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    Custom RNA, Stealth, Desalted
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    Use this tool for fast and easy online ordering of bench-tested Validated Stealth RNAi siRNA, ready-to-order Stealth Select RNAi siRNA, bench-tested BLOCK-iT Pol II miR Validated miRNA Vector Duopaks, and ready-to-clone BLOCK-iT miR RNAi Select.
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    Structured Review

    Thermo Fisher rna
    eNOS expression in thoracic aorta from SAMR and SAMP mice. ( A ) top: representative immunofluorescent merged images. Staining shows nucleus (blue, DAPI), actin fibers (red, phalloidin), eNOS (green). Bar graphs (bottom) show the results of densitometric analyses from pooled data. ( B ) Immunoblots analysis (top) in single aortas probed with antibodies against eNOS or GAPDH, as indicated. The lower graph shows the results of densitometric analyses from pooled data, plotted as optical densitometry relative to the signal obtained in by GAPDH. ( C ) eNOS mRNA expression in mice aorta normalized to the expression of ribosomal <t>RNA</t> subunit <t>18S,</t> which was used as an endogenous reference gene. OVX, ovariectomized; OVX+E2, ovariectomized treated with estrogen. Each data represents the mean ± SEM derived from 6–8 independent experiments. * p
    Use this tool for fast and easy online ordering of bench-tested Validated Stealth RNAi siRNA, ready-to-order Stealth Select RNAi siRNA, bench-tested BLOCK-iT Pol II miR Validated miRNA Vector Duopaks, and ready-to-clone BLOCK-iT miR RNAi Select.
    https://www.bioz.com/result/rna/product/Thermo Fisher
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    Images

    1) Product Images from "Aging Negatively Affects Estrogens-Mediated Effects on Nitric Oxide Bioavailability by Shifting ER?/ER? Balance in Female Mice"

    Article Title: Aging Negatively Affects Estrogens-Mediated Effects on Nitric Oxide Bioavailability by Shifting ER?/ER? Balance in Female Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025335

    eNOS expression in thoracic aorta from SAMR and SAMP mice. ( A ) top: representative immunofluorescent merged images. Staining shows nucleus (blue, DAPI), actin fibers (red, phalloidin), eNOS (green). Bar graphs (bottom) show the results of densitometric analyses from pooled data. ( B ) Immunoblots analysis (top) in single aortas probed with antibodies against eNOS or GAPDH, as indicated. The lower graph shows the results of densitometric analyses from pooled data, plotted as optical densitometry relative to the signal obtained in by GAPDH. ( C ) eNOS mRNA expression in mice aorta normalized to the expression of ribosomal RNA subunit 18S, which was used as an endogenous reference gene. OVX, ovariectomized; OVX+E2, ovariectomized treated with estrogen. Each data represents the mean ± SEM derived from 6–8 independent experiments. * p
    Figure Legend Snippet: eNOS expression in thoracic aorta from SAMR and SAMP mice. ( A ) top: representative immunofluorescent merged images. Staining shows nucleus (blue, DAPI), actin fibers (red, phalloidin), eNOS (green). Bar graphs (bottom) show the results of densitometric analyses from pooled data. ( B ) Immunoblots analysis (top) in single aortas probed with antibodies against eNOS or GAPDH, as indicated. The lower graph shows the results of densitometric analyses from pooled data, plotted as optical densitometry relative to the signal obtained in by GAPDH. ( C ) eNOS mRNA expression in mice aorta normalized to the expression of ribosomal RNA subunit 18S, which was used as an endogenous reference gene. OVX, ovariectomized; OVX+E2, ovariectomized treated with estrogen. Each data represents the mean ± SEM derived from 6–8 independent experiments. * p

    Techniques Used: Expressing, Mouse Assay, Staining, Western Blot, Derivative Assay

    NAD(P)H-dependent O 2 - production in aorta from young (SAMR) and aged (SAMP) females. (A) On top: Representative chemiluminescence tracing of NADPH-dependent O 2 − production in SAMR and SAMP mice. On the bottom bar graphs represent mean ± SEM of the areas under the curve obtained from 6–8 individual experiments. (B) mRNA expression of the NAD(P)H oxidase subunits in mice aorta normalized to the expression of ribosomal RNA subunit 18S, used as an endogenous control. OVX, ovariectomized; OVX+E2, ovariectomized treated with estrogen. Each data represents the mean ± SEM derived from 6–8 independent experiments. * p
    Figure Legend Snippet: NAD(P)H-dependent O 2 - production in aorta from young (SAMR) and aged (SAMP) females. (A) On top: Representative chemiluminescence tracing of NADPH-dependent O 2 − production in SAMR and SAMP mice. On the bottom bar graphs represent mean ± SEM of the areas under the curve obtained from 6–8 individual experiments. (B) mRNA expression of the NAD(P)H oxidase subunits in mice aorta normalized to the expression of ribosomal RNA subunit 18S, used as an endogenous control. OVX, ovariectomized; OVX+E2, ovariectomized treated with estrogen. Each data represents the mean ± SEM derived from 6–8 independent experiments. * p

    Techniques Used: Mouse Assay, Expressing, Derivative Assay

    2) Product Images from "The Characterization of GSDMB Splicing and Backsplicing Profiles Identifies Novel Isoforms and a Circular RNA That Are Dysregulated in Multiple Sclerosis"

    Article Title: The Characterization of GSDMB Splicing and Backsplicing Profiles Identifies Novel Isoforms and a Circular RNA That Are Dysregulated in Multiple Sclerosis

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms18030576

    GSDMB alternative splicing (AS) pattern. ( a ) Schematic representation of the GSDMB gene. Exons are represented by boxes and are approximately drawn to scale; untranslated regions (UTRs) are depicted by smaller boxes; introns are represented by lines. Constitutive exons are colored in light grey and alternative exons in dark grey. The three reverse-transcription (RT)-PCR assays (A, B, and C) performed to cover the GSDMB coding region are indicated; ( b ) An illustrative agarose gel (2%) shows the amplification products of the three RT-PCR assays. For “A” and “C” assays, the unique amplification product and their length, expressed as base pairs (bp), are schematized on the left. For the “B” assay, multiple specific amplification products were obtained, corresponding to several AS events involving exons 4 to 9, which were further analyzed by both fluorescent RT-PCR and direct sequencing (see further). MW: molecular-weight marker (pUC9- Hae III); ( c ) The left panel represents a demonstrative GeneMapper window showing the fluorescent RT-PCR products of the “B” assay (on RNA extracted from a healthy individual, heterozygous for the rs11078928 polymorphism), using the forward primer labelled with the HEX fluorophore. The filled peaks, shaded in grey, correspond to the RT-PCR products; empty peaks represent the size standard (ROX-500 HD). The vertical axis indicates the length of amplified products in bp, whereas the horizontal axis shows fluorescence units. The schematic representation and the name of the corresponding isoform product are indicated on the right side. The alternative exon 6* is depicted in black. F”: full length, “*” indicates the presence of exon 6* in the isoform, “∆” is followed by the skipped exon, separated by “-” or “,” if the exons are contiguous or not, respectively. The accession number of University of California, Santa Cruz (UCSC) Genome Browser-annotated isoforms is also indicated. The presence/absence in the transcript of a premature termination codon (PTC) is indicated by a “+”/“−”, respectively.
    Figure Legend Snippet: GSDMB alternative splicing (AS) pattern. ( a ) Schematic representation of the GSDMB gene. Exons are represented by boxes and are approximately drawn to scale; untranslated regions (UTRs) are depicted by smaller boxes; introns are represented by lines. Constitutive exons are colored in light grey and alternative exons in dark grey. The three reverse-transcription (RT)-PCR assays (A, B, and C) performed to cover the GSDMB coding region are indicated; ( b ) An illustrative agarose gel (2%) shows the amplification products of the three RT-PCR assays. For “A” and “C” assays, the unique amplification product and their length, expressed as base pairs (bp), are schematized on the left. For the “B” assay, multiple specific amplification products were obtained, corresponding to several AS events involving exons 4 to 9, which were further analyzed by both fluorescent RT-PCR and direct sequencing (see further). MW: molecular-weight marker (pUC9- Hae III); ( c ) The left panel represents a demonstrative GeneMapper window showing the fluorescent RT-PCR products of the “B” assay (on RNA extracted from a healthy individual, heterozygous for the rs11078928 polymorphism), using the forward primer labelled with the HEX fluorophore. The filled peaks, shaded in grey, correspond to the RT-PCR products; empty peaks represent the size standard (ROX-500 HD). The vertical axis indicates the length of amplified products in bp, whereas the horizontal axis shows fluorescence units. The schematic representation and the name of the corresponding isoform product are indicated on the right side. The alternative exon 6* is depicted in black. F”: full length, “*” indicates the presence of exon 6* in the isoform, “∆” is followed by the skipped exon, separated by “-” or “,” if the exons are contiguous or not, respectively. The accession number of University of California, Santa Cruz (UCSC) Genome Browser-annotated isoforms is also indicated. The presence/absence in the transcript of a premature termination codon (PTC) is indicated by a “+”/“−”, respectively.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, Sequencing, Molecular Weight, Marker, Fluorescence

    Analysis of exon 6/6* AS in multiple sclerosis (MS) cases and healthy controls. ( a ) Schematic representation of the GSDMB genomic region comprised between exons 4 and 8. Exons are approximately drawn to scale; alternative exons are depicted in dark grey, as in Figure 1 . The dashed arrows below the scheme indicate the primer couple used in the fluorescent-competitive RT-PCR assay; the forward primer is labelled with the HEX fluorophore; ( b ) The left panel shows three GeneMapper windows, representing an example of the fluorescent products obtained for each of the rs11078928 genotypes. The peaks shaded in grey correspond to the RT-PCR products; those not shaded represent the size standard (ROX-500 HD). The schematic representation of the obtained products for each genotype is shown on the right. Exon 6* is depicted in black; ( c ) Boxplots showing the percentage of the Δ6 isoform with respect to the sum of Δ6, F*, and F isoforms measured on RNA extracted from 30 relapsing remitting (RR)-MS cases and 30 controls, grouped on the basis of the rs11078928 genotype. Boxes define the interquartile range; the thick line refers to the median. The number of subjects belonging to each group is also indicated (N). Significance levels of t -tests is shown above the boxplots (** p
    Figure Legend Snippet: Analysis of exon 6/6* AS in multiple sclerosis (MS) cases and healthy controls. ( a ) Schematic representation of the GSDMB genomic region comprised between exons 4 and 8. Exons are approximately drawn to scale; alternative exons are depicted in dark grey, as in Figure 1 . The dashed arrows below the scheme indicate the primer couple used in the fluorescent-competitive RT-PCR assay; the forward primer is labelled with the HEX fluorophore; ( b ) The left panel shows three GeneMapper windows, representing an example of the fluorescent products obtained for each of the rs11078928 genotypes. The peaks shaded in grey correspond to the RT-PCR products; those not shaded represent the size standard (ROX-500 HD). The schematic representation of the obtained products for each genotype is shown on the right. Exon 6* is depicted in black; ( c ) Boxplots showing the percentage of the Δ6 isoform with respect to the sum of Δ6, F*, and F isoforms measured on RNA extracted from 30 relapsing remitting (RR)-MS cases and 30 controls, grouped on the basis of the rs11078928 genotype. Boxes define the interquartile range; the thick line refers to the median. The number of subjects belonging to each group is also indicated (N). Significance levels of t -tests is shown above the boxplots (** p

    Techniques Used: Mass Spectrometry, Reverse Transcription Polymerase Chain Reaction

    Characterization of the GSDMB exonic circular RNA (ecircRNA). ( a ) Schematic representation of the formation of the GSDMB ecircRNA through a backsplicing event between exons 4 and 5. Exons are approximately drawn to scale; exon 4 is depicted in grey, exon 5 in black. The curved arrow joins the 5′ splice site of exon 5 to 3′ splice site of exon 4. Arrows below exon 5 indicate the divergent primer couple used to detect the putative circular RNA (circRNA). On the right, a schematic representation of the circRNA is depicted, with the circular black line representing the product amplified by the primer couple. Below the scheme, direct-sequencing electropherograms show the head-to-tail splice junction, indicated by an arrow, located between GSDMB exons 5 and 4; ( b ) Schematic representation of the linear splicing involving exons 4–5. Arrows below exons 4–5 indicate the primer couple used in RT-PCR assays. Electropherograms showing the junction between exons 4 and 5 is also reported; ( c ) Agarose gel (2%) with the results of the RNase R treatment. The expression of the GSDMB ecircRNA was evaluated by RT-PCR in untreated (−) or RNase R-treated (+) RNA of a healthy control, using the divergent primer couple within exon 5. Expression of total GSDMB linear mRNA was also evaluated, using the RT-PCR assay shown in Figure 2 (linear product; exons 9–11), the assay reported in ( b ) (linear product; exons 4 and 5), as well as an assay using a divergent primer couple on exon 10 (negative control; no circRNA detected). MW: molecular-weight marker (pUC9- Hae III); ( d ) Boxplots show expression levels of the GSDMB ecircRNA measured by semi-quantitative real-time RT-PCR in PBMCs of 30 MS cases and 30 healthy controls. Boxes define the interquartile range; the thick line refers to the median. Results were normalized to expression levels of the HMBS housekeeping gene. The number of subjects belonging to each group is also indicated (N). The significance level of t -test analysis is shown. ** p
    Figure Legend Snippet: Characterization of the GSDMB exonic circular RNA (ecircRNA). ( a ) Schematic representation of the formation of the GSDMB ecircRNA through a backsplicing event between exons 4 and 5. Exons are approximately drawn to scale; exon 4 is depicted in grey, exon 5 in black. The curved arrow joins the 5′ splice site of exon 5 to 3′ splice site of exon 4. Arrows below exon 5 indicate the divergent primer couple used to detect the putative circular RNA (circRNA). On the right, a schematic representation of the circRNA is depicted, with the circular black line representing the product amplified by the primer couple. Below the scheme, direct-sequencing electropherograms show the head-to-tail splice junction, indicated by an arrow, located between GSDMB exons 5 and 4; ( b ) Schematic representation of the linear splicing involving exons 4–5. Arrows below exons 4–5 indicate the primer couple used in RT-PCR assays. Electropherograms showing the junction between exons 4 and 5 is also reported; ( c ) Agarose gel (2%) with the results of the RNase R treatment. The expression of the GSDMB ecircRNA was evaluated by RT-PCR in untreated (−) or RNase R-treated (+) RNA of a healthy control, using the divergent primer couple within exon 5. Expression of total GSDMB linear mRNA was also evaluated, using the RT-PCR assay shown in Figure 2 (linear product; exons 9–11), the assay reported in ( b ) (linear product; exons 4 and 5), as well as an assay using a divergent primer couple on exon 10 (negative control; no circRNA detected). MW: molecular-weight marker (pUC9- Hae III); ( d ) Boxplots show expression levels of the GSDMB ecircRNA measured by semi-quantitative real-time RT-PCR in PBMCs of 30 MS cases and 30 healthy controls. Boxes define the interquartile range; the thick line refers to the median. Results were normalized to expression levels of the HMBS housekeeping gene. The number of subjects belonging to each group is also indicated (N). The significance level of t -test analysis is shown. ** p

    Techniques Used: Amplification, Sequencing, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Expressing, Negative Control, Molecular Weight, Marker, Quantitative RT-PCR, Mass Spectrometry

    3) Product Images from "DEB025 (Alisporivir) Inhibits Hepatitis C Virus Replication by Preventing a Cyclophilin A Induced Cis-Trans Isomerisation in Domain II of NS5A"

    Article Title: DEB025 (Alisporivir) Inhibits Hepatitis C Virus Replication by Preventing a Cyclophilin A Induced Cis-Trans Isomerisation in Domain II of NS5A

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0013687

    Identification and characterization of mutations conferring resistance to DEB025. (A) Schematic representation of the selectable Con1 (GT1b) subgenomic replicon (Huh 9–13) that was used to select for DEB025 (and CsA) resistance. RNA isolated from drug resistant and control cell cultures (passaged in parallel), was sequenced. The mutations identified in the resistant cultures are depicted above their respective position in the polyprotein. (B) Prevalence of the variants, found in the replicon after DEB025 selection, in different genotypes in the European HCV database. 1 Variant identified in the DEB025 res replicon. 2 WT amino acid for that genotype (the amino acid with the highest prevalence). 3 del = deletion, [deletion, all of the genotype 2b and most of the genotype 2a and 4 sequences miss amino acid 262 (numbering according to genotype 1b) in NS5A]. (C) Mutations, that had been identified in the DEB025 res or CsA res replicon, were introduced (either alone or combined) in a WT background, after which the sensitivity to DEB025 and CsA was assessed. Data are expressed as fold reduction (based on EC 50 -values) in sensitivity to the drug as compared to WT replicon. (D) Dose-response curves for inhibition of replicon replication by DEB025 or (E) CsA in Huh7-Lunet cells transiently transfected with mutant replicon RNA (indicated on the right site of each panel). HCV replicon RNA was quantified by means of a luciferase assay and data are expressed as percentage of untreated controls. Data are mean values ± standard deviations for at least three independent experiments. (F) Replication fitness of the various mutant replicons. Huh7-Lunet cells were transiently transfected with the indicated mutant replicons and RNA replication was measured by means of a luciferase assay for 4 days post transfection. Data are normalized to the 4 h-value post electroporation to account for differences in electroporation efficiency and expressed as a percentage of the WT-value at 96 h post electroporation. Data are mean values ± standard deviations for at least two independent experiments.
    Figure Legend Snippet: Identification and characterization of mutations conferring resistance to DEB025. (A) Schematic representation of the selectable Con1 (GT1b) subgenomic replicon (Huh 9–13) that was used to select for DEB025 (and CsA) resistance. RNA isolated from drug resistant and control cell cultures (passaged in parallel), was sequenced. The mutations identified in the resistant cultures are depicted above their respective position in the polyprotein. (B) Prevalence of the variants, found in the replicon after DEB025 selection, in different genotypes in the European HCV database. 1 Variant identified in the DEB025 res replicon. 2 WT amino acid for that genotype (the amino acid with the highest prevalence). 3 del = deletion, [deletion, all of the genotype 2b and most of the genotype 2a and 4 sequences miss amino acid 262 (numbering according to genotype 1b) in NS5A]. (C) Mutations, that had been identified in the DEB025 res or CsA res replicon, were introduced (either alone or combined) in a WT background, after which the sensitivity to DEB025 and CsA was assessed. Data are expressed as fold reduction (based on EC 50 -values) in sensitivity to the drug as compared to WT replicon. (D) Dose-response curves for inhibition of replicon replication by DEB025 or (E) CsA in Huh7-Lunet cells transiently transfected with mutant replicon RNA (indicated on the right site of each panel). HCV replicon RNA was quantified by means of a luciferase assay and data are expressed as percentage of untreated controls. Data are mean values ± standard deviations for at least three independent experiments. (F) Replication fitness of the various mutant replicons. Huh7-Lunet cells were transiently transfected with the indicated mutant replicons and RNA replication was measured by means of a luciferase assay for 4 days post transfection. Data are normalized to the 4 h-value post electroporation to account for differences in electroporation efficiency and expressed as a percentage of the WT-value at 96 h post electroporation. Data are mean values ± standard deviations for at least two independent experiments.

    Techniques Used: Isolation, Selection, Variant Assay, Inhibition, Transfection, Mutagenesis, Luciferase, Electroporation

    Mutations in NS5A but not NS5B contribute to resistance against DEB025. Dose-response curves for inhibition of replicon replication by (A) DEB025 or (B) CsA in Huh7-Lunet cells transiently transfected with recombinant replicon RNA carrying either the NS5A or NS5B region from DEB025 res or control (WT) cultures. For comparative reasons also the original unmanipulated vector was included in the analysis. Cells were treated for 72 h with escalating concentrations of DEB025 or CsA and HCV replicon RNA was quantified by means of a luciferase assay and is expressed as percentage of the untreated controls. Data are mean values ± standard deviations for at least three independent experiments. (C) Replication fitness of the different recombinant replicons. Huh7-Lunet cells were transiently transfected with each of the indicated replicon constructs and replicon RNA replication was quantified by means of a luciferase assay for 4 days post transfection. Data are normalized to the 4 h-value post electroporation to account for differences in electroporation efficiency and is expressed as percentage of the WT-value at 96 h post electroporation. Data are mean values ± standard deviations for at least two independent experiments.
    Figure Legend Snippet: Mutations in NS5A but not NS5B contribute to resistance against DEB025. Dose-response curves for inhibition of replicon replication by (A) DEB025 or (B) CsA in Huh7-Lunet cells transiently transfected with recombinant replicon RNA carrying either the NS5A or NS5B region from DEB025 res or control (WT) cultures. For comparative reasons also the original unmanipulated vector was included in the analysis. Cells were treated for 72 h with escalating concentrations of DEB025 or CsA and HCV replicon RNA was quantified by means of a luciferase assay and is expressed as percentage of the untreated controls. Data are mean values ± standard deviations for at least three independent experiments. (C) Replication fitness of the different recombinant replicons. Huh7-Lunet cells were transiently transfected with each of the indicated replicon constructs and replicon RNA replication was quantified by means of a luciferase assay for 4 days post transfection. Data are normalized to the 4 h-value post electroporation to account for differences in electroporation efficiency and is expressed as percentage of the WT-value at 96 h post electroporation. Data are mean values ± standard deviations for at least two independent experiments.

    Techniques Used: Inhibition, Transfection, Recombinant, Plasmid Preparation, Luciferase, Construct, Electroporation

    4) Product Images from "Use of Recombinant Tobacco Mosaic Virus To Achieve RNA Interference in Plants against the Citrus Mealybug, Planococcus citri (Hemiptera: Pseudococcidae)"

    Article Title: Use of Recombinant Tobacco Mosaic Virus To Achieve RNA Interference in Plants against the Citrus Mealybug, Planococcus citri (Hemiptera: Pseudococcidae)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0073657

    Relative mRNA levels of CHS 1 (A) and V-ATPase (B) following microinjection of specific dsRNAs into third instar P. citri . qRT-PCR results of CHS 1 (A) and V-ATPase (B) mRNAs from P. citri following microinjection of elution buffer (EB), green fluorescent protein (GFP), CHS 1 or V-ATPase dsRNAs. Total RNA was extracted 4 days post injection and used for cDNA synthesis. qRT-PCR was performed using primers described in Table 3 . The P. citri 18S ribosomal RNA was used as an endogenous control in all experiments. Calculation of mRNA levels between control and treated groups was done by the comparative CT or 2 –ΔΔ Ct method and analyzed by ANOVA using statistix 8.1 software. Numbers followed by the same letter are not different at p
    Figure Legend Snippet: Relative mRNA levels of CHS 1 (A) and V-ATPase (B) following microinjection of specific dsRNAs into third instar P. citri . qRT-PCR results of CHS 1 (A) and V-ATPase (B) mRNAs from P. citri following microinjection of elution buffer (EB), green fluorescent protein (GFP), CHS 1 or V-ATPase dsRNAs. Total RNA was extracted 4 days post injection and used for cDNA synthesis. qRT-PCR was performed using primers described in Table 3 . The P. citri 18S ribosomal RNA was used as an endogenous control in all experiments. Calculation of mRNA levels between control and treated groups was done by the comparative CT or 2 –ΔΔ Ct method and analyzed by ANOVA using statistix 8.1 software. Numbers followed by the same letter are not different at p

    Techniques Used: Quantitative RT-PCR, Injection, Software

    Relative levels of CHS 1 (A) and V-ATPase (B) mRNAs in P. citri after feeding on N. benthamiana plants inoculated with recombinant TMV expressing P. citri CHS 1 and V-ATPase fragments. qRT-PCR results of CHS 1 (A) and V-ATPase (B) mRNA levels from P. citri after feeding on N. benthamiana plants infected with recombinant TMVs. TMV-GFP (pJL24) was used as a control and sense and antisense inserts of P. citri CHS 1 /V-ATPase sequences were compared. Total RNA was extracted after 12 days feeding on plants and used for cDNA synthesis. qRT-PCR was performed using primers described in Table 3 . The 18S ribosomal RNA was used as an endogenous control in all experiments. Calculation of mRNA levels between control and treated groups was carried out by the comparative CT or 2 –ΔΔ Ct method and analyzed by ANOVA using statistix 8.1 software. Numbers with the same letter indicate homogenous groups at p
    Figure Legend Snippet: Relative levels of CHS 1 (A) and V-ATPase (B) mRNAs in P. citri after feeding on N. benthamiana plants inoculated with recombinant TMV expressing P. citri CHS 1 and V-ATPase fragments. qRT-PCR results of CHS 1 (A) and V-ATPase (B) mRNA levels from P. citri after feeding on N. benthamiana plants infected with recombinant TMVs. TMV-GFP (pJL24) was used as a control and sense and antisense inserts of P. citri CHS 1 /V-ATPase sequences were compared. Total RNA was extracted after 12 days feeding on plants and used for cDNA synthesis. qRT-PCR was performed using primers described in Table 3 . The 18S ribosomal RNA was used as an endogenous control in all experiments. Calculation of mRNA levels between control and treated groups was carried out by the comparative CT or 2 –ΔΔ Ct method and analyzed by ANOVA using statistix 8.1 software. Numbers with the same letter indicate homogenous groups at p

    Techniques Used: Recombinant, Expressing, Quantitative RT-PCR, Infection, Software

    5) Product Images from "Hematopoietic Lineage Transcriptome Stability and Representation in PAXgene™ Collected Peripheral Blood Utilising SPIA Single-Stranded cDNA Probes for Microarray"

    Article Title: Hematopoietic Lineage Transcriptome Stability and Representation in PAXgene™ Collected Peripheral Blood Utilising SPIA Single-Stranded cDNA Probes for Microarray

    Journal: Biomarker Insights

    doi:

    Bioanalyser electropherograms of total RNA isolated from PAXgene ™ collected whole blood and subsequently synthesised sscDNA probes (a) Bioanalyzer electropherograms of a representative RNA from each experimental group are shown. (b) sscDNA quality assessed by distribution of size. The majority of sscDNA synthesised is in the order of approximately 1000 nucleotides. Bioanalyzer electropherograms of a representative sscDNA from each experimental group are shown. x-axis represents nucleotide length and y-axis is arbitrary signal intensity fluorescence units.
    Figure Legend Snippet: Bioanalyser electropherograms of total RNA isolated from PAXgene ™ collected whole blood and subsequently synthesised sscDNA probes (a) Bioanalyzer electropherograms of a representative RNA from each experimental group are shown. (b) sscDNA quality assessed by distribution of size. The majority of sscDNA synthesised is in the order of approximately 1000 nucleotides. Bioanalyzer electropherograms of a representative sscDNA from each experimental group are shown. x-axis represents nucleotide length and y-axis is arbitrary signal intensity fluorescence units.

    Techniques Used: Isolation, Fluorescence

    Experiment workflow utilised for storage and processing of PAXgene ™ -collected whole blood Peripheral whole blood was collected from a single donor on each of two separate days (Experiments 1 and 2). 2.5 ml whole blood was dispensed in each of 6 PAXgene ™ tubes for each experiment. Following inversion (10 times), tubes were stored at room temperature for 2 hrs or 24 hrs. All samples were then transferred to −20 °C overnight followed by −80 °C for storage until RNA isolation procedures were performed using the PAXgene ™ RNA isolation kit. sscDNA probes were synthesised by NuGEN Whole Blood SPIA amplification using the NuGEN Ovation ™ RNA amplification V2 and Whole Blood reagent system. sscDNA was then hybridised to Affymetrix Human Genome U133 Plus 2.0 arrays. WB—Whole Blood, HG = Human Genome, PAX′ = PAXgene ™ and RT = Room Temperature.
    Figure Legend Snippet: Experiment workflow utilised for storage and processing of PAXgene ™ -collected whole blood Peripheral whole blood was collected from a single donor on each of two separate days (Experiments 1 and 2). 2.5 ml whole blood was dispensed in each of 6 PAXgene ™ tubes for each experiment. Following inversion (10 times), tubes were stored at room temperature for 2 hrs or 24 hrs. All samples were then transferred to −20 °C overnight followed by −80 °C for storage until RNA isolation procedures were performed using the PAXgene ™ RNA isolation kit. sscDNA probes were synthesised by NuGEN Whole Blood SPIA amplification using the NuGEN Ovation ™ RNA amplification V2 and Whole Blood reagent system. sscDNA was then hybridised to Affymetrix Human Genome U133 Plus 2.0 arrays. WB—Whole Blood, HG = Human Genome, PAX′ = PAXgene ™ and RT = Room Temperature.

    Techniques Used: Isolation, Amplification, Western Blot

    Analyses of array data derived from hybridisation of sscDNA probes generated from PAXgene ™ collected whole blood RNA (a) Principal Component Analysis of those probesets remaining after filtering for (i) present call by MAS5.0 in at least 25% of samples and (ii) signal intensity greater than 6.5 (Log 2 ) in at least one sample following RMA processing of probesets derived from (i). Squares, experiment 1; triangles, experiment 2; blue, 2 hrs incubation; red, 24 hrs incubation. ( b ) Graphical representation of the number of probesets determined to be over- or under-represented in 24 hr compared to 2 hr stored samples following SAM analysis with an FDR of 0.05 and fold change ≥1.5. The number of probesets within each fold change range is indicated.
    Figure Legend Snippet: Analyses of array data derived from hybridisation of sscDNA probes generated from PAXgene ™ collected whole blood RNA (a) Principal Component Analysis of those probesets remaining after filtering for (i) present call by MAS5.0 in at least 25% of samples and (ii) signal intensity greater than 6.5 (Log 2 ) in at least one sample following RMA processing of probesets derived from (i). Squares, experiment 1; triangles, experiment 2; blue, 2 hrs incubation; red, 24 hrs incubation. ( b ) Graphical representation of the number of probesets determined to be over- or under-represented in 24 hr compared to 2 hr stored samples following SAM analysis with an FDR of 0.05 and fold change ≥1.5. The number of probesets within each fold change range is indicated.

    Techniques Used: Derivative Assay, Hybridization, Generated, Incubation

    6) Product Images from "Trim71 cooperates with microRNAs to repress Cdkn1a expression and promote embryonic stem cell proliferation"

    Article Title: Trim71 cooperates with microRNAs to repress Cdkn1a expression and promote embryonic stem cell proliferation

    Journal: Nature communications

    doi: 10.1038/ncomms1909

    Trim71 cooperates with miR-302 to inhibit Cdkn1a expression (a) Trim71 depletion leads to upregulation of Cdkn1a. The same cell extracts from Fig. 1(d) were blotted with α-Cdkn1a antibody. (b) TaqMan PCR for mature miR-295 and miR-302a levels (normalized to snoRNA142). Error bars represent mean +/− S.D. with n=3. (c) J1 ESCs transfected with Trim71 siRNA or control siRNA, together with anti-miR-302a or control inhibitor, as indicated. 60 hours post-transfection total RNA was analyzed by qRT-PCR for Cdkn1a mRNA levels (normalized to β-Actin). Error bars represent mean +/− S.D. with n=3. (d) Reporter assays were performed using a firefly luciferase that contains a miR-302 binding site from the mouse Cdkn1a 3′ UTR. A control pre-miRNA or pre-miR-302a was transfected into HeLa cells that do not express ESC-specific miRNAs. Signals were normalized to Renilla luciferase levels. Normalized signals for the pre-miR control were set to 100. (e) Reporter assays were performed 60 hours post-transfection using the same luciferase constructs as in (d) as well as with a mutant reporter (mut) in which nucleotides corresponding to the miR-302 seed sequence were mutated. Normalized signals for the mutant construct were set to 100 in each condition. Error bars represent mean +/− S.D. with n=3. The p values are from two-tailed t test.
    Figure Legend Snippet: Trim71 cooperates with miR-302 to inhibit Cdkn1a expression (a) Trim71 depletion leads to upregulation of Cdkn1a. The same cell extracts from Fig. 1(d) were blotted with α-Cdkn1a antibody. (b) TaqMan PCR for mature miR-295 and miR-302a levels (normalized to snoRNA142). Error bars represent mean +/− S.D. with n=3. (c) J1 ESCs transfected with Trim71 siRNA or control siRNA, together with anti-miR-302a or control inhibitor, as indicated. 60 hours post-transfection total RNA was analyzed by qRT-PCR for Cdkn1a mRNA levels (normalized to β-Actin). Error bars represent mean +/− S.D. with n=3. (d) Reporter assays were performed using a firefly luciferase that contains a miR-302 binding site from the mouse Cdkn1a 3′ UTR. A control pre-miRNA or pre-miR-302a was transfected into HeLa cells that do not express ESC-specific miRNAs. Signals were normalized to Renilla luciferase levels. Normalized signals for the pre-miR control were set to 100. (e) Reporter assays were performed 60 hours post-transfection using the same luciferase constructs as in (d) as well as with a mutant reporter (mut) in which nucleotides corresponding to the miR-302 seed sequence were mutated. Normalized signals for the mutant construct were set to 100 in each condition. Error bars represent mean +/− S.D. with n=3. The p values are from two-tailed t test.

    Techniques Used: Expressing, Polymerase Chain Reaction, Transfection, Quantitative RT-PCR, Luciferase, Binding Assay, Construct, Mutagenesis, Sequencing, Two Tailed Test

    Trim71 is highly expressed in undifferentiated cells and is a let-7 target (a)quantitative real time PCR (qRT-PCR) of Trim71 mRNA levels (normalized to β-Actin levels). (b) Western blot of Trim71 protein (arrowhead) using a rabbit polyclonal antibody. Flag-Trim71 expressed in HEK293 cells serves as a positive control. β-Tubulin was used as a loading control. (c) qRT-PCR for Oct-4 and Trim71 (normalized to β-Actin) and TaqMan qRT-PCR for mature let-7g and miR-295 (normalized to snoRNA142) during ESC differentiation to embryoid bodies (EB). RNA was prepared from V6.5 ESCs on day 0 (undifferentiated) and on day 5, 8, 10, and 12 of differentiation. (d) Whole cell extracts from J1 ESCs transfected with control, Lin28 or Trim71 siRNA were examined by Western blot 60 hours post-transfection. (e) TaqMan PCR of mature let-7 levels from samples in d. (f) Samples from J1 ESCs transfected with Lin28 siRNA, anti-let-7a, or both were examined by TaqMan PCR for mature let-7a (top) and by Western blot (bottom). (g) Luciferase reporter assays performed on HeLa cells using a firefly luciferase that contains two let-7 binding sites from the mouse Trim71 3′ UTR. All Signals were normalized to Renilla luciferase levels. Error bars represent mean +/− S.D. with n=3. The p value is from a two-tailed t test.
    Figure Legend Snippet: Trim71 is highly expressed in undifferentiated cells and is a let-7 target (a)quantitative real time PCR (qRT-PCR) of Trim71 mRNA levels (normalized to β-Actin levels). (b) Western blot of Trim71 protein (arrowhead) using a rabbit polyclonal antibody. Flag-Trim71 expressed in HEK293 cells serves as a positive control. β-Tubulin was used as a loading control. (c) qRT-PCR for Oct-4 and Trim71 (normalized to β-Actin) and TaqMan qRT-PCR for mature let-7g and miR-295 (normalized to snoRNA142) during ESC differentiation to embryoid bodies (EB). RNA was prepared from V6.5 ESCs on day 0 (undifferentiated) and on day 5, 8, 10, and 12 of differentiation. (d) Whole cell extracts from J1 ESCs transfected with control, Lin28 or Trim71 siRNA were examined by Western blot 60 hours post-transfection. (e) TaqMan PCR of mature let-7 levels from samples in d. (f) Samples from J1 ESCs transfected with Lin28 siRNA, anti-let-7a, or both were examined by TaqMan PCR for mature let-7a (top) and by Western blot (bottom). (g) Luciferase reporter assays performed on HeLa cells using a firefly luciferase that contains two let-7 binding sites from the mouse Trim71 3′ UTR. All Signals were normalized to Renilla luciferase levels. Error bars represent mean +/− S.D. with n=3. The p value is from a two-tailed t test.

    Techniques Used: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Positive Control, Transfection, Polymerase Chain Reaction, Luciferase, Binding Assay, Two Tailed Test

    7) Product Images from "Novel Intronic RNA Structures Contribute to Maintenance of Phenotype in Saccharomyces cerevisiae"

    Article Title: Novel Intronic RNA Structures Contribute to Maintenance of Phenotype in Saccharomyces cerevisiae

    Journal: Genetics

    doi: 10.1534/genetics.115.185363

    Properties of loci containing intronic novel RNA predictions or snoRNAs. Quantifications were based on RNA-seq data of the same total RNA as used in Figure 2 (GEO accession no. GSE58884) and on CRAC data (GEO accession no. GSE40046). (A) Box plots showing ribosomal protein intron transcript levels in RPKM without normalization and with normalization to host gene transcript levels. Values are shown for all RP gene introns and 12 RP gene introns with predictions. (B) Association of introns with the exosome. We reanalyzed 16 independent sequencing experiments by Schneider et al. (2012) of RNA fragments cross-linked to exosome components. Reads mapping to each intron were normalized by the host gene expression estimated by RNA-seq. For each CRAC experiment an intron was given a percentile value of how frequently it was bound to an exosome component compared to other introns. Values presented are averaged percentile derived from 16 experiments and are shown for all introns, the 19 introns with predictions, and the eight introns containing snoRNAs. The levels of significance for the Mann–Whitney U -tests are represented as follows: *** P
    Figure Legend Snippet: Properties of loci containing intronic novel RNA predictions or snoRNAs. Quantifications were based on RNA-seq data of the same total RNA as used in Figure 2 (GEO accession no. GSE58884) and on CRAC data (GEO accession no. GSE40046). (A) Box plots showing ribosomal protein intron transcript levels in RPKM without normalization and with normalization to host gene transcript levels. Values are shown for all RP gene introns and 12 RP gene introns with predictions. (B) Association of introns with the exosome. We reanalyzed 16 independent sequencing experiments by Schneider et al. (2012) of RNA fragments cross-linked to exosome components. Reads mapping to each intron were normalized by the host gene expression estimated by RNA-seq. For each CRAC experiment an intron was given a percentile value of how frequently it was bound to an exosome component compared to other introns. Values presented are averaged percentile derived from 16 experiments and are shown for all introns, the 19 introns with predictions, and the eight introns containing snoRNAs. The levels of significance for the Mann–Whitney U -tests are represented as follows: *** P

    Techniques Used: RNA Sequencing Assay, Sequencing, Expressing, Derivative Assay, MANN-WHITNEY

    Characterization of the size and expression of the ncRNA within GLC7 intron. (A) Northern blot of S. cerevisiae total RNA and oligos mimicking GLC7 intron. The blots were probed with strand-specific probes showing expression of ncRNA in antisense (left panel) and sense orientation (right panel) to the GLC7 gene. The estimation of the sizes is based on the 60-nt oligos visible on the film and a comparison with the markers visible on the membrane. (B) RT-PCR on total RNA and low-weight-enriched RNA using random priming showing the expression of ncRNA within the GLC7 intron. PCR of genomic DNA was used as positive control. Names of the primers are listed on the right and are the same as indicated in C. Gel annotation: G, genomic DNA; T, total cDNA; L, low-molecular-weight-enriched cDNA; and −, no template negative control. (C) Data uploaded into the University of California Santa Cruz genome browser for sequence annotation and data visualization presenting annotated GLC7 intron. Primer names used for RT-PCR are listed next to black, blue, and red boxes indicating their position with respect to the gene annotation below and the size of the corresponding PCR product. Lines joining primers mark the amplified sequences; the regions targeted for deletion by the loxP method are symbolized by the black box; the location of the strand-specific Northern probes are shown in green; and regions with the putative structure predicted by RNAz and CMfinder are shown in gray. All features map directly onto the fragment of the gene structure diagrams. The bottom panel represents the degree of conservation of gene regions among seven yeast species.
    Figure Legend Snippet: Characterization of the size and expression of the ncRNA within GLC7 intron. (A) Northern blot of S. cerevisiae total RNA and oligos mimicking GLC7 intron. The blots were probed with strand-specific probes showing expression of ncRNA in antisense (left panel) and sense orientation (right panel) to the GLC7 gene. The estimation of the sizes is based on the 60-nt oligos visible on the film and a comparison with the markers visible on the membrane. (B) RT-PCR on total RNA and low-weight-enriched RNA using random priming showing the expression of ncRNA within the GLC7 intron. PCR of genomic DNA was used as positive control. Names of the primers are listed on the right and are the same as indicated in C. Gel annotation: G, genomic DNA; T, total cDNA; L, low-molecular-weight-enriched cDNA; and −, no template negative control. (C) Data uploaded into the University of California Santa Cruz genome browser for sequence annotation and data visualization presenting annotated GLC7 intron. Primer names used for RT-PCR are listed next to black, blue, and red boxes indicating their position with respect to the gene annotation below and the size of the corresponding PCR product. Lines joining primers mark the amplified sequences; the regions targeted for deletion by the loxP method are symbolized by the black box; the location of the strand-specific Northern probes are shown in green; and regions with the putative structure predicted by RNAz and CMfinder are shown in gray. All features map directly onto the fragment of the gene structure diagrams. The bottom panel represents the degree of conservation of gene regions among seven yeast species.

    Techniques Used: Expressing, Northern Blot, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Positive Control, Molecular Weight, Negative Control, Sequencing, Amplification

    RT-PCR confirmation of intron fates conducted on total RNA and low-weight-enriched RNA using random priming. PCR of genomic DNA was used as positive control. (A) Agarose gel confirming ncRNAs expressed from introns and maintained in the cell. (B) Agarose gel confirming ncRNA expression accompanied by complete mRNA splicing. (C) Agarose gel confirming ncRNA expression accompanied by alternative splicing. Arrows indicate the expected size of the PCR products according to the key. Lane designations for DNA templates: G, Genomic DNA (positive control); T, total cDNA; L, low-molecular-weight-enriched cDNA; and −, no template negative control. With the exception of GLC7 ncRNA, which was run on a separate gel, the images of mRNAs, introns, and ncRNAs for each gene were cropped from the same agarose gel picture with brightness and contrast applied equally across the entire image (full images available in Figure S1 , Figure S2 , Figure S3 , Figure S4 , Figure S5 , and Figure S6 ). For small-size PCR products, cropping included primer dimers.
    Figure Legend Snippet: RT-PCR confirmation of intron fates conducted on total RNA and low-weight-enriched RNA using random priming. PCR of genomic DNA was used as positive control. (A) Agarose gel confirming ncRNAs expressed from introns and maintained in the cell. (B) Agarose gel confirming ncRNA expression accompanied by complete mRNA splicing. (C) Agarose gel confirming ncRNA expression accompanied by alternative splicing. Arrows indicate the expected size of the PCR products according to the key. Lane designations for DNA templates: G, Genomic DNA (positive control); T, total cDNA; L, low-molecular-weight-enriched cDNA; and −, no template negative control. With the exception of GLC7 ncRNA, which was run on a separate gel, the images of mRNAs, introns, and ncRNAs for each gene were cropped from the same agarose gel picture with brightness and contrast applied equally across the entire image (full images available in Figure S1 , Figure S2 , Figure S3 , Figure S4 , Figure S5 , and Figure S6 ). For small-size PCR products, cropping included primer dimers.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Positive Control, Agarose Gel Electrophoresis, Expressing, Molecular Weight, Negative Control

    8) Product Images from "RNA structures that resist degradation by Xrn1 produce a pathogenic Dengue virus RNA"

    Article Title: RNA structures that resist degradation by Xrn1 produce a pathogenic Dengue virus RNA

    Journal: eLife

    doi: 10.7554/eLife.01892

    Structural analyses of DVxrRNA1 mutant 6 (mutant in the three-way junction). ( A ) Normalized NMIA and DMS reactivity profiles of mutant 6 compared to WT RNA, depicted as in Figures 3 and 4 . Colored bars (green and blue) represent the WT RNA and black bars represent mutant 6. The location of the point mutation is shown with a purple shaded box. Reactivity changes are indicated by red boxes. ( B ) dPAGE of Xrn1 resistance by WT and mutant 6. ( C ) Non-denaturing (native) PAGE analyses of WT and mutant 6. ( D ) Secondary structure of the DVxrRNA1 with elements labeled. The purple shaded box shows the location of the mutation. Regions of the structure that show substantial changes in the chemical probing (from panel ( A )) are indicated by red boxes. DOI: http://dx.doi.org/10.7554/eLife.01892.017
    Figure Legend Snippet: Structural analyses of DVxrRNA1 mutant 6 (mutant in the three-way junction). ( A ) Normalized NMIA and DMS reactivity profiles of mutant 6 compared to WT RNA, depicted as in Figures 3 and 4 . Colored bars (green and blue) represent the WT RNA and black bars represent mutant 6. The location of the point mutation is shown with a purple shaded box. Reactivity changes are indicated by red boxes. ( B ) dPAGE of Xrn1 resistance by WT and mutant 6. ( C ) Non-denaturing (native) PAGE analyses of WT and mutant 6. ( D ) Secondary structure of the DVxrRNA1 with elements labeled. The purple shaded box shows the location of the mutation. Regions of the structure that show substantial changes in the chemical probing (from panel ( A )) are indicated by red boxes. DOI: http://dx.doi.org/10.7554/eLife.01892.017

    Techniques Used: Mutagenesis, Clear Native PAGE, Labeling

    Refined secondary structure of SL IV RNA (DVxrRNA2) in the DENV2 3′UTR. ( A ) Chemical probing profiles of (+31)-DVxrRNA2-MG from NMIA (blue) and DMS (green) -probed RNAs. The y-axis contains the sequence and nucleotide numbers, the x-axis depicts chemical reactivity. Data represent the average of three independent experiments, error bars show one standard deviation from the mean. Secondary structure elements are indicated to the right of the graphs. ( B – D ) Overlay of phylogenetic ( B ), NMIA ( C ), and DMS ( D ) data the support this refined secondary structure. In panel ( B ) we assign names to the secondary structure elements according to standard convention. The RNA shown here was quantitative resistance to Xrn1 degradation ( Figure 6D,E ). DOI: http://dx.doi.org/10.7554/eLife.01892.008
    Figure Legend Snippet: Refined secondary structure of SL IV RNA (DVxrRNA2) in the DENV2 3′UTR. ( A ) Chemical probing profiles of (+31)-DVxrRNA2-MG from NMIA (blue) and DMS (green) -probed RNAs. The y-axis contains the sequence and nucleotide numbers, the x-axis depicts chemical reactivity. Data represent the average of three independent experiments, error bars show one standard deviation from the mean. Secondary structure elements are indicated to the right of the graphs. ( B – D ) Overlay of phylogenetic ( B ), NMIA ( C ), and DMS ( D ) data the support this refined secondary structure. In panel ( B ) we assign names to the secondary structure elements according to standard convention. The RNA shown here was quantitative resistance to Xrn1 degradation ( Figure 6D,E ). DOI: http://dx.doi.org/10.7554/eLife.01892.008

    Techniques Used: Sequencing, Standard Deviation

    9) Product Images from "Yeast Helicase Pif1 Unwinds RNA:DNA Hybrids with Higher Processivity than DNA:DNA Duplexes"

    Article Title: Yeast Helicase Pif1 Unwinds RNA:DNA Hybrids with Higher Processivity than DNA:DNA Duplexes

    Journal:

    doi: 10.1074/jbc.M115.688648

    Single turnover unwinding of the 16bp DNA:DNA ( A ) and RNA:DNA ( B ) forked duplexes performed in the presence of heparin trap. The plot from F showing the unwinding of the same substrates in the presence of T 50 has been overlaid with the unwinding
    Figure Legend Snippet: Single turnover unwinding of the 16bp DNA:DNA ( A ) and RNA:DNA ( B ) forked duplexes performed in the presence of heparin trap. The plot from F showing the unwinding of the same substrates in the presence of T 50 has been overlaid with the unwinding

    Techniques Used:

    Helicase-catalyzed unwinding kinetics for DNA:DNA ( red circles ) and RNA:DNA ( blue squares ) forked duplexes under single turnover (single cycle) conditions. A , a schematic representation of a single cycle unwinding reaction. Pif1 takes a series of n sequential
    Figure Legend Snippet: Helicase-catalyzed unwinding kinetics for DNA:DNA ( red circles ) and RNA:DNA ( blue squares ) forked duplexes under single turnover (single cycle) conditions. A , a schematic representation of a single cycle unwinding reaction. Pif1 takes a series of n sequential

    Techniques Used:

    Measurement of the equilibrium dissociation constants ( K D ) for Pif1 interaction with DNA:DNA ( red circles ) and RNA:DNA ( blue squares ) partial duplex substrates. A and B , 1 n m 5′-FAM-labeled forked ( A ) and tailed ( B ) duplexes were titrated with
    Figure Legend Snippet: Measurement of the equilibrium dissociation constants ( K D ) for Pif1 interaction with DNA:DNA ( red circles ) and RNA:DNA ( blue squares ) partial duplex substrates. A and B , 1 n m 5′-FAM-labeled forked ( A ) and tailed ( B ) duplexes were titrated with

    Techniques Used: Labeling

    Helicase-catalyzed unwinding kinetics for DNA:DNA ( red circles ) and RNA:DNA ( blue squares ) forked substrates under multiple turnover conditions. A , a schematic representation of a multiple cycle unwinding reaction. ES , enzyme-substrate complex; EI , intermediate.
    Figure Legend Snippet: Helicase-catalyzed unwinding kinetics for DNA:DNA ( red circles ) and RNA:DNA ( blue squares ) forked substrates under multiple turnover conditions. A , a schematic representation of a multiple cycle unwinding reaction. ES , enzyme-substrate complex; EI , intermediate.

    Techniques Used:

    Measurement of the equilibrium dissociation constants ( K D ) for Pif1 interaction with DNA:DNA ( red circles ) and RNA:DNA ( blue squares ) partial duplex substrates in the presence of an ATP analog. A and B , 1 n m 5′-FAM-labeled forked ( A ) and tailed
    Figure Legend Snippet: Measurement of the equilibrium dissociation constants ( K D ) for Pif1 interaction with DNA:DNA ( red circles ) and RNA:DNA ( blue squares ) partial duplex substrates in the presence of an ATP analog. A and B , 1 n m 5′-FAM-labeled forked ( A ) and tailed

    Techniques Used: Labeling

    Stimulation of ATP hydrolysis of Pif1 (90 n m ) by ssDNA, forked, and tailed dsDNA as well as RNA:DNA hybrids. The rate of ATP hydrolysis by Pif1 was measured on ssT 50 (84.2 ± 7.6 s −1 ), 20-bp dsDNA forked duplex (78.1 ± 3.3 s −1
    Figure Legend Snippet: Stimulation of ATP hydrolysis of Pif1 (90 n m ) by ssDNA, forked, and tailed dsDNA as well as RNA:DNA hybrids. The rate of ATP hydrolysis by Pif1 was measured on ssT 50 (84.2 ± 7.6 s −1 ), 20-bp dsDNA forked duplex (78.1 ± 3.3 s −1

    Techniques Used:

    Model depicting the possible impact on Pif1 activity of the difference in processivity of Pif1 for unwinding DNA:DNA duplexes and RNA:DNA hybrids in various cellular activities. A , double-stranded DNA break repair. Pif1 prevents aberrant telomere addition
    Figure Legend Snippet: Model depicting the possible impact on Pif1 activity of the difference in processivity of Pif1 for unwinding DNA:DNA duplexes and RNA:DNA hybrids in various cellular activities. A , double-stranded DNA break repair. Pif1 prevents aberrant telomere addition

    Techniques Used: Activity Assay

    Measurement of the helicase dissociation rate from RNA:DNA versus DNA:DNA duplexes. A , an illustration of enzyme dissociation from nucleic acids is shown. Pif1 translocates unidirectionally (5′ to 3′) on DNA to unwind it, or it can dissociate
    Figure Legend Snippet: Measurement of the helicase dissociation rate from RNA:DNA versus DNA:DNA duplexes. A , an illustration of enzyme dissociation from nucleic acids is shown. Pif1 translocates unidirectionally (5′ to 3′) on DNA to unwind it, or it can dissociate

    Techniques Used:

    Measurement of the unwinding kinetics of dsDNA ( red circles ) and RNA:DNA ( blue squares ) tailed duplexes . A , shown is the schematic representation of Pif1-catalyzed melting of tailed substrates in the presence of ATP. B and C , 20-bp tailed dsDNA duplex
    Figure Legend Snippet: Measurement of the unwinding kinetics of dsDNA ( red circles ) and RNA:DNA ( blue squares ) tailed duplexes . A , shown is the schematic representation of Pif1-catalyzed melting of tailed substrates in the presence of ATP. B and C , 20-bp tailed dsDNA duplex

    Techniques Used:

    10) Product Images from "A New Model to Produce Infectious Hepatitis C Virus without the Replication Requirement"

    Article Title: A New Model to Produce Infectious Hepatitis C Virus without the Replication Requirement

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1001333

    BHK-WNV cells produce HCV infectious in Huh-7.5 cells. (A) Huh-7.5 cells were incubated for 3 hr with aliquots of each fraction of the sucrose gradient shown in Fig. 3B , then transfected with plasmid encoding core to NS2 (which enhanced the readout); cellular contents in HCV 5′-UTR RNA were measured at the indicated time points by RT-TaqMan qPCR, with in vitro transcripts as standards (cf. Materials and Methods ). (B) Huh-7.5 cells were incubated with HCVbp produced and harvested as described in Materials and Methods and, two days later, analyzed by confocal microscopy; NS5A was stained (red) with in-house rabbit polyclonal IgG; cells were counterstained with ERGIC-53 (green) and DAPI (blue). (C) Two days after infection with HCVbp, Huh-7.5 cells were incubated with BrUTP and NS5A antibody. Huh-7.5 cells with an established HCV subgenomic replicon were used as a positive control.
    Figure Legend Snippet: BHK-WNV cells produce HCV infectious in Huh-7.5 cells. (A) Huh-7.5 cells were incubated for 3 hr with aliquots of each fraction of the sucrose gradient shown in Fig. 3B , then transfected with plasmid encoding core to NS2 (which enhanced the readout); cellular contents in HCV 5′-UTR RNA were measured at the indicated time points by RT-TaqMan qPCR, with in vitro transcripts as standards (cf. Materials and Methods ). (B) Huh-7.5 cells were incubated with HCVbp produced and harvested as described in Materials and Methods and, two days later, analyzed by confocal microscopy; NS5A was stained (red) with in-house rabbit polyclonal IgG; cells were counterstained with ERGIC-53 (green) and DAPI (blue). (C) Two days after infection with HCVbp, Huh-7.5 cells were incubated with BrUTP and NS5A antibody. Huh-7.5 cells with an established HCV subgenomic replicon were used as a positive control.

    Techniques Used: Incubation, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, In Vitro, Produced, Confocal Microscopy, Staining, Infection, Positive Control

    HCV RNA is preferentially associated with in HCV bicistronic particles released by BHK-WNV cells. (A) Three days after transfection of BHK-WNV cells with HCVbp-coding plasmid, RNA was extracted from cells and pellets of supernatants ultracentrifuged through sucrose cushion (SN); WNV and HCV RNA were each quantified by RT-qPCR; a similar protocol was used for control (untransfected) cells. The values on the Y-axis represent the amounts of cell- and SN-associated RNA extracted from an equivalent number of cells. (B) HCVbp released by BHK-WNV cells were analyzed on a 20–60% sucrose gradient; HCV E1 was detected by Western blot (top panel) and HCV RNA 5′-UTR measured by RT-Taqman PCR (bottom panel).
    Figure Legend Snippet: HCV RNA is preferentially associated with in HCV bicistronic particles released by BHK-WNV cells. (A) Three days after transfection of BHK-WNV cells with HCVbp-coding plasmid, RNA was extracted from cells and pellets of supernatants ultracentrifuged through sucrose cushion (SN); WNV and HCV RNA were each quantified by RT-qPCR; a similar protocol was used for control (untransfected) cells. The values on the Y-axis represent the amounts of cell- and SN-associated RNA extracted from an equivalent number of cells. (B) HCVbp released by BHK-WNV cells were analyzed on a 20–60% sucrose gradient; HCV E1 was detected by Western blot (top panel) and HCV RNA 5′-UTR measured by RT-Taqman PCR (bottom panel).

    Techniques Used: Transfection, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Polymerase Chain Reaction

    11) Product Images from "Regulation of 4E-BP1 activity in the mammalian oocyte"

    Article Title: Regulation of 4E-BP1 activity in the mammalian oocyte

    Journal: Cell Cycle

    doi: 10.1080/15384101.2017.1295178

    Down-regulation of 4E-BP1 phosphorylation in oocytes results in defects in the MII spindle assembly. (A) Scheme of dominant negative mutant construct of 4E-BP1–4Ala used for in vitro transcription. (B) Scheme of experimental procedure to express 4E-BP1 RNA constructs in the oocyte. (C) Immunoblotting evaluation of expression of microinjected non-phosphorylable form (marked by arrowhead) of 4E-BP1 in the matured MII oocytes n = 2. GAPDH was used as a loading control. See Figure S5. (D) Confocal images of MII spindles of oocytes microinjected with 4E-BP1-Wt or dominant negative mutant 4E-BP1–4Ala, Tubulin (red) and DNA (blue). Scale bar = 10 µm. (E) Quantification of chromosome alignment in the metaphase plate, MII oocytes expressing 4E-BP1-Wt or 4E-BP1-Ala RNA. Data are presented as mean ± SD, Student's t-test, n ≥ 25.
    Figure Legend Snippet: Down-regulation of 4E-BP1 phosphorylation in oocytes results in defects in the MII spindle assembly. (A) Scheme of dominant negative mutant construct of 4E-BP1–4Ala used for in vitro transcription. (B) Scheme of experimental procedure to express 4E-BP1 RNA constructs in the oocyte. (C) Immunoblotting evaluation of expression of microinjected non-phosphorylable form (marked by arrowhead) of 4E-BP1 in the matured MII oocytes n = 2. GAPDH was used as a loading control. See Figure S5. (D) Confocal images of MII spindles of oocytes microinjected with 4E-BP1-Wt or dominant negative mutant 4E-BP1–4Ala, Tubulin (red) and DNA (blue). Scale bar = 10 µm. (E) Quantification of chromosome alignment in the metaphase plate, MII oocytes expressing 4E-BP1-Wt or 4E-BP1-Ala RNA. Data are presented as mean ± SD, Student's t-test, n ≥ 25.

    Techniques Used: Dominant Negative Mutation, Construct, In Vitro, Expressing

    4E-BP1 effects on protein synthesis in the oocytes. (A) Renilla luciferase reporter carrying 5′UTR TOP motive of Eef2 co-injected with 4E-BP1-Wt or 4E-BP1-Ala RNA. In the control no 4E-BP1 RNA was used and the IRES motive Firefly Luciferase was used as a loading control. Chemiluminescence was measured in the post NEBD stage (mean value ± 6 and 4 %, Student's t-test, NS = non-significant) and MII stage oocytes (mean values ± SD, Student's t-test). Data are presented as mean ± SD, n ≥ 10 replicates. (B) Measurement of in situ translation intensity in the chromosomal area (CTA, mean value ± SD, Student's t-test, NS = non-significant) and perispindular translational area (PTA, mean value ± SD, Student's t-test) in the post NEBD oocytes, HPG (red) and DNA (blue). Data are presented as mean ± SD, n ≥ 21. Scale bar = 20 µm.
    Figure Legend Snippet: 4E-BP1 effects on protein synthesis in the oocytes. (A) Renilla luciferase reporter carrying 5′UTR TOP motive of Eef2 co-injected with 4E-BP1-Wt or 4E-BP1-Ala RNA. In the control no 4E-BP1 RNA was used and the IRES motive Firefly Luciferase was used as a loading control. Chemiluminescence was measured in the post NEBD stage (mean value ± 6 and 4 %, Student's t-test, NS = non-significant) and MII stage oocytes (mean values ± SD, Student's t-test). Data are presented as mean ± SD, n ≥ 10 replicates. (B) Measurement of in situ translation intensity in the chromosomal area (CTA, mean value ± SD, Student's t-test, NS = non-significant) and perispindular translational area (PTA, mean value ± SD, Student's t-test) in the post NEBD oocytes, HPG (red) and DNA (blue). Data are presented as mean ± SD, n ≥ 21. Scale bar = 20 µm.

    Techniques Used: Luciferase, Injection, In Situ

    12) Product Images from "Translation of MMTV Gag requires nuclear events involving splicing motifs in addition to the viral Rem protein and RmRE"

    Article Title: Translation of MMTV Gag requires nuclear events involving splicing motifs in addition to the viral Rem protein and RmRE

    Journal: Retrovirology

    doi: 10.1186/1742-4690-9-8

    Schematic representation of gag mRNAs and their translation phenotypes . Full length viral mRNA is capped (7MeG) and polyadenylated (A n ), as are all other constructs. At the 5' and 3' ends of the viral genome length RNA, the R and U5 or U3 elements are indicated as grey (R) blue (U5) and purple boxes (U3). The viral open reading frames (ORF) gag , pol , and env are indicated while other genes are not presented to maintain clarity. The splice donors (SD) and splice acceptors (SA) are represented as green or orange triangles, respectively. The viral protein Rem (blue half moon) is shown bound to its cis RNA structural element, the RmRE. Three subviral mRNAs, derived from various CMV promoter constructs, are also depicted. The minimal expression construct contains only the cap, gag ORF, and poly-A tail. Also depicted is the mRNA from a gag bicistronic construct that contains an IRES element supporting cap-independent translation of eGFP. The second subviral construct includes the RmRE, to which Rem (provided in trans ) is shown to bind. The third subviral gag expression construct contains both a SD and SA positioning the gag ORF in an intron as well as the RmRE and Rem in trans . Expression of Gag protein from the different mRNAs is indicated as a purple polypeptide exiting the ribosome (red). For subviral constructs, loss of protein expression as a result of the included gag allele is indicated by dotted lines and by the absence of Gag polypeptide exiting the ribosome. Similarly, when the bicistronic constructs fail to support Gag expression, the lack of GFP production from the IRES is indicated by the dotted line and lack of the green polypeptide exiting the ribosome. The lightning bolt represents direct transfection of gag mRNAs where allele-independent Gag translation was detected.
    Figure Legend Snippet: Schematic representation of gag mRNAs and their translation phenotypes . Full length viral mRNA is capped (7MeG) and polyadenylated (A n ), as are all other constructs. At the 5' and 3' ends of the viral genome length RNA, the R and U5 or U3 elements are indicated as grey (R) blue (U5) and purple boxes (U3). The viral open reading frames (ORF) gag , pol , and env are indicated while other genes are not presented to maintain clarity. The splice donors (SD) and splice acceptors (SA) are represented as green or orange triangles, respectively. The viral protein Rem (blue half moon) is shown bound to its cis RNA structural element, the RmRE. Three subviral mRNAs, derived from various CMV promoter constructs, are also depicted. The minimal expression construct contains only the cap, gag ORF, and poly-A tail. Also depicted is the mRNA from a gag bicistronic construct that contains an IRES element supporting cap-independent translation of eGFP. The second subviral construct includes the RmRE, to which Rem (provided in trans ) is shown to bind. The third subviral gag expression construct contains both a SD and SA positioning the gag ORF in an intron as well as the RmRE and Rem in trans . Expression of Gag protein from the different mRNAs is indicated as a purple polypeptide exiting the ribosome (red). For subviral constructs, loss of protein expression as a result of the included gag allele is indicated by dotted lines and by the absence of Gag polypeptide exiting the ribosome. Similarly, when the bicistronic constructs fail to support Gag expression, the lack of GFP production from the IRES is indicated by the dotted line and lack of the green polypeptide exiting the ribosome. The lightning bolt represents direct transfection of gag mRNAs where allele-independent Gag translation was detected.

    Techniques Used: Construct, Derivative Assay, Expressing, Transfection

    Inhibition of expression occurs post-transcriptionally and does not affect mRNA transport and stability . A) Total RNA was extracted from HEK 293T cells transfected with Mtv-1, HBRV, and SM gag constructs. The gag and gapdh mRNAs were reverse transcribed into cDNA using an Oligo-dT primer and quantified by Real-Time PCR. The relative abundance of gag mRNA to gapdh is calculated as the reverse of the ratio of gag CT to gapdh CT value. B) Transfected HEK 293T cells were separated into nuclear and cytoplasmic fractions. The mRNA was extracted from each fraction, and gag and gapdh mRNAs were quantified by reverse transcription Real-Time PCR. The level of mRNA transport from the nucleus to the cytoplasm was quantified as the ratio of nuclear and cytoplasmic gag mRNA relative to gapdh . C) Cells were treated with actinomycin D, and RNA was extracted from the cells at 0, 2, 4 and 6 hrs post-treatment and quantified by reverse transcription Real-Time PCR. The relative abundance of gag mRNA to gapdh was calculated at each time point. Data are the average of at least three independent experiments ± SD.
    Figure Legend Snippet: Inhibition of expression occurs post-transcriptionally and does not affect mRNA transport and stability . A) Total RNA was extracted from HEK 293T cells transfected with Mtv-1, HBRV, and SM gag constructs. The gag and gapdh mRNAs were reverse transcribed into cDNA using an Oligo-dT primer and quantified by Real-Time PCR. The relative abundance of gag mRNA to gapdh is calculated as the reverse of the ratio of gag CT to gapdh CT value. B) Transfected HEK 293T cells were separated into nuclear and cytoplasmic fractions. The mRNA was extracted from each fraction, and gag and gapdh mRNAs were quantified by reverse transcription Real-Time PCR. The level of mRNA transport from the nucleus to the cytoplasm was quantified as the ratio of nuclear and cytoplasmic gag mRNA relative to gapdh . C) Cells were treated with actinomycin D, and RNA was extracted from the cells at 0, 2, 4 and 6 hrs post-treatment and quantified by reverse transcription Real-Time PCR. The relative abundance of gag mRNA to gapdh was calculated at each time point. Data are the average of at least three independent experiments ± SD.

    Techniques Used: Inhibition, Expressing, Transfection, Construct, Real-time Polymerase Chain Reaction

    13) Product Images from "CS-SELEX Generates High-Affinity ssDNA Aptamers as Molecular Probes for Hepatitis C Virus Envelope Glycoprotein E2"

    Article Title: CS-SELEX Generates High-Affinity ssDNA Aptamers as Molecular Probes for Hepatitis C Virus Envelope Glycoprotein E2

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0008142

    Aptamer ZE2 dramatically blocks HCVcc infection of hepatocytes. (A) Immunofluorescent detection of intracellular HCV mean fluorescence density (MFI) of HCVcc infected Huh-7.5.1 cells with anti-E2 antibody. The data shown were calculated as mean±SEM and data are from three independent experiments. (B) Western blot analysis of intracellular E2 protein expressions of HCVcc infected Huh7.5.1 in the presence of ZE2 or IFN-α with specific anti-E2 antibody; β-actin, a housekeeping gene with constant expression, was used as internal control. β-actin protein was detected with specific anti- β-actin antibody. (C) Quantitation of intracellular HCV RNA was detected using real-time quantitative RT-PCR and normalized to the levels of GAPDH mRNA. GAPDH, a housekeeping gene with constant mRNA expression, was used as internal control. The data shown were calculated as mean±SEM and data are from three independent experiments.
    Figure Legend Snippet: Aptamer ZE2 dramatically blocks HCVcc infection of hepatocytes. (A) Immunofluorescent detection of intracellular HCV mean fluorescence density (MFI) of HCVcc infected Huh-7.5.1 cells with anti-E2 antibody. The data shown were calculated as mean±SEM and data are from three independent experiments. (B) Western blot analysis of intracellular E2 protein expressions of HCVcc infected Huh7.5.1 in the presence of ZE2 or IFN-α with specific anti-E2 antibody; β-actin, a housekeeping gene with constant expression, was used as internal control. β-actin protein was detected with specific anti- β-actin antibody. (C) Quantitation of intracellular HCV RNA was detected using real-time quantitative RT-PCR and normalized to the levels of GAPDH mRNA. GAPDH, a housekeeping gene with constant mRNA expression, was used as internal control. The data shown were calculated as mean±SEM and data are from three independent experiments.

    Techniques Used: Infection, Fluorescence, Western Blot, Expressing, Quantitation Assay, Quantitative RT-PCR

    The ssDNA aptamer ZE2 specifically targets HCV particles. (A) HCVcc could be captured by the aptamer ZE2 in an aptamer dose-dependent manner by sandwich ELISA. Plates were coated with anti-E2 polyclonal antibody and blocked with 1% BSA. After extensive washing, the bound viral particles were added and then revealed using biotin-labeled aptamer ZE2, ZE3 or ZE2-mut as described in Materials and Methods . Data shown were calculated as mean±SEM, and data are from three independent experiments. (B) Comparison of HCV detection methods: HCV E2 antigen-aptamer method and HCV RNA quantification by real time fluorescence quantitative RT-PCR. The aptamer method was performed as above (A), except HCV patients and healthy donor serum samples were added into each 96-well ELISA plate instead of HCVcc. (C) Determination of E2 protein expressions of different genotypes of E1E2 gene stable-expressing HepG2 cells with anti-E2 antibody by western blot analysis. HepG2 was uesd as a control. (D) Different genotypes of E2 detected by aptamer ZE2. Data shown were calculated as mean±SEM and data are from three independent experiments.
    Figure Legend Snippet: The ssDNA aptamer ZE2 specifically targets HCV particles. (A) HCVcc could be captured by the aptamer ZE2 in an aptamer dose-dependent manner by sandwich ELISA. Plates were coated with anti-E2 polyclonal antibody and blocked with 1% BSA. After extensive washing, the bound viral particles were added and then revealed using biotin-labeled aptamer ZE2, ZE3 or ZE2-mut as described in Materials and Methods . Data shown were calculated as mean±SEM, and data are from three independent experiments. (B) Comparison of HCV detection methods: HCV E2 antigen-aptamer method and HCV RNA quantification by real time fluorescence quantitative RT-PCR. The aptamer method was performed as above (A), except HCV patients and healthy donor serum samples were added into each 96-well ELISA plate instead of HCVcc. (C) Determination of E2 protein expressions of different genotypes of E1E2 gene stable-expressing HepG2 cells with anti-E2 antibody by western blot analysis. HepG2 was uesd as a control. (D) Different genotypes of E2 detected by aptamer ZE2. Data shown were calculated as mean±SEM and data are from three independent experiments.

    Techniques Used: Sandwich ELISA, Labeling, Fluorescence, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

    14) Product Images from "Identification of Genes Responsive to Solar Simulated UV Radiation in Human Monocyte-Derived Dendritic Cells"

    Article Title: Identification of Genes Responsive to Solar Simulated UV Radiation in Human Monocyte-Derived Dendritic Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0006735

    Analysis of gene expression in UV-irradiated human DCs by RT-PCR. Real-time semiquantitative TaqMan RT-PCR was performed to validate differential gene expression induced by UV irradiation in human monocyte-derived dendritic cells (DCs) in culture. DCs (obtained from 3 donors) were untreated or exposed to solar-simulated UV radiation (3.7 J/cm 2 UVA+0.3 J/cm 2 UVB). Total RNA was extracted after a further 6 h in culture. Primer sequences are shown in Table S1 . Expression levels were normalized to 18s RNA. Bars correspond to log10 of fold down-regulation or up-regulation compared with non-irradiated cells.
    Figure Legend Snippet: Analysis of gene expression in UV-irradiated human DCs by RT-PCR. Real-time semiquantitative TaqMan RT-PCR was performed to validate differential gene expression induced by UV irradiation in human monocyte-derived dendritic cells (DCs) in culture. DCs (obtained from 3 donors) were untreated or exposed to solar-simulated UV radiation (3.7 J/cm 2 UVA+0.3 J/cm 2 UVB). Total RNA was extracted after a further 6 h in culture. Primer sequences are shown in Table S1 . Expression levels were normalized to 18s RNA. Bars correspond to log10 of fold down-regulation or up-regulation compared with non-irradiated cells.

    Techniques Used: Expressing, Irradiation, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

    Gene expression induced by UV irradiation in human primary DCs, MCs and KCs. Human primary monocyte-derived DCs (moDCs), melanocytes (MCs) and keratinocytes (KCs) were exposed to solar-simulated UV radiation (3.7 J/cm 2 UVA+0.3 J/cm 2 UVB) as in Fig. 1 and total RNA was extracted after a further 6 h in culture. Primer sequences are shown in Table S1 . Expression levels were normalized to 18s RNA. A. Genes encoding cytokines and chemokine receptors. B. Genes related to DNA damage and p53 response. C. Genes potentially involved in immunomodulation. Results are shown as log10 of fold up-regulation or down-regulation in UV-irradiated cells compared with expression in non-irradiated controls. Data correspond to arithmetic mean±SEM.
    Figure Legend Snippet: Gene expression induced by UV irradiation in human primary DCs, MCs and KCs. Human primary monocyte-derived DCs (moDCs), melanocytes (MCs) and keratinocytes (KCs) were exposed to solar-simulated UV radiation (3.7 J/cm 2 UVA+0.3 J/cm 2 UVB) as in Fig. 1 and total RNA was extracted after a further 6 h in culture. Primer sequences are shown in Table S1 . Expression levels were normalized to 18s RNA. A. Genes encoding cytokines and chemokine receptors. B. Genes related to DNA damage and p53 response. C. Genes potentially involved in immunomodulation. Results are shown as log10 of fold up-regulation or down-regulation in UV-irradiated cells compared with expression in non-irradiated controls. Data correspond to arithmetic mean±SEM.

    Techniques Used: Expressing, Irradiation, Derivative Assay

    15) Product Images from "A G-quadruplex structure at the 5′ end of the H19 coding region regulates H19 transcription"

    Article Title: A G-quadruplex structure at the 5′ end of the H19 coding region regulates H19 transcription

    Journal: Scientific Reports

    doi: 10.1038/srep45815

    H19 G-quadruplex suppresses transcription. ( A ) Schematic diagram of the H19 expression vectors used in this study. The full length H19 gene (WT- H19 ), G-quadruplex sequence-truncated H19 gene (∆G4- H19 ), or H19 gene with Mut2 or Mut3 G-quadruplex sequence (Mut2-G4- H19 , Mut3-G4- H19 ) were driven by the EF1α promoter. ( B ) qPCR analysis of H19 RNA levels in 293T cells transfected with the indicated plasmids. Values are normalized against G3PDH. ( C ) PCR analysis for transfection efficiency of the plasmids in ( B ) using a primer pair specific to the vector backbone. ( D ) Schematic diagram of the luciferase reporter plasmids. H19 G-quadruplex sequence was inserted just upstream of the luciferase gene (Luc). Luc or G-quadruplex-Luc (G4-Luc) was driven by the H19 promoter (the region between −840 to +14). ( E , F ) 293 T cells ( E ) or EpH4 cells ( F ) were transfected with the luciferase reporter plasmids together with the control RL-SV40 plasmid, and the luciferase activities measured. Values are normalized against the activity of co-transfected Renilla luciferase. ( B , E , F ) mean ± s.d. from three experiments; *P
    Figure Legend Snippet: H19 G-quadruplex suppresses transcription. ( A ) Schematic diagram of the H19 expression vectors used in this study. The full length H19 gene (WT- H19 ), G-quadruplex sequence-truncated H19 gene (∆G4- H19 ), or H19 gene with Mut2 or Mut3 G-quadruplex sequence (Mut2-G4- H19 , Mut3-G4- H19 ) were driven by the EF1α promoter. ( B ) qPCR analysis of H19 RNA levels in 293T cells transfected with the indicated plasmids. Values are normalized against G3PDH. ( C ) PCR analysis for transfection efficiency of the plasmids in ( B ) using a primer pair specific to the vector backbone. ( D ) Schematic diagram of the luciferase reporter plasmids. H19 G-quadruplex sequence was inserted just upstream of the luciferase gene (Luc). Luc or G-quadruplex-Luc (G4-Luc) was driven by the H19 promoter (the region between −840 to +14). ( E , F ) 293 T cells ( E ) or EpH4 cells ( F ) were transfected with the luciferase reporter plasmids together with the control RL-SV40 plasmid, and the luciferase activities measured. Values are normalized against the activity of co-transfected Renilla luciferase. ( B , E , F ) mean ± s.d. from three experiments; *P

    Techniques Used: Expressing, Sequencing, Real-time Polymerase Chain Reaction, Transfection, Polymerase Chain Reaction, Plasmid Preparation, Luciferase, Activity Assay

    L1H1-7OTD suppresses H19 transcription during mESC differentiation. ( A ) qRT-PCR analysis of RNA levels of H19, Oct4 and Sox1 during neural differentiation of mESCs by SFEBq at day 0, day 3, day 5 and day 7. ( B ) mESCs were differentiated in SFEBq medium in the presence or absence of L1H1-7OTD (5 μM). The qPCR analysis of H19, Oct4 and Sox1 RNA levels at differentiation Day 0 or Day 5 are shown. Values are normalized against β-actin. mean ± s.d. from three experiments; *P
    Figure Legend Snippet: L1H1-7OTD suppresses H19 transcription during mESC differentiation. ( A ) qRT-PCR analysis of RNA levels of H19, Oct4 and Sox1 during neural differentiation of mESCs by SFEBq at day 0, day 3, day 5 and day 7. ( B ) mESCs were differentiated in SFEBq medium in the presence or absence of L1H1-7OTD (5 μM). The qPCR analysis of H19, Oct4 and Sox1 RNA levels at differentiation Day 0 or Day 5 are shown. Values are normalized against β-actin. mean ± s.d. from three experiments; *P

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

    The genomic H19 G-quadruplex sequence regulates H19 expression. ( A , B ) Control mESCs (WT-G4 cell) or Mut2-G4-mESCs (Mut-G4-a cell)were cultured in mESC maintenance medium (undifferentiated) or differentiated in EB medium for 6 days (EB Day6) in the presence or absence of L1H1-7OTD (5 μM). The qPCR analyses of H19 RNA levels are shown. ( C ) qRT-PCR analysis of H19 RNA levels in WT- and Mut-G4-a, or -b cells in EB culture on day 6. ( D – F ) qRT-PCR analysis for the RNA levels of H19 ( D ), E2F1 ( E ) and Sp1 ( F ) in WT- and Mut2-G4 cells in mECS maintenance medium (undifferentiated) or in SFEBq culture on day 5. ( G , H ) qRT-PCR analysis for H19 RNA levels in WT- ( G ) and Mut2-G4 cells ( H ) transfected with or without the E2F1-coding plasmids. ( A – H ) Values are normalized against β-actin. mean ± s.d. from three experiments; *P
    Figure Legend Snippet: The genomic H19 G-quadruplex sequence regulates H19 expression. ( A , B ) Control mESCs (WT-G4 cell) or Mut2-G4-mESCs (Mut-G4-a cell)were cultured in mESC maintenance medium (undifferentiated) or differentiated in EB medium for 6 days (EB Day6) in the presence or absence of L1H1-7OTD (5 μM). The qPCR analyses of H19 RNA levels are shown. ( C ) qRT-PCR analysis of H19 RNA levels in WT- and Mut-G4-a, or -b cells in EB culture on day 6. ( D – F ) qRT-PCR analysis for the RNA levels of H19 ( D ), E2F1 ( E ) and Sp1 ( F ) in WT- and Mut2-G4 cells in mECS maintenance medium (undifferentiated) or in SFEBq culture on day 5. ( G , H ) qRT-PCR analysis for H19 RNA levels in WT- ( G ) and Mut2-G4 cells ( H ) transfected with or without the E2F1-coding plasmids. ( A – H ) Values are normalized against β-actin. mean ± s.d. from three experiments; *P

    Techniques Used: Sequencing, Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Transfection

    16) Product Images from "Mechanistic insights into type I toxin antitoxin systems in Helicobacter pylori: the importance of mRNA folding in controlling toxin expression"

    Article Title: Mechanistic insights into type I toxin antitoxin systems in Helicobacter pylori: the importance of mRNA folding in controlling toxin expression

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1343

    Structure probing of ( A ) AapA1_FL and ( B ) AapA1_Tr1 RNAs in presence or absence of IsoA1. The secondary structure of each in vitro transcribed RNA was probed by submitting ∼0.1 pmol 32 P- labeled RNA to partial enzymatic digestion either under native ( N ) or denaturing conditions ( D ) (RNase T1 cleaving single stranded G residues, lanes 3 and 4 ; RNase TA cleaving single stranded A residues, lanes 5 and 6). The interaction between AapA1 and IsoA1 was mapped using lead probing (lanes 7–11) in the absence (lane 7) or presence of either 2 or 10 times excess of wild-type IsoA1 (wt, lanes 8 and 10) or IsoA1 mutated in its apical loop (L1L2, lanes 9 and 11). Structure mapping, as well as secondary structures of IsoA1 wt and L1L2, are shown in Supplementary Figure S4 . Untreated RNA (lane 1, denoted C) and partially alkali digested RNA (lane 2, denoted OH) served as control and ladder, respectively. Cleaved fragments were analyzed on an 8% denaturing polyacrylamide gel. Positions of all G residues are indicated relative to the transcription start site of the aapA1 gene (left of the gel). Single stranded regions involved in the 5΄ structural rearrangement are indicated by vertical black bars (right of the gel). The internal loops I and II (IL I, IL II) present in AapA1_FL are replaced by an apical loop (AL II) and a 14-nucleotides linker (LK) in AapA1_Tr1 . Black stars on the right of the gel indicate other structural rearrangements observed following IsoA1 RNA binding.
    Figure Legend Snippet: Structure probing of ( A ) AapA1_FL and ( B ) AapA1_Tr1 RNAs in presence or absence of IsoA1. The secondary structure of each in vitro transcribed RNA was probed by submitting ∼0.1 pmol 32 P- labeled RNA to partial enzymatic digestion either under native ( N ) or denaturing conditions ( D ) (RNase T1 cleaving single stranded G residues, lanes 3 and 4 ; RNase TA cleaving single stranded A residues, lanes 5 and 6). The interaction between AapA1 and IsoA1 was mapped using lead probing (lanes 7–11) in the absence (lane 7) or presence of either 2 or 10 times excess of wild-type IsoA1 (wt, lanes 8 and 10) or IsoA1 mutated in its apical loop (L1L2, lanes 9 and 11). Structure mapping, as well as secondary structures of IsoA1 wt and L1L2, are shown in Supplementary Figure S4 . Untreated RNA (lane 1, denoted C) and partially alkali digested RNA (lane 2, denoted OH) served as control and ladder, respectively. Cleaved fragments were analyzed on an 8% denaturing polyacrylamide gel. Positions of all G residues are indicated relative to the transcription start site of the aapA1 gene (left of the gel). Single stranded regions involved in the 5΄ structural rearrangement are indicated by vertical black bars (right of the gel). The internal loops I and II (IL I, IL II) present in AapA1_FL are replaced by an apical loop (AL II) and a 14-nucleotides linker (LK) in AapA1_Tr1 . Black stars on the right of the gel indicate other structural rearrangements observed following IsoA1 RNA binding.

    Techniques Used: In Vitro, Labeling, RNA Binding Assay

    mRNA folding predictions of the different AapA1 transcripts. The sequences of the three different AapA1 mRNA species were inferred from the RNA-seq data ( 4 ) and from Northern Blot analyzed with a combination of various probes ( Supplementary Figure S3 ). Each secondary structure was predicted using RNAstructure 5.2 software ( 22 ) in agreement with the experimental data obtained by enzymatic and chemical footprinting (Figure 4 and Supplementary Figure S5 ). The secondary structures were visualized and designed with the VARNA applet ( 46 ). The AapA1-FL transcript corresponds to a 253 nt transcript in which most of the 5΄ UTR is engaged in a long distance interaction with the 3΄ end of the message. A 3΄ processing event removing the last 30 nt leads to the formation of a truncated transcript ( AapA1_Tr1 ) that undergoes a structural rearrangement, rendering the SD accessible to the ribosome. The first 76 nt of this translationally active message interact with IsoA1 to form an extended duplex (not shown here, see Supplementary Figure S5 ). This long duplex is then cleaved by RNase III to generate the AapA1_Tr2 transcript. The start and stop codons of the AapA1 ORF are indicated in orange and the SD sequence is in red. Apical loops (AL) are shown in blue. Two internal loops (IL) are shown in the AapA1_FL transcript.
    Figure Legend Snippet: mRNA folding predictions of the different AapA1 transcripts. The sequences of the three different AapA1 mRNA species were inferred from the RNA-seq data ( 4 ) and from Northern Blot analyzed with a combination of various probes ( Supplementary Figure S3 ). Each secondary structure was predicted using RNAstructure 5.2 software ( 22 ) in agreement with the experimental data obtained by enzymatic and chemical footprinting (Figure 4 and Supplementary Figure S5 ). The secondary structures were visualized and designed with the VARNA applet ( 46 ). The AapA1-FL transcript corresponds to a 253 nt transcript in which most of the 5΄ UTR is engaged in a long distance interaction with the 3΄ end of the message. A 3΄ processing event removing the last 30 nt leads to the formation of a truncated transcript ( AapA1_Tr1 ) that undergoes a structural rearrangement, rendering the SD accessible to the ribosome. The first 76 nt of this translationally active message interact with IsoA1 to form an extended duplex (not shown here, see Supplementary Figure S5 ). This long duplex is then cleaved by RNase III to generate the AapA1_Tr2 transcript. The start and stop codons of the AapA1 ORF are indicated in orange and the SD sequence is in red. Apical loops (AL) are shown in blue. Two internal loops (IL) are shown in the AapA1_FL transcript.

    Techniques Used: RNA Sequencing Assay, Northern Blot, Software, Footprinting, Sequencing

    17) Product Images from "Benchmarking of the Oxford Nanopore MinION sequencing for quantitative and qualitative assessment of cDNA populations"

    Article Title: Benchmarking of the Oxford Nanopore MinION sequencing for quantitative and qualitative assessment of cDNA populations

    Journal: Scientific Reports

    doi: 10.1038/srep31602

    Effect of the GC content and ERCC length on the estimation of the ERCC cDNA abundance with the ONT MinION platform. The figures present deviations of the ERCC expression level estimates with the ONT MinION platform from the Ambion RNA molecular counts ( A,C ) or from the Illumina HiSeq 2500/MiSeq estimated cDNA abundance ( B,D ) as a function of the GC content ( A,B ) and the ERCC length ( C,D ). We plot the log2 ratio of observed (ONT MinION) to expected (Ambion, Illumina) read counts for the ERCC spike-ins (y-axis, log) for each of the samples relative to their length or GC content (x-axis). Due to the variable sequencing depth from each ERCC MinION experiment, each point is the average value from different MinION flow cell runs if at least 5 reads have been detected for this point in the corresponding MinION runs. The points are colored differently based on the number of flow cell runs in which they were detected (red, green, cyan, purple correspond to values derived from one, two, three or four flow cell runs respectively). The standard deviation is also presented for points with values from two or more MinION runs.
    Figure Legend Snippet: Effect of the GC content and ERCC length on the estimation of the ERCC cDNA abundance with the ONT MinION platform. The figures present deviations of the ERCC expression level estimates with the ONT MinION platform from the Ambion RNA molecular counts ( A,C ) or from the Illumina HiSeq 2500/MiSeq estimated cDNA abundance ( B,D ) as a function of the GC content ( A,B ) and the ERCC length ( C,D ). We plot the log2 ratio of observed (ONT MinION) to expected (Ambion, Illumina) read counts for the ERCC spike-ins (y-axis, log) for each of the samples relative to their length or GC content (x-axis). Due to the variable sequencing depth from each ERCC MinION experiment, each point is the average value from different MinION flow cell runs if at least 5 reads have been detected for this point in the corresponding MinION runs. The points are colored differently based on the number of flow cell runs in which they were detected (red, green, cyan, purple correspond to values derived from one, two, three or four flow cell runs respectively). The standard deviation is also presented for points with values from two or more MinION runs.

    Techniques Used: Gas Chromatography, Expressing, Sequencing, Flow Cytometry, Derivative Assay, Standard Deviation

    Estimation of the ERCC cDNA abundance with the ONT MinION platform. We compared the ERCC cDNA abundance estimated from the ONT MinION ( A ) and the Illumina HiSeq 2500 or MiSeq platforms ( B ) against the expected number of RNA molecules as provided from the manufacturer (Ambion). The template reads from the ERCC MinION experiments number 1, 2, 3, 4 were pooled together and used for ( A ). Similarly, the corresponding Illumina data were pooled together for ( B ). The Illumina molecular counts data were derived using 5′ molecular tags at the RT step as described in material and methods. The total number of molecules presented on the x-axis corresponds to 3.5 pgs of ERCC RNA. In both axes the log10 transformation of the original count number is used.
    Figure Legend Snippet: Estimation of the ERCC cDNA abundance with the ONT MinION platform. We compared the ERCC cDNA abundance estimated from the ONT MinION ( A ) and the Illumina HiSeq 2500 or MiSeq platforms ( B ) against the expected number of RNA molecules as provided from the manufacturer (Ambion). The template reads from the ERCC MinION experiments number 1, 2, 3, 4 were pooled together and used for ( A ). Similarly, the corresponding Illumina data were pooled together for ( B ). The Illumina molecular counts data were derived using 5′ molecular tags at the RT step as described in material and methods. The total number of molecules presented on the x-axis corresponds to 3.5 pgs of ERCC RNA. In both axes the log10 transformation of the original count number is used.

    Techniques Used: Derivative Assay, Transformation Assay

    18) Product Images from "The chaperone-like activity of the hepatitis C virus IRES and CRE elements regulates genome dimerization"

    Article Title: The chaperone-like activity of the hepatitis C virus IRES and CRE elements regulates genome dimerization

    Journal: Scientific Reports

    doi: 10.1038/srep43415

    Differential dimerization ability of the 3′X-tail induced by the IRES and the CRE. ( a ) Left panel, dimerization process efficiency was monitored at different concentrations of 3′X, CU, I+CU and I+U. Dimeric products were resolved by native agarose (for CU, I+CU and I+U) or polyacrylamide gel electrophoresis (for 3′X). A representative image of the electrophoretic mobility shift assays is shown. Relative dimer formation for each transcript was quantified (right panel). Data represent the mean of at least three independent experiments ± standard deviation. ( b ) Core protein addition strongly promoted dimer formation for transcript 3′X, and to a lesser extent for CU, I+U and I+CU. Dimerization assays were performed in the presence of the core 2BD peptide at a protein to nucleotide molar ratio of 1:50. After 15 min incubation at 37 °C, the core protein was removed by digestion with proteinase K. RNA complexes were resolved by native polyacrylamide gels. A representative dimerization experiment for each transcript is shown in the left panel. Relative dimer formation was quantified (right panel). Data are the mean of three independent assays ± standard deviation. ( c ) Dimeric complexing reached maximum efficiency at high temperatures for all the transcripts tested. Fixed amounts of the transcripts were tested for their ability to dimerize at different temperatures. Reaction products were processed and analyzed as mentioned above. Values are the means of three independent assays ± standard deviation.
    Figure Legend Snippet: Differential dimerization ability of the 3′X-tail induced by the IRES and the CRE. ( a ) Left panel, dimerization process efficiency was monitored at different concentrations of 3′X, CU, I+CU and I+U. Dimeric products were resolved by native agarose (for CU, I+CU and I+U) or polyacrylamide gel electrophoresis (for 3′X). A representative image of the electrophoretic mobility shift assays is shown. Relative dimer formation for each transcript was quantified (right panel). Data represent the mean of at least three independent experiments ± standard deviation. ( b ) Core protein addition strongly promoted dimer formation for transcript 3′X, and to a lesser extent for CU, I+U and I+CU. Dimerization assays were performed in the presence of the core 2BD peptide at a protein to nucleotide molar ratio of 1:50. After 15 min incubation at 37 °C, the core protein was removed by digestion with proteinase K. RNA complexes were resolved by native polyacrylamide gels. A representative dimerization experiment for each transcript is shown in the left panel. Relative dimer formation was quantified (right panel). Data are the mean of three independent assays ± standard deviation. ( c ) Dimeric complexing reached maximum efficiency at high temperatures for all the transcripts tested. Fixed amounts of the transcripts were tested for their ability to dimerize at different temperatures. Reaction products were processed and analyzed as mentioned above. Values are the means of three independent assays ± standard deviation.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Electrophoretic Mobility Shift Assay, Standard Deviation, Incubation

    The IRES and the CRE control HCV dimerization efficiency. Data obtained from HMX assays were used to design a series of variants bearing different deletions or single-point mutations within the CRE and IRES. ( a ) Dimerization efficiency was monitored for deletion mutants of different RNA domains located throughout the CRE and the 3′UTR for I+CU. A defective dimerization mutant, the so-called_dimUU molecule 32 containing the point A9539U mutation in the DLS motif, was employed as a negative control. Dimeric and monomeric conformers were resolved by native agarose gel electrophoresis. The relative dimerization yield for each molecule tested was quantified and normalized to that obtained for the non-mutated control transcript. Data are the mean of at least three independent experiments ± standard deviation. ( b,c ) Single-point mutations were introduced in the IRES region as indicated, for both I+CU ( b ) and I+U ( c ). The effect on dimer formation was evaluated and quantified as noted above. Values represent the mean of three independent assays ± standard deviation.
    Figure Legend Snippet: The IRES and the CRE control HCV dimerization efficiency. Data obtained from HMX assays were used to design a series of variants bearing different deletions or single-point mutations within the CRE and IRES. ( a ) Dimerization efficiency was monitored for deletion mutants of different RNA domains located throughout the CRE and the 3′UTR for I+CU. A defective dimerization mutant, the so-called_dimUU molecule 32 containing the point A9539U mutation in the DLS motif, was employed as a negative control. Dimeric and monomeric conformers were resolved by native agarose gel electrophoresis. The relative dimerization yield for each molecule tested was quantified and normalized to that obtained for the non-mutated control transcript. Data are the mean of at least three independent experiments ± standard deviation. ( b,c ) Single-point mutations were introduced in the IRES region as indicated, for both I+CU ( b ) and I+U ( c ). The effect on dimer formation was evaluated and quantified as noted above. Values represent the mean of three independent assays ± standard deviation.

    Techniques Used: Mutagenesis, Negative Control, Agarose Gel Electrophoresis, Standard Deviation

    19) Product Images from "Protective Effects of CISD2 and Influence of Curcumin on CISD2 Expression in Aged Animals and Inflammatory Cell Model"

    Article Title: Protective Effects of CISD2 and Influence of Curcumin on CISD2 Expression in Aged Animals and Inflammatory Cell Model

    Journal: Nutrients

    doi: 10.3390/nu11030700

    Curcumin enhanced cell viability and attenuated LPS-challenge-driven cell death in non-stressed and LPS-challenged neural cells. The knockdown of CISD2 expression was shown to reduce the protective effects of curcumin. The eight groups of cells included the following: (i) scrambled RNA-transfected control cells, untreated with LPS and curcumin; (ii) scrambled RNA-transfected cells treated with curcumin; (iii) scramble RNA-transfected, LPS-challenged cells, without curcumin treatment; (iv) scramble RNA-transfected, LPS-challenged cells treated with curcumin; (v) siCID2-transfected control cells without LPS or curcumin treatment; (vi) siCID2-transfected cells treated with curcumin; (vii) siCID2-transfected, LPS-challenged cells without curcumin treatment; (viii) siCID2-transfected, LPS-challenged cells, treated with curcumin. Cell viability was measured using alamarBlue ® assay. Vertical bars indicate the mean ± SEM (n = 3). #, ,* p
    Figure Legend Snippet: Curcumin enhanced cell viability and attenuated LPS-challenge-driven cell death in non-stressed and LPS-challenged neural cells. The knockdown of CISD2 expression was shown to reduce the protective effects of curcumin. The eight groups of cells included the following: (i) scrambled RNA-transfected control cells, untreated with LPS and curcumin; (ii) scrambled RNA-transfected cells treated with curcumin; (iii) scramble RNA-transfected, LPS-challenged cells, without curcumin treatment; (iv) scramble RNA-transfected, LPS-challenged cells treated with curcumin; (v) siCID2-transfected control cells without LPS or curcumin treatment; (vi) siCID2-transfected cells treated with curcumin; (vii) siCID2-transfected, LPS-challenged cells without curcumin treatment; (viii) siCID2-transfected, LPS-challenged cells, treated with curcumin. Cell viability was measured using alamarBlue ® assay. Vertical bars indicate the mean ± SEM (n = 3). #, ,* p

    Techniques Used: Expressing, Transfection, Alamar Blue Assay

    Mitochondrial membrane potential [DeltaPsi(m)] of lipopolysaccharide (LPS)-challenged neural cells (with or without curcumin treatment), as determined using JC-1 staining and flow cytometry with or without CISD2 knockdown. Cells presenting a decrease in DeltaPsi(m) and impending cellular apoptosis are indicated by monomer green JC-1 fluorescence, compared to normal cells with intact DeltaPsi(m) (indicated by j-aggregated red fluorescence). All groups of cells were evaluated in terms of the ratio of PE+ to PE−. ( A ) Representative results of flow cytometry for the following groups of cells: (i) scramble RNA-transfected control cells untreated with LPS; (ii) scramble RNA-transfected, LPS-challenged cells, untreated with curcumin; (iii) scramble RNA-transfected, LPS-challenged cells following curcumin treatment; (iv) siCID2-transfected control cells without LPS or curcumin treatment; (v) siCID2-transfected, LPS-challenged cells without curcumin treatment; (vi) siCID2-transfected, LPS-challenged cells, treated with curcumin. ( B ) Bars indicate the mean ± SEM (n = 3). #, p
    Figure Legend Snippet: Mitochondrial membrane potential [DeltaPsi(m)] of lipopolysaccharide (LPS)-challenged neural cells (with or without curcumin treatment), as determined using JC-1 staining and flow cytometry with or without CISD2 knockdown. Cells presenting a decrease in DeltaPsi(m) and impending cellular apoptosis are indicated by monomer green JC-1 fluorescence, compared to normal cells with intact DeltaPsi(m) (indicated by j-aggregated red fluorescence). All groups of cells were evaluated in terms of the ratio of PE+ to PE−. ( A ) Representative results of flow cytometry for the following groups of cells: (i) scramble RNA-transfected control cells untreated with LPS; (ii) scramble RNA-transfected, LPS-challenged cells, untreated with curcumin; (iii) scramble RNA-transfected, LPS-challenged cells following curcumin treatment; (iv) siCID2-transfected control cells without LPS or curcumin treatment; (v) siCID2-transfected, LPS-challenged cells without curcumin treatment; (vi) siCID2-transfected, LPS-challenged cells, treated with curcumin. ( B ) Bars indicate the mean ± SEM (n = 3). #, p

    Techniques Used: Staining, Flow Cytometry, Cytometry, Fluorescence, Transfection

    20) Product Images from "Targeting of cadherin-11 decreases skin fibrosis in the tight skin-1 mouse model"

    Article Title: Targeting of cadherin-11 decreases skin fibrosis in the tight skin-1 mouse model

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0187109

    Anti-cadherin-11 antibody decreases mediators of fibrosis in Tsk-1 mice. Total RNA was isolated from skin biopsies from pa/pa control mice, Tsk-1 mice treated with isotype antibodies, and Tsk-1 mice treated with antibodies against CDH11 (13C2). Transcripts were determined for Col1 α 1 (a), α SMA (b), CCN2 (c), fibronectin (d), TGF- β (e), IL-6 (f) in parallel with 18S rRNA. Data are presented as mean of fold change transcripts ± SEM, n≥10 (*p ≤ 0.05 pa/pa vs. Tsk-1 isotype; #p ≤ 0.05 Tsk-1 isotype vs. Tsk-1 anti-CDH11).
    Figure Legend Snippet: Anti-cadherin-11 antibody decreases mediators of fibrosis in Tsk-1 mice. Total RNA was isolated from skin biopsies from pa/pa control mice, Tsk-1 mice treated with isotype antibodies, and Tsk-1 mice treated with antibodies against CDH11 (13C2). Transcripts were determined for Col1 α 1 (a), α SMA (b), CCN2 (c), fibronectin (d), TGF- β (e), IL-6 (f) in parallel with 18S rRNA. Data are presented as mean of fold change transcripts ± SEM, n≥10 (*p ≤ 0.05 pa/pa vs. Tsk-1 isotype; #p ≤ 0.05 Tsk-1 isotype vs. Tsk-1 anti-CDH11).

    Techniques Used: Mouse Assay, Isolation

    Characterization of Cadherin-11 expression in the Tsk-1 mouse model of skin fibrosis. (a) Skin biopsies from pa/pa and Tsk-1 mice were stained with H E demonstrating increased thickness of the hypodermis (arrow) in Tsk-1 mice compared to pa/pa mice. (10x magnification, scale bar 50 μm). These data were quantified in (b) (n = 10; * p ≤ 0.05 pa/pa vs. Tsk-1). (c) Tsk-1 mice have increased deposition of collagen using Sircol assay (n = 10; * p ≤ 0.05 pa/pa vs. Tsk-1). (d) Skin biopsies were used to isolate total RNA and transcripts were determined for Cdh11 . Transcripts were measured in parallel with 18S RNA and values are presented as mean of fold change transcripts ± SEM, n = 10 (* p ≤ 0.05 pa/pa vs. Tsk-1). (e) Immunofluorescence of CDH11 expression was determined in the hypodermal layer (arrows) in skin sections from pa/pa and Tsk-1 mice. Images are representative of 10 mice from each group. Top panels, Low magnification 20X, scale bars: 50 μm. Middle and lower panel, high magnification 100x, scale bars: 10 μm. (f) Dual color IF of hypodermis from pa/pa and Tsk-1 mice stained for α SMA (red) and CDH11 (green) or F4/80 (red) and CDH11 (green) demonstrate that CDH11 expression primarily localizes to α SMA myofibroblasts (100x, scale bars: 10 μm). (g, h) Quantification of α SMA and F4/80 positive cells that express CDH11 per high power field in the hypodermis of pa/pa and Tsk-1 mice (* p ≤ 0.05 pa/pa vs. Tsk-1).
    Figure Legend Snippet: Characterization of Cadherin-11 expression in the Tsk-1 mouse model of skin fibrosis. (a) Skin biopsies from pa/pa and Tsk-1 mice were stained with H E demonstrating increased thickness of the hypodermis (arrow) in Tsk-1 mice compared to pa/pa mice. (10x magnification, scale bar 50 μm). These data were quantified in (b) (n = 10; * p ≤ 0.05 pa/pa vs. Tsk-1). (c) Tsk-1 mice have increased deposition of collagen using Sircol assay (n = 10; * p ≤ 0.05 pa/pa vs. Tsk-1). (d) Skin biopsies were used to isolate total RNA and transcripts were determined for Cdh11 . Transcripts were measured in parallel with 18S RNA and values are presented as mean of fold change transcripts ± SEM, n = 10 (* p ≤ 0.05 pa/pa vs. Tsk-1). (e) Immunofluorescence of CDH11 expression was determined in the hypodermal layer (arrows) in skin sections from pa/pa and Tsk-1 mice. Images are representative of 10 mice from each group. Top panels, Low magnification 20X, scale bars: 50 μm. Middle and lower panel, high magnification 100x, scale bars: 10 μm. (f) Dual color IF of hypodermis from pa/pa and Tsk-1 mice stained for α SMA (red) and CDH11 (green) or F4/80 (red) and CDH11 (green) demonstrate that CDH11 expression primarily localizes to α SMA myofibroblasts (100x, scale bars: 10 μm). (g, h) Quantification of α SMA and F4/80 positive cells that express CDH11 per high power field in the hypodermis of pa/pa and Tsk-1 mice (* p ≤ 0.05 pa/pa vs. Tsk-1).

    Techniques Used: Expressing, Mouse Assay, Staining, Immunofluorescence

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    Amplification:

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    Stable Transfection:

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    Construct:

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    Adsorption:

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    Enzyme-linked Immunosorbent Assay:

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    Microarray:

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    Incubation:

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    Expressing:

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    Modification:

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    Electron Microscopy:

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    Chromatography:

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    Ligation:

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    Infection:

    Article Title: A cytoplasmic RNA virus generates functional viral small RNAs and regulates viral IRES activity in mammalian cells
    Article Snippet: After viral adsorption, the infected cells in each well were transfected with 160 pmol of synthetic mimic vsRNA1 (5′-GUAACUUAGAAGCUGUAAAUCAACGAUCA-3′) or scrambled vsRNA1 (5′-AAUGCUAUGAGACUAAUGAUACCAAGACU-3′) and 4 μl of Lipofectamine 2000 (Invitrogen). .. For the anti-vsRNA1 sponge RNA experiments, the infected cells were transfected with 1 μg of the anti-vsRNA1 sponge or scrambled RNA and 2 μl of Lipofectamine 2000 (Invitrogen). .. For the LNA experiment, the infected cells were transfected with 80 pmol/ml of LNA-vsRNA1 (5′ LNA-TUGAUUUACAGCUUCU) or LNA-control (5′ LNA-CAGUACUUUUGUGUAGUACAA) after virus adsorption.

    Confocal Laser Scanning Microscopy:

    Article Title: Design, synthesis and evaluation of VEGF-siRNA/CRS as a novel vector for gene delivery
    Article Snippet: To-Pro® -3 Iodide (642⁄661), diethylpyrocarbonate (DEPC) water, Lipofectamine™ 2000, TRIzol reagent, VEGF ELISA Kit, RNA to CDNA and TaqMan Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA). .. To-Pro® -3 Iodide (642⁄661), diethylpyrocarbonate (DEPC) water, Lipofectamine™ 2000, TRIzol reagent, VEGF ELISA Kit, RNA to CDNA and TaqMan Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Imaging:

    Article Title: Design, synthesis and evaluation of VEGF-siRNA/CRS as a novel vector for gene delivery
    Article Snippet: To-Pro® -3 Iodide (642⁄661), diethylpyrocarbonate (DEPC) water, Lipofectamine™ 2000, TRIzol reagent, VEGF ELISA Kit, RNA to CDNA and TaqMan Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA). .. To-Pro® -3 Iodide (642⁄661), diethylpyrocarbonate (DEPC) water, Lipofectamine™ 2000, TRIzol reagent, VEGF ELISA Kit, RNA to CDNA and TaqMan Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Transmission Assay:

    Article Title: Design, synthesis and evaluation of VEGF-siRNA/CRS as a novel vector for gene delivery
    Article Snippet: To-Pro® -3 Iodide (642⁄661), diethylpyrocarbonate (DEPC) water, Lipofectamine™ 2000, TRIzol reagent, VEGF ELISA Kit, RNA to CDNA and TaqMan Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA). .. To-Pro® -3 Iodide (642⁄661), diethylpyrocarbonate (DEPC) water, Lipofectamine™ 2000, TRIzol reagent, VEGF ELISA Kit, RNA to CDNA and TaqMan Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Sequencing:

    Article Title: Design, synthesis and evaluation of VEGF-siRNA/CRS as a novel vector for gene delivery
    Article Snippet: VEGF-siRNA, negative control (NC), fluorescein-labeled siRNA (Cy3/FAM-siRNA), VEGF primers (forward sequence: 5′-ATCGAGACCCTGGTGGACA-3′, reverse sequence: 5′-CCGCCTCGGCTTGTCACA-3′) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers (forward sequence: 5′-CAAATTCCATGGCACCGTCA-3′, reverse sequence: 5′-GGAGTGGGTGTCGCTGTTGA-3′) were synthesized and purified by GenePharma Co., Ltd. (Shanghai, China). .. To-Pro® -3 Iodide (642⁄661), diethylpyrocarbonate (DEPC) water, Lipofectamine™ 2000, TRIzol reagent, VEGF ELISA Kit, RNA to CDNA and TaqMan Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Article Title: The cholesterol biosynthesis pathway regulates IL-10 expression in human Th1 cells
    Article Snippet: Cell pellets for mRNA isolation were lysed in Trizol (Ambion, Life Technologies) and RNA was extracted by guanidium-isothescyanate phenol/chloroform extraction method following the manufacturer’s protocols. .. Cell pellets for mRNA isolation were lysed in Trizol (Ambion, Life Technologies) and RNA was extracted by guanidium-isothescyanate phenol/chloroform extraction method following the manufacturer’s protocols.

    Article Title: Regulatory consequences of neuronal ELAV-like protein binding to coding and non-coding RNAs in human brain
    Article Snippet: All scripts used in the analysis including the peak finding algorithm and more information can be publically obtained at http://zhanglab.c2b2.columbia.edu/index.php/Resources . .. RNA from human brain and IMR-32 neuroblastoma cell lines was Trizol (Invitrogen)-extracted, Ribo-Zero-selected (Epicentre, Madison, WI), DNase-treated (Roche), and prepared for sequencing, following the Illumina High-throughput TruSeq RNA Sample Preparation Guidelines. .. The libraries from subjects 6–8 and 15–17, as well Y RNA overexpression samples, were sequenced on an Illumina HiSeq 2500 at the New York Genome Center, generating 125-bp paired end reads.

    Sonication:

    Article Title: Assembly of AUF1 with eIF4G-poly(A) binding protein complex suggests a translation function in AU-rich mRNA decay
    Article Snippet: Cells were resuspended in lysis buffer (10 mM Tris-HCl at pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.7 mg/mL lysozyme, 1% NP-40, 1 mM DTT, 1 mM PMSF) and sonicated for 2 min. After addition of NaCl (final 450 mM), cell debris was removed by centrifugation at 15,000 rpm for 30 min at 4°C. .. Some preparations of PABP were treated with a ribonuclease cocktail to eliminate contaminating RNA (Ambion), as indicated.

    Recombinant:

    Article Title: Assembly of AUF1 with eIF4G-poly(A) binding protein complex suggests a translation function in AU-rich mRNA decay
    Article Snippet: Paragraph title: Recombinant proteins and antibodies ... Some preparations of PABP were treated with a ribonuclease cocktail to eliminate contaminating RNA (Ambion), as indicated.

    Molecular Weight:

    Article Title: Design, synthesis and evaluation of VEGF-siRNA/CRS as a novel vector for gene delivery
    Article Snippet: To-Pro® -3 Iodide (642⁄661), diethylpyrocarbonate (DEPC) water, Lipofectamine™ 2000, TRIzol reagent, VEGF ELISA Kit, RNA to CDNA and TaqMan Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA). .. To-Pro® -3 Iodide (642⁄661), diethylpyrocarbonate (DEPC) water, Lipofectamine™ 2000, TRIzol reagent, VEGF ELISA Kit, RNA to CDNA and TaqMan Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Marker:

    Article Title: Design, synthesis and evaluation of VEGF-siRNA/CRS as a novel vector for gene delivery
    Article Snippet: Cy3 marker was purchased from AAT Bioquest Inc. (Sunnyvale, CA, USA). .. To-Pro® -3 Iodide (642⁄661), diethylpyrocarbonate (DEPC) water, Lipofectamine™ 2000, TRIzol reagent, VEGF ELISA Kit, RNA to CDNA and TaqMan Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Isolation:

    Article Title: The cholesterol biosynthesis pathway regulates IL-10 expression in human Th1 cells
    Article Snippet: For IFNα-inducing experiments, CD4+ T cells (2.5 × 106 ml−1 ) were cultured with plate-bound CD3 (0.5 μgml−1 ), soluble CD28 (clone CD28.2, BD Biosciences, 1 μgml−1 ) and IFNα 2a (PBL Assay Science, 600 Uml−1 ) for 3 days . .. For primary cell experiments, cell pellets for mRNA isolation were lysed in Trizol (Ambion, Life Technologies) and RNA extracted by guanidium-isothiocyanate phenol/chloroform extraction method following the manufacturer’s instructions. .. Total RNA was quantified using the Nanodrop ND1000 and RNA integrity assessed with the Agilent Bioanalyzer 2100 using the RNA 6000 Nano Chips.

    Article Title: The cholesterol biosynthesis pathway regulates IL-10 expression in human Th1 cells
    Article Snippet: For IL-4 measurements, a BD Cytometric Bead Array kit (BD Biosciences) was used and data were analysed according to the manufacturer’s protocol. .. Cell pellets for mRNA isolation were lysed in Trizol (Ambion, Life Technologies) and RNA was extracted by guanidium-isothescyanate phenol/chloroform extraction method following the manufacturer’s protocols. .. Total RNA was quantified using the Nanodrop ND1000.

    Negative Control:

    Article Title: Design, synthesis and evaluation of VEGF-siRNA/CRS as a novel vector for gene delivery
    Article Snippet: VEGF-siRNA, negative control (NC), fluorescein-labeled siRNA (Cy3/FAM-siRNA), VEGF primers (forward sequence: 5′-ATCGAGACCCTGGTGGACA-3′, reverse sequence: 5′-CCGCCTCGGCTTGTCACA-3′) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers (forward sequence: 5′-CAAATTCCATGGCACCGTCA-3′, reverse sequence: 5′-GGAGTGGGTGTCGCTGTTGA-3′) were synthesized and purified by GenePharma Co., Ltd. (Shanghai, China). .. To-Pro® -3 Iodide (642⁄661), diethylpyrocarbonate (DEPC) water, Lipofectamine™ 2000, TRIzol reagent, VEGF ELISA Kit, RNA to CDNA and TaqMan Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Article Title: Adiponectin ameliorates angiotensin II-induced vascular endothelial damage
    Article Snippet: Cells were pretreated with adiponectin (Sigma-Aldrich, USA) at 1, 5, or 10 μg/ml for 4 h before adding 1-μM Ang II. .. Small interfering RNA (siRNA) for AdipoR1 and AdipoR2, APPL1, AMPK, and scrambled RNA (negative control) were from Invitrogen, USA. .. Cells were transfected with target siRNAs using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions (Shi H et al. ).

    Transfection:

    Article Title: A cytoplasmic RNA virus generates functional viral small RNAs and regulates viral IRES activity in mammalian cells
    Article Snippet: After viral adsorption, the infected cells in each well were transfected with 160 pmol of synthetic mimic vsRNA1 (5′-GUAACUUAGAAGCUGUAAAUCAACGAUCA-3′) or scrambled vsRNA1 (5′-AAUGCUAUGAGACUAAUGAUACCAAGACU-3′) and 4 μl of Lipofectamine 2000 (Invitrogen). .. For the anti-vsRNA1 sponge RNA experiments, the infected cells were transfected with 1 μg of the anti-vsRNA1 sponge or scrambled RNA and 2 μl of Lipofectamine 2000 (Invitrogen). .. For the LNA experiment, the infected cells were transfected with 80 pmol/ml of LNA-vsRNA1 (5′ LNA-TUGAUUUACAGCUUCU) or LNA-control (5′ LNA-CAGUACUUUUGUGUAGUACAA) after virus adsorption.

    Article Title: The influence of viral RNA secondary structure on interactions with innate host cell defences
    Article Snippet: Approximately 1.0 × 105 cells were seeded a day before transfections into 24 well plates. .. RNA was transfected using Lipofectamine 2000 (Invitrogen); RNA in 50 μl of DMEM was incubated for 5 min with 1 μl of Lipofectamine 2000, incubated for 20 min and added to wells containing target cells at 90% confluency. .. Transfected cells were incubated for 8 h and RNA was extracted and isolated using the RNeasy (Qiagen) or Illustra RNAspin kit (GE Healthcare) according to the manufacturer’s instructions, both including DNase treatment.

    Article Title: Genomic HIV RNA Induces Innate Immune Responses through RIG-I-Dependent Sensing of Secondary-Structured RNA
    Article Snippet: Human PBMCs were plated at 1×106 cells/ml overnight in RPMI containing 10% FCS onto 12 mm coverslips. .. Cells were transfected, with 1.2 µg/ml FAM-labelled HIV-Tar RNA at a RNA∶Lipofectamine 2000 (Invitrogen) ratio of 1∶2.5 (mass∶volume) for 180 min. .. Cells were subsequently fixed with methanol at −20°C for 5 min and stained with anti-Catalase (Abcam) to label peroxisomes or with DAPI to label nuclear DNA.

    Article Title: Adiponectin ameliorates angiotensin II-induced vascular endothelial damage
    Article Snippet: Paragraph title: Cell culture, treatment, and transfection ... Small interfering RNA (siRNA) for AdipoR1 and AdipoR2, APPL1, AMPK, and scrambled RNA (negative control) were from Invitrogen, USA.

    Microscopy:

    Article Title: Design, synthesis and evaluation of VEGF-siRNA/CRS as a novel vector for gene delivery
    Article Snippet: To-Pro® -3 Iodide (642⁄661), diethylpyrocarbonate (DEPC) water, Lipofectamine™ 2000, TRIzol reagent, VEGF ELISA Kit, RNA to CDNA and TaqMan Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA). .. To-Pro® -3 Iodide (642⁄661), diethylpyrocarbonate (DEPC) water, Lipofectamine™ 2000, TRIzol reagent, VEGF ELISA Kit, RNA to CDNA and TaqMan Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Article Title: Genomic HIV RNA Induces Innate Immune Responses through RIG-I-Dependent Sensing of Secondary-Structured RNA
    Article Snippet: Cells were transfected, with 1.2 µg/ml FAM-labelled HIV-Tar RNA at a RNA∶Lipofectamine 2000 (Invitrogen) ratio of 1∶2.5 (mass∶volume) for 180 min. .. Cells were transfected, with 1.2 µg/ml FAM-labelled HIV-Tar RNA at a RNA∶Lipofectamine 2000 (Invitrogen) ratio of 1∶2.5 (mass∶volume) for 180 min.

    Purification:

    Article Title: Design, synthesis and evaluation of VEGF-siRNA/CRS as a novel vector for gene delivery
    Article Snippet: VEGF-siRNA, negative control (NC), fluorescein-labeled siRNA (Cy3/FAM-siRNA), VEGF primers (forward sequence: 5′-ATCGAGACCCTGGTGGACA-3′, reverse sequence: 5′-CCGCCTCGGCTTGTCACA-3′) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) primers (forward sequence: 5′-CAAATTCCATGGCACCGTCA-3′, reverse sequence: 5′-GGAGTGGGTGTCGCTGTTGA-3′) were synthesized and purified by GenePharma Co., Ltd. (Shanghai, China). .. To-Pro® -3 Iodide (642⁄661), diethylpyrocarbonate (DEPC) water, Lipofectamine™ 2000, TRIzol reagent, VEGF ELISA Kit, RNA to CDNA and TaqMan Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Article Title: Manipulation of both virus- and cell-specific factors is required for robust transient replication of a hepatitis C virus genotype 3a sub-genomic replicon
    Article Snippet: Cells were mounted using Prolong Gold antifade mountant with DAPI and imaged using a Carl Zeiss LSM 700 inverted microscope and Zeiss Zen 2012 software. .. Stable SGR-harbouring cells were harvested in TRIZol (Invitrogen Life Technologies) and RNA purified according to the manufacturers’ instructions. .. One microgram of RNA was reverse-transcribed using Superscript II (Invitrogen) and random hexamer primers.

    Article Title: Structural Basis for NusA Stabilized Transcriptional Pausing
    Article Snippet: The final protein was concentrated to > 50 mg/ml, aliquots were flash frozen, and stored at −80°C. .. DNA (TriLink) and RNA (Dharmacon) oligonucleotides were chemically synthesized and gel purified by the manufacturer. .. RNA was deprotected following the protocols provided by the manufacturer.

    Article Title: Assembly of AUF1 with eIF4G-poly(A) binding protein complex suggests a translation function in AU-rich mRNA decay
    Article Snippet: His-tagged forms of proteins (AUF1, PABP) were purified similarly as described above except that cells were lysed in a His6 -extract buffer (50 mM NaH2 PO4 at pH 7.0, 300 mM NaCl, 0.5% NP-40, 0.7 mg/mL lysozyme, 1 mM PMSF). .. Some preparations of PABP were treated with a ribonuclease cocktail to eliminate contaminating RNA (Ambion), as indicated.

    Polymerase Chain Reaction:

    Article Title: The U1, U2 and U5 snRNAs crosslink to the 5? exon during yeast pre-mRNA splicing
    Article Snippet: CYH2 pre-mRNAs containing 4-thioU were produced by RNA ligation of a chemically synthesized 5′ RNA (Dharmacon) to an in vitro transcribed 3′ RNA ( ) by published procedures ( ). .. The 3′ RNA was made by in vitro transcription from PCR product containing a T7 promoter and primed with UpG (Sigma) to allow end labeling with 32 P. The RPS10B sequence was amplified from genomic DNA then inserted into pBluescript II KS+ (Stratagene) to produce plasmid pBS-RPS10B.

    Article Title: Manipulation of both virus- and cell-specific factors is required for robust transient replication of a hepatitis C virus genotype 3a sub-genomic replicon
    Article Snippet: Paragraph title: RNA extraction from cells and PCR ... Stable SGR-harbouring cells were harvested in TRIZol (Invitrogen Life Technologies) and RNA purified according to the manufacturers’ instructions.

    Article Title: Immune Activations and Viral Tissue Compartmentalization During Progressive HIV-1 Infection of Humanized Mice
    Article Snippet: The first round of the PCR was performed on a conventional PCR machine (T100 Thermal Cycler, BioRad, CA). .. The expression levels of tissue HIV-1 DNA and RNA were normalized to those for the human CD45 gene (Life Technology, CA).

    Article Title: HIV-1 cellular and tissue replication patterns in infected humanized mice
    Article Snippet: The amplicon sizes were 171 bp for the first PCR and 115 bp for the second (real-time) PCR of the msRNA assay. .. For sorted cells, levels of HIV-1 DNA and RNA were normalized to the expression of the housekeeping gene human GAPDH (Life Technology, California, USA).

    Article Title: The cholesterol biosynthesis pathway regulates IL-10 expression in human Th1 cells
    Article Snippet: Cell pellets for mRNA isolation were lysed in Trizol (Ambion, Life Technologies) and RNA was extracted by guanidium-isothescyanate phenol/chloroform extraction method following the manufacturer’s protocols. .. Cell pellets for mRNA isolation were lysed in Trizol (Ambion, Life Technologies) and RNA was extracted by guanidium-isothescyanate phenol/chloroform extraction method following the manufacturer’s protocols.

    Small Interfering RNA:

    Article Title: Adiponectin ameliorates angiotensin II-induced vascular endothelial damage
    Article Snippet: Cells were pretreated with adiponectin (Sigma-Aldrich, USA) at 1, 5, or 10 μg/ml for 4 h before adding 1-μM Ang II. .. Small interfering RNA (siRNA) for AdipoR1 and AdipoR2, APPL1, AMPK, and scrambled RNA (negative control) were from Invitrogen, USA. .. Cells were transfected with target siRNAs using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions (Shi H et al. ).

    Labeling:

    Article Title: The U1, U2 and U5 snRNAs crosslink to the 5? exon during yeast pre-mRNA splicing
    Article Snippet: Body labeled CYH2 pre-mRNA was produced as previously described ( ). .. CYH2 pre-mRNAs containing 4-thioU were produced by RNA ligation of a chemically synthesized 5′ RNA (Dharmacon) to an in vitro transcribed 3′ RNA ( ) by published procedures ( ).

    Article Title: Biochemical Characterization of Mycobacterium smegmatis RnhC (MSMEG_4305), a Bifunctional Enzyme Composed of Autonomous N-Terminal Type I RNase H and C-Terminal Acid Phosphatase Domains
    Article Snippet: RNA and DNA oligonucleotides were purchased from Dharmacon and Thermo Fisher Scientific, respectively. .. Oligonucleotides were 5′ 32 P labeled by reaction with T4 polynucleotide kinase and [γ32 P]ATP and then gel purified.

    Confocal Microscopy:

    Article Title: Genomic HIV RNA Induces Innate Immune Responses through RIG-I-Dependent Sensing of Secondary-Structured RNA
    Article Snippet: Paragraph title: Confocal microscopy ... Cells were transfected, with 1.2 µg/ml FAM-labelled HIV-Tar RNA at a RNA∶Lipofectamine 2000 (Invitrogen) ratio of 1∶2.5 (mass∶volume) for 180 min.

    Agarose Gel Electrophoresis:

    Article Title: Manipulation of both virus- and cell-specific factors is required for robust transient replication of a hepatitis C virus genotype 3a sub-genomic replicon
    Article Snippet: Stable SGR-harbouring cells were harvested in TRIZol (Invitrogen Life Technologies) and RNA purified according to the manufacturers’ instructions. .. Two microlitres of this cDNA were used as a template for PCR amplification of SEC14L2 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control.

    Chromatin Immunoprecipitation:

    Article Title: The cholesterol biosynthesis pathway regulates IL-10 expression in human Th1 cells
    Article Snippet: For primary cell experiments, cell pellets for mRNA isolation were lysed in Trizol (Ambion, Life Technologies) and RNA extracted by guanidium-isothiocyanate phenol/chloroform extraction method following the manufacturer’s instructions. .. For primary cell experiments, cell pellets for mRNA isolation were lysed in Trizol (Ambion, Life Technologies) and RNA extracted by guanidium-isothiocyanate phenol/chloroform extraction method following the manufacturer’s instructions.

    Plasmid Preparation:

    Article Title: The U1, U2 and U5 snRNAs crosslink to the 5? exon during yeast pre-mRNA splicing
    Article Snippet: CYH2 pre-mRNAs containing 4-thioU were produced by RNA ligation of a chemically synthesized 5′ RNA (Dharmacon) to an in vitro transcribed 3′ RNA ( ) by published procedures ( ). .. The 3′ RNA was made by in vitro transcription from PCR product containing a T7 promoter and primed with UpG (Sigma) to allow end labeling with 32 P. The RPS10B sequence was amplified from genomic DNA then inserted into pBluescript II KS+ (Stratagene) to produce plasmid pBS-RPS10B.

    Real-time Polymerase Chain Reaction:

    Article Title: Design, synthesis and evaluation of VEGF-siRNA/CRS as a novel vector for gene delivery
    Article Snippet: To-Pro® -3 Iodide (642⁄661), diethylpyrocarbonate (DEPC) water, Lipofectamine™ 2000, TRIzol reagent, VEGF ELISA Kit, RNA to CDNA and TaqMan Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA). .. To-Pro® -3 Iodide (642⁄661), diethylpyrocarbonate (DEPC) water, Lipofectamine™ 2000, TRIzol reagent, VEGF ELISA Kit, RNA to CDNA and TaqMan Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Article Title: Immune Activations and Viral Tissue Compartmentalization During Progressive HIV-1 Infection of Humanized Mice
    Article Snippet: The products were subsequently applied to the second round real-time PCR using TaqMan fluorescent probes on an ABI Prism 7000 real-time PCR machine (Applied Biosystems, MA). .. The expression levels of tissue HIV-1 DNA and RNA were normalized to those for the human CD45 gene (Life Technology, CA).

    Article Title: HIV-1 cellular and tissue replication patterns in infected humanized mice
    Article Snippet: Paragraph title: qPCR for viral RNA ... For sorted cells, levels of HIV-1 DNA and RNA were normalized to the expression of the housekeeping gene human GAPDH (Life Technology, California, USA).

    Article Title: The cholesterol biosynthesis pathway regulates IL-10 expression in human Th1 cells
    Article Snippet: Paragraph title: Quantitative real-time PCR ... Cell pellets for mRNA isolation were lysed in Trizol (Ambion, Life Technologies) and RNA was extracted by guanidium-isothescyanate phenol/chloroform extraction method following the manufacturer’s protocols.

    RNA Extraction:

    Article Title: Manipulation of both virus- and cell-specific factors is required for robust transient replication of a hepatitis C virus genotype 3a sub-genomic replicon
    Article Snippet: Paragraph title: RNA extraction from cells and PCR ... Stable SGR-harbouring cells were harvested in TRIZol (Invitrogen Life Technologies) and RNA purified according to the manufacturers’ instructions.

    Sample Prep:

    Article Title: Regulatory consequences of neuronal ELAV-like protein binding to coding and non-coding RNAs in human brain
    Article Snippet: All scripts used in the analysis including the peak finding algorithm and more information can be publically obtained at http://zhanglab.c2b2.columbia.edu/index.php/Resources . .. RNA from human brain and IMR-32 neuroblastoma cell lines was Trizol (Invitrogen)-extracted, Ribo-Zero-selected (Epicentre, Madison, WI), DNase-treated (Roche), and prepared for sequencing, following the Illumina High-throughput TruSeq RNA Sample Preparation Guidelines. .. The libraries from subjects 6–8 and 15–17, as well Y RNA overexpression samples, were sequenced on an Illumina HiSeq 2500 at the New York Genome Center, generating 125-bp paired end reads.

    Article Title: Hematopoietic Lineage Transcriptome Stability and Representation in PAXgene™ Collected Peripheral Blood Utilising SPIA Single-Stranded cDNA Probes for Microarray
    Article Snippet: Quality assessment by Agilent Bioanalyzer shows that the sscDNA fragments generated from whole blood RNA are of broad distribution in length, averaging approximately 1000 nucleotides, suggesting efficient sscDNA synthesis ( ). sscDNAs were fragmented and biotin-labelled in preparation for hybridisation and all resulted in fragments of ~50–200 nt (data not shown), similar to results previously reported using this method ( ). .. High quality control metrics for both RNA and sscDNA indicated successful sample preparation and all were subsequently hybridised to Affymetrix Human Genome U133 Plus 2.0 Arrays. .. All arrays within the study generated percentage present calls of between 60%–63%.

    In Vitro:

    Article Title: The U1, U2 and U5 snRNAs crosslink to the 5? exon during yeast pre-mRNA splicing
    Article Snippet: Body labeled CYH2 pre-mRNA was produced as previously described ( ). .. CYH2 pre-mRNAs containing 4-thioU were produced by RNA ligation of a chemically synthesized 5′ RNA (Dharmacon) to an in vitro transcribed 3′ RNA ( ) by published procedures ( ). .. The 3′ RNA was made by in vitro transcription from PCR product containing a T7 promoter and primed with UpG (Sigma) to allow end labeling with 32 P. The RPS10B sequence was amplified from genomic DNA then inserted into pBluescript II KS+ (Stratagene) to produce plasmid pBS-RPS10B.

    CCK-8 Assay:

    Article Title: Design, synthesis and evaluation of VEGF-siRNA/CRS as a novel vector for gene delivery
    Article Snippet: CCK-8 was purchased from Dojindo Molecular Technologies (Osaka, Japan). .. To-Pro® -3 Iodide (642⁄661), diethylpyrocarbonate (DEPC) water, Lipofectamine™ 2000, TRIzol reagent, VEGF ELISA Kit, RNA to CDNA and TaqMan Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Produced:

    Article Title: The U1, U2 and U5 snRNAs crosslink to the 5? exon during yeast pre-mRNA splicing
    Article Snippet: Body labeled CYH2 pre-mRNA was produced as previously described ( ). .. CYH2 pre-mRNAs containing 4-thioU were produced by RNA ligation of a chemically synthesized 5′ RNA (Dharmacon) to an in vitro transcribed 3′ RNA ( ) by published procedures ( ). .. The 3′ RNA was made by in vitro transcription from PCR product containing a T7 promoter and primed with UpG (Sigma) to allow end labeling with 32 P. The RPS10B sequence was amplified from genomic DNA then inserted into pBluescript II KS+ (Stratagene) to produce plasmid pBS-RPS10B.

    Concentration Assay:

    Article Title: The GBAP1 pseudogene acts as a ceRNA for the glucocerebrosidase gene GBA by sponging miR-22-3p
    Article Snippet: Expression profiles of GBA , GBAP1 , and miR-22-3p were determined using RNA from: a panel of 20 human tissues (First Choice total RNA; Ambion, Austin, USA), a panel of 24 human cerebral regions (Clontech Laboratories, Palo Alto, USA), 11 cell lines, iPSCs, and DA neurons differentiated from iPSCs (see below). .. RNA from cell lines, iPSCs, DA neurons, as well as transfected cells was isolated using the Eurozol kit (Euroclone), according to the manufacturer’s protocol.

    Transmission Electron Microscopy:

    Article Title: Design, synthesis and evaluation of VEGF-siRNA/CRS as a novel vector for gene delivery
    Article Snippet: To-Pro® -3 Iodide (642⁄661), diethylpyrocarbonate (DEPC) water, Lipofectamine™ 2000, TRIzol reagent, VEGF ELISA Kit, RNA to CDNA and TaqMan Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA). .. To-Pro® -3 Iodide (642⁄661), diethylpyrocarbonate (DEPC) water, Lipofectamine™ 2000, TRIzol reagent, VEGF ELISA Kit, RNA to CDNA and TaqMan Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    High Throughput Screening Assay:

    Article Title: Regulatory consequences of neuronal ELAV-like protein binding to coding and non-coding RNAs in human brain
    Article Snippet: All scripts used in the analysis including the peak finding algorithm and more information can be publically obtained at http://zhanglab.c2b2.columbia.edu/index.php/Resources . .. RNA from human brain and IMR-32 neuroblastoma cell lines was Trizol (Invitrogen)-extracted, Ribo-Zero-selected (Epicentre, Madison, WI), DNase-treated (Roche), and prepared for sequencing, following the Illumina High-throughput TruSeq RNA Sample Preparation Guidelines. .. The libraries from subjects 6–8 and 15–17, as well Y RNA overexpression samples, were sequenced on an Illumina HiSeq 2500 at the New York Genome Center, generating 125-bp paired end reads.

    Lysis:

    Article Title: Structural Basis for NusA Stabilized Transcriptional Pausing
    Article Snippet: NusA was eluted using a gradient over 10 CVs into lysis buffer plus 1 M NaCl. .. DNA (TriLink) and RNA (Dharmacon) oligonucleotides were chemically synthesized and gel purified by the manufacturer.

    Article Title: Assembly of AUF1 with eIF4G-poly(A) binding protein complex suggests a translation function in AU-rich mRNA decay
    Article Snippet: Cells were resuspended in lysis buffer (10 mM Tris-HCl at pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.7 mg/mL lysozyme, 1% NP-40, 1 mM DTT, 1 mM PMSF) and sonicated for 2 min. After addition of NaCl (final 450 mM), cell debris was removed by centrifugation at 15,000 rpm for 30 min at 4°C. .. Some preparations of PABP were treated with a ribonuclease cocktail to eliminate contaminating RNA (Ambion), as indicated.

    other:

    Article Title: Yeast Helicase Pif1 Unwinds RNA:DNA Hybrids with Higher Processivity than DNA:DNA Duplexes
    Article Snippet: DNA oligonucleotides were purchased from Integrated DNA Technologies, and RNA was from Dharmacon.

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    Thermo Fisher rna
    Confocal image (×10 4 ~10 5 ) of in vitro cellular uptakes of <t>VEGF-siRNA/CRS</t> at 2 h and 4 h. Abbreviation: siRNA, small interfering <t>RNA.</t>
    Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 74/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rna - by Bioz Stars, 2019-12
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    99
    Thermo Fisher total exosome rna
    Endothelial cell-derived exosomes mediate transmission of LGTV <t>RNA</t> from bEnd.3 cells to N2a neuronal cells. The bEnd.3 cells (1 x 10 5 ) were infected with 6 MOI of LGTV. QRT-PCR analysis showing total LGTV loads (A) or levels of LGTV positive-sense strand or negative-sense strand (B) in exosomes isolated from bEnd.3 cells at different time points (24, 48, 72, 96 and 120 h p.i.). (C) Viral loads were determined after 24 h p.i. by the presence of LGTV in N2a cells (1 x 10 5 ) treated with LGTV containing exosomes (20 μl) or supernatant (400 μl) fractions prepared from 24 or 48 h p.i. of bEnd.3 cells. (D) Viral loads at 48 h p.i. of N2a cells (plated in lower chamber) as determined by the presence of LGTV in a transwell assay performed with 1x 10 5 of bEnd.3 cells treated with 20 μl brain endothelial cell-derived exosomes (in upper chamber) for 4 h in the presence or absence of <t>exosome</t> inhibitor (5 μM) (in upper chamber) or infected with LGTV laboratory virus stocks is shown. LGTV transcript levels were normalized to mouse beta-actin. P value determined by Student’s two-tail t test is shown. Representative data is shown from three independent experiments.
    Total Exosome Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 90 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/total exosome rna/product/Thermo Fisher
    Average 99 stars, based on 90 article reviews
    Price from $9.99 to $1999.99
    total exosome rna - by Bioz Stars, 2019-12
    99/100 stars
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    81
    Thermo Fisher genomic rnas
    Relationships among H9 3′ NCR and CDS regions for viruses from different host species and geographic locations. The multi-dimensional analysis (MDS) scatter plots ( A ) are based on the pairwise genetic distance matrices from the multiple alignments of complete CDS regions and <t>3′NCRs.</t> Each dot represents a virus, colored according to their origins as indicated on the CDS-based phylogenetic tree ( B ). Alignments of the 3′ NCRs, shown corresponding to the genomic <t>RNAs,</t> from the viruses used in the analyses are next to the CDS-based neighbor-joining tree for these viruses.
    Genomic Rnas, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 81/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/genomic rnas/product/Thermo Fisher
    Average 81 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    genomic rnas - by Bioz Stars, 2019-12
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    Image Search Results


    Confocal image (×10 4 ~10 5 ) of in vitro cellular uptakes of VEGF-siRNA/CRS at 2 h and 4 h. Abbreviation: siRNA, small interfering RNA.

    Journal: Drug Design, Development and Therapy

    Article Title: Design, synthesis and evaluation of VEGF-siRNA/CRS as a novel vector for gene delivery

    doi: 10.2147/DDDT.S118461

    Figure Lengend Snippet: Confocal image (×10 4 ~10 5 ) of in vitro cellular uptakes of VEGF-siRNA/CRS at 2 h and 4 h. Abbreviation: siRNA, small interfering RNA.

    Article Snippet: To-Pro® -3 Iodide (642⁄661), diethylpyrocarbonate (DEPC) water, Lipofectamine™ 2000, TRIzol reagent, VEGF ELISA Kit, RNA to CDNA and TaqMan Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Techniques: In Vitro, Small Interfering RNA

    Release of VEGF-siRNA and CRS from VEGF-siRNA/CRS. Note: Data are presented as mean ± SD (n=3). Abbreviations: siRNA, small interfering RNA; SD, standard deviation.

    Journal: Drug Design, Development and Therapy

    Article Title: Design, synthesis and evaluation of VEGF-siRNA/CRS as a novel vector for gene delivery

    doi: 10.2147/DDDT.S118461

    Figure Lengend Snippet: Release of VEGF-siRNA and CRS from VEGF-siRNA/CRS. Note: Data are presented as mean ± SD (n=3). Abbreviations: siRNA, small interfering RNA; SD, standard deviation.

    Article Snippet: To-Pro® -3 Iodide (642⁄661), diethylpyrocarbonate (DEPC) water, Lipofectamine™ 2000, TRIzol reagent, VEGF ELISA Kit, RNA to CDNA and TaqMan Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Techniques: Small Interfering RNA, Standard Deviation

    Gene-silencing efficiency of VEGF-siRNA/CRS on HeLa cells. Note: Data are presented as mean ± SD (n=3). Abbreviations: NC, negative control; siRNA, small interfering RNA; SD, standard deviation.

    Journal: Drug Design, Development and Therapy

    Article Title: Design, synthesis and evaluation of VEGF-siRNA/CRS as a novel vector for gene delivery

    doi: 10.2147/DDDT.S118461

    Figure Lengend Snippet: Gene-silencing efficiency of VEGF-siRNA/CRS on HeLa cells. Note: Data are presented as mean ± SD (n=3). Abbreviations: NC, negative control; siRNA, small interfering RNA; SD, standard deviation.

    Article Snippet: To-Pro® -3 Iodide (642⁄661), diethylpyrocarbonate (DEPC) water, Lipofectamine™ 2000, TRIzol reagent, VEGF ELISA Kit, RNA to CDNA and TaqMan Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Techniques: Negative Control, Small Interfering RNA, Standard Deviation

    Nano-properties of CRS and VEGF-siRNA/CRS. Notes: ( A ) Particle size and ( B ) ζ potential of various CRS concentrations (n=3). Abbreviation: siRNA, small interfering RNA.

    Journal: Drug Design, Development and Therapy

    Article Title: Design, synthesis and evaluation of VEGF-siRNA/CRS as a novel vector for gene delivery

    doi: 10.2147/DDDT.S118461

    Figure Lengend Snippet: Nano-properties of CRS and VEGF-siRNA/CRS. Notes: ( A ) Particle size and ( B ) ζ potential of various CRS concentrations (n=3). Abbreviation: siRNA, small interfering RNA.

    Article Snippet: To-Pro® -3 Iodide (642⁄661), diethylpyrocarbonate (DEPC) water, Lipofectamine™ 2000, TRIzol reagent, VEGF ELISA Kit, RNA to CDNA and TaqMan Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Techniques: Small Interfering RNA

    The agarose gel electrophoresis of VEGF-siRNA and CRS with different concentrations. Notes: ( A ) 100% CRS, ( B ) 75% CRS, ( C ) 50% CRS and ( D ) 25% CRS (n=3). Abbreviation: siRNA, small interfering RNA.

    Journal: Drug Design, Development and Therapy

    Article Title: Design, synthesis and evaluation of VEGF-siRNA/CRS as a novel vector for gene delivery

    doi: 10.2147/DDDT.S118461

    Figure Lengend Snippet: The agarose gel electrophoresis of VEGF-siRNA and CRS with different concentrations. Notes: ( A ) 100% CRS, ( B ) 75% CRS, ( C ) 50% CRS and ( D ) 25% CRS (n=3). Abbreviation: siRNA, small interfering RNA.

    Article Snippet: To-Pro® -3 Iodide (642⁄661), diethylpyrocarbonate (DEPC) water, Lipofectamine™ 2000, TRIzol reagent, VEGF ELISA Kit, RNA to CDNA and TaqMan Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Techniques: Agarose Gel Electrophoresis, Small Interfering RNA

    ELISA results for VEGF protein expression of HeLa cells treated with VEGF-siRNA/CRS. Note: Data are presented as mean ± SD (n=3). Abbreviations: NC, negative control; siRNA, small interfering RNA; ELISA, enzyme-linked immunosorbent assay; SD, standard deviation.

    Journal: Drug Design, Development and Therapy

    Article Title: Design, synthesis and evaluation of VEGF-siRNA/CRS as a novel vector for gene delivery

    doi: 10.2147/DDDT.S118461

    Figure Lengend Snippet: ELISA results for VEGF protein expression of HeLa cells treated with VEGF-siRNA/CRS. Note: Data are presented as mean ± SD (n=3). Abbreviations: NC, negative control; siRNA, small interfering RNA; ELISA, enzyme-linked immunosorbent assay; SD, standard deviation.

    Article Snippet: To-Pro® -3 Iodide (642⁄661), diethylpyrocarbonate (DEPC) water, Lipofectamine™ 2000, TRIzol reagent, VEGF ELISA Kit, RNA to CDNA and TaqMan Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Negative Control, Small Interfering RNA, Standard Deviation

    In vivo antitumor effect of VEGF-siRNA/CRS. Note: Data are presented as mean ± SD (n=10). Abbreviations: DOX, doxorubicin; NS, normal saline; SD, standard deviation; siRNA, small interfering RNA.

    Journal: Drug Design, Development and Therapy

    Article Title: Design, synthesis and evaluation of VEGF-siRNA/CRS as a novel vector for gene delivery

    doi: 10.2147/DDDT.S118461

    Figure Lengend Snippet: In vivo antitumor effect of VEGF-siRNA/CRS. Note: Data are presented as mean ± SD (n=10). Abbreviations: DOX, doxorubicin; NS, normal saline; SD, standard deviation; siRNA, small interfering RNA.

    Article Snippet: To-Pro® -3 Iodide (642⁄661), diethylpyrocarbonate (DEPC) water, Lipofectamine™ 2000, TRIzol reagent, VEGF ELISA Kit, RNA to CDNA and TaqMan Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Techniques: In Vivo, Standard Deviation, Small Interfering RNA

    TEM ( A – D ) and SEM ( E – H ) images of VEGF-siRNA/CRS. Abbreviations: TEM, transmission electron microscopy; SEM, scanning electron microscopy; siRNA, small interfering RNA.

    Journal: Drug Design, Development and Therapy

    Article Title: Design, synthesis and evaluation of VEGF-siRNA/CRS as a novel vector for gene delivery

    doi: 10.2147/DDDT.S118461

    Figure Lengend Snippet: TEM ( A – D ) and SEM ( E – H ) images of VEGF-siRNA/CRS. Abbreviations: TEM, transmission electron microscopy; SEM, scanning electron microscopy; siRNA, small interfering RNA.

    Article Snippet: To-Pro® -3 Iodide (642⁄661), diethylpyrocarbonate (DEPC) water, Lipofectamine™ 2000, TRIzol reagent, VEGF ELISA Kit, RNA to CDNA and TaqMan Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Techniques: Transmission Electron Microscopy, Transmission Assay, Electron Microscopy, Small Interfering RNA

    In vitro inhibition of HeLa cells treated by VEGF-siRNA/CRS (n=3). Abbreviations: NC, negative control; siRNA, small interfering RNA.

    Journal: Drug Design, Development and Therapy

    Article Title: Design, synthesis and evaluation of VEGF-siRNA/CRS as a novel vector for gene delivery

    doi: 10.2147/DDDT.S118461

    Figure Lengend Snippet: In vitro inhibition of HeLa cells treated by VEGF-siRNA/CRS (n=3). Abbreviations: NC, negative control; siRNA, small interfering RNA.

    Article Snippet: To-Pro® -3 Iodide (642⁄661), diethylpyrocarbonate (DEPC) water, Lipofectamine™ 2000, TRIzol reagent, VEGF ELISA Kit, RNA to CDNA and TaqMan Mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Techniques: In Vitro, Inhibition, Negative Control, Small Interfering RNA

    Endothelial cell-derived exosomes mediate transmission of LGTV RNA from bEnd.3 cells to N2a neuronal cells. The bEnd.3 cells (1 x 10 5 ) were infected with 6 MOI of LGTV. QRT-PCR analysis showing total LGTV loads (A) or levels of LGTV positive-sense strand or negative-sense strand (B) in exosomes isolated from bEnd.3 cells at different time points (24, 48, 72, 96 and 120 h p.i.). (C) Viral loads were determined after 24 h p.i. by the presence of LGTV in N2a cells (1 x 10 5 ) treated with LGTV containing exosomes (20 μl) or supernatant (400 μl) fractions prepared from 24 or 48 h p.i. of bEnd.3 cells. (D) Viral loads at 48 h p.i. of N2a cells (plated in lower chamber) as determined by the presence of LGTV in a transwell assay performed with 1x 10 5 of bEnd.3 cells treated with 20 μl brain endothelial cell-derived exosomes (in upper chamber) for 4 h in the presence or absence of exosome inhibitor (5 μM) (in upper chamber) or infected with LGTV laboratory virus stocks is shown. LGTV transcript levels were normalized to mouse beta-actin. P value determined by Student’s two-tail t test is shown. Representative data is shown from three independent experiments.

    Journal: PLoS Pathogens

    Article Title: Exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral RNA and proteins to the vertebrate neuronal cells

    doi: 10.1371/journal.ppat.1006764

    Figure Lengend Snippet: Endothelial cell-derived exosomes mediate transmission of LGTV RNA from bEnd.3 cells to N2a neuronal cells. The bEnd.3 cells (1 x 10 5 ) were infected with 6 MOI of LGTV. QRT-PCR analysis showing total LGTV loads (A) or levels of LGTV positive-sense strand or negative-sense strand (B) in exosomes isolated from bEnd.3 cells at different time points (24, 48, 72, 96 and 120 h p.i.). (C) Viral loads were determined after 24 h p.i. by the presence of LGTV in N2a cells (1 x 10 5 ) treated with LGTV containing exosomes (20 μl) or supernatant (400 μl) fractions prepared from 24 or 48 h p.i. of bEnd.3 cells. (D) Viral loads at 48 h p.i. of N2a cells (plated in lower chamber) as determined by the presence of LGTV in a transwell assay performed with 1x 10 5 of bEnd.3 cells treated with 20 μl brain endothelial cell-derived exosomes (in upper chamber) for 4 h in the presence or absence of exosome inhibitor (5 μM) (in upper chamber) or infected with LGTV laboratory virus stocks is shown. LGTV transcript levels were normalized to mouse beta-actin. P value determined by Student’s two-tail t test is shown. Representative data is shown from three independent experiments.

    Article Snippet: We also isolated exosomes from N2a cells using the total exosome isolation reagent and extracted total RNA and proteins using total exosome RNA and Protein Isolation kit (Invitrogen/ThermoScientific) as per the manufacturer’s instruction.

    Techniques: Derivative Assay, Transmission Assay, Infection, Quantitative RT-PCR, Isolation, Transwell Assay

    Proposed model showing arthropod-borne flaviviruses transmission to human host via arthropod exosomes. Tick bite or injection/spit of saliva from infected ticks deposits exosomes containing viral infectious RNA or proteins securely to humans. Exosomes derived from tick cells/saliva infect human skin (keratinocytes) or may directly deposit saliva enriched with exosome containing tick-borne viruses into the blood capillaries/vessels. Exosomes derived from vascular endothelial cells containing flaviviruses infect neighboring endothelial cells leading into infection of the peripheral system. Higher viral loads in peripheral tissues increase viremia in blood and eventually allow entry and replication of these arthropod-borne viruses in brain microvascular endothelial cells that line the BBB. Early production and higher loads of flaviviruses in brain endothelial cells would allow these viruses to cross BBB and infect neurons. Infected neurons produce high numbers of exosomes containing infectious RNA and proteins that fuse with cell membranes of naïve neuronal cells thereby infecting neighboring neurons and leading to spread of infection and severe neuronal loss.

    Journal: PLoS Pathogens

    Article Title: Exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral RNA and proteins to the vertebrate neuronal cells

    doi: 10.1371/journal.ppat.1006764

    Figure Lengend Snippet: Proposed model showing arthropod-borne flaviviruses transmission to human host via arthropod exosomes. Tick bite or injection/spit of saliva from infected ticks deposits exosomes containing viral infectious RNA or proteins securely to humans. Exosomes derived from tick cells/saliva infect human skin (keratinocytes) or may directly deposit saliva enriched with exosome containing tick-borne viruses into the blood capillaries/vessels. Exosomes derived from vascular endothelial cells containing flaviviruses infect neighboring endothelial cells leading into infection of the peripheral system. Higher viral loads in peripheral tissues increase viremia in blood and eventually allow entry and replication of these arthropod-borne viruses in brain microvascular endothelial cells that line the BBB. Early production and higher loads of flaviviruses in brain endothelial cells would allow these viruses to cross BBB and infect neurons. Infected neurons produce high numbers of exosomes containing infectious RNA and proteins that fuse with cell membranes of naïve neuronal cells thereby infecting neighboring neurons and leading to spread of infection and severe neuronal loss.

    Article Snippet: We also isolated exosomes from N2a cells using the total exosome isolation reagent and extracted total RNA and proteins using total exosome RNA and Protein Isolation kit (Invitrogen/ThermoScientific) as per the manufacturer’s instruction.

    Techniques: Transmission Assay, Injection, Infection, Derivative Assay

    Detection of LGTV RNA and proteins in exosomes isolated from N2a neuronal cell line. (A) Cryo-EM images showing exosomes isolated from uninfected and LGTV-infected (MOI 6; 72 h p.i.), N2a cells (1 x 10 7 ). Scale indicates 100nm. Quantification of diameter or sizes for heterogenous population of exosomes from uninfected (B) or LGTV- infected (C) N2a cell-derived exosomes. Number of exosomes analyzed were n = 32 (uninfected) and n = 131 (infected) groups. (D) Comparison of exosome numbers per image from uninfected (n = 9) and infected (n = 13) groups is shown. (E) DG-Exos showing presence of enhanced LGTV envelope [E]-protein loads in fractions 1–6. Fractions 3–5 showed enriched amounts of exosomal markers CD9 and HSP70. E-protein detection in fraction 5 in 0.1 μm filtered samples processed for OptiPrep DG-isolation is shown. QRT-PCR analysis showing levels of total LGTV loads (F), copy numbers (G) and LGTV positive-sense strand or negative-sense strand (H) in exosomes isolated from N2a cells at different time points. N2a (1 x 10 5 ) cells were infected with 6 MOI of LGTV, and LGTV loads were analyzed at 72 h p.i. (I) Treatment of LGTV-infected (72 h p.i.) N2a cell-derived exosomes with RNase A is shown. The uninfected samples treated with RNase serve as control. LGTV transcript levels were normalized to mouse beta-actin. (J) Immunoblotting analysis showing detection of LGTV E glycoprotein and mammalian exosomal marker CD9 in exosome fractions and total lysates from whole cells prepared at 48 h p.i. from 2 x 10 6 uninfected (UI) or infected (I) N2a cells. Stain-free gels showing total protein profiles serve as the loading control. (K) Native-PAGE followed by immunoblotting analysis showing presence of LGTV E- and NS1 proteins from LGTV-infected (MOI 1; 72 h p.i.) or uninfected N2a cell-derived exosomes treated with Triton-X-100 (0.03%), or freeze-thaw cycles (3 times freezing at -80°C for 1 h each cycle) or untreated, held on ice. Coomassie stained gel image showing total protein profiles serve as a loading control. (L) ELISA performed on uninfected or LGTV-infected (MOI 6; 72 h p.i.) N2a cell-derived exosomes either untreated or treated with Triton-X-100 (0.1%). (M) Antibody-beads binding assay performed on LGTV infected (MOI 1, 72 h p.i.) N2a cell-derived exosomes (collected from N2a cells either untreated or treated with GW4869; 5 μM, exosome inhibitor) showing no differences in LGTV-E protein loads between 4G2 or isotype and untreated samples.

    Journal: PLoS Pathogens

    Article Title: Exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral RNA and proteins to the vertebrate neuronal cells

    doi: 10.1371/journal.ppat.1006764

    Figure Lengend Snippet: Detection of LGTV RNA and proteins in exosomes isolated from N2a neuronal cell line. (A) Cryo-EM images showing exosomes isolated from uninfected and LGTV-infected (MOI 6; 72 h p.i.), N2a cells (1 x 10 7 ). Scale indicates 100nm. Quantification of diameter or sizes for heterogenous population of exosomes from uninfected (B) or LGTV- infected (C) N2a cell-derived exosomes. Number of exosomes analyzed were n = 32 (uninfected) and n = 131 (infected) groups. (D) Comparison of exosome numbers per image from uninfected (n = 9) and infected (n = 13) groups is shown. (E) DG-Exos showing presence of enhanced LGTV envelope [E]-protein loads in fractions 1–6. Fractions 3–5 showed enriched amounts of exosomal markers CD9 and HSP70. E-protein detection in fraction 5 in 0.1 μm filtered samples processed for OptiPrep DG-isolation is shown. QRT-PCR analysis showing levels of total LGTV loads (F), copy numbers (G) and LGTV positive-sense strand or negative-sense strand (H) in exosomes isolated from N2a cells at different time points. N2a (1 x 10 5 ) cells were infected with 6 MOI of LGTV, and LGTV loads were analyzed at 72 h p.i. (I) Treatment of LGTV-infected (72 h p.i.) N2a cell-derived exosomes with RNase A is shown. The uninfected samples treated with RNase serve as control. LGTV transcript levels were normalized to mouse beta-actin. (J) Immunoblotting analysis showing detection of LGTV E glycoprotein and mammalian exosomal marker CD9 in exosome fractions and total lysates from whole cells prepared at 48 h p.i. from 2 x 10 6 uninfected (UI) or infected (I) N2a cells. Stain-free gels showing total protein profiles serve as the loading control. (K) Native-PAGE followed by immunoblotting analysis showing presence of LGTV E- and NS1 proteins from LGTV-infected (MOI 1; 72 h p.i.) or uninfected N2a cell-derived exosomes treated with Triton-X-100 (0.03%), or freeze-thaw cycles (3 times freezing at -80°C for 1 h each cycle) or untreated, held on ice. Coomassie stained gel image showing total protein profiles serve as a loading control. (L) ELISA performed on uninfected or LGTV-infected (MOI 6; 72 h p.i.) N2a cell-derived exosomes either untreated or treated with Triton-X-100 (0.1%). (M) Antibody-beads binding assay performed on LGTV infected (MOI 1, 72 h p.i.) N2a cell-derived exosomes (collected from N2a cells either untreated or treated with GW4869; 5 μM, exosome inhibitor) showing no differences in LGTV-E protein loads between 4G2 or isotype and untreated samples.

    Article Snippet: We also isolated exosomes from N2a cells using the total exosome isolation reagent and extracted total RNA and proteins using total exosome RNA and Protein Isolation kit (Invitrogen/ThermoScientific) as per the manufacturer’s instruction.

    Techniques: Isolation, Electron Microscopy, Infection, Derivative Assay, Quantitative RT-PCR, Marker, Staining, Clear Native PAGE, Enzyme-linked Immunosorbent Assay, Binding Assay

    Detection of LGTV RNA and proteins in exosomes isolated from primary cultures of mouse cortical neurons. LGTV (4 MOI) was used to infect 1 x 10 5 murine cortical neuronal cells. UI indicates uninfected and I indicates LGTV-infected. (A) QRT-PCR analysis showing LGTV infection kinetics in primary cultures of mouse cortical neurons at different time points (24, 48, 72, 96 h p.i.). Total LGTV loads in exosomes isolated from cortical neurons (B), copy numbers (C) and levels of LGTV positive-sense strand or negative-sense strand (D) in exosomes isolated from cortical neuronal cells at different time points (24, 48 and 72 h p.i.). (E) Immunoblotting analysis showing detection of LGTV E-protein and mammalian exosomal marker CD9 in exosome fraction and total lysates from whole cells prepared from uninfected (UI) or infected (I) cortical neuronal cells at 48 h p.i. Stain-free gel showing total protein profile serves as the loading control. (F) Immunoblotting analysis showing NS1 levels in total neuronal lysates and exosomes derived from LGTV-infected (MOI 4; 72 h p.i.) cortical neurons. Uninfected (UI) cells and exosomes derived from these cells serve as controls in addition to total protein profiles. For immunoblotting assays, 2 x 10 7 cortical neuronal cells were infected with 4 MOI of LGTV. (G) Quantitative assessment of number of plaques from exosomal and supernatant fractions is shown. (H) QRT-PCR analysis of viral loads in 1x 10 5 naïve cortical neuronal cells at 24 h p.i., infected through treatment with exosomes (20 μl) or supernatant fractions (400 μl) prepared from 24, 48 and 72 h p.i., LGTV-infected neurons show presence of LGTV. LGTV transcript levels were normalized to mouse beta-actin. P value determined by Student’s two-tailed t test is shown. Representative data is shown from at least three independent experiments.

    Journal: PLoS Pathogens

    Article Title: Exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral RNA and proteins to the vertebrate neuronal cells

    doi: 10.1371/journal.ppat.1006764

    Figure Lengend Snippet: Detection of LGTV RNA and proteins in exosomes isolated from primary cultures of mouse cortical neurons. LGTV (4 MOI) was used to infect 1 x 10 5 murine cortical neuronal cells. UI indicates uninfected and I indicates LGTV-infected. (A) QRT-PCR analysis showing LGTV infection kinetics in primary cultures of mouse cortical neurons at different time points (24, 48, 72, 96 h p.i.). Total LGTV loads in exosomes isolated from cortical neurons (B), copy numbers (C) and levels of LGTV positive-sense strand or negative-sense strand (D) in exosomes isolated from cortical neuronal cells at different time points (24, 48 and 72 h p.i.). (E) Immunoblotting analysis showing detection of LGTV E-protein and mammalian exosomal marker CD9 in exosome fraction and total lysates from whole cells prepared from uninfected (UI) or infected (I) cortical neuronal cells at 48 h p.i. Stain-free gel showing total protein profile serves as the loading control. (F) Immunoblotting analysis showing NS1 levels in total neuronal lysates and exosomes derived from LGTV-infected (MOI 4; 72 h p.i.) cortical neurons. Uninfected (UI) cells and exosomes derived from these cells serve as controls in addition to total protein profiles. For immunoblotting assays, 2 x 10 7 cortical neuronal cells were infected with 4 MOI of LGTV. (G) Quantitative assessment of number of plaques from exosomal and supernatant fractions is shown. (H) QRT-PCR analysis of viral loads in 1x 10 5 naïve cortical neuronal cells at 24 h p.i., infected through treatment with exosomes (20 μl) or supernatant fractions (400 μl) prepared from 24, 48 and 72 h p.i., LGTV-infected neurons show presence of LGTV. LGTV transcript levels were normalized to mouse beta-actin. P value determined by Student’s two-tailed t test is shown. Representative data is shown from at least three independent experiments.

    Article Snippet: We also isolated exosomes from N2a cells using the total exosome isolation reagent and extracted total RNA and proteins using total exosome RNA and Protein Isolation kit (Invitrogen/ThermoScientific) as per the manufacturer’s instruction.

    Techniques: Isolation, Infection, Quantitative RT-PCR, Marker, Staining, Derivative Assay, Two Tailed Test

    Arthropod exosomes mediate transmission of infectious LGTV RNA and proteins from tick to human cells. (A) Cryo-EM images showing exosomes isolated from uninfected and LGTV-infected (MOI 1; 72 h p.i.), ISE6 tick cells. Scale bar indicates 100nm. Quantification of diameter or sizes for heterogenous population of exosomes from uninfected (B) or LGTV- infected (C) tick cell-derived exosomes. Number of exosomes analyzed were n = 138 (uninfected) and n = 200 (infected) groups. (D) Comparison of exosome numbers per cryo-EM image from uninfected (n = 27) and infected (n = 14) groups is shown. (E) DG-Exos showing presence of LGTV envelope [E]-protein in fractions 1–6. QRT-PCR analysis showing total LGTV loads (F) and levels of LGTV positive-sense strand or negative-sense strand (G) in exosomes isolated from tick cells at 72 h (p.i.), Uninfected cells serve as control. LGTV transcript levels were normalized to tick beta-actin. (H) Immunoblotting analysis showing detection of LGTV E- glycoprotein in exosome fraction and total lysates from whole cells prepared from uninfected (UI) or infected (I) tick cells at 72 h (p.i.). Immunoblot detecting NS1 in both tick-cell derived exosomes and total cell lysates using monoclonal anti-Langat virus NS1 protein from LGTV-infected (MOI 1; 72 h p.i.) is also shown. HSP70 levels indicate enrichment of arthropod exosomal marker in exosome fractions. Uninfected samples and total protein profiles serve as controls. Tick cells (5 x 10 6 ) were infected with 1 MOI of LGTV in both QRT-PCR and immunoblotting analysis. I) Native-PAGE followed by immunoblotting analysis showing presence of E-protein from LGTV-infected (MOI 1; 72 h p.i.) or uninfected tick cell-derived exosomes treated with Triton-X-100 (0.03%), or freeze-thaw cycles (3 times freezing at -80°C for 1 h each cycle) or untreated samples held on ice. Coomassie stained gel showing total protein profiles serve as loading control (I). (J) Antibody-beads binding assay performed on LGTV infected (MOI 1) tick cell-derived exosomes (collected from tick cells either untreated or treated with GW4869; 5 μM, exosome inhibitor) showing no differences in Langat-E protein loads between 4G2 or isotype and untreated samples. (K) Quantitative assessment of number of plaques in exosomal and supernatant fractions is shown. TMTC indicates “too many to count”. (L) Viral re-infection kinetics as determined by the presence of total LGTV loads in HaCaT cells (1 x 10 5 cells) at different time points treated with exosomes (20 μl) or supernatant fractions (400 μl) prepared from 72 h p.i. LGTV-infected tick cells are shown. (M) Viral loads at 48 h p.i. was determined by the presence of LGTV in a transwell assay performed with 1 x 10 5 tick cells (in upper chamber) or 1 x 10 5 HaCaT cells (in lower chamber) treated with tick exosome fraction (20 μl, in upper chamber) for 4 h in the presence or absence of 5 μM exosome inhibitor GW4869. Tick cells infected with LGTV laboratory stocks were used as controls. LGTV transcript levels were normalized to human beta-actin in (L) and (M). P value determined by Student’s two-tail t test is shown. Representative data is shown from three independent experiments.

    Journal: PLoS Pathogens

    Article Title: Exosomes serve as novel modes of tick-borne flavivirus transmission from arthropod to human cells and facilitates dissemination of viral RNA and proteins to the vertebrate neuronal cells

    doi: 10.1371/journal.ppat.1006764

    Figure Lengend Snippet: Arthropod exosomes mediate transmission of infectious LGTV RNA and proteins from tick to human cells. (A) Cryo-EM images showing exosomes isolated from uninfected and LGTV-infected (MOI 1; 72 h p.i.), ISE6 tick cells. Scale bar indicates 100nm. Quantification of diameter or sizes for heterogenous population of exosomes from uninfected (B) or LGTV- infected (C) tick cell-derived exosomes. Number of exosomes analyzed were n = 138 (uninfected) and n = 200 (infected) groups. (D) Comparison of exosome numbers per cryo-EM image from uninfected (n = 27) and infected (n = 14) groups is shown. (E) DG-Exos showing presence of LGTV envelope [E]-protein in fractions 1–6. QRT-PCR analysis showing total LGTV loads (F) and levels of LGTV positive-sense strand or negative-sense strand (G) in exosomes isolated from tick cells at 72 h (p.i.), Uninfected cells serve as control. LGTV transcript levels were normalized to tick beta-actin. (H) Immunoblotting analysis showing detection of LGTV E- glycoprotein in exosome fraction and total lysates from whole cells prepared from uninfected (UI) or infected (I) tick cells at 72 h (p.i.). Immunoblot detecting NS1 in both tick-cell derived exosomes and total cell lysates using monoclonal anti-Langat virus NS1 protein from LGTV-infected (MOI 1; 72 h p.i.) is also shown. HSP70 levels indicate enrichment of arthropod exosomal marker in exosome fractions. Uninfected samples and total protein profiles serve as controls. Tick cells (5 x 10 6 ) were infected with 1 MOI of LGTV in both QRT-PCR and immunoblotting analysis. I) Native-PAGE followed by immunoblotting analysis showing presence of E-protein from LGTV-infected (MOI 1; 72 h p.i.) or uninfected tick cell-derived exosomes treated with Triton-X-100 (0.03%), or freeze-thaw cycles (3 times freezing at -80°C for 1 h each cycle) or untreated samples held on ice. Coomassie stained gel showing total protein profiles serve as loading control (I). (J) Antibody-beads binding assay performed on LGTV infected (MOI 1) tick cell-derived exosomes (collected from tick cells either untreated or treated with GW4869; 5 μM, exosome inhibitor) showing no differences in Langat-E protein loads between 4G2 or isotype and untreated samples. (K) Quantitative assessment of number of plaques in exosomal and supernatant fractions is shown. TMTC indicates “too many to count”. (L) Viral re-infection kinetics as determined by the presence of total LGTV loads in HaCaT cells (1 x 10 5 cells) at different time points treated with exosomes (20 μl) or supernatant fractions (400 μl) prepared from 72 h p.i. LGTV-infected tick cells are shown. (M) Viral loads at 48 h p.i. was determined by the presence of LGTV in a transwell assay performed with 1 x 10 5 tick cells (in upper chamber) or 1 x 10 5 HaCaT cells (in lower chamber) treated with tick exosome fraction (20 μl, in upper chamber) for 4 h in the presence or absence of 5 μM exosome inhibitor GW4869. Tick cells infected with LGTV laboratory stocks were used as controls. LGTV transcript levels were normalized to human beta-actin in (L) and (M). P value determined by Student’s two-tail t test is shown. Representative data is shown from three independent experiments.

    Article Snippet: We also isolated exosomes from N2a cells using the total exosome isolation reagent and extracted total RNA and proteins using total exosome RNA and Protein Isolation kit (Invitrogen/ThermoScientific) as per the manufacturer’s instruction.

    Techniques: Transmission Assay, Electron Microscopy, Isolation, Infection, Derivative Assay, Quantitative RT-PCR, Marker, Clear Native PAGE, Staining, Binding Assay, Transwell Assay

    Relationships among H9 3′ NCR and CDS regions for viruses from different host species and geographic locations. The multi-dimensional analysis (MDS) scatter plots ( A ) are based on the pairwise genetic distance matrices from the multiple alignments of complete CDS regions and 3′NCRs. Each dot represents a virus, colored according to their origins as indicated on the CDS-based phylogenetic tree ( B ). Alignments of the 3′ NCRs, shown corresponding to the genomic RNAs, from the viruses used in the analyses are next to the CDS-based neighbor-joining tree for these viruses.

    Journal: Veterinary Sciences

    Article Title: Analysis of the Variability in the Non-Coding Regions of Influenza A Viruses

    doi: 10.3390/vetsci5030076

    Figure Lengend Snippet: Relationships among H9 3′ NCR and CDS regions for viruses from different host species and geographic locations. The multi-dimensional analysis (MDS) scatter plots ( A ) are based on the pairwise genetic distance matrices from the multiple alignments of complete CDS regions and 3′NCRs. Each dot represents a virus, colored according to their origins as indicated on the CDS-based phylogenetic tree ( B ). Alignments of the 3′ NCRs, shown corresponding to the genomic RNAs, from the viruses used in the analyses are next to the CDS-based neighbor-joining tree for these viruses.

    Article Snippet: For the 3′ NCRs, genomic RNAs were polyadenylated with poly(A) polymerase (2 U µL−1 ) (Thermo Fisher Scientific) in the presence of MnCl2 (2.5 mM) and ATP (1 mM) in 1X reaction buffer and a final volume of 50 µL.

    Techniques:

    Relationships among NP 3′ NCR and CDS regions for viruses from different host species and geographic locations. Alignments of the 3′ NCRs, shown corresponding to the genomic RNAs, from the viruses used in the analyses are next to the CDS-based neighbor-joining tree for these viruses.

    Journal: Veterinary Sciences

    Article Title: Analysis of the Variability in the Non-Coding Regions of Influenza A Viruses

    doi: 10.3390/vetsci5030076

    Figure Lengend Snippet: Relationships among NP 3′ NCR and CDS regions for viruses from different host species and geographic locations. Alignments of the 3′ NCRs, shown corresponding to the genomic RNAs, from the viruses used in the analyses are next to the CDS-based neighbor-joining tree for these viruses.

    Article Snippet: For the 3′ NCRs, genomic RNAs were polyadenylated with poly(A) polymerase (2 U µL−1 ) (Thermo Fisher Scientific) in the presence of MnCl2 (2.5 mM) and ATP (1 mM) in 1X reaction buffer and a final volume of 50 µL.

    Techniques:

    Relationships among H13 3′ NCR and CDS regions for viruses from different host species and geographic locations. The MDS scatter plots ( A ) are based on the pairwise genetic distance matrices from the multiple alignments of complete CDS regions and 3′ NCRs. Each dot represents a virus, colored according to their origins as indicated on the CDS-based phylogenetic tree ( B ). Alignments of the 3′ NCRs, shown corresponding to the genomic RNAs, from the viruses used in the analyses are next to the CDS-based neighbor-joining tree for these viruses.

    Journal: Veterinary Sciences

    Article Title: Analysis of the Variability in the Non-Coding Regions of Influenza A Viruses

    doi: 10.3390/vetsci5030076

    Figure Lengend Snippet: Relationships among H13 3′ NCR and CDS regions for viruses from different host species and geographic locations. The MDS scatter plots ( A ) are based on the pairwise genetic distance matrices from the multiple alignments of complete CDS regions and 3′ NCRs. Each dot represents a virus, colored according to their origins as indicated on the CDS-based phylogenetic tree ( B ). Alignments of the 3′ NCRs, shown corresponding to the genomic RNAs, from the viruses used in the analyses are next to the CDS-based neighbor-joining tree for these viruses.

    Article Snippet: For the 3′ NCRs, genomic RNAs were polyadenylated with poly(A) polymerase (2 U µL−1 ) (Thermo Fisher Scientific) in the presence of MnCl2 (2.5 mM) and ATP (1 mM) in 1X reaction buffer and a final volume of 50 µL.

    Techniques:

    5′ NCRs of wild bird virus segments determined in this study. The multiple alignment of 5′ NCRs was generated with MUSCLE implemented in Geneious version 8. The RNA sequences are displayed in the 3′ to 5′ orientation corresponding to the packaged RNAs.

    Journal: Veterinary Sciences

    Article Title: Analysis of the Variability in the Non-Coding Regions of Influenza A Viruses

    doi: 10.3390/vetsci5030076

    Figure Lengend Snippet: 5′ NCRs of wild bird virus segments determined in this study. The multiple alignment of 5′ NCRs was generated with MUSCLE implemented in Geneious version 8. The RNA sequences are displayed in the 3′ to 5′ orientation corresponding to the packaged RNAs.

    Article Snippet: For the 3′ NCRs, genomic RNAs were polyadenylated with poly(A) polymerase (2 U µL−1 ) (Thermo Fisher Scientific) in the presence of MnCl2 (2.5 mM) and ATP (1 mM) in 1X reaction buffer and a final volume of 50 µL.

    Techniques: Generated

    Relationships among M 3′ NCR and CDS regions for viruses from different host species and geographic locations. Alignments of the 3′ NCRs, shown corresponding to the genomic RNAs, from the viruses used in the analyses are next to the CDS-based neighbor-joining tree for these viruses.

    Journal: Veterinary Sciences

    Article Title: Analysis of the Variability in the Non-Coding Regions of Influenza A Viruses

    doi: 10.3390/vetsci5030076

    Figure Lengend Snippet: Relationships among M 3′ NCR and CDS regions for viruses from different host species and geographic locations. Alignments of the 3′ NCRs, shown corresponding to the genomic RNAs, from the viruses used in the analyses are next to the CDS-based neighbor-joining tree for these viruses.

    Article Snippet: For the 3′ NCRs, genomic RNAs were polyadenylated with poly(A) polymerase (2 U µL−1 ) (Thermo Fisher Scientific) in the presence of MnCl2 (2.5 mM) and ATP (1 mM) in 1X reaction buffer and a final volume of 50 µL.

    Techniques:

    Relationships among H1 3′ NCR and CDS regions for viruses from different host species and geographic locations. Alignments of the 3′ NCRs, shown corresponding to the genomic RNAs, from the viruses used in the analyses are next to the CDS-based neighbor-joining tree for these viruses.

    Journal: Veterinary Sciences

    Article Title: Analysis of the Variability in the Non-Coding Regions of Influenza A Viruses

    doi: 10.3390/vetsci5030076

    Figure Lengend Snippet: Relationships among H1 3′ NCR and CDS regions for viruses from different host species and geographic locations. Alignments of the 3′ NCRs, shown corresponding to the genomic RNAs, from the viruses used in the analyses are next to the CDS-based neighbor-joining tree for these viruses.

    Article Snippet: For the 3′ NCRs, genomic RNAs were polyadenylated with poly(A) polymerase (2 U µL−1 ) (Thermo Fisher Scientific) in the presence of MnCl2 (2.5 mM) and ATP (1 mM) in 1X reaction buffer and a final volume of 50 µL.

    Techniques:

    Relationships among N6 3′ NCR and CDS regions for viruses from different host species and geographic locations. Alignments of the 3′ NCRs, shown corresponding to the genomic RNAs, from the viruses used in the analyses are next to the CDS-based neighbor-joining tree for these viruses. To facilitate the distinction of viruses from different hosts, gull viruses are in green, duck viruses are in orange, shorebird viruses are in purple, and chicken viruses are in brown.

    Journal: Veterinary Sciences

    Article Title: Analysis of the Variability in the Non-Coding Regions of Influenza A Viruses

    doi: 10.3390/vetsci5030076

    Figure Lengend Snippet: Relationships among N6 3′ NCR and CDS regions for viruses from different host species and geographic locations. Alignments of the 3′ NCRs, shown corresponding to the genomic RNAs, from the viruses used in the analyses are next to the CDS-based neighbor-joining tree for these viruses. To facilitate the distinction of viruses from different hosts, gull viruses are in green, duck viruses are in orange, shorebird viruses are in purple, and chicken viruses are in brown.

    Article Snippet: For the 3′ NCRs, genomic RNAs were polyadenylated with poly(A) polymerase (2 U µL−1 ) (Thermo Fisher Scientific) in the presence of MnCl2 (2.5 mM) and ATP (1 mM) in 1X reaction buffer and a final volume of 50 µL.

    Techniques:

    3′ NCRs of wild bird virus segments determined in this study. The multiple alignment of 3′ NCRs was generated with MUSCLE, implemented in Geneious version 8. The RNA sequences are displayed in the 3′ to 5′ orientation corresponding to the packaged RNAs.

    Journal: Veterinary Sciences

    Article Title: Analysis of the Variability in the Non-Coding Regions of Influenza A Viruses

    doi: 10.3390/vetsci5030076

    Figure Lengend Snippet: 3′ NCRs of wild bird virus segments determined in this study. The multiple alignment of 3′ NCRs was generated with MUSCLE, implemented in Geneious version 8. The RNA sequences are displayed in the 3′ to 5′ orientation corresponding to the packaged RNAs.

    Article Snippet: For the 3′ NCRs, genomic RNAs were polyadenylated with poly(A) polymerase (2 U µL−1 ) (Thermo Fisher Scientific) in the presence of MnCl2 (2.5 mM) and ATP (1 mM) in 1X reaction buffer and a final volume of 50 µL.

    Techniques: Generated