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. 2010 Oct 26;4(10):e848. doi: 10.1371/journal.pntd.0000848

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Scott et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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A) In vitro dicing assay. Biotinylated 500 bp β-gal dsRNA was added to Aag2 and C6/36 cell lysates, with (as indicated by +) or without human recombinant Dicer (reDicer), and aliquots were collected immediately (0) or after 18 hours. Lysate RNA was separated by electrophoresis on a polyacrylamide gel and transferred to a membrane for detection using the BrightStar detection system. C lane contains product of in vitro reaction of reDicer with 500 bp dsRNA, as size marker. (B) Analysis of dicing activity in Aag2 (a-d) and C6/36 (e-h) cell cultures. Aag2 cells were transfected with (a) pEGFP + EGFP siRNA, (b) pEGFP + WNV siRNA, (c) pEGFP + EGFP dsRNA, (d) pEGFP + WNV dsRNA. C6/36 cells were transfected with (e) pEGFP + EGFP siRNA, (f) pEGFP + WNV siRNA, (g) pEGFP + EGFP dsRNA, (h) pEGFP + WNV dsRNA. All cells were analyzed by fluorescent microscopy at 48 hr after transfection. (C) Immunoblot shows EGFP protein silencing. 1, pEGFP + EGFP siRNA, 2, pEGFP + WNV siRNA, 3, pEGFP + EGFP dsRNA, 4, pEGFP + WNV dsRNA.