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SERVICES

Adenovirus and AAV Service Customized Gene Knockdown Cells Customized Cells for Gene Overexpression shRNA Validation Service pVal shRNA Validation Platform Cell Line Development

PRODUCTS

AdenoONE TransMAX Premade Adenovirus for gene knockdown and overexpression

Industrial Co-Development

Project Opportunities Immortalization of Primary Cells - Co-Development

pVal shRNA Validation Platform - RNAiONE

Fields of Application

Identification of optimal shRNA sequences

for knockdown cell systems
for target validation
screening applications

Principle

Cloning of the cDNA of Your Gene of Interest (GOI) into the pVal vector (=pVal-GOI)
Cloning of 5, 10, 15 or more different shRNAs into pVal-GOI (=pVal-GOI-shRNA 1-x)
Transfection of 293 or NIH-3T3 cells with the resulting validation vectors
Quantification of target gene knockdown by qRT-PCR relative to control cells

Available pVal platforms

Vector System	Promoter	Transgene	Application	Standard moduls
pVal-U6	U6	shRNA	constitutive KD	5, 10 and 15 shRNAs
pVal-EF1a	EF1a	shRNA-mir	Inducible KD	5, 10 and 15 shRNAs

Prices available on request

Application Example

Screening of 10 shRNA designs against the human TKTL-1 gene. A partial cDNA of TKTL-1 was cloned and 10 shRNA designs tested. The vector was transfected into HEK293 cells and the level of TKTL-1 mRNA quantified by qPCR (Table 1).

Prevalidation Results

Among the 10 sequences checked, only shRNA 2 achieved a knockdown superior to 90%, and 2 more shRNAs knocked down the gene to a satisfactory degree of 80% (shRNAs 6+7).

Conformation of activity of prevalidated shRNAs in stable knockdown cell pools

3 knockdown cell pools in THP-1 cells were generated using the best 2 shRNA sequences (shRNA 2 and 7) and a NT shRNA sequence (control). The knockdown efficiency in the cell pools was determined on the mRNA level and with Western Blot.

Relative quantification of target gene expression in stable cell pools by qRT-PCR and Western Blot. Both cell pools display a high degree of knockdown superior to 90% both on mRNA and protein level. Expression of the NT-shRNA (Control) had no effect on gene expression.