Functional Genomics and RNAi

RNAi Question & Answer Archive

 

Experts in the field of RNAi have answered your questions. You will find your answers here.

I would like to know whether anyone has used shRNA on primary human monocytes and if so is there any advice?

I want to lyse plant cells at various stages and analyze the miRNA (siRNA) population. What vector can I clone the whole population into and how much starting material do I need to clone?

How can I trace with immunohistochemistry the incorporation of lentiviral vector-siRNA in the cells? Are there other methods that I can use to demonstrate the incorporation of the lentiviral vector?

Do you know from where antibodies against the puromycin resistance protein can be purchased?

Is the system GP2-293 and pVSV compatible with MISSION® RNAi Lentivirus?

How can we use the purified plasmid DNA format with the lentiviral packaging mix (Prod. No. SHP001) to produce lentiviral particles?

I have several of the Mission shRNA clones in the bacterial stocks. I was wondering what the competent cells used are and if you have any information on the stability of the shRNA plasmids in these cells?
  Can I resuspend dried siRNA duplex pellet in RNAse-free water instead of 1 x TE?

What is the best strategy to elucidate individual siRNA sequences from a heterogeneous population (pooled siRNA)?

Which transfection reagent is best suitable for siRNA transfection in to primary murine peritoneal macrophages?

For the plasmid DNA, is there a way to multiply the stock we've been given, using PCR or any other method? Also, are there any research papers or links to some literature that allow me to view the data on the genes that have been silenced?

What helper DNA are you using for packaging of the Lentivirus?

Are MISSION shRNA viral particles suitable for transduction of neurons? Does direct infusion of positive control viral solution into the mouse brain result in stable GFP expression? If so, can you provide any references?

I am wondering if you sell any RNAi for the plant GUS gene (GUSb)? Do you sell any GUS RNAi controls for plant cell work?

Q:
I would like to know whether anyone has used shRNA on primary human monocytes and if so is there any advice?

A:
Here is a reference describing how lentiviral shRNA has been used to stably transduce monocytic cells: Nat Biotechnol. 2004 Dec;22(12):1573-8. Epub 2004 Nov 28. Negative feedback inhibition of HIV-1 by TAT-inducible expression of siRNA. Unwalla HJ, Li MJ, Kim JD, Li HT, Ehsani A, Alluin J, Rossi JJ. . His main research focus is miRNA in plants.

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Q:
What is the best strategy to elucidate individual siRNA sequences from a heterogeneous population (pooled siRNA)?

A:
The best method would be to use a sequencing technology that gives high coverage and works well on short sequences. Another option is to subclone the population into a vector, followed by sequencing. If the constituents of the population are known, a chip-based detection method may also be used.

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Q:
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?

A:
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).

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Q:
Which transfection reagent is best suitable for siRNA transfection in to primary murine peritoneal macrophages?

A:
Non-adherent cells, such as macrophages, may only achieve highly efficient transfection by using electroporation; however, MISSION® shRNA lentiviral particles may be used to transduce primary cells, even non-adherent cells.

A protocol for transduction of suspension cultures may be found on the TRC Protocols Page

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Q:
You mention in a previous question that antibodies against the puromycin resistance protein can be used to detect virally infected cells. Do you know from where antibodies against the puromycin resistance protein can be purchased?

A:
Currently, there is no commercial supplier of anti-puromycin resistance protein antibody. Authors that reference using anti-puromycin resistance antibody to detect viral integration have actually produced the antibody in their labs.

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Q:
I am trying to decide between the three types of RNAi available through Sigma-Aldrich-- the bacterial stock, the plasmid DNA, and the lentiviral particle for a research project.

For the plasmid DNA, is there a way to multiply the stock we've been given, using PCR or any other method?

Also, are there any research papers or links to some literature that allow me to view the data on the genes that have been silenced?

A:
The MISSION® TRC shRNA library is composed of three formats to give you flexibility and convenience. The purified plasmid DNA format may be used directly for transient transfection assays or used with our lentiviral packaging mix (Prod. No. SHP001) to produce lentiviral particles. You may further propogate the DNA by transforming the plasmids into competent cells (we recommend GC5 cells) to create bacterial glycerol stocks.

The lentiviral particles offer the most convenience. They may be added directly to cells for stable transduction. You are correct; the cells that are transduced will continue to express the integrated shRNA. The cells may be expanded for many downstream assays.

Below are helpful links where you can find additional validation data. You may also download a copy of the Cell publication highlighting the use of the library in a high-content screen.
Application Data
RNAi Bibliographies

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Q:
I purchased several panels of MISSION® RNAi Lentiviral plasmids in bacterial stock. Using purified plasmids, GP2-293 cells, and pVSV plasmid, I tried to produce lentivirus, but in vain. The problem is that after infection, puromycin resistance clones have never arisen so far. Is that system (GP2-293 and pVSV) compatible with MISSION® RNAi Lentivirus?

A:
That cell line is NOT compatible and cannot be used to produce virus with the MISSION® shRNA library. You'll need to use packaging vectors derived from HIV. We recommend using our SHP001packaging mix for optimal high titer virus production.

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Q:
We are interested in your MISSION® shRNA, especially the lentivirus transduction. I am wondering what helper DNA are you using for packaging of the Lentivirus?

A:
Our packaging mix is an optimized formulation of two plasmids consisting of a packaging vector containing the minimal set of lentiviral genes required to generate the virion structural proteins and packaging functions, and an envelope plasmid containing the vesicular stomatitis virus (VSV) G-protein. The exact contents and nature of the formulation are proprietary.

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Q:
How can we use the purified plasmid DNA format with the lentiviral packaging mix (Prod. No. SHP001) to produce lentiviral particles?

A:
The MISSION® Lentiviral Packaging Mix can be used if you wish to produce more virus in your own lab since the viral particles we produce for purchase are replication-incompetent. The product number is SHP001. This product is an optimized formulation of two plasmids expressing the key HIV packaging genes and a heterologous viral envelope gene. The Lentiviral Packaging Mix is designed to be co-transfected along with a compatible lentiviral transfer vector into HEK293T cells in order to create high-titer pseudo-typed lentiviral particles used for downstream transduction applications. The procedure is available in the Product Information Sheet (286 Kb PDF) for product number SHP001. This does list the procedure.

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Q:
Are MISSION shRNA viral particles suitable for transduction of neurons? Does direct infusion of positive control viral solution into the mouse brain result in stable GFP expression? If so, can you provide any references?

A:
We haven't performed that exact experiment... but other with similar lentiviral particles and gene construction (i.e. CMV promoter driving transcription of GFP) have seen good results in mouse brain.

Transduction of normal brain cells table

LCMV GP and VSV G pseudotypes images

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Q:
I have several of the Mission shRNA clones in the bacterial stocks. I was wondering what the competent cells used are and if you have any information on the stability of the shRNA plasmids in these cells?

A:
I was wondering what the competent cells used are...
The E. coli strain is in DH5alphaT1R. Sigma sells the identical strain (i.e. identical genotype) called GC5™.

...and if you have any information on the stability of the shRNA plasmids in these cells?
The TRC has published several studies on plasmid stability of the library in that strain (see "Genome-scale loss-of-function screening with a lentiviral RNAi library" and "A Lentiviral RNAi Library for Human and Mouse Genes Applied to an Arrayed Viral High-Content Screen"). An early criteria when designing the plasmid vector was improved stability of the lentiviral sequences in E. coli over past retroviral /lentiviral libraries. That said, be aware that E. coli containing lentiviral plasmids do grow significantly slower when compared to bacterial strains containing standard cloning plasmids such as pUC18, pET vectors or pBR322. We routinely grow for 20–22 hours in TB mediums containing 100 µg/ml of carbenicillin (rather than ampicillin) as we see better stability using that antibiotic than we do with amp. Harvest and process pDNAs immediately post-growth (do not let them incubate at 4 °C for hours or even days, as that will result in lower quality pDNA). We also have some preliminary studies that suggests that the shRNA clones passaged in such strains such as STBL-3 tend to grow more consistently and produce higher plasmid yields then DH5aT1R. We have not performed a larger, more comprehensive study to confirm this observation to date however... therefore use this information at your own risk.

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Q:
I am wondering if you sell any RNAi for the plant GUS gene (GUSb)? Do you sell any GUS RNAi controls for plant cell work?

A:
While Sigma has no current pre-designed RNAi product against that particular gene target, it is possible for us to custom manufacture a synthetic siRNA for you. If you have a particular sequence in mind (i.e. sequence from a publication that was shown to elicit a good gene silencing response), or we could design a series of sequences for you based on our proprietary Rosetta design algorithm.........and then have those synthetic RNAi's made for you.
Learn more about siRNA

 

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