Once siRNAs are obtained, the next step in a typical gene silencing experiment is to assess the silencing efficiency of the selected siRNAs. siRNAs exert their effects at the mRNA level. Therefore, the preferred assay for siRNA validation is one that monitors target mRNA levels. The simplest and most sensitive assay for siRNA validation relies on qRT-PCR to measure target transcript levels in gene specific siRNA-treated cells versus negative control siRNA-treated cells.

Methods
To test the efficacy and potency of the three different Silencer® siRNAs targeting survivin, selected in Step 1, we individually reverse transfected HeLa cells with various concentrations of the survivin siRNAs or Silencer Negative Control #1 siRNA. 48 hours after transfection, RNA was isolated using the MagMAX™ Total RNA Isolation Kit, which simplified sample prep in our chosen 96-well format. The RNA was then converted to cDNA and the appropriate TaqMan® Gene Expression Assay was used to monitor down regulation of survivin mRNA using an Applied Biosystems 7900HT Fast Real-time PCR System.

Results
Figure 3 demonstrates that all three of the siRNAs provided excellent silencing of survivin. Indeed survivin mRNA levels were reduced >85% at 1 nM siRNA. We chose two Silencer survivin siRNAs that provided different silencing efficiency (#2646 and #12194) for further study to help determine if the differences in knockdown level impacted the biological response observed.

Figure 3. Validation of siRNAs Targeting Survivin. Three Silencer® Pre-designed siRNAs targeting human survivin and nontargeting Silencer Negative Control #1 siRNA (Ambion, Cat #AM4611) were reverse transfected into HeLa cells in triplicate at 6 different concentrations using 0.3 µL siPORT™ NeoFX™ Transfection Agent (Ambion, Cat. #AM4510). 48 hr post transfection, RNA was isolated using the MagMAX™-96 Total RNA Isolation Kit (Ambion, Cat #AM1830). cDNA was synthesized using MMLV Reverse Transcriptase (Ambion, Cat #AM2043) and 2 µL cDNA was used to amplify survivin mRNA in real time RT-PCR reactions with Survivin Hs00977611_g1 TaqMan® Gene Expression Assay (Applied Biosystems). Percent gene expression remaining is expressed as the relative amount of survivin mRNA in cultures transfected with survivin siRNAs vs cells transfected with the nontargeting control siRNA. TaqMan Gene Expression Assays against 18S rRNA were used to normalize for differences in total RNA concentration.

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