shRNA

Getting Started with MISSION® shRNA

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. siRNA has been shown to be effective for short-term gene inhibition in certain transformed mammalian cell lines, while shRNA offers an opportunity to potently and stably silence gene expression.

Long term, stable gene silencing distinguishes short hairpin RNA constructs (shRNA) from other RNAi tools. For genes with slow turnover, or potentially slow turnover, utilizing shRNA for long term silencing is not just a convenience, it is the only choice. For these proteins, siRNA may be diluted out before the cell can efficiently degrade the mRNA. Long term gene silencing can be achieved by standard clonal selection methodologies using the pLKO.1 plasmid followed by puromycin selection. The MISSION® bioproduction team can provide you with a far more effective and less biased solution than clonal selection, lentiviral delivery of shRNA MISSION TRC (what is TRC?) shRNA lentiviral constructs can easily transduce typically difficult cell lines, such as primary cells and non-dividing cells and readily integrate the shRNA into the genome of these cells for stable gene silencing.

Sigma-Aldrich guarantees that upon purchase of the defined clone set quantity on the shRNA detail page for your gene of interest (this varies, but is typically 5 clones), that at least one of those clones for a gene should yield greater than 70% knockdown.

View Application data from the Sigma® Research and Development Team as well as from independent investigators.

View References from people who have successfully utilized and published with the MISSION TRC library.

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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 the shRNA controls webpage and are closely aligned with the controls suggested in the Nature Cell Biology editorial.1



Reference:

1. Whither RNAi? Nature Cell Biology, 2003, 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. The vectors can be used directly for transient or stable transfection or used with packaging plasmids to produce lentiviral transduction particles.


Visit the  Vector map website

MISSION SHRNA Bacterial Glycerol Stock Technical Bulletin (583 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.

 

Visit the Vector map website

 

MISSION shRNA Plasmid DNA Technical Bulletin (561 Kb PDF)

MISSION TRC shRNA purified plasmid DNA 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 that cover many cell types.

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Starting with Lentiviral Transduction Particles


MISSION TRC shRNA lentiviral transduction particle format offers 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 shRNA Lentiviral Transduction Particles Technical Bulletin (110 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 experimental design and aids in interpretation of results. Transduction can be further enhanced with the ExpressMag® transduction system. This system utilizes a combination of nanoparticles and powerful rare earth magnets to enhance lentiviral transduction efficiencies.

Has your cell line been validated for lentiviral transduction?



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 and Proliferation
Cell Signaling


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.

  1. Plate 1.6 x 104 cells into wells of a 96-well plate with 120 µl fresh media.
  2. The next day add 500–10,000 ng/ml of puromycin to selected wells.
  3. Examine viability every 2 days.
  4. Culture for 10–14 days. Replace the media containing puromycin every 3 days.
  5. The minimum concentration of puromycin that causes complete cell death after 3–5 days should be used for that cell type.

We have a list of useful protocols shRNA protocols available for anyone at the MISSION protocols website.

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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 clones to your order cart.



More Help


 

FAQ center

Email MISSIONRNAi@sial.com for more assistance.

Technical Service


Contact Us


For questions about the library, pricing and quotes or other concerns, please e-mail us at: MISSIONRNAi@sial.com.



MISSION is a trademark of Sigma-Aldrich. Label License.

TurboGFP is a trademark of Evrogen.

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