TECHNICAL MANUAL

Dharmacon™ shMIMIC
Lentiviral microRNA

Contents
1 Introduction to shMIMIC Lentiviral microRNA..................................................................................................3
Recommended experimental controls............................................................................................................................................ 4
2 .Protocol for transduction of lentiviral particles.................................................................................................4
Workflow checklist.................................................................................................................................................................................. 4
Materials required.................................................................................................................................................................................... 5
Materials not supplied........................................................................................................................................................................... 5
Protocol using shMIMIC Lentiviral microRNA............................................................................................................................... 5.Determine functional titer in cells of interest......................................................................................................................... 5
General protocol............................................................................................................................................................................... 5
Establishing a stable cell population......................................................................................................................................... 7
3 Appendix................................................................................................................................................................8
Additional protocols............................................................................................................................................................................... 8
Identify the optimal promoter..................................................................................................................................................... 8
Determine cell density and transduction conditions........................................................................................................ 11
Determine functional titer........................................................................................................................................................... 15
Stability and storage............................................................................................................................................................................. 17
Quality assurance and control.......................................................................................................................................................... 17
Frequently asked questions (FAQs).................................................................................................................................................18
4 Lentiviral particle product safety level information........................................................................................21
5 Limited Use Licenses............................................................................................................................................22

shMIMIC Lentiviral shRNA
Technical Manual

1

Introduction to shMIMIC Lentiviral microRNA
The Dharmacon™ SMARTchoice lentiviral vector platform is an innovative system ideally suited for RNA interference(RNAi)-mediated studies. The purpose is to provide the researcher with the most effective tools for delivering andexpressing genetic content in their cells of interest. The SMARTvector™ shRNA and shMIMIC microRNA products, which
are built upon the SMARTchoice platform, utilize advanced options which include:••••

a microRNA scaffold for improved processing
seven RNA polymerase II promoter* options to enable experiments in a broad range of cell types with
two fluorescent reporter options [TurboGFP (tGFP), TurboRFP (tRFP), Evrogen, Moscow, Russia]
and a no-reporter option
an option of ultra-high titer of 2 × 109 TU/mL (±20%)

The SMARTchoice workflow incorporates a unique tool, the SMARTchoice Promoter Selection Plate (Cat #SP-001000-01).
This is a 96-well plate containing high-titer, transduction-ready lentiviral particles derived from seven unique lentiviralshRNA constructs, each containing a different transcriptional promoter. The SMARTchoice Promoter Selection Plate
allows the researcher to evaluate and then choose the vector configuration that is most effective for shRNA expression
in the particular cells of interest. This semi-customizable approach to vector design provides greater flexibility, and more
importantly, improved likelihood of experimental success relative to static systems, see SMARTchoice web page for more
information at https://dharmacon.horizondiscovery.com/brands/smartchoice/#all.

SMARTchoicepromoters

5’ LTR
Figure 1. Elements of the
SMARTvector shRNA and shMIMIC
microRNA lentiviral vector.

Ψ

RRE*Some promoter options may only
be available as custom products
or upon request.

hCMV
mCMV
hEF1α
mEF1α
PGK
CAG
UBC

tGFP IRES PuroR
ortRFP IRES
or

None

WPRE 3’ SIN LTR

SMARTvector universal scaffold

Vector element

Utility

5' LTR

5' Long Terminal Repeat is necessary for lentiviral particle production and integration into host cell genomeΨPsi packaging sequence allows viral genome packaging using lentiviral packaging systems

RRE

Rev Response Element enhances titer by increasing packaging efficiency of full-length viral genomes

tGFP or tRFP

TurboGFP or TurboRFP reporter for visual tracking of transduction and expression

None

No reporter option for use in applications where fluorescence is not required

IRES

Internal Ribosomal Entry Site allows expression of fluorescent reporter and puromycin resistance gene in a single transcript

Puro

Puromycin resistance permits antibiotic-selective pressure and propagation of stable integrants

SMARTvector universal scaffold

microRNA-adapted shRNA for gene knockdown or expression of a mature microRNA

WPRE

Woodchuck hepatitis Post-transcriptional Regulatory Element enhances transgene expression in the target cells

3' SIN LTR

3' Self-inactivating Long Terminal Repeat for increased lentivirus safetyR

3

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Technical Manual

Recommended Experimental Controls
Including both positive and negative controls in all experiments assures rigorous, high-confidence data, and is required
for accurate data interpretation. Additionally, negative and positive shRNA control lentiviral particles are cost-effective
reagents for experimental optimization prior to transductions with shMIMIC Lentiviral microRNA gene, such asdetermining optimal conditions for transduction.
The following SMARTvector Negative and Positive Controls are available with the above-mentioned array of promoter
and reporter options:
•
•
•

2

SMARTvector Non-targeting Control shRNA
SMARTvector GAPD Positive Control shRNA (human, mouse and rat)
SMARTvector PPIB Positive Control shRNA, hCMV/Turbo GFP only (human, mouse and rat)

Protocol for transduction of lentiviral particles
Workflow checklist
Before you order: Determine materials required for experiment Select promoter
		
If the optimal promoter for your cell type is unknown, empirically test using the SMARTchoice Promoter
		
Selection Plate (See Appendix: Identify the optimal promoter).
Order reagents with the optimal promoter/reporter combination for: Cells and microRNA(s) of interest
 Positive controls targeting GAPD or PPIB Non-targeting controls
After you order: Determine puromycin concentration
		
Prior to receiving your products, determine the minimum concentration of puromycin required to kill		
non-transduced cells. A separate antibiotic kill curve protocol is available online.
 Determine optimal transduction conditions
		Using the shRNA controls, determine optimal cell density and conditions for transduction (See Appendix:
		Determine cell density and transduction conditions) Determine functional titer		
Using shRNA controls, empirically identify the correct volume needed for each batch of shMIMIC Lentiviral		microRNA particles based upon your cells of interest (See Appendix: Determine functional titer).
 Evaluate cellular response to microRNA expression
		Using optimized conditions, transduce shMIMIC Lentiviral microRNAs and monitor phenotypic results with 				
assay of choice. See section below: Protocol using shMIMIC Lentiviral microRNAs.

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Technical Manual

Materials required•

shMIMIC Lentiviral microRNA for your microRNA of interest•

Positive and negative controls for optimization with matched promoter/reporter options»»

SMARTvector Non-targeting Control shRNA»»

SMARTvector Positive Control shRNA (GAPD or PPIB)

Materials not supplied•

SMARTchoice Promoter Selection Plate (Dharmacon, Cat # SP-001000-01)•

Polybrene (American Bioanalytical, Cat #AB01643)•

96-well tissue culture plates•

Deep-well 96-well plate (Nunc, Cat #12-565-553)•

Multichannel pipette (8-channel)•

Cells of interest•

Dulbecco’s Modified Eagle Medium (DMEM) High Glucose without L-Glut or Sodium Pyruvate(HyClone, Cat #SH30081.FS)•Base Medium: antibiotic-free cell culture medium (without supplements or serum)•

Growth Medium: antibiotic-free cell culture medium (with serum and/or supplements) recommended formaintenance of the cells•

Transduction Medium: the base cell culture medium containing lentiviral particles (with transduction additives
and serum (if necessary)•

Selection Medium: Growth Medium supplemented with the appropriate concentration of puromycin•

Resazurin Cell Viability Reagent

Protocol using shMIMIC Lentiviral microRNA
shMIMIC Lentiviral microRNAs are available as individual constructs supplied as concentrated lentiviral particles
and glycerol stocks. A well-constructed experiment should include a construct expressing the microRNA of interest
plus negative and positive controls and untransduced controls, and be performed as biological triplicates. Prior totransduction, cell line-specific optimization must be determined for cell density and transduction medium composition.
Determine functional titer in cells of interest
Lentiviral transduction efficiency can vary depending on factors such as target cell type, duration of exposure to lentiviralparticles, composition of transduction medium and experimental conditions. The titer of shMIMIC Lentiviral microRNA
particles reported on the Certificate of Analysis (C of A) was measured in HEK293T cells, so the relative transduction
efficiency of your cell type will likely be different. Therefore, it is recommended that the correct volume of lentiviralparticles should be empirically identified for each batch of shMIMIC lentiviral particles in the cell type of interest, andtesting a range of functionally adjusted multiplicities of infection (MOI) is recommended to ensure sufficient geneknockdown of positive controls (See Appendix: Determine functional titer).
General protocol
Follow the protocol described below for transducing cells with shMIMIC Lentiviral microRNA and SMARTvector controls.
Figure 2 depicts a suggested 96-well plate layout for a well-designed gene silencing experiment using shMIMIC LentiviralmicroRNA particles expressing the microRNA(s) of interest, along with the appropriate controls.
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Technical Manual

20 10 5 2.5 20 10 5 2.5 20 10 5 2.5

MOl =microRNA 1
GAPD
microRNA 2
Non-targeting
Figure 2. Example of plate layout for
typical experiment using shMIMIC LentiviralmicroRNA particles. Suggested 96-well plate
format using three different shMIMIC constructs(microRNA 1, microRNA 2 and microRNA 3) atfour MOl (20, 10, 5 and 2.5 TU/cell). Included are
positive (GAPD) and negative (non-targeting)controls and non-transduced cells.

microRNA 3
Non-transduced
empty

A
B
C
D
E
F
G
H

Day 1:
Plate cells in biological triplicate in a 96-well culture plate at previously determined density (See Appendix: Determine
cell density and transduction conditions).
Day 2:
1.

Remove your microRNA and control lentiviral particles from -80° C freezer and thaw on ice.

2.

Prepare 10 mL of pre-warmed Transduction Medium (Polybrene and serum concentrations as determined previously(See Appendix: Determine cell density and transduction conditions).

At all steps of these procedures, it is important to pipette slowly into wells to avoid dislodging adherent cells.

3.

Transduce cells at previously determined optimal conditions and MOIs ranging from 2.5-20 in a 96-well plate . We
recommend using a functionally adjusted titer for optimal scientific accuracy (See Appendix: Determine cell density
and transduction conditions and Determine functional titer).
a. Prepare dilutions of lentiviral particles (in pre-warmed Transduction Medium) such that 50 μL contains theappropriate number of lentiviral particles per cell (TU/cell) to obtain desired MOI. Dilutions should be preparedusing sterile 1.5 mL microfuge tubes (or deep-well plate). Use the following protocol to make a two-fold serial
dilution for each of the shMIMIC Lentiviral microRNA particles and SMARTvector controls.
i. Multiply the calculated volume for highest MOI (units in μL) by 7. The volume of lentiviral particles is multipliedby 7 to allow 3 volumes to be used to transduce cells in triplicate, 3 volumes to be used to make the next twofold dilution in series, and 1 volume to represent liquid overage required to accommodate small carryovers
during pipetting.
ii. Transfer calculated volume of lentiviral particles into sterile 1.5 mL microfuge tube. Label tube withappropriate MOI (for example “MOI = 20”).
iii. Bring volume up to 400 μL with pre-warmed Transduction Medium and mix gently by pipetting.
iv. With new pipette tip, transfer 200 μL from the MOI = 20 tube into another sterile 1.5 mL microfuge tube
labeled MOI = 10.
v. Bring volume up to 400 μL with pre-warmed Transduction Medium and mix gently by pipetting.

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vi. Repeat steps iv and v for MOI = 5 and MOI = 2.5.
vii. If Polybrene is present in Transduction Medium, allow Transduction Medium with lentiviral particles to
incubate for 10-20 minutes at room temperature before transducing cells. If Polybrene is not used, proceed
directly to transduction of cells.
viii. Remove culture plate(s) from incubator and replace medium with 50 μL of Transduction Medium withlentiviral particles. In the “non-transduced” wells, add an equivalent volume of Transduction Medium.
ix. Return plates to incubator.
x. After predetermined transduction time of 6-20 hours (See Appendix: Determine cell density andtransduction conditions), add 100 μL of Growth Medium directly to each well without removing medium.
xi. Return culture plate to incubator. Incubate for 24 hours.
Day 4:
Check for TurboGFP or TurboRFP expression, or for no reporter determine downstream gene knockdown as well as test for
toxicity using Resazurin or a similar assay for viability.
If you have a reporter, TurboGFP or TurboRFP expression will become visible after 24 hours and will reach a maximum at 48 to 72 hours
post-transduction. Similarly, microRNA-induced gene knockdown, at the mRNA level, is often detectable at 24 hours and reaches amaximum typically by 72-96 hours.

Establishing a stable cell population
For cell lines with longer proliferative capacity, stable cell lines may be established by transduction of shMIMIC LentiviralmicroRNA particles and subsequent puromycin selection. These cell lines may be maintained for long-term studies. Caremust be taken that cells be used within a limited number of passages from completion of selection; this will depend on
the characteristics of the particular cell type.
Days 1-4:
Follow the “General protocol” above until Day 4.
Day 4:
Change to Selection Medium containing appropriate concentration of puromycin.
Day 5 or 6:
Trypsinize cells and expand into a larger culture vessel with medium containing puromycin in order to select for cells withstably integrated shMIMIC construct.
Day 8 or later:
1.

Expand the culture until an adequate cell number has been achieved to freeze and archive the cell line.

2.

Check for TurboGFP or TurboRFP expression, or for no reporter determine downstream gene knockdown as wellas test for toxicity using Resazurin or a similar assay for viability.

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3

Appendix
Additional protocols
Identify the optimal promoter
The SMARTchoice Promoter Selection Plate is designed to allow data-driven promoter selection prior to ordering
SMARTvector Lentiviral shRNAs or shMIMIC Lentiviral microRNAs for specific genes or microRNAs of interest. This uniquetool permits the simultaneous evaluation of seven different promoters in order to select one which is most effective inyour cell line or cell type of interest. The Promoter Selection Plate contains transduction-ready lentiviral particles
representing seven different promoters arrayed in a 96-well plate (Figure 3).

Serial Dilutions
TU (× 105) =Promoter

empty
hCMV
Figure 3. SMARTchoice Promoter Selection Plate enables
straightforward, qualitative assessment of promotersthat actively drive expression. Lentiviral particles arearrayed in two-fold dilutions with the highest amountof particles at 8.0 × 105 transducing units (TU). Layout by
rows: A. empty wells (DMEM only); B. hCMV – humancytomegalovirus immediate early promoter; C. mCMV
– mouse cytomegalovirus immediate early promoter; D.
hEF1α – human elongation factor 1 alpha promoter; E.
mEF1α – mouse elongation factor 1 alpha promoter; F.
CAG – chicken beta actin hybrid promoter; G. PGK – mouse
phosphoglycerate kinase promoter; H. UBC – humanubiquitin C promoter.

mCMV
hEF1α
mEF1α
CAG
PGK
UBC

8.0 4.0 2.0 1.0 0.5 0.25 8.0 4.0 2.0 1.0 0.5 0.25

A
B
C
D
E
F
G
H
1

2

3

4

5

6

7

8

9 10 11 12

The arrayed SMARTvector Lentiviral shRNA particles co-express the TurboGFP reporter, a puromycin resistance selectable
marker and Non-targeting shRNA Control (NTC) in the context of the SMARTvector microRNA-adapted scaffold. The
seven promoters represented on the SMARTchoice Promoter Selection Plate are as follows:
hCMV – human cytomegalovirus immediate early promoter
mCMV – mouse cytomegalovirus immediate early promoter
hEF1α – human elongation factor 1 alpha promoter
mEF1α – mouse elongation factor 1 alpha promoter
CAG – chicken beta actin hybrid promoter
PGK – mouse phosphoglycerate kinase promoter
UBC – human ubiquitin C promoter
Columns 1-6 on the plate are duplicated in columns 7-12, allowing optimization experiments to be performed as
replicates, or using two different transduction conditions or two different cell lines. Each well contains lentiviralparticles suspended in 25 μL of DMEM containing no serum, with the exception of Row A, which contains DMEM only
and is intended for use as the untransduced control. Columns 1 and 7 have the highest number of lentiviral particles
8shMIMIC Lentiviral shRNA
Technical Manual[corresponding to 8 × 105 transducing units (TU)* in 25 μL] and serial two-fold dilutions are made in columns 2-6 and 8-12,
respectively.
For example, transduction of 10,000 cells with 25 μL of lentiviral particles from well B1 would represent an
MOI = 80, and the stepwise dilutions in wells B2, B3, B4, B5 and B6 would represent MOI equal to 40, 20, 10,
5 and 2.5, respectively.
*For an accurate assessment of promoter activity, all lentiviral particles are normalized to equivalent functional titers in
HEK293T cells using a p24 ELISA assay. A description of the titering method can be found in the FAQ section along with an
explanation of how p24 titers are converted to a functional titer.
Transduction of cells of interest using lentiviral particles from the SMARTchoice Promoter Selection Plate shouldbe performed using the cell number, Polybrene concentration, serum level and duration as determined previously.
(See Appendix: Determine cell density and transduction conditions). The specific formulation for the Transduction
Medium will reflect the optimal Polybrene and serum concentrations previously determined for the cells beingtransduced. Protocols for transduction of both adherent and suspension cells are included.
The SMARTchoice Promoter Selection Plate is not a cell culture plate. Cells should not be added to, nor should transductions be carriedout in the SMARTchoice Promoter Selection Plate.

SMARTchoice Promoter Selection Plate protocol for adherent cells
Day 1:
Seed appropriate number of cells in an appropriate cell culture plate as previously determined in a total volume of
100 μL per well in Growth Medium. Incubate plate overnight under appropriate conditions.
Day 2:
1.

Prepare the SMARTchoice Promoter Selection Plate as follows:a. Remove the SMARTchoice Promoter Selection Plate from freezer and thaw lentiviral particles on ice. It may take >30 minutes to thaw lentiviral particles in all wells. After lentiviral particles have thawed, spray the sealed plate with
70% ethanol and carefully wipe excess with a paper towel or Kimwipes®.
b. Quick-spin lentiviral particles to bottom of the wells by centrifuging (rcf = 300) for 20-40 seconds in a table top
centrifuge. Remove plate from centrifuge. Wipe the Promoter Selection Plate and clean the inside of centrifuge
using 70% ethanol. Carefully dry plate with paper towel or Kimwipes.
c. Within a Class II/Type A2 biological safety cabinet, carefully remove foil seal from Promoter Selection Plate being
careful not to splash lentiviral particles out of wells. Using forceps to hold edge of foil seal, immerse seal in liquid
waste reservoir and dispose of in biohazardous waste bag.

2.

Add Transduction Medium [with Polybrene and serum at optimal concentrations determined previously;(See Appendix: Determine cell density and transduction conditions)] to lentiviral particles:
a. If Polybrene was determined intolerable to cells and will not be used in transductions, skip this step andproceed directly to Step 3. Add Polybrene and serum to Base Medium at a 2x concentration determined during
optimization; this will result in 1x Polybrene and serum concentrations to generate 1x Transduction Medium withlentiviral particles.
b. To each well of the SMARTchoice Promoter Selection Plate, pipette 30 μL of Base Medium with 2x Polybrene andserum (as described above) creating ~ 55 μL total volume per well of 1x Transduction Medium with lentiviralparticles. An 8-channel pipette is recommended for all transfers to and from Promoter Selection Plate. Use newtips for each transfer and deactivate any lentiviral particles in pipette tips by aspirating and dispensing bleach or
Lysol® into and out of tips prior to disposal.

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shMIMIC Lentiviral shRNA
Technical Manualc. Place a new foil seal over the Promoter Selection Plate and quick-spin plate by centrifuging (rcf = 300) for
20-40 seconds in a table top centrifuge.
d. Incubate plate at room temperature for 10-20 minutes.
3.

Perform transduction of cells with SMARTvector Lentiviral shRNA particles
a. Take culture plate from incubator and place in biological safety cabinet. Using an 8-channel pipette, carefully
aspirate Growth Medium from each column of culture plate being careful not to dislodge cells from bottom ofwell. Dispense Growth Medium into liquid waste reservoir. Change tips between columns.
b. Carefully remove foil seal from Promoter Selection Plate. Transduce cells by transferring 50 μL of the Transduction
Medium with lentiviral particles from each well of Promoter Selection Plate to corresponding wells of culture
plate. It is recommended that an 8-channel pipette be used to transfer Transduction Medium with lentiviralparticles one column at a time. Tips should be changed following each transfer. After all columns (dilutions)of Transduction Medium with lentiviral particles are transferred to corresponding wells, place culture plate inincubator with appropriate conditions for the cell line.
c. Allow transduction to proceed for 6 hours or overnight as determined previously in the optimization of generalconditions, (See Appendix: Determine cell density and transduction conditions). After the transduction period,
add 100 μL of Growth Medium directly to each well. Adjust serum concentration accordingly.

4.

Culture transduced cells under appropriate conditions for 48 to 96 hours.

Day 4-6:
Visually inspect cells daily using fluorescence microscopy, flow cytometry or another method suitable for the observationof TurboGFP expression. A qualitative assessment of relative promoter activity can be made by comparing TurboGFP
fluorescence intensity among wells. Figure 4 depicts the fluorescent images of human A549 and HEK293T cells
transduced with lentiviral particles from the SMARTchoice Promoter Selection Plate.

Serial Dilutions
TU (× 105) =Figure 4. Fluorescense microscopy images ofTurboGFP-positive A549 and HEK293T cells forpromoter evaluation. Cells were transduced withlentiviral particles from the SMARTchoice Promoter
Selection Plate. Fluorescence intensity demonstrates
that the mouse CMV (mCMV) promoter is the optimal
promoter in A549 cells, whereas the human CMV (hCMV)
promoter is the optimal promoter in HEK293T cells.

8.0

4.0

2.0 1.0 0.5 0.25 8.0 4.0 2.0 1.0

0.5 0.25

Promoter
empty
hCMV
mCMV
hEF1α
mEF1α
CAG
PGK
UBC
A549

HEK293T10

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Technical Manual

For human A549 and HEK293T cells, the mCMV and hCMV promoter, respectively, consistently ranks highest influorescence intensity and therefore would be the optimal promoter choice for expression in these cell lines. Generally,
there will be a clear indication as to which promoter is the most active in the cells of interest. The most active promoter
for a particular cell line should be consistent across all six lentiviral particle concentrations.
SMARTchoice Promoter Selection Plate protocol for suspension cells
Day 1:
1.

Using ideal cell concentration (See Appendix: Determine cell density and transduction conditions), transfer
suspension cells into sterile centrifuge tube. Pellet cells by centrifuging (rcf = 300) for 6 minutes in a table top
centrifuge. Following centrifugation, carefully aspirate supernatant. Gently resuspend cell pellet in 3.75 mLof Base Medium, then plate 25 μL cells in Base Medium suspension into each well of 96-well culture plate.
For example, if the optimal density is 10,000 cells per well, pellet 1.5 × 106 cells and resuspend in 3.75 mLof Base Medium; 25 μL would then correspond to 10,000 cells per well.

2.

Prepare SMARTchoice Promoter Selection Plate for transduction in the same manner as described above for adherent
cells.

Use Polybrene and serum concentration such that the final concentration upon adding 50 μL of Transduction Medium with lentiviralparticles to 25 μL of cells (see next step) will give the 1x concentration as determined during the optimization step for your cells ofinterest.

3.

Transduce cells by transferring 50 μL of Transduction Medium with lentiviral particles from each well of Promoter
Selection Plate to corresponding wells of culture plate.

As suspension cells settle over time to the bottom of the well, it is recommended that you gently rock or tap the plate a few times (at30-60 minute intervals) for the first few hours to mix the cells with the lentiviral particles. For more thorough mixing, you may gently
pipette up and down 5-10 times using a multichannel pipette. Be sure to change pipette tips from one column to the next.

4.

Allow transduction to proceed for 6 hours or overnight as determined in optimization experiment. After transduction
period, add 75 μL of Growth Medium directly to each well. Adjust serum concentration accordingly.

5.

Culture transduced cells under appropriate conditions for 48 to 96 hours.

Day 3-5:
Visually inspect cells daily using fluorescence microscopy as described above in Day 3-4 of protocol above for adherent
cells. The optimal vector for your cell type is the one producing the highest level of fluorescence intensity whencomparing the promoter options at the same number of lentiviral particles.
Determine cell density and transduction conditions
Determine cell density and transduction conditions for adherent cells
Successful transduction of cells depends on cell type, cell density, passage number, MOI during transduction, purityof the lentiviral preparation and the presence and/or absence of reagents that facilitate transduction.
Polybrene can be toxic to certain cells. Although Polybrene is not generally necessary to achieve the MOIs recommended for lentiviralshRNAs, it may aid transduction for some difficult-to-transduce cell types.

The following optimization protocol allows you to determine lentiviral transduction conditions that are compatible
with the cell type of interest in one experiment in a 96-well plate format as depicted in Figure 5. This recommended
protocol will test cell density, Polybrene concentration, the presence or absence of serum, and the duration oftransduction (6 hours or overnight).
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Figure 5. Example of a 96-well plate layout forthe optimization of transduction conditions.
This plate layout tests three cell densities [5,000(5K), 7,500 (7.5K) and 10,000 (10K) cells per well),eight concentrations of Polybrene (0-14 μg/mL)
and medium with or without serum.

Polybrene - (0-14 µg/mL)

Cell Density

5K

0
2

A
B

4
6
8

C
D
E

10
12
14

F
G
H

7.5K

10K

without serum

5K

7.5K

10K

with serum

Day 1:
Seed cells into two 96-well culture plates at the appropriate cell density following the template depicted in Figure 5.
Use a total volume of 100 μL of the Growth Medium per well and place 96-well culture plates into incubator for overnight
culture under the appropriate conditions (temperature and CO2 concentration).
Day 2:
1.

Visually inspect each well under a microscope. Note the confluency for each of the three different cell concentrations
seeded on Day 1.

If all three cell concentrations seeded resulted in > 90% confluency, then it is necessary to re-seed the cells using a lower range of celldensities. Generally, transductions with lentiviral particles should be performed when cells are 40% to 80% confluent, depending on thecharacteristics of the cell line providing space for cells to replicate in culture. Because cells can differ significantly with respect to size andmorphology, the number of cells seeded should reflect this difference. The number of cells shown in Figure 5 (5,000, 7,500, and 10,000)
is specified only to depict a range of cell concentrations that should be seeded across the 96-well plate. The actual number of the cells
seeded for the specific cells of interest will vary depending on the size and morphology and will depend on previous experience.

2.

Prepare two sets of Transduction Medium, one without serum and one with serum.
a. Prepare Transduction Medium without serum:
i. Prepare 10 mL of Transduction Medium containing no serum by pipetting a 1:1 mixture of DMEM to
Base Medium

Lentiviral particles are provided in DMEM. Thus, testing a recipe for suitable transduction medium must include DMEM. Although, if thepreferred Base Medium for your cell line contains DMEM it is not necessary to test as the cells will tolerate DMEM.

ii. Transfer 700 μL aliquots of this mixture into eight wells of a sterile deep-well 96-well plate. Use these wells
to prepare a series of Transduction Medium formulations containing eight different Polybrene		 concentrations ranging from 0-14 μg/mL, increasing concentration in 2 μg/mL increments.
b.

Prepare Transduction Medium with serum:
i. Prepare a similar series of wells with Transduction Medium consisting of a 1:1 mixture of DMEM to
Base Medium and containing serum at the concentration that is appropriate for the cells of interest.

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ii. Transfer 700 μL aliquots of this mixture into eight wells of a sterile deep-well 96-well plate. Use thesewells to prepare a series of Transduction Medium formulations containing eight different Polybrene 		concentrations ranging from 0-14 μg/mL, increasing concentration in 2 μg/mL increments.
3.

Perform Transduction:
a. Remove culture plates from incubator and place in biological safety cabinet.
b. Using a multichannel pipette, carefully aspirate Growth Medium from each well of 96-well culture plates being
careful not to dislodge cells from bottom of well and dispense into a liquid waste reservoir.
c. Using a multichannel pipette, transfer 50 μL of Transduction Medium (with or without serum) containing therange of Polybrene concentrations (0-14 μg/mL) to cells seeded in 96-well culture plate. Transfer Transduction
Medium in format depicted in Figure 5.
d. Return culture plates to incubator and maintain under the appropriate conditions.
e. After 6 hours, remove one of the two replicate plates from the incubator.
f. Using a multichannel pipette add 100 μL of Growth Medium directly to each well of the replicate plate and return
to incubator for overnight culturing.

To adjust for the absence of serum (transductions without serum only) make up the difference by including additional serum to the
Growth Medium. F or example, if your cells require 10% serum, then increase the serum concentration of the Growth Medium to 15%.
Adding 100 μL of this culture medium to the 50 μL of Transduction Medium in the well will result in a final serum concentration of 10%.

Day 3:
After overnight incubation with Transduction Medium (16-20 hours) for the second replicate, add 100 μL of Growth
Medium directly to each well of plate. Adjust serum concentration as described above to the wells without serum.
Day 4:
Examine all wells of 96-well plates using a microscope and record the confluency and any morphological or phenotypicalterations that may be present.
Day 5:
1.

Examine cultures again for cell morphology or presence of phenotypic changes and record observations
followed by assaying the cells for viability using any commercially available kit, such as Resazurin or a similar assay
for viability.

2.

Select the optimal cell density and conditions that result in optimal phenotypic changes for the cell line.

Determine cell density and transduction conditions for suspension cells
Non-adherent or suspension cells should be counted and plated at the time of transduction and not incubatedovernight.
Based on observations from a variety of cell lines and cell types, the lowest concentration of serum generally results in the highest
transduction efficiency. Choose the highest concentration of Polybrene with acceptable viability for the cells of interest. If, in subsequent
protocols, it is found that transduction efficiency is low, Transduction Medium may be supplemented with this concentration of
Polybrene to enhance efficiency. Furthermore, if there is no evidence of significant cellular toxicity with overnight incubation (16-20
hours), then this incubation time may be used for subsequent transductions.

13shMIMIC Lentiviral shRNA
Technical ManualDay 1:
1.

Determine the number of suspension cells per mL.

2.

Transfer enough cells to seed two 96-well culture plates into two sterile centrifuge tubes (include additional
20% volume of cells to ensure adequate volumes for transfer).

3.

Centrifuge tubes at low speed to pellet cells.

4.

Pour off supernatant containing old culture medium.

5.

Tap bottom of centrifuge tube gently to dislodge cell pellets.

6.

Resuspend cells in one centrifuge tube with Base Medium containing no serum and cells in the other centrifuge tubein Base Medium containing appropriate concentration of serum for cells of interest. Resuspend in enough volume sothat appropriate number of cells are seeded in a total volume of 25 μL per well.

Transduction Medium formulations (with and without serum; 0-14 μg/mL Polybrene concentration) should be prepared essentiallyas described above. However, because Transduction Medium will be added to cells resuspended in 25 μL of Base Medium (with andwithout serum), Polybrene and serum concentrations of Transduction Medium will need to be adjusted accordingly.

7.

Prepare transduction medium without serum:a. Prepare 10 mL of Transduction Medium containing no serum by pipetting a 1:1 mixture of DMEM to
Base Medium.

Lentiviral particles are provided in DMEM. Thus, testing a recipe for suitable transduction medium must include DMEM. Although, if
the preferred Base Medium for your cell line contains DMEM it is not necessary to test as the cells will tolerate DMEM.

b. Transfer 700 μL aliquots of this mixture into eight wells of a sterile deep-well 96-well plate.
c. Use these wells to prepare a series of Transduction Medium formulations containing eight different Polybrene
concentrations ranging from 0-14 μg/mL, increasing concentration in 2 μg/mL increments (Figure 5). Polybrene
concentration is increased to account for the additional 25 μL of Base Medium, which does not already contain
Polybrene. For example, well (B1) contains 3 μg/mL Polybrene; adding 50 μL of Transduction Medium with a
Polybrene concentration of 3 μg/mL to 25 μL of Base Medium containing no Polybrene will result in a final
concentration of 2 μg/mL.
d. Use Table 1 to prepare the eight Transduction Medium formulations. Two sets of eight wells of Transduction
Medium should be prepared as before (Figure 5), one set without serum and one with serum.
8.

Perform Transduction
a. Remove culture plates from incubator and place in biological safety cabinet.

Table 1. Transduction Medium formulations for preparing media with eight different Polybrene concentrations.

Polybrene concentration ofTransduction Medium

Final Polybrene concentration after
adding to suspension cells

Well 1none

0 µg/mL

Well 23 µg/mL

2 µg/mL

Well 36 µg/mL

4 µg/mL

Well 49 µg/mL

6 µg/mL

Well 512 µg/mL

8 µg/mL

Well 615 µg/mL

10 µg/mL

Well 718 µg/mL

12 µg/mL

Well 821 µg/mL

14 µg/mL
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b. Using a multichannel pipette, transfer 50 μL of Transduction Medium (with or without serum) containing therange of Polybrene concentrations (0-14 μg/mL) to cells seeded in 96-well culture plate. Transfer Transduction
Medium in format depicted in Figure 5.
c. Return culture plates to incubator and maintain under the appropriate conditions.
d. After 6 hours, remove one of the two replicate plates from the incubator and place in biological safety cabinet.
e. Using a multichannel pipette add 75 μL of Growth Medium directly to each well of the replicate plate and return
to incubator for overnight culturing.
To adjust for the absence of serum (transductions without serum only) make up the difference by including additional serum to the
Growth Medium. For example, if your cells require 10% serum, then increase the serum concentration of the Growth Medium to 20%.
Adding 75 μL of this culture medium to the 75 μL of Transduction Medium in the well will result in a final serum concentration of 10%.

Day 2:
After overnight incubation with Transduction Medium (16-20 hours), add 75 μL of Growth Medium directly to each well inplate 2. Adjust serum concentration as described above to wells containing no serum.
Day 3:
Using a microscope, examine all wells of 96-well plate and note any morphological or phenotypic alterations present.
Day 4:
1.

Examine cultures for cell morphology or presence of phenotypic changes. Cell viability should be determined using
any commercially available kit, such as Resazurin or a similar assay for viability.

2.

Select optimal density and conditions that result in optimal phenotypic changes for the cell line.

Based on observations from a variety of cell lines and cell types, the lowest concentration of serum generally results in thehighest transduction efficiency. Choose the highest concentration of Polybrene with acceptable viability for the cells of interest.
If, in subsequent protocols, it is found that transduction efficiency is low, Transduction Medium may be supplemented with thisconcentration of Polybrene to enhance efficiency. Furthermore, if there is no evidence of significant cellular toxicity with overnightincubation (16-20 hours), then this incubation time may be used for subsequent transductions.

Determine functional titer
The functional titer of SMARTvector Lentiviral shRNA constructs in HEK293T cells is reported on the Certificate of Analysis (C of A).
The relative transduction efficiency of your cell type will likely be lower than that of HEK293T cells. The protocol below can be used todetermine your cell-line specific functional titer.

Functional titer can be determined either by counting fluorescence-positive colonies using fluorescence microscopy,
FACS analysis of fluorescent shRNA constructs, or resazurin for non-fluorescent shRNA constructs. The following protocol
describes how to estimate functional titer by using non-silencing control lentiviral particles and determining titer byfluorescence microscopy.
1.

The day before transduction, seed a 96-well cell culture plate (Destination Plate) with your cells at the density
determined during transduction optimization. Grow cells overnight.

2.

Make dilution medium using serum and Polybrene conditions determined during transduction optimization. Make
dilutions of non-silencing control lentiviral particles in a round-bottom 96-well plate (Dilution Plate). As shownin Figure 5 and Table 2, use one row of the plate for each replicate of the dilution series of the lentiviral stock. We
recommend performing two replicates. The procedure for dilution of the lentiviral stock is described below andresults in a series of five-fold dilutions to reach a final dilution of 390,625-fold.

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shMIMIC Lentiviral shRNA
Technical Manuala. Add 40 µL of dilution medium to wells A1 and B1. Add 80 µL of dilution medium to each well A2-A8 and B2-B8.
b. Thaw non-silencing control lentiviral particles on ice and then add 10 µL each to wells A1 and B1. Mix contents ofeach well by pipetting up and down 10-15 times. Discard pipette tip.
c. Transfer 20 µL from wells A1 and B1 to the corresponding wells in column 2. Mix contents of each well by pipetting
up and down 10-15 times. Discard pipette tip.
d. Repeat transfer of 20 µL for columns 2 through 8, mixing 10-15 times for each dilution.
e. Allow lentiviral-Polybrene complexes to form for 10-20 minutes at room temperature (only if using Polybrene).
3.

Remove culture medium from the cells in the 96-well plate Destination Plate.

4.

Transfer 25 µL of each dilution of lentiviral particles from the Dilution Plate to the corresponding wells in the
Destination Plate, being careful to not create bubbles.

5.

Incubate the cells for 6-20 hours (as determined during transduction optimization).

6.

Add 75 μL of normal growth medium to cells.

7.

Culture cells for 48-72 hours (as determined during transduction optimization).

8.

Choose one well in the Destination Plate for counting fluoresence-expressing colonies of cells. This should be a wellin which individual colonies of cells can be visualized and counted. Count each multi-cell colony as one transduction
event, as the cells have been dividing over the culture period (Figure 6). Calculate the average
number of fluorescent-positive colonies from the same destination well of each replicate.

Make five-fold dilutions into
Dilution Plate according toprotocol and Table 2.
1 2 3 4 5 6 7 8

1 2 3 4 5 6 7 8

A
B
C
D
E
F
G
H

Remove medium from
Destination Plate.
Add 25 µL diluted lentiviralparticles from each well in the
Dilution Plate to cells in thecorresponding well of the
Destination Plate

A
B
C
D
E
F
G
H

Dilution Plate

Figure 5. Diagram for dilution
series of lentiviral particles(Dilution Plate) and addition
to cells (Destination Plate).

Destination Plate

Table 2. Example setup for lentiviral particle dilution series.

Dilution Plate
Dilution factor

Volume of diluted lentiviralparticles used in transduction(Destination Plate)

40 µL

5

25 µL

80 µL

25

25 µL

20 µL (from A2)

80 µL

125

25 µL

A4

20 µL (from A3)

80 µL

625

25 µL

A5

20 µL (from A4)

80 µL

3125

25 µL

A6

20 µL (from A5)

80 µL

15625

25 µL

A7

20 µL (from A6)

80 µL

78125

25 µL

A8

20 µL (from A7)

80 µL

390625

25 µL

Well

Lentiviral particle serial dilution volume

Volume ofdilution medium

A1

10 µL (*control)

A2

20 µL (from A1)

A3*Control (non-silencing shRNA control lentiviral particles). Repeat identical dilution series in wells B1 to B8.

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Functional titer in transducing units per mL (TU/mL) can be determined using thefollowing formula:
Number of TurboGFP-positive colonies × Dilution factor (Table 2) ÷ 0.025 mL (Volume of diluted
lentiviral particles used) = Functional titer of non-silencing control lentiviral particles stock inyour cell line (TU/mL)
Relative transduction efficiency of your cell type can be determined by using the following formula:
Functional titer of non-silencing control in your cell line (TU/mL) ÷ Titer of non-silencing control
lentiviral particles stock as calculated by Dharmacon in HEK293T (TU/mL) (reported on the
C of A) = Relative transduction efficiency of your cell line

Figure 6. Example of individual colonies in
HEK293T cells 72 hour post-transduction.
Four colonies are circled. Imaged at 40x
magnification.

Use the calculated relative transduction efficiency of your cell line to calculate the anticipatedfunctional titer for each lentivirus using the following formula:
Relative transduction efficiency of your cell line × Titer of the lentivirus as calculated by in
HEK293T cells (TU/mL) = Anticipated functional titer in your cell line (TU/mL)

Calculating Examples
If you counted 58 fluorescent-positive colonies in well A7 of the destination plate, the titer of the non-silencing control
lentiviral particles in your cell line would be calculated as follows:
58 (transduction positive colonies) × 78,125 (dilution factor) ÷ 0.025 mL (volume of diluted lentiviral particles
used) = 1.8 × 108 TU/mL functional titer of non-silencing control in your cell line
If the titer for the non-silencing control lentiviral particles on the product insert was listed as 9.0 × 108 TU/mL, the relativetransduction efficiency of your cell type would be determined as follows:
1.8 × 108 TU/mL (functional titer in your cell line) ÷ 9.0 × 108 TU/mL (titer as indicated on product insert)= 0.2 relative transduction efficiency
If the relative transduction efficiency of your cell line is 0.2 and the titer of a lentiviral stock, as indicated on the C of A, is
5.0 × 108 TU/mL, the anticipated functional titer of the pool in your cell line would be determined as follows: 0.2 (relative transduction
efficiency) × 5.0 × 108 TU/mL (titer as indicated on product insert) = 1.0 × 108 TU/mL anticipated functional titer in your cell line

Stability and storage
SMARTvector Lentiviral shRNA particles and shMIMIC Lentiviral microRNA particles are shipped on dry ice as 25 μL
aliquots and must be stored at -80 °C. Under these conditions, lentiviral particles are stable for at least 12 months.
Repeated freeze-thaw cycles should be avoided, as this is expected to negatively affect titer. Once thawed, unused
lentiviral particles should be kept on ice, aliquoted into smaller volumes (if necessary) and immediately returned
to -80 °C.

Quality assurance and control
SMARTvector Lentiviral shRNA particles and shMIMIC Lentiviral microRNA particles are subject to stringent quality control
at multiple steps during the manufacturing process, including:
1.

Sanger sequencing of each clone to ensure integrity of construct sequence;2.

lentiviral particle titering using flow cytometry or p24 ELISA, followed by conversion to a functional titer basedon a matched-vector reference control of known functional titer in HEK293T cells;

3.

for fluorescent reporter options: confirmation of fluorescent reporter expression (TurboGFP or TurboRFP) following
transduction in HEK293T cells;

4.

examination of each batch to ensure preparations are free from mold and bacterial contamination; and5.

generation of Certificate of Analysis (C of A) with specified lentiviral titers for each batch, included witheach shipment.

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shMIMIC Lentiviral shRNA
Technical ManualFrequently asked questions (FAQs)
Can I use the SMARTchoice Promoter Selection Plate to optimize all of the transduction conditions for mycells of interest?
No. The Promoter Selection Plate is not meant to be used to establish fundamental lentiviral transduction conditions,such as optimal cell density, in your cells of interest. The purpose of the Promoter Selection Plate is to visually assess
relative promoter activity. Basic experimental conditions (such as cell density, with or without serum, Polybrene
concentration) should be established prior to transduction of viral particles using the SMARTchoice Promoter Selection
Plate. The Promoter Selection Plate contains duplicate wells of serially diluted lentiviral particles representing eachpromoter so that two separate conditions can be tested simultaneously. After identification of the optimal promoter foryour cells of interest, additional transduction optimization should be performed to identify appropriate MOIs for optimal
performance.
I think I know the best promoter for my cells of interest. Do I still need to evaluate promoter activity with the
SMARTchoice Promoter Selection Plate?
Promoter activity varies in different cell lines and cell types. We have demonstrated that this may affect the expressionof the construct and the level of expression that can be achieved. If you have previous data and/or knowledge that a
particular promoter is active in your cells, then lentiviral microRNAs can be ordered without testing the SMARTchoice
Promoter Selection Plate. If you are not sure which promoter to choose, then testing your cells with the SMARTchoice
Promoter Selection Plate prior to ordering lentiviral microRNAs as lentiviral particles can potentially save you a great dealof time and money.
I have used the human CMV promoter previously for my mouse cell line with low performance. Will a different
promoter increase shRNA functionality?
Poor performance may be due to low transduction efficiency or suboptimal promoter activity. Optimization oftransduction conditions is essential to successful results as well as understanding promoter activity. Promoter activity
varies in different cell lines and cell types, and we highly recommend using the SMARTchoice Promoter Selection Plate
to determine the most active promoter in your cells of interest. For example, the mouse CMV promoter in NIH/3T3 cells
results in significantly increased promoter activity and greater microRNA expression compared to the same constructexpressed from the human CMV promoter in this cell line.
I am using human cell lines in my research; should I always choose a human promoter for best activity?
In some instances promoter activity correlates with the species from which it is derived. However, promoter activity does
not always follow a species-specific expression pattern. For example, we have observed mouse promoters to be the mostactive in some human cell lines, whereas both human and mouse promoters were most active in some rat cells. Choosing
the most effective promoter in a particular cell line is not always predictable, and therefore should be determined
empirically.
Can I order shMIMIC or SMARTvector constructs with a promoter other than the seven SMARTchoicepromoter options?
No. SMARTchoice promoter options include human and mouse CMV, human and mouse EF1α, CAG, PGK and UBC.
Can I order shMIMIC or SMARTvector constructs without a fluorescent reporter?
Yes. SMARTchoice reporter options include TurboGFP, TurboRFP and a no reporter option.
Can I use the same promoter to express the microRNA in multiple cell lines?
As a general rule, we recommend using the SMARTchoice Promoter Selection Plate to identify the promoter with thehighest activity in each cell line or cell type. Promoter strength is important to the success of a microRNA expression
experiment, which can vary widely across cell lines and among species. The SMARTchoice Promoter Selection Plate
is designed to identify the optimal promoter specific for each cell line or cell type.
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What is titer?
Titer refers to the number of viral particles or transducing units present per milliliter of solution (TU/mL). The titer value isused in conjunction with the desired multiplicity of infection (MOI) to determine the number of viral particles (or volume
of viral particle suspension) required for transduction experiments.
What titers are achieved for shMIMIC Lentiviral microRNA Particles?
Our shMIMIC Lentiviral microRNA particles are provided at either a functional titer of 1 × 108 (± 20%) transducing units(TU) per milliliter (mL) or an ultra-high titer format of 2 × 109 TU/mL (± 20%), as determined by p24 ELISA of the lentiviralparticles. The p24 titer is then converted to a functional titer by correlating with values of a reference control of known
functional titer (SMARTvector Lentiviral shRNA particles containing matched promoter) that is included in the p24 ELISA.
What does transducing unit (TU) mean?
Transducing units are the number of functional viral particles in a solution that are capable of transducing a cell andexpressing the transgene.
What is MOI?
Multiplicity of infection (MOI) is the ratio of lentiviral transducing units (functional lentiviral particles) to cells. An MOIof 10 indicates that there are 10 transducing units for every cell in the well. It is important to note that different cell types
may require different MOIs for successful transduction. For instance, HEK293T cells are highly permissive to lentiviraltransduction (MOI of 5-20) while neuronal cells such as SH-SY5Y may require higher MOIs ranging from 10-40.
How do I transduce cells?
Once optimization studies have been performed to identify preferred Polybrene concentrations, cell densities, medium
conditions (See Appendix: Determine cell density and transduction conditions) and MOIs (See Appendix: Identify the
optimal promoter and MOI), the process of transduction requires nothing more than incubating lentiviral particles withyour target cell population for 4 to 20 hours.
How do I determine the transduction efficiency?
The transduction efficiency can be measured by assessing the fraction of cells expressing TurboGFP or TurboRFP after
transduction. This can be achieved by simple microscopic observation using a fluorescence microscope or by FACS
analysis 48-72 hours after transduction. For constructs that do not express a fluorescent protein reporter, we recommend
titering the lentiviral particles produced using a functional lentiviral titration protocol such as limiting dilution withcell viability assay by crystal violet staining or genomic qPCR assay.
How many cells can I transduce with the amount of shMIMIC Lentiviral microRNA particles provided?
The number of cells that can be transduced with each shMIMIC Lentiviral microRNA construct will depend upon the MOI
used during the transduction procedure. Procedures and formulas for optimizing transduction for your particular cell
type are provided in the Appendix.
How do I know if my target cell type can be transduced with lentiviral particles?
Lentiviral particles are manufactured with a VSVg envelope protein that provides broad tropism. Therefore, most cell
types are transduced, albeit, with varying degrees of efficiency. However, the efficiency of is not only a factor of successfultransduction, but is also dependent upon the efficiency of transgene expression; this can be assessed by utilizing the
SMARTchoice Promoter Selection Plate with the cells of interest. This assay will allow the simultaneous evaluation of seven
SMARTchoice promoters.

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What quality control is performed on shMIMIC Lentiviral microRNA particles?
shMIMIC Lentiviral microRNA constructs undergo multiple levels of analysis throughout production to ensure the qualityof the final product. Restriction enzyme analysis and sequencing are performed on all lentiviral vector constructs toensure integrity of the lentiviral vector backbone and of the targeting sequence, respectively. Lentiviral packaging cells
are carefully monitored for bacterial and/or fungal contaminants. Following packaging, lentiviral particles are titeredby p24 ELISA. The p24 titers are adjusted to a functional titer using reference control lentiviral particles titered by flow
cytometry to count GFP-positive cells.
Are Dharmacon lentiviral particle products safe to use in the laboratory? What precautions should be taken
when handling SMARTvector and shMIMIC lentiviral particles?
Lentiviral delivery systems have been employed in many research laboratories around the world without incident.
Handling of lentiviral reagents requires extensive experience with cell culture techniques. It is vital that the protocols andthe safety guidelines described in the Appendix for appropriate handling and storage are fully understood and followed
precisely. For additional guidance on containment of lentiviral vectors, we strongly recommend the user refer to the
Recombinant DNA Advisory Committee (RAC) guidelines for research with lentiviral vectors.
What is a SIN vector?
A self-inactivating (SIN) vector is a retroviral vector that contains a non-functional or modified 3' long terminal repeat(LTR) sequence. This sequence is copied to the 5' end of the vector genome during integration, resulting in theinactivation of promoter activity of both LTRs.
Can SMARTvector Lentiviral shRNA particles be further propagated in the lab?
No. SMARTvector products are engineered for maximum biosafety and are therefore replication incompetent.
What is Polybrene?
Polybrene, also known as hexadimethrine bromide, is a small, positively charged molecule that binds to cell surfaces
and neutralizes surface charge. Polybrene has been shown to enhance transduction of mammalian cells by 2-10 fold.
Alternatives include DEAE-Dextran.
What MOI and cell density should I be using?
The MOI used in your experiments will depend upon the cell type employed, the applicability of reagents thatenhance transduction (such as Polybrene), the appropriate medium conditions and the density at which cells are plated(See Appendix: for recommended optimization protocols).
How do I select for stably transduced cells?
The SMARTvector construct contains the puromycin resistance gene (PuroR) that allows for production of stable cell lines.
It is recommended to optimize the concentration of puromycin for your cells of interest to result in 100% cell death ofuntransduced cells after 4 to 6 days.
What are the maximum excitation and emission wavelengths for TurboGFP & TurboRFP?
Fluorescent reporter

Excitation wavelength

Emission wavelength

TurboGFP

482 nm502 nmTurboRFP

553 nm574 nm20

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Technical Manual

4

Lentiviral particle product safety level information
This Lentiviral Particle Product Safety Level Information constitutes Product Documentation according to clause
1 of the Product Terms and Conditions. It is applicable to the following lentiviral particle products:•

Dharmacon™ SMARTvector™ Lentiviral shRNAs•

Dharmacon™ SMARTvector™ Inducible Lentiviral shRNAs•

Dharmacon™ shRNAs•

Dharmacon™ Promoter Selection Plate•

Dharmacon™ Positive and Negative RNAi Controls•

Dharmacon™ Inducible Lentiviral shRNAs•

Dharmacon™ shMIMIC microRNAs•

Dharmacon™ Pooled Lentiviral shRNA Screening Libraries•

Dharmacon™GIPZ™ shRNAs•

Dharmacon™ Precision™ LentiORFs•

Dharmacon™ Decode™ Pooled RNAi Screening Libraries

The Products are solely for internal research use (as set forth in the Product Terms and Conditions) in laboratories where
the containment measures stated below and in applicable laws and regulations are met. Products may not
be used for diagnostic, therapeutic or other commercial purposes and may not to be administered to humans for any
purpose or to animals for therapeutic purposes. The Products are replication-incompetent, self-inactivating (SIN) andnon-pathogenic (do not cause infectious human disease).
Any investigator who purchases Dharmacon lentiviral particle products is responsible for consulting with their institution’s healthand biosafety personnel for specific guidelines on the handling of lentiviral vector particles. Furthermore, each investigator is fully
responsible for obtaining the required permissions for research using and the acceptance of replication incompetent SIN lentiviral
vectors and replication-defective lentiviral particles into their local jurisdiction and institution.

For questions concerning the design or production of the products, please contact our technical support team.
Horizon Discovery
2650 Crescent Drive
Lafayette, CO 80026 USA
Tel: 1-800-235-9880; 303-604-9499
Fax: 1-800-292-6088; 303-604-9680
Email: ts.dharmacon@horizondiscovery.com
In the US:
For US guidance on containment for lentiviral vectors, please refer to:
1.

The Recombinant DNA Advisory Committee (RAC) guidelines for research with lentiviral vectors(http://oba.od.nih.gov/oba/rac/Guidance/LentiVirus_Containment/pdf/Lenti)

2.

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);

3.

The NIH Guidelines For Research Involving Recombinant DNA Molecules (NIH Guidelines)(http://oba.od.nih.gov/rdna/nih_guidelines_oba.html).

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In the EU:
For the EU directives, please consult the following:
1.

Council Directive 2009/41/EC of the European Parliament and of the Council of 6 May 2009 on the contained use ofgenetically modified micro-organisms. (revised version of Directive 90/219/EEC of the European Parliament and of
the Council of 23 April 1990 on the contained use of genetically modified micro-organisms, amended by Council
Directive 98/81/ EC of 26 October 1998); and2.

Council Directive 2001/18/EC of the European Parliament and of the Council of 12 March on the deliberate release
into the environment of genetically modified organisms and repealing Council Directive 90/220/EEC.

In Germany:
Required Containment Measures: The containment requirements as stated in the German Genetic Safety Ordinance(Gentechnik-Sicherheitsverordnung) of Safety Level 2* or higher have been assigned to the handling of the abovementioned lentiviral vector particles. Please note a higher Security Level might be required if the lentiviral vector particles
are used for genetic engineering operations with other products which require a higher Security Level.
*Safety Level 2: activities of low risk for human health and the environment by the state of scientific knowledge(Stand der Wissenschaft).
For the German regulations, please consult the following:1.

German Genetic Engineering Act (Gentechnikgesetz - GenTG); and2.

Genetic Engineering Safety Ordinance (Gentechnik-Sicherheitsverordnung - GenTSV).

5 Limited Use Licenses
The shRNA and gene expression Products, use and applications, are covered by pending and issued patents. Certain
Label licenses govern the use of the products, these can be found at Dharmacon Licensing Statements. It is each Buyer’s
responsibility to determine which intellectual property rights held by third parties may restrict the use of Products for aparticular application. Please review the Label Licenses governing all use of the shRNA and gene expression Products.

If you have any questions, contact

Lysol is a registered trademark of Reckitt Benckiser Group. Tet-On is a trademark of Clontech Laboratories, Inc. TurboGFP and TurboRFP are trademarksof Evrogen Inc. All trademarks are the property of Horizon Discovery Company unless otherwise specified. ©2018 Horizon Discovery Group
Company—All rights reserved. First published June 2015. UK Registered Head Office: Building 8100, Cambridge Research Park, Cambridge, CB25 9TL,
United Kingdom.

V51218t +44 (0) 1223 976 000 (UK) or +1 800 235 9880 (USA); +1 303 604 9499 (USA)f + 44 (0)1223 655 581
w horizondiscovery.com/contact-us or dharmacon.horizondiscovery.com/service-and-support
Horizon Discovery, 8100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom
22