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. 2005 Mar 30;33(6):e54. doi: 10.1093/nar/gni052

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© The Author 2005. Published by Oxford University Press. All rights reserved

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RNAi-mediated knockdown of essential NMD factors results in the expected increase of NMD reporter mRNA. (A) HeLa cell lines stably expressing the HA-TCRβ-GFP reporter construct (PTC+) or the control construct lacking the JC intron (ΔJCin) were transfected with pSUPERpuro plasmids expressing shRNAs against hUpf1, or with the empty pSUPERpuro (mock) as a control. After elimination of the non-transfected cells by treatment with puromycin, RNA was isolated 4 days post-transfection and relative NMD reporter mRNA was measured by real-time RT–PCR and normalized to relative GAPDH mRNA levels. Average values of three PCR runs from a typical experiment are shown. (B) The hUpf1 knockdown in the cells used in (A) was monitored by a western blot using hUpf1-specific polyclonal antisera. Cell extract of untransfected HeLa cells was used as a reference in the first lane. Detection of Sm proteins B/B′ with the monoclonal antibody Y12 served as loading control. (C) Average reporter mRNA levels determined from three independent knockdowns of hUpf1 or hSmg6 in the cell line expressing the NMD reporter construct (PTC+). The relative reporter mRNA levels were measured by real-time RT–PCR and normalized to relative GAPDH mRNA levels. Relative hUpf1 and hSmg6 mRNA levels were also determined to monitor the efficiency of the knockdowns. hUpf1 mRNA was ∼10-fold reduced compared with the mock, and hSmg6 mRNA ∼3-fold (data of one typical experiment is shown).