Why choose shRNA
Using controls
Starting with Bacterial Glycerol Stocks
Starting with Purified Plasmid DNA
Starting with Lentiviral Transduction Particles
Ordering for the first time
RNAi Glossary
More help
Why Choose shRNA
Gene silencing and knockdown using RNA interference is becoming routine. The introduction
of small interfering RNAs (siRNAs) into cultured cells provides a fast and efficient means of
knocking down gene expression and has allowed siRNAs to quickly become a ubiquitous tool in
molecular biology. While siRNA has been shown to be effective for short-term gene inhibition
in certain transformed mammalian cell lines, there is a clear problem in its use in primary
cell cultures or for stable transcript knockdown.
Short hairpin RNA constructs (shRNA) have advantages over siRNA because the effects of
these constructs can lead to a more stable and long-term result. MISSION™ TRC shRNA lentiviral
constructs have additional benefits, they can be used to easily transduce typically difficult
cell lines, such as primary cells and non-dividing cells.
The MISSION TRC shRNA library can be used in a wide range of studies including single gene
analysis, pathway clarification or even genome-wide functional screens.
Application data
References
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Using Controls
When conducting experiments using MISSION TRC shRNA constructs, the proper controls should
be a key element of your experimental design to allow for accurate interpretation of knockdown results. All controls are available in both purified plasmid DNA and lentiviral particle format.
In addition, MISSION TurboGFP™ Control Transduction Particles (SHC003V) can serve to optimize
transduction efficiency when using a cell line for the first time.
Sigma’s recommended controls for any shRNA experiment are provided in this Control
Selection Table and are closely aligned with the controls suggested in the Nature Cell Biology editorial.1
| Recommended Control |
Objective |
Negative Control:
Untreated Cells |
Untreated cells will provide a reference point for comparing
all other samples. |
Negative Control:
Transduction or
Transfection with empty construct, containing no shRNA insert
(SHC001V or SHC001)
|
MISSION pLKO.1-puro Control Transduction Particles (Product No. SHC001V).
The empty viral particles, produced from pLKO.1-puro, are a useful negative control that will
not activate the RNAi pathway because they do not contain an shRNA insert. It will allow for
observation of cellular effects of the transduction process. Cells transduced with the empty
viral particles provide a useful reference point for comparing specific knockdown.
MISSION pLKO.1-puro Control Vector (Product No. SHC001).
The empty vector is a useful negative control that will not activate the RNAi pathway because
they do not contain an shRNA insert. It will allow for observation of cellular effects of the
transfection process and the delivery of the lentiviral vector. Cells transfected with the
empty vector provide a useful reference point for comparing specific knockdown.
|
Negative Control:
Transduction or
Transfection with non-targeting shRNA
(SHC002V or SHC002)
|
MISSION Non-Target shRNA Control Transduction Particles (Product No. SHC002V).
This non-targeting shRNA is a useful negative control that will activate RISC and the RNAi
pathway, but does not target any human or mouse genes. The short hairpin sequence contains 5
base pair mismatches to any known human or mouse gene. This allows for examination of the
effects of shRNA transduction on gene expression. Cells infected with the non-target shRNA
will also provide useful reference for interpretation of knockdown.
MISSION Non-Target shRNA Control Vector (Product No. SHC002).
The non-targeting shRNA vector is a useful negative control that will activate RISC and the
RNAi pathway, but does not target any human or mouse genes. The short hairpin sequence
contains 5 base pair mismatches to any known human or mouse gene. This allows for examination of the effects of shRNA transfection on gene expression. |
Positive Control:
Transduction or
Transfection with positive reporter vector or lentiviral particles
(SHC003V or SHC003)
|
MISSION TurboGFP Control Transduction Particles (Product No. SHC003V).
This is a useful positive control for measuring transduction efficiency and optimizing shRNA
delivery. The TurboGFP Control transduction particles are produced from the lentiviral
backbone vector, pLKO.1-puro, containing a gene encoding TurboGFP, driven by the CMV promoter.
Transfection of this control provides fast visual confirmation of successful
transduction.
MISSION TurboGFP Control Vector (Product No. SHC003).
This is a useful positive control for measuring transfection efficiency and optimizing shRNA
delivery. The TurboGFP Control vector consists of the lentiviral backbone vector,
pLKO.1-puro, containing a gene encoding TurboGFP, driven by the CMV promoter. Transfection of
this vector provides fast visual confirmation of successful transfection and
delivery. |
Reference:
- Whither RNAi? Nature Cell Biology, 5, 489-490 (2003).
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Starting with Bacterial Glycerol Stocks
MISSION TRC shRNA Bacterial Glycerol Stocks offer a renewable resource for shRNA
constructs. Target sets or individual clones may be propagated and prepped for plasmid DNA.
The vectors can be used directly for transient or stable transfection or used with packaging
plasmids in a packaging cell line to produce lentiviral transduction particles.
Vector map
MISSION SHRNA Bacterial Glycerol Stock Technical
Bulletin (135 Kb PDF)
Troubleshooting:
| Problem |
Cause |
Solution |
| No growth of bacterial culture on selection
plates |
Incorrect carbenicillin concentration |
Re-check the carbenicillin concentration or pour fresh plates
containing 100 µg/ml of carbenicillin. |
| Insufficient inoculum volume from frozen culture |
Remove a larger volume of culture from the frozen
glycerol. |
| Insufficient storage temperature of frozen culture |
Storage temperature must be -70°C or lower. Obtain new
stock. |
| Multiple freeze-thaw cycles |
Avoid freeze-thawing the culture more than 2 times. |
| Low plasmid yields |
Difficult construct |
Perform larger purifications (midi or maxi preps) on
constructs that produce low yields. |
| Failure to use a single colony for inoculation |
Use an isolated colony for inoculation of cultures for DNA
preps. |
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Starting with Purified Plasmid DNA
MISSION TRC shRNA purified plasmid DNA format takes the hassle out of preparing DNA from each construct. DNA may be used directly for transient or stable transfection or with packaging plasmids in a packaging cell line to produce lentiviral particles. 1 µg of DNA is provided for each construct in the target set.
Vector map
MISSION shRNA Plasmid DNA Technical Bulletin (550 Kb PDF)
MISSION TRC shRNA constructs are compatible with most commercially available transfection reagents. Some cell lines may be more sensitive/resistant to transfection than others. Transfection efficiency may be optimized using the MISSION TurboGFP Control Vector (SHC003).
We offer a wide variety of transfection reagents, the most broadly applicable being ESCORT V (121 Kb PDF).
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Starting with Lentiviral Transduction Particles
MISSION TRC shRNA target sets in lentiviral transduction particle format offer the ultimate in convenience. The transduction particles are ready to add directly to your cells. Virtually any mammalian cell line can be transduced, including primary and non-dividing cells.
High-Throughput Lentiviral Particle Production Results
MISSION Lentiviral Transduction Particles Technical Bulletin (67.7 Kb PDF)
Choosing Your Cell Line
The MISSION TRC lentiviral particles are pseudotyped with the VSV-G envelope protein. This
allows efficient transduction of lentiviral particles, containing the shRNA, into most
mammalian cell lines. It is recommended when working with a cell line for the first time to
optimize the amount of lentiviral particles needed for transduction using the MISSION TurboGFP
Control Transduction Particles (SHC003V). This essential positive
control expresses TurboGFP, a green fluorescent protein marker, which can be used to monitor
exerimental design and aids in interpretation of results.
Cell Type Table
The cell types listed below have been successfully infected by pLKO.1-puro based lentiviral
particles.
| Cells |
Cell Type |
| Cell Lines (human) |
| coll5 |
colorectal carcinoma |
| HEK293 |
embryonic kidney cells |
| HeLa |
cervical adenocarcinoma |
| A549 |
lung adenocarcinoma |
| H1299 |
lung carcinoma |
| HT29-D4 |
colon carcinoma |
| HepG2 |
hepatocellular carcinoma |
| MCF7 |
breast carcinoma |
| MCF10A |
breast carcinoma |
| HCT116 |
colon carcinoma |
| Panc-1 |
pancreatic epithelioid carcinoma |
| PC3 |
prostate carcinoma |
| DU145 |
prostate carcinoma |
| A431 |
epidermal carcinoma |
| THP1 |
monocytic cells |
| RAW264.7 |
macrophage cells |
| SH-SY5Y |
brain neuroblastoma |
| HCN-1A |
brain corticol neuron |
| SupT1 |
T cells |
| BJ-TERT |
diploid fibroblasts |
| Cell Lines (mouse) |
| NIH3T3 |
fibroblasts |
| Primary cells (human) |
| astrocytes |
normal |
| C3H10T1/2 |
mesenchymal |
| dendritic cells |
immature dendritic cells |
| T cells |
lymphocytes |
| epithelial cells |
prostate |
| fibroblasts |
primary mammary |
| Primary cells (other
species) |
| ESC |
mouse embryonic stem cells |
| fibroblasts |
mouse embryonic fibroblasts |
| MC3T3-E1 |
mouse bone marrow derived |
| molar mesenchymal |
mouse embryonic mesenchymal |
| cardiomyocytes |
rat neonatal cardiomyocytes |
Choosing Your Assay
It is essential to determine the assay you will perform to evaluate target gene expression and
knockdown. A variety of assays can be used to determine mRNA transcript level, protein level,
or phenotypic response. When determining your assay, keep in mind that knockdown of essential
genes may be lethal to your cells and impact the type of assay you perform.
mRNA Transcript Assays
Protein Assays
AQUA
Western Blotting
Phenotypic Assays
Cancer Markers
Cell Viability
Cell Proliferation
Cell Signaling
Control Selection Guide
When conducting experiments using MISSION TRC shRNA constructs, the proper controls should be
a key element of your experimental design to allow for accurate interpretation of knockdown
results.
In addition, MISSION TurboGFP Control Transduction Particles (SHC003V) can serve to optimize
transduction efficiency when using a cell line for the first time.
Sigma’s recommended controls for any shRNA experiment are provided in the Control
Selection Table and are closely aligned with the controls suggested in the Nature Cell
Biology editorial.1
Reference:
- Whither RNAi? Nature Cell Biology, 5, 489-490 (2003).
Multiplicity of Infection (MOI)
Multiplicity of Infection is the number of transducing lentiviral particles per cell. It is
highly recommended that for each new cell type to be transduced, a range of MOI be tested.
MOI can be adjusted by increasing/decreasing the number of cells per well or
increasing/decreasing the amount of supernatant added to the well. This will determine the
optimal amount of lentiviral supernatant needed for efficient transduction of each cell line
used. We recommend testing MOIs of .5, 1, 2, and 5.
To calculate:
(Total number of cells per well) x (Desired MOI) = Total transducing units needed
(TU)
(Total TU needed) / (TU/ml reported on C of A) = Total ml of lentiviral particles to add to
each well
Puromycin titration (when using selection)
Puromycin titration (kill curve) should be performed when working with a new cell type.
- Plate 1.6 x 104 cells into wells of a 96-well plate with 120 ml fresh
media.
- The next day add 500 – 10,000 ng/ml of puromycin to selected wells.
- Examine viability every 2 days.
- Culture for 10 – 14 days. Replace the media containing puromycin every 3 days.
- The minimum concentration of puromycin that causes complete cell death after 3-5 days
should be used for that cell type.
Transduction Efficiency In Cell Lines
Critical to the success of your shRNA lentiviral experiment is determining the transduction
efficiency of your cell line. This will allow you to add the optimal amount of lentiviral
particle supernatant that achieves efficient transduction while avoiding possible toxicity to
your cell line and saving reagents. This may be done by using the MISSION TurboGFP Control
Transduction Particles (SHC003V)
and a simple titration of lentiviral particle supernatant into your cells. The resulting
fluorescence can then be visualized under a fluorescent microscope.
Transduction protocol without selection
Lentiviral
Transduction Protocol (69.0 Kb)
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Ordering For The First Time
The MISSION TRC shRNA library is available for online ordering. You must first create your online profile and check the "request web ordering access" box on the form. Once your profile has been created and reviewed, you will be able to add target sets or individual clones to your order cart. We have created a helpful animated tutorial to assist you in the target search and order process.
More Help
FAQ center
Email RNAi@sial.com for more assistance.
Technical Service
Send this page to a colleague.
Contact Us
For questions about the library, pricing and quotes or other concerns, please e-mail us at: RNAi@sial.com.
MISSION is a registered trademark of Sigma-Aldrich Biotechnology LP and Sigma-Aldrich Co. Label License.
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