shRNA

Experimental Design and Analysis — Analysis FAQs




Can I knockdown highly stable proteins using shRNAs?
Does the MISSION TRC Library include a bar code that I could use for microarray analysis?
Do you guarantee knockdown using your clones?
Are there any issues associated with using the CMV promoter?
What is the correlation between mRNA and protein knockdown?
What kind of off-target effects do you see from shRNA use?
How do you deconvolute the results of a pooled shRNA experiment?
Why doesn't the non-target control vector affect gene expression?


Can I knockdown highly stable proteins using shRNAs?
Since the library is lentiviral based, it is ideal for knocking down both transcripts and proteins that are very stable. Stable integration of the shRNA into the host chromosome, allows for long-term silencing, thus allowing for longer turnover and degradation half-lives.


Does the MISSION TRC Library include a bar code that I could use for microarray analysis?
Currently, our vectors do not contain an internal barcode nor do we pool the clones for a target set. You may use the individual shRNA sequence to screen resulting clonal populations for stable integration.


Do you guarantee knockdown using your clones?
Yes. Based upon the design algorithm developed by The Broad Institute, if you purchase the defined clone set quantity on the shRNA detail page for your gene of interest (this varies, but is typically 5 clones), we guarantee that at least one of those clones for a gene should yield greater than 70% knockdown of the targeted transcript. Validation of the library is underway. Generally more than one clone in each gene set gives significant knockdown. Proper controls and conditions are required.


Gene Silencing using MISSION shRNA

Figure 1. Silencing of 40 different gene targets. MISSION shRNA lentiviral particles were used to transduce several human cell lines. Percent gene expression was determined by measuring mRNA transcript levels and comparing to the MISSION pLKO.1-puro Control Transduction Particles (SHC001V). Knockdown of all individual lentiviral constructs was greater than 70%.

Are there any issues associated with using the CMV promoter?
CMV-Driven gene products can, at times, not operate well in certain cell lines (such as primary and some murine cells). That being said, we are currently working on alternate promoters for these applications.

What is the correlation between mRNA and protein knockdown?
Generally, mRNA and protein roughly correlate with one another in a functional genomics experiment. That said, many times the efficacy/potency of silencing can differ between the mRNA and protein levels. Also, the half-life of the protein is generally much longer than that of its corresponding transcript. Protein knockdown is very dependant on the protein being studied.

What kind of off-target effects do you see from shRNA use?
There is a concern that inducing RNAi using shRNA can give unexpected non-specific effects the at mRNA and protein levels, such as off-target mRNA degradation and protein regulation, and induction of the interferon response. However, by optimizing transduction efficiency using lentiviruses and applying proper controls for the experiment, this will allow you to monitor the presence of non-specific effects.

How do you deconvolute the results of a pooled shRNA experiment?
Deconvolution after a pooled shRNA experiment can often be the most challenging aspect of an experiment. We currently suggest one of the two following methods to deconvolute which shRNAs are having an effect in your screen. (1) Deep-Sequencing or Next Generation sequencing methods to identify which shRNAs are affecting your cell population, or (2) After selection with puromycin, pick colonies, expand clones and sequence verify the integrated shRNA. We are aggressively searching out new means for our customers to deconvolute.

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.



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


MISSION is a registered trademark of Sigma-Aldrich Co. LLC Label License.

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