Journal: Nature structural & molecular biology

Article Title: Regulation of miRNA-mediated gene silencing by miRNA precursors

doi: 10.1038/nsmb.2862

Figure Lengend Snippet: Mmu-miR-151-5p cleaves E2f6 in the absence of a seed match. (a) Genomic locus of mmu-miR-151 encoded by a LINE2 repeat element. (b) Schematic of the binding site of mmu-miR-151-5p to E2f6 3′UTR. (c) Dual-luciferase reporter assay for the wildtype E2f6 3′UTR (wt) or other mutants (per 5p and mut 5p) in presence of miR-151-5p overexpression (sh-151-5p). (d) Western blot for E2f6in presence of sh-151-5p or a scrambled control (sh-scr). Actin serves as a loading control. Uncropped blot in . (e) E2f6 qPCR on miR-151-5p overexpression. Error bars, s.e.m. (n = 3 replicates). (f) Dual-luciferase reporter assay for E2f6 3′UTR or control ( Sox4 3′UTR) in presence of an increasing dosage of miR-151-5p inhibitor. (g) Dual-luciferase reporter assay in Ago2 −/− cells for E2f6 3′UTR in presence of sh-151-5p and a functional copy of Ago2 or cleavage deficient Ago2 (D597A) or Ago1. (h) 5′-RACE of E2f6 . Arrowhead in the E2f6 3′UTR sequence (schematic) indicates the 5′ end of majority of E2f6 cleavage products in mouse lung tissue. Agarose gel showing E2f6 cleaved products (shown by an asterisk) is shown in the top gel (uncropped gel in ). The bottom gel serves as a RACE reaction control to detect the presence of E2f6 and ARHGDIA cDNAs. (c,f, g) For reporter assays, normalization was done with respect to sh-scr. Error bars, s.e.m. (n = 2 biological replicates, each with 4 technical replicates), ns denotes not significant,*** P = 0.001 by two-tailed Student′s t test.

Article Snippet: Two micrograms of total RNA were reverse-transcribed using superscript II RT kit (Life technologies) and subjected to gene expression analyses using gene specific Taqman probes (Mm01270320_m1 for E2f6 and Mm03306373_pri for pri-miR-151).

Techniques: Binding Assay, Luciferase, Reporter Assay, Over Expression, Western Blot, Control, Functional Assay, Sequencing, Agarose Gel Electrophoresis, Two Tailed Test

Journal: Nature structural & molecular biology

Article Title: Regulation of miRNA-mediated gene silencing by miRNA precursors

doi: 10.1038/nsmb.2862

Figure Lengend Snippet: miR-151-3p suppresses E2f6 expression by binding to E2f6 3′UTR adjacent to where miR-151-5p binds. (a) Schematic of a putative binding site of the miR-151-3p to the E2f6 3′UTR region adjacent to where 5p strand binds, in both mice and humans. The seed regions (nucleotides 2-8) are indicated for both the 5p and 3p arms. (b) Dual-luciferase reporter assay for the wildtype E2f6 3′UTR (wt) or other mutants (mut 3p and seed 3p as shown in the schematic) in presence of a miR-151-3p overexpression (sh-151-3p) or a scrambled control(sh-scr). A reporter construct with deletion of the entire miR-151-3p binding site in E2f6 3′UTR was also included. (c) Dual-luciferase reporter assay in wildtype MEF and Ago2 −/− cells for E2f6 3′UTR in presence of sh-151-3p. For comparision, dual-luciferase reporter assay for E2f6 3′UTR in presence of sh-151-5p is also shown. (b,c) For reporter assays, normalization was done with respect to sh-scr. Error bars, s.e.m. (n = 2 biological replicates, each with 4 technical replicates).

Article Snippet: Two micrograms of total RNA were reverse-transcribed using superscript II RT kit (Life technologies) and subjected to gene expression analyses using gene specific Taqman probes (Mm01270320_m1 for E2f6 and Mm03306373_pri for pri-miR-151).

Techniques: Expressing, Binding Assay, Luciferase, Reporter Assay, Over Expression, Control, Construct

Journal: Nature structural & molecular biology

Article Title: Regulation of miRNA-mediated gene silencing by miRNA precursors

doi: 10.1038/nsmb.2862

Figure Lengend Snippet: Precursor miR-151 competes with the mature miR-151-5p for binding to E2f6 3′UTR. (a) Thermodynamics of pre-miR-151 binding to E2f6 . (b) Schematic of the stem-loop structure of the pre-miR-151 with the 5p arm (blue), 3p arm (purple) and two adenosines (green) substituted to guanosines (orange). (c) Northern analysis of miR-151 processing from pre-miR-151 overexpression plasmid (pEZX-151) or the double mutant form of pre-miR-151 (pEZX-DM). Let-7a serves as a loading control. (d) Dual-luciferase reporter assay for E2f6 3′UTR in presence of only the mature miR-151-5p (sh-miR-151-5p), or both the pre-miR-151 and mature miR-151-5p (pEZX-151 and pEZX-DM). (e) Schematic of the binding site of pre-miR-151 in E2f6 3′UTR and its modifications ( E2f6 3p del and E2f6 3p-5p swap). (f) In-vitro gel shift assay with radiolabed (denoted by an asterisk) synthetic pre-miR-151(I) or a control pre-miR-122 and increasing molar concentrations of wildtype E2f6 3′UTR (1, 10 and 100 nM) or its modified forms. (g) Dual-luciferase analysis for E2f6 3′UTR (wt), 3p del or 3p-5p swap reporters with pEZX-151 or pEZX-DM. (h) In-vitro gel shift assay of E2f6 3′UTR bound to miR-151-5p with increasing molar concentrations of a synthetic pre-miR-151 or a control pre-miR-122. Bands below the blue star and orange star represent radiolabeled pre-miR-151 and pre-miR-122 respectively. “*” denotes radiolabeled oligos. (d, g) For reporter assays, normalization was done with respect to a scrambled control (sh-scr). Error bars, s.e.m. (n = 3 biological replicates, each with 3 technical replicates). * P = 0.05, ** P = 0.01 by two-tailed Student′s t test.

Article Snippet: Two micrograms of total RNA were reverse-transcribed using superscript II RT kit (Life technologies) and subjected to gene expression analyses using gene specific Taqman probes (Mm01270320_m1 for E2f6 and Mm03306373_pri for pri-miR-151).

Techniques: Binding Assay, Northern Blot, Over Expression, Plasmid Preparation, Mutagenesis, Control, Luciferase, Reporter Assay, In Vitro, Gel Shift, Modification, Two Tailed Test

Journal: Nature structural & molecular biology

Article Title: Regulation of miRNA-mediated gene silencing by miRNA precursors

doi: 10.1038/nsmb.2862

Figure Lengend Snippet: Pre-miR-151 binds to E2f6 in vivo and may protect the E2f6 transcript in quiescent tissues. (a) Schematic of the ChIRP method used to pull-down E2f6 mRNA from mouse brain. Quantitative PCR of (b) E2f6 mRNA and a control Ctdnep1 mRNA, (c) pre-miR-151 and a control pre-miR-124, (d) mature miR-151-5p and a control miR-124, pulled down by the biotinylated tiling oligonucleotides against the E2f6 3′UTR or a control lacZ mRNA. Quantitative PCR of (e) E2f6 mRNA, (f) pri-miR-151, (g) mature miR-151-5p in quiescent and non-quiescent tissues. In each case ( e — g ), the data are presented as fold induction after normalization to the liver sample (value = 1). (h) Northern analysis of miR-151 processing in various tissues. The blot on the left was probed with a LNA probe against mature miR-151-5p as shown by the schematic above the blot. The primary or intermediate product in the miR-151 biogenesis pathway is indicated by an arrowhead (→) and the mature miR-151-5p is indicated by a circle (○). U6 serves as a loading control. The blot on the right was probed (sequence is provided in ) for a region just outside the annotated stem loop structure of mmu-miR-151 (as shown by the schematic above the blot). (i) Quantitative PCR analyses of E2f6 , pri-miR-151 and miR-151-5pduring differentiation of muscle cells (C2C12) ( b — g, i ) For qPCR data, error bars, s.e.m. (n = 2 biological replicates, each with 3 technical replicates).

Article Snippet: Two micrograms of total RNA were reverse-transcribed using superscript II RT kit (Life technologies) and subjected to gene expression analyses using gene specific Taqman probes (Mm01270320_m1 for E2f6 and Mm03306373_pri for pri-miR-151).

Techniques: In Vivo, Real-time Polymerase Chain Reaction, Control, Northern Blot, Sequencing

Journal: Arquivos Brasileiros de Cardiologia

Article Title: Congenital Heart Disease as a Warning Sign for the Diagnosis of the 22q11.2 Deletion

doi: 10.5935/abc.20140145

Figure Lengend Snippet: Demonstration of the deletion in region 22q11.2. A. FISH technique. B. MLPA technique.

Article Snippet: Commercial probes of unique sequences were used for the specific region in 22q11.2 (probes DGS/VCFS, TUPLE1 and N25 D22S75, Cytocell, Cambridge, UK) , and/or MLPA using various kits (P036-E1, P070-B2, P064-B3, MRC-Holland, Amsterdam, Netherlands - www.mlpa.com ).

Techniques:

Journal: Arquivos Brasileiros de Cardiologia

Article Title: Congenital Heart Disease as a Warning Sign for the Diagnosis of the 22q11.2 Deletion

doi: 10.5935/abc.20140145

Figure Lengend Snippet: Congenital heart diseases in 47 patients with the 22q11.2 deletion syndrome and the surgical corrections performed

Article Snippet: Commercial probes of unique sequences were used for the specific region in 22q11.2 (probes DGS/VCFS, TUPLE1 and N25 D22S75, Cytocell, Cambridge, UK) , and/or MLPA using various kits (P036-E1, P070-B2, P064-B3, MRC-Holland, Amsterdam, Netherlands - www.mlpa.com ).

Techniques:

Journal: Arquivos Brasileiros de Cardiologia

Article Title: Congenital Heart Disease as a Warning Sign for the Diagnosis of the 22q11.2 Deletion

doi: 10.5935/abc.20140145

Figure Lengend Snippet: Phenotypic characteristics of 60 patients with the 22q11.2 deletion syndrome

Article Snippet: Commercial probes of unique sequences were used for the specific region in 22q11.2 (probes DGS/VCFS, TUPLE1 and N25 D22S75, Cytocell, Cambridge, UK) , and/or MLPA using various kits (P036-E1, P070-B2, P064-B3, MRC-Holland, Amsterdam, Netherlands - www.mlpa.com ).

Techniques:

Journal: Arquivos Brasileiros de Cardiologia

Article Title: Congenital Heart Disease as a Warning Sign for the Diagnosis of the 22q11.2 Deletion

doi: 10.5935/abc.20140145

Figure Lengend Snippet: Main phenotypic characteristics of patients with the 22q11.2 deletion syndrome. A) Narrow palpebral fissure. B) Elongated face and/or nose. C) Thin lips.

Article Snippet: Commercial probes of unique sequences were used for the specific region in 22q11.2 (probes DGS/VCFS, TUPLE1 and N25 D22S75, Cytocell, Cambridge, UK) , and/or MLPA using various kits (P036-E1, P070-B2, P064-B3, MRC-Holland, Amsterdam, Netherlands - www.mlpa.com ).

Techniques:

Journal: Arquivos Brasileiros de Cardiologia

Article Title: Congenital Heart Disease as a Warning Sign for the Diagnosis of the 22q11.2 Deletion

doi: 10.5935/abc.20140145

Figure Lengend Snippet: Pictures showing evolving features of patients with the 22q11.2 deletion syndrome at different ages. A) Newborn with thin lips and dysplasic ears. These phenotypic features become more characteristic at school age. B) Newborn with facial dysmorphism (elongated face and nose, narrow palpebral fissure, thin lips). C) Infant with elongated face and nose more evident during development

Article Snippet: Commercial probes of unique sequences were used for the specific region in 22q11.2 (probes DGS/VCFS, TUPLE1 and N25 D22S75, Cytocell, Cambridge, UK) , and/or MLPA using various kits (P036-E1, P070-B2, P064-B3, MRC-Holland, Amsterdam, Netherlands - www.mlpa.com ).

Techniques:

Journal: Arquivos Brasileiros de Cardiologia

Article Title: Congenital Heart Disease as a Warning Sign for the Diagnosis of the 22q11.2 Deletion

doi: 10.5935/abc.20140145

Figure Lengend Snippet: Distribution of values of total lymphocytes, CD4 + , CD8 + and CD19 + in patients with the 22q11.2 deletion syndrome. A) Number of total lymphocytes. B) CD4 + count. C) CD8 + count. D) CD19 + count. Each dot ( • ) corresponds to an individual patient. Max: Maximum; Min: Mimum

Article Snippet: Commercial probes of unique sequences were used for the specific region in 22q11.2 (probes DGS/VCFS, TUPLE1 and N25 D22S75, Cytocell, Cambridge, UK) , and/or MLPA using various kits (P036-E1, P070-B2, P064-B3, MRC-Holland, Amsterdam, Netherlands - www.mlpa.com ).

Techniques: