rnase h Thermo Fisher Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher superscript iii reverse transcriptase
    Cell cycle state of ID4-EGFP + spermatogonial subpopulations. A ) Cell cycle state among P6 ID4-EGFP + subpopulations was characterized using flow cytometry and density dot plots demonstrate sequential cell pregating based on: light scatter characteristics (FSC-A × SSC-A; i ), viability (propidium iodide negative; ii ), single cells ( <t>iii</t> ), and ID4-EGFP + ( iv ). Cells stained with antibodies against TSPAN8 ( B – D ), EHPA2 ( E – G ), and PVR ( H – J ) were used for <t>DNA</t> content analysis with the Vybrant DyeCycle Violet Stain. Histograms indicating cell number (y-axis) and DNA content (x-axis) are shown for EGFP + spermatogonia with the top one-third (based on cell number) most intensely stained cells (B , E , and H) and marker-positive cells with the bottom one-third (based on cell number) weakest positive staining intensity (C , F , and I) . Insets in each panel show dot plots with EGFP intensity, antibody staining intensity, and selection gates for histograms. The percentage of ID4-EGFP + cells shown in each histogram is noted above the histogram. Transparent blue, red, and green curves in each histogram show the Gaussian functions corresponding to 2N (G1/G0), 4 > N > 2 (S), and (4N) G2/M fractions of each population, which serve as estimates of the proportion of gated cells in each cell cycle phase. D , G , and J) Mean ± SEM from results of four replicate staining experiments for each marker are shown in the stacked bar graphs. In each graph, significant differences in cell cycle state between subpopulations were determined by Student t -tests and are noted between bars (* P
    Superscript Iii Reverse Transcriptase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 98582 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii reverse transcriptase/product/Thermo Fisher
    Average 99 stars, based on 98582 article reviews
    Price from $9.99 to $1999.99
    superscript iii reverse transcriptase - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher trizol reagent
    MHV-ExoN(-) evolved WT-like genomic <t>RNA</t> accumulation and increased resistance to multiple nucleoside analogues over the passage. (A) Cells were infected with the indicated viruses at MOI = 1 PFU/cell, and intracellular RNA was harvested using <t>TRIzol</t> at the indicated times postinfection. MHV genomic RNA was detected using SYBR green and primers directed to nsp10, and values were normalized to intracellular GAPDH. (B) Same data as in panel A normalized to the RNA level for each virus at 4 hpi. Data represent means and standard errors of results for n = 9 (3 triplicate experiments). (C to F) Sensitivity of passaged viruses to nucleoside analogues at MOI = 0.01 PFU/cell. Cells were treated with the indicated concentrations of 5-FU (C), RBV (D), AZC (E), or CMeA (F) for 30 min prior to infection, supernatants were harvested at 24 hpi, and titers were determined by plaque assay. Data represent changes in titer relative to untreated control results and are plotted as means and standard errors of results from n = 6 (two triplicate experiments). For panels C to F, the statistical significance of changes in the titer of MHV-ExoN(-) P3 relative to MHV-ExoN(-) P250 was determined using the Mann-Whitney test (*, P
    Trizol Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 635164 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/trizol reagent/product/Thermo Fisher
    Average 99 stars, based on 635164 article reviews
    Price from $9.99 to $1999.99
    trizol reagent - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher superscript iii first strand synthesis system
    <t>HERV-K</t> gag and pol ORFs are more prevalent in ALS Both prototypical HERV-K genome in 5q33.3 (Panel A, i) and partial HERV-K genomes, such 7q36.1 (Panel A, <t>iii)</t> can encode for RT. In contrast, 7q34 has no ORF for RT, but contributes gag ORFs (Panel A, ii). HERV-K loci encode potential ORFs for retroviral proteins Gag, protease, RT, RNaseH, integrase and Env. Patients with ALS more frequently express combinations of loci encoding gag and pol ORFs, as compared to controls with systemic disease (Panel B).
    Superscript Iii First Strand Synthesis System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 53513 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii first strand synthesis system/product/Thermo Fisher
    Average 99 stars, based on 53513 article reviews
    Price from $9.99 to $1999.99
    superscript iii first strand synthesis system - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher first strand cdna synthesis
    Elevated gene 50 transcription is accompanied by hypomethylation of the gene 50 promoter at day 18. (A) Quantitative RT-PCR for total gene 50 (G50) (exon 2) transcripts on <t>cDNA</t> generated from total splenocyte <t>RNA.</t> Transcript levels are elevated in Cre-positive
    First Strand Cdna Synthesis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 39924 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/first strand cdna synthesis/product/Thermo Fisher
    Average 99 stars, based on 39924 article reviews
    Price from $9.99 to $1999.99
    first strand cdna synthesis - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher superscript first strand synthesis system
    Midkine expression was induced by FBS, growth factors and cytokines. A . LNCaP cells were cultured in serum-free DMEM and treated for 48 h with 10% FBS and the indicated agents: 10 ng/ml insulin, 10 ng/ml IGF-I, 10 ng/ml EGF, 10 ng/ml HGF, 10 ng/ml bFGF, 20 ng/ml T3, 10 nM R1881, 10 nM DHT, 10 ng/ml TNFα, 10 ng/ml IL-1β, 50 ng/ml IL-6, 50 ng/ml IL-17, and 33.3 μM RA. B . LNCaP cells were treated with different dosages (1 to 50 ng/ml) of TNFα for 48 h. C . LNCaP cells were also treated with 20 ng/ml TNFα for different time periods (8 to 48 h). 20 μl of each medium supernatant was subjected to Western blot analysis of midkine expression using rabbit anti-midkine antibodies, horseradish peroxidase-conjungated secondary antibodies and enhanced chemiluminescence reagents. D . Total RNA was extracted from LNCaP cells not treated or treated with 20 ng/ml TNFα, using RNeasy Mini Kit; cDNA was made from total RNA using <t>Superscript™</t> <t>First-Strand</t> <t>Synthesis</t> <t>System</t> with oligo dT primers; real-time quantitative PCR was done in triplicates with Sybr-Green reagents; results were normalized to GAPDH levels as described in Methods; the data (mean ± standard deviation of three independent experiments) were presented as fold change of midkine mRNA compared to the LNCaP cells without treatment for 8 h, where fold = 2 ΔΔCt ; solid bar, TNFα treated; open bar, control; * P
    Superscript First Strand Synthesis System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 42629 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript first strand synthesis system/product/Thermo Fisher
    Average 99 stars, based on 42629 article reviews
    Price from $9.99 to $1999.99
    superscript first strand synthesis system - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher m mlv reverse transcriptase
    <t>Gadd45a</t> binds RNA in vitro . A, RNA filter binding assay using the indicated proteins and 32 P-labeled RNA (multiple cloning site transcript, MCS). Co, no protein; BSA, bovine serum albumin; <t>M-MLV</t> RT - Moloney murine leukemia virus reverse transcriptase. B, C, RNA filter binding assays using 32 P-labeled MCS RNA were performed with recombinant Gadd45a in the presence of the indicated unlabeled competitor nucleic acids. Data are shown as percentage of 32 P bound in the absence of the competitor. Each sample was done in triplicate; average and standard deviation was generated; A representative experiment out of three is shown. U, unmethylated; M, methylated; U/U, unmethylated; U/M, hemimethylated; M/M, holomethylated; PolyA, polyC, polyG, polyU, homopolyribonucleotides; total RNA, RNA isolated from HEK293T cells; tRNA, yeast tRNA; MCS RNA, multiple cloning site RNA. Error bars, s.e.m. (n = 3). A representative experiment out of three is shown.
    M Mlv Reverse Transcriptase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 26041 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m mlv reverse transcriptase/product/Thermo Fisher
    Average 99 stars, based on 26041 article reviews
    Price from $9.99 to $1999.99
    m mlv reverse transcriptase - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher superscript vilo cdna synthesis kit
    mtDNA increases SOCE and expression of Sphk1 in PMN. (A) Human PMN were treated with 20 µg/mL of E.coli DNA or mtDNA for 60 min then loaded with fura-2. Thapsigargin (1 µM) was applied to under nominally calcium-free conditions and then 1.8 mM extracellular calcium was applied at the indicated times. Calcium influx was calculated as described elsewhere [9] as the area under the curve for [Ca 2+ ] i (AUC) over 120 sec. Data were analyzed using medium-treated PMN as 100%. Mean and SE values are shown. At least 3 experiment were done per condition. * denotes a significant difference by student t-test (SigmaPlot 11) compared to time “0” value. Experiments were repeated at least three times. (7B) Freshly isolated human PMN (5 million cells in 2 mL) were incubated with medium, mtDNA (10 µg/mL), E.coli DNA (10 µg/mL), or LPS (100 ng/mL) for 60 min. Then RNA and <t>cDNA</t> were prepared using the RNesay mini kit (Qiagen) and SuperScript <t>VILO</t> cDNA Synthesis kit (Life technologies), respectively. 200 ng of cDNA per reaction was used for TaqMan qPCR assay for Sphk1 and GAPDH to evaluate expression levels. Sphk1 expression levels were then further normalized by GAPDH using the medium control result as 100%. Four different PMN preparations were used for stimulation and qPCR assays were done in triplicates. Mean and SE values from four different experiments are shown. Data were analyzed by One Way ANOVA. * denotes a significant difference between mtDNA treatment and the medium control (p
    Superscript Vilo Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 17594 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript vilo cdna synthesis kit/product/Thermo Fisher
    Average 99 stars, based on 17594 article reviews
    Price from $9.99 to $1999.99
    superscript vilo cdna synthesis kit - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher total rna
    Illustrations of real-time PCR of OT and VP RNAs using cDNA derived from the total <t>RNA</t> of the same rat <t>SON.</t> Representative real-time PCR growth curves for OT ( A ) and VP ( B ) specific PCR primers using cDNA template from total RNA of the same rat SON are
    Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 475535 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/total rna/product/Thermo Fisher
    Average 99 stars, based on 475535 article reviews
    Price from $9.99 to $1999.99
    total rna - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher nacl
    The hyphal growth defects of the Δ cnaA or Δ cnaB strains could be suppressed by the dysfunction of CchA under salt stress. (A) Colony morphology of the Δ cnaA , Δ cnaA cchA re , Δ cnaB , and Δ cnaB Δ cchA strains and the reference strain. The conidia were spotted on solid <t>MMPDRUU</t> and MMPDRUU supplemented with 800 mM <t>NaCl,</t> 600 mM KCl, or 1 M sorbitol, respectively, at 37°C for 2.5 days. (B) Graphic representation of radial growth rates of the Δ cnaA and Δ cnaA cchA re strains and the reference strain. The values are means ± standard deviations (SD) from three independent experiments. (C) Differential interference contrast images of hyphae grown on MMPDRUU in the presence or absence of salt stress at 37°C for 16 h. Bar, 10 μm. (D) Analysis of branching frequencies in hyphal filaments of the Δ cnaA and Δ cnaA cchA re strains and the reference strain. The values are means ± SD from three independent experiments.
    Nacl, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 14230 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nacl/product/Thermo Fisher
    Average 99 stars, based on 14230 article reviews
    Price from $9.99 to $1999.99
    nacl - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher t7 rna polymerase
    Dominant-negative effect of L-protein mutants. (A) Inactive mutants were transfected in a ratio of 1:1 with wild-type L protein. As a control, a PCR fragment of the L gene lacking the upstream <t>T7</t> RNA polymerase promoter was transfected 1:1 with the complete
    T7 Rna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12287 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t7 rna polymerase/product/Thermo Fisher
    Average 99 stars, based on 12287 article reviews
    Price from $9.99 to $1999.99
    t7 rna polymerase - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher dnase i
    Flow cytometric analysis of apoptotic cells with fragmented DNA in RSV-infected cells. A549 cells in a semimicroplate were trypsinized pretreatment (pre) and at 36 and 48 h after RSV infection, labeled by TUNEL, and analyzed by flow cytometry. As a positive control, DNase I-treated A549 cells were used; around 20% of these cells were shown to be positive.
    Dnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 73125 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnase i/product/Thermo Fisher
    Average 99 stars, based on 73125 article reviews
    Price from $9.99 to $1999.99
    dnase i - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher superscript iii first strand synthesis supermix
    Flow cytometric analysis of apoptotic cells with fragmented DNA in RSV-infected cells. A549 cells in a semimicroplate were trypsinized pretreatment (pre) and at 36 and 48 h after RSV infection, labeled by TUNEL, and analyzed by flow cytometry. As a positive control, DNase I-treated A549 cells were used; around 20% of these cells were shown to be positive.
    Superscript Iii First Strand Synthesis Supermix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 11389 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii first strand synthesis supermix/product/Thermo Fisher
    Average 99 stars, based on 11389 article reviews
    Price from $9.99 to $1999.99
    superscript iii first strand synthesis supermix - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher rnase h
    Polyadenylation state of Fmr1 mRNA variants in the CGG KI mouse brain. ( A ) Left panel: schematic view of the polyadenylation assay (PAT). According to di Penta et al . ( 42 ), the poly(A) tails are tagged by incubating the RNA with a (T) 12 -tag oligonucleotide, blocked at the 3′-end, in the presence of dNTPs and Klenow enzyme to fill in the complementary tag sequence. The RNA is then denatured and annealed to a DNA primer, identical to the tag, to start a reverse transcription (RT). The cDNA is then amplified using a gene-specific forward primer and the reverse tag oligo. Right panel: cartoon of a polyadenylation profile obtained with a PAT assay. The PCR of a polyadenylated mRNA gives rise to a smear while the same mRNA deadenylated with oligodT and <t>RNase</t> H prior to poly(A) tagging is used as a negative control and gives a sharp band. ( B ) Upper panel: β-actin mRNA in WT (lane 1) and CGG KI (lane 3). Deadenylated RNA is shown as negative control (lane 2) and the deadenylated form is indicated by black arrows. Lower panel: dispersion graph representing the distribution of the β-actin polyadenylated transcripts in WT (black line) and CGG KI (grey line). The signal intensity along the lane has been plotted against the poly(A) tail length, estimated from the molecular markers loaded on the same gel. ( C ) PAT for all three poly(A) Fmr1 mRNA variants. Because of close proximity, the transcripts containing sites V and VI cannot be discriminated and therefore they are not taken into exam (upper panel). Black arrows points to the deadenylated form. The polyadenylation of transcripts using site IV from WT (lane 1) and CGG KI (lane 3) has been independently acquired and highlighted in the box below. Deadenylated RNA treated as mentioned above, is shown as negative control (lane 2). Right panel: dispersion graph for Fmr1 variants using site IV in WT (black line) and CGG KI (grey line). ( D ) Left panel: PAT for Fmr1 variants using site VI in WT (lane 1) and CGG KI (lane 3) brain. Deadenylated RNA as above is used as negative control (lane 2). Right panel: Dispersion graph representing the distribution of the polyadenylated transcripts using site VI in WT (black line) and CGG KI (grey line).
    Rnase H, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9985 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase h/product/Thermo Fisher
    Average 99 stars, based on 9985 article reviews
    Price from $9.99 to $1999.99
    rnase h - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher revertaid h minus first strand cdna synthesis kit
    Polyadenylation state of Fmr1 mRNA variants in the CGG KI mouse brain. ( A ) Left panel: schematic view of the polyadenylation assay (PAT). According to di Penta et al . ( 42 ), the poly(A) tails are tagged by incubating the RNA with a (T) 12 -tag oligonucleotide, blocked at the 3′-end, in the presence of dNTPs and Klenow enzyme to fill in the complementary tag sequence. The RNA is then denatured and annealed to a DNA primer, identical to the tag, to start a reverse transcription (RT). The cDNA is then amplified using a gene-specific forward primer and the reverse tag oligo. Right panel: cartoon of a polyadenylation profile obtained with a PAT assay. The PCR of a polyadenylated mRNA gives rise to a smear while the same mRNA deadenylated with oligodT and <t>RNase</t> H prior to poly(A) tagging is used as a negative control and gives a sharp band. ( B ) Upper panel: β-actin mRNA in WT (lane 1) and CGG KI (lane 3). Deadenylated RNA is shown as negative control (lane 2) and the deadenylated form is indicated by black arrows. Lower panel: dispersion graph representing the distribution of the β-actin polyadenylated transcripts in WT (black line) and CGG KI (grey line). The signal intensity along the lane has been plotted against the poly(A) tail length, estimated from the molecular markers loaded on the same gel. ( C ) PAT for all three poly(A) Fmr1 mRNA variants. Because of close proximity, the transcripts containing sites V and VI cannot be discriminated and therefore they are not taken into exam (upper panel). Black arrows points to the deadenylated form. The polyadenylation of transcripts using site IV from WT (lane 1) and CGG KI (lane 3) has been independently acquired and highlighted in the box below. Deadenylated RNA treated as mentioned above, is shown as negative control (lane 2). Right panel: dispersion graph for Fmr1 variants using site IV in WT (black line) and CGG KI (grey line). ( D ) Left panel: PAT for Fmr1 variants using site VI in WT (lane 1) and CGG KI (lane 3) brain. Deadenylated RNA as above is used as negative control (lane 2). Right panel: Dispersion graph representing the distribution of the polyadenylated transcripts using site VI in WT (black line) and CGG KI (grey line).
    Revertaid H Minus First Strand Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9555 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/revertaid h minus first strand cdna synthesis kit/product/Thermo Fisher
    Average 99 stars, based on 9555 article reviews
    Price from $9.99 to $1999.99
    revertaid h minus first strand cdna synthesis kit - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    Cell cycle state of ID4-EGFP + spermatogonial subpopulations. A ) Cell cycle state among P6 ID4-EGFP + subpopulations was characterized using flow cytometry and density dot plots demonstrate sequential cell pregating based on: light scatter characteristics (FSC-A × SSC-A; i ), viability (propidium iodide negative; ii ), single cells ( iii ), and ID4-EGFP + ( iv ). Cells stained with antibodies against TSPAN8 ( B – D ), EHPA2 ( E – G ), and PVR ( H – J ) were used for DNA content analysis with the Vybrant DyeCycle Violet Stain. Histograms indicating cell number (y-axis) and DNA content (x-axis) are shown for EGFP + spermatogonia with the top one-third (based on cell number) most intensely stained cells (B , E , and H) and marker-positive cells with the bottom one-third (based on cell number) weakest positive staining intensity (C , F , and I) . Insets in each panel show dot plots with EGFP intensity, antibody staining intensity, and selection gates for histograms. The percentage of ID4-EGFP + cells shown in each histogram is noted above the histogram. Transparent blue, red, and green curves in each histogram show the Gaussian functions corresponding to 2N (G1/G0), 4 > N > 2 (S), and (4N) G2/M fractions of each population, which serve as estimates of the proportion of gated cells in each cell cycle phase. D , G , and J) Mean ± SEM from results of four replicate staining experiments for each marker are shown in the stacked bar graphs. In each graph, significant differences in cell cycle state between subpopulations were determined by Student t -tests and are noted between bars (* P

    Journal: Biology of Reproduction

    Article Title: TSPAN8 Expression Distinguishes Spermatogonial Stem Cells in the Prepubertal Mouse Testis 1

    doi: 10.1095/biolreprod.116.144220

    Figure Lengend Snippet: Cell cycle state of ID4-EGFP + spermatogonial subpopulations. A ) Cell cycle state among P6 ID4-EGFP + subpopulations was characterized using flow cytometry and density dot plots demonstrate sequential cell pregating based on: light scatter characteristics (FSC-A × SSC-A; i ), viability (propidium iodide negative; ii ), single cells ( iii ), and ID4-EGFP + ( iv ). Cells stained with antibodies against TSPAN8 ( B – D ), EHPA2 ( E – G ), and PVR ( H – J ) were used for DNA content analysis with the Vybrant DyeCycle Violet Stain. Histograms indicating cell number (y-axis) and DNA content (x-axis) are shown for EGFP + spermatogonia with the top one-third (based on cell number) most intensely stained cells (B , E , and H) and marker-positive cells with the bottom one-third (based on cell number) weakest positive staining intensity (C , F , and I) . Insets in each panel show dot plots with EGFP intensity, antibody staining intensity, and selection gates for histograms. The percentage of ID4-EGFP + cells shown in each histogram is noted above the histogram. Transparent blue, red, and green curves in each histogram show the Gaussian functions corresponding to 2N (G1/G0), 4 > N > 2 (S), and (4N) G2/M fractions of each population, which serve as estimates of the proportion of gated cells in each cell cycle phase. D , G , and J) Mean ± SEM from results of four replicate staining experiments for each marker are shown in the stacked bar graphs. In each graph, significant differences in cell cycle state between subpopulations were determined by Student t -tests and are noted between bars (* P

    Article Snippet: Complementary DNA was synthesized from DNase-treated RNA, as described previously [ ], using SuperScript III reverse transcriptase (ThermoFisher Scientific) and oligo-dT18 priming.

    Techniques: Flow Cytometry, Cytometry, Staining, Marker, Selection

    CBLC ubiquitin ligase activity is required for Golgi but not SRC levels regulation. (A) Requirement of CBLC RING domain. HeLa wild type (WT) or stably expressing siRNA-resistant-CBLC (CBLC-GFP) or siRNA-resistant-CBLC RING mutant (C351A-GFP) cells were transfected with deconvoluted CBLC-7 siRNA. Golgi stained with Giantin. Top right: Schematic diagram, exogenous CBLC contains three-point mutations at CBLC-7 siRNA-binding site. Bottom right: Fragmentation Index measured in quadruplicates on at least 400 cells per condition. Error bars show SD statistical significance (p) measured by unpaired Student’s t-test. (**) represents p

    Journal: PLoS ONE

    Article Title: The Ubiquitin Ligase CBLC Maintains the Network Organization of the Golgi Apparatus

    doi: 10.1371/journal.pone.0138789

    Figure Lengend Snippet: CBLC ubiquitin ligase activity is required for Golgi but not SRC levels regulation. (A) Requirement of CBLC RING domain. HeLa wild type (WT) or stably expressing siRNA-resistant-CBLC (CBLC-GFP) or siRNA-resistant-CBLC RING mutant (C351A-GFP) cells were transfected with deconvoluted CBLC-7 siRNA. Golgi stained with Giantin. Top right: Schematic diagram, exogenous CBLC contains three-point mutations at CBLC-7 siRNA-binding site. Bottom right: Fragmentation Index measured in quadruplicates on at least 400 cells per condition. Error bars show SD statistical significance (p) measured by unpaired Student’s t-test. (**) represents p

    Article Snippet: Total RNA was extracted from HeLa cells and full-length CBLC (GenBank/EMBL/DDBJ accession no. NM_012116) was synthesized using SuperScript-III (Invitrogen).

    Techniques: Activity Assay, Stable Transfection, Expressing, Mutagenesis, Transfection, Staining, Binding Assay

    Shu maps to the voltage-gated sodium channel gene paralytic. A , Mapping positions of the Shu mutation on the X chromosome. Red triangles and horizontal lines represent pairs of molecularly defined P-element insertions used to estimate the Shu mutation site; red X’s indicate the mutation sites deduced from the recombination rates between the corresponding P-element insertion and Shu . The estimated sites all reside within the para locus (CG9907). Boxes designate annotated genes near the para locus (based on FlyBase). B , DNA sequencing chromatogram identifying a G-to-A transition mutation (arrowheads) in the Shu genome at the position corresponding to the nucleotide 4249 in the para-RE cDNA (FlyBase). This mutation results in a methionine-to-isoleucine substitution at the amino acid position 1327. C , Schematic structural diagram of a Drosophila voltage-gated sodium channel. Arrow indicates the Shu mutation in the transmembrane segment S2 in homology domain III. The para GEFS+ mutation K1330T, which corresponds to a SCN1A mutation K1270T causing GEFS+ in humans ( Sun et al., 2012 ), lies three codons away from that of Shu . Also shown are the para DS mutation S1291R and the para bss1 mutation L1676F. D , Amino acid sequence alignment of Na v channels of different animal species. Note that the methionine residue, which is mutated to isoleucine in Shu , is present in all Na v channels.

    Journal: eNeuro

    Article Title: Lithium-Responsive Seizure-Like Hyperexcitability Is Caused by a Mutation in the Drosophila Voltage-Gated Sodium Channel Gene paralytic

    doi: 10.1523/ENEURO.0221-16.2016

    Figure Lengend Snippet: Shu maps to the voltage-gated sodium channel gene paralytic. A , Mapping positions of the Shu mutation on the X chromosome. Red triangles and horizontal lines represent pairs of molecularly defined P-element insertions used to estimate the Shu mutation site; red X’s indicate the mutation sites deduced from the recombination rates between the corresponding P-element insertion and Shu . The estimated sites all reside within the para locus (CG9907). Boxes designate annotated genes near the para locus (based on FlyBase). B , DNA sequencing chromatogram identifying a G-to-A transition mutation (arrowheads) in the Shu genome at the position corresponding to the nucleotide 4249 in the para-RE cDNA (FlyBase). This mutation results in a methionine-to-isoleucine substitution at the amino acid position 1327. C , Schematic structural diagram of a Drosophila voltage-gated sodium channel. Arrow indicates the Shu mutation in the transmembrane segment S2 in homology domain III. The para GEFS+ mutation K1330T, which corresponds to a SCN1A mutation K1270T causing GEFS+ in humans ( Sun et al., 2012 ), lies three codons away from that of Shu . Also shown are the para DS mutation S1291R and the para bss1 mutation L1676F. D , Amino acid sequence alignment of Na v channels of different animal species. Note that the methionine residue, which is mutated to isoleucine in Shu , is present in all Na v channels.

    Article Snippet: Reverse-transcription PCR analysis For semiquantitative and real-time reverse-transcription (RT)-PCR, RNA was extracted using the methods described above, and single-strand cDNA libraries were synthesized with DNase I-treated RNA using Superscript III reverse transcriptase kit (Invitrogen) or, in the real-time experiments, iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA).

    Techniques: Mutagenesis, DNA Sequencing, Sequencing

    Analysis of tumor samples for expression of antigens from stable cell lines confirmed expression in the tumors. (a) Protein was extracted from primary tumor tissues, and the concentration was calculated. Tissues were homogenized by using a tissue tearer prior to processing for protein extraction. Portions (100 μg) of lysate were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. Equal loading of samples was confirmed with Ponceau-S staining of the membrane in all cases. The EBNA1 and Myc-tagged Nm23-H1 or EBNA3C were analyzed as reported with the use of anti-EBV human serum and anti-Myc antibody, respectively. (b) Total RNA was isolated from tissues by using TRIZOL reagent. Tissues were homogenized by using a tissue tearer prior to processing for RNA isolation. RT was carried performed with SuperScript II RNase H reverse transcriptase. followed by PCR with specific primers to detect the desired transcript. The Nm23-H1-Myc transcript was amplified by using the forward primer 5′-GATTACACGAGCTGTGCTCA-3′ and the reverse primer 5′-TTCGCTAGCCAAGTCTTCTT-3′ designed to amplify the junction sequence between Nm23-H1 and Myc tag. The EBNA3C-Myc transcript was amplified by using the forward primer 5′-CGGGATCCGGAAGGAACCATGGCCA-3′ and the reverse primer 5′-GAATTCTCCTGTCATTTCATAGATCCA-3′. The EBNA1 transcript was amplified by using the forward primer 5′-CGGGATCCGGAAGGAACCATGGCCA-3′ and the reverse primer 5′-GAATTCTCCTGTCATTTCATAGATCCA-3′. Amplification products were resolved in 1.5% agarose gels.

    Journal: Journal of Virology

    Article Title: Epstein-Barr Virus Latent Nuclear Antigens Can Induce Metastasis in a Nude Mouse Model ▿

    doi: 10.1128/JVI.00886-07

    Figure Lengend Snippet: Analysis of tumor samples for expression of antigens from stable cell lines confirmed expression in the tumors. (a) Protein was extracted from primary tumor tissues, and the concentration was calculated. Tissues were homogenized by using a tissue tearer prior to processing for protein extraction. Portions (100 μg) of lysate were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. Equal loading of samples was confirmed with Ponceau-S staining of the membrane in all cases. The EBNA1 and Myc-tagged Nm23-H1 or EBNA3C were analyzed as reported with the use of anti-EBV human serum and anti-Myc antibody, respectively. (b) Total RNA was isolated from tissues by using TRIZOL reagent. Tissues were homogenized by using a tissue tearer prior to processing for RNA isolation. RT was carried performed with SuperScript II RNase H reverse transcriptase. followed by PCR with specific primers to detect the desired transcript. The Nm23-H1-Myc transcript was amplified by using the forward primer 5′-GATTACACGAGCTGTGCTCA-3′ and the reverse primer 5′-TTCGCTAGCCAAGTCTTCTT-3′ designed to amplify the junction sequence between Nm23-H1 and Myc tag. The EBNA3C-Myc transcript was amplified by using the forward primer 5′-CGGGATCCGGAAGGAACCATGGCCA-3′ and the reverse primer 5′-GAATTCTCCTGTCATTTCATAGATCCA-3′. The EBNA1 transcript was amplified by using the forward primer 5′-CGGGATCCGGAAGGAACCATGGCCA-3′ and the reverse primer 5′-GAATTCTCCTGTCATTTCATAGATCCA-3′. Amplification products were resolved in 1.5% agarose gels.

    Article Snippet: RT was carried out with SuperScript II RNase H-reverse transcriptase (Life Technologies, Gaithersburg, MD).

    Techniques: Expressing, Stable Transfection, Concentration Assay, Protein Extraction, Polyacrylamide Gel Electrophoresis, Staining, Isolation, Polymerase Chain Reaction, Amplification, Sequencing

    MHV-ExoN(-) evolved WT-like genomic RNA accumulation and increased resistance to multiple nucleoside analogues over the passage. (A) Cells were infected with the indicated viruses at MOI = 1 PFU/cell, and intracellular RNA was harvested using TRIzol at the indicated times postinfection. MHV genomic RNA was detected using SYBR green and primers directed to nsp10, and values were normalized to intracellular GAPDH. (B) Same data as in panel A normalized to the RNA level for each virus at 4 hpi. Data represent means and standard errors of results for n = 9 (3 triplicate experiments). (C to F) Sensitivity of passaged viruses to nucleoside analogues at MOI = 0.01 PFU/cell. Cells were treated with the indicated concentrations of 5-FU (C), RBV (D), AZC (E), or CMeA (F) for 30 min prior to infection, supernatants were harvested at 24 hpi, and titers were determined by plaque assay. Data represent changes in titer relative to untreated control results and are plotted as means and standard errors of results from n = 6 (two triplicate experiments). For panels C to F, the statistical significance of changes in the titer of MHV-ExoN(-) P3 relative to MHV-ExoN(-) P250 was determined using the Mann-Whitney test (*, P

    Journal: mBio

    Article Title: Proofreading-Deficient Coronaviruses Adapt for Increased Fitness over Long-Term Passage without Reversion of Exoribonuclease-Inactivating Mutations

    doi: 10.1128/mBio.01503-17

    Figure Lengend Snippet: MHV-ExoN(-) evolved WT-like genomic RNA accumulation and increased resistance to multiple nucleoside analogues over the passage. (A) Cells were infected with the indicated viruses at MOI = 1 PFU/cell, and intracellular RNA was harvested using TRIzol at the indicated times postinfection. MHV genomic RNA was detected using SYBR green and primers directed to nsp10, and values were normalized to intracellular GAPDH. (B) Same data as in panel A normalized to the RNA level for each virus at 4 hpi. Data represent means and standard errors of results for n = 9 (3 triplicate experiments). (C to F) Sensitivity of passaged viruses to nucleoside analogues at MOI = 0.01 PFU/cell. Cells were treated with the indicated concentrations of 5-FU (C), RBV (D), AZC (E), or CMeA (F) for 30 min prior to infection, supernatants were harvested at 24 hpi, and titers were determined by plaque assay. Data represent changes in titer relative to untreated control results and are plotted as means and standard errors of results from n = 6 (two triplicate experiments). For panels C to F, the statistical significance of changes in the titer of MHV-ExoN(-) P3 relative to MHV-ExoN(-) P250 was determined using the Mann-Whitney test (*, P

    Article Snippet: For each passage, supernatants were harvested at 24 h. RNA was extracted from 100 μl of supernatant using 900 μl of TRIzol reagent and PureLink RNA minikit columns (Thermo Scientific, Waltham, MA), and 150 μl of supernatant was used to infect fresh cells in a 24-well plate (total MOI estimated at 1 PFU/cell).

    Techniques: Infection, SYBR Green Assay, Plaque Assay, MANN-WHITNEY

    a 3-BrPA potentiates Prima-1 toxicity against A549 cells in 5 mM glucose. A549 cells (4X10 3 ) were seeded in tissue culture 96 well plates in complete medium containing 20 mM glucose and 10% fetal bovine serum, then washed 3X with PBS and treated as indicated in each case, in medium supplemented with physiological 5 mM glucose, 2 mM glutamine and 5% dialyzed serum for 48 h. Relative proliferation /toxicity was assayed fluorometrically in octuplicate by the Alamar Blue method by quantitating the conversion of resazurin to fluorescent resorufin [ 8 ]. This revealed that 50 μM Prima-1 cooperated with 3-BrPA rather than with CHC to suppress A549 cell growth. b Prima-1 decreases SLC2A1-GLUT1 in A549 cells. Sparse cells were seeded in 5 cm tissue culture plates (5 × 10 5 cells per plate) in complete Dulbecco’s medium containing 20 mM glucose supplemented with 10% serum for 18 h, then washed 3X with PBS and treated as indicated in each case, in medium supplemented with physiological 5 mM glucose, 2 mM glutamine and 5% dialyzed serum whenever indicated (+) for 24 h. After RNA extraction with TRIZOL and quantification in a Qubit® 2.0 Fluorometer, cDNAs were prepared for end-point PCR analysis as indicated under Methods.essentially similar results were obtained in cells treated with Prima-1 in 5 mM glucose (not shown). Cells treated in parallel with those used for RNA analysis were used for GLUT1 protein immune blot [ 40 ]. c Prima-1 activates p21CDKN1A gene expression in A549 cells in 5 mM glucose. qPCR was used to determine relative expression of the p21CDK1N1 gene in control and treated cells, after RNA extraction, cDNA preparation and qPCR, as indicated under Methods. *denotes significance between treated cells relative to controls

    Journal: BMC Cancer

    Article Title: Hypoxic resistance of KRAS mutant tumor cells to 3-Bromopyruvate is counteracted by Prima-1 and reversed by N-acetylcysteine

    doi: 10.1186/s12885-016-2930-9

    Figure Lengend Snippet: a 3-BrPA potentiates Prima-1 toxicity against A549 cells in 5 mM glucose. A549 cells (4X10 3 ) were seeded in tissue culture 96 well plates in complete medium containing 20 mM glucose and 10% fetal bovine serum, then washed 3X with PBS and treated as indicated in each case, in medium supplemented with physiological 5 mM glucose, 2 mM glutamine and 5% dialyzed serum for 48 h. Relative proliferation /toxicity was assayed fluorometrically in octuplicate by the Alamar Blue method by quantitating the conversion of resazurin to fluorescent resorufin [ 8 ]. This revealed that 50 μM Prima-1 cooperated with 3-BrPA rather than with CHC to suppress A549 cell growth. b Prima-1 decreases SLC2A1-GLUT1 in A549 cells. Sparse cells were seeded in 5 cm tissue culture plates (5 × 10 5 cells per plate) in complete Dulbecco’s medium containing 20 mM glucose supplemented with 10% serum for 18 h, then washed 3X with PBS and treated as indicated in each case, in medium supplemented with physiological 5 mM glucose, 2 mM glutamine and 5% dialyzed serum whenever indicated (+) for 24 h. After RNA extraction with TRIZOL and quantification in a Qubit® 2.0 Fluorometer, cDNAs were prepared for end-point PCR analysis as indicated under Methods.essentially similar results were obtained in cells treated with Prima-1 in 5 mM glucose (not shown). Cells treated in parallel with those used for RNA analysis were used for GLUT1 protein immune blot [ 40 ]. c Prima-1 activates p21CDKN1A gene expression in A549 cells in 5 mM glucose. qPCR was used to determine relative expression of the p21CDK1N1 gene in control and treated cells, after RNA extraction, cDNA preparation and qPCR, as indicated under Methods. *denotes significance between treated cells relative to controls

    Article Snippet: Real-time and end-point RT-PCR Cells were seeded in 5 cm-well plates (3 × 105 cells per plate) in complete Dulbecco’s medium containing 20 mM glucose supplemented with 10% serum for 24 h. Cells were washed 3X with PBS and treated as indicated in medium supplemented with physiological 5 mM glucose and 5% dialyzed serum for 24 h. RNA extraction was performed using TRIZOL® (Life Technologies, Cat # 15596–026) and quantification was determined using a Qubit® 2.0 Fluorometer (Life Technologies, Cat #Q32866) with a Qubit™ RNA Assay Kit (Life Technologies, Cat # Q32852).

    Techniques: RNA Extraction, Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction

    HERV-K gag and pol ORFs are more prevalent in ALS Both prototypical HERV-K genome in 5q33.3 (Panel A, i) and partial HERV-K genomes, such 7q36.1 (Panel A, iii) can encode for RT. In contrast, 7q34 has no ORF for RT, but contributes gag ORFs (Panel A, ii). HERV-K loci encode potential ORFs for retroviral proteins Gag, protease, RT, RNaseH, integrase and Env. Patients with ALS more frequently express combinations of loci encoding gag and pol ORFs, as compared to controls with systemic disease (Panel B).

    Journal: Annals of neurology

    Article Title: Identification of Active Loci of a Human Endogenous Retrovirus in Neurons of Patients with Amyotrophic Lateral Sclerosis

    doi: 10.1002/ana.22149

    Figure Lengend Snippet: HERV-K gag and pol ORFs are more prevalent in ALS Both prototypical HERV-K genome in 5q33.3 (Panel A, i) and partial HERV-K genomes, such 7q36.1 (Panel A, iii) can encode for RT. In contrast, 7q34 has no ORF for RT, but contributes gag ORFs (Panel A, ii). HERV-K loci encode potential ORFs for retroviral proteins Gag, protease, RT, RNaseH, integrase and Env. Patients with ALS more frequently express combinations of loci encoding gag and pol ORFs, as compared to controls with systemic disease (Panel B).

    Article Snippet: DNase I digested RNA samples were reverse transcribed using random hexamers and a gene-specific HERV-K pol primer (5′-GTTGAAGAGCTCGACCTACAAAA- 3′) using SuperScript III First-Strand Synthesis System for real-time-PCR (Invitrogen).

    Techniques:

    RT-LAMP assay in solution. (a) Real-time RT-LAMP amplification of in vitro transcribed H1N1 RNA standards from 10 10 cp/mL down to 10 5 cp/mL. NTC = no template control. (b) 2% Agarose gel electrophoresis of RT-LAMP products. L = 100bp DNA ladder, 10 = 10 10 cp/mL, 9 = 10 9 cp/mL, etc. NTC = no template control. M1 = M1 gene in vitro transcribed standards, 10 10 cp/mL. (c) Representative lateral flow strips from three independent experiments show detection of RT-LAMP products. The top line is the flow strip control line; the bottom line is a test line. Test line intensity as percentage of control line intensity for three experiments is plotted. (* p

    Journal: Analytical chemistry

    Article Title: Paper-Based RNA Extraction, in Situ Isothermal Amplification, and Lateral Flow Detection for Low-Cost, Rapid Diagnosis of Influenza A (H1N1) from Clinical Specimens

    doi: 10.1021/acs.analchem.5b01594

    Figure Lengend Snippet: RT-LAMP assay in solution. (a) Real-time RT-LAMP amplification of in vitro transcribed H1N1 RNA standards from 10 10 cp/mL down to 10 5 cp/mL. NTC = no template control. (b) 2% Agarose gel electrophoresis of RT-LAMP products. L = 100bp DNA ladder, 10 = 10 10 cp/mL, 9 = 10 9 cp/mL, etc. NTC = no template control. M1 = M1 gene in vitro transcribed standards, 10 10 cp/mL. (c) Representative lateral flow strips from three independent experiments show detection of RT-LAMP products. The top line is the flow strip control line; the bottom line is a test line. Test line intensity as percentage of control line intensity for three experiments is plotted. (* p

    Article Snippet: Genomic RNA from the patient sample was extracted via the QIAamp Viral RNA Mini Kit (Qiagen) and reverse-transcribed with the Superscript III First-Strand Synthesis Kit (Invitrogen) with a gene-specific reverse primer.

    Techniques: RT Lamp Assay, Amplification, In Vitro, Agarose Gel Electrophoresis, Flow Cytometry, Stripping Membranes

    RT-LAMP in situ with in vitro transcribed H1N1 RNA standards. (a) Method schematic of paper RNA extraction followed by in situ RT-LAMP and immediate downstream lateral flow detection. (b) Representative lateral flow detection strips. 10 = 10 10 cp/mL, etc. NTC = no template control. (c) Lateral flow detection strip test line intensities from three independent experiments are plotted as a percentage of control line intensities. (* p

    Journal: Analytical chemistry

    Article Title: Paper-Based RNA Extraction, in Situ Isothermal Amplification, and Lateral Flow Detection for Low-Cost, Rapid Diagnosis of Influenza A (H1N1) from Clinical Specimens

    doi: 10.1021/acs.analchem.5b01594

    Figure Lengend Snippet: RT-LAMP in situ with in vitro transcribed H1N1 RNA standards. (a) Method schematic of paper RNA extraction followed by in situ RT-LAMP and immediate downstream lateral flow detection. (b) Representative lateral flow detection strips. 10 = 10 10 cp/mL, etc. NTC = no template control. (c) Lateral flow detection strip test line intensities from three independent experiments are plotted as a percentage of control line intensities. (* p

    Article Snippet: Genomic RNA from the patient sample was extracted via the QIAamp Viral RNA Mini Kit (Qiagen) and reverse-transcribed with the Superscript III First-Strand Synthesis Kit (Invitrogen) with a gene-specific reverse primer.

    Techniques: In Situ, In Vitro, RNA Extraction, Flow Cytometry, Stripping Membranes

    Elevated gene 50 transcription is accompanied by hypomethylation of the gene 50 promoter at day 18. (A) Quantitative RT-PCR for total gene 50 (G50) (exon 2) transcripts on cDNA generated from total splenocyte RNA. Transcript levels are elevated in Cre-positive

    Journal: Journal of Virology

    Article Title: The De Novo Methyltransferases DNMT3a and DNMT3b Target the Murine Gammaherpesvirus Immediate-Early Gene 50 Promoter during Establishment of Latency ▿

    doi: 10.1128/JVI.00060-10

    Figure Lengend Snippet: Elevated gene 50 transcription is accompanied by hypomethylation of the gene 50 promoter at day 18. (A) Quantitative RT-PCR for total gene 50 (G50) (exon 2) transcripts on cDNA generated from total splenocyte RNA. Transcript levels are elevated in Cre-positive

    Article Snippet: Twenty microliters of DNase-treated RNA was subsequently used for first-strand cDNA synthesis by use of SuperScript II reverse transcriptase (Invitrogen).

    Techniques: Quantitative RT-PCR, Generated

    Analysis of C-rich tails. ( A ) Boxplot analysis of the length of C-rich tails observed by 3′ RACE. ( B ) and ( D ) 3′-tails at the end of nad2 and rrnS2 , respectively (red, with a blue line marking the next gene). Browser view of WTSS 3′-soft-clipped reads; coverage is plotted in log scale above the reads and only soft-clipped bases are colored (C: blue, U: red; A: green; G: brown). ( C ) and ( E ) the corresponding proportion-based sequence logos and length profiles for the 3′-tails using WTSS are shown. ( F ) RT-PCR to detect C-rich tails on mt transcripts by oligo(dG) priming. The 3′ RACE experiment serves as a control. ( G ) RT-PCR showing absence of C-rich tails on cytosolic and chloroplast transcripts. First strand cDNA synthesis was primed with oligo(dG) (top) or random hexamers (bottom).

    Journal: Nucleic Acids Research

    Article Title: Polycytidylation of mitochondrial mRNAs in Chlamydomonas reinhardtii

    doi: 10.1093/nar/gkx903

    Figure Lengend Snippet: Analysis of C-rich tails. ( A ) Boxplot analysis of the length of C-rich tails observed by 3′ RACE. ( B ) and ( D ) 3′-tails at the end of nad2 and rrnS2 , respectively (red, with a blue line marking the next gene). Browser view of WTSS 3′-soft-clipped reads; coverage is plotted in log scale above the reads and only soft-clipped bases are colored (C: blue, U: red; A: green; G: brown). ( C ) and ( E ) the corresponding proportion-based sequence logos and length profiles for the 3′-tails using WTSS are shown. ( F ) RT-PCR to detect C-rich tails on mt transcripts by oligo(dG) priming. The 3′ RACE experiment serves as a control. ( G ) RT-PCR showing absence of C-rich tails on cytosolic and chloroplast transcripts. First strand cDNA synthesis was primed with oligo(dG) (top) or random hexamers (bottom).

    Article Snippet: For 5′-RACE, the first-strand cDNA synthesized above was C-tailed with the terminal deoxynucleotidyl-transferase (Invitrogen) in the presence of dCTP.

    Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction

    Calponin-3-GFP is expressed from the endogenous Cnn3 locus. A. RT-PCR analysis of the Cnn3 mRNA splicing in targeted cells. Total RNA from the pre-B cell line Oct as well as from pre-B cells derived from a Cnn3 ki f/f mouse was transcribed into cDNA and analyzed by PCR. Primer pairs for PCRs 1 and 2 are depicted in the schematic illustration of the targeted allele. A sample lacking cDNA served as a control. B. Western blot analysis for expression of the calponin-3-GFP fusion protein from the targeted Cnn3 locus. Pre-B cells derived from a Cnn3 ki f/f mouse or a non-targeted littermate were lysed and subjected to SDS-PAGE and blotting. Oct pre-B cells expressing GFP and calponin-3-GFP were used as a control. Equal loading was demonstrated by anti-eIF4α. C. Flow cytometric analysis of cells from a Cnn3 ki f/f mouse or a non-targeted +/+ littermate. Cells were isolated from the bone marrow of the respective mice, stained with an anti-IgM antibody and analyzed for expression of IgM and GFP by flow cytometry. Numbers indicate the percentage of cells in the respective gate.

    Journal: PLoS ONE

    Article Title: A Conditional Knockout Mouse Model Reveals That Calponin-3 Is Dispensable for Early B Cell Development

    doi: 10.1371/journal.pone.0128385

    Figure Lengend Snippet: Calponin-3-GFP is expressed from the endogenous Cnn3 locus. A. RT-PCR analysis of the Cnn3 mRNA splicing in targeted cells. Total RNA from the pre-B cell line Oct as well as from pre-B cells derived from a Cnn3 ki f/f mouse was transcribed into cDNA and analyzed by PCR. Primer pairs for PCRs 1 and 2 are depicted in the schematic illustration of the targeted allele. A sample lacking cDNA served as a control. B. Western blot analysis for expression of the calponin-3-GFP fusion protein from the targeted Cnn3 locus. Pre-B cells derived from a Cnn3 ki f/f mouse or a non-targeted littermate were lysed and subjected to SDS-PAGE and blotting. Oct pre-B cells expressing GFP and calponin-3-GFP were used as a control. Equal loading was demonstrated by anti-eIF4α. C. Flow cytometric analysis of cells from a Cnn3 ki f/f mouse or a non-targeted +/+ littermate. Cells were isolated from the bone marrow of the respective mice, stained with an anti-IgM antibody and analyzed for expression of IgM and GFP by flow cytometry. Numbers indicate the percentage of cells in the respective gate.

    Article Snippet: 1 μg of RNA was reverse-transcribed into cDNA using the First Strand cDNA Synthesis Kit (Fermentas) according to the manufacturers’ instructions.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Polymerase Chain Reaction, Western Blot, Expressing, SDS Page, Flow Cytometry, Isolation, Mouse Assay, Staining, Cytometry

    Midkine expression was induced by FBS, growth factors and cytokines. A . LNCaP cells were cultured in serum-free DMEM and treated for 48 h with 10% FBS and the indicated agents: 10 ng/ml insulin, 10 ng/ml IGF-I, 10 ng/ml EGF, 10 ng/ml HGF, 10 ng/ml bFGF, 20 ng/ml T3, 10 nM R1881, 10 nM DHT, 10 ng/ml TNFα, 10 ng/ml IL-1β, 50 ng/ml IL-6, 50 ng/ml IL-17, and 33.3 μM RA. B . LNCaP cells were treated with different dosages (1 to 50 ng/ml) of TNFα for 48 h. C . LNCaP cells were also treated with 20 ng/ml TNFα for different time periods (8 to 48 h). 20 μl of each medium supernatant was subjected to Western blot analysis of midkine expression using rabbit anti-midkine antibodies, horseradish peroxidase-conjungated secondary antibodies and enhanced chemiluminescence reagents. D . Total RNA was extracted from LNCaP cells not treated or treated with 20 ng/ml TNFα, using RNeasy Mini Kit; cDNA was made from total RNA using Superscript™ First-Strand Synthesis System with oligo dT primers; real-time quantitative PCR was done in triplicates with Sybr-Green reagents; results were normalized to GAPDH levels as described in Methods; the data (mean ± standard deviation of three independent experiments) were presented as fold change of midkine mRNA compared to the LNCaP cells without treatment for 8 h, where fold = 2 ΔΔCt ; solid bar, TNFα treated; open bar, control; * P

    Journal: BMC Medical Genomics

    Article Title: Midkine is a NF-?B-inducible gene that supports prostate cancer cell survival

    doi: 10.1186/1755-8794-1-6

    Figure Lengend Snippet: Midkine expression was induced by FBS, growth factors and cytokines. A . LNCaP cells were cultured in serum-free DMEM and treated for 48 h with 10% FBS and the indicated agents: 10 ng/ml insulin, 10 ng/ml IGF-I, 10 ng/ml EGF, 10 ng/ml HGF, 10 ng/ml bFGF, 20 ng/ml T3, 10 nM R1881, 10 nM DHT, 10 ng/ml TNFα, 10 ng/ml IL-1β, 50 ng/ml IL-6, 50 ng/ml IL-17, and 33.3 μM RA. B . LNCaP cells were treated with different dosages (1 to 50 ng/ml) of TNFα for 48 h. C . LNCaP cells were also treated with 20 ng/ml TNFα for different time periods (8 to 48 h). 20 μl of each medium supernatant was subjected to Western blot analysis of midkine expression using rabbit anti-midkine antibodies, horseradish peroxidase-conjungated secondary antibodies and enhanced chemiluminescence reagents. D . Total RNA was extracted from LNCaP cells not treated or treated with 20 ng/ml TNFα, using RNeasy Mini Kit; cDNA was made from total RNA using Superscript™ First-Strand Synthesis System with oligo dT primers; real-time quantitative PCR was done in triplicates with Sybr-Green reagents; results were normalized to GAPDH levels as described in Methods; the data (mean ± standard deviation of three independent experiments) were presented as fold change of midkine mRNA compared to the LNCaP cells without treatment for 8 h, where fold = 2 ΔΔCt ; solid bar, TNFα treated; open bar, control; * P

    Article Snippet: Analysis of MDK mRNA expression by real-time quantitative RT-PCR Total RNA was extracted from LNCaP cells not treated or treated with 20 ng/ml TNFα, using RNeasy Mini Kit (QIAGEN, Valencia, CA) with on-membrane DNase I digestion to avoid genomic DNA contamination. cDNA was made from total RNA using Superscript™ First-Strand Synthesis System with oligo dT primers (Invitrogen, Carlsbad, CA).

    Techniques: Expressing, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction, SYBR Green Assay, Standard Deviation

    Gadd45a binds RNA in vitro . A, RNA filter binding assay using the indicated proteins and 32 P-labeled RNA (multiple cloning site transcript, MCS). Co, no protein; BSA, bovine serum albumin; M-MLV RT - Moloney murine leukemia virus reverse transcriptase. B, C, RNA filter binding assays using 32 P-labeled MCS RNA were performed with recombinant Gadd45a in the presence of the indicated unlabeled competitor nucleic acids. Data are shown as percentage of 32 P bound in the absence of the competitor. Each sample was done in triplicate; average and standard deviation was generated; A representative experiment out of three is shown. U, unmethylated; M, methylated; U/U, unmethylated; U/M, hemimethylated; M/M, holomethylated; PolyA, polyC, polyG, polyU, homopolyribonucleotides; total RNA, RNA isolated from HEK293T cells; tRNA, yeast tRNA; MCS RNA, multiple cloning site RNA. Error bars, s.e.m. (n = 3). A representative experiment out of three is shown.

    Journal: PLoS ONE

    Article Title: Gadd45a Is an RNA Binding Protein and Is Localized in Nuclear Speckles

    doi: 10.1371/journal.pone.0014500

    Figure Lengend Snippet: Gadd45a binds RNA in vitro . A, RNA filter binding assay using the indicated proteins and 32 P-labeled RNA (multiple cloning site transcript, MCS). Co, no protein; BSA, bovine serum albumin; M-MLV RT - Moloney murine leukemia virus reverse transcriptase. B, C, RNA filter binding assays using 32 P-labeled MCS RNA were performed with recombinant Gadd45a in the presence of the indicated unlabeled competitor nucleic acids. Data are shown as percentage of 32 P bound in the absence of the competitor. Each sample was done in triplicate; average and standard deviation was generated; A representative experiment out of three is shown. U, unmethylated; M, methylated; U/U, unmethylated; U/M, hemimethylated; M/M, holomethylated; PolyA, polyC, polyG, polyU, homopolyribonucleotides; total RNA, RNA isolated from HEK293T cells; tRNA, yeast tRNA; MCS RNA, multiple cloning site RNA. Error bars, s.e.m. (n = 3). A representative experiment out of three is shown.

    Article Snippet: Recombinant proteins used were bovine serum albumin (Fraction V, Sigma), His-Gadd45a and M-MLV-reverse transcriptase (Invitrogen).

    Techniques: In Vitro, Filter-binding Assay, Labeling, Clone Assay, Binding Assay, Recombinant, Standard Deviation, Generated, Methylation, Isolation

    mtDNA increases SOCE and expression of Sphk1 in PMN. (A) Human PMN were treated with 20 µg/mL of E.coli DNA or mtDNA for 60 min then loaded with fura-2. Thapsigargin (1 µM) was applied to under nominally calcium-free conditions and then 1.8 mM extracellular calcium was applied at the indicated times. Calcium influx was calculated as described elsewhere [9] as the area under the curve for [Ca 2+ ] i (AUC) over 120 sec. Data were analyzed using medium-treated PMN as 100%. Mean and SE values are shown. At least 3 experiment were done per condition. * denotes a significant difference by student t-test (SigmaPlot 11) compared to time “0” value. Experiments were repeated at least three times. (7B) Freshly isolated human PMN (5 million cells in 2 mL) were incubated with medium, mtDNA (10 µg/mL), E.coli DNA (10 µg/mL), or LPS (100 ng/mL) for 60 min. Then RNA and cDNA were prepared using the RNesay mini kit (Qiagen) and SuperScript VILO cDNA Synthesis kit (Life technologies), respectively. 200 ng of cDNA per reaction was used for TaqMan qPCR assay for Sphk1 and GAPDH to evaluate expression levels. Sphk1 expression levels were then further normalized by GAPDH using the medium control result as 100%. Four different PMN preparations were used for stimulation and qPCR assays were done in triplicates. Mean and SE values from four different experiments are shown. Data were analyzed by One Way ANOVA. * denotes a significant difference between mtDNA treatment and the medium control (p

    Journal: PLoS ONE

    Article Title: Mitochondrial DAMPs Increase Endothelial Permeability through Neutrophil Dependent and Independent Pathways

    doi: 10.1371/journal.pone.0059989

    Figure Lengend Snippet: mtDNA increases SOCE and expression of Sphk1 in PMN. (A) Human PMN were treated with 20 µg/mL of E.coli DNA or mtDNA for 60 min then loaded with fura-2. Thapsigargin (1 µM) was applied to under nominally calcium-free conditions and then 1.8 mM extracellular calcium was applied at the indicated times. Calcium influx was calculated as described elsewhere [9] as the area under the curve for [Ca 2+ ] i (AUC) over 120 sec. Data were analyzed using medium-treated PMN as 100%. Mean and SE values are shown. At least 3 experiment were done per condition. * denotes a significant difference by student t-test (SigmaPlot 11) compared to time “0” value. Experiments were repeated at least three times. (7B) Freshly isolated human PMN (5 million cells in 2 mL) were incubated with medium, mtDNA (10 µg/mL), E.coli DNA (10 µg/mL), or LPS (100 ng/mL) for 60 min. Then RNA and cDNA were prepared using the RNesay mini kit (Qiagen) and SuperScript VILO cDNA Synthesis kit (Life technologies), respectively. 200 ng of cDNA per reaction was used for TaqMan qPCR assay for Sphk1 and GAPDH to evaluate expression levels. Sphk1 expression levels were then further normalized by GAPDH using the medium control result as 100%. Four different PMN preparations were used for stimulation and qPCR assays were done in triplicates. Mean and SE values from four different experiments are shown. Data were analyzed by One Way ANOVA. * denotes a significant difference between mtDNA treatment and the medium control (p

    Article Snippet: RNA and cDNA Preparation and Quantitative PCR RNA and cDNA were prepared using the RNesay mini kit (Qiagen, Valencia, CA) and the SuperScript VILO cDNA Synthesis kit (Life Technologies, Carlsbad, CA), respectively.

    Techniques: Expressing, Size-exclusion Chromatography, Isolation, Incubation, Real-time Polymerase Chain Reaction

    Illustrations of real-time PCR of OT and VP RNAs using cDNA derived from the total RNA of the same rat SON. Representative real-time PCR growth curves for OT ( A ) and VP ( B ) specific PCR primers using cDNA template from total RNA of the same rat SON are

    Journal:

    Article Title: Oxytocin and vasopressin gene expression and RNA splicing patterns in the rat supraoptic nucleus

    doi: 10.1152/physiolgenomics.90218.2008

    Figure Lengend Snippet: Illustrations of real-time PCR of OT and VP RNAs using cDNA derived from the total RNA of the same rat SON. Representative real-time PCR growth curves for OT ( A ) and VP ( B ) specific PCR primers using cDNA template from total RNA of the same rat SON are

    Article Snippet: Reverse transcription was performed using 200 ng total RNA extracted and purified from the SON or SCN of each animal, 2 pmol of either OT or VP primers, and 200 units of SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA) in a total volume of 20 μl.

    Techniques: Real-time Polymerase Chain Reaction, Derivative Assay, Polymerase Chain Reaction

    Comparisons of OT and VP hnRNA and mRNA levels in supraoptic nuclei (SONs) of acutely hyperosmotically stimulated male rats vs. SONs in isotonic controls. Real-time PCR analysis of the various OT and VP RNA species in SON total RNA extracted from male

    Journal:

    Article Title: Oxytocin and vasopressin gene expression and RNA splicing patterns in the rat supraoptic nucleus

    doi: 10.1152/physiolgenomics.90218.2008

    Figure Lengend Snippet: Comparisons of OT and VP hnRNA and mRNA levels in supraoptic nuclei (SONs) of acutely hyperosmotically stimulated male rats vs. SONs in isotonic controls. Real-time PCR analysis of the various OT and VP RNA species in SON total RNA extracted from male

    Article Snippet: Reverse transcription was performed using 200 ng total RNA extracted and purified from the SON or SCN of each animal, 2 pmol of either OT or VP primers, and 200 units of SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA) in a total volume of 20 μl.

    Techniques: Real-time Polymerase Chain Reaction

    Induction of interferon regulatory factor-9 ( IRF-9 ) in the mutant IFNAR-2c (FF) cells by interferon-β (IFN-β). ( A ) RNA was isolated from untreated cells ( C ) or cells treated with 1,000 IU/mL of recombinant IFN-β for 1 and 3 h,

    Journal: Journal of Interferon & Cytokine Research

    Article Title: STAT-Phosphorylation-Independent Induction of Interferon Regulatory Factor-9 by Interferon-?

    doi: 10.1089/jir.2009.0032

    Figure Lengend Snippet: Induction of interferon regulatory factor-9 ( IRF-9 ) in the mutant IFNAR-2c (FF) cells by interferon-β (IFN-β). ( A ) RNA was isolated from untreated cells ( C ) or cells treated with 1,000 IU/mL of recombinant IFN-β for 1 and 3 h,

    Article Snippet: To identify genes induced in the absence of STAT phosphorylation by IFN-β, RNA was isolated from FF cells treated with and without IFN-β for 1 or 3 h. Oligonucleotide array analysis (Affymetrix™ ) identified a small subset of ISGs induced by IFN-β.

    Techniques: Mutagenesis, Isolation, Recombinant

    The hyphal growth defects of the Δ cnaA or Δ cnaB strains could be suppressed by the dysfunction of CchA under salt stress. (A) Colony morphology of the Δ cnaA , Δ cnaA cchA re , Δ cnaB , and Δ cnaB Δ cchA strains and the reference strain. The conidia were spotted on solid MMPDRUU and MMPDRUU supplemented with 800 mM NaCl, 600 mM KCl, or 1 M sorbitol, respectively, at 37°C for 2.5 days. (B) Graphic representation of radial growth rates of the Δ cnaA and Δ cnaA cchA re strains and the reference strain. The values are means ± standard deviations (SD) from three independent experiments. (C) Differential interference contrast images of hyphae grown on MMPDRUU in the presence or absence of salt stress at 37°C for 16 h. Bar, 10 μm. (D) Analysis of branching frequencies in hyphal filaments of the Δ cnaA and Δ cnaA cchA re strains and the reference strain. The values are means ± SD from three independent experiments.

    Journal: Applied and Environmental Microbiology

    Article Title: Calcineurin and Calcium Channel CchA Coordinate the Salt Stress Response by Regulating Cytoplasmic Ca2+ Homeostasis in Aspergillus nidulans

    doi: 10.1128/AEM.00330-16

    Figure Lengend Snippet: The hyphal growth defects of the Δ cnaA or Δ cnaB strains could be suppressed by the dysfunction of CchA under salt stress. (A) Colony morphology of the Δ cnaA , Δ cnaA cchA re , Δ cnaB , and Δ cnaB Δ cchA strains and the reference strain. The conidia were spotted on solid MMPDRUU and MMPDRUU supplemented with 800 mM NaCl, 600 mM KCl, or 1 M sorbitol, respectively, at 37°C for 2.5 days. (B) Graphic representation of radial growth rates of the Δ cnaA and Δ cnaA cchA re strains and the reference strain. The values are means ± standard deviations (SD) from three independent experiments. (C) Differential interference contrast images of hyphae grown on MMPDRUU in the presence or absence of salt stress at 37°C for 16 h. Bar, 10 μm. (D) Analysis of branching frequencies in hyphal filaments of the Δ cnaA and Δ cnaA cchA re strains and the reference strain. The values are means ± SD from three independent experiments.

    Article Snippet: A total of 1 × 106 spores were cultured on either MMPDRUU or MMPDRUU with 800 mM NaCl in a 96-well microtiter plate (Thermo Fischer, United Kingdom) and then incubated at 37°C for 18 h. The medium was then removed, and the mycelia were rinsed twice with PGM: 20 mM PIPES [piperazine- N , N ′-bis(2-ethanesulfonic acid)] (pH 6.7), 50 mM glucose, and 1 mM MgCl2 .

    Techniques:

    Colony morphologies of the Δ yvcA , Δ cnaA , and Δ cnaA Δ yvcA strains grown on MMPDRUU in the presence or absence of 800 mM NaCl or 800 mM NaCl plus 3 mM EGTA at 37°C for 2.5 days.

    Journal: Applied and Environmental Microbiology

    Article Title: Calcineurin and Calcium Channel CchA Coordinate the Salt Stress Response by Regulating Cytoplasmic Ca2+ Homeostasis in Aspergillus nidulans

    doi: 10.1128/AEM.00330-16

    Figure Lengend Snippet: Colony morphologies of the Δ yvcA , Δ cnaA , and Δ cnaA Δ yvcA strains grown on MMPDRUU in the presence or absence of 800 mM NaCl or 800 mM NaCl plus 3 mM EGTA at 37°C for 2.5 days.

    Article Snippet: A total of 1 × 106 spores were cultured on either MMPDRUU or MMPDRUU with 800 mM NaCl in a 96-well microtiter plate (Thermo Fischer, United Kingdom) and then incubated at 37°C for 18 h. The medium was then removed, and the mycelia were rinsed twice with PGM: 20 mM PIPES [piperazine- N , N ′-bis(2-ethanesulfonic acid)] (pH 6.7), 50 mM glucose, and 1 mM MgCl2 .

    Techniques:

    Expression analysis of Ca 2+ -signaling-related and salt-stress-induced genes in response to salt stress by quantitative PCR. (A) Fold changes in mRNA levels, including vcxA , yvcA , pmrA , and pmcA , after incubation with MMPDRUU with addition of 800 mM NaCl compared to results with MMPDRUU alone using real-time RT-PCR. (B) Fold changes in mRNA levels, including enaA , nhaA , and trkA , after incubation with MMPDRUU with addition of 800 mM NaCl compared to results with MMPDRUU alone using real-time RT-PCR. Data representing the indicated strains' mRNA levels from salt stress pretreatment were normalized to the non-salt-pretreatment condition. The error bars indicate the standard deviations from three independent replicates.

    Journal: Applied and Environmental Microbiology

    Article Title: Calcineurin and Calcium Channel CchA Coordinate the Salt Stress Response by Regulating Cytoplasmic Ca2+ Homeostasis in Aspergillus nidulans

    doi: 10.1128/AEM.00330-16

    Figure Lengend Snippet: Expression analysis of Ca 2+ -signaling-related and salt-stress-induced genes in response to salt stress by quantitative PCR. (A) Fold changes in mRNA levels, including vcxA , yvcA , pmrA , and pmcA , after incubation with MMPDRUU with addition of 800 mM NaCl compared to results with MMPDRUU alone using real-time RT-PCR. (B) Fold changes in mRNA levels, including enaA , nhaA , and trkA , after incubation with MMPDRUU with addition of 800 mM NaCl compared to results with MMPDRUU alone using real-time RT-PCR. Data representing the indicated strains' mRNA levels from salt stress pretreatment were normalized to the non-salt-pretreatment condition. The error bars indicate the standard deviations from three independent replicates.

    Article Snippet: A total of 1 × 106 spores were cultured on either MMPDRUU or MMPDRUU with 800 mM NaCl in a 96-well microtiter plate (Thermo Fischer, United Kingdom) and then incubated at 37°C for 18 h. The medium was then removed, and the mycelia were rinsed twice with PGM: 20 mM PIPES [piperazine- N , N ′-bis(2-ethanesulfonic acid)] (pH 6.7), 50 mM glucose, and 1 mM MgCl2 .

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Incubation, Quantitative RT-PCR

    Dominant-negative effect of L-protein mutants. (A) Inactive mutants were transfected in a ratio of 1:1 with wild-type L protein. As a control, a PCR fragment of the L gene lacking the upstream T7 RNA polymerase promoter was transfected 1:1 with the complete

    Journal: Journal of Virology

    Article Title: Mutational Evidence for a Structural Model of the Lassa Virus RNA Polymerase Domain and Identification of Two Residues, Gly1394 and Asp1395, That Are Critical for Transcription but Not Replication of the Genome ▿

    doi: 10.1128/JVI.00220-08

    Figure Lengend Snippet: Dominant-negative effect of L-protein mutants. (A) Inactive mutants were transfected in a ratio of 1:1 with wild-type L protein. As a control, a PCR fragment of the L gene lacking the upstream T7 RNA polymerase promoter was transfected 1:1 with the complete

    Article Snippet: BSR T7/5 cells ( ) stably expressing T7 RNA polymerase and BHK-21 cells were grown in Glasgow modified Eagle medium (MEM; Gibco) supplemented with 5% fetal calf serum (FCS).

    Techniques: Dominant Negative Mutation, Transfection, Polymerase Chain Reaction

    Flow cytometric analysis of apoptotic cells with fragmented DNA in RSV-infected cells. A549 cells in a semimicroplate were trypsinized pretreatment (pre) and at 36 and 48 h after RSV infection, labeled by TUNEL, and analyzed by flow cytometry. As a positive control, DNase I-treated A549 cells were used; around 20% of these cells were shown to be positive.

    Journal: Journal of Virology

    Article Title: Respiratory Syncytial Virus Infection of Human Alveolar Epithelial Cells Enhances Interferon Regulatory Factor 1 and Interleukin-1?-Converting Enzyme Gene Expression but Does Not Cause Apoptosis

    doi:

    Figure Lengend Snippet: Flow cytometric analysis of apoptotic cells with fragmented DNA in RSV-infected cells. A549 cells in a semimicroplate were trypsinized pretreatment (pre) and at 36 and 48 h after RSV infection, labeled by TUNEL, and analyzed by flow cytometry. As a positive control, DNase I-treated A549 cells were used; around 20% of these cells were shown to be positive.

    Article Snippet: For positive control of DNA fragmentation, A549 cells treated with DNase I (GIBCO BRL) (10 μg/ml for 10 min) were employed ( ).

    Techniques: Flow Cytometry, Infection, Labeling, TUNEL Assay, Cytometry, Positive Control

    Polyadenylation state of Fmr1 mRNA variants in the CGG KI mouse brain. ( A ) Left panel: schematic view of the polyadenylation assay (PAT). According to di Penta et al . ( 42 ), the poly(A) tails are tagged by incubating the RNA with a (T) 12 -tag oligonucleotide, blocked at the 3′-end, in the presence of dNTPs and Klenow enzyme to fill in the complementary tag sequence. The RNA is then denatured and annealed to a DNA primer, identical to the tag, to start a reverse transcription (RT). The cDNA is then amplified using a gene-specific forward primer and the reverse tag oligo. Right panel: cartoon of a polyadenylation profile obtained with a PAT assay. The PCR of a polyadenylated mRNA gives rise to a smear while the same mRNA deadenylated with oligodT and RNase H prior to poly(A) tagging is used as a negative control and gives a sharp band. ( B ) Upper panel: β-actin mRNA in WT (lane 1) and CGG KI (lane 3). Deadenylated RNA is shown as negative control (lane 2) and the deadenylated form is indicated by black arrows. Lower panel: dispersion graph representing the distribution of the β-actin polyadenylated transcripts in WT (black line) and CGG KI (grey line). The signal intensity along the lane has been plotted against the poly(A) tail length, estimated from the molecular markers loaded on the same gel. ( C ) PAT for all three poly(A) Fmr1 mRNA variants. Because of close proximity, the transcripts containing sites V and VI cannot be discriminated and therefore they are not taken into exam (upper panel). Black arrows points to the deadenylated form. The polyadenylation of transcripts using site IV from WT (lane 1) and CGG KI (lane 3) has been independently acquired and highlighted in the box below. Deadenylated RNA treated as mentioned above, is shown as negative control (lane 2). Right panel: dispersion graph for Fmr1 variants using site IV in WT (black line) and CGG KI (grey line). ( D ) Left panel: PAT for Fmr1 variants using site VI in WT (lane 1) and CGG KI (lane 3) brain. Deadenylated RNA as above is used as negative control (lane 2). Right panel: Dispersion graph representing the distribution of the polyadenylated transcripts using site VI in WT (black line) and CGG KI (grey line).

    Journal: Nucleic Acids Research

    Article Title: Differential usage of transcriptional start sites and polyadenylation sites in FMR1 premutation alleles †

    doi: 10.1093/nar/gkr100

    Figure Lengend Snippet: Polyadenylation state of Fmr1 mRNA variants in the CGG KI mouse brain. ( A ) Left panel: schematic view of the polyadenylation assay (PAT). According to di Penta et al . ( 42 ), the poly(A) tails are tagged by incubating the RNA with a (T) 12 -tag oligonucleotide, blocked at the 3′-end, in the presence of dNTPs and Klenow enzyme to fill in the complementary tag sequence. The RNA is then denatured and annealed to a DNA primer, identical to the tag, to start a reverse transcription (RT). The cDNA is then amplified using a gene-specific forward primer and the reverse tag oligo. Right panel: cartoon of a polyadenylation profile obtained with a PAT assay. The PCR of a polyadenylated mRNA gives rise to a smear while the same mRNA deadenylated with oligodT and RNase H prior to poly(A) tagging is used as a negative control and gives a sharp band. ( B ) Upper panel: β-actin mRNA in WT (lane 1) and CGG KI (lane 3). Deadenylated RNA is shown as negative control (lane 2) and the deadenylated form is indicated by black arrows. Lower panel: dispersion graph representing the distribution of the β-actin polyadenylated transcripts in WT (black line) and CGG KI (grey line). The signal intensity along the lane has been plotted against the poly(A) tail length, estimated from the molecular markers loaded on the same gel. ( C ) PAT for all three poly(A) Fmr1 mRNA variants. Because of close proximity, the transcripts containing sites V and VI cannot be discriminated and therefore they are not taken into exam (upper panel). Black arrows points to the deadenylated form. The polyadenylation of transcripts using site IV from WT (lane 1) and CGG KI (lane 3) has been independently acquired and highlighted in the box below. Deadenylated RNA treated as mentioned above, is shown as negative control (lane 2). Right panel: dispersion graph for Fmr1 variants using site IV in WT (black line) and CGG KI (grey line). ( D ) Left panel: PAT for Fmr1 variants using site VI in WT (lane 1) and CGG KI (lane 3) brain. Deadenylated RNA as above is used as negative control (lane 2). Right panel: Dispersion graph representing the distribution of the polyadenylated transcripts using site VI in WT (black line) and CGG KI (grey line).

    Article Snippet: As a negative control, RNAs were deadenylated by incubating with Oligo(dT)12–18 (Invitrogen) and RNase H (Fermentas) at 37°C for 90 min, purified by phenol-chloroform and subjected to the same tagging reaction.

    Techniques: Sequencing, Amplification, Polymerase Chain Reaction, Negative Control