If a custom shRNA is required to knockdown your gene of interest, Sirion can use the RNAiONE platform to find the most efficient shRNA available. Sirion identifies the 10 most promising shRNA sequences in silico using their proprietary in-house algorithms and validates the knockdown efficiency for each via qRT-PCR in an optimized cell-based system.
Screening of 10 shRNA sequences (shRNA 1-10) for the silencing of Hsp90b.
The process consists of three potential phases. Initially, a proprietary in-house bioinformatics algorithm is used to generate a set of promising shRNA sequences which in step 2 are cloned into a plasmid vector that also expresses the target gene to be silenced. The knockdown efficiency of each shRNA is then validated in cells transfected with the plasmids by measuring reduction in vector-driven target gene expression on the mRNA level. To rule out possible side-effects of endogenous target gene mRNA expression on the validation results, different cellular backgrounds are used (e.g murine NIH-3T3 cells for human targets and human HEK293 for non-human targets). Customers can then receive a report containing the performance of the optimal sequence, or if they prefer, Sirion can provide the shRNA plasmid in Lentiviral particles to customers or can proceed to delivery of a cell line stably expressing their validated shRNA.
|shRNA design using
||shRNA validation with RNAiONE technology (mRNA level)
||Cloning of best perfomer shRNA into SIRION’s inducible lentiviral vector platform followed by cell pool generation
GPCR expression in NIH-3T3 cells transfected with plasmid shRNA sequences (1-15) or non-target shRNA.
This approach has proven to be highly effective. In over 80% of projects, Sirion’s proprietary RNAi platform has identified shRNA sequences with knockdown efficiencies above 90%. The example below illustrates the results of 30 separate RNAiONE projects, and is highly indicative of Sirion’s rate of success.
Sirion’s success rate with RNAiONE.
The results can be delivered as a nucleotide sequence in a report, as transduction-ready lentivirus or as knockdown cell pools.
RNAiONE consists of:
- A feasibility test to determine if the target is adaptable to the RNAiONE platform*
- Cloning of target cDNA and 10 shRNA cassettes in the RNAiONE validation vector
- Determination of knockdown efficiency for each shRNA via qRT-PCR in a cell-based system
*If the project is not viable, Sirion will contact you and you will not be charged for your order.
Lentivirus generation consists of:
- Cloning of a shRNA into a lentiviral expression vector
- Verification of cloning success by sequencing
- Lentivirus generation from 1 x106 HEK-293T cells
- Lentivirus titration by qRT-PCR
Cell line generation+ consists of:
- Transduction of cells with lentiviral particles
- Generation of stable cell pools using antibiotic selection
- Verification of transgene expression by qRT-PCR relative to control cells
- Qualitiy control: viability, sterility and mycoplasma testing, exclusion of replication competent lentiviruses via qRT-PCR based assay
+Sirion can treat close to any cell type. Standard cells can be taken directly into cell pool generation, whereas all other cell types require a test transduction to define and adapt the transduction/ selection protocol.
Standard cells are: