COMPREHENSIVE GENE SILENCING LABORATORY SERVICES
RNAi Services at Altogen Labs has experience in RNAi applications both for research and industrial environments. We provide all-encompassing gene silencing services and related analytical services in a GLP facility, including:
- production of siRNA to induce specific effects
- construction of shRNA-inducing vectors
- modification of DNA and RNA for desired effects, including the following modifications for durability, improved binding, and stability
- Locked Nucleic Acids (LNA)
- Peptide Nucleic Acid (PNA)
- 2′-O-methyl bases
- determination of stability of RNAi over long-term, both in vitro and in vivo
- quantification of RNAi effects with qRT-PCR
- cell-based assays to determine functionality
- determination of protein expression via Western blot
- incorporation of dyes for research/imaging purposes
- formulation assistance, including production of multiple liposomal formulations, to improve delivery
- inducible (Tet-on / Tet-off) RNAi effects
- production of libraries to screening genes for loss-of-function and identify genes associated with particular phenotypes
Transient transfection is a process that delivers RNAi into a cell resulting in the reduction of target gene expression for a finite period of time. The RNAi is ultimately lost from the cell and target gene expression returns to normal. Transient transfection of RNAi results in efficient gene knockdown for 36-72 hours.
There are two strategies for RNAi transient transfection:
- liposome encapsulationof in vitro manufactured siRNA
- Virus or plasmidbased expression of short haripin RNAs (shRNAs)
Transient RNAi transfection services includes: Design, synthesis, HPLC purification, and annealing of 5 different siRNA molecules targeting gene of interest. siRNAs are tested for gene silencing potency. Two out of five siRNAs are finally selected (siRNAs are guaranteed to provide at least 80% mRNA expression knockdown). Transfection conditions are developed for a cell line(s) of choice. Validation of gene silencing (RNAi) effect by real-time qRT-PCR (mRNA expression level) or Western Blot (protein expression level).
Virus and plasmid expression services include: synthesis and cloning of shRNA construct targeting gene of interest into plasmid DNA, Stable cell line generation: transfection of plasmid DNA into a cell line of choice, drug selection and generation of stably-expressing clones, validation of shRNA construct expression and RNAi gene silencing by real-time qRT-PCR (mRNA expression) or Western Blot (protein expression).
Long term gene knock-down experiments are accomplished using cell lines engineered to stably express shRNAs using viral or plasmid expression vectors. First, gene-expression reduction by the shRNA expression vector is validated by transient expression. Transfected cells are subjected to selection for an antibiotic resistance marker to generate stable shRNA expressing cells. Selection for antibiotic resistance requires 10-14 days and 2-3 weeks of expansion and screening. Therefore, generation of cell lines with stable expression will take up to 5 weeks.
A potential pitfall of stable RNAi expression is failure to generate clones that have significant reduction of target gene expression. This is possible even after validation by transient transfection. The most likely reason for this is that long-term reduction of expression of the specific gene has a deleterious effect on the cell. This results in selection against expression of the shRNA. In this case inducible shRNA is advisable using the tet-inducible system.
Several analyses will be performed with cells expressing RNAi. Analysis of gene expression at both the RNA and protein level should be done. Effects on mRNA levels will be done suing real-time qRT-PCR and protein expression levels will be done by Western blot or ELISA. It is possible for the mRNA levels to be significantly reduced with no reduction of protein levels. This can be observed, especially in transient expression assays, with proteins that have long half-lives. If this is observed then stable shRNA expression should be used.
Altogen Labs has a variety of services to study the biological effects once your gene knock-down system has been established and verified. Request formal project quotation from Altogen Labs CRO.
- Altogen Labs pharmacology and toxicologytesting services can be used with both transient and stable RNAi expression systems.
- Altogen Labs will develop cell based assaysto help you understand the biological effect of your system.
- Altogen Labs xenograft services will help you understand the biological effect of specific gene knock-down in a biologically relevant microenvironment. Xenograft studies especially benefit cancer researchers that want to determine whether a gene of interest is involved in tumor growth and to determine whether a specific gene is a target of a newly developed therapeutic drug.