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    Structured Review

    Thermo Fisher rna targets
    Hierarchical clustering of subjects and micro <t>RNA</t> s (mi RNA s). The levels of 55 serum mi RNA s that passed false discovery rate‐corrected ANCOVA s ( α = 0.05) were used for analyses of the 30 subjects. An “A” followed by a number designates all the alcohol‐consuming subjects, while a “C” designates control subjects. Subjects were clustered into 2 groups with most alcohol‐consuming and control individuals present in separate clusters. mi RNA s were clustered into 2 groups, those that were increased in the alcohol group and those that were decreased in this group.
    Rna Targets, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Alcohol Use During Pregnancy is Associated with Specific Alterations in Micro RNA Levels in Maternal Serum"

    Article Title: Alcohol Use During Pregnancy is Associated with Specific Alterations in Micro RNA Levels in Maternal Serum

    Journal: Alcoholism, Clinical and Experimental Research

    doi: 10.1111/acer.13026

    Hierarchical clustering of subjects and micro RNA s (mi RNA s). The levels of 55 serum mi RNA s that passed false discovery rate‐corrected ANCOVA s ( α = 0.05) were used for analyses of the 30 subjects. An “A” followed by a number designates all the alcohol‐consuming subjects, while a “C” designates control subjects. Subjects were clustered into 2 groups with most alcohol‐consuming and control individuals present in separate clusters. mi RNA s were clustered into 2 groups, those that were increased in the alcohol group and those that were decreased in this group.
    Figure Legend Snippet: Hierarchical clustering of subjects and micro RNA s (mi RNA s). The levels of 55 serum mi RNA s that passed false discovery rate‐corrected ANCOVA s ( α = 0.05) were used for analyses of the 30 subjects. An “A” followed by a number designates all the alcohol‐consuming subjects, while a “C” designates control subjects. Subjects were clustered into 2 groups with most alcohol‐consuming and control individuals present in separate clusters. mi RNA s were clustered into 2 groups, those that were increased in the alcohol group and those that were decreased in this group.

    Techniques Used:

    Reverse transcription quantitative polymerase chain reaction (RT‐qPCR) confirms results of microarray analysis. ( A ) Relative levels by microarray of 4 micro RNA s (mi RNA s) that passed false discovery rate‐corrected ANCOVA s ( α = 0.05), compared to control (dotted line). ( B ) Relative levels by RT ‐ qPCR of the same mi RNA s. Spike‐in synthetic C aenorhabditis elegans mi R ‐39 was used for normalization for RT ‐q PCR . Data represent mean ± SEM . * p
    Figure Legend Snippet: Reverse transcription quantitative polymerase chain reaction (RT‐qPCR) confirms results of microarray analysis. ( A ) Relative levels by microarray of 4 micro RNA s (mi RNA s) that passed false discovery rate‐corrected ANCOVA s ( α = 0.05), compared to control (dotted line). ( B ) Relative levels by RT ‐ qPCR of the same mi RNA s. Spike‐in synthetic C aenorhabditis elegans mi R ‐39 was used for normalization for RT ‐q PCR . Data represent mean ± SEM . * p

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

    Red blood cell ( RBC ) and white blood cell ( WBC ) micro RNA (mi RNA ) levels in serum. Relative levels of RBC mi RNA s (mi R ‐451, mi R ‐486‐5p, mi R ‐92a, let‐7a, and mi R ‐16) ( A ), and in particular mi R ‐451 ( B ), showed no difference between groups, indicating that hemolysis did not affect the results. There was also no difference between groups for WBC mi RNA s, mi R ‐223 ( C ) and mi R ‐125b ( D ). Data represent microarray signal intensities normalized to the average values of the control group (mean ± SEM ).
    Figure Legend Snippet: Red blood cell ( RBC ) and white blood cell ( WBC ) micro RNA (mi RNA ) levels in serum. Relative levels of RBC mi RNA s (mi R ‐451, mi R ‐486‐5p, mi R ‐92a, let‐7a, and mi R ‐16) ( A ), and in particular mi R ‐451 ( B ), showed no difference between groups, indicating that hemolysis did not affect the results. There was also no difference between groups for WBC mi RNA s, mi R ‐223 ( C ) and mi R ‐125b ( D ). Data represent microarray signal intensities normalized to the average values of the control group (mean ± SEM ).

    Techniques Used: Microarray

    Identification of the top 10 most discriminant micro RNA s (mi RNA s). The 55 significantly altered mi RNA s were subjected to orthogonal partial least‐squares discriminant analysis to determine which mi RNA s contributed most to the segregation of the 2 groups. The top panel shows the separation of subjects according to the alcohol consumption. The top 10 contributors are labeled in the bottom panel.
    Figure Legend Snippet: Identification of the top 10 most discriminant micro RNA s (mi RNA s). The 55 significantly altered mi RNA s were subjected to orthogonal partial least‐squares discriminant analysis to determine which mi RNA s contributed most to the segregation of the 2 groups. The top panel shows the separation of subjects according to the alcohol consumption. The top 10 contributors are labeled in the bottom panel.

    Techniques Used: Labeling

    Bar graphs illustrating the difference between alcohols and controls for the top 10 discriminating micro RNA s (mi RNA s) identified by discriminant analysis. Data represent batch‐corrected and log 2 ‐normalized microarray signal intensities (mean ± SEM ). ** p
    Figure Legend Snippet: Bar graphs illustrating the difference between alcohols and controls for the top 10 discriminating micro RNA s (mi RNA s) identified by discriminant analysis. Data represent batch‐corrected and log 2 ‐normalized microarray signal intensities (mean ± SEM ). ** p

    Techniques Used: Microarray

    2) Product Images from "Reverse engineering a hierarchical regulatory network downstream of oncogenic KRAS"

    Article Title: Reverse engineering a hierarchical regulatory network downstream of oncogenic KRAS

    Journal: Molecular Systems Biology

    doi: 10.1038/msb.2012.32

    Effects of transcription factor silencing on cell morphology and the transcriptional profile of RAS-ROSE cells. ( A ) Morphological characteristics of normal ROSE versus RAS-ROSE cells 48 h after treatment with scrambled siRNA-duplex (Sc), transfection reagents only (Mock) and two independent specific siRNAs targeting Fosl1, Hmga2, Klf6, JunB, Otx1, Gfi1 and RelA, respectively. Phase contrast, magnification: 450-fold. ( B ) Quantification of lateral processes of RAS-ROSE and transfectants, in which transcription factor expression was silenced by siRNA. ( C ) Effects of transcription factor silencing on the transcriptome of RAS-ROSE cells. Each transcription factor controlled expression of hundreds of regulated genes as indicated by hybridization of target RNA to probe sets on the microarray (cutoff for significant upregulation or downregulation: ±0.7 on log2 scale). Total number of probe sets indicating expression alterations as a response to transcription factor silencing: 5971 (3247 and 3054 upregulated and downregulated by knockdown, respectively; few probe sets indicated inconsistent target regulation among knockdowns); average number of probe sets indicating response to silencing of an individual transcription factor: 3300; number of probe sets indicating response to scrambled (sc) siRNA: 319. ( D ) Specificity of transcription factor silencing as determined by profiling of target genes. Transcription factor knockdowns induced broadly overlapping gene expression responses. Each probe set indicating expression alterations was classified according to the exact number of transcription factor knockdowns (1–7) affecting gene expression. Cutoff used for significant deregulation as in C. In total, expression levels of genes represented by 4832 (81%) probe sets were altered by two or more transcription factor knockdowns, while genes represented by only 1139 probe sets (19%) indicated specifically responded to one of the knockdowns. Source data is available for this figure in the Supplementary Information .
    Figure Legend Snippet: Effects of transcription factor silencing on cell morphology and the transcriptional profile of RAS-ROSE cells. ( A ) Morphological characteristics of normal ROSE versus RAS-ROSE cells 48 h after treatment with scrambled siRNA-duplex (Sc), transfection reagents only (Mock) and two independent specific siRNAs targeting Fosl1, Hmga2, Klf6, JunB, Otx1, Gfi1 and RelA, respectively. Phase contrast, magnification: 450-fold. ( B ) Quantification of lateral processes of RAS-ROSE and transfectants, in which transcription factor expression was silenced by siRNA. ( C ) Effects of transcription factor silencing on the transcriptome of RAS-ROSE cells. Each transcription factor controlled expression of hundreds of regulated genes as indicated by hybridization of target RNA to probe sets on the microarray (cutoff for significant upregulation or downregulation: ±0.7 on log2 scale). Total number of probe sets indicating expression alterations as a response to transcription factor silencing: 5971 (3247 and 3054 upregulated and downregulated by knockdown, respectively; few probe sets indicated inconsistent target regulation among knockdowns); average number of probe sets indicating response to silencing of an individual transcription factor: 3300; number of probe sets indicating response to scrambled (sc) siRNA: 319. ( D ) Specificity of transcription factor silencing as determined by profiling of target genes. Transcription factor knockdowns induced broadly overlapping gene expression responses. Each probe set indicating expression alterations was classified according to the exact number of transcription factor knockdowns (1–7) affecting gene expression. Cutoff used for significant deregulation as in C. In total, expression levels of genes represented by 4832 (81%) probe sets were altered by two or more transcription factor knockdowns, while genes represented by only 1139 probe sets (19%) indicated specifically responded to one of the knockdowns. Source data is available for this figure in the Supplementary Information .

    Techniques Used: Transfection, Expressing, Hybridization, Microarray

    3) Product Images from "Discriminating cellular heterogeneity using microwell-based RNA cytometry"

    Article Title: Discriminating cellular heterogeneity using microwell-based RNA cytometry

    Journal: Nature communications

    doi: 10.1038/ncomms4451

    The endpoint fluorescence of the ~20 pL microwell PCR reaction is proportional to the logarithm of the initial amount of target DNA (i.e. log-linear). (A) A representative image of a 15,000 well array before PCR. Scale bar represents 40 µm.(B) The clear log-linear trend between well intensity at cycle 40 and the number of cells per well after amplifying the promoter region of the Nanog gene suggests that the well intensity is a semi-quantitative indicator of the initial amount of target DNA. Error bars represent standard error (n = 50 for 0–3 cells/well and n = 4 for 4 cells/well). Note that the initial background fluorescence coming from the nuclear DNA before PCR has been subtracted and the mean intensity is the signal from the free in-solution fluorescence. (C) Representative image from the micro-well array after 40 cycles for PCR. Scale bar represents 40 µm. (D) Magnified image of a representative region with wells containing, 0, 1, 2, 3 and 4 cells. The array was loaded with ~500 cell/µL in a total volume of 60 µL. The dashed lines represent the well boundaries. Note that all pixels that correspond to the fluorescent intensity from the cells are discarded from the quantification, thus only the area corresponding to the free, in-solution fluorescence is quantified. Scale bar represents 20 µm. (E) Log-linear relation between endpoint fluorescence (40 cycles) and starting number of spiked in pAW109 RNA templates. Error bars represent standard error (n = 20).
    Figure Legend Snippet: The endpoint fluorescence of the ~20 pL microwell PCR reaction is proportional to the logarithm of the initial amount of target DNA (i.e. log-linear). (A) A representative image of a 15,000 well array before PCR. Scale bar represents 40 µm.(B) The clear log-linear trend between well intensity at cycle 40 and the number of cells per well after amplifying the promoter region of the Nanog gene suggests that the well intensity is a semi-quantitative indicator of the initial amount of target DNA. Error bars represent standard error (n = 50 for 0–3 cells/well and n = 4 for 4 cells/well). Note that the initial background fluorescence coming from the nuclear DNA before PCR has been subtracted and the mean intensity is the signal from the free in-solution fluorescence. (C) Representative image from the micro-well array after 40 cycles for PCR. Scale bar represents 40 µm. (D) Magnified image of a representative region with wells containing, 0, 1, 2, 3 and 4 cells. The array was loaded with ~500 cell/µL in a total volume of 60 µL. The dashed lines represent the well boundaries. Note that all pixels that correspond to the fluorescent intensity from the cells are discarded from the quantification, thus only the area corresponding to the free, in-solution fluorescence is quantified. Scale bar represents 20 µm. (E) Log-linear relation between endpoint fluorescence (40 cycles) and starting number of spiked in pAW109 RNA templates. Error bars represent standard error (n = 20).

    Techniques Used: Fluorescence, Polymerase Chain Reaction

    Verification of specific mRNA amplification. (A) Single-cell RT-PCR of ActB mRNA in BB88 cells with reverse transcription omitted and only PCR thermo-cycling performed. A histogram of the well intensities showing no amplification signal over the empty well threshold f tr (well intensities over f tr represented by the red dotted line). This demonstrates that DNA is not amplified. (B) Single-cell RT-PCR of ActB mRNA in BB88 cells after removing the RT and polymerase enzymes from the reaction mix. A histogram of the well intensities showing no amplification signal over the empty well threshold f tr (well intensities over f tr represented by the red dotted line). No DNA or RNA is amplified. (C) Single-cell RT-PCR of ActB mRNA in BB88 cells, positive control where RT and polymerase enzymes are included and the standard thermo-cycling protocol is performed. Histogram of the well intensities showing a clear presence of amplification signal over the empty well threshold f tr (signal above the red dotted line). Note all reactions are done using the CellsDirect, Invitrogen kit as described in the Methods, RT-PCR Amplification section. Abbreviations: f tr indicates threshold fluorescence; μ norm , mean; and σ, standard deviation.
    Figure Legend Snippet: Verification of specific mRNA amplification. (A) Single-cell RT-PCR of ActB mRNA in BB88 cells with reverse transcription omitted and only PCR thermo-cycling performed. A histogram of the well intensities showing no amplification signal over the empty well threshold f tr (well intensities over f tr represented by the red dotted line). This demonstrates that DNA is not amplified. (B) Single-cell RT-PCR of ActB mRNA in BB88 cells after removing the RT and polymerase enzymes from the reaction mix. A histogram of the well intensities showing no amplification signal over the empty well threshold f tr (well intensities over f tr represented by the red dotted line). No DNA or RNA is amplified. (C) Single-cell RT-PCR of ActB mRNA in BB88 cells, positive control where RT and polymerase enzymes are included and the standard thermo-cycling protocol is performed. Histogram of the well intensities showing a clear presence of amplification signal over the empty well threshold f tr (signal above the red dotted line). Note all reactions are done using the CellsDirect, Invitrogen kit as described in the Methods, RT-PCR Amplification section. Abbreviations: f tr indicates threshold fluorescence; μ norm , mean; and σ, standard deviation.

    Techniques Used: Amplification, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Positive Control, Fluorescence, Standard Deviation

    4) Product Images from "HAPIscreen, a method for high-throughput aptamer identification"

    Article Title: HAPIscreen, a method for high-throughput aptamer identification

    Journal: Journal of Nanobiotechnology

    doi: 10.1186/1477-3155-9-25

    HAPIscreen - proof of concept . ( A ) Scheme of the assay setup using a digoxigenin-tagged aptamer (R06) and a biotinylated target RNA hairpin (TAR). The association of the two components is detected by using both Donor streptavidin (D) and Acceptor anti-digoxigenin (A) coated AlphaScreen ® beads. The production of singlet oxygen upon laser excitation by D-phtalocyanin is monitored by the fluorescence emission of A-rubrene beads. ( B ) Results obtained when increasing concentrations of dig-R06 were added to A and D beads for different biot-TAR concentrations (from 0 to 40 nM as indicated on the right). ( C ) Secondary structures and/or sequences of the top part of the trans -activating responsive (TAR) RNA element of HIV-1 (biot-TAR), 5' end-extended TAR (rA-TAR), RNA aptamer R06 (dig-R06), domain II of the HCV Internal Ribosome Entry Site (biot-DII), primer anchor (dig-primer). The latter was synthesized with 2'-O-methyl residues except at two positions (underlined) where Locked Nucleic Acid residues were introduced in order to promote hybridization and to increase complex stability. Oligod(T 3 CT 21 ) anchor was used for capturing rA-TAR or the candidates from the M1 or M2 SELEX. The former were captured with biot-dT and the latter with dig-dT oligonucleotides, respectively.
    Figure Legend Snippet: HAPIscreen - proof of concept . ( A ) Scheme of the assay setup using a digoxigenin-tagged aptamer (R06) and a biotinylated target RNA hairpin (TAR). The association of the two components is detected by using both Donor streptavidin (D) and Acceptor anti-digoxigenin (A) coated AlphaScreen ® beads. The production of singlet oxygen upon laser excitation by D-phtalocyanin is monitored by the fluorescence emission of A-rubrene beads. ( B ) Results obtained when increasing concentrations of dig-R06 were added to A and D beads for different biot-TAR concentrations (from 0 to 40 nM as indicated on the right). ( C ) Secondary structures and/or sequences of the top part of the trans -activating responsive (TAR) RNA element of HIV-1 (biot-TAR), 5' end-extended TAR (rA-TAR), RNA aptamer R06 (dig-R06), domain II of the HCV Internal Ribosome Entry Site (biot-DII), primer anchor (dig-primer). The latter was synthesized with 2'-O-methyl residues except at two positions (underlined) where Locked Nucleic Acid residues were introduced in order to promote hybridization and to increase complex stability. Oligod(T 3 CT 21 ) anchor was used for capturing rA-TAR or the candidates from the M1 or M2 SELEX. The former were captured with biot-dT and the latter with dig-dT oligonucleotides, respectively.

    Techniques Used: Amplified Luminescent Proximity Homogenous Assay, Fluorescence, Synthesized, Hybridization

    5) Product Images from "Genome-wide analysis of plant nat-siRNAs reveals insights into their distribution, biogenesis and function"

    Article Title: Genome-wide analysis of plant nat-siRNAs reveals insights into their distribution, biogenesis and function

    Journal: Genome Biology

    doi: 10.1186/gb-2012-13-3-r20

    Expression of the NAT transcripts is increased in dcl1 - 7 fwf2 and dcl3 - 1 mutants . Expression was examined by quantitative RT-PCR and Actin2 was used as an internal control. Total RNA (5 μg) was treated with DNaseI and then subjected to reverse transcription. Error bars indicate standard deviations derived from three technical replicates. Similar results were obtained from two biological replicates.
    Figure Legend Snippet: Expression of the NAT transcripts is increased in dcl1 - 7 fwf2 and dcl3 - 1 mutants . Expression was examined by quantitative RT-PCR and Actin2 was used as an internal control. Total RNA (5 μg) was treated with DNaseI and then subjected to reverse transcription. Error bars indicate standard deviations derived from three technical replicates. Similar results were obtained from two biological replicates.

    Techniques Used: Expressing, Quantitative RT-PCR, Derivative Assay

    Related Articles

    Amplification:

    Article Title: Discriminating cellular heterogeneity using microwell-based RNA cytometry
    Article Snippet: .. RT-PCR reactions of pAW109 RNA were performed by loading the microwells with a known number of RNA targets obtained from the GeneAmp RNA PCR controls kit (cat # 4308238, Life Technologies, Foster City, CA, USA), and reverse transcribed into cDNA and PCR amplified using the primer set for pAW109 RNA included in the kit. .. Fluorescence signal was achieved by adding 20× Evagreen dye from Biotium (Hayward, CA, USA) to the RT-PCR mix.

    Synthesized:

    Article Title: HAPIscreen, a method for high-throughput aptamer identification
    Article Snippet: .. All RNA targets and the digoxygenin 2'-O-methyl-LNA anchor (dig-primer) were chemically synthesized on an Expedite 8908 synthesizer (Applied Biosystems, USA) and purified by electrophoresis on denaturing 20% polyacrylamide, 7M urea gels. .. RNA candidates were synthesized by in vitro transcription using T7 RNA polymerase.

    Quantitative RT-PCR:

    Article Title: Genome-wide analysis of plant nat-siRNAs reveals insights into their distribution, biogenesis and function
    Article Snippet: .. Quantitative RT-PCR analysis of small RNA targets For QRT-PCR analysis, 5 μg total DNaseI-treated (Invitrogen, Grand Island, NY, USA) RNA was used for synthesizing cDNA using Oligo dT and SuperScript II (Invitrogen). .. Amplification of small RNA targets was carried out using a real-time PCR machine (MyiQ, Bio-Rad, Hercules, CA, USA).

    Purification:

    Article Title: HAPIscreen, a method for high-throughput aptamer identification
    Article Snippet: .. All RNA targets and the digoxygenin 2'-O-methyl-LNA anchor (dig-primer) were chemically synthesized on an Expedite 8908 synthesizer (Applied Biosystems, USA) and purified by electrophoresis on denaturing 20% polyacrylamide, 7M urea gels. .. RNA candidates were synthesized by in vitro transcription using T7 RNA polymerase.

    Electrophoresis:

    Article Title: HAPIscreen, a method for high-throughput aptamer identification
    Article Snippet: .. All RNA targets and the digoxygenin 2'-O-methyl-LNA anchor (dig-primer) were chemically synthesized on an Expedite 8908 synthesizer (Applied Biosystems, USA) and purified by electrophoresis on denaturing 20% polyacrylamide, 7M urea gels. .. RNA candidates were synthesized by in vitro transcription using T7 RNA polymerase.

    Microarray:

    Article Title: Reverse engineering a hierarchical regulatory network downstream of oncogenic KRAS
    Article Snippet: .. Whole genome microarray analysis Labelling of RNA targets, hybridization and post-hybridization procedures for Affymetrix microarrays were essentially as previously described ( ). .. Normalization and determining the expression values were done using Robust Multi-array Average (RMA) available in the bioconductor R-package.

    Polymerase Chain Reaction:

    Article Title: Discriminating cellular heterogeneity using microwell-based RNA cytometry
    Article Snippet: .. RT-PCR reactions of pAW109 RNA were performed by loading the microwells with a known number of RNA targets obtained from the GeneAmp RNA PCR controls kit (cat # 4308238, Life Technologies, Foster City, CA, USA), and reverse transcribed into cDNA and PCR amplified using the primer set for pAW109 RNA included in the kit. .. Fluorescence signal was achieved by adding 20× Evagreen dye from Biotium (Hayward, CA, USA) to the RT-PCR mix.

    Hybridization:

    Article Title: Transcriptional monitoring of steady state and effects of anaerobic phases in chemostat cultures of the filamentous fungus Trichoderma reesei
    Article Snippet: .. The steps following hybridization, including affinity capture, washing and elution, were automated with a magnetic bead particle processor KingFisher 96 (Thermo Electron, Vantaa, Finland) in 96-well plates, as follows: 1) affinity capture of hybridized RNA targets to 50 μg of streptavidin-coated MyOne DynaBeads (Dynal, Oslo, Norway) at room temperature for 30 min, 2) washing of the beads two times for 1.5 min in 150 μl of 1 × SSC, 0.1% (w/v) SDS at room temperature, 3) washing twice for 1.5 min in 150 μl of 0.5 × SSC, 0.1% (w/v) SDS at RT, 4) washing once for 1.5 min in 150 μl of 0.1 × SSC, 0.1% (w/v) SDS at RT and 5) elution of probes to 10 μl deionised formamide (Sigma) for 20 min at 37°C. .. The eluents were analyzed by capillary electrophoresis with an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA).

    Article Title: Reverse engineering a hierarchical regulatory network downstream of oncogenic KRAS
    Article Snippet: .. Whole genome microarray analysis Labelling of RNA targets, hybridization and post-hybridization procedures for Affymetrix microarrays were essentially as previously described ( ). .. Normalization and determining the expression values were done using Robust Multi-array Average (RMA) available in the bioconductor R-package.

    Plex Assay:

    Article Title: Validation of whole-blood transcriptome signature during microdose recombinant human erythropoietin (rHuEpo) administration
    Article Snippet: .. QuantiGene Plex experiment and data analysis Two hundred nanogram RNA was run in duplicate for quantification of the 45 selected RNA targets and 5 reference genes (ACTB , ACTR10 , MRFAP1 , PPIB and RAB11A ) in subjects participating in the MDS and ATS, using the QuantiGene Plex Assay (Affymetrix, Santa Clara, CA, USA). .. The resulting fluorescence signal was measured on the MAGPIX (Luminex, Austin, TX, USA).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Discriminating cellular heterogeneity using microwell-based RNA cytometry
    Article Snippet: .. RT-PCR reactions of pAW109 RNA were performed by loading the microwells with a known number of RNA targets obtained from the GeneAmp RNA PCR controls kit (cat # 4308238, Life Technologies, Foster City, CA, USA), and reverse transcribed into cDNA and PCR amplified using the primer set for pAW109 RNA included in the kit. .. Fluorescence signal was achieved by adding 20× Evagreen dye from Biotium (Hayward, CA, USA) to the RT-PCR mix.

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    Thermo Fisher p labeled target rna
    Target <t>RNA</t> cleavage by the <t>Csm</t> complex. Wild-type (WT), Csm3 D35A variant (C3), or cyclase variant (Cy) Csm were incubated with 5’- 32 P-target RNA under single-turnover conditions for 0.5, 2, 5, 30, 90 min and analysed by denaturing PAGE.
    P Labeled Target Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher target rnas
    PR100 down-regulates the expression of ribosomal <t>RNAs.</t> a , b NSC-34 cells were transfected with the empty vector or the FLAG-PR100-encoding vector. At 48 h after the transfection, the cell lysates were immunoprecipitated (IP) with normal mouse IgG (Cont.) or FLAG antibody. Precipitates were then used for <t>RNA</t> immunoprecipitation (RIP) assay ( a ) and dot blotting analysis using FLAG antibody ( b ). RT (−) was used as negative control to monitor the PCR amplification from genomic DNA. Priming sites of primers are shown in Fig. S 1a . c NSC-34 cells were infected with adenovirus encoding LacZ or FLAG-PR100 at an MOI of 800 together with adenovirus encoding Cre-recombinase at an MOI of 40. At 48 h after the infection, quantitative real time PCR analysis of 45S pre-rRNA, 18S rRNA, and 28S rRNA was performed. Means ± SD, N = 3 biological replicates. Statistical analysis was determined by unpaired t test
    Target Rnas, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rna interference small interfering rna sirna oligonucleotides targeting tlr2
    Saturated fatty acid-induced CSF3 expression via Toll-like receptors . (A) CSF3 mRNA expression was measured in human myotubes pretreated for 6 h with TAK-242 (1 μg/mL) prior to 24-hour treatment with palmitate (0.5 mmol/L). (B) Human myotubes were pretreated for 24 h with <t>siRNA</t> for <t>TLR2</t> or TLR4 , respectively, prior to 24-hour treatment with palmitate (0.5 mmol/L), and TLR2 , TLR4 , and CSF3 mRNA contents were quantified. Data are given as means ± SEM. Two-group comparisons were performed using matched-pairs Student's t -test (N ≥ 3; *p
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    Thermo Fisher rna interference targeting particle particle knockdown
    In vivo <t>PARTICLE</t> and DNA methyltransferase 1 (DNMT1) interaction. ( A ) Linearized dbroccoli in pUC57 (2956 bp; p.db.) after E coR I/H ind III restriction enzyme digestion resolved on a 1.8% agarose gel (left). Plasmid pGEM-T encoding dbroccoli - PARTICLE (p.db. PT ) restriction digested with N co I/H ind III to produce 3728 bp and 950 bp products (upper and lower arrows). DL3000 (Genscript) and Kb ladder (Ld. left and right gels respectively). ( B ) dbroccoli - PARTICLE (1678 bp; db. PT ) in vitro transcript resolved through a 12% NuPAGE gel alongside a high range <t>RNA</t> ladder (Ld. Thermo Fisher RiboRuler) before and after staining in DFHBI-1T ((Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-1-(2,2,2-trifluoroethyl)-1 H -imidazol-5(4 H )-one; left and right gels respectively). ( A,B ) Cropped images of gels shown. ( C ) Time-lapse fluorescence images of a U2OS cell transfected with db. PT and expressing a chromobody to DNMT1-V H H fused to TagRFP (DNMT1_RFP) before (minus time point) and after (plus time points) DFHBI-1T (20 μM) addition. Scale bar 2 μm. ( D ) Histograms of arbitrary units (AU) of overall fluorescence intensity with time in U2OS transfected with db. PT dose range in the presence of DFHBI-1T (20 μM). Data are represented as mean ± SEM from n = 3 experiments per dose. ( E ) Summary co-localization plots for DNMT_RFP (ChR = red channel) and db. PT (ChG = green channel) from ROIs in the nucleus with time. Data are represented as mean ± SEM. ( F,G ) Plots of AU of fluorescence intensity with time from DNMT_RFP ( F ) and in db. PT ( G ) in ROIs (n = 10) within nuclear (encircling dashed line) and extranuclear (Extranuc.) cellular compartments.
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    Target RNA cleavage by the Csm complex. Wild-type (WT), Csm3 D35A variant (C3), or cyclase variant (Cy) Csm were incubated with 5’- 32 P-target RNA under single-turnover conditions for 0.5, 2, 5, 30, 90 min and analysed by denaturing PAGE.

    Journal: bioRxiv

    Article Title: Cyclic oligoadenylate signalling mediates Mycobacterium tuberculosis CRISPR defence

    doi: 10.1101/667758

    Figure Lengend Snippet: Target RNA cleavage by the Csm complex. Wild-type (WT), Csm3 D35A variant (C3), or cyclase variant (Cy) Csm were incubated with 5’- 32 P-target RNA under single-turnover conditions for 0.5, 2, 5, 30, 90 min and analysed by denaturing PAGE.

    Article Snippet: Target RNA cleavage by the Csm complex The ribonuclease activity of the Csm complex was assessed by adding 25 nM [5’-32 P]-labeled target RNA ( ) to 0.8 µM Csm, 5 mM MgCl2 , 0.1 U µl-1 SUPERase•In™ (Thermo Scientific), 50 mM Tris-HCl, 50 mM NaCl, pH 8.0, and incubating at 30 °C for up to 2 h. The reaction was stopped by phenol-chloroform-isoamyl alcohol extraction to remove proteins.

    Techniques: Variant Assay, Incubation, Polyacrylamide Gel Electrophoresis

    cOA production and Csm6 activation ( A ) Extracted ion chromatograms of oligoadenylates produced by wild type (WT) and Csm3 D35A variant (C3) Csm complex. Identical y -scaling throughout. The identity of the cyclic OAs (cA n ) and respective 5’-triphosphates (P 3 -A n ) is indicated by the number of AMP subunits. Production of cOAs, cA 5 and cA 6 in particular, was significantly increased for the Csm3 D35A (C3) Csm complex. ( B ) Csm6 ribonuclease activation by cyclic oligoadenylates. Mtb Csm6 (125 nM) was incubated with 200 nM 5’-[ 32 P]-substrate RNA at 37 °C for 30 min in the presence of synthetic oligoadenylates (cA 4 , cA 6 ) or a 10-fold dilution of Csm-derived cOAs. Csm wild-type (WT, 800 nM), cyclase variant (Cy, 800 nM), or Csm3 D35A (C3, 200 nM) was incubated with 1 mM ATP and 200 nM target (T), non-target (NT), or anti-tag (AT) RNA for 0.5, 2 (WT, C3), or 90 min (all others) at 30 °C. Only cA 6 activated Csm6. For WT and C3 Csm, sufficient cA 6 was produced within 0.5 min to switch on Csm6 ribonuclease activity; whereas no detectable amount of cA 6 was produced under all other conditions even after prolonged reaction time.

    Journal: bioRxiv

    Article Title: Cyclic oligoadenylate signalling mediates Mycobacterium tuberculosis CRISPR defence

    doi: 10.1101/667758

    Figure Lengend Snippet: cOA production and Csm6 activation ( A ) Extracted ion chromatograms of oligoadenylates produced by wild type (WT) and Csm3 D35A variant (C3) Csm complex. Identical y -scaling throughout. The identity of the cyclic OAs (cA n ) and respective 5’-triphosphates (P 3 -A n ) is indicated by the number of AMP subunits. Production of cOAs, cA 5 and cA 6 in particular, was significantly increased for the Csm3 D35A (C3) Csm complex. ( B ) Csm6 ribonuclease activation by cyclic oligoadenylates. Mtb Csm6 (125 nM) was incubated with 200 nM 5’-[ 32 P]-substrate RNA at 37 °C for 30 min in the presence of synthetic oligoadenylates (cA 4 , cA 6 ) or a 10-fold dilution of Csm-derived cOAs. Csm wild-type (WT, 800 nM), cyclase variant (Cy, 800 nM), or Csm3 D35A (C3, 200 nM) was incubated with 1 mM ATP and 200 nM target (T), non-target (NT), or anti-tag (AT) RNA for 0.5, 2 (WT, C3), or 90 min (all others) at 30 °C. Only cA 6 activated Csm6. For WT and C3 Csm, sufficient cA 6 was produced within 0.5 min to switch on Csm6 ribonuclease activity; whereas no detectable amount of cA 6 was produced under all other conditions even after prolonged reaction time.

    Article Snippet: Target RNA cleavage by the Csm complex The ribonuclease activity of the Csm complex was assessed by adding 25 nM [5’-32 P]-labeled target RNA ( ) to 0.8 µM Csm, 5 mM MgCl2 , 0.1 U µl-1 SUPERase•In™ (Thermo Scientific), 50 mM Tris-HCl, 50 mM NaCl, pH 8.0, and incubating at 30 °C for up to 2 h. The reaction was stopped by phenol-chloroform-isoamyl alcohol extraction to remove proteins.

    Techniques: Activation Assay, Produced, Variant Assay, Incubation, Derivative Assay, Activity Assay

    RNA backbone cleavage by Csm interference complex. (A ) 5’- 32 P-labeled target RNA or non-target (NT) RNA were treated with 0.8 µM Csm effector complex for 5, 15, 30, 60, 120 min. Reactions were analysed by denaturing PAGE alongside alkaline hydrolysis ladders prepared from target RNA. ( B ) Target RNA and anti-tag RNA (full-length complementarity to crRNA including repeat-derived 5’-handle) are both cleaved by Csm to give identical products. Reaction time: 0.5, 2, 5, 30, 100 min; substrate RNA is indicated by unfilled triangles; the five cleavage sites (B1 – B5) with the characteristic 6 nt spacing are indicated by filled triangles. Target RNA with a 4 nt truncation at the 3’-end was used, leading to a different distribution of cleavage products compared to Figure 2 .

    Journal: bioRxiv

    Article Title: Cyclic oligoadenylate signalling mediates Mycobacterium tuberculosis CRISPR defence

    doi: 10.1101/667758

    Figure Lengend Snippet: RNA backbone cleavage by Csm interference complex. (A ) 5’- 32 P-labeled target RNA or non-target (NT) RNA were treated with 0.8 µM Csm effector complex for 5, 15, 30, 60, 120 min. Reactions were analysed by denaturing PAGE alongside alkaline hydrolysis ladders prepared from target RNA. ( B ) Target RNA and anti-tag RNA (full-length complementarity to crRNA including repeat-derived 5’-handle) are both cleaved by Csm to give identical products. Reaction time: 0.5, 2, 5, 30, 100 min; substrate RNA is indicated by unfilled triangles; the five cleavage sites (B1 – B5) with the characteristic 6 nt spacing are indicated by filled triangles. Target RNA with a 4 nt truncation at the 3’-end was used, leading to a different distribution of cleavage products compared to Figure 2 .

    Article Snippet: Target RNA cleavage by the Csm complex The ribonuclease activity of the Csm complex was assessed by adding 25 nM [5’-32 P]-labeled target RNA ( ) to 0.8 µM Csm, 5 mM MgCl2 , 0.1 U µl-1 SUPERase•In™ (Thermo Scientific), 50 mM Tris-HCl, 50 mM NaCl, pH 8.0, and incubating at 30 °C for up to 2 h. The reaction was stopped by phenol-chloroform-isoamyl alcohol extraction to remove proteins.

    Techniques: Labeling, Polyacrylamide Gel Electrophoresis, Derivative Assay

    The CRISPR system of M. tuberculosis A. The CRISPR locus of M. tuberculosis includes genes encoding Cas6 (crRNA processing), Csm1-5 (type III-A interference complex), Csm6 (ancillary ribonuclease), Cas1 and Cas2 (Adaptation). Cas6 cleaves the CRISPR RNA at the base of a short hairpin to generate mature crRNA that is bound by the Csm complex. On target RNA binding, the Csm complex is expected to display three enzymatic activities: target RNA cleavage ( 1 ), DNA cleavage by the HD domain ( 2 ) and cOA production by the cyclase domain ( 3 ). B. Purified, recombinant CRISPR-associated proteins of M. tuberculosis . M: PageRuler Unstained (Thermo Scientific); 1: Csm1-5 interference complex; 2: Csm1-5, Csm1 D630A, D631A (Cy variant); 3: Csm1-5, Csm3 D35A (C3 variant); 4: Csm6; 5: Cas6.

    Journal: bioRxiv

    Article Title: Cyclic oligoadenylate signalling mediates Mycobacterium tuberculosis CRISPR defence

    doi: 10.1101/667758

    Figure Lengend Snippet: The CRISPR system of M. tuberculosis A. The CRISPR locus of M. tuberculosis includes genes encoding Cas6 (crRNA processing), Csm1-5 (type III-A interference complex), Csm6 (ancillary ribonuclease), Cas1 and Cas2 (Adaptation). Cas6 cleaves the CRISPR RNA at the base of a short hairpin to generate mature crRNA that is bound by the Csm complex. On target RNA binding, the Csm complex is expected to display three enzymatic activities: target RNA cleavage ( 1 ), DNA cleavage by the HD domain ( 2 ) and cOA production by the cyclase domain ( 3 ). B. Purified, recombinant CRISPR-associated proteins of M. tuberculosis . M: PageRuler Unstained (Thermo Scientific); 1: Csm1-5 interference complex; 2: Csm1-5, Csm1 D630A, D631A (Cy variant); 3: Csm1-5, Csm3 D35A (C3 variant); 4: Csm6; 5: Cas6.

    Article Snippet: Target RNA cleavage by the Csm complex The ribonuclease activity of the Csm complex was assessed by adding 25 nM [5’-32 P]-labeled target RNA ( ) to 0.8 µM Csm, 5 mM MgCl2 , 0.1 U µl-1 SUPERase•In™ (Thermo Scientific), 50 mM Tris-HCl, 50 mM NaCl, pH 8.0, and incubating at 30 °C for up to 2 h. The reaction was stopped by phenol-chloroform-isoamyl alcohol extraction to remove proteins.

    Techniques: CRISPR, RNA Binding Assay, Purification, Recombinant, Variant Assay

    In vitro analysis of Csm HD mutant. ( A ) SDS-PAGE of purified Csm3 D35A (C3) and Cas10 H18A/D19A (HD) variant interference complexes; *: contaminants; M: PageRuler Unstained (Fisher Scientific). ( B ) Target RNA backbone cleavage by Csm3 D35A (C3) and Cas10 H18A/D19A (HD) variant interference complexes; 5’-radiolabeled target RNA was incubated with HD or C3 in the presence of Mg 2+ for 5, 10, 30, 60 min at 30 °C; as expected the characteristic cleavage products B2 – B5 are produced by HD but not the C3 variant. ( C ) The ssDNase activity of Csm interference complex is not dependent on RNA substrate or active site mutations; 5’-radiolabeled ssDNA was incubated with Csm wild type (WT), HD, Cas10 D630A/D631A (Cy), or Csm3 D35A (C3) in the presence of Mg 2+ for 90 min at 30 °C; cold RNA was added as indicated. Mn 2+ and Co 2+ also supported the observed activity, Zn 2+ less so, and Cu 2+ did not stimulate DNase activity (data not shown). T: target RNA, NT: non-target RNA, AT: anti-tag RNA.

    Journal: bioRxiv

    Article Title: Cyclic oligoadenylate signalling mediates Mycobacterium tuberculosis CRISPR defence

    doi: 10.1101/667758

    Figure Lengend Snippet: In vitro analysis of Csm HD mutant. ( A ) SDS-PAGE of purified Csm3 D35A (C3) and Cas10 H18A/D19A (HD) variant interference complexes; *: contaminants; M: PageRuler Unstained (Fisher Scientific). ( B ) Target RNA backbone cleavage by Csm3 D35A (C3) and Cas10 H18A/D19A (HD) variant interference complexes; 5’-radiolabeled target RNA was incubated with HD or C3 in the presence of Mg 2+ for 5, 10, 30, 60 min at 30 °C; as expected the characteristic cleavage products B2 – B5 are produced by HD but not the C3 variant. ( C ) The ssDNase activity of Csm interference complex is not dependent on RNA substrate or active site mutations; 5’-radiolabeled ssDNA was incubated with Csm wild type (WT), HD, Cas10 D630A/D631A (Cy), or Csm3 D35A (C3) in the presence of Mg 2+ for 90 min at 30 °C; cold RNA was added as indicated. Mn 2+ and Co 2+ also supported the observed activity, Zn 2+ less so, and Cu 2+ did not stimulate DNase activity (data not shown). T: target RNA, NT: non-target RNA, AT: anti-tag RNA.

    Article Snippet: Target RNA cleavage by the Csm complex The ribonuclease activity of the Csm complex was assessed by adding 25 nM [5’-32 P]-labeled target RNA ( ) to 0.8 µM Csm, 5 mM MgCl2 , 0.1 U µl-1 SUPERase•In™ (Thermo Scientific), 50 mM Tris-HCl, 50 mM NaCl, pH 8.0, and incubating at 30 °C for up to 2 h. The reaction was stopped by phenol-chloroform-isoamyl alcohol extraction to remove proteins.

    Techniques: In Vitro, Mutagenesis, SDS Page, Purification, Variant Assay, Incubation, Produced, Activity Assay

    Re-programming the Mtb Csm system for cA 4 -responsive immunity. ( A ) In vitro activity of TsuCsx1. The reaction contained 0.5 µM TsuCsx1 dimer, 100 nM 5’- 32 P-labeled RNA A1, 20 mM Tris, 150 mM NaCl, 1 mM DTT, pH 7.5 and was conducted at 35 °C for 1, 5, 15, 30 min. Activators were added as indicated; Csm-derived cOAs were from a 2 h reaction, otherwise as described for Figure 3 . TsuCsx1 was activated by cA 4 but not cA 6 , and Csm-derived cOAs are able to induce Csx1 ribonuclease activity in vitro . ( B ) Plasmid immunity assay using pUC19 lacZα -targeting Csm effector complex in E. coli C43 (pCsm1-5_Csm6/tsuCsx1 and pCRISPR, Figure S4 ). The ribonuclease was either the cognate Csm6 or TsuCsx1. Cells were transformed with pRAT (control plasmid) or pRAT-Target (target plasmid) and 10-fold serial dilutions were plated on selective plates containing arabinose for induction of target transcription. TsuCsx1 confers the same level of plasmid immunity as the cognate Mtb Csm6.

    Journal: bioRxiv

    Article Title: Cyclic oligoadenylate signalling mediates Mycobacterium tuberculosis CRISPR defence

    doi: 10.1101/667758

    Figure Lengend Snippet: Re-programming the Mtb Csm system for cA 4 -responsive immunity. ( A ) In vitro activity of TsuCsx1. The reaction contained 0.5 µM TsuCsx1 dimer, 100 nM 5’- 32 P-labeled RNA A1, 20 mM Tris, 150 mM NaCl, 1 mM DTT, pH 7.5 and was conducted at 35 °C for 1, 5, 15, 30 min. Activators were added as indicated; Csm-derived cOAs were from a 2 h reaction, otherwise as described for Figure 3 . TsuCsx1 was activated by cA 4 but not cA 6 , and Csm-derived cOAs are able to induce Csx1 ribonuclease activity in vitro . ( B ) Plasmid immunity assay using pUC19 lacZα -targeting Csm effector complex in E. coli C43 (pCsm1-5_Csm6/tsuCsx1 and pCRISPR, Figure S4 ). The ribonuclease was either the cognate Csm6 or TsuCsx1. Cells were transformed with pRAT (control plasmid) or pRAT-Target (target plasmid) and 10-fold serial dilutions were plated on selective plates containing arabinose for induction of target transcription. TsuCsx1 confers the same level of plasmid immunity as the cognate Mtb Csm6.

    Article Snippet: Target RNA cleavage by the Csm complex The ribonuclease activity of the Csm complex was assessed by adding 25 nM [5’-32 P]-labeled target RNA ( ) to 0.8 µM Csm, 5 mM MgCl2 , 0.1 U µl-1 SUPERase•In™ (Thermo Scientific), 50 mM Tris-HCl, 50 mM NaCl, pH 8.0, and incubating at 30 °C for up to 2 h. The reaction was stopped by phenol-chloroform-isoamyl alcohol extraction to remove proteins.

    Techniques: In Vitro, Activity Assay, Labeling, Derivative Assay, Plasmid Preparation, Transformation Assay

    PR100 down-regulates the expression of ribosomal RNAs. a , b NSC-34 cells were transfected with the empty vector or the FLAG-PR100-encoding vector. At 48 h after the transfection, the cell lysates were immunoprecipitated (IP) with normal mouse IgG (Cont.) or FLAG antibody. Precipitates were then used for RNA immunoprecipitation (RIP) assay ( a ) and dot blotting analysis using FLAG antibody ( b ). RT (−) was used as negative control to monitor the PCR amplification from genomic DNA. Priming sites of primers are shown in Fig. S 1a . c NSC-34 cells were infected with adenovirus encoding LacZ or FLAG-PR100 at an MOI of 800 together with adenovirus encoding Cre-recombinase at an MOI of 40. At 48 h after the infection, quantitative real time PCR analysis of 45S pre-rRNA, 18S rRNA, and 28S rRNA was performed. Means ± SD, N = 3 biological replicates. Statistical analysis was determined by unpaired t test

    Journal: Cell Death & Disease

    Article Title: The proline–arginine repeat protein linked to C9-ALS/FTD causes neuronal toxicity by inhibiting the DEAD-box RNA helicase-mediated ribosome biogenesis

    doi: 10.1038/s41419-018-1028-5

    Figure Lengend Snippet: PR100 down-regulates the expression of ribosomal RNAs. a , b NSC-34 cells were transfected with the empty vector or the FLAG-PR100-encoding vector. At 48 h after the transfection, the cell lysates were immunoprecipitated (IP) with normal mouse IgG (Cont.) or FLAG antibody. Precipitates were then used for RNA immunoprecipitation (RIP) assay ( a ) and dot blotting analysis using FLAG antibody ( b ). RT (−) was used as negative control to monitor the PCR amplification from genomic DNA. Priming sites of primers are shown in Fig. S 1a . c NSC-34 cells were infected with adenovirus encoding LacZ or FLAG-PR100 at an MOI of 800 together with adenovirus encoding Cre-recombinase at an MOI of 40. At 48 h after the infection, quantitative real time PCR analysis of 45S pre-rRNA, 18S rRNA, and 28S rRNA was performed. Means ± SD, N = 3 biological replicates. Statistical analysis was determined by unpaired t test

    Article Snippet: The pairs of primers and the Taqman probes for target RNAs were designed based on mouse RNA sequences using Taqman Gene Expression Assays (Thermo Fisher Scientific).

    Techniques: Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Negative Control, Polymerase Chain Reaction, Amplification, Infection, Real-time Polymerase Chain Reaction

    Saturated fatty acid-induced CSF3 expression via Toll-like receptors . (A) CSF3 mRNA expression was measured in human myotubes pretreated for 6 h with TAK-242 (1 μg/mL) prior to 24-hour treatment with palmitate (0.5 mmol/L). (B) Human myotubes were pretreated for 24 h with siRNA for TLR2 or TLR4 , respectively, prior to 24-hour treatment with palmitate (0.5 mmol/L), and TLR2 , TLR4 , and CSF3 mRNA contents were quantified. Data are given as means ± SEM. Two-group comparisons were performed using matched-pairs Student's t -test (N ≥ 3; *p

    Journal: Molecular Metabolism

    Article Title: Granulocyte colony-stimulating factor (G-CSF): A saturated fatty acid-induced myokine with insulin-desensitizing properties in humans

    doi: 10.1016/j.molmet.2016.02.001

    Figure Lengend Snippet: Saturated fatty acid-induced CSF3 expression via Toll-like receptors . (A) CSF3 mRNA expression was measured in human myotubes pretreated for 6 h with TAK-242 (1 μg/mL) prior to 24-hour treatment with palmitate (0.5 mmol/L). (B) Human myotubes were pretreated for 24 h with siRNA for TLR2 or TLR4 , respectively, prior to 24-hour treatment with palmitate (0.5 mmol/L), and TLR2 , TLR4 , and CSF3 mRNA contents were quantified. Data are given as means ± SEM. Two-group comparisons were performed using matched-pairs Student's t -test (N ≥ 3; *p

    Article Snippet: 1.3 RNA interference Small interfering RNA (siRNA) oligonucleotides targeting TLR2 , TLR4 , NFKB1 , NFKB2 , REL , RELA , RELB , JUN , and CEBPA were purchased as siGENOME-SMART-pools (Thermo Scientific, Rockford, IL, USA).

    Techniques: Expressing

    In vivo PARTICLE and DNA methyltransferase 1 (DNMT1) interaction. ( A ) Linearized dbroccoli in pUC57 (2956 bp; p.db.) after E coR I/H ind III restriction enzyme digestion resolved on a 1.8% agarose gel (left). Plasmid pGEM-T encoding dbroccoli - PARTICLE (p.db. PT ) restriction digested with N co I/H ind III to produce 3728 bp and 950 bp products (upper and lower arrows). DL3000 (Genscript) and Kb ladder (Ld. left and right gels respectively). ( B ) dbroccoli - PARTICLE (1678 bp; db. PT ) in vitro transcript resolved through a 12% NuPAGE gel alongside a high range RNA ladder (Ld. Thermo Fisher RiboRuler) before and after staining in DFHBI-1T ((Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-1-(2,2,2-trifluoroethyl)-1 H -imidazol-5(4 H )-one; left and right gels respectively). ( A,B ) Cropped images of gels shown. ( C ) Time-lapse fluorescence images of a U2OS cell transfected with db. PT and expressing a chromobody to DNMT1-V H H fused to TagRFP (DNMT1_RFP) before (minus time point) and after (plus time points) DFHBI-1T (20 μM) addition. Scale bar 2 μm. ( D ) Histograms of arbitrary units (AU) of overall fluorescence intensity with time in U2OS transfected with db. PT dose range in the presence of DFHBI-1T (20 μM). Data are represented as mean ± SEM from n = 3 experiments per dose. ( E ) Summary co-localization plots for DNMT_RFP (ChR = red channel) and db. PT (ChG = green channel) from ROIs in the nucleus with time. Data are represented as mean ± SEM. ( F,G ) Plots of AU of fluorescence intensity with time from DNMT_RFP ( F ) and in db. PT ( G ) in ROIs (n = 10) within nuclear (encircling dashed line) and extranuclear (Extranuc.) cellular compartments.

    Journal: Scientific Reports

    Article Title: Long non-coding RNA PARTICLE bridges histone and DNA methylation

    doi: 10.1038/s41598-017-01875-1

    Figure Lengend Snippet: In vivo PARTICLE and DNA methyltransferase 1 (DNMT1) interaction. ( A ) Linearized dbroccoli in pUC57 (2956 bp; p.db.) after E coR I/H ind III restriction enzyme digestion resolved on a 1.8% agarose gel (left). Plasmid pGEM-T encoding dbroccoli - PARTICLE (p.db. PT ) restriction digested with N co I/H ind III to produce 3728 bp and 950 bp products (upper and lower arrows). DL3000 (Genscript) and Kb ladder (Ld. left and right gels respectively). ( B ) dbroccoli - PARTICLE (1678 bp; db. PT ) in vitro transcript resolved through a 12% NuPAGE gel alongside a high range RNA ladder (Ld. Thermo Fisher RiboRuler) before and after staining in DFHBI-1T ((Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-1-(2,2,2-trifluoroethyl)-1 H -imidazol-5(4 H )-one; left and right gels respectively). ( A,B ) Cropped images of gels shown. ( C ) Time-lapse fluorescence images of a U2OS cell transfected with db. PT and expressing a chromobody to DNMT1-V H H fused to TagRFP (DNMT1_RFP) before (minus time point) and after (plus time points) DFHBI-1T (20 μM) addition. Scale bar 2 μm. ( D ) Histograms of arbitrary units (AU) of overall fluorescence intensity with time in U2OS transfected with db. PT dose range in the presence of DFHBI-1T (20 μM). Data are represented as mean ± SEM from n = 3 experiments per dose. ( E ) Summary co-localization plots for DNMT_RFP (ChR = red channel) and db. PT (ChG = green channel) from ROIs in the nucleus with time. Data are represented as mean ± SEM. ( F,G ) Plots of AU of fluorescence intensity with time from DNMT_RFP ( F ) and in db. PT ( G ) in ROIs (n = 10) within nuclear (encircling dashed line) and extranuclear (Extranuc.) cellular compartments.

    Article Snippet: RNA interference targeting PARTICLE PARTICLE knockdown was undertaken with Silencer® Select siRNA interference technology (siRNA id: n307629; Part # 4390771, Thermo Fisher Scientific).

    Techniques: In Vivo, Agarose Gel Electrophoresis, Plasmid Preparation, In Vitro, Staining, Fluorescence, Transfection, Expressing