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. Author manuscript; available in PMC: 2011 Jan 19.
Published in final edited form as: Anal Biochem. 2008 Jun 20;381(1):166–168. doi: 10.1016/j.ab.2008.06.026

Figure 1. Experimental validation of the PCR-based strategy designed to generate regulatory elements bearing a various number of contiguous miRNA binding sites.

Figure 1

(A) Schematic representation of the PCR amplification strategy. (B–C) N2a and NIH 3T3 cells were cotransfected with a pre-miR-328 expression vector and either (B) the Rluc/miR-328 reporter construct harboring 1 or 3 copies of the miR-328 binding site (BS) or a sequence perfectly complementary (PC) to miR-328 (n = 2 experiments), or (C) the Rluc/miR-328 reporter construct harboring 3 copies of the miR-328 BS (n = 2 experiments). (D) Cells were cotransfected with the synthetic miR-298 duplex and an Rluc/miR-298 reporter construct harboring 1 or 3 copies of the miR-298 BS (n = 2 experiments). (B–D) The results of Rluc activity were normalized with Fluc reporter activity and expressed as a percentage of the results obtained with the empty reporter vector (set at 100%). Results are expressed as mean ± standard error of the mean.