human polya enriched brain rna  (TaKaRa)

Journal: PLoS ONE

Article Title: Identification of a Novel Modulator of Thyroid Hormone Receptor-Mediated Action

doi: 10.1371/journal.pone.0001183

Figure Lengend Snippet: Panel A. Amino acid sequence of human, mouse and rat DOR proteins (sequences 1, 2 and 3, respectively). Multi-alignment done using the CLUSTALW Sequence Alignment programme . Amino acids differing from the consensus are inverse. The amino acid residues used to generate the polyclonal antibodies are shown in bold. The C-terminal basic motif, indicated by a line of “+”, is predicted to form an alpha-helix structure whereas the N-terminal half is unstructured (GLOBPLOT 2). Panel B. PolyA + -RNA membrane containing human adult tissues was probed with 32 P-labelled rat DOR cDNA and washed in stringent conditions. The probe hybridises to a transcript of approximately 4.5 kb. Hybridisation with human glycerol-3-phosphate dehydrogenase (GPDH) cDNA was used as a control probe. Br, brain; He, heart; SK, skeletal muscle; Co, colon; Th, thymus; Sp, spleen; Ki, kidney; Li, liver; Sl, small intestine; Pl, placenta; Lu, lung; Leu, leukocytes. Panels C. Total RNA was purified from several rat tissues and subjected to Northern blot analysis. Ethidium bromide staining of the ribosomal 28S subunit was used as a control of the relative amounts of RNA loaded in each lane and to check the integrity of RNA in each sample. SK, skeletal muscle; He, heart; WAT, white adipose tissue; Ki, kidney; Br, brain; Lu, lung. Panel D. Total RNA was purified from skeletal muscle from non-diabetic and ZDF rats, and RNA was subjected to Northern blot analysis. The mean±SD of 6 separate observations is shown. * difference compared to the control group, at P<0.01 (Student's t test).

Article Snippet: Northern blot assays on 20 µg of total RNA or with human polyA + -RNA obtained from several tissues (Human 12-Lane MTN Blot, Clontech) were performed as described using the 32 P-labelled C42 cDNA fragment or a 0.5 kb cDNA labelled fragment of human glycerol-3-phosphate dehydrogenase (as a control).

Techniques: Sequencing, Hybridization, Purification, Northern Blot, Staining

Journal: PLoS ONE

Article Title: Identification of a Novel Modulator of Thyroid Hormone Receptor-Mediated Action

doi: 10.1371/journal.pone.0001183

Figure Lengend Snippet: Panel A. C2C12 myoblasts previously infected with lentiviruses encoding scrambled RNA (open bar) or DOR siRNA (black bar) were cultured. Cell extracts and total RNA were obtained and DOR protein and mRNA levels were assayed by Western blot and real-time PCR. Relative amounts of proteins in each sample were checked by expression of the nonmuscle-specific protein β-actin. * difference compared to the scrambled group, at P<0.05 (Student's t test). Panel B. Scramble (open bars) or DOR siRNA C2C12 muscle cells (black bars) were transfected with a reporter vector driven by a TRE, and with or without a expression vector for TR α1 . Cells were then incubated in the presence or absence of thyroid hormone for 16 h. Results are mean±SD of triplicates and are representative of three independent experiments. * difference compared to the scrambled group, at P<0.05 (post hoc t test). Panels C–H. Scrambled (open bars) or DOR siRNA C2C12 muscle cells (black bars) were incubated in 5% horse serum-containing medium either in the absence or in the presence of 100 nM T 3 . Total RNA obtained at 48 h of T 3 t treatment were assayed by real-time PCR to measure the expression of several genes. Results are mean±SD of a representative experiment. * difference compared to the control group, at P<0.05 (post hoc t test).

Article Snippet: Northern blot assays on 20 µg of total RNA or with human polyA + -RNA obtained from several tissues (Human 12-Lane MTN Blot, Clontech) were performed as described using the 32 P-labelled C42 cDNA fragment or a 0.5 kb cDNA labelled fragment of human glycerol-3-phosphate dehydrogenase (as a control).

Techniques: Infection, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Transfection, Plasmid Preparation, Incubation