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Validation of siRNA Induced Silencing

TaqMan® Protein Assays can be used as an effective method for monitoring protein knockdown in cell lines with siRNA-mediated inhibition of OCT3/4, SOX2, and LIN28, using Ambion® Silencer® Select siRNAs. It is faster and uses significantly less material than western blotting. In addition, analysis of the effect on other proteins can be performed on the same sample, which is of high biological potential. Moreover, correlation analysis with mRNA levels (performed by TaqMan® real-time RT-PCR assay) may be easily included.

The results for analysis of testicular germ cell tumours (TGCT) and derived cell lines with known expression of OCT3/4 and SOX2 showed that these pluripotency markers are indeed an informative tool for identification of malignant stem cells (seminoma and embryonal carcinoma cells, and their common precursor, carcinoma in situ cells). The findings corroborated observations made using IHC (Figure 1), while enabling quantitation, speed, and the ability to be performed with much smaller sample inputs.

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Figure 1. TaqMan® Protein Assays Corroborate IHC Staining Results.

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