Fig. 2.

Promoter-associated RNA variant and mRNA expression in siRNA treated cultures. (A) The EF1a promoter-associated RNA variant is reduced (P = 0.352) along with EF1a mRNA expression (P = 0.038) when the promoter is targeted by the EF52 siRNA (EF1a) relative to the CCR5-specific siRNA (Control). Measurements of mRNA or promoter-associated RNA levels from three independent transfections standardized to GAPDH expression with the standard error of the means (SEM) are shown with P values reported for a single-sided F test (mRNA) and double-sided t test (promoter-associated RNA). (B) Treatment with an EF1a-specific (EF1a) antisense phosphorothioate ODN suppresses EF1a promoter-associated RNA variant expression whereas the CCR5-specific ODN (Control) does not. 293T cells were transfected with either an ODN targeted to the EF1a promoter (EF1a) or to the CCR5 promoter (CCR5) (100 nM Lipofectamine 2000). Twenty-four hours later, the cultures were collected, cell RNA was isolated, Dnase was treated, and EF1a promoter-associated RNAs were assessed by qRT-PCR relative to GAPDH expression. Triplicate treated cultures were assessed and the standard error of the means is shown. (C) Treatment of cells with the EF1a promoter-targeted ODN (EF1a) suppresses siRNA (EF52)-mediated transcriptional silencing of the EF1a gene, whereas the control CCR5 ODN (Control) does not. EF1a mRNA expression was measured by qRT-PCR at 18 h after siRNA transfection in untreated (Mock) and cells treated with either EF52 (EF52) or control (CCR5) siRNAs, which was 42 h after ODN transfection and standardized to GAPDH expression. Results are from triplicate transfections, and the standard deviations are shown.