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  • 99
    Thermo Fisher superscript vilo cdna synthesis kit
    mtDNA increases SOCE and expression of Sphk1 in PMN. (A) Human PMN were treated with 20 µg/mL of E.coli DNA or mtDNA for 60 min then loaded with fura-2. Thapsigargin (1 µM) was applied to under nominally calcium-free conditions and then 1.8 mM extracellular calcium was applied at the indicated times. Calcium influx was calculated as described elsewhere [9] as the area under the curve for [Ca 2+ ] i (AUC) over 120 sec. Data were analyzed using medium-treated PMN as 100%. Mean and SE values are shown. At least 3 experiment were done per condition. * denotes a significant difference by student t-test (SigmaPlot 11) compared to time “0” value. Experiments were repeated at least three times. (7B) Freshly isolated human PMN (5 million cells in 2 mL) were incubated with medium, mtDNA (10 µg/mL), E.coli DNA (10 µg/mL), or LPS (100 ng/mL) for 60 min. Then RNA and <t>cDNA</t> were prepared using the RNesay mini kit (Qiagen) and SuperScript <t>VILO</t> cDNA Synthesis kit (Life technologies), respectively. 200 ng of cDNA per reaction was used for TaqMan qPCR assay for Sphk1 and GAPDH to evaluate expression levels. Sphk1 expression levels were then further normalized by GAPDH using the medium control result as 100%. Four different PMN preparations were used for stimulation and qPCR assays were done in triplicates. Mean and SE values from four different experiments are shown. Data were analyzed by One Way ANOVA. * denotes a significant difference between mtDNA treatment and the medium control (p
    Superscript Vilo Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 17827 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    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 7107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript double stranded cdna synthesis kit/product/Thermo Fisher
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    99
    Thermo Fisher superscript iii first strand cdna synthesis kit
    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 <t>cDNA</t> 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 <t>three</t> 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)
    Superscript Iii First Strand Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3113 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript iii cdna synthesis kit
    Isolation and Purification of Mx, ORS, DP, DF, and Mc Populations (A) Schematic of isolation procedure. After removing subcutaneous fat by dissection, and epidermis/upper follicle segment by enzymatic digestion, single-cell suspensions were prepared from pure dermis and subjected to <t>three</t> FACS schemes to purify five populations of cells: Mx, GFP low RFP − ; ORS, GFP high RFP − ; DP, RFP high GFP − CD34 − CD45 − CD117 − ; DF, RFP − GFP − CD34 − CD45 − CD117 − ; Mc, RFP high GFP − CD117 + . (B) Immunofluorescence analyses of FACS isolated cell populations. Frozen skin sections (hair bulb) and relevant cytospin populations were stained with Abs as color-coded and indicated. At the right of each set is quantification of percentage of cells that expressed the marker. Note: ~10% of DP and DF cells lysed on cytospin. and hence did not stain with any markers. β4, β4 integrin; Tyr, tyrosinase; Vim, vimentin; white line, basement membrane. (C) RT-PCR: <t>cDNA</t> fragments were resolved by agarose gel electrophoresis, and the gene detected is denoted at left. All fragments were of the expected size. Expression of Msx2, vimentin, and β4 in multiple populations was later confirmed. (D) Cell cycle differences in cell populations. Profiles of the five purified populations were performed by FACS. Anti-BrdU immunofluorescence is from a P4 backskin follicle from a mouse injected intraperitoneally with 50 μg/g 5-bromo-2′-deoxyuridine (BrdU) (Sigma-Aldrich) and analyzed 4 h later. Note greatest incorporation in Mx and ORS.
    Superscript Iii Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2773 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript iii first strand cdna kit
    qRT-PCR analysis of AβO-induced differential gene expression. <t>cDNA</t> from the <t>three</t> donors used in the microarray analysis plus four samples from additional independent donors were used. Expression levels were determined by relative expression,
    Superscript Iii First Strand Cdna Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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

    ( A ) Allele-specific expression of Inpp5f transcript variants, assessed by RT-PCR sequencing assays. PCR primers were designed to specifically amplify each transcript (Figure 1A) over regions containing SNPs between two inbred strains of mice: C57BL/6J (B6) and Mus mus castaneus (cast). Allele-specific expression can be determined in F1 hybrids by the relative height of the sequence trace corresponding to each parental allele at the polymorphic site. The strain of the mother is given first in each cross. ( B ). Tissue-specific expression of Inpp5f_v2 transcript variants, assessed by qualitative RT-PCR. All primers were designed to span introns (Figure 1A), and each reaction was conducted using cDNA prepared in the presence (RT+) or absence (RT–) of reverse transcriptase, to eliminate the possibility of template contamination. cDNA was prepared from 1 μg of RNA extracted from B6 × cast F1 hybrid tissues. Adult material was prepared from animals sacrificed at 6 weeks. In every case, Inpp5f_v2 and Inpp5f_v3 underwent 33 cycles of amplification, whereas Gapdh was amplified for 30 cycles. ( C ) Developmental expression screen for Inpp5f_v2 and Inpp5f_v3 transcript variants under the same experimental conditions as panel B.

    Journal: Nucleic Acids Research

    Article Title: Allele-specific demethylation at an imprinted mammalian promoter

    doi: 10.1093/nar/gkm742

    Figure Lengend Snippet: ( A ) Allele-specific expression of Inpp5f transcript variants, assessed by RT-PCR sequencing assays. PCR primers were designed to specifically amplify each transcript (Figure 1A) over regions containing SNPs between two inbred strains of mice: C57BL/6J (B6) and Mus mus castaneus (cast). Allele-specific expression can be determined in F1 hybrids by the relative height of the sequence trace corresponding to each parental allele at the polymorphic site. The strain of the mother is given first in each cross. ( B ). Tissue-specific expression of Inpp5f_v2 transcript variants, assessed by qualitative RT-PCR. All primers were designed to span introns (Figure 1A), and each reaction was conducted using cDNA prepared in the presence (RT+) or absence (RT–) of reverse transcriptase, to eliminate the possibility of template contamination. cDNA was prepared from 1 μg of RNA extracted from B6 × cast F1 hybrid tissues. Adult material was prepared from animals sacrificed at 6 weeks. In every case, Inpp5f_v2 and Inpp5f_v3 underwent 33 cycles of amplification, whereas Gapdh was amplified for 30 cycles. ( C ) Developmental expression screen for Inpp5f_v2 and Inpp5f_v3 transcript variants under the same experimental conditions as panel B.

    Article Snippet: Expression studies RNA was prepared using the RNAeasy kit (Qiagen) according to the manufacturer's standard protocol, and then quantified using an Agilent 2100 Bioanalyser. cDNA was prepared from 1 μg of total RNA using the superscript II cDNA synthesis kit (Invitrogen).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Sequencing, Polymerase Chain Reaction, Mouse Assay, Amplification

    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

    Upregulation of expression of the Grg2 (RDA1) and Grg6 (RDA3) genes induced by E2A-HLF. (A) Northern blot analysis of poly(A) + RNA (1 μg per lane) prepared from FL5.12 cells stably transfected with the pMT empty vector (lanes 1 to 4) or with metallothionein promoter-regulated constructs containing cDNAs for E2A-HLF (lanes 5 to 8), the ΔAD1 E2A-HLF mutant lacking the AD1 transactivation domain of E2A (lanes 9 and 10), the BX E2A-HLF mutant with a disabled HLF DNA-binding domain (lanes 11 and 12), E2A (E12 [lanes 13 and 14] and E47 [lanes 15 and 16]), HLF (lanes 17 and 18), or TEF (lanes 19 and 20). Cells were cultured in IL-3-containing medium with (+) or without (−) 100 μM ZnSO 4 for 18 h to induce expression of the transfected cDNA and then continued in the same concentration of zinc and either maintained in IL-3 (+) or deprived of the cytokine (−) for an additional 12 h before RNA extraction. The blot was hybridized with the Grg2 (RDA1), Grg6 (RDA3), or GAPDH cDNA probe as indicated. The mobilities of the 18S and 28S rRNAs are shown. (B) Immunoblot analysis of FL5.12 cells transfected with E2A-HLF, E12, E47, HLF, or TEF protein. To verify that expression of the indicated genes was upregulated by zinc addition, lysates were prepared from FL5.12 cells and analyzed by immunoblotting with E2A rabbit antisera (αE2A) (top) and HLF(C) rabbit antisera (αHLF) (bottom). Cells were stably transfected with the pMT empty vector (pMT-CB6+) or with metallothionein promoter-regulated constructs containing cDNAs for E2A-HLF, E2A (E12 and E47), HLF, or TEF, with or without the addition of zinc as described for panel A.

    Journal: Molecular and Cellular Biology

    Article Title: The E2A-HLF Oncoprotein Activates Groucho-Related Genes and Suppresses Runx1

    doi: 10.1128/MCB.21.17.5935-5945.2001

    Figure Lengend Snippet: Upregulation of expression of the Grg2 (RDA1) and Grg6 (RDA3) genes induced by E2A-HLF. (A) Northern blot analysis of poly(A) + RNA (1 μg per lane) prepared from FL5.12 cells stably transfected with the pMT empty vector (lanes 1 to 4) or with metallothionein promoter-regulated constructs containing cDNAs for E2A-HLF (lanes 5 to 8), the ΔAD1 E2A-HLF mutant lacking the AD1 transactivation domain of E2A (lanes 9 and 10), the BX E2A-HLF mutant with a disabled HLF DNA-binding domain (lanes 11 and 12), E2A (E12 [lanes 13 and 14] and E47 [lanes 15 and 16]), HLF (lanes 17 and 18), or TEF (lanes 19 and 20). Cells were cultured in IL-3-containing medium with (+) or without (−) 100 μM ZnSO 4 for 18 h to induce expression of the transfected cDNA and then continued in the same concentration of zinc and either maintained in IL-3 (+) or deprived of the cytokine (−) for an additional 12 h before RNA extraction. The blot was hybridized with the Grg2 (RDA1), Grg6 (RDA3), or GAPDH cDNA probe as indicated. The mobilities of the 18S and 28S rRNAs are shown. (B) Immunoblot analysis of FL5.12 cells transfected with E2A-HLF, E12, E47, HLF, or TEF protein. To verify that expression of the indicated genes was upregulated by zinc addition, lysates were prepared from FL5.12 cells and analyzed by immunoblotting with E2A rabbit antisera (αE2A) (top) and HLF(C) rabbit antisera (αHLF) (bottom). Cells were stably transfected with the pMT empty vector (pMT-CB6+) or with metallothionein promoter-regulated constructs containing cDNAs for E2A-HLF, E2A (E12 and E47), HLF, or TEF, with or without the addition of zinc as described for panel A.

    Article Snippet: Double-stranded cDNAs were synthesized with the Superscript cDNA synthesis kit (GIBCO-BRL) according to the manufacturer's protocol.

    Techniques: Expressing, Northern Blot, Stable Transfection, Transfection, Plasmid Preparation, Construct, Mutagenesis, Binding Assay, Cell Culture, Concentration Assay, RNA Extraction

    Bioenergetics analysis of macrophages challenged with S. aureus Mouse BMDM (5 × 10 4 ) were plated in XF 96 V3-PS cell culture plates and treated with AICAR (1mM) one hour before challenge with S. aureus (MOI 10:1) (A) and HKSA (10 8 cfu) (B) for 8h. The extracellular acidification rate (ECAR) was measured by the sequential addition of glucose, oligomycin and 2DG. In a separate experiment, macrophages were plated in 6 well plate (5× 10 6 cells/well) and treated with AICAR (1mM) one hour before S. aureus infection for 8h. Cells were collected, RNA was extracted, cDNA was synthesized, and qRT PCR was performed for the glycolytic pathway genes HK2 and Glut1 and fold change was calculated using GAPDH ( C ). Each data point represents mean ± SD (n = 6/condition/experiment), and results are representative of at least 3 independent experiments. Statistical analysis was performed using one-way ANOVA with Bonferroni’s multiple-comparison test. * P

    Journal: Cellular microbiology

    Article Title: AICAR-mediated AMPK activation induces protective innate responses in bacterial endophthalmitis

    doi: 10.1111/cmi.12625

    Figure Lengend Snippet: Bioenergetics analysis of macrophages challenged with S. aureus Mouse BMDM (5 × 10 4 ) were plated in XF 96 V3-PS cell culture plates and treated with AICAR (1mM) one hour before challenge with S. aureus (MOI 10:1) (A) and HKSA (10 8 cfu) (B) for 8h. The extracellular acidification rate (ECAR) was measured by the sequential addition of glucose, oligomycin and 2DG. In a separate experiment, macrophages were plated in 6 well plate (5× 10 6 cells/well) and treated with AICAR (1mM) one hour before S. aureus infection for 8h. Cells were collected, RNA was extracted, cDNA was synthesized, and qRT PCR was performed for the glycolytic pathway genes HK2 and Glut1 and fold change was calculated using GAPDH ( C ). Each data point represents mean ± SD (n = 6/condition/experiment), and results are representative of at least 3 independent experiments. Statistical analysis was performed using one-way ANOVA with Bonferroni’s multiple-comparison test. * P

    Article Snippet: RNA was reversed transcribed to cDNA using a cDNA synthesis kit (Superscript, Invitrogen Carlsbad, CA, USA).

    Techniques: Cell Culture, Infection, Synthesized, Quantitative RT-PCR

    S. aureus -infected microglia and retinal tissue showed increased glycolysis and AICAR treatment inhibited this response BV2 cells (3 × 10 4 cells/well) were plated in XF 96 V3-PS 96 cell culture plates (Seahorse Bioscience) and treated with AICAR (1mM) one hour before challenged with live S. aureus (MOI 10:1) (A) or HKSA (10 8 cfu) for 8h (B). The extracellular acidification rate (ECAR) was measured by the sequential addition of glucose, oligomycin and 2 deoxyglucose (2-DG). To examine the contribution of bacteria alone in the total response of the cells, live S. aureus was added in media only. In the another experiment, BV2 mouse microglia cells were plated in 6 well plate (1× 10 6 cells/well) and treated with AICAR (1mM) one hour before S. aureus infection for 8h. Cells were collected, RNA was extracted, cDNA was synthesized, and qRT-PCR was performed for glycolytic pathway genes HK2 and Glut1 and fold changes were calculated using housekeeping gene GAPDH ( C ). Eyes of WT (C57BL/6) (n = 5) were infected with S. aureus (5000 cfu/eye) followed by AICAR treatment (30 μg/eye) for 24h. Retinas were removed and pooled for RNA extraction. cDNA was prepared, and qPCR was performed for the glycolytic pathway genes HK2 and Glut1 and fold change was calculated using GAPDH (D) . Each data point represents mean ± SD (n = 6/condition/experiment), and results are representative of at least 3 independent experiments. The results represent the mean ± SD of triplicates from three independent experiments. Statistical analysis was performed using one-way ANOVA with Bonferroni’s multiple-comparison test. * P

    Journal: Cellular microbiology

    Article Title: AICAR-mediated AMPK activation induces protective innate responses in bacterial endophthalmitis

    doi: 10.1111/cmi.12625

    Figure Lengend Snippet: S. aureus -infected microglia and retinal tissue showed increased glycolysis and AICAR treatment inhibited this response BV2 cells (3 × 10 4 cells/well) were plated in XF 96 V3-PS 96 cell culture plates (Seahorse Bioscience) and treated with AICAR (1mM) one hour before challenged with live S. aureus (MOI 10:1) (A) or HKSA (10 8 cfu) for 8h (B). The extracellular acidification rate (ECAR) was measured by the sequential addition of glucose, oligomycin and 2 deoxyglucose (2-DG). To examine the contribution of bacteria alone in the total response of the cells, live S. aureus was added in media only. In the another experiment, BV2 mouse microglia cells were plated in 6 well plate (1× 10 6 cells/well) and treated with AICAR (1mM) one hour before S. aureus infection for 8h. Cells were collected, RNA was extracted, cDNA was synthesized, and qRT-PCR was performed for glycolytic pathway genes HK2 and Glut1 and fold changes were calculated using housekeeping gene GAPDH ( C ). Eyes of WT (C57BL/6) (n = 5) were infected with S. aureus (5000 cfu/eye) followed by AICAR treatment (30 μg/eye) for 24h. Retinas were removed and pooled for RNA extraction. cDNA was prepared, and qPCR was performed for the glycolytic pathway genes HK2 and Glut1 and fold change was calculated using GAPDH (D) . Each data point represents mean ± SD (n = 6/condition/experiment), and results are representative of at least 3 independent experiments. The results represent the mean ± SD of triplicates from three independent experiments. Statistical analysis was performed using one-way ANOVA with Bonferroni’s multiple-comparison test. * P

    Article Snippet: RNA was reversed transcribed to cDNA using a cDNA synthesis kit (Superscript, Invitrogen Carlsbad, CA, USA).

    Techniques: Infection, Cell Culture, Synthesized, Quantitative RT-PCR, RNA Extraction, Real-time Polymerase Chain Reaction

    Agarose gel electrophoresis of RDA products from RNA extracted from bovine parainfluenza virus 3-infected cells. Double-stranded cDNA was synthesized from RNA of bovine parainfluenza virus 3-infected MDBK cells and subjected to RDA. Mock-infected cells were used for the synthesis of driver amplicons for RDA. One-twentieth of the volume of the amplified products was separated on 3% agarose gel and stained with ethidium bromide. RDA product from the uninfected control cells was used as a negative control.

    Journal: Nucleic Acids Research

    Article Title: Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription

    doi: 10.1093/nar/gni064

    Figure Lengend Snippet: Agarose gel electrophoresis of RDA products from RNA extracted from bovine parainfluenza virus 3-infected cells. Double-stranded cDNA was synthesized from RNA of bovine parainfluenza virus 3-infected MDBK cells and subjected to RDA. Mock-infected cells were used for the synthesis of driver amplicons for RDA. One-twentieth of the volume of the amplified products was separated on 3% agarose gel and stained with ethidium bromide. RDA product from the uninfected control cells was used as a negative control.

    Article Snippet: cDNA RDA First-strand cDNA was synthesized from the mixed RNA with non-ribosomal hexanucleotides by using a double-stranded cDNA synthesis kit (Invitrogen) according to the manufacturer's protocol, i.e. the total RNA was diluted to 1 μg per μl and mixed with dNTPs, the non-ribosomal hexanucleotides, 5× reaction buffer, 0.1 M DTT and an RNase inhibitor.

    Techniques: Agarose Gel Electrophoresis, Infection, Synthesized, Amplification, Staining, Negative Control

    Autoradiogram of 32 P-labelled double-stranded cDNA synthesized from mixtures consisting of artificial RNA and total cellular RNA. In vitro transcribed RNA was synthesized from pCIneo plasmid and mixed with total cellular RNA extracted from rat2 cells in weight proportions 1:0 (lanes 1 and 8), 1:1 (lanes 2 and 9), 1:10 (lanes 3 and 10), 1:100 (lanes 4 and 11), 1:300 (lanes 5 and 12), 1:1000 (lanes 6 and 13) and 0:1 (lanes 7 and 14). One microgram of mixed RNA was reverse transcribed using random (lanes 1–7) or non-ribosomal (lanes 8–14) hexanucleotides and a second-strand cDNA was then synthesized with RNaseH, DNA polymerase and DNA ligase according to the method described in Materials and Methods. One-tenth of the volume of synthesized cDNAs was loaded on agarose gel ( A ). Loaded volumes were corrected to include the same amounts of 32 P in each sample ( B ). Positions and sizes (bp) of markers are present on the left.

    Journal: Nucleic Acids Research

    Article Title: Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription

    doi: 10.1093/nar/gni064

    Figure Lengend Snippet: Autoradiogram of 32 P-labelled double-stranded cDNA synthesized from mixtures consisting of artificial RNA and total cellular RNA. In vitro transcribed RNA was synthesized from pCIneo plasmid and mixed with total cellular RNA extracted from rat2 cells in weight proportions 1:0 (lanes 1 and 8), 1:1 (lanes 2 and 9), 1:10 (lanes 3 and 10), 1:100 (lanes 4 and 11), 1:300 (lanes 5 and 12), 1:1000 (lanes 6 and 13) and 0:1 (lanes 7 and 14). One microgram of mixed RNA was reverse transcribed using random (lanes 1–7) or non-ribosomal (lanes 8–14) hexanucleotides and a second-strand cDNA was then synthesized with RNaseH, DNA polymerase and DNA ligase according to the method described in Materials and Methods. One-tenth of the volume of synthesized cDNAs was loaded on agarose gel ( A ). Loaded volumes were corrected to include the same amounts of 32 P in each sample ( B ). Positions and sizes (bp) of markers are present on the left.

    Article Snippet: cDNA RDA First-strand cDNA was synthesized from the mixed RNA with non-ribosomal hexanucleotides by using a double-stranded cDNA synthesis kit (Invitrogen) according to the manufacturer's protocol, i.e. the total RNA was diluted to 1 μg per μl and mixed with dNTPs, the non-ribosomal hexanucleotides, 5× reaction buffer, 0.1 M DTT and an RNase inhibitor.

    Techniques: Synthesized, In Vitro, Plasmid Preparation, Agarose Gel Electrophoresis

    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

    Effects of Arf6 mutants on Tac recycling in MDA-MB-231 cells. MDA-MB-231 cells were transfected with Tac cDNA, together with Arf6(Q67L) or Arf6(T27N) cDNAs as described, and subjected to internalization ( A ) and recycling-back ( B ) assays of Tac antigen. The control included cotransfection of Tac with EGFP cDNA (mock). Results are means ± SEM from three independent experiments.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Requirement for Arf6 in breast cancer invasive activities

    doi: 10.1073/pnas.0401753101

    Figure Lengend Snippet: Effects of Arf6 mutants on Tac recycling in MDA-MB-231 cells. MDA-MB-231 cells were transfected with Tac cDNA, together with Arf6(Q67L) or Arf6(T27N) cDNAs as described, and subjected to internalization ( A ) and recycling-back ( B ) assays of Tac antigen. The control included cotransfection of Tac with EGFP cDNA (mock). Results are means ± SEM from three independent experiments.

    Article Snippet: Random-primed cDNAs, prepared from 2 μg of cellular RNAs by using the SuperScript first-strand cDNA synthesis kit (Invitrogen), were subjected to real-time PCR amplification analysis using the LightCycler FastStart DNA Master SYBR Green I (Roche) and the LightCycler (Roche), according to the manufacturer's instructions.

    Techniques: Multiple Displacement Amplification, Transfection, Cotransfection

    Inhibition of iNOS expression and NO production by tryptophan deficiency in IFN-γ+LPS stimulated RAW264.7 cells. (A) Q-PCR analysis of iNOS mRNA expression. Raw264.7 cells were pre-incubated in RPMI, tryptophan-deficient medium (Trp-D), tryptophan-deficient medium supplemented with 50 µg/ml tryptophan or RPMI supplemented with increasing concentrations of kynurenine (10, 25, and 50 µg/ml) for an hour. Cells were then stimulated with IFN-γ+LPS for 12 hours. Cells were collected, and iNOS expression was determined by Q-PCR after RNA extraction and cDNA synthesis. GAPDH was used as the reference gene. The relative expression of iNOS mRNA is evaluated by Q-PCR in Raw264.7 cells cultured and treated in different conditions. The iNOS expression was normalized to the GAPDH expression level (B) Raw264.7 cells were cultured and stimulated for 24 hours as described above. The concentration of released nitric oxide was evaluated in the cell supernatant by the Griess method. Data is mean±SEM of four independent experiments (**P-value

    Journal: PLoS ONE

    Article Title: Immuno-Regulatory Function of Indoleamine 2,3 Dioxygenase through Modulation of Innate Immune Responses

    doi: 10.1371/journal.pone.0071044

    Figure Lengend Snippet: Inhibition of iNOS expression and NO production by tryptophan deficiency in IFN-γ+LPS stimulated RAW264.7 cells. (A) Q-PCR analysis of iNOS mRNA expression. Raw264.7 cells were pre-incubated in RPMI, tryptophan-deficient medium (Trp-D), tryptophan-deficient medium supplemented with 50 µg/ml tryptophan or RPMI supplemented with increasing concentrations of kynurenine (10, 25, and 50 µg/ml) for an hour. Cells were then stimulated with IFN-γ+LPS for 12 hours. Cells were collected, and iNOS expression was determined by Q-PCR after RNA extraction and cDNA synthesis. GAPDH was used as the reference gene. The relative expression of iNOS mRNA is evaluated by Q-PCR in Raw264.7 cells cultured and treated in different conditions. The iNOS expression was normalized to the GAPDH expression level (B) Raw264.7 cells were cultured and stimulated for 24 hours as described above. The concentration of released nitric oxide was evaluated in the cell supernatant by the Griess method. Data is mean±SEM of four independent experiments (**P-value

    Article Snippet: Following DNase treatment, total RNA (5 µg) was reverse transcribed into cDNA using a Superscript II First Strand cDNA Synthesis kit (Invitrogen).

    Techniques: Inhibition, Expressing, Polymerase Chain Reaction, Incubation, RNA Extraction, Cell Culture, Concentration Assay

    Isolation and Purification of Mx, ORS, DP, DF, and Mc Populations (A) Schematic of isolation procedure. After removing subcutaneous fat by dissection, and epidermis/upper follicle segment by enzymatic digestion, single-cell suspensions were prepared from pure dermis and subjected to three FACS schemes to purify five populations of cells: Mx, GFP low RFP − ; ORS, GFP high RFP − ; DP, RFP high GFP − CD34 − CD45 − CD117 − ; DF, RFP − GFP − CD34 − CD45 − CD117 − ; Mc, RFP high GFP − CD117 + . (B) Immunofluorescence analyses of FACS isolated cell populations. Frozen skin sections (hair bulb) and relevant cytospin populations were stained with Abs as color-coded and indicated. At the right of each set is quantification of percentage of cells that expressed the marker. Note: ~10% of DP and DF cells lysed on cytospin. and hence did not stain with any markers. β4, β4 integrin; Tyr, tyrosinase; Vim, vimentin; white line, basement membrane. (C) RT-PCR: cDNA fragments were resolved by agarose gel electrophoresis, and the gene detected is denoted at left. All fragments were of the expected size. Expression of Msx2, vimentin, and β4 in multiple populations was later confirmed. (D) Cell cycle differences in cell populations. Profiles of the five purified populations were performed by FACS. Anti-BrdU immunofluorescence is from a P4 backskin follicle from a mouse injected intraperitoneally with 50 μg/g 5-bromo-2′-deoxyuridine (BrdU) (Sigma-Aldrich) and analyzed 4 h later. Note greatest incorporation in Mx and ORS.

    Journal: PLoS Biology

    Article Title: Molecular Dissection of Mesenchymal-Epithelial Interactions in the Hair FollicleMolecular Signatures of the Developing Hair Follicle

    doi: 10.1371/journal.pbio.0030331

    Figure Lengend Snippet: Isolation and Purification of Mx, ORS, DP, DF, and Mc Populations (A) Schematic of isolation procedure. After removing subcutaneous fat by dissection, and epidermis/upper follicle segment by enzymatic digestion, single-cell suspensions were prepared from pure dermis and subjected to three FACS schemes to purify five populations of cells: Mx, GFP low RFP − ; ORS, GFP high RFP − ; DP, RFP high GFP − CD34 − CD45 − CD117 − ; DF, RFP − GFP − CD34 − CD45 − CD117 − ; Mc, RFP high GFP − CD117 + . (B) Immunofluorescence analyses of FACS isolated cell populations. Frozen skin sections (hair bulb) and relevant cytospin populations were stained with Abs as color-coded and indicated. At the right of each set is quantification of percentage of cells that expressed the marker. Note: ~10% of DP and DF cells lysed on cytospin. and hence did not stain with any markers. β4, β4 integrin; Tyr, tyrosinase; Vim, vimentin; white line, basement membrane. (C) RT-PCR: cDNA fragments were resolved by agarose gel electrophoresis, and the gene detected is denoted at left. All fragments were of the expected size. Expression of Msx2, vimentin, and β4 in multiple populations was later confirmed. (D) Cell cycle differences in cell populations. Profiles of the five purified populations were performed by FACS. Anti-BrdU immunofluorescence is from a P4 backskin follicle from a mouse injected intraperitoneally with 50 μg/g 5-bromo-2′-deoxyuridine (BrdU) (Sigma-Aldrich) and analyzed 4 h later. Note greatest incorporation in Mx and ORS.

    Article Snippet: Quality was assessed by RNA 6000 Pico Assay (Agilent Technologies, Palo Alto, California, United States), and 800 ng were primed with oligo(dT)-T7 primer and reverse transcribed (Superscript III cDNA synthesis kit; Invitrogen, Carlsbad, California, United States).

    Techniques: Isolation, Purification, Dissection, FACS, Immunofluorescence, Staining, Marker, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Expressing, Injection

    Implementation of Array Anal yses to Examine Characteristics and Dynamics of the Follicle DP Niche (A) Semi-quantitative RT-PCR on mRNAs isolated from each population. Shown are representative data from molecular signature genes (see Figure 4 ) whose expression patterns in the DP niche environment had been previously uncharacterized. In this case, categories were consolidated into three groups: Signal transduction, transcription/nuclear, and cytoskeleton/ECM/cell adhesion. For each primer set, at least three different cycles were employed, and the resulting cDNA fragments were resolved by agarose gel electrophoresis along with DNA size markers to confirm that bands were of the expected sizes. For each gene, the data presented were from the cycle that provided the most meaningful comparisons. Note: bands seen in > 1 fraction accurately reflect mRNA expression at the differences in levels shown. (B) Immunohistochemistry and in situ hybridizations. Skin sections were taken from 2-mo-old K14-GFPactin mice [ 49 ] whose follicles were at the transition from the resting to growing (telogen to anagen) stage of the hair cycle (8 wk) or from P4 WT mice (full anagen follicles) (all others). Sections were subjected to either immunofluorescence using color-coded Abs as indicated or in situ hybridization using the indicated biotinylated cRNA probes (sense controls were negative).

    Journal: PLoS Biology

    Article Title: Molecular Dissection of Mesenchymal-Epithelial Interactions in the Hair FollicleMolecular Signatures of the Developing Hair Follicle

    doi: 10.1371/journal.pbio.0030331

    Figure Lengend Snippet: Implementation of Array Anal yses to Examine Characteristics and Dynamics of the Follicle DP Niche (A) Semi-quantitative RT-PCR on mRNAs isolated from each population. Shown are representative data from molecular signature genes (see Figure 4 ) whose expression patterns in the DP niche environment had been previously uncharacterized. In this case, categories were consolidated into three groups: Signal transduction, transcription/nuclear, and cytoskeleton/ECM/cell adhesion. For each primer set, at least three different cycles were employed, and the resulting cDNA fragments were resolved by agarose gel electrophoresis along with DNA size markers to confirm that bands were of the expected sizes. For each gene, the data presented were from the cycle that provided the most meaningful comparisons. Note: bands seen in > 1 fraction accurately reflect mRNA expression at the differences in levels shown. (B) Immunohistochemistry and in situ hybridizations. Skin sections were taken from 2-mo-old K14-GFPactin mice [ 49 ] whose follicles were at the transition from the resting to growing (telogen to anagen) stage of the hair cycle (8 wk) or from P4 WT mice (full anagen follicles) (all others). Sections were subjected to either immunofluorescence using color-coded Abs as indicated or in situ hybridization using the indicated biotinylated cRNA probes (sense controls were negative).

    Article Snippet: Quality was assessed by RNA 6000 Pico Assay (Agilent Technologies, Palo Alto, California, United States), and 800 ng were primed with oligo(dT)-T7 primer and reverse transcribed (Superscript III cDNA synthesis kit; Invitrogen, Carlsbad, California, United States).

    Techniques: Quantitative RT-PCR, Isolation, Expressing, Transduction, Agarose Gel Electrophoresis, Immunohistochemistry, In Situ, Mouse Assay, Immunofluorescence, In Situ Hybridization

    Analysis of inflammatory gene and protein expression in tongue tissues.A. Two chemokines (CXCL1, CXCL2), a neutrophil-specific antigen (CD177) and an epithelial cell-derived neutrophil-activating cytokine (IL-17C) were tested in the same RNA samples surveyed by microarray analysis on day 5 post infection. After cDNA synthesis, equal amounts from three mice per group were mixed and analysed in triplicate by RT-qPCR. All genes were significantly ( P

    Journal: Cellular Microbiology

    Article Title: Streptococcal co-infection augments Candida pathogenicity by amplifying the mucosal inflammatory response

    doi: 10.1111/cmi.12216

    Figure Lengend Snippet: Analysis of inflammatory gene and protein expression in tongue tissues.A. Two chemokines (CXCL1, CXCL2), a neutrophil-specific antigen (CD177) and an epithelial cell-derived neutrophil-activating cytokine (IL-17C) were tested in the same RNA samples surveyed by microarray analysis on day 5 post infection. After cDNA synthesis, equal amounts from three mice per group were mixed and analysed in triplicate by RT-qPCR. All genes were significantly ( P

    Article Snippet: RNA concentrations and quality were determined by measuring the absorbance at 260 nm and 280 nm using the NanoDrop device. cDNA was synthesized by using SuperScript III CellsDirect cDNA Synthesis Kit® (Invitrogen), according to the manual.

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

    E-cadherin is downregulated in migratory PGCs. (A) Relative amount of E-cadherin ( cdh1 ) in PGCs and somatic endodermal cells (Som) isolated from stage 28–30 embryos (migratory PGCs) normalized to E-cadherin level in the corresponding cell type isolated from stage 17–19 embryos (pre-migratory PGCs) measured by quantitative RT PCR. Relative amount was calculated by ΔΔCt method (see Material and Methods) using three independent cDNA preparations. Error bars represent standard deviation. * corresponds to P

    Journal: Biology Open

    Article Title: Migratory and adhesive properties of Xenopus laevis primordial germ cells in vitro

    doi: 10.1242/bio.20135140

    Figure Lengend Snippet: E-cadherin is downregulated in migratory PGCs. (A) Relative amount of E-cadherin ( cdh1 ) in PGCs and somatic endodermal cells (Som) isolated from stage 28–30 embryos (migratory PGCs) normalized to E-cadherin level in the corresponding cell type isolated from stage 17–19 embryos (pre-migratory PGCs) measured by quantitative RT PCR. Relative amount was calculated by ΔΔCt method (see Material and Methods) using three independent cDNA preparations. Error bars represent standard deviation. * corresponds to P

    Article Snippet: 30 cells from each cell population were used for the preparation of cDNA using SuperScript III CellsDirect cDNA Synthesis System (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol.

    Techniques: Isolation, Quantitative RT-PCR, Standard Deviation

    qRT-PCR analysis of AβO-induced differential gene expression. cDNA from the three donors used in the microarray analysis plus four samples from additional independent donors were used. Expression levels were determined by relative expression,

    Journal: The Journal of Biological Chemistry

    Article Title: Amyloid-? Oligomers Induce Differential Gene Expression in Adult Human Brain Slices *

    doi: 10.1074/jbc.M111.298471

    Figure Lengend Snippet: qRT-PCR analysis of AβO-induced differential gene expression. cDNA from the three donors used in the microarray analysis plus four samples from additional independent donors were used. Expression levels were determined by relative expression,

    Article Snippet: For rat neuronal cultures, template cDNA was prepared from total RNA (1 μg) using 50 pmol of oligo dT20 and the Superscript III First Strand cDNA kit (Invitrogen), and ACTB or GAPDH was used as the endogenous control.

    Techniques: Quantitative RT-PCR, Expressing, Microarray