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  • 99
    Zymo Research total rna
    <t>FACS,</t> <t>RNA-seq</t> library preparation and characterization of iN cell and endogenous neuron populations .a , Representative immunofluorescence labelling of tau–EGFP + iN cell population ( Ascl2/Pou4f2 ) on day 12 post-induction using neuronal antibodies TUJ1 and MAP2. Scale bars, 100 μm. Pou4f2 is also known as Brn3b . b , Quantification of co-labelling of tau–eGFP and MAP2 in Tuj1 + cells on day 12 post-induction calculated from various reprogramming transcription factor pairs. Data are presented as mean ± s.d. from n = 4 independent experiments and n = 574 cells. c , d , Representative FACS gates of an Ascl2/Pou4f2 iN cell population (500,000 cells shown) ( c ) and a negative rtTA-only control (40,000 cells shown) ( d ) sorted on day 16 post-induction. Live tau–eGFP + cells were enriched by first gating DRAQ5 + DAPI – cells, then collecting only those that were GFP + . For Ascl2/Pou4f2, n = 2 independent experiments showed similar results, while for rtTA only, n = 40 independent experiments showed similar results. For all other iN cell populations, at least n = 2 independent experiments were performed to obtain biological replicates. e , Per cent of tau–eGFP + cells out of total number of cells collected post-FACS, presented as mean ± s.d. ( n = 4 sorts, > 100 cells per sort). f , g , Correlation plots between aligned counts from single sequenced libraries of a Neurog3/Pou3f2-iN cell population generated from 10 ng versus 5 ng input RNA ( f ) and 10 ng versus 1 ng input RNA ( g ). Pou3f2 is also known as Brn2 . r , Pearson correlation coefficient. h , Correlation plots between aligned counts from single sequenced libraries of a Neurog3/Pou3f2 (10 ng input RNA) population and an Ascl1/Pou3f2 (10 ng input RNA) population. i – n , Representative images taken while dissecting tissue from various brain regions of appropriate mouse reporter strains used to isolate specific endogenous cell-type populations used for RNA-seq: cerebellum (CER) ( i ), DRG ( j ), cortex (CTX) ( k ), olfactory bulb mitral and tufted cells (OB-MT) and olfactory bulb granule cells (OB-GC) ( l ), hippocampus (HIP) ( m ), and dorsal-medial habenula (MHb-d) and ventral-medial habenula (MHb-v) ( n ). n = 2 independent RNA-seq experiments. o , Characteristics of the endogenous neuron populations used for RNA-seq.
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    Thermo Fisher total rna extraction
    <t>FACS,</t> <t>RNA-seq</t> library preparation and characterization of iN cell and endogenous neuron populations .a , Representative immunofluorescence labelling of tau–EGFP + iN cell population ( Ascl2/Pou4f2 ) on day 12 post-induction using neuronal antibodies TUJ1 and MAP2. Scale bars, 100 μm. Pou4f2 is also known as Brn3b . b , Quantification of co-labelling of tau–eGFP and MAP2 in Tuj1 + cells on day 12 post-induction calculated from various reprogramming transcription factor pairs. Data are presented as mean ± s.d. from n = 4 independent experiments and n = 574 cells. c , d , Representative FACS gates of an Ascl2/Pou4f2 iN cell population (500,000 cells shown) ( c ) and a negative rtTA-only control (40,000 cells shown) ( d ) sorted on day 16 post-induction. Live tau–eGFP + cells were enriched by first gating DRAQ5 + DAPI – cells, then collecting only those that were GFP + . For Ascl2/Pou4f2, n = 2 independent experiments showed similar results, while for rtTA only, n = 40 independent experiments showed similar results. For all other iN cell populations, at least n = 2 independent experiments were performed to obtain biological replicates. e , Per cent of tau–eGFP + cells out of total number of cells collected post-FACS, presented as mean ± s.d. ( n = 4 sorts, > 100 cells per sort). f , g , Correlation plots between aligned counts from single sequenced libraries of a Neurog3/Pou3f2-iN cell population generated from 10 ng versus 5 ng input RNA ( f ) and 10 ng versus 1 ng input RNA ( g ). Pou3f2 is also known as Brn2 . r , Pearson correlation coefficient. h , Correlation plots between aligned counts from single sequenced libraries of a Neurog3/Pou3f2 (10 ng input RNA) population and an Ascl1/Pou3f2 (10 ng input RNA) population. i – n , Representative images taken while dissecting tissue from various brain regions of appropriate mouse reporter strains used to isolate specific endogenous cell-type populations used for RNA-seq: cerebellum (CER) ( i ), DRG ( j ), cortex (CTX) ( k ), olfactory bulb mitral and tufted cells (OB-MT) and olfactory bulb granule cells (OB-GC) ( l ), hippocampus (HIP) ( m ), and dorsal-medial habenula (MHb-d) and ventral-medial habenula (MHb-v) ( n ). n = 2 independent RNA-seq experiments. o , Characteristics of the endogenous neuron populations used for RNA-seq.
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    Thermo Fisher rna extraction total rna
    <t>FACS,</t> <t>RNA-seq</t> library preparation and characterization of iN cell and endogenous neuron populations .a , Representative immunofluorescence labelling of tau–EGFP + iN cell population ( Ascl2/Pou4f2 ) on day 12 post-induction using neuronal antibodies TUJ1 and MAP2. Scale bars, 100 μm. Pou4f2 is also known as Brn3b . b , Quantification of co-labelling of tau–eGFP and MAP2 in Tuj1 + cells on day 12 post-induction calculated from various reprogramming transcription factor pairs. Data are presented as mean ± s.d. from n = 4 independent experiments and n = 574 cells. c , d , Representative FACS gates of an Ascl2/Pou4f2 iN cell population (500,000 cells shown) ( c ) and a negative rtTA-only control (40,000 cells shown) ( d ) sorted on day 16 post-induction. Live tau–eGFP + cells were enriched by first gating DRAQ5 + DAPI – cells, then collecting only those that were GFP + . For Ascl2/Pou4f2, n = 2 independent experiments showed similar results, while for rtTA only, n = 40 independent experiments showed similar results. For all other iN cell populations, at least n = 2 independent experiments were performed to obtain biological replicates. e , Per cent of tau–eGFP + cells out of total number of cells collected post-FACS, presented as mean ± s.d. ( n = 4 sorts, > 100 cells per sort). f , g , Correlation plots between aligned counts from single sequenced libraries of a Neurog3/Pou3f2-iN cell population generated from 10 ng versus 5 ng input RNA ( f ) and 10 ng versus 1 ng input RNA ( g ). Pou3f2 is also known as Brn2 . r , Pearson correlation coefficient. h , Correlation plots between aligned counts from single sequenced libraries of a Neurog3/Pou3f2 (10 ng input RNA) population and an Ascl1/Pou3f2 (10 ng input RNA) population. i – n , Representative images taken while dissecting tissue from various brain regions of appropriate mouse reporter strains used to isolate specific endogenous cell-type populations used for RNA-seq: cerebellum (CER) ( i ), DRG ( j ), cortex (CTX) ( k ), olfactory bulb mitral and tufted cells (OB-MT) and olfactory bulb granule cells (OB-GC) ( l ), hippocampus (HIP) ( m ), and dorsal-medial habenula (MHb-d) and ventral-medial habenula (MHb-v) ( n ). n = 2 independent RNA-seq experiments. o , Characteristics of the endogenous neuron populations used for RNA-seq.
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    <t>FACS,</t> <t>RNA-seq</t> library preparation and characterization of iN cell and endogenous neuron populations .a , Representative immunofluorescence labelling of tau–EGFP + iN cell population ( Ascl2/Pou4f2 ) on day 12 post-induction using neuronal antibodies TUJ1 and MAP2. Scale bars, 100 μm. Pou4f2 is also known as Brn3b . b , Quantification of co-labelling of tau–eGFP and MAP2 in Tuj1 + cells on day 12 post-induction calculated from various reprogramming transcription factor pairs. Data are presented as mean ± s.d. from n = 4 independent experiments and n = 574 cells. c , d , Representative FACS gates of an Ascl2/Pou4f2 iN cell population (500,000 cells shown) ( c ) and a negative rtTA-only control (40,000 cells shown) ( d ) sorted on day 16 post-induction. Live tau–eGFP + cells were enriched by first gating DRAQ5 + DAPI – cells, then collecting only those that were GFP + . For Ascl2/Pou4f2, n = 2 independent experiments showed similar results, while for rtTA only, n = 40 independent experiments showed similar results. For all other iN cell populations, at least n = 2 independent experiments were performed to obtain biological replicates. e , Per cent of tau–eGFP + cells out of total number of cells collected post-FACS, presented as mean ± s.d. ( n = 4 sorts, > 100 cells per sort). f , g , Correlation plots between aligned counts from single sequenced libraries of a Neurog3/Pou3f2-iN cell population generated from 10 ng versus 5 ng input RNA ( f ) and 10 ng versus 1 ng input RNA ( g ). Pou3f2 is also known as Brn2 . r , Pearson correlation coefficient. h , Correlation plots between aligned counts from single sequenced libraries of a Neurog3/Pou3f2 (10 ng input RNA) population and an Ascl1/Pou3f2 (10 ng input RNA) population. i – n , Representative images taken while dissecting tissue from various brain regions of appropriate mouse reporter strains used to isolate specific endogenous cell-type populations used for RNA-seq: cerebellum (CER) ( i ), DRG ( j ), cortex (CTX) ( k ), olfactory bulb mitral and tufted cells (OB-MT) and olfactory bulb granule cells (OB-GC) ( l ), hippocampus (HIP) ( m ), and dorsal-medial habenula (MHb-d) and ventral-medial habenula (MHb-v) ( n ). n = 2 independent RNA-seq experiments. o , Characteristics of the endogenous neuron populations used for RNA-seq.
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    Promega eastep super total rna extraction kit
    <t>FACS,</t> <t>RNA-seq</t> library preparation and characterization of iN cell and endogenous neuron populations .a , Representative immunofluorescence labelling of tau–EGFP + iN cell population ( Ascl2/Pou4f2 ) on day 12 post-induction using neuronal antibodies TUJ1 and MAP2. Scale bars, 100 μm. Pou4f2 is also known as Brn3b . b , Quantification of co-labelling of tau–eGFP and MAP2 in Tuj1 + cells on day 12 post-induction calculated from various reprogramming transcription factor pairs. Data are presented as mean ± s.d. from n = 4 independent experiments and n = 574 cells. c , d , Representative FACS gates of an Ascl2/Pou4f2 iN cell population (500,000 cells shown) ( c ) and a negative rtTA-only control (40,000 cells shown) ( d ) sorted on day 16 post-induction. Live tau–eGFP + cells were enriched by first gating DRAQ5 + DAPI – cells, then collecting only those that were GFP + . For Ascl2/Pou4f2, n = 2 independent experiments showed similar results, while for rtTA only, n = 40 independent experiments showed similar results. For all other iN cell populations, at least n = 2 independent experiments were performed to obtain biological replicates. e , Per cent of tau–eGFP + cells out of total number of cells collected post-FACS, presented as mean ± s.d. ( n = 4 sorts, > 100 cells per sort). f , g , Correlation plots between aligned counts from single sequenced libraries of a Neurog3/Pou3f2-iN cell population generated from 10 ng versus 5 ng input RNA ( f ) and 10 ng versus 1 ng input RNA ( g ). Pou3f2 is also known as Brn2 . r , Pearson correlation coefficient. h , Correlation plots between aligned counts from single sequenced libraries of a Neurog3/Pou3f2 (10 ng input RNA) population and an Ascl1/Pou3f2 (10 ng input RNA) population. i – n , Representative images taken while dissecting tissue from various brain regions of appropriate mouse reporter strains used to isolate specific endogenous cell-type populations used for RNA-seq: cerebellum (CER) ( i ), DRG ( j ), cortex (CTX) ( k ), olfactory bulb mitral and tufted cells (OB-MT) and olfactory bulb granule cells (OB-GC) ( l ), hippocampus (HIP) ( m ), and dorsal-medial habenula (MHb-d) and ventral-medial habenula (MHb-v) ( n ). n = 2 independent RNA-seq experiments. o , Characteristics of the endogenous neuron populations used for RNA-seq.
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    Thermo Fisher total rna extraction total rna
    Global gene expression analysis during neuronal differentiation a , Principal component (PC) analysis plot of embryonic stem cells (ES), induced pluripotent stem cells (iPS), neuronal progenitor cells (NPC) and neurons (NE) for TD, WS and pWS88. c , Euclidian matrix distance-based heatmap and hierarchical clustering-based dendrogram of ES, NPC and NE cells for WD, WS and pWS88 samples. Expression variability between samples is indicated by Z-score, varying from green (negative variation) to red (positive variation). c , Euclidian matrix distance-based heatmap and hierarchical clustering-based dendrogram of pluripotency gene markers for ES, NPC and NE cells for TD, WS and pWS88 samples. d , Euclidian matrix distance-based heatmap and hierarchical clustering-based dendrogram of neuronal gene markers for iPS, NPC and NE cells for TD, WS and pWS88 samples. Expression variability between samples is indicated by Z-score, varying from green (negative variation) to red (positive variation). e , Specific cell type-based clustering analysis of biological replicates subjected to <t>RNA-seq</t> for the WS-related genes in three stages during differentiation (iPS, NPC and NE). f , Fold change variation of WS-related genes in different cell lines. Ideogram of chromosome 7 (band 7q11.23) corresponding to the commonly deleted region with the WS-related genes. Fold change variation of normalized WS-related gene expression in <t>NPCs</t> and neurons (NE) compared to TDs. Non-represented fold change corresponds to those genes having high expression variability between biological replicates, or having very low expression values. g , Expression of FZD9 gene in iPSC, NPCs and neurons from TD and WS. Error bars are represented by standard error. h , Venn diagram showing correlation of significant differentially expressed genes between TD, pWS88 and WS during neuronal differentiation. Significantly enriched GO terms found for down-regulated (red histogram) and up-regulated (blue histogram) differentially expressed genes between TD and WS in NPC. Significantly enriched GO terms found for down-regulated (red histogram) and up-regulated (blue histogram) differentially expressed genes between TD and WS in neurons (NE). Vertical line (black) corresponds to significant p-value (0.05). i , Enriched GO metabolic process terms found in NPC of WS samples correlated with the GO found by a similar comparison performed by Adamo et al. (2014) 13 .
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    iNtRON Biotechnology easy blue total rna extraction kit
    Retrograde vacuole trafficking controls the pathogenicity of Cryptococcus neoformans . Various tests were performed using WT strain (H99S) and vps15 Δ mutants (YSB1500 and YSB1501) ( a ) Vps15 is required for virulence of C. neoformans . WT and PBS were used as positive and negative virulence controls, respectively. ( b ) vps15 Δ mutants display enlarged vacuole morphology. Scale bars,10 μm. ( c ) vps15 Δ mutants show significant growth defect under ER stresses. Overnight cultured cells were serially diluted tenfold (undiluted to 10 4 -fold dilution), spotted on the solid YPD medium containing 15 mM dithiothreitol (DTT) or 0.3 μg ml −1 tunicamycin (TM), further incubated at 30 °C for 3 days, and photographed. ( d ) vps15 Δ mutants show significant growth defects at high temperature and under cell membrane/wall stresses. Overnight cultured cells were spotted on the YPD medium and further incubated at indicated temperature (upper panel) or the YPD medium containing 0.03% SDS or 5 mg ml −1 calcofluor white (CFW) and further incubated at 30 °C (lower panel). Plates were photographed after 3 days. ( e ) Vps15 is not involved in the regulation of the calcineurin pathway in C. neoformans . For quantitative RT–PCR (qRT–PCR), <t>RNA</t> was extracted from three biological replicates with three technical replicates of WT and vps15 Δ mutants. CNA1 , CNB1 , CRZ1 , UTR2 expression levels were normalized by ACT1 expression levels as controls. Error bars represent s.e.m. ( f ) Vps15 negatively regulates the HXL1 splicing. For RT–PCR, total RNA was extracted from WT and vps15 Δ mutants and <t>cDNA</t> was synthesized. HXL1 and ACT1 -specific primer pairs were used for RT–PCR. This experiment was repeated twice and one representative experiment is presented. The whole-gel images were displayed on Supplementary Fig. 6e .
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    Inhibiting sirt1 decreased the sirt1 increase caused by EGCG Sirt1 small interfering <t>RNA</t> (siSirt1) or negative control siRNA (NC) transfected <t>SH-SY5Y</t> cells were incubated with EGCG (10 μM) for 30 hr. Western blot for sirt1 and acetyl-p53 proteins was conducted from SH-SY5Y cells. β-actin was used as the loading control A . Relative sirt1 mRNA expression levels were analyzed using quantitative real-time polymerase chain reaction. The indicated relative gene expression level shows expression levels that were normalized to β-actin expression as the standard B . siSirt1 or NC transfected SH-SY5Y cells were pre-incubated with EGCG (10 μM) for 1 hr and then exposed to PrP (106-126) for 30 hr. Sirt1 deacetylase activities were analyzed in SH-SY5Y cell nuclei. C . Bars indicate mean ± standard error ( n = 4). * p
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    tiangen biotech co total rna extraction kit
    Overexpression of SiMYB56 significantly improves ABA accumulation in transgenic rice seeds and activates the expression of ABA signaling pathway related genes under drought condition. (A) Seeds of wild-type controls and transgenic rice plants were germinated on plates containing different ABA concentrations. Germination rates of SiMYB56-overexpressing and wide-type seeds under 0 μM (B) , 2.5 μM (C) , and 5 μM (D) ABA treatments ( n = 3, 16 seeds in each replicate). (E) ABA content of sprouts from germinated seeds of transgenic rice plants and wide-type controls under 0 μM ABA treatments. Values shown are means ± SD ( n = 3). (F) Expression analysis of ABA signaling pathway related genes in wild-type controls and transgenic rice plants. Entire 2-week-old SiMYB56- overexpressing and wild-type seedlings, grown under normal condition and 10% PEG6000 treatments, were harvested for <t>RNA</t> isolation. Transcript level was quantified by <t>qRT-PCR.</t> Values shown are means ± SD ( n = 3).
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    Expression of MMP13 in ovaries from 60-, 90-, 123-, and 159-d-old hens. (A) Messenger <t>RNA</t> expression of MMP13 was analyzed by real-time <t>PCR.</t> (B) Expression of MMP13 protein was analyzed by Western blot analysis. A band of approximately 48 kDa corresponding to the molecular mass of MMP13 was detected. β-actin (41 kDa) was used as the loading control. Data are presented as mean ± SEM from at least four independent experiments. Bars with different superscript letters are significantly different ( P
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    Thermo Fisher total rna extractions
    Expression of MMP13 in ovaries from 60-, 90-, 123-, and 159-d-old hens. (A) Messenger <t>RNA</t> expression of MMP13 was analyzed by real-time <t>PCR.</t> (B) Expression of MMP13 protein was analyzed by Western blot analysis. A band of approximately 48 kDa corresponding to the molecular mass of MMP13 was detected. β-actin (41 kDa) was used as the loading control. Data are presented as mean ± SEM from at least four independent experiments. Bars with different superscript letters are significantly different ( P
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    Illumina Inc total rna extraction
    Phylogenetic analysis of the bat bornavirus-related sequences. (A) Schematic representation of the L gene (almost 5,140 nt encoding the <t>RNA-dependent</t> RNA polymerase of almost 1,710 aa) of the genome of Borna disease virus isolate bil (GenBank number ACG59353), with black and dark gray bars corresponding to the three bat bornavirus HSPs identified in samples b1 and b6, respectively. The position of the genome region amplified by <t>PCR</t> is represented by a dashed bar, and the sequence used for phylogenetic analysis is indicated with an asterisk. (B) Phylogenetic tree produced from the nucleotide alignment based on a selected region (214–216 nt, approximate position 3233–3446 of the L gene sequence of Borna disease virus isolate bil) of the sequence obtained from the PCR product. Bat bornavirus-related sequences are indicated in bold. The scale bar indicates branch length, and bootstrap values ≥70% are shown next to the relevant nodes. The tree is midpoint-rooted for purposes of clarity only.
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    Qiagen rneasy total rna extraction kit
    Phylogenetic analysis of the bat bornavirus-related sequences. (A) Schematic representation of the L gene (almost 5,140 nt encoding the <t>RNA-dependent</t> RNA polymerase of almost 1,710 aa) of the genome of Borna disease virus isolate bil (GenBank number ACG59353), with black and dark gray bars corresponding to the three bat bornavirus HSPs identified in samples b1 and b6, respectively. The position of the genome region amplified by <t>PCR</t> is represented by a dashed bar, and the sequence used for phylogenetic analysis is indicated with an asterisk. (B) Phylogenetic tree produced from the nucleotide alignment based on a selected region (214–216 nt, approximate position 3233–3446 of the L gene sequence of Borna disease virus isolate bil) of the sequence obtained from the PCR product. Bat bornavirus-related sequences are indicated in bold. The scale bar indicates branch length, and bootstrap values ≥70% are shown next to the relevant nodes. The tree is midpoint-rooted for purposes of clarity only.
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    Inhibitory effect of MUPCDH on cell proliferation. ( a ) Knockdown of the MUPCDH gene increased cell survival in human renal cortical epithelial (HRCE) cells. ( b ) Overexpression of the MUPCDH gene decreased cell growth of WT9-7 cells. ( c ) 5-bromo-2′-deoxyuridine (BrdU) incorporation assays were carried out using HRCE cells that had been transfected with MUPCDH short interfering <t>RNA</t> (siRNA) or control siRNA. ( d ) Proliferating cell nuclear antigen (PCNA) staining carried out using HEK293T cells that overexpressed the MUPCDH gene. The level of PCNA expression was also measured by real-time quantitative reverse transcription polymerase chain reaction <t>(qRT-PCR).</t> β-Actin was used as an internal control in real-time qRT-PCR assays. ( e ) Cell proliferation rate was measured using a cell proliferation assay kit (WST-1) following treatment of WT9-7 cells with 5-aza-2′-deoxycytidine (5 μM). The experiment was performed in triplicate. * P
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    CHIKV mos has reduced attachment to host cells than CHIKV vero. ( A), Equal PFUs of CHIKV vero or CHIKV mos (Ross strain) were added to Vero cells (left), NIH3T3 cells (middle) and L929 cells (right) for plaque development. (B), Plaque counts of CHIKV vero and CHIKV mos in the indicated cells were quantified. (C) L929, NIH3T3 and HFF cells were inoculated with CHIKV vero or CHIKV mos (Ross strain, MOI = 1) at 4°C for 1 h and attached viruses were quantified by <t>RT-qPCR</t> and presented as the ratio of CHIKV E1 copy number per 1000 copy of cellular β-actin . (D) L929, NIH3T3 and HFF cells were inoculated with CHIKV vero or CHIKV mos (LR OPY1, MOI = 1) at 4°C for 1 h and viruses attached to cells were quantified by RT-qPCR. (E) NIH3T3 cells were infected with CHIKV vero or CHIKV mos (Ross strain, MOI = 2.5) at 4°C for 45 min and the virus-bound cells were quantified by flow cytometry. (F) CHIKV vero or CHIKV mos (100 PFUs) were added to monolayer of L929 cells at 4°C for 1 h and both attached and unattached viruses were quantified by plaque assay to calculate percentage attachment. (G) CHIKV vero or CHIKV mos (Ross strain, 100 PFUs) were added to the L929 cell monolayer at 4°C for 1 h. After removing unattached virus, cells were replaced with fresh medium (control), or treated with acidic medium (pH 5.5) for 2 minutes before adding fresh medium, or replaced with NH 4 Cl (20 mM) containing media. Viruses that entered into cells were analyzed by allowing plaque development for 48 h and normalized to controls. (H) Viral <t>RNA</t> copies in L929 cells infected with CHIKV vero or CHIKV mos (Ross strain, MOI = 1) with or without NH 4 Cl (20 mM) were measured by RT-qPCR at 24 h.p.i. Data represent at least two independent experiments performed in triplicates with the similar results. ND denotes not detected, and ns denotes not significant ( p ≥ 0.05).
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    RpoE1 regulates positively the expression of hrp gene cluster ( hrpA - F ), type III secreted effector-encoding genes ( XC_0241 and XC_1553 ) and the hrp master regulator hrpX . (A) Quantitative real-time PCR analysis of the transcription of hrpA - F, hrpG, hrpX, XC_0241 , and XC_1553 in the <t>Xcc</t> wild-type strain 8004 and the rpoE1 deletion mutant strain Δ rpoE1 . <t>RNA</t> was isolated from cultures of the strains grown in XCM1 medium for 24 h. The relative mRNA level was calculated with respect to the level of the corresponding transcript in the wild-type strain 8004. (B) GUS activity of hrpB, hrpF, hrpG, hrpX, XC_0241 , and XC_1553 promoter- gusA reporters (pLgushrpB, pLgushrpF, pLgushrpG, pLgushrpX, pLgus0241, and pLgus1553) in the wild-type strain 8004 and the mutant strain Δ rpoE1 . The strains were cultured in XCM1 medium for 24 h and GUS activity in the total culture was determined by using ρ-nitrophenyl-β-D-glucuronide as substrate. Values given are means ± standard deviations of triplicate measurements from a representative experiment; similar results were obtained in two other independent experiments. Asterisks indicate statistically significant difference, compared with the wild type (Student's t -test). ** P
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    Global organization and functionality of the newly identified 28-kb GI harboring β-myrcene core-code. ( A ) Genomic locus organization and functional elements detected using in silico prediction. Genes transcribed in forward orientation are represented by rightward arrows, whereas genes located in antisense strand are represented by leftward arrows. Genes are organized in TU, promoter regions (P) are illustrated by blue arrows, and terminator sites indicated by red circles. ( B ) Detailed analysis of the <t>RNA-seq</t> data mapping with the GI. In the top panel, read mapping coverage per base was plotted for samples derived from <t>M1</t> growth in lactate (gray line) and in β-myrcene (blue line). Prediction of the operon-like transcripts (horizontal orange bars) and transcription starting sites (TSS) (red arrows) was carried out with ReadXplorer ( Hilker et al. 2014 ). Expression levels for each gene are shown, in the bottom panel, as the ratio (FC) of normalized transcripts between cells grown in lactate (L) and cells grown in β-myrcene (M).
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    Global organization and functionality of the newly identified 28-kb GI harboring β-myrcene core-code. ( A ) Genomic locus organization and functional elements detected using in silico prediction. Genes transcribed in forward orientation are represented by rightward arrows, whereas genes located in antisense strand are represented by leftward arrows. Genes are organized in TU, promoter regions (P) are illustrated by blue arrows, and terminator sites indicated by red circles. ( B ) Detailed analysis of the <t>RNA-seq</t> data mapping with the GI. In the top panel, read mapping coverage per base was plotted for samples derived from <t>M1</t> growth in lactate (gray line) and in β-myrcene (blue line). Prediction of the operon-like transcripts (horizontal orange bars) and transcription starting sites (TSS) (red arrows) was carried out with ReadXplorer ( Hilker et al. 2014 ). Expression levels for each gene are shown, in the bottom panel, as the ratio (FC) of normalized transcripts between cells grown in lactate (L) and cells grown in β-myrcene (M).
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    Image Search Results


    FACS, RNA-seq library preparation and characterization of iN cell and endogenous neuron populations .a , Representative immunofluorescence labelling of tau–EGFP + iN cell population ( Ascl2/Pou4f2 ) on day 12 post-induction using neuronal antibodies TUJ1 and MAP2. Scale bars, 100 μm. Pou4f2 is also known as Brn3b . b , Quantification of co-labelling of tau–eGFP and MAP2 in Tuj1 + cells on day 12 post-induction calculated from various reprogramming transcription factor pairs. Data are presented as mean ± s.d. from n = 4 independent experiments and n = 574 cells. c , d , Representative FACS gates of an Ascl2/Pou4f2 iN cell population (500,000 cells shown) ( c ) and a negative rtTA-only control (40,000 cells shown) ( d ) sorted on day 16 post-induction. Live tau–eGFP + cells were enriched by first gating DRAQ5 + DAPI – cells, then collecting only those that were GFP + . For Ascl2/Pou4f2, n = 2 independent experiments showed similar results, while for rtTA only, n = 40 independent experiments showed similar results. For all other iN cell populations, at least n = 2 independent experiments were performed to obtain biological replicates. e , Per cent of tau–eGFP + cells out of total number of cells collected post-FACS, presented as mean ± s.d. ( n = 4 sorts, > 100 cells per sort). f , g , Correlation plots between aligned counts from single sequenced libraries of a Neurog3/Pou3f2-iN cell population generated from 10 ng versus 5 ng input RNA ( f ) and 10 ng versus 1 ng input RNA ( g ). Pou3f2 is also known as Brn2 . r , Pearson correlation coefficient. h , Correlation plots between aligned counts from single sequenced libraries of a Neurog3/Pou3f2 (10 ng input RNA) population and an Ascl1/Pou3f2 (10 ng input RNA) population. i – n , Representative images taken while dissecting tissue from various brain regions of appropriate mouse reporter strains used to isolate specific endogenous cell-type populations used for RNA-seq: cerebellum (CER) ( i ), DRG ( j ), cortex (CTX) ( k ), olfactory bulb mitral and tufted cells (OB-MT) and olfactory bulb granule cells (OB-GC) ( l ), hippocampus (HIP) ( m ), and dorsal-medial habenula (MHb-d) and ventral-medial habenula (MHb-v) ( n ). n = 2 independent RNA-seq experiments. o , Characteristics of the endogenous neuron populations used for RNA-seq.

    Journal: Nature

    Article Title: Diverse reprogramming codes for neuronal identity

    doi: 10.1038/s41586-018-0103-5

    Figure Lengend Snippet: FACS, RNA-seq library preparation and characterization of iN cell and endogenous neuron populations .a , Representative immunofluorescence labelling of tau–EGFP + iN cell population ( Ascl2/Pou4f2 ) on day 12 post-induction using neuronal antibodies TUJ1 and MAP2. Scale bars, 100 μm. Pou4f2 is also known as Brn3b . b , Quantification of co-labelling of tau–eGFP and MAP2 in Tuj1 + cells on day 12 post-induction calculated from various reprogramming transcription factor pairs. Data are presented as mean ± s.d. from n = 4 independent experiments and n = 574 cells. c , d , Representative FACS gates of an Ascl2/Pou4f2 iN cell population (500,000 cells shown) ( c ) and a negative rtTA-only control (40,000 cells shown) ( d ) sorted on day 16 post-induction. Live tau–eGFP + cells were enriched by first gating DRAQ5 + DAPI – cells, then collecting only those that were GFP + . For Ascl2/Pou4f2, n = 2 independent experiments showed similar results, while for rtTA only, n = 40 independent experiments showed similar results. For all other iN cell populations, at least n = 2 independent experiments were performed to obtain biological replicates. e , Per cent of tau–eGFP + cells out of total number of cells collected post-FACS, presented as mean ± s.d. ( n = 4 sorts, > 100 cells per sort). f , g , Correlation plots between aligned counts from single sequenced libraries of a Neurog3/Pou3f2-iN cell population generated from 10 ng versus 5 ng input RNA ( f ) and 10 ng versus 1 ng input RNA ( g ). Pou3f2 is also known as Brn2 . r , Pearson correlation coefficient. h , Correlation plots between aligned counts from single sequenced libraries of a Neurog3/Pou3f2 (10 ng input RNA) population and an Ascl1/Pou3f2 (10 ng input RNA) population. i – n , Representative images taken while dissecting tissue from various brain regions of appropriate mouse reporter strains used to isolate specific endogenous cell-type populations used for RNA-seq: cerebellum (CER) ( i ), DRG ( j ), cortex (CTX) ( k ), olfactory bulb mitral and tufted cells (OB-MT) and olfactory bulb granule cells (OB-GC) ( l ), hippocampus (HIP) ( m ), and dorsal-medial habenula (MHb-d) and ventral-medial habenula (MHb-v) ( n ). n = 2 independent RNA-seq experiments. o , Characteristics of the endogenous neuron populations used for RNA-seq.

    Article Snippet: Total RNA was isolated from FACS-sorted cells using Direct-zol RNA MiniPrep Kit (Zymo Rsearch) according to the manufacturer’s protocol, except linearized acrylamide (1 μg) was added to each sample before the first step and Zymo-Spin IC columns were used in replacement of Zymo-Spin IIC columns.

    Techniques: FACS, RNA Sequencing Assay, Immunofluorescence, Generated

    Global gene expression analysis during neuronal differentiation a , Principal component (PC) analysis plot of embryonic stem cells (ES), induced pluripotent stem cells (iPS), neuronal progenitor cells (NPC) and neurons (NE) for TD, WS and pWS88. c , Euclidian matrix distance-based heatmap and hierarchical clustering-based dendrogram of ES, NPC and NE cells for WD, WS and pWS88 samples. Expression variability between samples is indicated by Z-score, varying from green (negative variation) to red (positive variation). c , Euclidian matrix distance-based heatmap and hierarchical clustering-based dendrogram of pluripotency gene markers for ES, NPC and NE cells for TD, WS and pWS88 samples. d , Euclidian matrix distance-based heatmap and hierarchical clustering-based dendrogram of neuronal gene markers for iPS, NPC and NE cells for TD, WS and pWS88 samples. Expression variability between samples is indicated by Z-score, varying from green (negative variation) to red (positive variation). e , Specific cell type-based clustering analysis of biological replicates subjected to RNA-seq for the WS-related genes in three stages during differentiation (iPS, NPC and NE). f , Fold change variation of WS-related genes in different cell lines. Ideogram of chromosome 7 (band 7q11.23) corresponding to the commonly deleted region with the WS-related genes. Fold change variation of normalized WS-related gene expression in NPCs and neurons (NE) compared to TDs. Non-represented fold change corresponds to those genes having high expression variability between biological replicates, or having very low expression values. g , Expression of FZD9 gene in iPSC, NPCs and neurons from TD and WS. Error bars are represented by standard error. h , Venn diagram showing correlation of significant differentially expressed genes between TD, pWS88 and WS during neuronal differentiation. Significantly enriched GO terms found for down-regulated (red histogram) and up-regulated (blue histogram) differentially expressed genes between TD and WS in NPC. Significantly enriched GO terms found for down-regulated (red histogram) and up-regulated (blue histogram) differentially expressed genes between TD and WS in neurons (NE). Vertical line (black) corresponds to significant p-value (0.05). i , Enriched GO metabolic process terms found in NPC of WS samples correlated with the GO found by a similar comparison performed by Adamo et al. (2014) 13 .

    Journal: Nature

    Article Title: A human neurodevelopmental model for Williams syndrome

    doi: 10.1038/nature19067

    Figure Lengend Snippet: Global gene expression analysis during neuronal differentiation a , Principal component (PC) analysis plot of embryonic stem cells (ES), induced pluripotent stem cells (iPS), neuronal progenitor cells (NPC) and neurons (NE) for TD, WS and pWS88. c , Euclidian matrix distance-based heatmap and hierarchical clustering-based dendrogram of ES, NPC and NE cells for WD, WS and pWS88 samples. Expression variability between samples is indicated by Z-score, varying from green (negative variation) to red (positive variation). c , Euclidian matrix distance-based heatmap and hierarchical clustering-based dendrogram of pluripotency gene markers for ES, NPC and NE cells for TD, WS and pWS88 samples. d , Euclidian matrix distance-based heatmap and hierarchical clustering-based dendrogram of neuronal gene markers for iPS, NPC and NE cells for TD, WS and pWS88 samples. Expression variability between samples is indicated by Z-score, varying from green (negative variation) to red (positive variation). e , Specific cell type-based clustering analysis of biological replicates subjected to RNA-seq for the WS-related genes in three stages during differentiation (iPS, NPC and NE). f , Fold change variation of WS-related genes in different cell lines. Ideogram of chromosome 7 (band 7q11.23) corresponding to the commonly deleted region with the WS-related genes. Fold change variation of normalized WS-related gene expression in NPCs and neurons (NE) compared to TDs. Non-represented fold change corresponds to those genes having high expression variability between biological replicates, or having very low expression values. g , Expression of FZD9 gene in iPSC, NPCs and neurons from TD and WS. Error bars are represented by standard error. h , Venn diagram showing correlation of significant differentially expressed genes between TD, pWS88 and WS during neuronal differentiation. Significantly enriched GO terms found for down-regulated (red histogram) and up-regulated (blue histogram) differentially expressed genes between TD and WS in NPC. Significantly enriched GO terms found for down-regulated (red histogram) and up-regulated (blue histogram) differentially expressed genes between TD and WS in neurons (NE). Vertical line (black) corresponds to significant p-value (0.05). i , Enriched GO metabolic process terms found in NPC of WS samples correlated with the GO found by a similar comparison performed by Adamo et al. (2014) 13 .

    Article Snippet: Total RNA extraction Total RNA of DPCs, iPSCs, NPCs and neurons was extracted using TRIzol reagent (Life Technologies) according to the manufacturer’s protocols.

    Techniques: Expressing, RNA Sequencing Assay

    Retrograde vacuole trafficking controls the pathogenicity of Cryptococcus neoformans . Various tests were performed using WT strain (H99S) and vps15 Δ mutants (YSB1500 and YSB1501) ( a ) Vps15 is required for virulence of C. neoformans . WT and PBS were used as positive and negative virulence controls, respectively. ( b ) vps15 Δ mutants display enlarged vacuole morphology. Scale bars,10 μm. ( c ) vps15 Δ mutants show significant growth defect under ER stresses. Overnight cultured cells were serially diluted tenfold (undiluted to 10 4 -fold dilution), spotted on the solid YPD medium containing 15 mM dithiothreitol (DTT) or 0.3 μg ml −1 tunicamycin (TM), further incubated at 30 °C for 3 days, and photographed. ( d ) vps15 Δ mutants show significant growth defects at high temperature and under cell membrane/wall stresses. Overnight cultured cells were spotted on the YPD medium and further incubated at indicated temperature (upper panel) or the YPD medium containing 0.03% SDS or 5 mg ml −1 calcofluor white (CFW) and further incubated at 30 °C (lower panel). Plates were photographed after 3 days. ( e ) Vps15 is not involved in the regulation of the calcineurin pathway in C. neoformans . For quantitative RT–PCR (qRT–PCR), RNA was extracted from three biological replicates with three technical replicates of WT and vps15 Δ mutants. CNA1 , CNB1 , CRZ1 , UTR2 expression levels were normalized by ACT1 expression levels as controls. Error bars represent s.e.m. ( f ) Vps15 negatively regulates the HXL1 splicing. For RT–PCR, total RNA was extracted from WT and vps15 Δ mutants and cDNA was synthesized. HXL1 and ACT1 -specific primer pairs were used for RT–PCR. This experiment was repeated twice and one representative experiment is presented. The whole-gel images were displayed on Supplementary Fig. 6e .

    Journal: Nature Communications

    Article Title: Systematic functional analysis of kinases in the fungal pathogen Cryptococcus neoformans

    doi: 10.1038/ncomms12766

    Figure Lengend Snippet: Retrograde vacuole trafficking controls the pathogenicity of Cryptococcus neoformans . Various tests were performed using WT strain (H99S) and vps15 Δ mutants (YSB1500 and YSB1501) ( a ) Vps15 is required for virulence of C. neoformans . WT and PBS were used as positive and negative virulence controls, respectively. ( b ) vps15 Δ mutants display enlarged vacuole morphology. Scale bars,10 μm. ( c ) vps15 Δ mutants show significant growth defect under ER stresses. Overnight cultured cells were serially diluted tenfold (undiluted to 10 4 -fold dilution), spotted on the solid YPD medium containing 15 mM dithiothreitol (DTT) or 0.3 μg ml −1 tunicamycin (TM), further incubated at 30 °C for 3 days, and photographed. ( d ) vps15 Δ mutants show significant growth defects at high temperature and under cell membrane/wall stresses. Overnight cultured cells were spotted on the YPD medium and further incubated at indicated temperature (upper panel) or the YPD medium containing 0.03% SDS or 5 mg ml −1 calcofluor white (CFW) and further incubated at 30 °C (lower panel). Plates were photographed after 3 days. ( e ) Vps15 is not involved in the regulation of the calcineurin pathway in C. neoformans . For quantitative RT–PCR (qRT–PCR), RNA was extracted from three biological replicates with three technical replicates of WT and vps15 Δ mutants. CNA1 , CNB1 , CRZ1 , UTR2 expression levels were normalized by ACT1 expression levels as controls. Error bars represent s.e.m. ( f ) Vps15 negatively regulates the HXL1 splicing. For RT–PCR, total RNA was extracted from WT and vps15 Δ mutants and cDNA was synthesized. HXL1 and ACT1 -specific primer pairs were used for RT–PCR. This experiment was repeated twice and one representative experiment is presented. The whole-gel images were displayed on Supplementary Fig. 6e .

    Article Snippet: Total RNAs were extracted using easy-BLUE (Total RNA Extraction Kit, Intron Biotechnology) and subsequently complementary DNA (cDNA) was synthesized using an MMLV reverse transcriptase (Invitrogen).

    Techniques: Cell Culture, Incubation, Quantitative RT-PCR, Expressing, Reverse Transcription Polymerase Chain Reaction, Synthesized

    Inhibiting sirt1 decreased the sirt1 increase caused by EGCG Sirt1 small interfering RNA (siSirt1) or negative control siRNA (NC) transfected SH-SY5Y cells were incubated with EGCG (10 μM) for 30 hr. Western blot for sirt1 and acetyl-p53 proteins was conducted from SH-SY5Y cells. β-actin was used as the loading control A . Relative sirt1 mRNA expression levels were analyzed using quantitative real-time polymerase chain reaction. The indicated relative gene expression level shows expression levels that were normalized to β-actin expression as the standard B . siSirt1 or NC transfected SH-SY5Y cells were pre-incubated with EGCG (10 μM) for 1 hr and then exposed to PrP (106-126) for 30 hr. Sirt1 deacetylase activities were analyzed in SH-SY5Y cell nuclei. C . Bars indicate mean ± standard error ( n = 4). * p

    Journal: Oncotarget

    Article Title: EGCG-mediated autophagy flux has a neuroprotection effect via a class III histone deacetylase in primary neuron cells

    doi:

    Figure Lengend Snippet: Inhibiting sirt1 decreased the sirt1 increase caused by EGCG Sirt1 small interfering RNA (siSirt1) or negative control siRNA (NC) transfected SH-SY5Y cells were incubated with EGCG (10 μM) for 30 hr. Western blot for sirt1 and acetyl-p53 proteins was conducted from SH-SY5Y cells. β-actin was used as the loading control A . Relative sirt1 mRNA expression levels were analyzed using quantitative real-time polymerase chain reaction. The indicated relative gene expression level shows expression levels that were normalized to β-actin expression as the standard B . siSirt1 or NC transfected SH-SY5Y cells were pre-incubated with EGCG (10 μM) for 1 hr and then exposed to PrP (106-126) for 30 hr. Sirt1 deacetylase activities were analyzed in SH-SY5Y cell nuclei. C . Bars indicate mean ± standard error ( n = 4). * p

    Article Snippet: Quantitative real-time polymerase chain reaction (qRT-PCR) Total ribonucleic acid (RNA) was extracted from SH-SY5Y cells using the Easy-spin™ Total RNA Extraction kit (iNtRON Biotechnology, Seoul, Korea).

    Techniques: Small Interfering RNA, Negative Control, Transfection, Incubation, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Histone Deacetylase Assay

    EGCG increases autophagy through the sirt1 pathway The SH-SY5Y cells were transfected by overexpressing adenovirus (Ad-Sirt1) or lacZ-bearing adenovirus (Ad-lacz). A Western blot of the LC3-II, ATG5 and p62 proteins was conducted in SH-SY5Y cells. Beta-actin was used as the loading control A . Sirt1 small interfering RNA (siSirt1) or negative control siRNA (NC) transfected SH-SY5Y cells were incubated with EGCG (10 μM) for 30 hr. A Western blot for the LC3-II, ATG5, and p62 proteins was conducted with SH-SY5Y cells. Beta-actin was used as the loading control B . SH-SY5Y cells were treated as described in Figure 3A , and then ICC for p62 was analyzed C .

    Journal: Oncotarget

    Article Title: EGCG-mediated autophagy flux has a neuroprotection effect via a class III histone deacetylase in primary neuron cells

    doi:

    Figure Lengend Snippet: EGCG increases autophagy through the sirt1 pathway The SH-SY5Y cells were transfected by overexpressing adenovirus (Ad-Sirt1) or lacZ-bearing adenovirus (Ad-lacz). A Western blot of the LC3-II, ATG5 and p62 proteins was conducted in SH-SY5Y cells. Beta-actin was used as the loading control A . Sirt1 small interfering RNA (siSirt1) or negative control siRNA (NC) transfected SH-SY5Y cells were incubated with EGCG (10 μM) for 30 hr. A Western blot for the LC3-II, ATG5, and p62 proteins was conducted with SH-SY5Y cells. Beta-actin was used as the loading control B . SH-SY5Y cells were treated as described in Figure 3A , and then ICC for p62 was analyzed C .

    Article Snippet: Quantitative real-time polymerase chain reaction (qRT-PCR) Total ribonucleic acid (RNA) was extracted from SH-SY5Y cells using the Easy-spin™ Total RNA Extraction kit (iNtRON Biotechnology, Seoul, Korea).

    Techniques: Transfection, Western Blot, Small Interfering RNA, Negative Control, Incubation, Immunocytochemistry

    Inhibiting autophagy with ATG5 siRNA reduced the increase in autophagy caused by EGCG ATG5 small interfering RNA (siATG5) or negative control siRNA (NC) transfected primary neuron cells were incubated with 50 μM PrP (106-126) for 36 h in the presence of EGCG. Cell viability was measured by annexin V assay A , B. siATG5 or NC transfected primary neuron cells were incubated with EGCG (10 μM) for 30 h. Western blot for ATG5, LC3 and p62 proteins was analyzed from SH-SY5Y cells. β-actin was used as loading control C. siATG5 or NC transfected SH-SY5Y cells were incubated with 50 μM PrP (106-126) for 36 hr in the presence of EGCG. Cell viability was measured by Annexin V assay D , E. siATG5 or NC transfected SH-SY5Y cells were incubated with EGCG (10 μM) for 30 hr. Western blot for ATG5, LC3-II and p62 proteins was analyzed from SH-SY5Y cells. Beta-actin was used as the loading control F. Bars indicate mean ± standard error (n = 4). * p

    Journal: Oncotarget

    Article Title: EGCG-mediated autophagy flux has a neuroprotection effect via a class III histone deacetylase in primary neuron cells

    doi:

    Figure Lengend Snippet: Inhibiting autophagy with ATG5 siRNA reduced the increase in autophagy caused by EGCG ATG5 small interfering RNA (siATG5) or negative control siRNA (NC) transfected primary neuron cells were incubated with 50 μM PrP (106-126) for 36 h in the presence of EGCG. Cell viability was measured by annexin V assay A , B. siATG5 or NC transfected primary neuron cells were incubated with EGCG (10 μM) for 30 h. Western blot for ATG5, LC3 and p62 proteins was analyzed from SH-SY5Y cells. β-actin was used as loading control C. siATG5 or NC transfected SH-SY5Y cells were incubated with 50 μM PrP (106-126) for 36 hr in the presence of EGCG. Cell viability was measured by Annexin V assay D , E. siATG5 or NC transfected SH-SY5Y cells were incubated with EGCG (10 μM) for 30 hr. Western blot for ATG5, LC3-II and p62 proteins was analyzed from SH-SY5Y cells. Beta-actin was used as the loading control F. Bars indicate mean ± standard error (n = 4). * p

    Article Snippet: Quantitative real-time polymerase chain reaction (qRT-PCR) Total ribonucleic acid (RNA) was extracted from SH-SY5Y cells using the Easy-spin™ Total RNA Extraction kit (iNtRON Biotechnology, Seoul, Korea).

    Techniques: Small Interfering RNA, Negative Control, Transfection, Incubation, Annexin V Assay, Western Blot

    EGCG prevents neuronal cells from PrP (106-126)-induced cell death through the sirt1 pathway Sirt1 small interfering RNA (siSirt1) or negative control siRNA (NC) transfected SH-SY5Y cells were incubated with 50 μM PrP (106-126) for 36 hr in the presence of EGCG. Cell viability was measured by Annexin V assay A , B . SH-SY5Y cells were pretreated with sirtinol (10 μM) and EGCG (10 μM) for 1 h and then exposed to 50 μM PrP (106-126) for 36 hr. Cell viability was measured by Annexin V assay C . The cells were measured for JC-1 mono form (green) by flow cytometry. M1 represents the population of JC-1 monomeric cells D , E . Bars indicates mean ± standard error ( n = 4). * p

    Journal: Oncotarget

    Article Title: EGCG-mediated autophagy flux has a neuroprotection effect via a class III histone deacetylase in primary neuron cells

    doi:

    Figure Lengend Snippet: EGCG prevents neuronal cells from PrP (106-126)-induced cell death through the sirt1 pathway Sirt1 small interfering RNA (siSirt1) or negative control siRNA (NC) transfected SH-SY5Y cells were incubated with 50 μM PrP (106-126) for 36 hr in the presence of EGCG. Cell viability was measured by Annexin V assay A , B . SH-SY5Y cells were pretreated with sirtinol (10 μM) and EGCG (10 μM) for 1 h and then exposed to 50 μM PrP (106-126) for 36 hr. Cell viability was measured by Annexin V assay C . The cells were measured for JC-1 mono form (green) by flow cytometry. M1 represents the population of JC-1 monomeric cells D , E . Bars indicates mean ± standard error ( n = 4). * p

    Article Snippet: Quantitative real-time polymerase chain reaction (qRT-PCR) Total ribonucleic acid (RNA) was extracted from SH-SY5Y cells using the Easy-spin™ Total RNA Extraction kit (iNtRON Biotechnology, Seoul, Korea).

    Techniques: Small Interfering RNA, Negative Control, Transfection, Incubation, Annexin V Assay, Flow Cytometry, Cytometry

    Overexpression of SiMYB56 significantly improves ABA accumulation in transgenic rice seeds and activates the expression of ABA signaling pathway related genes under drought condition. (A) Seeds of wild-type controls and transgenic rice plants were germinated on plates containing different ABA concentrations. Germination rates of SiMYB56-overexpressing and wide-type seeds under 0 μM (B) , 2.5 μM (C) , and 5 μM (D) ABA treatments ( n = 3, 16 seeds in each replicate). (E) ABA content of sprouts from germinated seeds of transgenic rice plants and wide-type controls under 0 μM ABA treatments. Values shown are means ± SD ( n = 3). (F) Expression analysis of ABA signaling pathway related genes in wild-type controls and transgenic rice plants. Entire 2-week-old SiMYB56- overexpressing and wild-type seedlings, grown under normal condition and 10% PEG6000 treatments, were harvested for RNA isolation. Transcript level was quantified by qRT-PCR. Values shown are means ± SD ( n = 3).

    Journal: Frontiers in Plant Science

    Article Title: SiMYB56 Confers Drought Stress Tolerance in Transgenic Rice by Regulating Lignin Biosynthesis and ABA Signaling Pathway

    doi: 10.3389/fpls.2020.00785

    Figure Lengend Snippet: Overexpression of SiMYB56 significantly improves ABA accumulation in transgenic rice seeds and activates the expression of ABA signaling pathway related genes under drought condition. (A) Seeds of wild-type controls and transgenic rice plants were germinated on plates containing different ABA concentrations. Germination rates of SiMYB56-overexpressing and wide-type seeds under 0 μM (B) , 2.5 μM (C) , and 5 μM (D) ABA treatments ( n = 3, 16 seeds in each replicate). (E) ABA content of sprouts from germinated seeds of transgenic rice plants and wide-type controls under 0 μM ABA treatments. Values shown are means ± SD ( n = 3). (F) Expression analysis of ABA signaling pathway related genes in wild-type controls and transgenic rice plants. Entire 2-week-old SiMYB56- overexpressing and wild-type seedlings, grown under normal condition and 10% PEG6000 treatments, were harvested for RNA isolation. Transcript level was quantified by qRT-PCR. Values shown are means ± SD ( n = 3).

    Article Snippet: RNA Isolation and Quantitative Real-Time PCR Total RNA was extracted from seedlings using the Total RNA Extraction Kit (TIANGEN, China).

    Techniques: Over Expression, Transgenic Assay, Expressing, Isolation, Quantitative RT-PCR

    SiMYB56 activates the expression of lignin synthesis related genes under drought conditions. (A) Expression analysis of lignin biosynthesis related genes in wild-type controls and transgenic rice plants. Entire 2-week-old SiMYB56 -overexpressing and wild-type seedlings, growing under normal condition and 10% PEG6000 treatment, were harvested for RNA isolation. Transcript level was quantified by qRT-PCR. Values shown are means ± SD ( n = 3). (B) Transcriptional activity assays in Nicotiana benthamiana leaves. SiMYB56 can activate the transcription of 4CL5 and F5H1. Four-week-old Nicotiana benthamiana plants were prepared for Agrobacterium -mediated transient expression, and generally the third through fifth leaf of each plant were analyzed. In each analysis, five independent Nicotiana benthamiana leaves were infiltrated and analyzed, and totally three biological replications were performed with quantification.

    Journal: Frontiers in Plant Science

    Article Title: SiMYB56 Confers Drought Stress Tolerance in Transgenic Rice by Regulating Lignin Biosynthesis and ABA Signaling Pathway

    doi: 10.3389/fpls.2020.00785

    Figure Lengend Snippet: SiMYB56 activates the expression of lignin synthesis related genes under drought conditions. (A) Expression analysis of lignin biosynthesis related genes in wild-type controls and transgenic rice plants. Entire 2-week-old SiMYB56 -overexpressing and wild-type seedlings, growing under normal condition and 10% PEG6000 treatment, were harvested for RNA isolation. Transcript level was quantified by qRT-PCR. Values shown are means ± SD ( n = 3). (B) Transcriptional activity assays in Nicotiana benthamiana leaves. SiMYB56 can activate the transcription of 4CL5 and F5H1. Four-week-old Nicotiana benthamiana plants were prepared for Agrobacterium -mediated transient expression, and generally the third through fifth leaf of each plant were analyzed. In each analysis, five independent Nicotiana benthamiana leaves were infiltrated and analyzed, and totally three biological replications were performed with quantification.

    Article Snippet: RNA Isolation and Quantitative Real-Time PCR Total RNA was extracted from seedlings using the Total RNA Extraction Kit (TIANGEN, China).

    Techniques: Expressing, Transgenic Assay, Isolation, Quantitative RT-PCR, Activity Assay

    Expression of MMP13 in ovaries from 60-, 90-, 123-, and 159-d-old hens. (A) Messenger RNA expression of MMP13 was analyzed by real-time PCR. (B) Expression of MMP13 protein was analyzed by Western blot analysis. A band of approximately 48 kDa corresponding to the molecular mass of MMP13 was detected. β-actin (41 kDa) was used as the loading control. Data are presented as mean ± SEM from at least four independent experiments. Bars with different superscript letters are significantly different ( P

    Journal: G3: Genes|Genomes|Genetics

    Article Title: Characterization of Chicken MMP13 Expression and Genetic Effect on Egg Production Traits of Its Promoter Polymorphisms

    doi: 10.1534/g3.116.027755

    Figure Lengend Snippet: Expression of MMP13 in ovaries from 60-, 90-, 123-, and 159-d-old hens. (A) Messenger RNA expression of MMP13 was analyzed by real-time PCR. (B) Expression of MMP13 protein was analyzed by Western blot analysis. A band of approximately 48 kDa corresponding to the molecular mass of MMP13 was detected. β-actin (41 kDa) was used as the loading control. Data are presented as mean ± SEM from at least four independent experiments. Bars with different superscript letters are significantly different ( P

    Article Snippet: Total RNA extraction and real-time quantitative PCR Total RNA was extracted from all tissues using an RNA extraction kit DP419 (Tiangen).

    Techniques: Expressing, RNA Expression, Real-time Polymerase Chain Reaction, Western Blot

    Phylogenetic analysis of the bat bornavirus-related sequences. (A) Schematic representation of the L gene (almost 5,140 nt encoding the RNA-dependent RNA polymerase of almost 1,710 aa) of the genome of Borna disease virus isolate bil (GenBank number ACG59353), with black and dark gray bars corresponding to the three bat bornavirus HSPs identified in samples b1 and b6, respectively. The position of the genome region amplified by PCR is represented by a dashed bar, and the sequence used for phylogenetic analysis is indicated with an asterisk. (B) Phylogenetic tree produced from the nucleotide alignment based on a selected region (214–216 nt, approximate position 3233–3446 of the L gene sequence of Borna disease virus isolate bil) of the sequence obtained from the PCR product. Bat bornavirus-related sequences are indicated in bold. The scale bar indicates branch length, and bootstrap values ≥70% are shown next to the relevant nodes. The tree is midpoint-rooted for purposes of clarity only.

    Journal: PLoS ONE

    Article Title: A Preliminary Study of Viral Metagenomics of French Bat Species in Contact with Humans: Identification of New Mammalian Viruses

    doi: 10.1371/journal.pone.0087194

    Figure Lengend Snippet: Phylogenetic analysis of the bat bornavirus-related sequences. (A) Schematic representation of the L gene (almost 5,140 nt encoding the RNA-dependent RNA polymerase of almost 1,710 aa) of the genome of Borna disease virus isolate bil (GenBank number ACG59353), with black and dark gray bars corresponding to the three bat bornavirus HSPs identified in samples b1 and b6, respectively. The position of the genome region amplified by PCR is represented by a dashed bar, and the sequence used for phylogenetic analysis is indicated with an asterisk. (B) Phylogenetic tree produced from the nucleotide alignment based on a selected region (214–216 nt, approximate position 3233–3446 of the L gene sequence of Borna disease virus isolate bil) of the sequence obtained from the PCR product. Bat bornavirus-related sequences are indicated in bold. The scale bar indicates branch length, and bootstrap values ≥70% are shown next to the relevant nodes. The tree is midpoint-rooted for purposes of clarity only.

    Article Snippet: Total RNA extraction following by sequence-independent PCR amplification process were realized, and high-throughput sequencing was performed with Illumina technology.

    Techniques: Amplification, Polymerase Chain Reaction, Sequencing, Produced

    Phylogenetic analysis of the bat nairovirus-related sequences. (A) Schematic representation of the large (L) segment (almost 12,260 nt encoding the RNA-dependent RNA polymerase of almost 4,040 aa) from the genome of Dugbe virus (GenBank number U15018), with black and gray bars corresponding to the longest contig sequences ( > 300 nt) of the bat nairovirus (named Ahun nairovirus) identified in specimens b8 and b9, respectively. The genomic region amplified by PCR is represented by a dashed bar, and the sequence used for phylogenetic analysis is indicated with an asterisk. (B) Phylogenetic tree produced from the amino-acid alignment of the partial polymerase fragment (396-nt sequence, translated into a 132-aa sequence, aa positions 2317 to 2448 of the L protein of Dugbe virus, and aligned in accordance with previous studies [86] , [87] ). The name of the bat nairovirus is indicated in bold. The various serogroups of nairoviruses and prototype species of the other genera belonging to the family Bunyaviridae are indicated on the right of the tree. The scale bar indicates branch length, and bootstrap values ≥70% are shown next to the relevant nodes. The tree is midpoint-rooted for purposes of clarity only.

    Journal: PLoS ONE

    Article Title: A Preliminary Study of Viral Metagenomics of French Bat Species in Contact with Humans: Identification of New Mammalian Viruses

    doi: 10.1371/journal.pone.0087194

    Figure Lengend Snippet: Phylogenetic analysis of the bat nairovirus-related sequences. (A) Schematic representation of the large (L) segment (almost 12,260 nt encoding the RNA-dependent RNA polymerase of almost 4,040 aa) from the genome of Dugbe virus (GenBank number U15018), with black and gray bars corresponding to the longest contig sequences ( > 300 nt) of the bat nairovirus (named Ahun nairovirus) identified in specimens b8 and b9, respectively. The genomic region amplified by PCR is represented by a dashed bar, and the sequence used for phylogenetic analysis is indicated with an asterisk. (B) Phylogenetic tree produced from the amino-acid alignment of the partial polymerase fragment (396-nt sequence, translated into a 132-aa sequence, aa positions 2317 to 2448 of the L protein of Dugbe virus, and aligned in accordance with previous studies [86] , [87] ). The name of the bat nairovirus is indicated in bold. The various serogroups of nairoviruses and prototype species of the other genera belonging to the family Bunyaviridae are indicated on the right of the tree. The scale bar indicates branch length, and bootstrap values ≥70% are shown next to the relevant nodes. The tree is midpoint-rooted for purposes of clarity only.

    Article Snippet: Total RNA extraction following by sequence-independent PCR amplification process were realized, and high-throughput sequencing was performed with Illumina technology.

    Techniques: Amplification, Polymerase Chain Reaction, Sequencing, Produced

    Phylogenetic analysis of VP1 bat rotavirus-related sequences. (A) Schematic representation of the VP1 segment (almost 3,300 nt encoding the RNA-dependent RNA polymerase of almost 1,090 aa) of the genome of the lamb rotavirus strain Lamb-NT (GenBank number FJ031024), with black bars corresponding to the longest contig sequences ( > 300 nt) of the bat rotavirus (named Maule rotavirus) identified in specimen b8 ( Myotis mystacinus ). The genomic region amplified by PCR is represented by a dashed bar, and the sequence used for phylogenetic analysis is indicated with an asterisk. B) Phylogenetic tree produced from the amino-acid alignment based on the partial VP1 sequence (119 aa, positions 964 to 1082 of the VP1 protein of lamb rotavirus strain Lamb-NT) translated from one of the longest HSPs. The bat rotavirus-related sequence is indicated in bold within the various rotavirus groups. The scale bar indicates branch length, and bootstrap values ≥70% are shown next to the relevant nodes. The tree is midpoint-rooted for purposes of clarity only.

    Journal: PLoS ONE

    Article Title: A Preliminary Study of Viral Metagenomics of French Bat Species in Contact with Humans: Identification of New Mammalian Viruses

    doi: 10.1371/journal.pone.0087194

    Figure Lengend Snippet: Phylogenetic analysis of VP1 bat rotavirus-related sequences. (A) Schematic representation of the VP1 segment (almost 3,300 nt encoding the RNA-dependent RNA polymerase of almost 1,090 aa) of the genome of the lamb rotavirus strain Lamb-NT (GenBank number FJ031024), with black bars corresponding to the longest contig sequences ( > 300 nt) of the bat rotavirus (named Maule rotavirus) identified in specimen b8 ( Myotis mystacinus ). The genomic region amplified by PCR is represented by a dashed bar, and the sequence used for phylogenetic analysis is indicated with an asterisk. B) Phylogenetic tree produced from the amino-acid alignment based on the partial VP1 sequence (119 aa, positions 964 to 1082 of the VP1 protein of lamb rotavirus strain Lamb-NT) translated from one of the longest HSPs. The bat rotavirus-related sequence is indicated in bold within the various rotavirus groups. The scale bar indicates branch length, and bootstrap values ≥70% are shown next to the relevant nodes. The tree is midpoint-rooted for purposes of clarity only.

    Article Snippet: Total RNA extraction following by sequence-independent PCR amplification process were realized, and high-throughput sequencing was performed with Illumina technology.

    Techniques: Amplification, Polymerase Chain Reaction, Sequencing, Produced

    Inhibitory effect of MUPCDH on cell proliferation. ( a ) Knockdown of the MUPCDH gene increased cell survival in human renal cortical epithelial (HRCE) cells. ( b ) Overexpression of the MUPCDH gene decreased cell growth of WT9-7 cells. ( c ) 5-bromo-2′-deoxyuridine (BrdU) incorporation assays were carried out using HRCE cells that had been transfected with MUPCDH short interfering RNA (siRNA) or control siRNA. ( d ) Proliferating cell nuclear antigen (PCNA) staining carried out using HEK293T cells that overexpressed the MUPCDH gene. The level of PCNA expression was also measured by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). β-Actin was used as an internal control in real-time qRT-PCR assays. ( e ) Cell proliferation rate was measured using a cell proliferation assay kit (WST-1) following treatment of WT9-7 cells with 5-aza-2′-deoxycytidine (5 μM). The experiment was performed in triplicate. * P

    Journal: Scientific Reports

    Article Title: Epigenetic silencing of the MUPCDH gene as a possible prognostic biomarker for cyst growth in ADPKD

    doi: 10.1038/srep15238

    Figure Lengend Snippet: Inhibitory effect of MUPCDH on cell proliferation. ( a ) Knockdown of the MUPCDH gene increased cell survival in human renal cortical epithelial (HRCE) cells. ( b ) Overexpression of the MUPCDH gene decreased cell growth of WT9-7 cells. ( c ) 5-bromo-2′-deoxyuridine (BrdU) incorporation assays were carried out using HRCE cells that had been transfected with MUPCDH short interfering RNA (siRNA) or control siRNA. ( d ) Proliferating cell nuclear antigen (PCNA) staining carried out using HEK293T cells that overexpressed the MUPCDH gene. The level of PCNA expression was also measured by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). β-Actin was used as an internal control in real-time qRT-PCR assays. ( e ) Cell proliferation rate was measured using a cell proliferation assay kit (WST-1) following treatment of WT9-7 cells with 5-aza-2′-deoxycytidine (5 μM). The experiment was performed in triplicate. * P

    Article Snippet: Total RNA extraction and real-time qRT-PCR Total RNA was extracted using the NucleoSpin® TriPrep Extract kit (MACHEREY-NAGEL), according to the manufacturer’s protocol.

    Techniques: Over Expression, BrdU Incorporation Assay, Transfection, Small Interfering RNA, Staining, Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Proliferation Assay

    The regulation of MUPCDH transcription via transcription factor AP-2α. ( a ) As assayed by chromatin immunoprecipitation, together with quantitative reverse transcription polymerase chain reaction (qRT-PCR), AP-2α was highly enriched in the MUPCDH promoter region of human renal cortical epithelial cells. RNA polymerase II was used as a positive control. ( b , c ) Knockdown of AP-2α decreased the mRNA and protein expression levels of MUPCDH , as confirmed by real-time qRT-PCR and western blot analyses, respectively. The band density was measured using the MultiGauge software. ( d ) The luciferase activity was measured following knockdown of AP-2α in HEK293T cells. The ratio of Renilla luciferase to Firefly luciferase was calculated for each experiment, and the values from triplicate experiments were averaged. The mean value for each test construct was normalized to the activity of the empty vector. Bars represent the mean of the normalized values with error bars indicating the range. Each experiment was performed in triplicate. * P

    Journal: Scientific Reports

    Article Title: Epigenetic silencing of the MUPCDH gene as a possible prognostic biomarker for cyst growth in ADPKD

    doi: 10.1038/srep15238

    Figure Lengend Snippet: The regulation of MUPCDH transcription via transcription factor AP-2α. ( a ) As assayed by chromatin immunoprecipitation, together with quantitative reverse transcription polymerase chain reaction (qRT-PCR), AP-2α was highly enriched in the MUPCDH promoter region of human renal cortical epithelial cells. RNA polymerase II was used as a positive control. ( b , c ) Knockdown of AP-2α decreased the mRNA and protein expression levels of MUPCDH , as confirmed by real-time qRT-PCR and western blot analyses, respectively. The band density was measured using the MultiGauge software. ( d ) The luciferase activity was measured following knockdown of AP-2α in HEK293T cells. The ratio of Renilla luciferase to Firefly luciferase was calculated for each experiment, and the values from triplicate experiments were averaged. The mean value for each test construct was normalized to the activity of the empty vector. Bars represent the mean of the normalized values with error bars indicating the range. Each experiment was performed in triplicate. * P

    Article Snippet: Total RNA extraction and real-time qRT-PCR Total RNA was extracted using the NucleoSpin® TriPrep Extract kit (MACHEREY-NAGEL), according to the manufacturer’s protocol.

    Techniques: Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Positive Control, Expressing, Western Blot, Software, Luciferase, Activity Assay, Construct, Plasmid Preparation

    CHIKV mos has reduced attachment to host cells than CHIKV vero. ( A), Equal PFUs of CHIKV vero or CHIKV mos (Ross strain) were added to Vero cells (left), NIH3T3 cells (middle) and L929 cells (right) for plaque development. (B), Plaque counts of CHIKV vero and CHIKV mos in the indicated cells were quantified. (C) L929, NIH3T3 and HFF cells were inoculated with CHIKV vero or CHIKV mos (Ross strain, MOI = 1) at 4°C for 1 h and attached viruses were quantified by RT-qPCR and presented as the ratio of CHIKV E1 copy number per 1000 copy of cellular β-actin . (D) L929, NIH3T3 and HFF cells were inoculated with CHIKV vero or CHIKV mos (LR OPY1, MOI = 1) at 4°C for 1 h and viruses attached to cells were quantified by RT-qPCR. (E) NIH3T3 cells were infected with CHIKV vero or CHIKV mos (Ross strain, MOI = 2.5) at 4°C for 45 min and the virus-bound cells were quantified by flow cytometry. (F) CHIKV vero or CHIKV mos (100 PFUs) were added to monolayer of L929 cells at 4°C for 1 h and both attached and unattached viruses were quantified by plaque assay to calculate percentage attachment. (G) CHIKV vero or CHIKV mos (Ross strain, 100 PFUs) were added to the L929 cell monolayer at 4°C for 1 h. After removing unattached virus, cells were replaced with fresh medium (control), or treated with acidic medium (pH 5.5) for 2 minutes before adding fresh medium, or replaced with NH 4 Cl (20 mM) containing media. Viruses that entered into cells were analyzed by allowing plaque development for 48 h and normalized to controls. (H) Viral RNA copies in L929 cells infected with CHIKV vero or CHIKV mos (Ross strain, MOI = 1) with or without NH 4 Cl (20 mM) were measured by RT-qPCR at 24 h.p.i. Data represent at least two independent experiments performed in triplicates with the similar results. ND denotes not detected, and ns denotes not significant ( p ≥ 0.05).

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Loss of Glycosaminoglycan Receptor Binding after Mosquito Cell Passage Reduces Chikungunya Virus Infectivity

    doi: 10.1371/journal.pntd.0004139

    Figure Lengend Snippet: CHIKV mos has reduced attachment to host cells than CHIKV vero. ( A), Equal PFUs of CHIKV vero or CHIKV mos (Ross strain) were added to Vero cells (left), NIH3T3 cells (middle) and L929 cells (right) for plaque development. (B), Plaque counts of CHIKV vero and CHIKV mos in the indicated cells were quantified. (C) L929, NIH3T3 and HFF cells were inoculated with CHIKV vero or CHIKV mos (Ross strain, MOI = 1) at 4°C for 1 h and attached viruses were quantified by RT-qPCR and presented as the ratio of CHIKV E1 copy number per 1000 copy of cellular β-actin . (D) L929, NIH3T3 and HFF cells were inoculated with CHIKV vero or CHIKV mos (LR OPY1, MOI = 1) at 4°C for 1 h and viruses attached to cells were quantified by RT-qPCR. (E) NIH3T3 cells were infected with CHIKV vero or CHIKV mos (Ross strain, MOI = 2.5) at 4°C for 45 min and the virus-bound cells were quantified by flow cytometry. (F) CHIKV vero or CHIKV mos (100 PFUs) were added to monolayer of L929 cells at 4°C for 1 h and both attached and unattached viruses were quantified by plaque assay to calculate percentage attachment. (G) CHIKV vero or CHIKV mos (Ross strain, 100 PFUs) were added to the L929 cell monolayer at 4°C for 1 h. After removing unattached virus, cells were replaced with fresh medium (control), or treated with acidic medium (pH 5.5) for 2 minutes before adding fresh medium, or replaced with NH 4 Cl (20 mM) containing media. Viruses that entered into cells were analyzed by allowing plaque development for 48 h and normalized to controls. (H) Viral RNA copies in L929 cells infected with CHIKV vero or CHIKV mos (Ross strain, MOI = 1) with or without NH 4 Cl (20 mM) were measured by RT-qPCR at 24 h.p.i. Data represent at least two independent experiments performed in triplicates with the similar results. ND denotes not detected, and ns denotes not significant ( p ≥ 0.05).

    Article Snippet: Real time-quantitative PCR (RT-qPCR) CHIKV infected cells were subjected to total RNA extraction using TRI-reagent (Molecular Research Center, Inc.).

    Techniques: Quantitative RT-PCR, Infection, Flow Cytometry, Cytometry, Plaque Assay

    CHIKV mos infectivity can be enhanced after replication in mammalian cells. The viral stocks of CHIKV vero-NIH3T3 and CHIKV mos-NIH3T3 were prepared after CHIKV vero or CHIKV mos (Ross strain, MOI = 1) replicated in NIH3T3 for 48 h. (A) NIH3T3 and HFF cells (B) were infected with CHIKV vero , CHIKV mos , CHIKV vero-NIH3T3 and CHIKV mos-NIH3T3 (MOI = 1), and viral RNA copy numbers were measured at 24 h by RT-qPCR. Data represents ratio of copy number of CHIKV E1 to β-actin . (C) NIH3T3 cells were infected with CHIKV vero-L929 and CHIKV mos-L929 (MOI = 1), and viral RNA copy numbers were measured by RT-qPCR at 24 h. (D) NIH3T3 cells were infected with CHIKV vero-VERO and CHIKV mos-VERO (MOI = 1), and viral RNA copy numbers were measured at 24 h by RT-qPCR. (E) Attachment of CHIKV vero-NIH and CHIKV mos-NIH (MOI = 1) were performed in NIH3T3 cells at 4°C for 1 h and attached viruses were measured by RT-qPCR. (F) GAGs neutralization of CHIKV vero-NIH3T3 and CHIKV mos-NIH3T3 attachment was performed with heparin (1000 U/mL), chondroitin sulfate A (CSA, 1000 μg/mL) and dermatan sulfate (DS, 1000 μg/mL) in NIH3T3 cells, and the attached viruses were quantified by RT-qPCR and normalized to the respective untreated controls. All data sets represent two independent experiments performed in triplicates. GAG-treated samples were compared to the respective controls (without GAGs) and analyzed using a one-way ANOVA (**** denotes p

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Loss of Glycosaminoglycan Receptor Binding after Mosquito Cell Passage Reduces Chikungunya Virus Infectivity

    doi: 10.1371/journal.pntd.0004139

    Figure Lengend Snippet: CHIKV mos infectivity can be enhanced after replication in mammalian cells. The viral stocks of CHIKV vero-NIH3T3 and CHIKV mos-NIH3T3 were prepared after CHIKV vero or CHIKV mos (Ross strain, MOI = 1) replicated in NIH3T3 for 48 h. (A) NIH3T3 and HFF cells (B) were infected with CHIKV vero , CHIKV mos , CHIKV vero-NIH3T3 and CHIKV mos-NIH3T3 (MOI = 1), and viral RNA copy numbers were measured at 24 h by RT-qPCR. Data represents ratio of copy number of CHIKV E1 to β-actin . (C) NIH3T3 cells were infected with CHIKV vero-L929 and CHIKV mos-L929 (MOI = 1), and viral RNA copy numbers were measured by RT-qPCR at 24 h. (D) NIH3T3 cells were infected with CHIKV vero-VERO and CHIKV mos-VERO (MOI = 1), and viral RNA copy numbers were measured at 24 h by RT-qPCR. (E) Attachment of CHIKV vero-NIH and CHIKV mos-NIH (MOI = 1) were performed in NIH3T3 cells at 4°C for 1 h and attached viruses were measured by RT-qPCR. (F) GAGs neutralization of CHIKV vero-NIH3T3 and CHIKV mos-NIH3T3 attachment was performed with heparin (1000 U/mL), chondroitin sulfate A (CSA, 1000 μg/mL) and dermatan sulfate (DS, 1000 μg/mL) in NIH3T3 cells, and the attached viruses were quantified by RT-qPCR and normalized to the respective untreated controls. All data sets represent two independent experiments performed in triplicates. GAG-treated samples were compared to the respective controls (without GAGs) and analyzed using a one-way ANOVA (**** denotes p

    Article Snippet: Real time-quantitative PCR (RT-qPCR) CHIKV infected cells were subjected to total RNA extraction using TRI-reagent (Molecular Research Center, Inc.).

    Techniques: Infection, Quantitative RT-PCR, Neutralization

    RpoE1 regulates positively the expression of hrp gene cluster ( hrpA - F ), type III secreted effector-encoding genes ( XC_0241 and XC_1553 ) and the hrp master regulator hrpX . (A) Quantitative real-time PCR analysis of the transcription of hrpA - F, hrpG, hrpX, XC_0241 , and XC_1553 in the Xcc wild-type strain 8004 and the rpoE1 deletion mutant strain Δ rpoE1 . RNA was isolated from cultures of the strains grown in XCM1 medium for 24 h. The relative mRNA level was calculated with respect to the level of the corresponding transcript in the wild-type strain 8004. (B) GUS activity of hrpB, hrpF, hrpG, hrpX, XC_0241 , and XC_1553 promoter- gusA reporters (pLgushrpB, pLgushrpF, pLgushrpG, pLgushrpX, pLgus0241, and pLgus1553) in the wild-type strain 8004 and the mutant strain Δ rpoE1 . The strains were cultured in XCM1 medium for 24 h and GUS activity in the total culture was determined by using ρ-nitrophenyl-β-D-glucuronide as substrate. Values given are means ± standard deviations of triplicate measurements from a representative experiment; similar results were obtained in two other independent experiments. Asterisks indicate statistically significant difference, compared with the wild type (Student's t -test). ** P

    Journal: Frontiers in Microbiology

    Article Title: Systematic Functional Analysis of Sigma (σ) Factors in the Phytopathogen Xanthomonas campestris Reveals Novel Roles in the Regulation of Virulence and Viability

    doi: 10.3389/fmicb.2018.01749

    Figure Lengend Snippet: RpoE1 regulates positively the expression of hrp gene cluster ( hrpA - F ), type III secreted effector-encoding genes ( XC_0241 and XC_1553 ) and the hrp master regulator hrpX . (A) Quantitative real-time PCR analysis of the transcription of hrpA - F, hrpG, hrpX, XC_0241 , and XC_1553 in the Xcc wild-type strain 8004 and the rpoE1 deletion mutant strain Δ rpoE1 . RNA was isolated from cultures of the strains grown in XCM1 medium for 24 h. The relative mRNA level was calculated with respect to the level of the corresponding transcript in the wild-type strain 8004. (B) GUS activity of hrpB, hrpF, hrpG, hrpX, XC_0241 , and XC_1553 promoter- gusA reporters (pLgushrpB, pLgushrpF, pLgushrpG, pLgushrpX, pLgus0241, and pLgus1553) in the wild-type strain 8004 and the mutant strain Δ rpoE1 . The strains were cultured in XCM1 medium for 24 h and GUS activity in the total culture was determined by using ρ-nitrophenyl-β-D-glucuronide as substrate. Values given are means ± standard deviations of triplicate measurements from a representative experiment; similar results were obtained in two other independent experiments. Asterisks indicate statistically significant difference, compared with the wild type (Student's t -test). ** P

    Article Snippet: The total RNA of Xcc strains was extracted with a total-RNA extraction kit (Promega, Madison, Wisconsin, USA) and reverse transcription was performed using a cDNA synthesis kit (Takara, Dalian, China), according to the manufacturer's instructions.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Mutagenesis, Isolation, Activity Assay, Cell Culture

    RpoE1 enhances the expression of hrpX in vivo and in vitro . (A) Quantitative real-time PCR (qRT-PCR) showed that overexpression of RpoE1 in Xcc enhanced hrpX transcription. For the qRT-PCR, Xcc wild-type strain 8004 and its derivative strain 8004/pJrpoE1 which overexpresses RpoE1 in the wild-type background were grown in the minimal medium XCM1. RNA was isolated from the cultures after incubation for 24 h. The relative mRNA level of hrpX was analyzed by qRT-PCR and calculated with respect to the transcript level in strain 8004. Data are means ± standard deviations of triplicate measurements from a representative experiment; similar results were obtained in two other independent experiments. Asterisks indicate statistically significant difference (Student's t -test). ** P

    Journal: Frontiers in Microbiology

    Article Title: Systematic Functional Analysis of Sigma (σ) Factors in the Phytopathogen Xanthomonas campestris Reveals Novel Roles in the Regulation of Virulence and Viability

    doi: 10.3389/fmicb.2018.01749

    Figure Lengend Snippet: RpoE1 enhances the expression of hrpX in vivo and in vitro . (A) Quantitative real-time PCR (qRT-PCR) showed that overexpression of RpoE1 in Xcc enhanced hrpX transcription. For the qRT-PCR, Xcc wild-type strain 8004 and its derivative strain 8004/pJrpoE1 which overexpresses RpoE1 in the wild-type background were grown in the minimal medium XCM1. RNA was isolated from the cultures after incubation for 24 h. The relative mRNA level of hrpX was analyzed by qRT-PCR and calculated with respect to the transcript level in strain 8004. Data are means ± standard deviations of triplicate measurements from a representative experiment; similar results were obtained in two other independent experiments. Asterisks indicate statistically significant difference (Student's t -test). ** P

    Article Snippet: The total RNA of Xcc strains was extracted with a total-RNA extraction kit (Promega, Madison, Wisconsin, USA) and reverse transcription was performed using a cDNA synthesis kit (Takara, Dalian, China), according to the manufacturer's instructions.

    Techniques: Expressing, In Vivo, In Vitro, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Over Expression, Isolation, Incubation

    Gene expression of Sp_0149 during infection. ( A ) Ethidium stained agarose gel showing equal amplification efficiency for the primer pairs used for PCR of psaA and metQ when using S. pneumoniae 0100993 genomic DNA as the template. ( B ) Ethidium stained agarose gel of cDNA products generated by RT-PCR after 26 and 30 cycles using S. pneumoniae 0100993 total RNA extracted from the blood of infected mice and primers for 16S rRNA (internal control), psaA (positive control) and metQ . (C) Relative mRNA concentrations of selected genes of S. pneumoniae WCH43 (serotype 4) in various in vivo niches at 72 h post-intranasal infection of mice. Transcript abundance for each gene was obtained by microarray analysis, and normalized against those obtained for the 16S rRNA control. Quantitative fold differences for each transcript were determined as a ratio of its abundance to that of the 16S rRNA control. Data are means ± SEM for each gene transcript from three replicate challenge experiments.

    Journal: PLoS ONE

    Article Title: The Effects of Methionine Acquisition and Synthesis on Streptococcus Pneumoniae Growth and Virulence

    doi: 10.1371/journal.pone.0049638

    Figure Lengend Snippet: Gene expression of Sp_0149 during infection. ( A ) Ethidium stained agarose gel showing equal amplification efficiency for the primer pairs used for PCR of psaA and metQ when using S. pneumoniae 0100993 genomic DNA as the template. ( B ) Ethidium stained agarose gel of cDNA products generated by RT-PCR after 26 and 30 cycles using S. pneumoniae 0100993 total RNA extracted from the blood of infected mice and primers for 16S rRNA (internal control), psaA (positive control) and metQ . (C) Relative mRNA concentrations of selected genes of S. pneumoniae WCH43 (serotype 4) in various in vivo niches at 72 h post-intranasal infection of mice. Transcript abundance for each gene was obtained by microarray analysis, and normalized against those obtained for the 16S rRNA control. Quantitative fold differences for each transcript were determined as a ratio of its abundance to that of the 16S rRNA control. Data are means ± SEM for each gene transcript from three replicate challenge experiments.

    Article Snippet: Nucleic acid manipulations, reverse transcriptase PCR and transcriptome analysis S. pneumoniae genomic DNA was extracted from bacteria grown in THY using a modified Wizard genomic DNA kit (Promega) and RNA using SV total RNA extraction kit (Promega) as previously described .

    Techniques: Expressing, Infection, Staining, Agarose Gel Electrophoresis, Amplification, Polymerase Chain Reaction, Generated, Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Positive Control, In Vivo, Microarray

    Global organization and functionality of the newly identified 28-kb GI harboring β-myrcene core-code. ( A ) Genomic locus organization and functional elements detected using in silico prediction. Genes transcribed in forward orientation are represented by rightward arrows, whereas genes located in antisense strand are represented by leftward arrows. Genes are organized in TU, promoter regions (P) are illustrated by blue arrows, and terminator sites indicated by red circles. ( B ) Detailed analysis of the RNA-seq data mapping with the GI. In the top panel, read mapping coverage per base was plotted for samples derived from M1 growth in lactate (gray line) and in β-myrcene (blue line). Prediction of the operon-like transcripts (horizontal orange bars) and transcription starting sites (TSS) (red arrows) was carried out with ReadXplorer ( Hilker et al. 2014 ). Expression levels for each gene are shown, in the bottom panel, as the ratio (FC) of normalized transcripts between cells grown in lactate (L) and cells grown in β-myrcene (M).

    Journal: Genome Biology and Evolution

    Article Title: Deciphering the Genome Repertoire of Pseudomonas sp. M1 toward β-Myrcene Biotransformation

    doi: 10.1093/gbe/evu254

    Figure Lengend Snippet: Global organization and functionality of the newly identified 28-kb GI harboring β-myrcene core-code. ( A ) Genomic locus organization and functional elements detected using in silico prediction. Genes transcribed in forward orientation are represented by rightward arrows, whereas genes located in antisense strand are represented by leftward arrows. Genes are organized in TU, promoter regions (P) are illustrated by blue arrows, and terminator sites indicated by red circles. ( B ) Detailed analysis of the RNA-seq data mapping with the GI. In the top panel, read mapping coverage per base was plotted for samples derived from M1 growth in lactate (gray line) and in β-myrcene (blue line). Prediction of the operon-like transcripts (horizontal orange bars) and transcription starting sites (TSS) (red arrows) was carried out with ReadXplorer ( Hilker et al. 2014 ). Expression levels for each gene are shown, in the bottom panel, as the ratio (FC) of normalized transcripts between cells grown in lactate (L) and cells grown in β-myrcene (M).

    Article Snippet: Total RNA Extraction from Pseudomonas sp. M1 Total RNA samples were obtained using the Aurum Total RNA Mini kit (Bio-Rad), as described by the manufacturer’s instructions.

    Techniques: Functional Assay, In Silico, RNA Sequencing Assay, Derivative Assay, Expressing