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T. +49 89 700 961 99 13
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pVal shRNA Validation Platform - RNAiONE - Your success guraranty

Fields of Application 

 Identification of optimal shRNA sequences 

  • for knockdown cell systems 
  • for target validation 
  • screening applications 

Project Statistics & Customer Satisfaction Guaranty

Over the years SIRION BIOTECH has developed its base technology into an art: in 80% of recent projects SIRION BIOTECH managed to identify shRNAs with knockdown results of above 80%. Such experience makes it possible to issue warranties with its quotes. 

 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.