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  • 99
    Thermo Fisher revertaid first strand cdna synthesis kit
    IL-37 + T-cells play a protective role in DSS-induced chronic colitis in mice. ( A ) Relative weight curves of WT mice, DSS-induced WT mice, DSS-induced WT mice treated with PBS, and DSS-induced WT mice injected with IL-37 + T-cells in chronic colitis. The statistical significance between the relative weights of DSS-induced mice injected with IL-37 + T-cells versus DSS-induced mice treated with PBS at the end of the experiment is shown. ( B ) The disease activity index (DAI) was determined at day 63 of the study period, based on body weight loss (0, none; 1, 1–5%; 2, 5–10%; 3, 10–20%; 4, > 20%), stool consistency (0, normal; 2, loose stool; 4, diarrhea), and stool blood (0, negative; 2, fecal occult blood test positive; 4, gross bleeding). ( C ) Representative pictures of the colon from indicated treatment cohorts. ( D ) Macroscopic damage score of the colon in the four groups. ( E ) Representative haematoxylin and eosin (H E) staining of colon sections of mice. ( F ) Histological inflammation score. This score comprises the sum of architecture of the bowel, infiltration of neutrophils and mononuclear cells, gobleT-cell depletion, and epithelial cell erosion. Three sections per animal were evaluated. ( G ) Expressions of IFN-γ, IL-1β, TNF-α, and IL-10 mRNAs within colonic tissues in the four groups. Total RNA with the RNAiso Plusi Kit. cDNAs were synthesized by using the <t>RevertAid</t> First Strand <t>cDNA</t> Synthesis Kit according to the manufacturer’s instructions. qPCR amplification reactions were prepared with the SYBR Green PCR Kit and performed using the CFX96 Real-Time System. Data are means ± SEM ( n = 6). * p
    Revertaid First Strand Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 40345 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad iscript cdna synthesis kit
    RNA immunoprecipitation of (TMG)-capped primary miRNAs (Pri-miRNAs) in quiescent human foreskin fibroblasts (HFFs) RT-PCR data shows the RNA immunoprecipitation of Pri-miR-34a and Pri-miR-3188 in quiescent HFFs (as well as the positive control snRNA U7), but not Pri-miR-423 using an antibody against (TMG)-capped RNAs. Total RNA was extracted using TRIzol Reagent, and 10 μg of RNA was diluted in NET-2 buffer, precleared and incubated with Protein G Sepharose 4 Fast Flow beads loaded with 15 μl of control antibody (Normal Rabbit Serum, EMD-Millipore) or with antibody recognizing the (TMG)-cap (Anti-m3G-cap, rabbit polyclonal, Synaptic Systems). Beads were rinsed five times with NET-2 buffer and were resuspended in G-50 buffer. RNA was extracted from the beads by phenol-chloroform-isoamyl alcohol extraction and resuspended in 20 μl of nuclease-free water. Immunoprecipitated tri-methylated capped RNA was converted to <t>cDNA</t> using <t>iScript</t> cDNA synthesis kit (Bio-Rad), followed by RT-PCR, and visualized after gel electrophoresis.
    Iscript Cdna Synthesis Kit, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 95772 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 40724 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    Thermo Fisher cdna synthesis
    Expression levels of type I collagen, Smad2/3, and TGF-β1 genes after ES-P treatment in aged mice. (A) The left ears of fourteen-week old mice were treated daily with 30 μg T . spiralis ES-P for 14 days (red color). The right ears of were treated daily with PBS for 14 days as control (blue color). After 14 days, ears were collected. Total <t>RNA</t> was extracted from each ear tissue and <t>cDNA</t> was constructed. (B) The gene expression levels of collagen I , TGF-β1 , and Smad2/3 were analyzed by real-time PCR. The gene expression value of each group was normalized by control value, 14 weeks group. (**; P
    Cdna Synthesis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 106131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher cdna synthesis kit
    Gene expression profiles in GANT61-treated SKOV3 cells. ( A ) Identification of differentially expressed genes (DEGs) in GANT61-treated SKOV3 cells. Cells were treated with GANT61 (20 µM) or DMSO for 60 hr, and total <t>RNA</t> was extracted as described in Methods . Changes in gene expression were determined by <t>cDNA</t> microarray gene profiling using the Illumina Human HT expression BeadChip V4 (Illumina Inc., San Diego, CA). Differential expressions for the DEGs are shown. ( B ) The top 11 canonical signaling pathways influenced by inhibition of Gli1/Gli2 function in SKOV3 cells. The top 11 canonical signaling pathways, determined by IPA, that were significantly up-regulated or down-regulated by GANT61 treatment in SKOV3 cells, are shown. The 412 DEGs were mapped to the IPA-defined network. The significant P -values that determine the probability that the association between the genes in the dataset and the canonical pathway is by chance alone were calculated by Fisher's exact test, and are expressed as −log ( P -value). ( C ) Heat map of DEGs in the focal adhesion signal pathway in GANT61-treated SKOV3 cells. The heat map shows that 17 genes were significantly differentially expressed, including seven up-regulated genes and 12 down-regulated genes, in the GANT61-treated compared to control SKOV3 cells. ( D ) Selected DEGs from cDNA array gene expression profiling analyzed by real-time PCR. SKOV3 cells were treated with DMSO or GANT61 for the indicated times, total RNA was extracted for real-time PCR as described in Methods using the primer sets in Table 1 . The data represent the mean ± SD of three determinations, and GAPDH was used to normalize the relative mRNA levels.
    Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10713 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher revertaid h minus first strand cdna synthesis kit
    Gene expression profiles in GANT61-treated SKOV3 cells. ( A ) Identification of differentially expressed genes (DEGs) in GANT61-treated SKOV3 cells. Cells were treated with GANT61 (20 µM) or DMSO for 60 hr, and total <t>RNA</t> was extracted as described in Methods . Changes in gene expression were determined by <t>cDNA</t> microarray gene profiling using the Illumina Human HT expression BeadChip V4 (Illumina Inc., San Diego, CA). Differential expressions for the DEGs are shown. ( B ) The top 11 canonical signaling pathways influenced by inhibition of Gli1/Gli2 function in SKOV3 cells. The top 11 canonical signaling pathways, determined by IPA, that were significantly up-regulated or down-regulated by GANT61 treatment in SKOV3 cells, are shown. The 412 DEGs were mapped to the IPA-defined network. The significant P -values that determine the probability that the association between the genes in the dataset and the canonical pathway is by chance alone were calculated by Fisher's exact test, and are expressed as −log ( P -value). ( C ) Heat map of DEGs in the focal adhesion signal pathway in GANT61-treated SKOV3 cells. The heat map shows that 17 genes were significantly differentially expressed, including seven up-regulated genes and 12 down-regulated genes, in the GANT61-treated compared to control SKOV3 cells. ( D ) Selected DEGs from cDNA array gene expression profiling analyzed by real-time PCR. SKOV3 cells were treated with DMSO or GANT61 for the indicated times, total RNA was extracted for real-time PCR as described in Methods using the primer sets in Table 1 . The data represent the mean ± SD of three determinations, and GAPDH was used to normalize the relative mRNA levels.
    Revertaid H Minus First Strand Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 9555 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bio-Rad cdna synthesis
    Adora2b -deficient mice have increased inflammation with impaired induction of Tregs after LPS-induced lung injury. LPS-induced inflammation was initiated in 9–12 wk-old male Adora2b +/+ (C57BL/6J) or age and weight matched Adora2b −/− mice by intratracheal instillation of LPS or saline. Bronchoalveolar lavage (BAL) fluids were collected 1 d post treatment. A) Leukocyte cell counts of BAL fluid were determined utilizing a cellometer (mean ± SEM, n = 2–4). B) Protein content of BAL fluid was determined with BCA assay. Fold increase of protein concentration in LPS treated BAL samples over respective saline BAL samples is displayed (mean ± SEM, n = 3–10). C) Analysis of cell infiltration into the lung airspace (BAL) of mice exposed to inhaled LPS. Mice ( Adora2b +/+ or Adora2b −/−) were exposed to nebulized saline or lipopolysaccharide (LPS); 24 hours post-exposure, mice were euthanized, and BAL was harvested. Cells were then subjected to flow cytometric analysis to identify neutrophils (highly granular, Gr1+ cells). D) Analysis of FoxP3 gene expression in lungs of mice exposed to inhaled LPS. Mice ( Adora2b +/+ or −/−) were exposed to nebulized saline or lipopolysaccharide (LPS); 24 hours post-exposure, mice were euthanized, and perfused lungs were harvested. Total <t>RNA</t> was harvested from lungs, <t>cDNA</t> prepared and analyzed by real-time PCR. Data depict mean (± SEM) fold changes in indicated genes. Fold change was calculated based on primer efficiencies, standardized to changes in actin, with Adora2b +/+ Saline mean value defined as 1. E) Fold change in cell number of either regulatory T cells (lymphocyte size, CD3+ CD4+ FoxP3+) in the lung airspace (BAL) of mice exposed to inhaled saline or LPS as above. Data are from 2–6 mice; 2 independent experiments. F) Relative abundance of CD4 effector T cells (Teffs, defined as lymphocyte size, CD3+ CD4+ CD44high cells) to Tregs based on cell counts from either Adora2b +/+ or Adora2b −/− mice, with pie charts depicting mean cell count for the indicated cell populations from 2–6 mice; 2 independent expts. Analysis of total cellular infiltration into airways and protein leakage (panels A–B) was done in animals treated with intratracheal LPS. Analysis of neutrophil infiltration and Tregs (panels C–F) was done in animals treated with inhaled LPS. Statistically significant differences are indicated and were calculated either by one-way ANOVA (A) or by unpaired t test (B–E) with a focus on whether Adora2b −/− LPS treated animals were statistically different from either saline treated or Adora2b +/+ LPS treated mice.
    Cdna Synthesis, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 14206 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa primescript 1st strand cdna synthesis kit
    Adora2b -deficient mice have increased inflammation with impaired induction of Tregs after LPS-induced lung injury. LPS-induced inflammation was initiated in 9–12 wk-old male Adora2b +/+ (C57BL/6J) or age and weight matched Adora2b −/− mice by intratracheal instillation of LPS or saline. Bronchoalveolar lavage (BAL) fluids were collected 1 d post treatment. A) Leukocyte cell counts of BAL fluid were determined utilizing a cellometer (mean ± SEM, n = 2–4). B) Protein content of BAL fluid was determined with BCA assay. Fold increase of protein concentration in LPS treated BAL samples over respective saline BAL samples is displayed (mean ± SEM, n = 3–10). C) Analysis of cell infiltration into the lung airspace (BAL) of mice exposed to inhaled LPS. Mice ( Adora2b +/+ or Adora2b −/−) were exposed to nebulized saline or lipopolysaccharide (LPS); 24 hours post-exposure, mice were euthanized, and BAL was harvested. Cells were then subjected to flow cytometric analysis to identify neutrophils (highly granular, Gr1+ cells). D) Analysis of FoxP3 gene expression in lungs of mice exposed to inhaled LPS. Mice ( Adora2b +/+ or −/−) were exposed to nebulized saline or lipopolysaccharide (LPS); 24 hours post-exposure, mice were euthanized, and perfused lungs were harvested. Total <t>RNA</t> was harvested from lungs, <t>cDNA</t> prepared and analyzed by real-time PCR. Data depict mean (± SEM) fold changes in indicated genes. Fold change was calculated based on primer efficiencies, standardized to changes in actin, with Adora2b +/+ Saline mean value defined as 1. E) Fold change in cell number of either regulatory T cells (lymphocyte size, CD3+ CD4+ FoxP3+) in the lung airspace (BAL) of mice exposed to inhaled saline or LPS as above. Data are from 2–6 mice; 2 independent experiments. F) Relative abundance of CD4 effector T cells (Teffs, defined as lymphocyte size, CD3+ CD4+ CD44high cells) to Tregs based on cell counts from either Adora2b +/+ or Adora2b −/− mice, with pie charts depicting mean cell count for the indicated cell populations from 2–6 mice; 2 independent expts. Analysis of total cellular infiltration into airways and protein leakage (panels A–B) was done in animals treated with intratracheal LPS. Analysis of neutrophil infiltration and Tregs (panels C–F) was done in animals treated with inhaled LPS. Statistically significant differences are indicated and were calculated either by one-way ANOVA (A) or by unpaired t test (B–E) with a focus on whether Adora2b −/− LPS treated animals were statistically different from either saline treated or Adora2b +/+ LPS treated mice.
    Primescript 1st Strand Cdna Synthesis Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 7752 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Quanta Biosciences qscript cdna synthesis kit
    Adora2b -deficient mice have increased inflammation with impaired induction of Tregs after LPS-induced lung injury. LPS-induced inflammation was initiated in 9–12 wk-old male Adora2b +/+ (C57BL/6J) or age and weight matched Adora2b −/− mice by intratracheal instillation of LPS or saline. Bronchoalveolar lavage (BAL) fluids were collected 1 d post treatment. A) Leukocyte cell counts of BAL fluid were determined utilizing a cellometer (mean ± SEM, n = 2–4). B) Protein content of BAL fluid was determined with BCA assay. Fold increase of protein concentration in LPS treated BAL samples over respective saline BAL samples is displayed (mean ± SEM, n = 3–10). C) Analysis of cell infiltration into the lung airspace (BAL) of mice exposed to inhaled LPS. Mice ( Adora2b +/+ or Adora2b −/−) were exposed to nebulized saline or lipopolysaccharide (LPS); 24 hours post-exposure, mice were euthanized, and BAL was harvested. Cells were then subjected to flow cytometric analysis to identify neutrophils (highly granular, Gr1+ cells). D) Analysis of FoxP3 gene expression in lungs of mice exposed to inhaled LPS. Mice ( Adora2b +/+ or −/−) were exposed to nebulized saline or lipopolysaccharide (LPS); 24 hours post-exposure, mice were euthanized, and perfused lungs were harvested. Total <t>RNA</t> was harvested from lungs, <t>cDNA</t> prepared and analyzed by real-time PCR. Data depict mean (± SEM) fold changes in indicated genes. Fold change was calculated based on primer efficiencies, standardized to changes in actin, with Adora2b +/+ Saline mean value defined as 1. E) Fold change in cell number of either regulatory T cells (lymphocyte size, CD3+ CD4+ FoxP3+) in the lung airspace (BAL) of mice exposed to inhaled saline or LPS as above. Data are from 2–6 mice; 2 independent experiments. F) Relative abundance of CD4 effector T cells (Teffs, defined as lymphocyte size, CD3+ CD4+ CD44high cells) to Tregs based on cell counts from either Adora2b +/+ or Adora2b −/− mice, with pie charts depicting mean cell count for the indicated cell populations from 2–6 mice; 2 independent expts. Analysis of total cellular infiltration into airways and protein leakage (panels A–B) was done in animals treated with intratracheal LPS. Analysis of neutrophil infiltration and Tregs (panels C–F) was done in animals treated with inhaled LPS. Statistically significant differences are indicated and were calculated either by one-way ANOVA (A) or by unpaired t test (B–E) with a focus on whether Adora2b −/− LPS treated animals were statistically different from either saline treated or Adora2b +/+ LPS treated mice.
    Qscript Cdna Synthesis Kit, supplied by Quanta Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 4783 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad iscripttm cdna synthesis kit
    Superoxide dismutase 2 (SOD2) and NF-κB levels are increased in BRAF pathway inhibitor-resistant cells. ( A ) An equal number of WM-115 (parental, DR, and TDR) cells were plated. Total RNA was extracted from these cell lines using the RNA extraction mini kit, according to the manufacturer’s instructions. Two-step real time-qPCR was performed to assess the mRNA level of SOD2 and PRDX1. First strand <t>cDNA</t> was synthesized using the <t>iScriptTM</t> cDNA synthesis kit qPCR, which was set up using the CFX96 real-time system. Actin was used as an internal control. Relative SOD2 and PRDX1 expression was presented by the 2 −ΔΔCT method (left panel). In separate experiments, whole cell lysates were prepared and separated in a 12.5% sodium dodecyl sulfate (SDS) gel, followed by immunoblot analysis with indicated antibodies. Western blot data is shown on the right panel. ( B ) An equal number of WM-983, WM-983 DR, and WM-983 TDR cells were plated. Two-step real time-qPCR and immunoblot analysis with indicated antibodies were performed under the above conditions (left and right panels). ( C ) Nuclear extracts were collected and prepared from WM-115 and WM-983 (parental and TDR). * p
    Iscripttm Cdna Synthesis Kit, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 4755 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare first strand cdna synthesis kit
    Northern blot analysis of rasGEFM expression in parental and mutant strains . ( A ) Total <t>RNA</t> was extracted from AX2 cells developed in suspension for 0 to 9 hours or on filters. In the latter case the cells were harvested at mound (12 hours), first finger (16 hours) and preculminant (20 hours) stages. The membrane was hybridized to a radiolabelled rasGEFM specific probe (probe a ) corresponding to bp 760–1518 of the <t>cDNA</t> clone and to the actin gene used as a loading control. ( B ) Total RNA was extracted from different developmental Dictyostelium mutants starved in shaking suspension up to 6 hours. LW6 (G protein β subunit minus), HSB1 (PIA ts -mutant, defective in the G-protein adenylyl cyclase activation) and HSB50 (mutant blocked at mound stage).
    First Strand Cdna Synthesis Kit, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 5249 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript double stranded cdna synthesis kit
    Overview of Cufflinks. The algorithm takes as input <t>cDNA</t> fragment sequences that have been ( a ) aligned to the genome by software capable of producing spliced alignments, such as TopHat. With paired-end RNA-Seq, Cufflinks treats each pair of fragment reads as a single alignment. The algorithm assembles overlapping ‘bundles’ of fragment alignments ( b-c ) separately, which reduces running time and memory use because each bundle typically contains the fragments from no more than a few genes. Cufflinks then estimates the abundances of the assembled transcripts ( d-e ). ( b ) The first step in fragment assembly is to identify pairs of ‘incompatible’ fragments that must have originated from distinct spliced <t>mRNA</t> isoforms. Fragments are connected in an ‘overlap graph’ when they are compatible and their alignments overlap in the genome. Each fragment has one node in the graph, and an edge, directed from left to right along the genome, is placed between each pair of compatible fragments. In this example, the yellow, blue, and red fragments must have originated from separate isoforms, but any other fragment could have come from the same transcript as one of these three. ( c ) Assembling isoforms from the overlap graph. Paths through the graph correspond to sets of mutually compatible fragments that could be merged into complete isoforms. The overlap graph here can be minimally ‘covered’ by three paths, each representing a different isoform. Dilworth's Theorem states that the number of mutually incompatible reads is the same as the minimum number of transcripts needed to “explain” all the fragments. Cufflinks implements a proof of Dilworth's Theorem that produces a minimal set of paths that cover all the fragments in the overlap graph by finding the largest set of reads with the property that no two could have originated from the same isoform. ( d ) Estimating transcript abundance. Fragments are matched (denoted here using color) to the transcripts from which they could have originated. The violet fragment could have originated from the blue or red isoform. Gray fragments could have come from any of the three shown. Cufflinks estimates transcript abundances using a statistical model in which the probability of observing each fragment is a linear function of the abundances of the transcripts from which it could have originated. Because only the ends of each fragment are sequenced, the length of each may be unknown. Assigning a fragment to different isoforms often implies a different length for it. Cufflinks can incorporate the distribution of fragment lengths to help assign fragments to isoforms. For example, the violet fragment would be much longer, and very improbable according to Cufflinks' model, if it were to come from the red isoform instead of the blue isoform. ( e ) The program then numerically maximizes a function that assigns a likelihood to all possible sets of relative abundances of the yellow, red and blue isoforms (γ 1 ,γ 2 ,γ 3 ), producing the abundances that best explain the observed fragments, shown as a pie chart.
    Superscript Double Stranded Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6021 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher high capacity cdna synthesis kit
    Interaction with K562 CML cells and its paracrine factors modified cell surface antigen expression in MSC. a Cell surface expression of CD13, CD73, CD90, CD95 and CD105 in control-MSC (CON-MSC) and CML-MSC was analyzed by flow cytometry. Mean (geometric) fluorescent intensity (MFI) was calculated for each marker against its isotype control. b , c Control-MSC were cultured in conditioned media derived from K562 cells for one week and their cell surface gene expression in control-MSC (CON) and conditioned media treated MSC (MSC + CM) was analyzed by flow cytometry. MFI of analyzed markers was normalized to control-MSC. c Representative flow cytometry histogram showing cell surface antigen expression levels in CON and MSC + CM conditions. Grey line represents the isotype control, blue and red line represents the stained cells. d , e Control-MSC were co-cultured without (CON) or with K562 cells (MSC + K) for one week and their cell surface gene expression profile was determined by flow cytometry. MFI was calculated for each antigen and normalized to control-MSC. e Representative flow cytometry histograms showing the cell surface antigen expression in CON (blue line), MSC + K (red line) and isotype control (grey line). f , g <t>RNA</t> was extracted from control-MSC (CON) and K562 co-cultured MSC (MSC + K) and reverse transcribed into <t>cDNA.</t> mRNA expression levels of ( f ) CD90, ( g ) CAT and MnSOD was analyzed by real-time PCR. (h) Mitochondrial ROS levels in CON and MSC + K were analyzed by staining with mitosox red and MFI of mitosox was normalized to unstained cells. Values are mean ± SE, *p
    High Capacity Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2972 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Meridian Life Science tetro cdna synthesis kit
    Interaction with K562 CML cells and its paracrine factors modified cell surface antigen expression in MSC. a Cell surface expression of CD13, CD73, CD90, CD95 and CD105 in control-MSC (CON-MSC) and CML-MSC was analyzed by flow cytometry. Mean (geometric) fluorescent intensity (MFI) was calculated for each marker against its isotype control. b , c Control-MSC were cultured in conditioned media derived from K562 cells for one week and their cell surface gene expression in control-MSC (CON) and conditioned media treated MSC (MSC + CM) was analyzed by flow cytometry. MFI of analyzed markers was normalized to control-MSC. c Representative flow cytometry histogram showing cell surface antigen expression levels in CON and MSC + CM conditions. Grey line represents the isotype control, blue and red line represents the stained cells. d , e Control-MSC were co-cultured without (CON) or with K562 cells (MSC + K) for one week and their cell surface gene expression profile was determined by flow cytometry. MFI was calculated for each antigen and normalized to control-MSC. e Representative flow cytometry histograms showing the cell surface antigen expression in CON (blue line), MSC + K (red line) and isotype control (grey line). f , g <t>RNA</t> was extracted from control-MSC (CON) and K562 co-cultured MSC (MSC + K) and reverse transcribed into <t>cDNA.</t> mRNA expression levels of ( f ) CD90, ( g ) CAT and MnSOD was analyzed by real-time PCR. (h) Mitochondrial ROS levels in CON and MSC + K were analyzed by staining with mitosox red and MFI of mitosox was normalized to unstained cells. Values are mean ± SE, *p
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    IL-37 + T-cells play a protective role in DSS-induced chronic colitis in mice. ( A ) Relative weight curves of WT mice, DSS-induced WT mice, DSS-induced WT mice treated with PBS, and DSS-induced WT mice injected with IL-37 + T-cells in chronic colitis. The statistical significance between the relative weights of DSS-induced mice injected with IL-37 + T-cells versus DSS-induced mice treated with PBS at the end of the experiment is shown. ( B ) The disease activity index (DAI) was determined at day 63 of the study period, based on body weight loss (0, none; 1, 1–5%; 2, 5–10%; 3, 10–20%; 4, > 20%), stool consistency (0, normal; 2, loose stool; 4, diarrhea), and stool blood (0, negative; 2, fecal occult blood test positive; 4, gross bleeding). ( C ) Representative pictures of the colon from indicated treatment cohorts. ( D ) Macroscopic damage score of the colon in the four groups. ( E ) Representative haematoxylin and eosin (H E) staining of colon sections of mice. ( F ) Histological inflammation score. This score comprises the sum of architecture of the bowel, infiltration of neutrophils and mononuclear cells, gobleT-cell depletion, and epithelial cell erosion. Three sections per animal were evaluated. ( G ) Expressions of IFN-γ, IL-1β, TNF-α, and IL-10 mRNAs within colonic tissues in the four groups. Total RNA with the RNAiso Plusi Kit. cDNAs were synthesized by using the RevertAid First Strand cDNA Synthesis Kit according to the manufacturer’s instructions. qPCR amplification reactions were prepared with the SYBR Green PCR Kit and performed using the CFX96 Real-Time System. Data are means ± SEM ( n = 6). * p

    Journal: International Journal of Molecular Sciences

    Article Title: Anti-Inflammatory Effect of IL-37-Producing T-Cell Population in DSS-Induced Chronic Inflammatory Bowel Disease in Mice

    doi: 10.3390/ijms19123884

    Figure Lengend Snippet: IL-37 + T-cells play a protective role in DSS-induced chronic colitis in mice. ( A ) Relative weight curves of WT mice, DSS-induced WT mice, DSS-induced WT mice treated with PBS, and DSS-induced WT mice injected with IL-37 + T-cells in chronic colitis. The statistical significance between the relative weights of DSS-induced mice injected with IL-37 + T-cells versus DSS-induced mice treated with PBS at the end of the experiment is shown. ( B ) The disease activity index (DAI) was determined at day 63 of the study period, based on body weight loss (0, none; 1, 1–5%; 2, 5–10%; 3, 10–20%; 4, > 20%), stool consistency (0, normal; 2, loose stool; 4, diarrhea), and stool blood (0, negative; 2, fecal occult blood test positive; 4, gross bleeding). ( C ) Representative pictures of the colon from indicated treatment cohorts. ( D ) Macroscopic damage score of the colon in the four groups. ( E ) Representative haematoxylin and eosin (H E) staining of colon sections of mice. ( F ) Histological inflammation score. This score comprises the sum of architecture of the bowel, infiltration of neutrophils and mononuclear cells, gobleT-cell depletion, and epithelial cell erosion. Three sections per animal were evaluated. ( G ) Expressions of IFN-γ, IL-1β, TNF-α, and IL-10 mRNAs within colonic tissues in the four groups. Total RNA with the RNAiso Plusi Kit. cDNAs were synthesized by using the RevertAid First Strand cDNA Synthesis Kit according to the manufacturer’s instructions. qPCR amplification reactions were prepared with the SYBR Green PCR Kit and performed using the CFX96 Real-Time System. Data are means ± SEM ( n = 6). * p

    Article Snippet: The Nanodrop spectrophotometer (Thermo Scientific, Waltham, MA, USA) was used to measure absorbance. cDNAs were synthesized by using the RevertAid First Strand cDNA Synthesis Kit (ThermoFisher, Waltham, MA, USA) according to manufacturer’s instructions.

    Techniques: Mouse Assay, Injection, Activity Assay, Staining, Synthesized, Real-time Polymerase Chain Reaction, Amplification, SYBR Green Assay, Polymerase Chain Reaction

    Long non-coding RNA LINC01021 was downregulated by Da0324 treatment in gastric cancer cells. A. Volcano plots of lncRNAs differentially expressed between the control and Da0324 treatment groups. SGC7901 cells were treated with DMSO (control) or 4 μM Da0324 for 48 h and high-throughput sequencing assay was performed. The X-axis represents log 2 of fold changes. The Y-axis represents -log 10 of P values. Red spots denote upregulated lncRNAs, blue spots denote downregulated lncRNAs. B. Heatmap of the top 40 lncRNAs most significantly differentially expressed in SGC7901 cells with Da0324 treatment. C. Ten differentially expressed lncRNAs regulated by Da0324 were validated by qRT-PCR. SGC7901 cells were treated with DMSO (control) or 4 μM Da0324 for 48 h. RNAs were isolated and converted to cDNA. Quantitative real-time PCR was performed to determine the expression level of lncRNAs. GAPDH was used as a housekeeping gene. All bars represent relative expression levels and data represent mean ± SD. ***, P

    Journal: American Journal of Translational Research

    Article Title: Downregulation of LINC01021 by curcumin analog Da0324 inhibits gastric cancer progression through activation of P53

    doi:

    Figure Lengend Snippet: Long non-coding RNA LINC01021 was downregulated by Da0324 treatment in gastric cancer cells. A. Volcano plots of lncRNAs differentially expressed between the control and Da0324 treatment groups. SGC7901 cells were treated with DMSO (control) or 4 μM Da0324 for 48 h and high-throughput sequencing assay was performed. The X-axis represents log 2 of fold changes. The Y-axis represents -log 10 of P values. Red spots denote upregulated lncRNAs, blue spots denote downregulated lncRNAs. B. Heatmap of the top 40 lncRNAs most significantly differentially expressed in SGC7901 cells with Da0324 treatment. C. Ten differentially expressed lncRNAs regulated by Da0324 were validated by qRT-PCR. SGC7901 cells were treated with DMSO (control) or 4 μM Da0324 for 48 h. RNAs were isolated and converted to cDNA. Quantitative real-time PCR was performed to determine the expression level of lncRNAs. GAPDH was used as a housekeeping gene. All bars represent relative expression levels and data represent mean ± SD. ***, P

    Article Snippet: Total RNA was reverse transcribed to cDNA using the Revert Aid First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific).

    Techniques: Next-Generation Sequencing, Quantitative RT-PCR, Isolation, Real-time Polymerase Chain Reaction, Expressing

    RNA immunoprecipitation of (TMG)-capped primary miRNAs (Pri-miRNAs) in quiescent human foreskin fibroblasts (HFFs) RT-PCR data shows the RNA immunoprecipitation of Pri-miR-34a and Pri-miR-3188 in quiescent HFFs (as well as the positive control snRNA U7), but not Pri-miR-423 using an antibody against (TMG)-capped RNAs. Total RNA was extracted using TRIzol Reagent, and 10 μg of RNA was diluted in NET-2 buffer, precleared and incubated with Protein G Sepharose 4 Fast Flow beads loaded with 15 μl of control antibody (Normal Rabbit Serum, EMD-Millipore) or with antibody recognizing the (TMG)-cap (Anti-m3G-cap, rabbit polyclonal, Synaptic Systems). Beads were rinsed five times with NET-2 buffer and were resuspended in G-50 buffer. RNA was extracted from the beads by phenol-chloroform-isoamyl alcohol extraction and resuspended in 20 μl of nuclease-free water. Immunoprecipitated tri-methylated capped RNA was converted to cDNA using iScript cDNA synthesis kit (Bio-Rad), followed by RT-PCR, and visualized after gel electrophoresis.

    Journal: Bio-protocol

    Article Title: Immunoprecipitation of Tri-methylated Capped RNA

    doi: 10.21769/BioProtoc.2717

    Figure Lengend Snippet: RNA immunoprecipitation of (TMG)-capped primary miRNAs (Pri-miRNAs) in quiescent human foreskin fibroblasts (HFFs) RT-PCR data shows the RNA immunoprecipitation of Pri-miR-34a and Pri-miR-3188 in quiescent HFFs (as well as the positive control snRNA U7), but not Pri-miR-423 using an antibody against (TMG)-capped RNAs. Total RNA was extracted using TRIzol Reagent, and 10 μg of RNA was diluted in NET-2 buffer, precleared and incubated with Protein G Sepharose 4 Fast Flow beads loaded with 15 μl of control antibody (Normal Rabbit Serum, EMD-Millipore) or with antibody recognizing the (TMG)-cap (Anti-m3G-cap, rabbit polyclonal, Synaptic Systems). Beads were rinsed five times with NET-2 buffer and were resuspended in G-50 buffer. RNA was extracted from the beads by phenol-chloroform-isoamyl alcohol extraction and resuspended in 20 μl of nuclease-free water. Immunoprecipitated tri-methylated capped RNA was converted to cDNA using iScript cDNA synthesis kit (Bio-Rad), followed by RT-PCR, and visualized after gel electrophoresis.

    Article Snippet: Protein G Sepharose® 4 Fast Flow Beads (GE Healthcare, catalog number: 17061801) Normal rabbit serum (control, EMD Millipore, catalog number: NS01L-1ML) Sodium chloride (NaCl) (Fisher Scientific, catalog number: S671-3) NP-40 (Thermo Fisher Scientific, catalog number: 28324) Tris base (Fisher Scientific, catalog number: BP152-5) Hydrochloric acid (HCl) (VWR, catalog number: BDH7204-1) RNasin™ Plus RNase inhibitor (Promega, catalog number: N2611) NaOAc (AMRESCO, catalog number: 0602) Ethylenediaminetetraacetate acid (EDTA), pH 8 (Thermo Fisher Scientific, catalog number: AM9260G) Ethylenediaminetetraacetate acid (EDTA) (AMRESCO, catalog number: 0105) Sodium dodecyl sulfate (SDS) (Thermo Fisher Scientific, catalog number: AM9822) Phenol/Chloroform/Isoamyl Alcohol; 125:24:1 mixture, pH 4.5 (Thermo Fisher Scientific, catalog number: AM9720) Agarose LE (Denville Scientific, catalog number: CA3510-8) iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, catalog number: 1708891) Sso Advanced™ Universal SYBR® Green Supermix (Bio-Rad Laboratories, catalog number: 1725274) PARIS™ Kit (Thermo Fisher Scientific, catalog number: AM1921) Boric acid (Fisher Scientific, catalog number: A73-1) NET-2 Buffer (see Recipes) G-50 Buffer (see Recipes) 1× TBE (see Recipes)

    Techniques: Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Positive Control, Incubation, Flow Cytometry, Methylation, Nucleic Acid Electrophoresis

    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

    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

    A Zinc-dependent metalloproteinase of B. abortus is an operon that forms a putative type II toxin-antitoxin. Identification of the transcriptional unit (operon) constituted by ORF BAB1_0270 and a transcriptional regulator in B. abortus 2308 expressed in (A) the genomic DNA and (B) the cDNA from total RNA. MW: Molecular weight; lane 1: Transcriptional regulator (357 bp); lane 2: BAB1_0270 (549 bp); and lane 3: operon constituted by ORF BAB1_0270-transcriptional regulator (906 bp). (C) Hypothetical Type II toxin-antitoxin (TA) model for operon constitute by ZnMP and transcriptional regulator. Toxin (ZnMP) and anti-toxin (transcriptional regulator) are transcribed to mRNA together. Proteases are activated under stress conditions, cleaving the anti-toxin, which increases the levels of toxin-free, inducing various biological functions in bacteria. Predicted promoter at site -35 and -10 binding by RNA polymerase sigma factor rpoD. ATG A: nucleotides shared between final part of transcriptional factor and metalloproteinase codified for ORF BAB1_0270.

    Journal: Frontiers in Microbiology

    Article Title: A Zinc-Dependent Metalloproteinase of Brucella abortus Is Required in the Intracellular Adaptation of Macrophages

    doi: 10.3389/fmicb.2020.01586

    Figure Lengend Snippet: A Zinc-dependent metalloproteinase of B. abortus is an operon that forms a putative type II toxin-antitoxin. Identification of the transcriptional unit (operon) constituted by ORF BAB1_0270 and a transcriptional regulator in B. abortus 2308 expressed in (A) the genomic DNA and (B) the cDNA from total RNA. MW: Molecular weight; lane 1: Transcriptional regulator (357 bp); lane 2: BAB1_0270 (549 bp); and lane 3: operon constituted by ORF BAB1_0270-transcriptional regulator (906 bp). (C) Hypothetical Type II toxin-antitoxin (TA) model for operon constitute by ZnMP and transcriptional regulator. Toxin (ZnMP) and anti-toxin (transcriptional regulator) are transcribed to mRNA together. Proteases are activated under stress conditions, cleaving the anti-toxin, which increases the levels of toxin-free, inducing various biological functions in bacteria. Predicted promoter at site -35 and -10 binding by RNA polymerase sigma factor rpoD. ATG A: nucleotides shared between final part of transcriptional factor and metalloproteinase codified for ORF BAB1_0270.

    Article Snippet: Complementary DNA (cDNA) was obtained from RNA by reverse transcription using the Maxima First Strand cDNA Synthesis kit for RT-PCR (Thermo Fisher Scientific Inc., MA, United States) and the relative expression of the genes of interest ( ) was quantified using the Takyon q-PCR kit for SYBR assays by means of the AriaMx Real Time PCR system (Agilent Technologies, CA, United States). gyrA and 16s housekeeping genes were used as reference genes for all assays.

    Techniques: Molecular Weight, Binding Assay

    Effect of TLR9 deletion on cytokine mRNA expression pattern in leukocytes isolated from C. neoformans infected lungs. Lung leukocytes were isolated from uninfected (week 0) and infected TLR9+/+ and TLR9−/−mice at 3 wpi. Total leukocyte RNA was extracted, converted to cDNA, and analyzed by real-time RT-PCR for the expression of selected “polarizing” cytokines. The values were normalized to GAPDH mRNA levels and were expressed as relative gene expression ± SEM. Data were pooled from two parallel experiments. N = 3 for uninfected controls and at least seven for infected mice at each of the analyzed time points. For the comparisons between TLR9+/+ versus TLR9−/− mice, * P

    Journal: The American Journal of Pathology

    Article Title: TLR9 Signaling Is Required for Generation of the Adaptive Immune Protection in Cryptococcus neoformans-Infected Lungs

    doi: 10.2353/ajpath.2010.091104

    Figure Lengend Snippet: Effect of TLR9 deletion on cytokine mRNA expression pattern in leukocytes isolated from C. neoformans infected lungs. Lung leukocytes were isolated from uninfected (week 0) and infected TLR9+/+ and TLR9−/−mice at 3 wpi. Total leukocyte RNA was extracted, converted to cDNA, and analyzed by real-time RT-PCR for the expression of selected “polarizing” cytokines. The values were normalized to GAPDH mRNA levels and were expressed as relative gene expression ± SEM. Data were pooled from two parallel experiments. N = 3 for uninfected controls and at least seven for infected mice at each of the analyzed time points. For the comparisons between TLR9+/+ versus TLR9−/− mice, * P

    Article Snippet: Total RNA was prepared using RNeasy Plus Mini Kit (Qiagen, Valencia, CA) and first-strand cDNA was synthesized using SuperScriptIII (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

    Techniques: Expressing, Isolation, Infection, Mouse Assay, Quantitative RT-PCR

    Effect of TLR9 deletion on pulmonary lymph node and spleen polarization. A: Pulmonary lymph nodes were collected from uninfected (week 0) and infected TLR9+/+ and TLR9−/−mice at 3 wpi. Total RNA was extracted, converted to cDNA, and analyzed by real-time RT-PCR for the expression of selected “polarizing” cytokines. The values were normalized to GAPDH mRNA levels and were expressed as relative mRNA expression ± SEM. Data were pooled from two parallel experiments. N = 2 for uninfected controls and 7 for infected mice; * P

    Journal: The American Journal of Pathology

    Article Title: TLR9 Signaling Is Required for Generation of the Adaptive Immune Protection in Cryptococcus neoformans-Infected Lungs

    doi: 10.2353/ajpath.2010.091104

    Figure Lengend Snippet: Effect of TLR9 deletion on pulmonary lymph node and spleen polarization. A: Pulmonary lymph nodes were collected from uninfected (week 0) and infected TLR9+/+ and TLR9−/−mice at 3 wpi. Total RNA was extracted, converted to cDNA, and analyzed by real-time RT-PCR for the expression of selected “polarizing” cytokines. The values were normalized to GAPDH mRNA levels and were expressed as relative mRNA expression ± SEM. Data were pooled from two parallel experiments. N = 2 for uninfected controls and 7 for infected mice; * P

    Article Snippet: Total RNA was prepared using RNeasy Plus Mini Kit (Qiagen, Valencia, CA) and first-strand cDNA was synthesized using SuperScriptIII (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

    Techniques: Infection, Mouse Assay, Quantitative RT-PCR, Expressing

    Gene expression in pigs with or without worms at day 53 after inoculation with Trichuris suis eggs. Tissue sections from the small intestine ileum (IL), large intestine proximal colon (PCM) and distal colon (DCM), mesenteric (MLN), colonic (CLN), and tracheal-bronchial lymph nodes (TBLN), jejunum Peyer’s patch (JJ PP), ileal-cecal Peyer’s patch (IC PP), and lympho-glandular complexes (LGC) were collected at necropsy. Quantitative real time PCR was performed on cDNA synthesized from each sample using 10 μg of total RNA. Up-regulation is shown in green, down-regulation in red, and low lever modulation in yellow. The high end of the right triangle symbol represents high worm burden and the low end represents low worm burden. Data is derived from three pigs/group (Supplemental Table S1 ).

    Journal: Scientific Reports

    Article Title: Molecular and metabolomic changes in the proximal colon of pigs infected with Trichuris suis

    doi: 10.1038/s41598-020-69462-5

    Figure Lengend Snippet: Gene expression in pigs with or without worms at day 53 after inoculation with Trichuris suis eggs. Tissue sections from the small intestine ileum (IL), large intestine proximal colon (PCM) and distal colon (DCM), mesenteric (MLN), colonic (CLN), and tracheal-bronchial lymph nodes (TBLN), jejunum Peyer’s patch (JJ PP), ileal-cecal Peyer’s patch (IC PP), and lympho-glandular complexes (LGC) were collected at necropsy. Quantitative real time PCR was performed on cDNA synthesized from each sample using 10 μg of total RNA. Up-regulation is shown in green, down-regulation in red, and low lever modulation in yellow. The high end of the right triangle symbol represents high worm burden and the low end represents low worm burden. Data is derived from three pigs/group (Supplemental Table S1 ).

    Article Snippet: Total RNA was used for first strand cDNA synthesis using SuperScript II (Invitrogen, Life Technologies) and oligo(dT).

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

    Topoisomerase-I-specific CD4+ T cells show a Th17 polarized functional phenotype. a CD4+ T cell subsets were identified by flow cytometry using the combined surface expression of chemokine receptors CCR6, CXCR3, and CCR4. b After sorting, the different T helper subsets were stimulated with anti-CD3/CD28 beads for 48 h and secretion of IFNγ, IL-4, and IL-17A was measured in their supernatants by enzyme-linked immunosorbent assay ( bars represent mean ± SE, n = 3). c The distribution of polarized T helper subsets within topo-I-specific CD4+ T cells ( closed circles ) is shown in comparison to the general CD4+ population ( open circles ). T test with Bonferroni correction was used for multiple comparisons. Lines indicate mean ± SD. d After sorting topo-I-specific CD4+ T cells according to their polarized T helper phenotype (range 16–2064 cells), mRNA expression levels of IFNγ, IL-4, and IL-17A were measured by qPCR using the Arcturus® PicoPure® RNA Isolation system, which is designed to recover high-quality RNA from a very low number of cells (10–500 cells) and to accomplish cDNA synthesis from minimal amounts of cDNA. IFNγ and IL-17A expression was calculated as fold change in reference to a mix of topo-I-reactive CD4+ T cells from three patients; IL-4 expression is reported as delta Ct from actin expression since it was not detectable in the reference population. Data are representative of four separate patients (mean ± SE)

    Journal: Arthritis Research & Therapy

    Article Title: Frequency of circulating topoisomerase-I-specific CD4 T cells predicts presence and progression of interstitial lung disease in scleroderma

    doi: 10.1186/s13075-016-0993-2

    Figure Lengend Snippet: Topoisomerase-I-specific CD4+ T cells show a Th17 polarized functional phenotype. a CD4+ T cell subsets were identified by flow cytometry using the combined surface expression of chemokine receptors CCR6, CXCR3, and CCR4. b After sorting, the different T helper subsets were stimulated with anti-CD3/CD28 beads for 48 h and secretion of IFNγ, IL-4, and IL-17A was measured in their supernatants by enzyme-linked immunosorbent assay ( bars represent mean ± SE, n = 3). c The distribution of polarized T helper subsets within topo-I-specific CD4+ T cells ( closed circles ) is shown in comparison to the general CD4+ population ( open circles ). T test with Bonferroni correction was used for multiple comparisons. Lines indicate mean ± SD. d After sorting topo-I-specific CD4+ T cells according to their polarized T helper phenotype (range 16–2064 cells), mRNA expression levels of IFNγ, IL-4, and IL-17A were measured by qPCR using the Arcturus® PicoPure® RNA Isolation system, which is designed to recover high-quality RNA from a very low number of cells (10–500 cells) and to accomplish cDNA synthesis from minimal amounts of cDNA. IFNγ and IL-17A expression was calculated as fold change in reference to a mix of topo-I-reactive CD4+ T cells from three patients; IL-4 expression is reported as delta Ct from actin expression since it was not detectable in the reference population. Data are representative of four separate patients (mean ± SE)

    Article Snippet: Single-strand cDNAs were synthesized with the Superscript III first-strand cDNA synthesis kit following the manufacturer’s protocol (Invitrogen, Life Technologies, Waltham, MA, USA).

    Techniques: Functional Assay, Flow Cytometry, Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, 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

    Expression levels of type I collagen, Smad2/3, and TGF-β1 genes after ES-P treatment in aged mice. (A) The left ears of fourteen-week old mice were treated daily with 30 μg T . spiralis ES-P for 14 days (red color). The right ears of were treated daily with PBS for 14 days as control (blue color). After 14 days, ears were collected. Total RNA was extracted from each ear tissue and cDNA was constructed. (B) The gene expression levels of collagen I , TGF-β1 , and Smad2/3 were analyzed by real-time PCR. The gene expression value of each group was normalized by control value, 14 weeks group. (**; P

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Identification of a host collagen inducing factor from the excretory secretory proteins of Trichinella spiralis

    doi: 10.1371/journal.pntd.0006516

    Figure Lengend Snippet: Expression levels of type I collagen, Smad2/3, and TGF-β1 genes after ES-P treatment in aged mice. (A) The left ears of fourteen-week old mice were treated daily with 30 μg T . spiralis ES-P for 14 days (red color). The right ears of were treated daily with PBS for 14 days as control (blue color). After 14 days, ears were collected. Total RNA was extracted from each ear tissue and cDNA was constructed. (B) The gene expression levels of collagen I , TGF-β1 , and Smad2/3 were analyzed by real-time PCR. The gene expression value of each group was normalized by control value, 14 weeks group. (**; P

    Article Snippet: Real-time PCR Homogenized ear tissues were mixed with TRIzol (Invitrogen, Germany), and total RNA extraction and cDNA synthesis (Invitrogen, Germany) was performed in accordance with the manufacturer’s protocols.

    Techniques: Expressing, Mouse Assay, Construct, Real-time Polymerase Chain Reaction

    Gene expression profiles in GANT61-treated SKOV3 cells. ( A ) Identification of differentially expressed genes (DEGs) in GANT61-treated SKOV3 cells. Cells were treated with GANT61 (20 µM) or DMSO for 60 hr, and total RNA was extracted as described in Methods . Changes in gene expression were determined by cDNA microarray gene profiling using the Illumina Human HT expression BeadChip V4 (Illumina Inc., San Diego, CA). Differential expressions for the DEGs are shown. ( B ) The top 11 canonical signaling pathways influenced by inhibition of Gli1/Gli2 function in SKOV3 cells. The top 11 canonical signaling pathways, determined by IPA, that were significantly up-regulated or down-regulated by GANT61 treatment in SKOV3 cells, are shown. The 412 DEGs were mapped to the IPA-defined network. The significant P -values that determine the probability that the association between the genes in the dataset and the canonical pathway is by chance alone were calculated by Fisher's exact test, and are expressed as −log ( P -value). ( C ) Heat map of DEGs in the focal adhesion signal pathway in GANT61-treated SKOV3 cells. The heat map shows that 17 genes were significantly differentially expressed, including seven up-regulated genes and 12 down-regulated genes, in the GANT61-treated compared to control SKOV3 cells. ( D ) Selected DEGs from cDNA array gene expression profiling analyzed by real-time PCR. SKOV3 cells were treated with DMSO or GANT61 for the indicated times, total RNA was extracted for real-time PCR as described in Methods using the primer sets in Table 1 . The data represent the mean ± SD of three determinations, and GAPDH was used to normalize the relative mRNA levels.

    Journal: PLoS ONE

    Article Title: Down-Regulation of Gli Transcription Factor Leads to the Inhibition of Migration and Invasion of Ovarian Cancer Cells via Integrin ?4-Mediated FAK Signaling

    doi: 10.1371/journal.pone.0088386

    Figure Lengend Snippet: Gene expression profiles in GANT61-treated SKOV3 cells. ( A ) Identification of differentially expressed genes (DEGs) in GANT61-treated SKOV3 cells. Cells were treated with GANT61 (20 µM) or DMSO for 60 hr, and total RNA was extracted as described in Methods . Changes in gene expression were determined by cDNA microarray gene profiling using the Illumina Human HT expression BeadChip V4 (Illumina Inc., San Diego, CA). Differential expressions for the DEGs are shown. ( B ) The top 11 canonical signaling pathways influenced by inhibition of Gli1/Gli2 function in SKOV3 cells. The top 11 canonical signaling pathways, determined by IPA, that were significantly up-regulated or down-regulated by GANT61 treatment in SKOV3 cells, are shown. The 412 DEGs were mapped to the IPA-defined network. The significant P -values that determine the probability that the association between the genes in the dataset and the canonical pathway is by chance alone were calculated by Fisher's exact test, and are expressed as −log ( P -value). ( C ) Heat map of DEGs in the focal adhesion signal pathway in GANT61-treated SKOV3 cells. The heat map shows that 17 genes were significantly differentially expressed, including seven up-regulated genes and 12 down-regulated genes, in the GANT61-treated compared to control SKOV3 cells. ( D ) Selected DEGs from cDNA array gene expression profiling analyzed by real-time PCR. SKOV3 cells were treated with DMSO or GANT61 for the indicated times, total RNA was extracted for real-time PCR as described in Methods using the primer sets in Table 1 . The data represent the mean ± SD of three determinations, and GAPDH was used to normalize the relative mRNA levels.

    Article Snippet: Real-time PCR Total RNA (1 µg) was employed to prepare cDNA via reverse transcription using cDNA synthesis kit (Invitrogen) according to manufacturer's instructions and analyzed using an Applied Biosystems 7500 PCR Detection System (Applied Biosystems Inc.).

    Techniques: Expressing, Microarray, Inhibition, Indirect Immunoperoxidase Assay, Real-time Polymerase Chain Reaction

    Differential expressions of genes modulating the mechanism of resistance to A. alternata in naturally infected mango fruits. (A) Effect of HWB on alternaria black spot (ABS) symptom development on mango cvs. Palmer, Kent, Tommy Atkins, Keitt, Lily and Shelly. (B) ABS symptom development on naturally infected fruits cv. Keitt following HWB treatment. (C) qRT-PCR differential expression profiling of genes Syn121 , glutardoxin , IT1K2 and AOS of cv. Shelly. Fruit peel tissues were sampled at four different time points after HWB treatment. RNA was extracted and served as a template for cDNA followed by qRT-PCR analysis of the genes of interest. Proportional increases in relative expression values were normalized against the samples of untreated mango fruits at 0 h. Expression data are means of two replicates. ABS-covered area was evaluated after 4 weeks of storage at 12°C. Average values followed by different letters differ significantly at P

    Journal: BMC Genomics

    Article Title: De-novo assembly of mango fruit peel transcriptome reveals mechanisms of mango response to hot water treatment

    doi: 10.1186/1471-2164-15-957

    Figure Lengend Snippet: Differential expressions of genes modulating the mechanism of resistance to A. alternata in naturally infected mango fruits. (A) Effect of HWB on alternaria black spot (ABS) symptom development on mango cvs. Palmer, Kent, Tommy Atkins, Keitt, Lily and Shelly. (B) ABS symptom development on naturally infected fruits cv. Keitt following HWB treatment. (C) qRT-PCR differential expression profiling of genes Syn121 , glutardoxin , IT1K2 and AOS of cv. Shelly. Fruit peel tissues were sampled at four different time points after HWB treatment. RNA was extracted and served as a template for cDNA followed by qRT-PCR analysis of the genes of interest. Proportional increases in relative expression values were normalized against the samples of untreated mango fruits at 0 h. Expression data are means of two replicates. ABS-covered area was evaluated after 4 weeks of storage at 12°C. Average values followed by different letters differ significantly at P

    Article Snippet: Analysis by qRT-PCR Single-stranded cDNA was synthesized from 1 μg of total RNA by means of the Verso cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Infection, Quantitative RT-PCR, Expressing

    EK transcriptional activation affects CMT1 downstream expression. a Schematic representation of the CMT1 gene and the insertion of EK within exon 13. CMT1 exons are numbered and shown as blue boxes and introns as black lines. Primers used to amplify upstream and downstream CMT1 sequences from cDNA are marked by arrowheads and numbered (P1–P7). The red broken line indicates the splice acceptor site of exon 14 (based on 27). EK LTRs are marked by red arrowheads. b CMT1 upstream transcript is alternatively spliced showing intron 12 retention. cDNA was prepared from RNA extracted from flowers derived from WT Col, WT Ler, ddm1, kyp2 , cmt3, ago4 and rdr2 and subjected to PCR to amplify CMT1 RNA sequences using primer set 1 + 2 (ex10-F + ex11-R) or primer set 3 + 4 (ex11-F + ex13-R). Note that primer set 3 + 4 yielded two PCR fragments SP1 and SP2. Actin was used as a reference. PCRs were performed for 35 cycles except for actin (30 cycles). M indicates DNA molecular size markers. The predicted alternative spliced variants SP1 and SP2 are schematically shown below. c CMT1 downstream expression using primer set 5 + 6 (ex14-F + ex16-R). WT Ler gDNA was used as a reference for PCR product derived from RNA. Note the enhanced downstream CMT1 expression in kyp2 and cmt3 mutants. Actin was used as a reference. Ler genomic DNA (gDNA) was used to confirm amplification from RNA. PCRs were for 35 cycles except for actin (30 cycles). d Analysis of splicing out of the entire EK retroelement using primer set 6 + 7 (ex13-F + ex16-R) on both sides of the EK insertion site. Note lack of a PCR product in kyp2 and cmt3 mutant. Actin was used as a reference RNA. Col genomic DNA (gDNA) was used to confirm amplification from RNA. PCR was for 42 cycles except for actin (30 cycles)

    Journal: Epigenetics & Chromatin

    Article Title: CMT3 and SUVH4/KYP silence the exonic Evelknievel retroelement to allow for reconstitution of CMT1 mRNA

    doi: 10.1186/s13072-018-0240-y

    Figure Lengend Snippet: EK transcriptional activation affects CMT1 downstream expression. a Schematic representation of the CMT1 gene and the insertion of EK within exon 13. CMT1 exons are numbered and shown as blue boxes and introns as black lines. Primers used to amplify upstream and downstream CMT1 sequences from cDNA are marked by arrowheads and numbered (P1–P7). The red broken line indicates the splice acceptor site of exon 14 (based on 27). EK LTRs are marked by red arrowheads. b CMT1 upstream transcript is alternatively spliced showing intron 12 retention. cDNA was prepared from RNA extracted from flowers derived from WT Col, WT Ler, ddm1, kyp2 , cmt3, ago4 and rdr2 and subjected to PCR to amplify CMT1 RNA sequences using primer set 1 + 2 (ex10-F + ex11-R) or primer set 3 + 4 (ex11-F + ex13-R). Note that primer set 3 + 4 yielded two PCR fragments SP1 and SP2. Actin was used as a reference. PCRs were performed for 35 cycles except for actin (30 cycles). M indicates DNA molecular size markers. The predicted alternative spliced variants SP1 and SP2 are schematically shown below. c CMT1 downstream expression using primer set 5 + 6 (ex14-F + ex16-R). WT Ler gDNA was used as a reference for PCR product derived from RNA. Note the enhanced downstream CMT1 expression in kyp2 and cmt3 mutants. Actin was used as a reference. Ler genomic DNA (gDNA) was used to confirm amplification from RNA. PCRs were for 35 cycles except for actin (30 cycles). d Analysis of splicing out of the entire EK retroelement using primer set 6 + 7 (ex13-F + ex16-R) on both sides of the EK insertion site. Note lack of a PCR product in kyp2 and cmt3 mutant. Actin was used as a reference RNA. Col genomic DNA (gDNA) was used to confirm amplification from RNA. PCR was for 42 cycles except for actin (30 cycles)

    Article Snippet: One microgram of total RNA was used for cDNA production using the Verso cDNA Kit (Thermo Scientific) according to the manufacturer’s protocol.

    Techniques: Activation Assay, Expressing, Derivative Assay, Polymerase Chain Reaction, Amplification, Mutagenesis

    Adora2b -deficient mice have increased inflammation with impaired induction of Tregs after LPS-induced lung injury. LPS-induced inflammation was initiated in 9–12 wk-old male Adora2b +/+ (C57BL/6J) or age and weight matched Adora2b −/− mice by intratracheal instillation of LPS or saline. Bronchoalveolar lavage (BAL) fluids were collected 1 d post treatment. A) Leukocyte cell counts of BAL fluid were determined utilizing a cellometer (mean ± SEM, n = 2–4). B) Protein content of BAL fluid was determined with BCA assay. Fold increase of protein concentration in LPS treated BAL samples over respective saline BAL samples is displayed (mean ± SEM, n = 3–10). C) Analysis of cell infiltration into the lung airspace (BAL) of mice exposed to inhaled LPS. Mice ( Adora2b +/+ or Adora2b −/−) were exposed to nebulized saline or lipopolysaccharide (LPS); 24 hours post-exposure, mice were euthanized, and BAL was harvested. Cells were then subjected to flow cytometric analysis to identify neutrophils (highly granular, Gr1+ cells). D) Analysis of FoxP3 gene expression in lungs of mice exposed to inhaled LPS. Mice ( Adora2b +/+ or −/−) were exposed to nebulized saline or lipopolysaccharide (LPS); 24 hours post-exposure, mice were euthanized, and perfused lungs were harvested. Total RNA was harvested from lungs, cDNA prepared and analyzed by real-time PCR. Data depict mean (± SEM) fold changes in indicated genes. Fold change was calculated based on primer efficiencies, standardized to changes in actin, with Adora2b +/+ Saline mean value defined as 1. E) Fold change in cell number of either regulatory T cells (lymphocyte size, CD3+ CD4+ FoxP3+) in the lung airspace (BAL) of mice exposed to inhaled saline or LPS as above. Data are from 2–6 mice; 2 independent experiments. F) Relative abundance of CD4 effector T cells (Teffs, defined as lymphocyte size, CD3+ CD4+ CD44high cells) to Tregs based on cell counts from either Adora2b +/+ or Adora2b −/− mice, with pie charts depicting mean cell count for the indicated cell populations from 2–6 mice; 2 independent expts. Analysis of total cellular infiltration into airways and protein leakage (panels A–B) was done in animals treated with intratracheal LPS. Analysis of neutrophil infiltration and Tregs (panels C–F) was done in animals treated with inhaled LPS. Statistically significant differences are indicated and were calculated either by one-way ANOVA (A) or by unpaired t test (B–E) with a focus on whether Adora2b −/− LPS treated animals were statistically different from either saline treated or Adora2b +/+ LPS treated mice.

    Journal: PLoS ONE

    Article Title: Adora2b Adenosine Receptor Engagement Enhances Regulatory T Cell Abundance during Endotoxin-Induced Pulmonary Inflammation

    doi: 10.1371/journal.pone.0032416

    Figure Lengend Snippet: Adora2b -deficient mice have increased inflammation with impaired induction of Tregs after LPS-induced lung injury. LPS-induced inflammation was initiated in 9–12 wk-old male Adora2b +/+ (C57BL/6J) or age and weight matched Adora2b −/− mice by intratracheal instillation of LPS or saline. Bronchoalveolar lavage (BAL) fluids were collected 1 d post treatment. A) Leukocyte cell counts of BAL fluid were determined utilizing a cellometer (mean ± SEM, n = 2–4). B) Protein content of BAL fluid was determined with BCA assay. Fold increase of protein concentration in LPS treated BAL samples over respective saline BAL samples is displayed (mean ± SEM, n = 3–10). C) Analysis of cell infiltration into the lung airspace (BAL) of mice exposed to inhaled LPS. Mice ( Adora2b +/+ or Adora2b −/−) were exposed to nebulized saline or lipopolysaccharide (LPS); 24 hours post-exposure, mice were euthanized, and BAL was harvested. Cells were then subjected to flow cytometric analysis to identify neutrophils (highly granular, Gr1+ cells). D) Analysis of FoxP3 gene expression in lungs of mice exposed to inhaled LPS. Mice ( Adora2b +/+ or −/−) were exposed to nebulized saline or lipopolysaccharide (LPS); 24 hours post-exposure, mice were euthanized, and perfused lungs were harvested. Total RNA was harvested from lungs, cDNA prepared and analyzed by real-time PCR. Data depict mean (± SEM) fold changes in indicated genes. Fold change was calculated based on primer efficiencies, standardized to changes in actin, with Adora2b +/+ Saline mean value defined as 1. E) Fold change in cell number of either regulatory T cells (lymphocyte size, CD3+ CD4+ FoxP3+) in the lung airspace (BAL) of mice exposed to inhaled saline or LPS as above. Data are from 2–6 mice; 2 independent experiments. F) Relative abundance of CD4 effector T cells (Teffs, defined as lymphocyte size, CD3+ CD4+ CD44high cells) to Tregs based on cell counts from either Adora2b +/+ or Adora2b −/− mice, with pie charts depicting mean cell count for the indicated cell populations from 2–6 mice; 2 independent expts. Analysis of total cellular infiltration into airways and protein leakage (panels A–B) was done in animals treated with intratracheal LPS. Analysis of neutrophil infiltration and Tregs (panels C–F) was done in animals treated with inhaled LPS. Statistically significant differences are indicated and were calculated either by one-way ANOVA (A) or by unpaired t test (B–E) with a focus on whether Adora2b −/− LPS treated animals were statistically different from either saline treated or Adora2b +/+ LPS treated mice.

    Article Snippet: RNA isolation and Real-time PCR Total RNA was extracted from cells or tissue by Trizol, followed by cDNA synthesis using iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Inc, Munich, Germany) according to the manufacturer's instructions.

    Techniques: Mouse Assay, BIA-KA, Protein Concentration, Flow Cytometry, Expressing, Real-time Polymerase Chain Reaction, Cell Counting

    Superoxide dismutase 2 (SOD2) and NF-κB levels are increased in BRAF pathway inhibitor-resistant cells. ( A ) An equal number of WM-115 (parental, DR, and TDR) cells were plated. Total RNA was extracted from these cell lines using the RNA extraction mini kit, according to the manufacturer’s instructions. Two-step real time-qPCR was performed to assess the mRNA level of SOD2 and PRDX1. First strand cDNA was synthesized using the iScriptTM cDNA synthesis kit qPCR, which was set up using the CFX96 real-time system. Actin was used as an internal control. Relative SOD2 and PRDX1 expression was presented by the 2 −ΔΔCT method (left panel). In separate experiments, whole cell lysates were prepared and separated in a 12.5% sodium dodecyl sulfate (SDS) gel, followed by immunoblot analysis with indicated antibodies. Western blot data is shown on the right panel. ( B ) An equal number of WM-983, WM-983 DR, and WM-983 TDR cells were plated. Two-step real time-qPCR and immunoblot analysis with indicated antibodies were performed under the above conditions (left and right panels). ( C ) Nuclear extracts were collected and prepared from WM-115 and WM-983 (parental and TDR). * p

    Journal: Cancers

    Article Title: BRAF Mutant Melanoma Adjusts to BRAF/MEK Inhibitors via Dependence on Increased Antioxidant SOD2 and Increased Reactive Oxygen Species Levels

    doi: 10.3390/cancers12061661

    Figure Lengend Snippet: Superoxide dismutase 2 (SOD2) and NF-κB levels are increased in BRAF pathway inhibitor-resistant cells. ( A ) An equal number of WM-115 (parental, DR, and TDR) cells were plated. Total RNA was extracted from these cell lines using the RNA extraction mini kit, according to the manufacturer’s instructions. Two-step real time-qPCR was performed to assess the mRNA level of SOD2 and PRDX1. First strand cDNA was synthesized using the iScriptTM cDNA synthesis kit qPCR, which was set up using the CFX96 real-time system. Actin was used as an internal control. Relative SOD2 and PRDX1 expression was presented by the 2 −ΔΔCT method (left panel). In separate experiments, whole cell lysates were prepared and separated in a 12.5% sodium dodecyl sulfate (SDS) gel, followed by immunoblot analysis with indicated antibodies. Western blot data is shown on the right panel. ( B ) An equal number of WM-983, WM-983 DR, and WM-983 TDR cells were plated. Two-step real time-qPCR and immunoblot analysis with indicated antibodies were performed under the above conditions (left and right panels). ( C ) Nuclear extracts were collected and prepared from WM-115 and WM-983 (parental and TDR). * p

    Article Snippet: First strand cDNA was synthesized using the iScriptTM cDNA Synthesis Kit (Biorad, 1708890, Hercules, CA, USA). qPCR was set up using the CFX96 Real-Time System (Biorad, USA).

    Techniques: RNA Extraction, Real-time Polymerase Chain Reaction, Synthesized, Expressing, SDS-Gel, Western Blot

    Northern blot analysis of rasGEFM expression in parental and mutant strains . ( A ) Total RNA was extracted from AX2 cells developed in suspension for 0 to 9 hours or on filters. In the latter case the cells were harvested at mound (12 hours), first finger (16 hours) and preculminant (20 hours) stages. The membrane was hybridized to a radiolabelled rasGEFM specific probe (probe a ) corresponding to bp 760–1518 of the cDNA clone and to the actin gene used as a loading control. ( B ) Total RNA was extracted from different developmental Dictyostelium mutants starved in shaking suspension up to 6 hours. LW6 (G protein β subunit minus), HSB1 (PIA ts -mutant, defective in the G-protein adenylyl cyclase activation) and HSB50 (mutant blocked at mound stage).

    Journal: BMC Cell Biology

    Article Title: A novel Dictyostelium RasGEF required for chemotaxis and development

    doi: 10.1186/1471-2121-6-43

    Figure Lengend Snippet: Northern blot analysis of rasGEFM expression in parental and mutant strains . ( A ) Total RNA was extracted from AX2 cells developed in suspension for 0 to 9 hours or on filters. In the latter case the cells were harvested at mound (12 hours), first finger (16 hours) and preculminant (20 hours) stages. The membrane was hybridized to a radiolabelled rasGEFM specific probe (probe a ) corresponding to bp 760–1518 of the cDNA clone and to the actin gene used as a loading control. ( B ) Total RNA was extracted from different developmental Dictyostelium mutants starved in shaking suspension up to 6 hours. LW6 (G protein β subunit minus), HSB1 (PIA ts -mutant, defective in the G-protein adenylyl cyclase activation) and HSB50 (mutant blocked at mound stage).

    Article Snippet: As template, cDNA isolated with "First strand cDNA synthesis kit" (Amersham Pharmacia Biotech), from RNA of cells developed for 5 hours on solid substrata, was used.

    Techniques: Northern Blot, Expressing, Mutagenesis, Activation Assay

    Disruption of the rasGEFM gene . ( A ) Southern blot of genomic DNA from parental strain (AX2) and rasGEFM null mutant (HSB61). Genomic DNA was digested with Eco RI, separated in 0.8% agarose gel, blotted onto nylon membrane and probed with probe a ( rasGEFM N-terminal fragment corresponding to bp 0–617 of the cDNA clone), probe b (corresponding to bp 760–1518 of the rasGEFM cDNA clone) and probe c ( bsr cassette). Three different probes were used to characterize the genomic locus of the mutant and the parental strain. In the rasGEFM null strain the central part of the gene (recognized by probe b ), was replaced by the blasticidin cassette (recognized by probe c ), which has the same size of the replaced fragment. Because of that, the size of the locus remains unchanged, but the central part is recognized specifically by bsr (probe c ) or probe b in HSB61 or AX2, respectively. Both AX2 and HSB61 genomic loci are recognized by the probe a . ( B ) Northern blot of total RNA extracted from HSB61 cells at the indicated time points of growth and development. The membrane was hybridized to the rasGEFM and to the actin gene.

    Journal: BMC Cell Biology

    Article Title: A novel Dictyostelium RasGEF required for chemotaxis and development

    doi: 10.1186/1471-2121-6-43

    Figure Lengend Snippet: Disruption of the rasGEFM gene . ( A ) Southern blot of genomic DNA from parental strain (AX2) and rasGEFM null mutant (HSB61). Genomic DNA was digested with Eco RI, separated in 0.8% agarose gel, blotted onto nylon membrane and probed with probe a ( rasGEFM N-terminal fragment corresponding to bp 0–617 of the cDNA clone), probe b (corresponding to bp 760–1518 of the rasGEFM cDNA clone) and probe c ( bsr cassette). Three different probes were used to characterize the genomic locus of the mutant and the parental strain. In the rasGEFM null strain the central part of the gene (recognized by probe b ), was replaced by the blasticidin cassette (recognized by probe c ), which has the same size of the replaced fragment. Because of that, the size of the locus remains unchanged, but the central part is recognized specifically by bsr (probe c ) or probe b in HSB61 or AX2, respectively. Both AX2 and HSB61 genomic loci are recognized by the probe a . ( B ) Northern blot of total RNA extracted from HSB61 cells at the indicated time points of growth and development. The membrane was hybridized to the rasGEFM and to the actin gene.

    Article Snippet: As template, cDNA isolated with "First strand cDNA synthesis kit" (Amersham Pharmacia Biotech), from RNA of cells developed for 5 hours on solid substrata, was used.

    Techniques: Southern Blot, Mutagenesis, Agarose Gel Electrophoresis, Northern Blot

    Overview of Cufflinks. The algorithm takes as input cDNA fragment sequences that have been ( a ) aligned to the genome by software capable of producing spliced alignments, such as TopHat. With paired-end RNA-Seq, Cufflinks treats each pair of fragment reads as a single alignment. The algorithm assembles overlapping ‘bundles’ of fragment alignments ( b-c ) separately, which reduces running time and memory use because each bundle typically contains the fragments from no more than a few genes. Cufflinks then estimates the abundances of the assembled transcripts ( d-e ). ( b ) The first step in fragment assembly is to identify pairs of ‘incompatible’ fragments that must have originated from distinct spliced mRNA isoforms. Fragments are connected in an ‘overlap graph’ when they are compatible and their alignments overlap in the genome. Each fragment has one node in the graph, and an edge, directed from left to right along the genome, is placed between each pair of compatible fragments. In this example, the yellow, blue, and red fragments must have originated from separate isoforms, but any other fragment could have come from the same transcript as one of these three. ( c ) Assembling isoforms from the overlap graph. Paths through the graph correspond to sets of mutually compatible fragments that could be merged into complete isoforms. The overlap graph here can be minimally ‘covered’ by three paths, each representing a different isoform. Dilworth's Theorem states that the number of mutually incompatible reads is the same as the minimum number of transcripts needed to “explain” all the fragments. Cufflinks implements a proof of Dilworth's Theorem that produces a minimal set of paths that cover all the fragments in the overlap graph by finding the largest set of reads with the property that no two could have originated from the same isoform. ( d ) Estimating transcript abundance. Fragments are matched (denoted here using color) to the transcripts from which they could have originated. The violet fragment could have originated from the blue or red isoform. Gray fragments could have come from any of the three shown. Cufflinks estimates transcript abundances using a statistical model in which the probability of observing each fragment is a linear function of the abundances of the transcripts from which it could have originated. Because only the ends of each fragment are sequenced, the length of each may be unknown. Assigning a fragment to different isoforms often implies a different length for it. Cufflinks can incorporate the distribution of fragment lengths to help assign fragments to isoforms. For example, the violet fragment would be much longer, and very improbable according to Cufflinks' model, if it were to come from the red isoform instead of the blue isoform. ( e ) The program then numerically maximizes a function that assigns a likelihood to all possible sets of relative abundances of the yellow, red and blue isoforms (γ 1 ,γ 2 ,γ 3 ), producing the abundances that best explain the observed fragments, shown as a pie chart.

    Journal: Nature biotechnology

    Article Title: Transcript assembly and abundance estimation from RNA-Seq reveals thousands of new transcripts and switching among isoforms

    doi: 10.1038/nbt.1621

    Figure Lengend Snippet: Overview of Cufflinks. The algorithm takes as input cDNA fragment sequences that have been ( a ) aligned to the genome by software capable of producing spliced alignments, such as TopHat. With paired-end RNA-Seq, Cufflinks treats each pair of fragment reads as a single alignment. The algorithm assembles overlapping ‘bundles’ of fragment alignments ( b-c ) separately, which reduces running time and memory use because each bundle typically contains the fragments from no more than a few genes. Cufflinks then estimates the abundances of the assembled transcripts ( d-e ). ( b ) The first step in fragment assembly is to identify pairs of ‘incompatible’ fragments that must have originated from distinct spliced mRNA isoforms. Fragments are connected in an ‘overlap graph’ when they are compatible and their alignments overlap in the genome. Each fragment has one node in the graph, and an edge, directed from left to right along the genome, is placed between each pair of compatible fragments. In this example, the yellow, blue, and red fragments must have originated from separate isoforms, but any other fragment could have come from the same transcript as one of these three. ( c ) Assembling isoforms from the overlap graph. Paths through the graph correspond to sets of mutually compatible fragments that could be merged into complete isoforms. The overlap graph here can be minimally ‘covered’ by three paths, each representing a different isoform. Dilworth's Theorem states that the number of mutually incompatible reads is the same as the minimum number of transcripts needed to “explain” all the fragments. Cufflinks implements a proof of Dilworth's Theorem that produces a minimal set of paths that cover all the fragments in the overlap graph by finding the largest set of reads with the property that no two could have originated from the same isoform. ( d ) Estimating transcript abundance. Fragments are matched (denoted here using color) to the transcripts from which they could have originated. The violet fragment could have originated from the blue or red isoform. Gray fragments could have come from any of the three shown. Cufflinks estimates transcript abundances using a statistical model in which the probability of observing each fragment is a linear function of the abundances of the transcripts from which it could have originated. Because only the ends of each fragment are sequenced, the length of each may be unknown. Assigning a fragment to different isoforms often implies a different length for it. Cufflinks can incorporate the distribution of fragment lengths to help assign fragments to isoforms. For example, the violet fragment would be much longer, and very improbable according to Cufflinks' model, if it were to come from the red isoform instead of the blue isoform. ( e ) The program then numerically maximizes a function that assigns a likelihood to all possible sets of relative abundances of the yellow, red and blue isoforms (γ 1 ,γ 2 ,γ 3 ), producing the abundances that best explain the observed fragments, shown as a pie chart.

    Article Snippet: After removal of hydrolysis ions by G50 Sephadex filtration (USA Scientific catalog # 1415-1602), the fragmented mRNA was random primed with hexamers and reverse-transcribed using the Super Script II cDNA synthesis kit (Invitrogen catalog # 11917010).

    Techniques: Software, RNA Sequencing Assay

    Interaction with K562 CML cells and its paracrine factors modified cell surface antigen expression in MSC. a Cell surface expression of CD13, CD73, CD90, CD95 and CD105 in control-MSC (CON-MSC) and CML-MSC was analyzed by flow cytometry. Mean (geometric) fluorescent intensity (MFI) was calculated for each marker against its isotype control. b , c Control-MSC were cultured in conditioned media derived from K562 cells for one week and their cell surface gene expression in control-MSC (CON) and conditioned media treated MSC (MSC + CM) was analyzed by flow cytometry. MFI of analyzed markers was normalized to control-MSC. c Representative flow cytometry histogram showing cell surface antigen expression levels in CON and MSC + CM conditions. Grey line represents the isotype control, blue and red line represents the stained cells. d , e Control-MSC were co-cultured without (CON) or with K562 cells (MSC + K) for one week and their cell surface gene expression profile was determined by flow cytometry. MFI was calculated for each antigen and normalized to control-MSC. e Representative flow cytometry histograms showing the cell surface antigen expression in CON (blue line), MSC + K (red line) and isotype control (grey line). f , g RNA was extracted from control-MSC (CON) and K562 co-cultured MSC (MSC + K) and reverse transcribed into cDNA. mRNA expression levels of ( f ) CD90, ( g ) CAT and MnSOD was analyzed by real-time PCR. (h) Mitochondrial ROS levels in CON and MSC + K were analyzed by staining with mitosox red and MFI of mitosox was normalized to unstained cells. Values are mean ± SE, *p

    Journal: Journal of Cell Communication and Signaling

    Article Title: K562 chronic myeloid leukemia cells modify osteogenic differentiation and gene expression of bone marrow stromal cells

    doi: 10.1007/s12079-017-0412-8

    Figure Lengend Snippet: Interaction with K562 CML cells and its paracrine factors modified cell surface antigen expression in MSC. a Cell surface expression of CD13, CD73, CD90, CD95 and CD105 in control-MSC (CON-MSC) and CML-MSC was analyzed by flow cytometry. Mean (geometric) fluorescent intensity (MFI) was calculated for each marker against its isotype control. b , c Control-MSC were cultured in conditioned media derived from K562 cells for one week and their cell surface gene expression in control-MSC (CON) and conditioned media treated MSC (MSC + CM) was analyzed by flow cytometry. MFI of analyzed markers was normalized to control-MSC. c Representative flow cytometry histogram showing cell surface antigen expression levels in CON and MSC + CM conditions. Grey line represents the isotype control, blue and red line represents the stained cells. d , e Control-MSC were co-cultured without (CON) or with K562 cells (MSC + K) for one week and their cell surface gene expression profile was determined by flow cytometry. MFI was calculated for each antigen and normalized to control-MSC. e Representative flow cytometry histograms showing the cell surface antigen expression in CON (blue line), MSC + K (red line) and isotype control (grey line). f , g RNA was extracted from control-MSC (CON) and K562 co-cultured MSC (MSC + K) and reverse transcribed into cDNA. mRNA expression levels of ( f ) CD90, ( g ) CAT and MnSOD was analyzed by real-time PCR. (h) Mitochondrial ROS levels in CON and MSC + K were analyzed by staining with mitosox red and MFI of mitosox was normalized to unstained cells. Values are mean ± SE, *p

    Article Snippet: The resulting RNA was reverse transcribed using high capacity cDNA synthesis kit and oligodT primers at 37°C for 120 min. Real-time PCR was performed using Power SyBr Green reagents in an ABI 7500 system (Thermo Fisher Scientific).

    Techniques: Modification, Expressing, Flow Cytometry, Cytometry, Marker, Cell Culture, Derivative Assay, Staining, Real-time Polymerase Chain Reaction