Experimental Design and Analysis — Experimental Design FAQs

What controls do you recommend?
Why doesn't the non-target control vector affect gene expression?
How many cells can I infect with the amount of lentivirus provided?
What is the method you recommend for screening the stable clones and is there a pair of primers that can be used for screening them by PCR?
After selection with puromycin, why are there no surviving cells?
How should a Puromycin titration (kill curve) be set up?
How much virus will I need?
How do I screen with pooled shRNA libraries/panels?
How do I screen arrayed shRNA libraries?
Where can antibodies against puromycin can be purchased?
What are the advantages of selection with antibiotics over fluorescent sorting using GFP?
What alternatives are available in the event of a polybrene-sensitive cell line?
Can I silence multiple gene targets simultaneously?
How many copies/cell can I expect?
When transducing, is it best to reduce the volume of media on my cells?
What duration of knockdown can I expect with shRNA?
How can I visualize cells and infect with shRNA?
How can I trace with immunohistochemistry the incorporation of lentiviral vector-siRNA in the cells? Are there any antibodies against part of the virus that I can use? Do you have any reporter genes like GFP or siRNA labeling like Cy3? Are there other methods that I can use to demonstrate the incorporation of the lentiviral vector?
Can I pool shRNAs to the same gene?

What controls do you recommend?
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 (Product No. SHC003V) can serve to optimize transduction efficiency when using a cell line for the first time.

Our recommended controls for shRNA experiments are provided in the Control Selection table.

The best negative control to use is either the MISSION^® non-target shRNA or empty vector controls (Product Nos. SHC002/SHC002V or SHC001/SHC001V, respectively).

Why doesn't the non-target control vector affect gene expression?
The non-target control vector (Product Nos. SHC002, SHC002V) has at least 5 bp mismatches within the shRNA to any known human or mouse genes, limiting its ability to target any human or mouse genes. Please refer to the product listing for hairpin sequence and vector map.

How many cells can I infect with the amount of lentivirus provided?
The amount of cells that can be infected depends upon the cell line being used. For HeLa, A549 and HEK293T cells we have used 4 µl (1 x 10⁶ TU/ml via p24 assay) for each well containing ~16,000 cells in a 96-well plate. Primary or other difficult cells may require more lentiviral supernatant, but even at a 10 µl level, it would give you enough for 20 x 96-well reactions. We suggest decreasing the number of cells plated to increase the Multiplicity of Infection (MOI) if necessary. Also, performing a limiting dilution titer on your cell line will give you the optimal amount of viral particles needed for each assay.

What is the method you recommend for screening the stable clones and is there a pair of primers that can be used for screening them by PCR?
The best approach is to screen knockdown by using primers designed against the gene of interest. Puromycin resistant clones may be screened via qRT-PCR using primers designed against PAC or the target gene of interest.

After selection with puromycin, why are there no surviving cells?
The cells you are working with may be sensitive to puromycin. The concentration of puromycin added to the cells may be too high. For each new cell type used, it is recommended that a puromycin titration be performed to determine the lowest concentration of puromycin needed to efficiently select transfected or transduced cells. Also, be sure to wait 48 hours post-transduction before applying antibiotics to the cells. Alternatively, a higher MOI or transduction enhancement methods, such as ExpressMag^® may be required for an effective transduction.

How should a Puromycin titration (kill curve) be set up?

1. Plate 1.6 x 10⁴ cells into wells of a 96-well plate with 120 ml 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.

How much virus will I need?
The amount of virus necessary to effectively transduce any given cell line must be determined empirically. We've attempted to make this easier on our customers by providing a table of suggested transduction conditions for a variety of cell lines/types. If your cell line isn't included on this list, we suggest titrating the virus to determine the most appropriate amount necessary for your cells. We recommend initial attempts to optimize virus at an MOI range of 0.5 - 20x. Beyond this range, the lentivirus may become toxic to some cell lines. That said, there have been some reports of researches using very high concentrations of virus of MOIs up to 100x. Often these extreme MOIs require higher titer virus, which we can accommodate upon request.

How do I screen with pooled shRNA libraries/panels?
When screening it is important to have redundant shRNAs showing the same phenotype, thus mitigating the potential complication of off-target effects. There are five primary steps required for utilizing the LentiPlex pooled library.

1. Optimization Of Puromycin Selection Conditionsm
2. Optimization Of Transduction Efficiency
3. Transduction And Selection Of Target Cells With The Mission^® LentiPlex Pooled shRNA Library
4. PCR Amplification
5. Identification Of Positive Hits by sequncing

Information on the TRC shRNA sequence(s) identified and the corresponding gene targets can be readily found on Your Favorite Gene (YFG).

How do I screen arrayed shRNA libraries?
When screening it's important to have redundant shRNAs showing the same phenotype, thus mitigating the potential complication of off-target effects. Other RNAi providers may offer smaller libraries of pooled shRNAs targeting single genes. However, our research has shown that pooling at the gene level poses three potential dangers: (1) Muted functional or phenotypic effect, (2) Generation of a larger number of false positive leads and (3) Inability to rule out possible off-target effects.

Where can antibodies against puromycin can be purchased?
If a reference indicates that anti-PAC has been used to detect viral integration, they will have had produced the antibody in their labs.

What are the advantages of selection with antibiotics over fluorescent sorting using GFP?
Selection of transduced cells can be accomplished using either puromycin or FACS selecting for tGFP expressers. That being said, either option is effective. However, for completely pure, transduced cell populations, selection with puromycin is suggested.

What alternatives are available in the event of a polybrene-sensitive cell line?
Yes, some cell lines may find polybrene toxic. Some labs have begun using fibronectin instead. Then again, many times, polybrene merely enhances knockdown, and may not be completely requisite. ExpressMag is an alternative to using polybrene and can greatly enhance transduction efficiency. If the cells are suspension cells, some version of Spinfection might also work well.

Can I silence multiple gene targets simultaneously?
There would be several approaches to try and target multiple genes at once, but keep in mind, all the constructs contain the same selection marker (puromycin). Because of this, there is not a clear way to determine (while still in culture) which cells got both virus types. You could transduce with a mixture of two shRNA targets, or you could do serial transductions, where you transduce with one shRNA, followed the next day by another. The only way to screen for positives would be qPCR or other analysis method (selection would not be sufficient).

How many copies/cell can I expect?
The answer isn't cut and dry. Some cell lines require a larger dosage of virus (multiplicity of infection). Many researchers aim to only insert a single copy per cell, and this must be determined empirically for each cell line. We have already validated a host of cells, see cell table.

When transducing, is it best to reduce the volume of media on my cells?
Many researchers prefer to reduce the volume of media on their cells when performing a transduction given their cell line is not adversely affected. A reduction in volume can boost the overall concentration of the lentivirus on the cells, thus potentially increasing transduction efficiency.

What duration of knockdown can I expect with shRNA?
Theoretically, production of the shRNA and knockdown should be a permanent condition. We see stable and permanent knockdown in cells that were transduced and cultured for over 1 year. These cultures were grown from a single resistance cell (clonal selection). We have seen populations of cells that have not undergone clonal selection shift over time, which is sometimes interpreted as a change in knockdown. What is actually happening is that within the heterogenous population of cells, there are varying effects from the knockdown. As you can imagine, some cells may outpace others in their growth, and as a result the percentage of knockdown seen varies over time as the population of the cell culture shifts.

How can I visualize cells and infect with shRNA?
We have a wonderful vector, which expresses both tGFP and a U6-driven shRNA that we can clone any TRC or customer-derived shRNA into. This customization, along with many other vectors, is available on our improved website.

How can I trace with immunohistochemistry the incorporation of lentiviral vector-siRNA in the cells? Are there any antibodies against part of the virus that I can use? Do you have any reporter genes like GFP or siRNA labeling like Cy3? Are there other methods that I can use to demonstrate the incorporation of the lentiviral vector?
There are many ways to verify the shRNA has been incorporated into your cells. Each of the MISSION^®-TRC constructs carries the puromycin resistance gene (PAC). Cells may be traced using anti-PAC antibody or selected using puromycin. Surviving cells express the shRNA. Cells may also be traced using qRT-PCR using probes against the PAC gene. We do offer a reporter control for fluorescence visualization of cells (MISSION^® TurboGFP Control Transduction Particles – SHC003V).

Can I pool shRNAs to the same gene?
Yes, you can pool shRNAs at the gene, family or even genome level. In fact, we offer a genome-wide pooled screen called LentiPlex that is ready for use. That said, lower level pooling (i.e. at the gene level) is not recommended. Single, poor performing clones can drastically change expression and phenotype, possibly leading to false negative.