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Cellular off-target cleavage is observed only with RNase H1 overexpression.

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posted on 2014-07-29, 02:53 authored by Walt F. Lima, Timothy A. Vickers, Josh Nichols, Cheryl Li, Stanley T. Crooke

A modified RLM-RACE protocol was used to determine sites of target-specific and off-target cleavage. A) Exon 5 target-specific RACE cleavage products. (B) Quantitative RACE of exon 5 target-specific cleavage products for SOD/TO cells (black bars) and SOD/TO-RHA cells (gray bars). Results are given as threshold cycle (cT) for the amplification reaction with or without overexpression of RNase H. C) Exon 4 off-target RACE cleavage products. D) Quantitative RACE of exon 4 off-target cleavage products (cT). E) Human RNase H1 was overexpressed in HeLa SOD/TO cells by infecting with adenoviral human RNase H1 for 48 hours [4]. Cells were then treated with 50 nM ASO 38, and RLM_RACE was performed as described above. 293 cells with or without E. coli RNase H; HeLa cells with or without human RNase H1. F) RACE products were gel purified and sequenced using gene specific RACE primers. Arrows indicate the predominant cleavage site.