Journal: Journal of Advanced Research

Article Title: A reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy for the cost-effective construction of RNA sequencing libraries

doi: 10.1016/j.jare.2019.12.005

Figure Lengend Snippet: Comparison of human rRNA removal specificity and efficiency between the RTR2D procedure and NEBNext® rRNA Depletion kit. Human total RNA (1.0 µg) was subjected to the RTR2D (R2D) (with the pooled rRNA probes) or the NEBNext® rRNA Depletion (NEB) protocol. The rRNA-depleted products were subjected to the Bioanalyzer. Representative gel image ( a ) and electropherograms ( b ) are shown. “Input” and “No probes (NP)” groups were used as controls. ( B ) TqPCR analysis of the residual rRNA species ( a ) and removal efficiency ( b ). “**” p < 0.001, compared with that of the NP group or the Input group. ( C ) Effect of rRNA removal protocols on the expression of housekeeping genes GAPDH and β-ACTIN ( a ) and genes/lncRNA with different abundances, c-MYC , TP53 and lncRNA HOTAIR ( b ). All qPCR reactions were done in triplicate.

Article Snippet: Commercially available pull-out kits include Ribo-Zero (Illumina, USA) and RiboMinus (Thermo Fisher, USA), while the RNase H-based rRNA degradation of oligo-DNA:RNA hybrids includes RiboErase (Kapa Biosystems, USA) and NEBNext rRNA Depletion (New England Biolabs, NEB), which depletes both cytoplasmic (5S rRNA, 5.8S rRNA, 18S rRNA and 28S rRNA) and mitochondrial ribosomal RNA (12S rRNA and 16S rRNA) from human, mouse and rat total RNA preparations.

Techniques: Expressing

Journal: Journal of Advanced Research

Article Title: A reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy for the cost-effective construction of RNA sequencing libraries

doi: 10.1016/j.jare.2019.12.005

Figure Lengend Snippet: Comparison of mouse rRNA removal specificity and efficiency between the RTR2D procedure and NEB Next rRNA Depletion kit. Mouse total RNA (1.0 µg) was subjected to the RTR2D (R2D) or the NEBNext® rRNA Depletion (NEB) protocol. The rRNA-depleted products were subjected to the Bioanalyzer. Representative gel image ( a ) and electropherograms ( b ) are shown. “Input” and “No probes (NP)” groups were used as controls. ( B ) TqPCR analysis of the residual rRNA species ( a ) and removal efficiency ( b ). “**” p < 0.001, compared with that of the NP group or the Input group. ( C ) Effect of rRNA removal protocols on the expression of housekeeping genes Gapdh and β-Actin ( a ) and genes/lncRNA with different abundances, c-Myc , Tp53 and lncRNA Hotair ( b ). All qPCR reactions were done in triplicate.

Article Snippet: Commercially available pull-out kits include Ribo-Zero (Illumina, USA) and RiboMinus (Thermo Fisher, USA), while the RNase H-based rRNA degradation of oligo-DNA:RNA hybrids includes RiboErase (Kapa Biosystems, USA) and NEBNext rRNA Depletion (New England Biolabs, NEB), which depletes both cytoplasmic (5S rRNA, 5.8S rRNA, 18S rRNA and 28S rRNA) and mitochondrial ribosomal RNA (12S rRNA and 16S rRNA) from human, mouse and rat total RNA preparations.

Techniques: Expressing

Journal: Journal of Advanced Research

Article Title: A reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy for the cost-effective construction of RNA sequencing libraries

doi: 10.1016/j.jare.2019.12.005

Figure Lengend Snippet: Transcriptomic comparison of the RNA-seq libraries prepared with the RTR2D and the NEBNext rRNA Depletion protocols. Human total RNA (1.0 µg) was subjected to the rRNA removal process by using the RTR2D procedure (R2D) and the NEBNext rRNA Depletion kit (NEB). The rRNA-depleted samples were used for RNA-seq library preparations using the Illumina protocol and subjected to NGS analysis (in replicate). ( A ) The average valid reads (RPKM) for the RTR2D ( a ) and the NEB kit ( b ) are depicted in various categories of transcripts. ( B ) Scatter plots and correlations of mRNA transcripts between the RTR2D and the NEB protocols in two different batches of preparations ( a & b ). ( C ) Scatter plots and correlations of lncRNA transcripts between the RTR2D and the NEB protocols in two different batches of preparations ( a & b ).

Article Snippet: Commercially available pull-out kits include Ribo-Zero (Illumina, USA) and RiboMinus (Thermo Fisher, USA), while the RNase H-based rRNA degradation of oligo-DNA:RNA hybrids includes RiboErase (Kapa Biosystems, USA) and NEBNext rRNA Depletion (New England Biolabs, NEB), which depletes both cytoplasmic (5S rRNA, 5.8S rRNA, 18S rRNA and 28S rRNA) and mitochondrial ribosomal RNA (12S rRNA and 16S rRNA) from human, mouse and rat total RNA preparations.

Techniques: RNA Sequencing Assay

Journal: Journal of Advanced Research

Article Title: A reverse transcriptase-mediated ribosomal RNA depletion (RTR2D) strategy for the cost-effective construction of RNA sequencing libraries

doi: 10.1016/j.jare.2019.12.005

Figure Lengend Snippet: Nutlin3A-induced transcriptomic changes determined by RNA-seq analysis of the NGS libraries prepared with the RTR2D and the NEBNext rRNA Depletion protocols . Exponentially growing human osteosarcoma line SJSA1 cells were treated with 2 µM Nutlin3A or DMSO control for 24 h and subjected to total RNA isolation. Human total RNA (1.0 µg) was subjected to rRNA removal by using the RTR2D procedure (R2D) and the NEBNext rRNA Depletion kit (NEB). The rRNA-depleted samples were used for RNA-seq library preparations using the Illumina protocol and subjected to NGS analysis. ( A ) Scatter plots of mRNA transcripts affected by DMSO (Y-axis) and Nutlin3A (X-axis) identified in the RNA-seq libraries prepared with the NEB protocol ( a ) or the RTR2D protocol ( b ). Venn diagrams were generated by using more stringent criteria for the differentially expressed transcripts for up-regulated ( c ) (total relative reads > 54) and down-regulated transcripts ( d ) (total relative reads > 13). ( B ) Scatter plots of lncRNA transcripts affected by DMSO (Y-axis) and Nutlin3A (X-axis) identified in the RNA-seq libraries prepared with the NEB protocol ( a ) or the RTR2D protocol ( b ). Significant up and down-regulated transcripts were defined as log2FoldChange > 1 and < −1, respectively, with a false detection rate (FDR) < 0.001. Venn diagrams were generated by using more stringent criteria for the differentially expressed transcripts for up-regulated ( c ) (total relative reads > 540) and down-regulated transcripts ( d ) (total relative reads > 20).

Article Snippet: Commercially available pull-out kits include Ribo-Zero (Illumina, USA) and RiboMinus (Thermo Fisher, USA), while the RNase H-based rRNA degradation of oligo-DNA:RNA hybrids includes RiboErase (Kapa Biosystems, USA) and NEBNext rRNA Depletion (New England Biolabs, NEB), which depletes both cytoplasmic (5S rRNA, 5.8S rRNA, 18S rRNA and 28S rRNA) and mitochondrial ribosomal RNA (12S rRNA and 16S rRNA) from human, mouse and rat total RNA preparations.

Techniques: RNA Sequencing Assay, Isolation, Generated

Journal: Cell Proliferation

Article Title: Establishment of a porcine pancreatic stem cell line using T‐REx ™ system‐inducible Wnt3a expression

doi: 10.1111/cpr.12188

Figure Lengend Snippet: The expression of GFP and Wnt3a was regulated by Dox in a time‐ and dose‐dependent manner. (a) The expression of GFP in PSCs was observed under fluorescence microscope after treatment with different concentrations of Dox (0, 0.1, 1, 5, 10 μg/ml) for different times (0, 24, 48 and 72 h) (bar = 200 μm). (b) The relative Wnt3a mRNA expression level in PSCs after Dox treatment for different times analyzed by QRT‐PCR. (c) The relative Wnt3a mRNA expression level in PSCs after treatment with different concentrations of Dox analysed by QRT‐PCR. (d) GFP‐positive PSCs were quantified by flow cytometric after induced by Dox. (e) The histogram of statistical result for Fig. d. (f) The Wnt3a protein expression in PSCs after Dox addition for different times detected by Western blotting. (g) The Wnt3a protein expression in the supernate of cultured PSC after Dox treatment for 72 h. (h) Immunostaining of Wnt3a and active β‐catenin in PSCs after treatment with or without Dox (bar = 100 μm).

Article Snippet: Wnt3a was amplified from mouse tail vein‐derived samples using Vazyme Blood Direct PCR Kit (Vazyme, Nanjing, China), by PCR.

Techniques: Expressing, Fluorescence, Microscopy, Quantitative RT-PCR, Western Blot, Cell Culture, Immunostaining

Journal: The Journal of Neuroscience

Article Title: A Destructive Interaction Mechanism Accounts for Dominant-Negative Effects of Misfolded Mutants of Voltage-Gated Calcium Channels

doi: 10.1523/JNEUROSCI.2844-07.2008

Figure Lengend Snippet: Inhibition of Cav2.1 Ca2+ currents by the R1279X EA2-truncated form of Cav2.1 protein. A, Schematic cartoon representing the main Cav2.1 constructs used in this study. B, The Ca2+ current density in HEK293 cells expressing the Cav2.1 channel protein. The cells were cotransfected with plasmids encoding the human Cav2.1 subunit, the β1b subunit, and the α2/δ1 subunit. Mean ± SEM values of the current density for cells expressing the Cav2.1 subunit alone (white bar; n = 18) or in the presence of the R1279X mutant (+R1279X) (black bar; n = 22) are presented (***p < 0.001; Student's t test). The inset shows representative current traces at TP −40 and 0 mV [holding potential (HP), −80 mV]. C, Same experiments as in B performed in the neuroblastoma cell line NG108-15. The HP was −50 mV (***p < 0.001; Student's t test). Cav2.1 subunit alone (white bar; n = 15) or in the presence of the R1279X mutant (+R1279X) (black bar; n = 22). D, Measurements of native T-type and HVA Ca2+ current densities in R1279X-transfected NG108-15 cells. The T-current density (left) was measured in proliferative NG108-15 cells (Prolif.; 2–3 d after transfection; n = 9) and in differentiated NG108-15 cells (Diff.; 6 d after transfection; 3–4 d after switching to differentiation medium; n = 11) using HP −100 mV and TP −30 mV. The HVA current density, mainly corresponding to L- and N-type channel activities, was measured using HP −50 mV and TP 0 mV (right).

Article Snippet: GFP-Ca v 2.1 and Ca v 2.1-HA cDNA were transferred in pCIneo expression vector (Promega, Madison, WI).

Techniques: Inhibition, Construct, Expressing, Mutagenesis, Transfection

Journal: The Journal of Neuroscience

Article Title: A Destructive Interaction Mechanism Accounts for Dominant-Negative Effects of Misfolded Mutants of Voltage-Gated Calcium Channels

doi: 10.1523/JNEUROSCI.2844-07.2008

Figure Lengend Snippet: Effect of R1279X mutant on surface expression of Cav2.1 channel. A, HEK293 cells were cotransfected with the HA-tagged wild-type Cav2.1 subunit alone (with free GFP) or together with the R1279X mutant (fused to GFP). Left column, GFP fluorescence. Middle column, Staining of cells with monoclonal rat anti-HA antibody (primary antibody) and Alexa594 (secondary antibody). NP, Nonpermeabilized; P, permeabilized. Images were acquired using a Leica SP2 confocal microscope, with a 63× oil immersion objective. B, Luminometric ELISA assays to quantify surface expression of the HA-tagged Cav2.1 in the presence of the R1279X mutant and auxiliary subunits (**p < 0.01, ***p < 0.001; Student's t test). C, Western blots performed on HEK293 cells expressing Cav2.1-HA alone (−) or in the presence of R1279X (+) without (left) or with (right) auxiliary subunits. β1b-HA was used in these experiments (right).

Article Snippet: GFP-Ca v 2.1 and Ca v 2.1-HA cDNA were transferred in pCIneo expression vector (Promega, Madison, WI).

Techniques: Mutagenesis, Expressing, Fluorescence, Staining, Microscopy, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: The Journal of Neuroscience

Article Title: A Destructive Interaction Mechanism Accounts for Dominant-Negative Effects of Misfolded Mutants of Voltage-Gated Calcium Channels

doi: 10.1523/JNEUROSCI.2844-07.2008

Figure Lengend Snippet: Channel misfolding and instability induced by the R1279X mutant. A, B, Pulse-chase experiments were performed on HEK293 cells 48 h after transfection (see Materials and Methods). Cells were labeled with 35S-methionine-cysteine for 20 min. The chase was performed for 0, 1, 2, or 4 h as indicated. After lysis, the Cav2.1 subunit was immunoprecipitated with an anti-HA antibody. The arrow indicates the band corresponding to the EA2 mutant that coimmunoprecipitates with the wild-type Cav2.1 subunit. In B (left), the β1b is detected (arrow). C, Quantification of three independent experiments using GE Healthcare software. D, Pulse-chase analyses performed on HEK293 cells transfected with CD4-AAXX alone or in the presence of R1279X. Quantification was performed as described above. E, Immunoprecipitations performed on cells coexpressing various subunit arrangements (as indicated) in the absence or presence of an untagged R1279X mutant (right) using standard SDS-PAGE (6%). Western blots (WBs) were performed with the indicated antibodies. F, Immunoprecipitations were performed between β1b-HA and GFP-R1279X as described in E. MW, Molecular weight.

Article Snippet: GFP-Ca v 2.1 and Ca v 2.1-HA cDNA were transferred in pCIneo expression vector (Promega, Madison, WI).

Techniques: Mutagenesis, Pulse Chase, Transfection, Labeling, Lysis, Immunoprecipitation, Software, SDS Page, Western Blot, Molecular Weight

Journal: The Journal of Neuroscience

Article Title: A Destructive Interaction Mechanism Accounts for Dominant-Negative Effects of Misfolded Mutants of Voltage-Gated Calcium Channels

doi: 10.1523/JNEUROSCI.2844-07.2008

Figure Lengend Snippet: EA2 missense mutants act in a dominant-negative manner by misfolding and instability induction. A, Histograms of the mean Ca2+ current density (± SEM) obtained in a representative batch of NG108-15 cells expressing wild-type subunit alone (Cav2.1-HA, n = 18); the EA2 mutants alone (Cav2.1-HA-G293R, n = 33, 5 with detectable current; Cav2.1-HA-AY1593/94D, n = 6); and the wild-type and EA2 mutants together (Cav2.1-HA+Cav2.1-HA-G293R, n = 23, 3 with detectable current; Cav2.1-HA+Cav2.1-HA-AY1593/94D, n = 10). These experiments were conducted in the presence of the auxiliary β1b and α2/δ1 subunits. B, Normalized native T-type Ca2+ current densities (mean ± SEM) in the NG108-15 cells analyzed in A. The differences are not statistically significant between the various conditions tested (Student's t test). C, Western blots with the indicated antibodies (WB) were performed on HEK293 cells coexpressing Cav2.1-HA alone or in the presence of G293R and AY1593/94D mutants together with auxiliary subunits. The bottom panel shows the Western blot from cells expressing missense mutants alone. D, Pulse-chase analyses were performed on HEK293 cells transfected with wild-type Cav2.1 alone or in the presence of G293R. Quantification of three independents experiment was performed as described above. MW, Molecular weight.

Article Snippet: GFP-Ca v 2.1 and Ca v 2.1-HA cDNA were transferred in pCIneo expression vector (Promega, Madison, WI).

Techniques: Dominant Negative Mutation, Expressing, Western Blot, Pulse Chase, Transfection, Molecular Weight

Journal: The Journal of Neuroscience

Article Title: A Destructive Interaction Mechanism Accounts for Dominant-Negative Effects of Misfolded Mutants of Voltage-Gated Calcium Channels

doi: 10.1523/JNEUROSCI.2844-07.2008

Figure Lengend Snippet: Endoplasmic reticulum retention and proteasomal degradation of the dominant-negative Cav mutants. A, Confocal images of nonpermeabilized NG108-15 cells expressing EA2 mutants and truncated Cav3.2 subunits. Alexa 594-coupled CT was used as plasma membrane marker (0.5 μg/ml). B, Confocal images of immunofluorescence staining performed on permeabilized NG108-15 cells expressing indicated EA2 mutants and truncated Cav3.2. Polyclonal anti-protein disulfide isomerase (Assay Designs, Ann Arbor, MI) was used as ER marker. C, Pulse-chase experiments were performed as in Figure 3 on cells transfected with EA2 mutants (R1279X and G293R) and Cav3.2 truncated forms. Chase was done for the indicated time (hours) with or without MG-132 proteasome inhibitor (50 μm). After lysis, the truncated Cav channels were immunoprecipitated with anti-GFP antibody. D, Representative pulse chase performed on HEK293 cells transfected with Cav2.1 and the R1279X mutant and the corresponding quantification (n = 3). During the chase, cells were treated with MG-132 (50 μm), leupeptin (20 μm), and NH4Cl (10 mm).

Article Snippet: GFP-Ca v 2.1 and Ca v 2.1-HA cDNA were transferred in pCIneo expression vector (Promega, Madison, WI).

Techniques: Dominant Negative Mutation, Expressing, Marker, Immunofluorescence, Staining, Pulse Chase, Transfection, Lysis, Immunoprecipitation, Mutagenesis