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  • 99
    Thermo Fisher superscript iii ssiii first strand synthesis system
    Effect of glaucocalyxin-A (GLA) on cyclooxygenase-2 (COX-2) and prostaglandin E 2 (PGE 2 ) expression in lipopolysaccharide (LPS)-stimulated microglia. Cells were pre-treated with the indicated concentrations of GLA for 1 h before incubating with LPS (25 ng/mL or 100 ng/mL) for 18 h A: Primary Microglia, B: BV-2 cells. Results are expressed as a ratio of COX-2 to β-actin. Representative quantification data was shown in the lower panel. C: Cells were pre-treated with the indicated concentrations of GLA for 1 h and then stimulated with LPS (100 ng/mL) for 6 h. Total <t>RNA</t> was prepared and COX-2 mRNA level was determined by RT-PCR. Results are expressed as the ratio of COX-2 to GAPDH. Quantification data are shown in the lower panel. D: PGE 2 levels were analyzed with an enzyme immunoassay kit. Absorbance was measured at 420 nm spectrophotometrically. Data are mean ± S.E.M. (n = 3) for <t>three</t> independent experiments. # P
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    Thermo Fisher superscript iii first strand synthesis ssiii supermix
    Effect of glaucocalyxin-A (GLA) on cyclooxygenase-2 (COX-2) and prostaglandin E 2 (PGE 2 ) expression in lipopolysaccharide (LPS)-stimulated microglia. Cells were pre-treated with the indicated concentrations of GLA for 1 h before incubating with LPS (25 ng/mL or 100 ng/mL) for 18 h A: Primary Microglia, B: BV-2 cells. Results are expressed as a ratio of COX-2 to β-actin. Representative quantification data was shown in the lower panel. C: Cells were pre-treated with the indicated concentrations of GLA for 1 h and then stimulated with LPS (100 ng/mL) for 6 h. Total <t>RNA</t> was prepared and COX-2 mRNA level was determined by RT-PCR. Results are expressed as the ratio of COX-2 to GAPDH. Quantification data are shown in the lower panel. D: PGE 2 levels were analyzed with an enzyme immunoassay kit. Absorbance was measured at 420 nm spectrophotometrically. Data are mean ± S.E.M. (n = 3) for <t>three</t> independent experiments. # P
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    Thermo Fisher superscript iii rt system
    Lifespan extension in <t>sft-1(RNAi)</t> and oxa-1(RNAi) animals. A : Lifespan curves for N2, oxa-1(RNAi) and sft-1(RNAi) animals (recorded as number of days of survival post L4, or the time at which controls reached L4, in the case of oxa-1(RNAi) animals). Lifespan is prolonged in sft-1(RNAi) and oxa-1(RNAi) animals compared with WT controls, with oxa-1 having the greatest effect. B : Lifespan analysis of sft-1(RNAi) animals performed in a daf-16(m26) mutant background, in which sft-1 RNAi fails to extend lifespan. C : Lifespan analysis of oxa-1(RNAi) animals performed in a daf-16(m26) mutant background. oxa-1 RNAi still extends lifespan under these circumstances. The data presented combine <t>three</t> independent data sets (the data for individual replicate experiments can be found in Additional file 1 ).
    Superscript Iii Rt System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 196 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ssiii reverse transcriptase kit
    Lifespan extension in <t>sft-1(RNAi)</t> and oxa-1(RNAi) animals. A : Lifespan curves for N2, oxa-1(RNAi) and sft-1(RNAi) animals (recorded as number of days of survival post L4, or the time at which controls reached L4, in the case of oxa-1(RNAi) animals). Lifespan is prolonged in sft-1(RNAi) and oxa-1(RNAi) animals compared with WT controls, with oxa-1 having the greatest effect. B : Lifespan analysis of sft-1(RNAi) animals performed in a daf-16(m26) mutant background, in which sft-1 RNAi fails to extend lifespan. C : Lifespan analysis of oxa-1(RNAi) animals performed in a daf-16(m26) mutant background. oxa-1 RNAi still extends lifespan under these circumstances. The data presented combine <t>three</t> independent data sets (the data for individual replicate experiments can be found in Additional file 1 ).
    Ssiii Reverse Transcriptase Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript ssiii kit
    Lifespan extension in <t>sft-1(RNAi)</t> and oxa-1(RNAi) animals. A : Lifespan curves for N2, oxa-1(RNAi) and sft-1(RNAi) animals (recorded as number of days of survival post L4, or the time at which controls reached L4, in the case of oxa-1(RNAi) animals). Lifespan is prolonged in sft-1(RNAi) and oxa-1(RNAi) animals compared with WT controls, with oxa-1 having the greatest effect. B : Lifespan analysis of sft-1(RNAi) animals performed in a daf-16(m26) mutant background, in which sft-1 RNAi fails to extend lifespan. C : Lifespan analysis of oxa-1(RNAi) animals performed in a daf-16(m26) mutant background. oxa-1 RNAi still extends lifespan under these circumstances. The data presented combine <t>three</t> independent data sets (the data for individual replicate experiments can be found in Additional file 1 ).
    Superscript Ssiii Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher superscript iii vilo kit
    Lifespan extension in <t>sft-1(RNAi)</t> and oxa-1(RNAi) animals. A : Lifespan curves for N2, oxa-1(RNAi) and sft-1(RNAi) animals (recorded as number of days of survival post L4, or the time at which controls reached L4, in the case of oxa-1(RNAi) animals). Lifespan is prolonged in sft-1(RNAi) and oxa-1(RNAi) animals compared with WT controls, with oxa-1 having the greatest effect. B : Lifespan analysis of sft-1(RNAi) animals performed in a daf-16(m26) mutant background, in which sft-1 RNAi fails to extend lifespan. C : Lifespan analysis of oxa-1(RNAi) animals performed in a daf-16(m26) mutant background. oxa-1 RNAi still extends lifespan under these circumstances. The data presented combine <t>three</t> independent data sets (the data for individual replicate experiments can be found in Additional file 1 ).
    Superscript Iii Vilo Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 110 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ssiii reverse transcribing kit
    Lifespan extension in <t>sft-1(RNAi)</t> and oxa-1(RNAi) animals. A : Lifespan curves for N2, oxa-1(RNAi) and sft-1(RNAi) animals (recorded as number of days of survival post L4, or the time at which controls reached L4, in the case of oxa-1(RNAi) animals). Lifespan is prolonged in sft-1(RNAi) and oxa-1(RNAi) animals compared with WT controls, with oxa-1 having the greatest effect. B : Lifespan analysis of sft-1(RNAi) animals performed in a daf-16(m26) mutant background, in which sft-1 RNAi fails to extend lifespan. C : Lifespan analysis of oxa-1(RNAi) animals performed in a daf-16(m26) mutant background. oxa-1 RNAi still extends lifespan under these circumstances. The data presented combine <t>three</t> independent data sets (the data for individual replicate experiments can be found in Additional file 1 ).
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    Image Search Results


    Effect of glaucocalyxin-A (GLA) on cyclooxygenase-2 (COX-2) and prostaglandin E 2 (PGE 2 ) expression in lipopolysaccharide (LPS)-stimulated microglia. Cells were pre-treated with the indicated concentrations of GLA for 1 h before incubating with LPS (25 ng/mL or 100 ng/mL) for 18 h A: Primary Microglia, B: BV-2 cells. Results are expressed as a ratio of COX-2 to β-actin. Representative quantification data was shown in the lower panel. C: Cells were pre-treated with the indicated concentrations of GLA for 1 h and then stimulated with LPS (100 ng/mL) for 6 h. Total RNA was prepared and COX-2 mRNA level was determined by RT-PCR. Results are expressed as the ratio of COX-2 to GAPDH. Quantification data are shown in the lower panel. D: PGE 2 levels were analyzed with an enzyme immunoassay kit. Absorbance was measured at 420 nm spectrophotometrically. Data are mean ± S.E.M. (n = 3) for three independent experiments. # P

    Journal: PLoS ONE

    Article Title: Regulation of Microglia Activity by Glaucocalyxin-A: Attenuation of Lipopolysaccharide-Stimulated Neuroinflammation through NF-?B and p38 MAPK Signaling Pathways

    doi: 10.1371/journal.pone.0055792

    Figure Lengend Snippet: Effect of glaucocalyxin-A (GLA) on cyclooxygenase-2 (COX-2) and prostaglandin E 2 (PGE 2 ) expression in lipopolysaccharide (LPS)-stimulated microglia. Cells were pre-treated with the indicated concentrations of GLA for 1 h before incubating with LPS (25 ng/mL or 100 ng/mL) for 18 h A: Primary Microglia, B: BV-2 cells. Results are expressed as a ratio of COX-2 to β-actin. Representative quantification data was shown in the lower panel. C: Cells were pre-treated with the indicated concentrations of GLA for 1 h and then stimulated with LPS (100 ng/mL) for 6 h. Total RNA was prepared and COX-2 mRNA level was determined by RT-PCR. Results are expressed as the ratio of COX-2 to GAPDH. Quantification data are shown in the lower panel. D: PGE 2 levels were analyzed with an enzyme immunoassay kit. Absorbance was measured at 420 nm spectrophotometrically. Data are mean ± S.E.M. (n = 3) for three independent experiments. # P

    Article Snippet: RNA (2.5 µg) was reverse-transcribed using a Superscript™-III kit (Invitrogen), according to the manufacturer’s instruction.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Effect of glaucocalyxin-A (GLA) on inducible nitric oxide synthase (iNOS) expression and enzyme activity in lipopolysaccharide (LPS)-stimulated microglia. Cells were pre-treated with the indicated concentrations of GLA for 1 h before incubating with LPS (25 ng/mL or 100 ng/mL) for 18 h A: Primary microglia, B: BV-2 microglia. Lysates were analyzed by immunoblotting with an anti-iNOS antibody. Representative quantification data were shown in the lower panel. Results are expressed as a ratio of iNOS to β-actin. C: BV-2 cells were pre-treated with the indicated concentrations of GLA for 1 h before incubating with LPS (100 ng/mL) for 6 h. The total RNA was isolated and iNOS mRNA level was determined by RT-PCR. Quantification data are shown in the lower panel. Results are expressed as a ratio of iNOS to GAPDH. D: BV-2 microglial cells were pre-treated with the indicated concentrations of GLA for 1 h before incubating with LPS (100 ng/mL) for 18 h. iNOS activity was analyzed with enzyme immunoassay kit. Data are mean ± S.E.M. (n = 3) for three independent experiments. # P

    Journal: PLoS ONE

    Article Title: Regulation of Microglia Activity by Glaucocalyxin-A: Attenuation of Lipopolysaccharide-Stimulated Neuroinflammation through NF-?B and p38 MAPK Signaling Pathways

    doi: 10.1371/journal.pone.0055792

    Figure Lengend Snippet: Effect of glaucocalyxin-A (GLA) on inducible nitric oxide synthase (iNOS) expression and enzyme activity in lipopolysaccharide (LPS)-stimulated microglia. Cells were pre-treated with the indicated concentrations of GLA for 1 h before incubating with LPS (25 ng/mL or 100 ng/mL) for 18 h A: Primary microglia, B: BV-2 microglia. Lysates were analyzed by immunoblotting with an anti-iNOS antibody. Representative quantification data were shown in the lower panel. Results are expressed as a ratio of iNOS to β-actin. C: BV-2 cells were pre-treated with the indicated concentrations of GLA for 1 h before incubating with LPS (100 ng/mL) for 6 h. The total RNA was isolated and iNOS mRNA level was determined by RT-PCR. Quantification data are shown in the lower panel. Results are expressed as a ratio of iNOS to GAPDH. D: BV-2 microglial cells were pre-treated with the indicated concentrations of GLA for 1 h before incubating with LPS (100 ng/mL) for 18 h. iNOS activity was analyzed with enzyme immunoassay kit. Data are mean ± S.E.M. (n = 3) for three independent experiments. # P

    Article Snippet: RNA (2.5 µg) was reverse-transcribed using a Superscript™-III kit (Invitrogen), according to the manufacturer’s instruction.

    Techniques: Expressing, Activity Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    Activity of the Cch1 channel in C. neoformans is dependent on a C-terminal modulatory region of the subunit, Mid1. (A) Three truncated mutants of Mid1, each lacking a portion of the C terminus, were constructed. Mid1-CA-124 lacked 124 amino acids (aa), Mid1-CB-91 lacked 91 amino acids, and Mid1-CC-24 lacked 24 amino acids. Each mutant was constitutively expressed in the mid1 Δ deletion background of C. neoformans ). (B) Sensitivity spot assays were used to identify regions of the Mid1 protein critical for Cch1 activity. The Mid1-CC-24 truncated mutant did not rescue the sensitivity of the mid1 Δ deletion strain on low, free [Ca 2+ ] medium, suggesting that this region is critical for Cch1 activity. (C) A wild-type yeast ( Saccharomyces cerevisiae ) strain was transformed with full-length CnMid1. Two representative transformants expressing CnMid1 did not restore growth of the Scmid1 Δ deletion strain on low, free [Ca 2+ ] medium. (B and C) Serially diluted cells (10 6 , 10 5 , 10 4 , 10 3 , 10 2 ) from wild-type and mid1 Δ deletion strains of C. neoformans and yeast were added to YPD agar plates supplemented with BAPTA (cell impermeative), and spot assays were performed as explained above.

    Journal: Eukaryotic Cell

    Article Title: Activity of the Calcium Channel Pore Cch1 Is Dependent on a Modulatory Region of the Subunit Mid1 in Cryptococcus neoformans

    doi: 10.1128/EC.00130-12

    Figure Lengend Snippet: Activity of the Cch1 channel in C. neoformans is dependent on a C-terminal modulatory region of the subunit, Mid1. (A) Three truncated mutants of Mid1, each lacking a portion of the C terminus, were constructed. Mid1-CA-124 lacked 124 amino acids (aa), Mid1-CB-91 lacked 91 amino acids, and Mid1-CC-24 lacked 24 amino acids. Each mutant was constitutively expressed in the mid1 Δ deletion background of C. neoformans ). (B) Sensitivity spot assays were used to identify regions of the Mid1 protein critical for Cch1 activity. The Mid1-CC-24 truncated mutant did not rescue the sensitivity of the mid1 Δ deletion strain on low, free [Ca 2+ ] medium, suggesting that this region is critical for Cch1 activity. (C) A wild-type yeast ( Saccharomyces cerevisiae ) strain was transformed with full-length CnMid1. Two representative transformants expressing CnMid1 did not restore growth of the Scmid1 Δ deletion strain on low, free [Ca 2+ ] medium. (B and C) Serially diluted cells (10 6 , 10 5 , 10 4 , 10 3 , 10 2 ) from wild-type and mid1 Δ deletion strains of C. neoformans and yeast were added to YPD agar plates supplemented with BAPTA (cell impermeative), and spot assays were performed as explained above.

    Article Snippet: Briefly, the MID1 amplicon was generated from cDNA synthesized by a SuperScript III first-strand synthesis system for reverse transcriptase PCR (RT-PCR) (Invitrogen) using total RNA as the template with the following primers: MID1-cDNA-F and MID1-cDNA-R ( ).

    Techniques: Activity Assay, Construct, Mutagenesis, Transformation Assay, Expressing

    C-terminal-truncated mutants of Mid1 are mislocalized in HEK293 cells. Confocal microscopy was used to examine the localization of three truncated mutants of Mid1 in HEK293 cells. (A) Full-length Mid1-GFP under the control of the CMV promoter was expressed in HEK293 and observed on the cell surface. (B, C, and D) However, expression of the Mid1 truncated mutants lacking specified regions of the C terminus (Mid1-CA-124, Mid1-CB-91, and Mid1-CC-24) displayed a diffuse and predominately cytosolic localization pattern unlike that of full-length Mid1. The Mid1-CA-124 mutant lacked 124 residues, Mid1-CB-91 lacked 91 residues, and Mid1-CC-24 lacked 24 residues.

    Journal: Eukaryotic Cell

    Article Title: Activity of the Calcium Channel Pore Cch1 Is Dependent on a Modulatory Region of the Subunit Mid1 in Cryptococcus neoformans

    doi: 10.1128/EC.00130-12

    Figure Lengend Snippet: C-terminal-truncated mutants of Mid1 are mislocalized in HEK293 cells. Confocal microscopy was used to examine the localization of three truncated mutants of Mid1 in HEK293 cells. (A) Full-length Mid1-GFP under the control of the CMV promoter was expressed in HEK293 and observed on the cell surface. (B, C, and D) However, expression of the Mid1 truncated mutants lacking specified regions of the C terminus (Mid1-CA-124, Mid1-CB-91, and Mid1-CC-24) displayed a diffuse and predominately cytosolic localization pattern unlike that of full-length Mid1. The Mid1-CA-124 mutant lacked 124 residues, Mid1-CB-91 lacked 91 residues, and Mid1-CC-24 lacked 24 residues.

    Article Snippet: Briefly, the MID1 amplicon was generated from cDNA synthesized by a SuperScript III first-strand synthesis system for reverse transcriptase PCR (RT-PCR) (Invitrogen) using total RNA as the template with the following primers: MID1-cDNA-F and MID1-cDNA-R ( ).

    Techniques: Confocal Microscopy, Expressing, Mutagenesis

    Effect of TcKnk2 dsRNA-treatment on the development of T. castaneum . (A) Injection of dsRNA for TcKnk2 into penultimate instar larvae, last instar larvae and pharate pupae (n = 60) led to lethal phenotype at pupal-adult molt (∼55%) and ∼15% adult hypomorphic phenotype with split elytra. (B) Specific down-regulation of TcKnk2 transcripts by RNAi. dsRNAs (200 ng per insect) for TcKnk2 were injected into pharate pupae. Three days after injection, total RNA was extracted from whole insects of at pupal stage day 3 (n = 3) and used for cDNA synthesis. (C) Specificity of TcKnk2 dsRNA-treatment. Effect of TcKnk2 dsRNA-treatment on transcripts for TcKnk , TcKnk3 and other genes involved in chitin metabolism such as TcChs-A and TcCht5 was checked by RT-PCR. T. castaneum ribosomal protein-S6 ( TcRPS6 ) was used as internal loading control. dsRNA for T. castaneum Vermilion ( TcVer ) and TcKnk was injected as a control.

    Journal: PLoS Genetics

    Article Title: Functional Specialization Among Members Of Knickkopf Family Of Proteins In Insect Cuticle Organization

    doi: 10.1371/journal.pgen.1004537

    Figure Lengend Snippet: Effect of TcKnk2 dsRNA-treatment on the development of T. castaneum . (A) Injection of dsRNA for TcKnk2 into penultimate instar larvae, last instar larvae and pharate pupae (n = 60) led to lethal phenotype at pupal-adult molt (∼55%) and ∼15% adult hypomorphic phenotype with split elytra. (B) Specific down-regulation of TcKnk2 transcripts by RNAi. dsRNAs (200 ng per insect) for TcKnk2 were injected into pharate pupae. Three days after injection, total RNA was extracted from whole insects of at pupal stage day 3 (n = 3) and used for cDNA synthesis. (C) Specificity of TcKnk2 dsRNA-treatment. Effect of TcKnk2 dsRNA-treatment on transcripts for TcKnk , TcKnk3 and other genes involved in chitin metabolism such as TcChs-A and TcCht5 was checked by RT-PCR. T. castaneum ribosomal protein-S6 ( TcRPS6 ) was used as internal loading control. dsRNA for T. castaneum Vermilion ( TcVer ) and TcKnk was injected as a control.

    Article Snippet: The Superscript III first–strand synthesis system for RT-PCR (Invitrogen) was used to synthesize first-strand cDNA according to the manufacturer's instructions.

    Techniques: Injection, Reverse Transcription Polymerase Chain Reaction

    Requirement for helicase-competent DDX5 and its associated lncRNA Rmrp in induction of Th17 cell cytokines a , Cytokine production in DDX5-Tko cells transduced with WT or helicase-mutant DDX5 and subjected to sub-optimal Th17 cell polarization. b , Results from four independent experiments shown (a). c , IGV browser view of Rmrp showing coverage of mapped RNA reads from total Th17 lysate, Ribosome TRAP-seq (described in Methods), DDX5 RIP-seq, and RORγt RIP-seq. d , Effect of mouse Rmrp-specific ASO. Results are representative of three independent experiments with two technical replicates. e , IL-17A production following Rmrp knockdown in in vitro polarized human Th17 cells. Each symbol (right panel) represents cells from a healthy donor (n=5). Graphs show mean ± s.d. CTL, control; ** p

    Journal: Nature

    Article Title: DDX5 and its associated lncRNA Rmrp modulate Th17 cell effector functions

    doi: 10.1038/nature16193

    Figure Lengend Snippet: Requirement for helicase-competent DDX5 and its associated lncRNA Rmrp in induction of Th17 cell cytokines a , Cytokine production in DDX5-Tko cells transduced with WT or helicase-mutant DDX5 and subjected to sub-optimal Th17 cell polarization. b , Results from four independent experiments shown (a). c , IGV browser view of Rmrp showing coverage of mapped RNA reads from total Th17 lysate, Ribosome TRAP-seq (described in Methods), DDX5 RIP-seq, and RORγt RIP-seq. d , Effect of mouse Rmrp-specific ASO. Results are representative of three independent experiments with two technical replicates. e , IL-17A production following Rmrp knockdown in in vitro polarized human Th17 cells. Each symbol (right panel) represents cells from a healthy donor (n=5). Graphs show mean ± s.d. CTL, control; ** p

    Article Snippet: Complementary DNAs (cDNAs) were synthesized from TRIzol (Invitrogen) isolated RNA, using Superscript III kits (Invitrogen).

    Techniques: Transduction, Mutagenesis, Allele-specific Oligonucleotide, In Vitro, CTL Assay

    Requirement for DDX5 in Th17 cytokine production in vitro and at steady state in vivo a , Selective Th17 cell differentiation defect in DDX5-deficient T cells after polarization for 96 h. Representative of three independent experiments. b , Volcano plot of RNA-seq of cultured Th17 cells from DDX5-Tko mice and littermate controls. Black dots: differentially expressed genes (minimum fold change of two with p-value

    Journal: Nature

    Article Title: DDX5 and its associated lncRNA Rmrp modulate Th17 cell effector functions

    doi: 10.1038/nature16193

    Figure Lengend Snippet: Requirement for DDX5 in Th17 cytokine production in vitro and at steady state in vivo a , Selective Th17 cell differentiation defect in DDX5-deficient T cells after polarization for 96 h. Representative of three independent experiments. b , Volcano plot of RNA-seq of cultured Th17 cells from DDX5-Tko mice and littermate controls. Black dots: differentially expressed genes (minimum fold change of two with p-value

    Article Snippet: Complementary DNAs (cDNAs) were synthesized from TRIzol (Invitrogen) isolated RNA, using Superscript III kits (Invitrogen).

    Techniques: In Vitro, In Vivo, Cell Differentiation, RNA Sequencing Assay, Cell Culture, Mouse Assay

    Expression variations between SG and SYN obtained using NormFinder software over the blood-feeding duration. The data are the average of three biological replications. The circles are for intergroup variation and error bars are for intragroup variation

    Journal: Journal of medical entomology

    Article Title: Validation of Internal Reference Genes for Real-Time Quantitative Polymerase Chain Reaction Studies in the Tick, Ixodes scapularis (Acari: Ixodidae)

    doi:

    Figure Lengend Snippet: Expression variations between SG and SYN obtained using NormFinder software over the blood-feeding duration. The data are the average of three biological replications. The circles are for intergroup variation and error bars are for intragroup variation

    Article Snippet: Total RNA, 340 ng from SG and 40–60 ng from SYN, was used to synthesize first-strand cDNA using the SuperScript III system (Invitrogen, Life sciences, Grand Island, NY)and an oligo-dT-based reverse transcriptase protocol.

    Techniques: Expressing, Software

    Detection of processed transcripts of E. coli CRISPR I cassette A. The E. coli CRISPR I locus is schematically shown. Genes are indicated by arrows and identified. In CRISPR I cassette, repeats are indicated by black rhombuses, spacers – by white rectangles. Numbers below the scheme indicate spacer numbers. The last repeat of the cassette is colored grey, to indicate that its sequence differs from other repeats. The leader sequence is located between the CRISPR I cassette and the cas2 gene and is indicated by a thicker black line. B. A Northern blot showing changes in abundance of spacer 4 transcript in E. coli strains with indicated cas genes disruptions. The probe was designed to reveal RNA produced by rightward (see Fig. 1A) transcription. C. Northern blots with total RNA purified from E. coli cells with disrupted casA gene were performed with probes specific for spacers indicated (direction of transcription the same as in Fig. 1B). In each panel, the first three lanes contained increasing amounts of DNA oligonucleotides complementary to probes used for blotting. Approximate calculated numbers of processed CRISPR transcript per cell are shown below.

    Journal: Molecular microbiology

    Article Title: Transcription, Processing, and Function of CRISPR Cassettes in Escherichia coli

    doi: 10.1111/j.1365-2958.2010.07265.x

    Figure Lengend Snippet: Detection of processed transcripts of E. coli CRISPR I cassette A. The E. coli CRISPR I locus is schematically shown. Genes are indicated by arrows and identified. In CRISPR I cassette, repeats are indicated by black rhombuses, spacers – by white rectangles. Numbers below the scheme indicate spacer numbers. The last repeat of the cassette is colored grey, to indicate that its sequence differs from other repeats. The leader sequence is located between the CRISPR I cassette and the cas2 gene and is indicated by a thicker black line. B. A Northern blot showing changes in abundance of spacer 4 transcript in E. coli strains with indicated cas genes disruptions. The probe was designed to reveal RNA produced by rightward (see Fig. 1A) transcription. C. Northern blots with total RNA purified from E. coli cells with disrupted casA gene were performed with probes specific for spacers indicated (direction of transcription the same as in Fig. 1B). In each panel, the first three lanes contained increasing amounts of DNA oligonucleotides complementary to probes used for blotting. Approximate calculated numbers of processed CRISPR transcript per cell are shown below.

    Article Snippet: For a single extension reaction up to 0.5 µg of in vitro transcribed RNA was reverse-transcribed with 100 U of SuperScript III enzyme of First-Strand Synthesis Kit for RT-PCR (Invitrogen) according to the manufacturer’s protocol in the presence of 1 pmoles of [32 P] 5’-end-labelled primers.

    Techniques: CRISPR, Sequencing, Northern Blot, Produced, Purification

    HbCIPK2 interacted with HbFd1. (A) Examination of the interaction between HbCIPK2 and HbFd1 in yeast. The interactions were verified by the yeast growing on selective medium (SC/-Leu-Trp-His-Ura with 35 mM 3-AT) and conducting ß-Gal assays. Bait vector with HbCIPK2 or prey vector with HbFd1 was transformed with empty vector as negative control, and the plasmids pPC97-Fos and pPC86-Jun provided by Invitrogen system were used as positive control. (B) Co-BiFC assay to verify the interaction of HbCIPK2 and HbFd1 in Arabidopsis protoplasts. Construct AtCBF1::RFP was used as reference to co-localize the interaction of HbFd1 with HbCIPK2, and transformants expressing SPYNE/HbFd1::SPYCE and SPYNE::HbCIPK2/SPYCE were used as negative controls. (C) Co-IP assay to show the interaction between HbCIPK2 and mature HbFd1 in cell line HEK293, co-expressing HbCIPK2-Myc and HbFd1△cTP-Flag. Cell proteins before (Input) and after (IP) immunoprecipitation were separated in SDS-PAGE gels, transferred onto the nitrocellulose membranes, and analyzed by protein gel blotting with antibodies as indicated. All assays repeated three times.

    Journal: PLoS ONE

    Article Title: Identification and Expression Analysis of a Novel HbCIPK2-Interacting Ferredoxin from Halophyte H. brevisubulatum

    doi: 10.1371/journal.pone.0144132

    Figure Lengend Snippet: HbCIPK2 interacted with HbFd1. (A) Examination of the interaction between HbCIPK2 and HbFd1 in yeast. The interactions were verified by the yeast growing on selective medium (SC/-Leu-Trp-His-Ura with 35 mM 3-AT) and conducting ß-Gal assays. Bait vector with HbCIPK2 or prey vector with HbFd1 was transformed with empty vector as negative control, and the plasmids pPC97-Fos and pPC86-Jun provided by Invitrogen system were used as positive control. (B) Co-BiFC assay to verify the interaction of HbCIPK2 and HbFd1 in Arabidopsis protoplasts. Construct AtCBF1::RFP was used as reference to co-localize the interaction of HbFd1 with HbCIPK2, and transformants expressing SPYNE/HbFd1::SPYCE and SPYNE::HbCIPK2/SPYCE were used as negative controls. (C) Co-IP assay to show the interaction between HbCIPK2 and mature HbFd1 in cell line HEK293, co-expressing HbCIPK2-Myc and HbFd1△cTP-Flag. Cell proteins before (Input) and after (IP) immunoprecipitation were separated in SDS-PAGE gels, transferred onto the nitrocellulose membranes, and analyzed by protein gel blotting with antibodies as indicated. All assays repeated three times.

    Article Snippet: Total RNA from H . brevisubulatum seedling treated with various stresses (350 mM NaCl, 350 mM mannitol and 10% PEG6000 stressed for 6 hrs, and 4°C for 12 hrs, respectively) upon the emergence of the third shoot was extracted using RNeasy Plant Mini kit (QIAGEN, Stockach, Germany) and first strand cDNA was synthesized using the SuperScript III First-Strand (Invitrogen).

    Techniques: Plasmid Preparation, Transformation Assay, Negative Control, Positive Control, Bimolecular Fluorescence Complementation Assay, Construct, Expressing, Co-Immunoprecipitation Assay, Immunoprecipitation, SDS Page

    Ag-specific CD4 + T cells are not recruited to the infected kidney. Leukocytes from naive and infected kidneys ( A ) or small intestine ( B ) of mice, treated as in Fig. 1 , were analyzed by flow cytometry for the presence of OT.II (Vα2 + CD45.1 + ) cells. Example plots are gated on CD4 + lymphocytes and are representative of ≥12 animals from four experiments. Graph in (A) shows the frequency of OT.II cells in the kidney in naive ( n = 4) and infected ( n = 15) mice at day 3 postinfection. ( C ) Frequency of OT.I (Vα2 + CD45.1 + ) cells in naive ( n = 2) and infected ( n = 10) kidneys at day 3 postinfection. Example plot is representative of 10 animals from two experiments and is gated on CD8 + lymphocytes. ( D ) pAls3-Tet staining of the draining LNs from an animal immunized with 20 μg recombinant Als3 in CFA 7 d postimmunization. ( E ) pAls3-Tet also was used to stain splenocytes ( upper panels ) and kidney-isolated leukocytes ( lower panels ) from infected mice at 7 d postinfection. Plots are representative of at least three animals from two independent experiments and are gated on B220 − CD11b − CD11c − CD3 + CD4 + CD8 − T cells. Data are pooled from two to four independent experiments.

    Journal: The Journal of Immunology Author Choice

    Article Title: Cutting Edge: Failure of Antigen-Specific CD4+ T Cell Recruitment to the Kidney during Systemic Candidiasis

    doi: 10.4049/jimmunol.1401675

    Figure Lengend Snippet: Ag-specific CD4 + T cells are not recruited to the infected kidney. Leukocytes from naive and infected kidneys ( A ) or small intestine ( B ) of mice, treated as in Fig. 1 , were analyzed by flow cytometry for the presence of OT.II (Vα2 + CD45.1 + ) cells. Example plots are gated on CD4 + lymphocytes and are representative of ≥12 animals from four experiments. Graph in (A) shows the frequency of OT.II cells in the kidney in naive ( n = 4) and infected ( n = 15) mice at day 3 postinfection. ( C ) Frequency of OT.I (Vα2 + CD45.1 + ) cells in naive ( n = 2) and infected ( n = 10) kidneys at day 3 postinfection. Example plot is representative of 10 animals from two experiments and is gated on CD8 + lymphocytes. ( D ) pAls3-Tet staining of the draining LNs from an animal immunized with 20 μg recombinant Als3 in CFA 7 d postimmunization. ( E ) pAls3-Tet also was used to stain splenocytes ( upper panels ) and kidney-isolated leukocytes ( lower panels ) from infected mice at 7 d postinfection. Plots are representative of at least three animals from two independent experiments and are gated on B220 − CD11b − CD11c − CD3 + CD4 + CD8 − T cells. Data are pooled from two to four independent experiments.

    Article Snippet: Multiplex PCR RNA was isolated from OT.II cells positively purified from the renal LNs using anti-CD45.1–biotin Ab and anti-biotin MicroBeads (Miltenyi Biotec), converted to cDNA using the SuperScript III synthesis system (Invitrogen), and used as a template in multiplex PCR reactions (Maxim Biotech), as per the manufacturers’ instructions.

    Techniques: Infection, Mouse Assay, Flow Cytometry, Cytometry, Staining, Recombinant, Isolation

    Lifespan extension in sft-1(RNAi) and oxa-1(RNAi) animals. A : Lifespan curves for N2, oxa-1(RNAi) and sft-1(RNAi) animals (recorded as number of days of survival post L4, or the time at which controls reached L4, in the case of oxa-1(RNAi) animals). Lifespan is prolonged in sft-1(RNAi) and oxa-1(RNAi) animals compared with WT controls, with oxa-1 having the greatest effect. B : Lifespan analysis of sft-1(RNAi) animals performed in a daf-16(m26) mutant background, in which sft-1 RNAi fails to extend lifespan. C : Lifespan analysis of oxa-1(RNAi) animals performed in a daf-16(m26) mutant background. oxa-1 RNAi still extends lifespan under these circumstances. The data presented combine three independent data sets (the data for individual replicate experiments can be found in Additional file 1 ).

    Journal: Longevity & Healthspan

    Article Title: The SFT-1 and OXA-1 respiratory chain complex assembly factors influence lifespan by distinct mechanisms in C. elegans

    doi: 10.1186/2046-2395-2-9

    Figure Lengend Snippet: Lifespan extension in sft-1(RNAi) and oxa-1(RNAi) animals. A : Lifespan curves for N2, oxa-1(RNAi) and sft-1(RNAi) animals (recorded as number of days of survival post L4, or the time at which controls reached L4, in the case of oxa-1(RNAi) animals). Lifespan is prolonged in sft-1(RNAi) and oxa-1(RNAi) animals compared with WT controls, with oxa-1 having the greatest effect. B : Lifespan analysis of sft-1(RNAi) animals performed in a daf-16(m26) mutant background, in which sft-1 RNAi fails to extend lifespan. C : Lifespan analysis of oxa-1(RNAi) animals performed in a daf-16(m26) mutant background. oxa-1 RNAi still extends lifespan under these circumstances. The data presented combine three independent data sets (the data for individual replicate experiments can be found in Additional file 1 ).

    Article Snippet: RT-PCR Reduction of the target transcripts following RNAi treatment was confirmed by gene-specific RT-PCR using the Superscript III RT system (Life Technologies (Invitrogen Division).

    Techniques: Mutagenesis