Journal: PLoS ONE

Article Title: MicroRNA-96 Directly Inhibits γ-Globin Expression in Human Erythropoiesis

doi: 10.1371/journal.pone.0022838

Figure Lengend Snippet: (A) Western blot analysis of AGO1, 2, 3, 4, γ-globin and β-globin in reticulocytes purified from umbilical cord blood ( CB ) and blood from adults ( AB ). Actin was included as loading control. (B) AGO2-containing RNA-protein complexes were immunoprecipitated from equal amounts of cord blood reticulocyte ( CB ) and adult blood reticulocyte ( AB ) lysates using rat monoclonal anti-AGO2 antibodies; as isotype control, non-specific rat immuno-globulin ( IgG ) as well as rat monoclonal anti-AGO1 antibodies were used. Immunoprecipitations were compared to lysate ( input ) and subjected to immunoblot analysis using anti-AGO2 antibodies. (C) The amounts of α-globin ( HBA ), β-globin ( HBB ), γ-globin ( HBG ) and δ-globin ( HBD ) mRNA immunoprecipitated together with AGO2 from CB and AB were compared to amounts of globin mRNAs that were non-specifically immunoprecipitated with control IgG. As control GAPDH and DNMT3a mRNAs were analyzed. For every sample the same amount of precipitated RNA was analyzed. The values are expressed as fold-enrichment over IgG immunoprecipitations, and represent mean±SEM (n = 3).

Article Snippet: Retroviral particles were prepared from a mixture of 4 HuSH 29mer shRNA constructs against the γ-globin mRNA (Origene Technologies, Inc., Rockville, MD, USA) and used as control.

Techniques: Western Blot, Purification, Control, Immunoprecipitation

Journal: PLoS ONE

Article Title: MicroRNA-96 Directly Inhibits γ-Globin Expression in Human Erythropoiesis

doi: 10.1371/journal.pone.0022838

Figure Lengend Snippet: (A) Represented are the 5′ untranslated region (5′UTR, light blue), open reading frame (ORF, dark blue) and 3′UTR (gray) of γ-globin mRNA (HBG). By using RNA hybrid , the free energy of the individual miRNA:mRNA hybridization was determined and the corresponding base pairing shown. The hybridizations of γ-globin mRNA with miR-96, miR-146a and let-7a (black squares) showed either well-defined mRNA:miRNA pairing (miR-146a and let-7a) consisting of a seed region containing eight base pairs, followed by a four-base pair bulge region and a 3′ complementary region as described or seedless base pairing (miR-96), all within the open reading frame (ORF) of HBG. (B) The amounts of miR-96, miR-146a and let-7a immunoprecipitated together with AGO2 from CB and AB were compared to amounts of miR-96, miR-146a and let-7a that were non-specifically immunoprecipitated with control IgG. For every sample the same amount of precipitated RNA was analyzed. The values are expressed as 1000-fold-enrichment over IgG immunoprecipitations, and represent mean±SEM (n = 3). P values were determined by the Student's t-test.

Article Snippet: Retroviral particles were prepared from a mixture of 4 HuSH 29mer shRNA constructs against the γ-globin mRNA (Origene Technologies, Inc., Rockville, MD, USA) and used as control.

Techniques: Hybridization, Immunoprecipitation, Control