FIG. 2.

Analysis of stable mRNA decay by Northern blot and Invader RNA assay. (A) Log-phase cells expressing a tetracycline-regulated β-globin gene received tetracycline at time zero to turn off reporter gene transcription. RNA isolated at the indicated times over the next 8 h was assayed by Northern blotting for human β-globin mRNA and murine GAPDH mRNA. (B) The RNAs in panel A were analyzed by Invader assay using probe sets specific to exon 1, exon 2, and exon 3. Each sample was normalized to 30 ng of input RNA and quantified against a standard curve of in vitro-transcribed full-length β-globin mRNA. (C) The cells used in the preceding experiments were transfected 24 and 48 h prior to harvest with siRNAs to luciferase (GL2), Dcp2, or PM/Scl-100. Total protein isolated 24 h after the second round of transfection (time zero) was analyzed by Western blotting (wb) with antibodies to Dcp2, PM/Scl-100, or β-actin. (D) Tetracycline was added to cells 24 h after the second transfection with Dcp2 siRNA (time zero), and RNA isolated at the indicated times was analyzed by Northern blotting. (E) The RNA examined in panel D was analyzed by Invader assay as described above for panel B. (F) Tetracycline was added to cells 24 h after the second transfection with PM/Scl-100 siRNA (time zero), and RNA isolated at the indicated times was analyzed by Northern blotting. (G) The RNA examined in panel F was analyzed by Invader assay as described above for panel B. The points in each graph represent the means ± standard deviations (error bars) for triplicate determinations. To avoid bias introduced by curve-fitting programs, the actual decay lines are plotted.