTR

pTRIPZpSMARTTRIPZ
2.0 Inducible shRNA clones grown for 18 hours at 37 °C and
Figure 3. Representative
the plasmid isolated. Clones were then digested with SalI and run on an agarose gel. The
expected band sizes are 7104 bp, 4028 bp, 2188 bp. No recombinant products are visible.
10 kb molecular weight ladder
R (10 kb, 7 kb, 5 kb, 4 kb, 3 kb, 2.5 kb, 2 kb, 1.5 kb, 1 kb).
5ʹ LTR

Ψ

shRNA

Amp

pUC ori

SV40 ori

Additional safety information
Historically, the greatest safety risk associated with a lentiviral delivery
platform stems from the potential generation of recombinant lentiviruses
that are capable of autonomous replication. The TRIPZ Lentiviral shRNA
platform minimizes these hazards to the greatest degree by combining
a disabled viral genome with the proprietary Trans-Lentiviral packaging
process. Starting with the HXB2 clone of HIV-1 (GenBank Accession Number
K03455), the lentiviral backbone has been modified to eliminate all but themost 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-inactivating (SIN) vector greatly reduces theprobability of producing recombinant particles and limits cellular toxicity
often associated with expression of HIV genes.
Additional safety features can be incorporated by the packaging processitself. Generation of TRIPZ Lentiviral shRNA particles requires a packaging
step during which the expression construct containing the silencing
sequence is enclosed in a viral capsid. Gene functions that facilitate
this process (such as encoded by the structural genes gag, pol, env,etc.) are distributed amongst multiple helper plasmids which do notcontain significant regions of homology. This tactic further minimizes
the probability of recombination events that might otherwise generate
lentiviruses capable of autonomous replication. Among commerciallyavailable lentiviral vector systems, the Dharmacon™ Trans-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 tetracyclineresponsive promoter element (TRE), limiting expression of these viralcomponents strictly to the packaging cell line. A detailed description of theTrans-Lentiviral Packaging System has been published (Wu 2000).
With these safety measures in place, TRIPZ lentiviral shRNA particles can be
employed in standard Biosafety Level 2 tissue culture facilities.
Any investigator who purchases Dharmacon viral vector products is
responsible for consulting with their institution’s health and biosafety groupfor specific guidelines on the handling of lentiviral vector particles. Further,each investigator is fully responsible for obtaining the required permissions
for the acceptance of lentiviral particles into their local geography andinstitution.
• 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, Dec 2009 here.
• See also: NIH Guidelines For Research Involving Recombinant DNA
Molecules (NIH Guidelines), Apr 2016, downloadable here.
• For Biosafety Considerations for Research with Lentiviral Vectors, here.

Protocol I – Replication
For archive replication, grow all TRIPZ Inducible shRNA clones at 37 °C
in 2x LB broth (low salt) medium plus 25 μg/mL Zeocin and 100 μg/mL
carbenicillin in order to provide maximum stability of the clones.
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 Zeocin™ and 100 μg/mL carbenicillin, Table 2).
Prepare source plates
1. Remove foil seals while the source plates are still frozen to minimize
cross-contamination.
2. Thaw the source plates with the lid on. Wipe any condensation
underneath the lid with a paper wipe soaked in ethanol.

Replicate
1. Gently place a disposable replicator in the thawed source plate andlightly move the replicator around inside the well to mix the culture.
Make sure to scrape the bottom of the wells.
2. Carefully remove the replicator from the source plate and gently place inthe target 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. Reseal and 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 37 °C incubator for 18-19 hours.
Freeze at -80 °C for long-term storage. Avoid long periods of storage at room
temperature or higher 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. *Glycerol should beomitted from the medium if you are culturing for plasmid
preparation. If making copies of the constructs for longterm storage at -80 °C, 8% glycerol is required.
Table 2. Materials for plate replication.

Item

VendorCat #LB-Lennox 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

Zeocin™

Invivogenant-zn-5p

Puromycin

Fisher ScientificBP2956-100

96-well microplates

Fisher Scientific12-565-363

Aluminum seals

Fisher Scientific12-565-475

Disposable replicators

Fisher Scientific

NC9584102

Protocol II – Plasmid preparation
Culture conditions for individual plasmid preparations
For plasmid preparation, grow all TRIPZ Inducible shRNA clones at 37 °C in LB
broth (low salt) medium plus 100 μg/mL carbenicillin only.
Most plasmid mini-prep kits recommend a culture volume of 1-10 mL for goodyield. For shRNA constructs, 3-5 mL of culture can be used for one plasmidmini-prep generally producing 5-10 µg of plasmid DNA.
Upon receiving your glycerol stock(s) containing the shRNA of interest store at-80 °C until ready to begin.
1. 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.
2. 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.
3. Incubate at 37 °C for 18-19 hours with vigorous shaking.
If a larger culture volume is desired, use the 3-5 mL
culture as a starter inoculum. Allow the starter culture to
incubate for 8 hours at 37 ˚C then dilute it 1:500 to 1:1000
into a larger culture volume. Incubate at 37 °C for 18-19
hours with vigorous shaking.
Due to the tendency of viral vectors to recombine, we
recommend keeping the incubation times as short as
possible and avoid subculturing.

4. Pellet the culture and follow the instructions for your plasmid prep kit
to obtain the plasmid DNA. Plasmid DNA can be isolated using Thermo
Scientific™ GeneJET™ Plasmid Miniprep Kit (Cat #K0502 ) or similar.
5. Run 3-5 μL of the plasmid DNA on a 1% agarose gel. The pTRIPZ plasmidwith shRNA is 13,320 bp (empty vector) or 13,362 with hairpin shRNA.
Return to your original glycerol stock 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.
We use the above 96-well bio-block plasmid preparation
protocol in conjunction with a Qiagen™ Turbo™ Kit (Cat#27191). We use 2 bio-blocks combined. Do not performthe optional wash and elute the DNA in water.

Protocol III – Restriction digest
The following is a sample protocol for restriction enzyme digestion using
Thermo Scientific™ FastDigest™ SalI (Cat# FD0644) for diagnostic quality
control of TRIPZ Inducible Lentiviral shRNA vectors.
1. Using filtered pipette tips and sterile conditions, add the components
for a FastDigest SalI restriction digest (as listed in Table 3) to a sterile PCR
thin-wall tube. Mix gently by pipetting.
2. Incubate at 37 °C for 5 minutes to digest.
3. Load the gel with 20 μL of each of the digested samples (using SalI) on
a 1% agarose gel. Run uncut sample alongside the digested samples.
Expected band sizes for the empty vector are: 7104 bp, 4028 bp, 2188 bp(Figure 3). For a TRIPZ Inducible shRNA vector with a hairpin the 7104 bp
band will be slightly larger. You should also be aware of the possibility
that any particular shRNA may contain a SalI site of its own, generating
a fourth band.

Protocol IV – Cloning
Moving shRNA constructs from pGIPZ to pTRIPZ
1. Isolate plasmid from your GIPZ shRNA clone according to Protocol II –Plasmid Preparation.
2. Set up a restriction digest of the prepared plasmid using MluI and XhoI
restriction enzymes according to Table 3, adding 1µL of each enzyme tothe tube. Incubate reaction at 37 ˚C for 5 minutes.
You will need a large amount of DNA digested in order tovisualize the small 345 bp band on an agarose gel. If it isdifficult to see this band you will need to scale up the digest
for more than 5 μg of DNA.
3. Run the entire digest on a 1.2%-1.5% agarose gel. Two bands should beseen (345 bp and a large band near 11.4 kb). Two bands will appear only
if both MluI and XhoI have cut. If one or the other of these enzymes does
not cut, you will not see the 345 bp band but only a band at ~ 11.7 kb.
4. Excise the 345 bp band containing the shRNA of interest and purify using
the Thermo Scientific™ GeneJET™ Gel Extraction Kit (Cat #K0691) column
according to the kit directions. Elute in 50 μL nuclease-free water.
5. Quantify the insert fragment.
6. Prepare the pTRIPZ empty vector for ligation to the shRNA insert bydigesting 3 μg of plasmid with restriction enzymes MluI and XhoI as
above.
7. Run the entire digest on a 0.8% agarose gel. Make sure to run thegel through no less than 3 cm length of agarose. This will also aid indecreasing contamination of uncut vector in your vector preparation.
8. Gel isolate the 13,061 bp band using the GeneJET Gel Extraction Kit(Cat #K0691) according to the kit directions. Elute in 50 μL nuclease-freewater. You will likely not see a band representing the excised portion of
the vector as it is too small.
9. Quantify the amount of isolated cut vector per μL.
10. Ligate 7.4 ng of the shRNA insert and 250 ng of cut pTRIPZ vector.
This yields a molar ratio of 1 vector to 1 insert. Prepare ano-insert ligation control in parallel.
11. Transform 5 μL of the diluted ligation mix into competent E.coli cells.
Follow the transformation protocol for the competent cells. Plate thetransformed cells onto agar plates containing 100 μg/mL carbenicillinand 25 μg/mL Zeocin™. Be sure to transform the same volume ofligation mix and plate the same volume of cells for both the control
and the experimental sample. Plating 100 μL, 50 μL, and 10 μL aliquots
is recommended.
12. Incubate plates at 37 ˚C overnight. Count colonies and determine theratio of colonies on the control plate versus the experimental plates.
Determine the number of colonies to sequence verify.
13. Sequence verify clones. The pTRIPZ sequencing primer is as follows:
5’-GGAAAGAATCAAGGAGG-3’

Figure 3. Representative TRIPZ Inducible shRNA clones grown for 18 hours at 37 °C andthe plasmid isolated. Clones were then digested with SalI and run on an agarose gel. The
expected band sizes are 7104 bp, 4028 bp, 2188 bp. No recombinant products are visible.
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).

This primer runs in the forward direction. The melting
temperature of this 17mer = 46.7 °C.

Table 3. Restriction Digest Components.

Component

AmountProtocol V – Puromycin selection

Water, Nuclease-free

X μL

Puromycin kill curve and puromycin selection

10x FastDigest Buffer

2 μL

DNA sample (up to 1 µg) in water

X μL

In order to generate stable cell lines expressing the transgene of interest, it isimportant to determine the minimum amount of antibiotic required to kill
non-transfected cells. A simple procedure to test this is as follows:

Recommended FastDigest Enzyme(s)1 μLFinal Volume

20 μL

Day 1: Using the same cell type and relative cell densities to be used insubsequent transfection procedures, plate cells and culture overnight.

Day 2: Replace complete growth medium with growth medium supplemented
with a range of antibiotic concentrations, for example puromycin (0-15 μg/mL), including untreated control cells with no antibiotic added.

2. Dilute 1 µg of DNA in 50 µL of serum-free DMEM or other serum-free
growth medium.
3. Gently mix DharmaFECT kb reagent and add 3 µL of it to the diluted
DNA. Mix immediately by pipetting.
4. Incubate 10 minutes at room temperature. Remove medium from welland replace with 0.45 mL fresh growth medium.

Day 4: Refresh medium and assess viability.
• Replace medium with fresh medium supplemented with theappropriate concentration of puromycin every 2-3 days depending on
the growth of cells.
• Examine cells daily and identify the minimal concentration of antibiotic
that efficiently kills all non-transfected cells between 4-6 days following
addition of antibiotic.

Prepare immediately prior to transfection. We recommend
starting with 1 µg of DNA and 3 µL of DharmaFECT kb per
well in a 24-well plate (see scale-up Table 4). Subsequent
optimization may further increase transfection efﬁciency
depending on the cell line and transgene used.

Protocol VI – Transfection

5. Gently add 50 µL of the DharmaFECT kb/DNA mixture to each well.
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.

General protocol for transfection of adherent and suspension cells in a
24-well plate
Quantities and volumes should be scaled-up according to the number of cells/
wells to be transfected
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 dayof transfection is 70-90%. Suspension cells should be inlogarithmic growth phase at the time of transfection.

Table 4. Scale-up ratios for transfection of adherent and suspension cells 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.

Induction of TurboRFP/shRNA expression In transfected cells
using doxycycline
Optimization within a range of 0.1-2 µg/mL doxycycline is recommended fortransfected cells as amounts of doxycycline will vary due to the transfectablilityof the cell line, amounts of DNA used to transfect, as well as other variables.

Cells grown in suspension
Transfection of cells in suspension would follow all the above principles andthe protocol would largely remain the same, except that the DNA/DharmaFECT
kb reagent mixture should be added to cells after incubation for complex
formation, to a total volume of 250 µL serum-free medium with antibiotics.

After 24 hours of incubation post-transfection, add medium containing theappropriate amount of doxycycline. Examine the cells microscopically for thepresence of TurboRFP expression 24-48 hours after doxycycline addition, as
this will be your first indication of transfection efficiency. Then assay cells for
reduction in gene expression by reverse transcription quantitative real-time
qPCR (RT-qPCR), western blot, or other appropriate functional assay; compare
to untreated, non-silencing shRNA or other negative controls.

Transfection optimization using Dharmacon DharmaFECT
kb transfection reagent
It is essential to optimize transfection conditions to achieve the highest
transfection efficiencies and lowest toxicity with your cells. The most importantparameters for optimization are DNA to transfection reagent ratio, DNAconcentrations, and cell confluency. We recommend that you initially begin
with the DharmaFECT kb reagent and DNA amount indicated in Table 4, andextrapolate the number of cells needed for your vessel size from the numberof cells used in a well of a 24-well plate, as listed in step 1 of the protocol fordelivery of plasmid DNA.

The half-life of doxycycline in culture is 24 hours. It istherefore advisable to refresh the doxycycline-containing
medium on the cells regularly to avoid fluctuation in thetranscription levels from the inducible promoter.
Optimal length of incubation from the start of transfection to analysis isdependent on cell type, gene of interest, and the stability of the mRNA and/or protein being analyzed. RT-qPCR generally gives the best indicationof expression knock-down. The use of western blots to determine
knockdown is very dependent on quantity and quality of the protein sample,
its half-life, and the sensitivity and specificity of the antibody and detection
systems used.

Protocol VII – packaging lentivirus
The pTRIPZ 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 System (Cat #TLP5912,
TLP5913, TLP5914, or TLP5917). The Trans-Lentiviral Packaging System allows
creation of a replication-incompetent (Shimada 1995), HIV-1-based lentiviralparticles which can be used to deliver and express your gene or shRNA ofinterest in either dividing or non-dividing mammalian cells. The Trans-

Lentiviral shRNA/ORF Packaging System generates a replication-incompetentparticle based on the trans-lentiviral system developed by Kappes (Kappes andWu 2001). For protocols and information on packaging TRIPZ Inducible shRNA
with our Trans-Lentiviral shRNA/ORF Packaging System Kit, please see theproduct manual available here.

Protocol VIII – Titering
Follow the procedure below to determine the titer of your lentiviral stockusing your mammalian cell line of choice. This protocol uses the HEK293T
packaging cell line that is available as part of our Trans-Lentiviral shRNA
Packaging Kit (Cat #TLP5917).
If you have generated a lentiviral stock of the expression
control (such as pTRIPZ Inducible shRNA Non-silencing
control), we recommend titering this stock as well.
1.

2.

The day before transduction, seed a 24-well tissue culture plate with
HEK293T cells at 5 × 104 cells per well in DMEM (10% FBS, 1% pen-strep).
The following day, the well should be no more than 40-50% confluent.
Make dilutions of the viral stock in a round bottom 96-well plate using
serum-free medium. Utilize the plate as shown (Figure 4) using one row
for each viral stock to be tested. The goal is to produce a series of five-folddilutions to reach a final dilution of 390,625 fold.

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

1

Virus stock 2

B

Virus stock 3

CVirus stock 4

D

2

3

4

5

6

4.

5.

6.

Volume Diluted
Virus Used

Dilution Factor

25 μL

5*

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 toinclude either well 8 or well 1 respectively.

Figure 5. Twenty four well tissue culture plate,seeded with HEK293T cells, used to titer the virus.

3.

Destination(24-well plate)

A1

Dilution Plate

A

Table 5. Example of set up for dilutions.

Originating(96-wellplate)

Figure 4. Five-fold serial dilutions of virus stock.

Virus stock 1

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 forgene silencing.

Well (Row A, B, C, or D)

A
B
C
D
E
F
G
H

Virus stock 1

Note: It is strongly recommended that you use a high quality multichannel
pipettor when performing multiple dilutions.
7. Label the 24-well plate as shown in Figure 5 using one row for each viral
stock to be tested.
8. Remove culture medium from the cells in the 24-well plate.
9. Add 225 µL of serum-free medium to each well.
10. Transduce cells by adding 25 µL of diluted lentiviral particles from theoriginal 96-well plate (Figure 4) to a well on the 24-well destination plate(Figure 5) containing the cells. For example, transfer 25μL from well A2 ofthe 96-well plate into well A1 in the 24-well plate (Table 5).
11. Incubate transduced cultures at 37 °C for 4 hours.
12. Remove transduction mix from cultures and gently rinse cells with PBS.
13. Add 1 mL of DMEM (10% FBS, 1% pen-strep) containing 1 µg/mL of
doxycycline.
14. Culture cells for 72 hours.
15. Count the TurboRFP expressing cells or colonies of cells (Figure 6).
Note: Count each multi-cell colony as 1 transduced cell, as the cells will be
dividing over the 72 hour culture period.
16. Transducing units per mL (TU/mL) can be determined using the following
formula:# of TurboRFP positive colonies counted × dilution factor × 40 = # TU/mL.
Example: 55 TurboRFP positive colonies counted in well A3.
5 (TurboGFP positive colonies) × 625 (dilution factor) × 40 = 1.38 × 106 TU/mL.

Add 80 μL ofserumfree medium to each well.
Add 20 µL of thawed viral stock to each corresponding well in column
1 (five-fold dilution). Pipette contents of well up and down 10-15 times.
Discard pipette tips.
With new pipette tips, transfer 20 µL from each well of column 1 to thecorresponding well in column 2. Pipette contents of well up and down
10-15 times. Discard pipette tips.
Repeat transfers of 20 µL from columns 2 through 8, pipetting up anddown 10-15 times and changing pipette tips between each dilution.

Figure 6. Examples of individual colonies.

Multiplicity of Infection (MOI)
To obtain optimal silencing of your gene of interest, you will need to transduce
the lentiviral 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 ofyour gene of interest. If you are transducing your lentiviral construct intothe mammalian cell line of choice for the first time, after you have titered thelentiviral particles, we recommend using a range of MOIs (for example, 0, 0.5,
1, 2, 5, 10, 20) to determine the MOI required to obtain optimal expression foryour particular application. It should be noted that to achieve single copy
knockdown, an MOI of 0.3 is generally used, as less than 4% of your cells will
have more than one insert.

That the higher the MOI you have chosen the more copiesof the shRNA and puromycin resistance gene you will
have per cell. When selecting on 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
select for the population of transduced cells you wish to
select for, without going below the minimum antibiotic
concentration you have established in your “kill curve.”

Protocol IX – 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
medium. 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 6). It is strongly recommended that you optimize transduction
conditions to suit your target cell line for the highest transduction efficiency
possible.
It is preferable that transduction be carried out in medium that is serumfree 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 need to optimize the protocol according to
your needs.

Protocol X – Induction of TurboRFP/shRNA
expression
Induction of TurboRFP and shRNA in transduced cells using doxycycline
1.
Remove medium and gently rinse cells with PBS.
2.
Add medium containing doxycycline at a concentration of 0.5 µg/mL.
TurboRFP will become visible within 24 hours and will be at full intensity
by 72 hours.
Doxycycline concentrations ranging from 0.1 µg/mL to 2 µg/
mL can be used. We recommend you optimize the doxycycline
concentration according to your experimental needs. Cells
transduced with pTRIPZ Inducible shRNA at single copy arecapable of producing visible TurboRFP at concentrations as
low as 0.1 µg/mL. Increasing TurboRFP signal will be seen
with both increasing doxycycline concentrations as well as
increased TRIPZ shRNA copy number.

Table 6. Suggested volumes of medium per surface area per well of adherent cells.

Suggested Total
Serum-free Medium
Volume Per Well (mL)

Tissue Culture Dish

Surface Area
Per Well (cm2)

100 mm

56.0

5.0

60 mm

20.0

2.0

35 mm

8.0

1.0

6-well9.4

1.0

12-well

3.8

0.5

24-well

1.9

0.25

96-well

0.3

0.1

1. On day 0, plate 5-8 × 104 cells per well for complete medium (including
serum) in a 24-well plate. Incubate overnight.
2. The next day (day 1), remove the medium and add the virus to the MOI
you wish to use. Bring the total volume of liquid up so that it just covers
the cells efficiently with serum-free medium (See Table 6 for guidelines). If
you are using concentrated virus you are likely to use very little virus volume
and a lot of serum-free medium; if you are using unconcentrated virus you
will find you need much more virus volume.
3. Approximately 6-8 hours post-transduction, add 1 mL of complete
medium (serum plus pen-strep if you are using it) to your cells andincubate overnight. We have experienced low toxicity with transductionin the cell lines tested, therefore removal of virus is not required for manycell lines. In our experience, higher transduction efficiencies have beenachieved if the virus is not removed after 6 hours. However, if toxicity is
a problem, aspirate the mixture after 3-6 hours and replace with fresh
growth medium. Additionally, fresh growth medium should be replenished
as required for continued cell growth.
4. At 48 hours post-transduction, replace the current medium withcomplete medium containing puromycin into the appropriate wells.
When adding puromycin, use the appropriate concentration as
determined based on the above “kill curve.” Incubate.
a. Approximately every 2-3 days replace with freshly preparedselective medium.
b. Monitor the cells daily and observe the percentage of surviving
cells. At some time point almost all of the cells surviving selection
will be harboring the shRNA. Optimum effectiveness should bereached in 3-10 days with puromycin.

3.

Maintain cells in doxycycline for the duration of the experiment or as
desired. The half-life of doxycycline in culture is 24 hours. It is therefore
advisable to refresh the doxycycline containing medium on the cells
regularly to avoid fluctuation in the transcription levels from theinducible promoter.
Induction of TurboRFP can be conducted exclusive of, in
conjunction with, or sequential to puromycin selection.

4.

Proceed to extract RNA for knockdown evaluation RT-qPCR.
The optimal length of incubation from the start ofinduction to analysis is dependent on cell type, yourgene of interest and the stability of the mRNA and/or
protein being analyzed.

Turning TurboRFP and shRNA Expression off after doxycycline induction
1.
Split and replate the cells into a fresh plate/well at a ratio suitable foryour experimental purposes.
Wash the cells in PBS before splitting and use medium that
does NOT contain doxycycline during the split. Doxycycline
is inclined to adhere to the cells so precautions should betaken to make sure that no residual doxycycline getscarried over.
2.
3.
4.

After splitting the cells into fresh medium without doxycycline,
incubate for 3 hours.
Wash the cells with PBS one to three times.
Add medium without doxycycline. The TurboRFP protein will be turned
over in approximately 72 hours to the point it will no longer be visible
under the microscope.

A

B

Non-silencing shRNANo
doxycycline (dox) 48 hours

0.5 µg/mL dox 48 hours

DC

0.5 µg/mL dox 72 hours

E

Post dox 24 hours

10. 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
internal control.
11. Cq values less than 11 for the qPCR internal 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 internal
control as well as your experimental target to determine an optimum
cDNA concentration.
12. It may be necessary to change internal controls if conditions in steps 10
and 11 cannot be simultaneously met.

Post dox 72 hours

Table 7. Related Reagents.
Figure 7. Induction of TRIPZ shRNA (tracked by TurboRFP) with doxycycline is tightly
regulated. HEK293T cells were transduced at an MOI of 0.3, puromycin selected (2 µL/mL) for 96 hours after which 0.5 µg/mL of doxycycline was added to the cells andTurboRFP expression was assessed at 48-72 hours (B,C). Post-doxycycline samples (D,E)
were photographed at times indicated. At 72 hours after doxycycline removal TurboRFP
expression is reduced to background levels.

Protocol XI – qPCR
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
and trustworthy.
1.

2.
3.
4.
5.

6.

7.

8.

9.

Experimental samples should be run in no less than duplicate. It
should be noted that with duplicate experiments it will not be possible
to assign error bars to indicate consistency from experimental sample
to experimental sample. Using triplicate samples or higher will enableerror bars to be assigned indicating the level of experimental variation.
We have found that normalizing the RNA concentration prior to cDNA
synthesis will increase consistency downstream.
Make sure the mRNA you are using as your internal control for qPCR isexpressed at a level higher than your target mRNA.
Use only high-quality calibrated pipettes, in conjunction with wellfitting barrier tips.
When pipetting, take the time to visually inspect the fluid in the tip(s)for accuracy and lack of bubbles, especially when using a multichannel pipette.
Be sure to spin your qPCR plate prior to loading in the instrument
in order to collect the sample at the bottom of the well, as well as
eliminate any bubbles that may have developed.
With regard to knockdown experiments using shRNA, it is importantthat you greatly reduce, if not eliminate entirely, those cells which are
not transduced or transfected from the population (cells that are notexpressing the fluorescent reporter). This can be done in several ways:
increase the efficiency of your transfection, use a higher multiplicity
of infection (MOI) for your transduction, or utilize the puromycin
selection marker and drug select against those cells that do notcontain the shRNA.
Always utilize the non-silencing control as a reference for target gene
expression, as opposed to an untreated sample. The non-silencing
control treated samples will most accurately reproduce the conditions
in your experimental samples.
You may also use an untreated sample to indicate substantial changes
in target gene expression as seen in the non-silencing control
due to generic consequences of viral infection/transfection reagentsetc. However, it should be noted that small changes in expression
levels between an untreated sample and the non-silencing control
are to be expected.

Related reagentsCat #TRIPZ Human GAPDH Inducible LentiviralshRNA Positive Control

RHS4744

TRIPZ Inducible Lentiviral Non-silencing shRNA Control

RHS4743

TRIPZ Inducible Lentiviral Empty Vector shRNA Control

RHS4750

Trans-Lentiviral shRNA Packaging System

TLP5912,
TLP5913, TLP5914

Trans-Lentiviral shRNA Packaging Systemwith HEK293T Cells

TLP5917Frequently Asked Questions (FAQs)
What clones are part of my 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 (dharmacon.horizondiscovery.com). Enterthe 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 theresults. Click on the plus sign to expand the details for this clone and select theSequence tab.
Which antibiotic should I use?
You should grow all TRIPZ constructs in both 25 μg/mL zeocin and 100 μg/mL
carbenicillin for archive replication. You should grow the constructs in media
containing only 100 μg/mL carbenicillin for plasmid preparation.
What packaging cell line should I use for making lentivirus?
For packaging our lentiviral shRNA constructs, we recommend the TransLentiviral shRNA Packaging Kit (see Table 7). The Trans-Lentiviral Packaging
Kit allows creation of a replication-incompetent (Shimada 1995), lentiviralparticles which can be used to deliver and express your gene or shRNAof interest in either dividing or non-dividing mammalian cells. The TransLentiviral Packaging Kit generates replication-incompetent particles based on
the trans-lentiviral system developed by Kappes (Kappes and Wu 2001). For
protocols and information on packaging TRIPZ 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 pTRIPZ vector?
Packaging on the TRIPZ shRNA into lentiviral particles is tat-dependent, so you
must use a packaging system that expresses the tat gene.
What does the number 40 refer to in the formula for the calculationof titer?
The titer units are given in transducing units (TU) per mL, so the number
40 is used to convert the 25 µL used in the titration (“volume of diluted viralparticles” Table 5) to one milliliter.

What is the sequencing primer for use with TRIPZ shRNA?
The TRIPZ sequencing primer is 5’-GGAAAGAATCAAGGAGG-3’.
Note: This primer runs in the forward direction.
How can I make a stable cell line?
In order to generate stable cell lines, it is important to determine theminimum amount of puromycin required to kill non-transfected/transduced
cells. This can be done by generating a puromycin kill curve (see Protocol
V – Puromycin Selection). After you have determined the appropriate
concentration of puromycin to use, you can transfect or transduce your cellswith the shRNA construct and culture with puromycin to select for those cells
that have a stable integrant.
If transfection in to 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 ofantibiotics can adversely affect the transfection efficiency and leadto increased toxicity levels in some cell types. It is recommended that
antibiotics be excluded until transfection has mostly occurred (5-6
hours) and then be added together with the full medium.
4. Cell history, density, and passage number — it is very important touse 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 torecover from the passaging (generally > 12 hours). Plate cells at
a consistent density to minimize experimental variation. If transfection
efficiencies are low or reduction occurs over time, thawing a newbatch of cells or using cells with a 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 lentiviral particles you have produced. Factors to consider
when transducing include MOI (related to accurate titer in the target cellline), the presence of serum in the medium, the use of Polybrene™ in the
media, length of exposure to lentiviral particles, and viral toxicity to yourparticular 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 3-4 for factors influencing successfultransfection (above).

References
Cited references and additional suggested reading
1. Bartel, D. P. (2004). “MicroRNAs: genomics, biogenesis, mechanism,and function.” Cell 116(2): 281-97.
2. Boden, D., O. Pusch, et al. (2004). “Enhanced gene silencing of HIV-1
specific siRNA 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. Cullen, B. R. (2005). “RNAi the naturalway.” Nat Genet 37(11): 1163-5.
6. Das, A. T., X. Zhou, et al. (2004). “Viral evolution as a tool to improve thetetracycline-regulated gene expression system.” J Biol Chem 279(18):18776-82.
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 interferenceby 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. Gregory, R. I., K. P. Yan, et al. (2004). “The Microprocessor complex
mediates the genesis of microRNAs.” Nature 432 (7014): 235-40.
13. Hannon, G. J. (2002). “RNA interference.” Nature 418(6894): 244-51.
14. Hannon, G. J. and J. J. Rossi (2004). “Unlocking the potential of thehuman genome with RNA interference.” Nature 431(7006): 371-8.
15. He, L. and G. J. Hannon (2004). “MicroRNAs: small RNAs with a big rolein gene regulation.” Nat Rev Genet 5(7): 522-31.
16. Kappes, J. C. and X. Wu (2001). “Safety considerations in vector
development.” Somat Cell Mol Genet 26(1-6): 147-58.
17. Kappes, J. C., X. Wu, et al. (2003). “Production of trans-lentiviral vector
with predictable safety.” Methods Mol Med 76: 449-65.
18. Lee, Y., C. Ahn, et al. (2003). “The nuclear RNase III Drosha initiates
microRNA processing.” Nature 425(6956): 415-9.
19. Lee, Y., K. Jeon, et al. (2002). “MicroRNA maturation: stepwise
processing and subcellular localization.” Embo J 21(17): 4663-70.
20. Nakahara, K. and R. W. Carthew (2004). “Expanding roles for miRNAs
and siRNAs in cell regulation.” Curr Opin Cell Biol 16(2): 127-33.
21. Paddison, P. J., J. M. Silva, et al. (2004). “A resource for large-scale RNAinterference-based screens in mammals.” Nature 428(6981): 427-31.
22. Pollard, H., J. S. Remy, et al. (1998). “Polyethylenimine but not cationiclipids promotes transgene delivery to the nucleus in mammalian cells.”
J Biol Chem 273(13): 7507-11.
23. Shimada, T., et. al. (1995). “Development of Vectors Utilized for Gene
Therapy for AIDS”. AIDS 4.
24. Stegmeier, F., G. Hu, et al. (2005). “A lentiviral microRNA-based
system for single-copy polymerase II-regulated RNA interference inmammalian cells.” Proc Natl Acad Sci U S A 102(37): 13212-7.
25. 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.
26. Zeng, Y. and B. R. Cullen (2003). “Sequence requirements for micro RNA
processing and function in human cells.” Rna 9(1): 112-23.
27. Zeng, Y., R. Yi, et al. (2005). “Recognition and cleavage of primary
microRNA precursors by the nuclear processing enzyme Drosha.”
Embo J 24(1): 138-48.

FAQs/troubleshooting
For answers to questions that are not addressed here, please email
technical support at ts.dharmacon@horizondiscovery.com with yourquestion, your sales order or purchase order number and the catalog
number or clone ID of the construct or collection with which you arehaving trouble.

Label licenses
The shRNA and gene expression Products, use and applications, arecovered by pending and issued patents. Certain Label licenses govern
the use of the products, these can be found at dharmacon-licensingstatements. It is each Buyer’s responsibility to determine 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.

If you have any questions

Illumina, HiSeq, MiSeq, and TruSeq are trademarks or registered trademarks of Illumina, Inc. Limited permission to copy or distributesequence information. Oligonucleotide sequences ©2007-2011 Illumina, Inc. All rights reserved. Derivative works created by Illumina
customers are authorized for use with Illumina Instruments and products only. All other uses are strictly prohibited. Tet-On 3G is a trademarkof Clontech Laboratories, Inc. TurboGFP and TurboRFP are trademarks of Evrogen Inc. HXB2 is a registered trademark of GenBank. Polybrene
is a registered trademark of Santa Cruz Biotechnology, Inc. Turbo Kit is a registered trademark of Qiagen. Zeocin is a registered trademarkof Invitrogen. Phusion is a trademark of Thermo Fisher Scientific, Inc. ©2018 Horizon Discovery Group Company—All rights reserved. All othertrademarks are the property of Horizon Discovery Company unless otherwise specified. First published July 2016. UK Registered Head Office:
Building 8100, Cambridge Research Park, Cambridge, CB25 9TL, United Kingdom.

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