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RNA INTERFERENCE 7
psiTEST System - Prevalidation of siRNAs and shRNAs
The psiTEST system enables you to screen for effective siRNAs and shRNAs without using time-consuming and costly reagents that are specific
to your target gene, such as antibodies. The psiTEST system can be used to evaluate RNAi on virtually any gene of any species for which the
sequence is known. The silencing efficiency of a given siRNA or shRNA is evaluated by monitoring the reduction of expression of a chimeric
gene consisting of a secreted alkaline phosphatase (SEAP) reporter gene transcriptionally fused to the target gene. Transfection of effective
siRNAs or shRNAs triggers the RNAi process resulting in the degradation of the chimeric mRNA and therefore the absence of SEAP activity.
Description
The psiTEST system is based on a transcriptional fusion between a
reporter gene and your target gene. The reporter gene is an optimized
alkaline phosphatase gene engineered to be secreted (SEAP). The efficacy
of an siRNA or shRNA to induce RNAi on your target gene is
determined by measuring the expression levels of the SEAP reporter
gene (see figure). Since SEAP is a secreted protein, the expression level
can be evaluated from supernatants therefore allowing to perform kinetics
of the silencing process.
Features and Benefits
The psiTEST system contains two major components, the psiTEST plasmid
and QUANTI-Blue .
psiTEST plasmid
The psiTEST plasmid features the SEAP gene fused at the 3’ end after its
Stop codon to a multiple cloning site (MCS). This MCS contains many
common restriction sites compatible with many other restriction sites.
psiTEST also features a GFP::zeo fusion gene: the zeo moiety allows to
amplify the plasmid in E. coli and select stable mammalian clones while
the GFP moiety enables the monitoring of transfection efficiency.
psiTEST can be used to clone fragments of genes or entire genes, the
optimum size being 1.5 kb, although genes up to 3.5 kb can be targeted.
QUANTI-Blue
QUANTI-Blue is a SEAP detection medium that turns purple/blue in
the presence of phosphatase activity. Functional siRNAs or shRNAs will
induce the degradation of the SEAP mRNA resulting in the reduction or
absence of SEAP activity.
Silencing efficiencies of the siRNAs or shRNAs can be observed visually
and unlike fluorescent reporters can also be easily quantified using a
microplate reader or spectrophotometer.
Contents and Storage
The psiTEST system is provided with several control plasmids: psiTESThp53,
psiRNA-hp53 and psiRNA-Luc. psiTEST-hp53 contains a fusion
between the SEAP and human p53 genes. psiRNA-hp53 produces an
shRNA that functionally silences the human p53 resulting in the absence
of phosphatase activity. psiRNA-Luc expresses an shRNA insert that
targets luciferase GL3 and therefore will not affect the expression of the
phosphatase reporter gene.
The psiTEST system contains the following components:
- 20 μg of psiTEST and 20 μg of psiTEST-hp53 (control plasmid)
- 20 μg of psiRNA-hp53 and 20 μg of psiRNA-Luc
- 2 pouches of QUANTI-Blue (100 ml each)
- 4 pouches of Fast-Media ® Zeo (2 TB, 2 Agar)
Products are shipped at room temperature. Store Fast-Media ® at room
temperature. Store all other components at -20°C.
Procedure
146 www.invivogen.com/psitest-system
1- Clone your target gene (or a fragment) into the psiTEST
plasmid.
2- Cotransfect mammalian cells with recombinant psiTEST
and siRNA or shRNA-producing plasmid, such as psiRNA .
3- Incubate at 37°C with 5% CO2 for 72 hours.
4- Collect supernatant and add to a QUANTI-Blue -
containing well.
5- Incubate at 37°C for 30 min to 3 hours.
6- Determine the functionality of your siRNA or shRNA by
observing the coloration of QUANTI-Blue visually and/or
with a spectrophotometer at 620-655 nm.
PRODUCT QTY CAT. CODE
psiTEST 1 kit ksitest
QUANTI-Blue 5 pouches rep-qb1