turbo dnase  (Thermo Fisher)


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    Name:
    TURBO DNase 2 U µL
    Description:
    TURBO DNase cleaves double stranded DNA nonspecifically to leave 5 phosphorylated oligodeoxynucleotides It has increased affinity for DNA binding and remains active in the presence of salt Note this product is just the enzyme If you would like this enzyme plus reagents to inactivate the enzyme and remove divalent cations post digestion please see TURBO DNA free Kit Features of TURBO DNase include • Up to 50x more activity and 350 greater catalytic efficiency• Efficiently degrades DNA in solutions containing up to 0 25 M salt• Efficiently digests DNA to oligonucleotides• Vastly superior in clearing DNA templates from in vitro transcription reactions• RNase free and recombinant in originUsing TURBO DNaseDNase I is commonly used to clear DNA contamination from RNA samples prior to RT PCR Conventional DNase I has a poor affinity for DNA and cleaves DNA of low concentration very inefficiently In addition DNase I is very salt sensitive as little as 20 mM NaCl can reduce the activity of the enzyme by 30 Finally DNase I is purified from bovine pancreas one of the richest natural sources of RNase A The threat of contaminating RNase activity in DNase I preparations requires that the enzyme be exhaustively purified In spite of these limitations the DNase I that researchers use today is the very same enzyme that was first characterized by Kunitz more than a half century ago A different DNase with superior properties to wild type DNase ITURBO DNase was developed using a protein engineering approach that introduced amino acid changes into the DNA binding pocket of wild type DNase I These changes markedly increase the affinity of the protein for DNA The result is a versatile enzyme that has a 6 fold lower Km for DNA and an ability to maintain at least 50 of peak activity in solutions approaching 200 mM monovalent salt even when the DNA concentration is in the nanomolar nM range When in vitro transcription reactions are treated with either DNase I or TURBO DNase TURBO DNase removes 63x more of the input plasmid DNA template than the wild type enzyme The proficiency of TURBO DNase in binding very low concentrations of DNA means that the enzyme is particularly effective in removing trace quantities of DNA contamination This becomes important for complete removal of DNA from a sample since the cleavable DNA substrate is reduced as the DNase reaction proceeds TURBO DNase thus has a functional advantage over wild type DNase due to its superior affinity for DNA This is best exploited in RT PCR applications where even a few copies of DNA can lead to a false positive outcome by PCR
    Catalog Number:
    am2238
    Price:
    None
    Applications:
    General Real-Time PCR Reagents|PCR & Real-Time PCR|Real Time PCR (qPCR)|Reverse Transcription
    Category:
    Proteins Enzymes Peptides
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    Structured Review

    Thermo Fisher turbo dnase
    Characterization of short SatIII RNAs. ( A ) Short RNAs were prepared from heat-shocked HeLa cells allowed to recover at 37 °C for 42 h. RNAs were digested with <t>DNase</t> I or with RNase A and then analyzed using Northern blotting with the LNA oligo specific for SatIII repeats. ( B ) Short RNAs were prepared from heat-shocked hamster B14-150 cells and from the hamster > human somatic cell hybrid GM-10611A containing human chromosome 9. RNAs were then analyzed using Northern blotting as in panel A. Hybridization to Mir16 was used as a loading control. The histogram shows the quantitation of long SatIII RNAs in unstressed (C) and in heat-shocked GM10611A cells allowed to recover at 37 °C for the indicated time periods. Data are represented as mean fold increases ± standard deviation of three independent experiments. ( C ) Short RNAs were prepared from unstressed (C) and from heat-shocked (1 h at 42 °C) HeLa cells allowed to recover for 30 h at 37 °C in the presence or in the absence of Actinomycin D (5 μg/mL) during the last 6 h of recovery. The same membrane was hybridized with the LNA probe against SatIII RNAs and with a probe against Mir16 (loading control). M: molecular size marker. Total RNAs prepared from the same cells were analyzed using RT-PCR to assess the level of c-myc RNAs to control the efficacy of Act D treatment. RT-PCR analysis of hP0 <t>RNA</t> was used as a loading control. ( D ) Unstressed (37) and heat-shocked HeLa cells were collected after 40 h of recovery at 37 °C and fractionated in nuclear pellet (N-P), nucleoplasm (Nu) and cytoplasm (Cy) fractions. Total and short RNAs were prepared from each fraction. Short RNAs were analyzed using Northern blotting with an LNA probe against SatIII repeats. As a control of loading and fractionation quality, the same filter was hybridized to a probe against Mir16 (mainly cytoplasmic) and U6 RNAs. Total RNAs from nuclear pellet, nucleoplasm and cytoplasm fractions were prepared from heat-shocked HeLa cells allowed to recover for 40 h at 37 °C. RNAs were analyzed using quantitative RT-PCR. The histogram shows the relative abundance of SatIII RNAs in the three factions as determined in three independent experiments. Data are mean ± standard deviation.
    TURBO DNase cleaves double stranded DNA nonspecifically to leave 5 phosphorylated oligodeoxynucleotides It has increased affinity for DNA binding and remains active in the presence of salt Note this product is just the enzyme If you would like this enzyme plus reagents to inactivate the enzyme and remove divalent cations post digestion please see TURBO DNA free Kit Features of TURBO DNase include • Up to 50x more activity and 350 greater catalytic efficiency• Efficiently degrades DNA in solutions containing up to 0 25 M salt• Efficiently digests DNA to oligonucleotides• Vastly superior in clearing DNA templates from in vitro transcription reactions• RNase free and recombinant in originUsing TURBO DNaseDNase I is commonly used to clear DNA contamination from RNA samples prior to RT PCR Conventional DNase I has a poor affinity for DNA and cleaves DNA of low concentration very inefficiently In addition DNase I is very salt sensitive as little as 20 mM NaCl can reduce the activity of the enzyme by 30 Finally DNase I is purified from bovine pancreas one of the richest natural sources of RNase A The threat of contaminating RNase activity in DNase I preparations requires that the enzyme be exhaustively purified In spite of these limitations the DNase I that researchers use today is the very same enzyme that was first characterized by Kunitz more than a half century ago A different DNase with superior properties to wild type DNase ITURBO DNase was developed using a protein engineering approach that introduced amino acid changes into the DNA binding pocket of wild type DNase I These changes markedly increase the affinity of the protein for DNA The result is a versatile enzyme that has a 6 fold lower Km for DNA and an ability to maintain at least 50 of peak activity in solutions approaching 200 mM monovalent salt even when the DNA concentration is in the nanomolar nM range When in vitro transcription reactions are treated with either DNase I or TURBO DNase TURBO DNase removes 63x more of the input plasmid DNA template than the wild type enzyme The proficiency of TURBO DNase in binding very low concentrations of DNA means that the enzyme is particularly effective in removing trace quantities of DNA contamination This becomes important for complete removal of DNA from a sample since the cleavable DNA substrate is reduced as the DNase reaction proceeds TURBO DNase thus has a functional advantage over wild type DNase due to its superior affinity for DNA This is best exploited in RT PCR applications where even a few copies of DNA can lead to a false positive outcome by PCR
    https://www.bioz.com/result/turbo dnase/product/Thermo Fisher
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    Images

    1) Product Images from "Heat Shock Affects Mitotic Segregation of Human Chromosomes Bound to Stress-Induced Satellite III RNAs"

    Article Title: Heat Shock Affects Mitotic Segregation of Human Chromosomes Bound to Stress-Induced Satellite III RNAs

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21082812

    Characterization of short SatIII RNAs. ( A ) Short RNAs were prepared from heat-shocked HeLa cells allowed to recover at 37 °C for 42 h. RNAs were digested with DNase I or with RNase A and then analyzed using Northern blotting with the LNA oligo specific for SatIII repeats. ( B ) Short RNAs were prepared from heat-shocked hamster B14-150 cells and from the hamster > human somatic cell hybrid GM-10611A containing human chromosome 9. RNAs were then analyzed using Northern blotting as in panel A. Hybridization to Mir16 was used as a loading control. The histogram shows the quantitation of long SatIII RNAs in unstressed (C) and in heat-shocked GM10611A cells allowed to recover at 37 °C for the indicated time periods. Data are represented as mean fold increases ± standard deviation of three independent experiments. ( C ) Short RNAs were prepared from unstressed (C) and from heat-shocked (1 h at 42 °C) HeLa cells allowed to recover for 30 h at 37 °C in the presence or in the absence of Actinomycin D (5 μg/mL) during the last 6 h of recovery. The same membrane was hybridized with the LNA probe against SatIII RNAs and with a probe against Mir16 (loading control). M: molecular size marker. Total RNAs prepared from the same cells were analyzed using RT-PCR to assess the level of c-myc RNAs to control the efficacy of Act D treatment. RT-PCR analysis of hP0 RNA was used as a loading control. ( D ) Unstressed (37) and heat-shocked HeLa cells were collected after 40 h of recovery at 37 °C and fractionated in nuclear pellet (N-P), nucleoplasm (Nu) and cytoplasm (Cy) fractions. Total and short RNAs were prepared from each fraction. Short RNAs were analyzed using Northern blotting with an LNA probe against SatIII repeats. As a control of loading and fractionation quality, the same filter was hybridized to a probe against Mir16 (mainly cytoplasmic) and U6 RNAs. Total RNAs from nuclear pellet, nucleoplasm and cytoplasm fractions were prepared from heat-shocked HeLa cells allowed to recover for 40 h at 37 °C. RNAs were analyzed using quantitative RT-PCR. The histogram shows the relative abundance of SatIII RNAs in the three factions as determined in three independent experiments. Data are mean ± standard deviation.
    Figure Legend Snippet: Characterization of short SatIII RNAs. ( A ) Short RNAs were prepared from heat-shocked HeLa cells allowed to recover at 37 °C for 42 h. RNAs were digested with DNase I or with RNase A and then analyzed using Northern blotting with the LNA oligo specific for SatIII repeats. ( B ) Short RNAs were prepared from heat-shocked hamster B14-150 cells and from the hamster > human somatic cell hybrid GM-10611A containing human chromosome 9. RNAs were then analyzed using Northern blotting as in panel A. Hybridization to Mir16 was used as a loading control. The histogram shows the quantitation of long SatIII RNAs in unstressed (C) and in heat-shocked GM10611A cells allowed to recover at 37 °C for the indicated time periods. Data are represented as mean fold increases ± standard deviation of three independent experiments. ( C ) Short RNAs were prepared from unstressed (C) and from heat-shocked (1 h at 42 °C) HeLa cells allowed to recover for 30 h at 37 °C in the presence or in the absence of Actinomycin D (5 μg/mL) during the last 6 h of recovery. The same membrane was hybridized with the LNA probe against SatIII RNAs and with a probe against Mir16 (loading control). M: molecular size marker. Total RNAs prepared from the same cells were analyzed using RT-PCR to assess the level of c-myc RNAs to control the efficacy of Act D treatment. RT-PCR analysis of hP0 RNA was used as a loading control. ( D ) Unstressed (37) and heat-shocked HeLa cells were collected after 40 h of recovery at 37 °C and fractionated in nuclear pellet (N-P), nucleoplasm (Nu) and cytoplasm (Cy) fractions. Total and short RNAs were prepared from each fraction. Short RNAs were analyzed using Northern blotting with an LNA probe against SatIII repeats. As a control of loading and fractionation quality, the same filter was hybridized to a probe against Mir16 (mainly cytoplasmic) and U6 RNAs. Total RNAs from nuclear pellet, nucleoplasm and cytoplasm fractions were prepared from heat-shocked HeLa cells allowed to recover for 40 h at 37 °C. RNAs were analyzed using quantitative RT-PCR. The histogram shows the relative abundance of SatIII RNAs in the three factions as determined in three independent experiments. Data are mean ± standard deviation.

    Techniques Used: Northern Blot, Hybridization, Quantitation Assay, Standard Deviation, Marker, Reverse Transcription Polymerase Chain Reaction, Fractionation, Quantitative RT-PCR

    2) Product Images from "Distinct properties of proteases and nucleases in the gut, salivary gland and saliva of southern green stink bug, Nezara viridula"

    Article Title: Distinct properties of proteases and nucleases in the gut, salivary gland and saliva of southern green stink bug, Nezara viridula

    Journal: Scientific Reports

    doi: 10.1038/srep27587

    DNase and RNase activities and degradation of DNA by Nezara viridula nucleases. ( a ) Total and specific nuclease activities in gut, salivary gland and saliva (letters indicate significantly different groups; P ≤ 0.05; Student’s t -test). ( b ) Degradation of DNA by DNases from gut, salivary gland and saliva. Samples (gut, salivary gland or saliva) were incubated with DNA for 5, 10 and 30 min. Samples were then run on 2% agarose gels and DNA visualized by ethidium bromide staining. TURBO™ DNase (DNase) was used as a positive control.
    Figure Legend Snippet: DNase and RNase activities and degradation of DNA by Nezara viridula nucleases. ( a ) Total and specific nuclease activities in gut, salivary gland and saliva (letters indicate significantly different groups; P ≤ 0.05; Student’s t -test). ( b ) Degradation of DNA by DNases from gut, salivary gland and saliva. Samples (gut, salivary gland or saliva) were incubated with DNA for 5, 10 and 30 min. Samples were then run on 2% agarose gels and DNA visualized by ethidium bromide staining. TURBO™ DNase (DNase) was used as a positive control.

    Techniques Used: Incubation, Staining, Positive Control

    3) Product Images from "One-plasmid double-expression His-tag system for rapid production and easy purification of MS2 phage-like particles"

    Article Title: One-plasmid double-expression His-tag system for rapid production and easy purification of MS2 phage-like particles

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-17951-5

    His-tagged MS2 phage-like (His-tagged MS2 PLP) particles stability testing. Agarose gel electrophoresis of His-tagged MS2 PLP, DNA and RNA treated (+) and not treated (−) with DNase and/or RNase; M, marker, 2-log DNA ladder (New England Biolabs, UK); the marker values are in kb. The figure was cropped, full-length gel is presented in Supplementary Figure 5 .
    Figure Legend Snippet: His-tagged MS2 phage-like (His-tagged MS2 PLP) particles stability testing. Agarose gel electrophoresis of His-tagged MS2 PLP, DNA and RNA treated (+) and not treated (−) with DNase and/or RNase; M, marker, 2-log DNA ladder (New England Biolabs, UK); the marker values are in kb. The figure was cropped, full-length gel is presented in Supplementary Figure 5 .

    Techniques Used: Plasmid Purification, Agarose Gel Electrophoresis, Marker

    4) Product Images from "N6-methyladenosine modification and the YTHDF2 reader protein play cell type specific roles in lytic viral gene expression during Kaposi's sarcoma-associated herpesvirus infection"

    Article Title: N6-methyladenosine modification and the YTHDF2 reader protein play cell type specific roles in lytic viral gene expression during Kaposi's sarcoma-associated herpesvirus infection

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006995

    KSHV mRNA contains m 6 A modifications. (A) Two independent replicates of iSLK.219 cells containing latent KSHV were treated with dox for 5 days to induce the viral lytic cycle (induced) or left untreated to preserve viral latency (uninduced). DNase-treated RNA was isolated and subjected to m 6 Aseq. Displayed are peaks with a fold change of four or higher, comparing reads in the m 6 A-IP to the corresponding input. Numbers above peaks correspond to the base position within the KSHV genome. (B) Overview of sequencing reads from induced and uninduced m 6 A IP samples, aligned to the ORF50 transcript and the annotated GG(m 6 A)C consensus motifs found in exon 2 of ORF50. Numbers to the left of sequencing reads indicate the scale of the read count. (C) Cells were induced as in (A), and total RNA was subjected to m 6 A RIP, followed by RT-qPCR using primers for the indicated viral and cellular genes. Values are displayed as fold change over input, normalized to GAPDH. (D) Quantification of cellular m 6 A peaks from m 6 Aseq analysis.
    Figure Legend Snippet: KSHV mRNA contains m 6 A modifications. (A) Two independent replicates of iSLK.219 cells containing latent KSHV were treated with dox for 5 days to induce the viral lytic cycle (induced) or left untreated to preserve viral latency (uninduced). DNase-treated RNA was isolated and subjected to m 6 Aseq. Displayed are peaks with a fold change of four or higher, comparing reads in the m 6 A-IP to the corresponding input. Numbers above peaks correspond to the base position within the KSHV genome. (B) Overview of sequencing reads from induced and uninduced m 6 A IP samples, aligned to the ORF50 transcript and the annotated GG(m 6 A)C consensus motifs found in exon 2 of ORF50. Numbers to the left of sequencing reads indicate the scale of the read count. (C) Cells were induced as in (A), and total RNA was subjected to m 6 A RIP, followed by RT-qPCR using primers for the indicated viral and cellular genes. Values are displayed as fold change over input, normalized to GAPDH. (D) Quantification of cellular m 6 A peaks from m 6 Aseq analysis.

    Techniques Used: Isolation, Sequencing, Quantitative RT-PCR

    5) Product Images from "Integrated Analysis of Dysregulated lncRNA Expression in Fetal Cardiac Tissues with Ventricular Septal Defect"

    Article Title: Integrated Analysis of Dysregulated lncRNA Expression in Fetal Cardiac Tissues with Ventricular Septal Defect

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0077492

    Bioinformatics analysis of ENST00000513542. ( A ) ENST00000513542 is a natural antisense lncRNA, transcribed from 2,105 bp downstream of the second exon of the Smad1 gene. ( B ) Several tracks of interest, including conservation, histone markings, DNase hypersensitivity, and TFBS are displayed. Integrated Regulation track data for a region spanning the ENST0000051354 are shown. The red box shows a region of overlap between the various tracks. ( C ) Transcription factor binding sites (TFBS) prediction indicated that ENST0000051354 loci combine with AP-1 as a cis-acting element. ( D ) Further catRAPID analysis indicated a strong RNA-protein interaction between ENST0000051354 and TCF-4, which is the TF of SMAD1.
    Figure Legend Snippet: Bioinformatics analysis of ENST00000513542. ( A ) ENST00000513542 is a natural antisense lncRNA, transcribed from 2,105 bp downstream of the second exon of the Smad1 gene. ( B ) Several tracks of interest, including conservation, histone markings, DNase hypersensitivity, and TFBS are displayed. Integrated Regulation track data for a region spanning the ENST0000051354 are shown. The red box shows a region of overlap between the various tracks. ( C ) Transcription factor binding sites (TFBS) prediction indicated that ENST0000051354 loci combine with AP-1 as a cis-acting element. ( D ) Further catRAPID analysis indicated a strong RNA-protein interaction between ENST0000051354 and TCF-4, which is the TF of SMAD1.

    Techniques Used: Binding Assay

    6) Product Images from "Mitochondrial double-stranded RNA triggers antiviral signalling in humans"

    Article Title: Mitochondrial double-stranded RNA triggers antiviral signalling in humans

    Journal: Nature

    doi: 10.1038/s41586-018-0363-0

    Characterization of anti-dsRNA J2 antibody and mtDNA depletion results in loss of mtdsRNA formation. a , RT–qPCR analysis of L-mRNA expression in encephalomyocarditis virus (EMCV) infected HeLa cells at MOI 1 at the indicated time points after infection. Data are from two independent experiments. b , Confocal microscopy images of uninfected or EMCV-infected HeLa cells at multiplicity of infection (MOI) of 1, 8 h after infection stained with anti-dsRNA (J2) antibody (green) and DAPI (nuclei stained blue). Images are representative of two experiments. Scale bars, 10 μm. c , Immunostaining of dsRNA (green) and DNA (red) in HeLa cells treated with indicated nucleases before staining. Signal from J2 antibody is specific for RNA but not for DNA and is sensitive only to RNase III treatment. Images are representative of three experiments. Scale bars, 10 μm. d , Quantification of fluorescence signal from HeLa cells treated as in c . Data are mean ± s.e.m. from 4,095, 1,755, 4,766 and 5,585 cells for the untreated, RNase T1, RNase III and DNase Turbo groups, respectively. e , Autoradiogram showing substrate specificity of J2 on the basis of immunoprecipitation efficiency for uniformly 32 . f , Chromosome-wise coverage plot of dsRNA-seq reads. Inset, read distribution of dsRNA-seq on the basis of RNA class biotypes. g , Left, dsRNA and DNA staining of HeLa cells transfected with constructs encoding the indicated proteins, the expression of which results in mtDNA depletion. Plasmids encoding mtDNA-depletion factors co-express EGFP from an independent promoter, which enables identification of transfected cells. Mitochondria were stained using anti-OXA1L antibody. Scale bars, 10 μm. Right, quantitative analysis of fluorescence signal from HeLa cells. Data are mean ± s.e.m. from ten cells.
    Figure Legend Snippet: Characterization of anti-dsRNA J2 antibody and mtDNA depletion results in loss of mtdsRNA formation. a , RT–qPCR analysis of L-mRNA expression in encephalomyocarditis virus (EMCV) infected HeLa cells at MOI 1 at the indicated time points after infection. Data are from two independent experiments. b , Confocal microscopy images of uninfected or EMCV-infected HeLa cells at multiplicity of infection (MOI) of 1, 8 h after infection stained with anti-dsRNA (J2) antibody (green) and DAPI (nuclei stained blue). Images are representative of two experiments. Scale bars, 10 μm. c , Immunostaining of dsRNA (green) and DNA (red) in HeLa cells treated with indicated nucleases before staining. Signal from J2 antibody is specific for RNA but not for DNA and is sensitive only to RNase III treatment. Images are representative of three experiments. Scale bars, 10 μm. d , Quantification of fluorescence signal from HeLa cells treated as in c . Data are mean ± s.e.m. from 4,095, 1,755, 4,766 and 5,585 cells for the untreated, RNase T1, RNase III and DNase Turbo groups, respectively. e , Autoradiogram showing substrate specificity of J2 on the basis of immunoprecipitation efficiency for uniformly 32 . f , Chromosome-wise coverage plot of dsRNA-seq reads. Inset, read distribution of dsRNA-seq on the basis of RNA class biotypes. g , Left, dsRNA and DNA staining of HeLa cells transfected with constructs encoding the indicated proteins, the expression of which results in mtDNA depletion. Plasmids encoding mtDNA-depletion factors co-express EGFP from an independent promoter, which enables identification of transfected cells. Mitochondria were stained using anti-OXA1L antibody. Scale bars, 10 μm. Right, quantitative analysis of fluorescence signal from HeLa cells. Data are mean ± s.e.m. from ten cells.

    Techniques Used: Quantitative RT-PCR, Expressing, Infection, Confocal Microscopy, Staining, Immunostaining, Fluorescence, Immunoprecipitation, Transfection, Construct

    7) Product Images from "Mutational Analysis of Vaccinia Virus E3 Protein: the Biological Functions Do Not Correlate with Its Biochemical Capacity To Bind Double-Stranded RNA"

    Article Title: Mutational Analysis of Vaccinia Virus E3 Protein: the Biological Functions Do Not Correlate with Its Biochemical Capacity To Bind Double-Stranded RNA

    Journal: Journal of Virology

    doi: 10.1128/JVI.03288-14

    Modulation of cytokine expression by selected E3 mutants. HeLa cells were infected with selected E3 mutant recombinant viruses at an MOI of 10, and the total RNA was collected at 8 h postinfection. Trace DNA contamination was removed with DNase. The mRNA
    Figure Legend Snippet: Modulation of cytokine expression by selected E3 mutants. HeLa cells were infected with selected E3 mutant recombinant viruses at an MOI of 10, and the total RNA was collected at 8 h postinfection. Trace DNA contamination was removed with DNase. The mRNA

    Techniques Used: Expressing, Infection, Mutagenesis, Recombinant

    8) Product Images from "C9orf72 frontotemporal lobar degeneration is characterised by frequent neuronal sense and antisense RNA foci"

    Article Title: C9orf72 frontotemporal lobar degeneration is characterised by frequent neuronal sense and antisense RNA foci

    Journal: Acta Neuropathologica

    doi: 10.1007/s00401-013-1200-z

    Antisense RNA foci are a consistent and specific feature of C9FTLD. RNA FISH for antisense foci ( green ) was combined with immunostaining for neurons with NeuN ( red ) and nuclear DNA staining with DAPI ( blue ). a Representative images from the frontal cortex, hippocampus and cerebellum from a neurologically normal control and heterozygous (C9 Het) and homozygous (C9 Hom) C9FTLD cases. Blind quantification of antisense RNA foci in frontal cortical neurons in C9FTLD cases and controls ( b ) and granule cell neurons of the hippocampus and cerebellum in C9FTLD cases only ( c ). d Representative images from a heterozygous C9FTLD case show antisense RNA foci are present in astrocytes, microglia, and oligodendrocytes. e RNA FISH for antisense foci was combined with RNase or DNase treatment, confirming the antisense probe detects RNA and not DNA. Scale bar represents 2 μm in all panels. In b , c , each dot represents an individual case with the homozygous C9FTLD case shown in red , and the average and SEM of heterozygous cases shown as long and short horizontal bars, respectively
    Figure Legend Snippet: Antisense RNA foci are a consistent and specific feature of C9FTLD. RNA FISH for antisense foci ( green ) was combined with immunostaining for neurons with NeuN ( red ) and nuclear DNA staining with DAPI ( blue ). a Representative images from the frontal cortex, hippocampus and cerebellum from a neurologically normal control and heterozygous (C9 Het) and homozygous (C9 Hom) C9FTLD cases. Blind quantification of antisense RNA foci in frontal cortical neurons in C9FTLD cases and controls ( b ) and granule cell neurons of the hippocampus and cerebellum in C9FTLD cases only ( c ). d Representative images from a heterozygous C9FTLD case show antisense RNA foci are present in astrocytes, microglia, and oligodendrocytes. e RNA FISH for antisense foci was combined with RNase or DNase treatment, confirming the antisense probe detects RNA and not DNA. Scale bar represents 2 μm in all panels. In b , c , each dot represents an individual case with the homozygous C9FTLD case shown in red , and the average and SEM of heterozygous cases shown as long and short horizontal bars, respectively

    Techniques Used: Fluorescence In Situ Hybridization, Immunostaining, Staining

    DNase and RNase treatments confirm specificity of RNA foci. a RNA FISH for sense foci ( red ) or CTG repeats [(CAG) 7 probe, bottom right panel ] was combined with nuclear DNA staining with DAPI ( blue ) in the hippocampus of the homozygous C9FTLD case. RNA foci were not detected after RNase treatment but were still present after DNase treatment, which was confirmed by blind quantification. b Loss of DAPI staining ( bottom left panel ) confirmed the DNase was effective. No signal was observed for the (CAG) 7 probe, suggesting the sense RNA foci are not due to non-specific binding of the RNA probe. Scale bar represents 5 μm
    Figure Legend Snippet: DNase and RNase treatments confirm specificity of RNA foci. a RNA FISH for sense foci ( red ) or CTG repeats [(CAG) 7 probe, bottom right panel ] was combined with nuclear DNA staining with DAPI ( blue ) in the hippocampus of the homozygous C9FTLD case. RNA foci were not detected after RNase treatment but were still present after DNase treatment, which was confirmed by blind quantification. b Loss of DAPI staining ( bottom left panel ) confirmed the DNase was effective. No signal was observed for the (CAG) 7 probe, suggesting the sense RNA foci are not due to non-specific binding of the RNA probe. Scale bar represents 5 μm

    Techniques Used: Fluorescence In Situ Hybridization, CTG Assay, Staining, Binding Assay

    9) Product Images from "Programmed synthesis of 3D tissues"

    Article Title: Programmed synthesis of 3D tissues

    Journal: Nature methods

    doi: 10.1038/nmeth.3553

    Programming the reconstitution of fully ECM-embedded 3D microtissues by DNA-programmed assembly (DPAC) (a) Scheme showing the relationship between DNA spots (colored squares), DNA-programmed connectivity (colored lines), and multistep assembly. (b) Incubation of cells with lipid-modified oligonucleotides results in chemical remodeling of cell surfaces. Combining cells bearing complementary cell-surface oligonucleotides forms a temporary chemical adhesion. (c) 7 μm amino-modified DNA spots are patterned onto aldehyde-coated glass slides and covalently linked to the surface by reductive amination. Cells bearing complementary cell-surface oligonucleotides are introduced above the patterned substrate at high concentration and at controlled flow rate using a flow cell. Cells adhere to the appropriate DNA spot, and excess cells are removed by gentle washing. Iteration of this process assembles the microtissue into the third dimension. Addition of liquid ECM incorporating DNase releases the assembled microtissues from the template where they are trapped in the embedding ECM as it gels. The gel is peeled off the glass, releasing the tissues. Underlay of the gel with additional ECM results in a fully embedded 3D culture. Cells interact with each other and their microenvironment as they condense into 3D microtissues. (d) Implementation of the scheme described in Figure 1a–c using MCF10A mammary epithelial cells showing (i) DNA spots, (ii) cells in flow cell, and (iii) single cell array followed by additional rounds of programmed assembly. X,Z reconstructions show an unstained MCF10A cell aggregate embedded between Alexa Fluor-488 and Alexa Fluor 555-stained layers of Matrigel at (iv) 0 and (v) 24 hr. All scale bars are 100 μm.
    Figure Legend Snippet: Programming the reconstitution of fully ECM-embedded 3D microtissues by DNA-programmed assembly (DPAC) (a) Scheme showing the relationship between DNA spots (colored squares), DNA-programmed connectivity (colored lines), and multistep assembly. (b) Incubation of cells with lipid-modified oligonucleotides results in chemical remodeling of cell surfaces. Combining cells bearing complementary cell-surface oligonucleotides forms a temporary chemical adhesion. (c) 7 μm amino-modified DNA spots are patterned onto aldehyde-coated glass slides and covalently linked to the surface by reductive amination. Cells bearing complementary cell-surface oligonucleotides are introduced above the patterned substrate at high concentration and at controlled flow rate using a flow cell. Cells adhere to the appropriate DNA spot, and excess cells are removed by gentle washing. Iteration of this process assembles the microtissue into the third dimension. Addition of liquid ECM incorporating DNase releases the assembled microtissues from the template where they are trapped in the embedding ECM as it gels. The gel is peeled off the glass, releasing the tissues. Underlay of the gel with additional ECM results in a fully embedded 3D culture. Cells interact with each other and their microenvironment as they condense into 3D microtissues. (d) Implementation of the scheme described in Figure 1a–c using MCF10A mammary epithelial cells showing (i) DNA spots, (ii) cells in flow cell, and (iii) single cell array followed by additional rounds of programmed assembly. X,Z reconstructions show an unstained MCF10A cell aggregate embedded between Alexa Fluor-488 and Alexa Fluor 555-stained layers of Matrigel at (iv) 0 and (v) 24 hr. All scale bars are 100 μm.

    Techniques Used: Incubation, Modification, Concentration Assay, Flow Cytometry, Staining

    10) Product Images from "Toll-Like Receptor 3/TRIF-Dependent IL-12p70 Secretion Mediated by Streptococcus pneumoniae RNA and Its Priming by Influenza A Virus Coinfection in Human Dendritic Cells"

    Article Title: Toll-Like Receptor 3/TRIF-Dependent IL-12p70 Secretion Mediated by Streptococcus pneumoniae RNA and Its Priming by Influenza A Virus Coinfection in Human Dendritic Cells

    Journal: mBio

    doi: 10.1128/mBio.00168-16

    RNA is required as a pneumococcal stimulus to induce IL-12p70 production. DCs were challenged with live, UV-killed, or heat-killed (HK) T4R (A), with live or UV-killed T4R at the indicated MOI (B), with UV-killed T4R (MOI, 10), with LPS pretreated with a cocktail of RNase A (200 to 20 U/ml) and RNase T1 (8,000 to 800 U/ml) (C), or with UV-killed T4R (MOI, 10) pretreated with DNase I (1,000 to 250 U/ml) (D). IL-12p70 production in the cell supernatant was measured in an ELISA (A to D), and DC viability was measured by flow cytometry (B). Values represent means ± standard errors of the means for results from 4 (A), 3 (B), 7 (C), or 4 (D) experiments. Statistical analysis was performed using a one-way analysis of variance and a Bonferroni posttest. *, P
    Figure Legend Snippet: RNA is required as a pneumococcal stimulus to induce IL-12p70 production. DCs were challenged with live, UV-killed, or heat-killed (HK) T4R (A), with live or UV-killed T4R at the indicated MOI (B), with UV-killed T4R (MOI, 10), with LPS pretreated with a cocktail of RNase A (200 to 20 U/ml) and RNase T1 (8,000 to 800 U/ml) (C), or with UV-killed T4R (MOI, 10) pretreated with DNase I (1,000 to 250 U/ml) (D). IL-12p70 production in the cell supernatant was measured in an ELISA (A to D), and DC viability was measured by flow cytometry (B). Values represent means ± standard errors of the means for results from 4 (A), 3 (B), 7 (C), or 4 (D) experiments. Statistical analysis was performed using a one-way analysis of variance and a Bonferroni posttest. *, P

    Techniques Used: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry

    11) Product Images from "Sam68 marks the transcriptionally active stages of spermatogenesis and modulates alternative splicing in male germ cells"

    Article Title: Sam68 marks the transcriptionally active stages of spermatogenesis and modulates alternative splicing in male germ cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr085

    Nuclear localization of Sam68 requires the integrity of nucleic acids. Purified pachytene spermatocytes were permeabilized on microscope slides in a buffer containing 0.1% Triton X-100 and incubated for 15 min with medium alone (Control) or DNase or Rnase as indicated. At the end of the incubation, cells were washed three times with PBS and fixed for immunofluorescence analysis with the anti-Sam68 and H5 ( A ) or H14 ( B ) antibodies. DNA was stained by Hoechst dye. ( C ) The western blot analysis of Sam68 and β-tubulin in control or treated (DNase or RNase) pachytene spermatocytes after sequential extractions with the indicated buffers.
    Figure Legend Snippet: Nuclear localization of Sam68 requires the integrity of nucleic acids. Purified pachytene spermatocytes were permeabilized on microscope slides in a buffer containing 0.1% Triton X-100 and incubated for 15 min with medium alone (Control) or DNase or Rnase as indicated. At the end of the incubation, cells were washed three times with PBS and fixed for immunofluorescence analysis with the anti-Sam68 and H5 ( A ) or H14 ( B ) antibodies. DNA was stained by Hoechst dye. ( C ) The western blot analysis of Sam68 and β-tubulin in control or treated (DNase or RNase) pachytene spermatocytes after sequential extractions with the indicated buffers.

    Techniques Used: Purification, Microscopy, Incubation, Immunofluorescence, Staining, Western Blot

    Sam68 regulates alternative splicing of Sgce exon 8 in male germ cells. RT–PCR analysis of Sgce exon 8 inclusion in total RNA extracted from Sam68 wild-type or knockout testes at 8, 16 and 30 days post partum (dpp) ( A ) or from isolated wild-type or knockout spermatocytes and spermatids. ( B ) Bands corresponding to mRNAs containing or not, exon 8 are indicated on the right of the panels. ( C and D ) Chromatin immunoprecipitation (ChIP) analysis of Sam68 wild-type and knockout germ cells. Sonicated chromatin (100 µg of DNA/sample) was immunoprecipitated with 2µg of H5, H14 antibodies or control rabbit IgGs and co-precipitated DNA was analysed by real time PCR with primers (black arrows in the scheme) spanning the Sgce transcription unit as indicated. ( E ) CLIP analysis of the binding of Sam68 to Sgce pre-mRNA. After UV crosslink, Sam68 wild-type and knockout germ cell extracts were sonicated and treated with DNase and RNase to yield RNA fragments of ∼200 to 250 nt. Wild-type and knockout germ cell extracts were immunoprecipitated with 2 µg of rabbit IgGs or anti-Sam68 antibodies and co-precipitated RNA was analysed by real time PCR with primers (black arrows in the scheme) spanning the Sgce transcription unit as indicated. ( F ) Electrophoretic mobility shift assays (EMSAs) of the binding of purified GST-Sam68 1–277 to a labelled Sgce probe containing the sequences encoded at the intron 7/exon 8 boundary. The position of the free probe and the probe complexed with GST-Sam68 1–277 are shown on the left side. Competition with the wild-type (middle panel) or mutated (right panel) cold probes is shown. The scheme above the gels shows the wild-type and mutated sequence used in the EMSAs. The mutated bases that interfere with the Sam68 consensus are shown in violet. ( G ) The western blot analysis of RNA pulldown assay of U2AF65 binding to Sgce exon 8. Biotinylated RNAs encoding the Sgce wild-type and mutated exon 8 sequences (from −32 to +63) were bound to Streptavidine agarose beads and incubated with testicular nuclear extract (100 µg) in the presence of 1 µg of purified GST or GST-Sam68 1–277, as indicated.
    Figure Legend Snippet: Sam68 regulates alternative splicing of Sgce exon 8 in male germ cells. RT–PCR analysis of Sgce exon 8 inclusion in total RNA extracted from Sam68 wild-type or knockout testes at 8, 16 and 30 days post partum (dpp) ( A ) or from isolated wild-type or knockout spermatocytes and spermatids. ( B ) Bands corresponding to mRNAs containing or not, exon 8 are indicated on the right of the panels. ( C and D ) Chromatin immunoprecipitation (ChIP) analysis of Sam68 wild-type and knockout germ cells. Sonicated chromatin (100 µg of DNA/sample) was immunoprecipitated with 2µg of H5, H14 antibodies or control rabbit IgGs and co-precipitated DNA was analysed by real time PCR with primers (black arrows in the scheme) spanning the Sgce transcription unit as indicated. ( E ) CLIP analysis of the binding of Sam68 to Sgce pre-mRNA. After UV crosslink, Sam68 wild-type and knockout germ cell extracts were sonicated and treated with DNase and RNase to yield RNA fragments of ∼200 to 250 nt. Wild-type and knockout germ cell extracts were immunoprecipitated with 2 µg of rabbit IgGs or anti-Sam68 antibodies and co-precipitated RNA was analysed by real time PCR with primers (black arrows in the scheme) spanning the Sgce transcription unit as indicated. ( F ) Electrophoretic mobility shift assays (EMSAs) of the binding of purified GST-Sam68 1–277 to a labelled Sgce probe containing the sequences encoded at the intron 7/exon 8 boundary. The position of the free probe and the probe complexed with GST-Sam68 1–277 are shown on the left side. Competition with the wild-type (middle panel) or mutated (right panel) cold probes is shown. The scheme above the gels shows the wild-type and mutated sequence used in the EMSAs. The mutated bases that interfere with the Sam68 consensus are shown in violet. ( G ) The western blot analysis of RNA pulldown assay of U2AF65 binding to Sgce exon 8. Biotinylated RNAs encoding the Sgce wild-type and mutated exon 8 sequences (from −32 to +63) were bound to Streptavidine agarose beads and incubated with testicular nuclear extract (100 µg) in the presence of 1 µg of purified GST or GST-Sam68 1–277, as indicated.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Knock-Out, Isolation, Chromatin Immunoprecipitation, Sonication, Immunoprecipitation, Real-time Polymerase Chain Reaction, Cross-linking Immunoprecipitation, Binding Assay, Electrophoretic Mobility Shift Assay, Purification, Sequencing, Western Blot, Incubation

    12) Product Images from "Toll-Like Receptor 7 Mediates Early Innate Immune Responses to Malaria"

    Article Title: Toll-Like Receptor 7 Mediates Early Innate Immune Responses to Malaria

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00923-13

    TLR7 recognizes diverse malaria species. (A, B) Mice of the specified genotypes were infected for 24 h with the indicated Plasmodium species. (A) A total of 2.5 × 10 6 splenocytes were quantified by flow cytometry for Ifnb-Yfp expression. Numbers represent YFP + cells per million live cells. (B) Splenic IFN-α was measured by ELISA. (C) A total of 2 × 10 6 bone marrow cells from B6 MOB or Tlr7 −/− MOB mice were stimulated in vitro with 10 7 uninfected or P. falciparum -infected erythrocytes for 24 h, and Ifnb-Yfp expression was assessed by flow cytometry. Numbers represent YFP + cells per million live cells. (D) B6 or Tlr7 −/− MOB mice were injected with DOTAP alone or in complex with 10 μg DNase-treated RNA isolated from P. falciparum . Ifnb-Yfp expression in splenocytes was measured by flow cytometry 18 h postinjection. A representative experiment of two independent experiments is shown.
    Figure Legend Snippet: TLR7 recognizes diverse malaria species. (A, B) Mice of the specified genotypes were infected for 24 h with the indicated Plasmodium species. (A) A total of 2.5 × 10 6 splenocytes were quantified by flow cytometry for Ifnb-Yfp expression. Numbers represent YFP + cells per million live cells. (B) Splenic IFN-α was measured by ELISA. (C) A total of 2 × 10 6 bone marrow cells from B6 MOB or Tlr7 −/− MOB mice were stimulated in vitro with 10 7 uninfected or P. falciparum -infected erythrocytes for 24 h, and Ifnb-Yfp expression was assessed by flow cytometry. Numbers represent YFP + cells per million live cells. (D) B6 or Tlr7 −/− MOB mice were injected with DOTAP alone or in complex with 10 μg DNase-treated RNA isolated from P. falciparum . Ifnb-Yfp expression in splenocytes was measured by flow cytometry 18 h postinjection. A representative experiment of two independent experiments is shown.

    Techniques Used: Mouse Assay, Infection, Flow Cytometry, Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, In Vitro, Injection, Isolation

    13) Product Images from "H-IPSE Is a Pathogen-Secreted Host Nucleus-Infiltrating Protein (Infiltrin) Expressed Exclusively by the Schistosoma haematobium Egg Stage"

    Article Title: H-IPSE Is a Pathogen-Secreted Host Nucleus-Infiltrating Protein (Infiltrin) Expressed Exclusively by the Schistosoma haematobium Egg Stage

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00301-17

    Stage-specific expression of H-IPSE mRNA. Shown are RT-PCR results for H-IPSE obtained from cDNAs prepared by reverse transcription of DNase-treated RNAs isolated at various life stages of S. haematobium . Ladder, 100-bp DNA ladder; egg, S. haematobium egg cDNA; mir, miracidial cDNA; cer, cercarial cDNA; som, in vitro mechanically transformed schistosomulum cDNA; AdF, AdM, and Ad mix, mixed cDNAs from female, male, and mixed adult worms, respectively; ShTub, S. haematobium tubulin (control housekeeping gene).
    Figure Legend Snippet: Stage-specific expression of H-IPSE mRNA. Shown are RT-PCR results for H-IPSE obtained from cDNAs prepared by reverse transcription of DNase-treated RNAs isolated at various life stages of S. haematobium . Ladder, 100-bp DNA ladder; egg, S. haematobium egg cDNA; mir, miracidial cDNA; cer, cercarial cDNA; som, in vitro mechanically transformed schistosomulum cDNA; AdF, AdM, and Ad mix, mixed cDNAs from female, male, and mixed adult worms, respectively; ShTub, S. haematobium tubulin (control housekeeping gene).

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, In Vitro, Transformation Assay

    14) Product Images from "Enhanced methods for unbiased deep sequencing of Lassa and Ebola RNA viruses from clinical and biological samples"

    Article Title: Enhanced methods for unbiased deep sequencing of Lassa and Ebola RNA viruses from clinical and biological samples

    Journal: Genome Biology

    doi: 10.1186/s13059-014-0519-7

    RNase H selective depletion of poly(rA) carrier from Lassa samples. (A) Native polyacrylamide gel depicting library PCR and side products of LASV preparations with poly(rA) carrier present (middle) or depleted (right panel). No free poly(rA) was present in control library (left). (B) Median base qualities per MiSeq cycle of poly(rA)-contaminated LASV libraries (solid line) and control (no carrier observed in library, dashed) from FastQC report. Both read 1 and read 2 of paired end reads are merged in the library BAM file and the quality scores are shown at each base. (C) Schematic of carrier RNA selective depletion and DNase treatment of oligo (dT).
    Figure Legend Snippet: RNase H selective depletion of poly(rA) carrier from Lassa samples. (A) Native polyacrylamide gel depicting library PCR and side products of LASV preparations with poly(rA) carrier present (middle) or depleted (right panel). No free poly(rA) was present in control library (left). (B) Median base qualities per MiSeq cycle of poly(rA)-contaminated LASV libraries (solid line) and control (no carrier observed in library, dashed) from FastQC report. Both read 1 and read 2 of paired end reads are merged in the library BAM file and the quality scores are shown at each base. (C) Schematic of carrier RNA selective depletion and DNase treatment of oligo (dT).

    Techniques Used: Polymerase Chain Reaction

    15) Product Images from "TOX4 and NOVA1 Proteins Are Partners of the LEDGF PWWP Domain and Affect HIV-1 Replication"

    Article Title: TOX4 and NOVA1 Proteins Are Partners of the LEDGF PWWP Domain and Affect HIV-1 Replication

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0081217

    Interaction Of Tox4 And Nova1 Pirs With Ledgf Pwwp By Gst Pull-Down. GST pull down were performed using purified GST-PWWP protein and Flag-TOX4 PIR or Flag-NOVA1 PIR expressed and present in 293T cells extracts (A and B) or with His-TOX4 PIR or Flag-NOVA1 PIR expressed in E coli and purified (C). Eluted proteins following pull down were separated through 10% PA SDS-PAGE and analyzed by western blot using M2 anti-Flag antibody (A and B), H1029 anti-His antibody (C) and 4C10 anti-GST antibody (A to C). Purified GST was used as negative control for each experiment. B) Effect of DNA and RNA on interaction with PIRs in extracts was studied by DNAse or RNAse treatment of these extracts. C) Effect of DNA or PN on interaction with purified PIRs was studied by addition of a 2.6 kbp 5SG5E4 DNA fragment or a polynucleosome (PN) asssembled on it.
    Figure Legend Snippet: Interaction Of Tox4 And Nova1 Pirs With Ledgf Pwwp By Gst Pull-Down. GST pull down were performed using purified GST-PWWP protein and Flag-TOX4 PIR or Flag-NOVA1 PIR expressed and present in 293T cells extracts (A and B) or with His-TOX4 PIR or Flag-NOVA1 PIR expressed in E coli and purified (C). Eluted proteins following pull down were separated through 10% PA SDS-PAGE and analyzed by western blot using M2 anti-Flag antibody (A and B), H1029 anti-His antibody (C) and 4C10 anti-GST antibody (A to C). Purified GST was used as negative control for each experiment. B) Effect of DNA and RNA on interaction with PIRs in extracts was studied by DNAse or RNAse treatment of these extracts. C) Effect of DNA or PN on interaction with purified PIRs was studied by addition of a 2.6 kbp 5SG5E4 DNA fragment or a polynucleosome (PN) asssembled on it.

    Techniques Used: Purification, SDS Page, Western Blot, Negative Control

    Interaction Of Tox4 And Nova1, Full Length Or Pir, With Ledgf/P75 By Co-Immunoprecipitation. Total extracts of cells transiently expressing HA-LEDGF and either 3×Flag-TOX4, 3xFlag-NOVA1, PIR or full length, Flag-HIV Integrase or Flag-Brd4 were immunoprecipitated with anti-Flag M2 coupled agarose beads. Immunoprecipitated proteins were separated on 10% or 7.5% PA-SDS gels and revealed by immunoblot using antibodies indicated on the left side of the panels and more precisely described in Material and Methods section. A) HA-tagged LEDGF co-immunoprecipitates with 3×Flag tagged full-length and PIR constructs of TOX4 and NOVA1 but not with Flag Brd4. B) HA-tagged LEDGF co-immunoprecipitates Flag-HIV1 integrase. C) DNAse (but not RNAse) treatment of cell extracts abolishes HA-tagged LEDGF co-IP with 3×Flag tagged PIR of TOX4 and NOVA1. Cell extracts were digested by nothing (lane 1), DNAse (lane 2) or RNAse (lane 3) before the co-IP protocol (IP (n > 3))
    Figure Legend Snippet: Interaction Of Tox4 And Nova1, Full Length Or Pir, With Ledgf/P75 By Co-Immunoprecipitation. Total extracts of cells transiently expressing HA-LEDGF and either 3×Flag-TOX4, 3xFlag-NOVA1, PIR or full length, Flag-HIV Integrase or Flag-Brd4 were immunoprecipitated with anti-Flag M2 coupled agarose beads. Immunoprecipitated proteins were separated on 10% or 7.5% PA-SDS gels and revealed by immunoblot using antibodies indicated on the left side of the panels and more precisely described in Material and Methods section. A) HA-tagged LEDGF co-immunoprecipitates with 3×Flag tagged full-length and PIR constructs of TOX4 and NOVA1 but not with Flag Brd4. B) HA-tagged LEDGF co-immunoprecipitates Flag-HIV1 integrase. C) DNAse (but not RNAse) treatment of cell extracts abolishes HA-tagged LEDGF co-IP with 3×Flag tagged PIR of TOX4 and NOVA1. Cell extracts were digested by nothing (lane 1), DNAse (lane 2) or RNAse (lane 3) before the co-IP protocol (IP (n > 3))

    Techniques Used: Immunoprecipitation, Expressing, Construct, Co-Immunoprecipitation Assay

    16) Product Images from "Murine and related chapparvoviruses are nephro-tropic and produce novel accessory proteins in infected kidneys"

    Article Title: Murine and related chapparvoviruses are nephro-tropic and produce novel accessory proteins in infected kidneys

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008262

    Map of the MKPV genome. (A-B) Maps of the MKPV/MuCPV strains from Centenary Institute (CI, accession MH670587), Memorial Sloan Kettering Cancer Center (MSKCC, accession MH670588) and New York City basements (wild-NY, MF175078). “Bowties” indicate terminal repeats (TR). (A) Single nucleotide variations (SNV) between the CI, MSKCC and wild-NY accessions. Vertical lines—differences between accessions. Half height vertical lines—polymorphisms within an accession. ▼; 2 bp insertion in the CI strain. ▲; 1 bp insertion in a CI sub-strain. Dashed lines—missing extremities in MSKCC and wild-NY accessions, which consist of the exterior inverted repeats in the full-length CI sequence. (B-C) Alternative splicing allows production of the polypeptides p10, p15, NS1, NS2, NP and VP. Black, brown or blue shading indicate the relative reading frames of ORFs. p15, p10 and NP could theoretically be produced from multiple transcripts. Orange or red indicate peptides present in LC-MS/MS datasets PXD014938 (this paper) or PXD010540 [ 9 ], respectively. Exon or intron sequences flanking splice sites are shown in black or red text, respectively. (C) Quantitation of spliced MKPV reads in RNAseq data pooled from two MKPV-infected kidneys. Columns indicate splice site usage (left y-axis); heights of arcs (right y-axis) indicate the abundance of specific splice combinations. See S4 Table for more information. (D-E) Detection of spliced transcripts by RT-dependent PCR, using primers mapped in A-B. Input templates were MKPV-infected (D) kidney DNA or (E) DNAse/ExoI-treated kidney RNA, converted (+RT) or mock-converted (-RT) to cDNA. RT-PCR products corresponding to transcripts 1 to 4 are indicated by white numbers. (F) Mapping of transcription start and stop sites by RACE. See S1 Fig for RACE details. Major 5’ and 3’ RACE products, indicated by black arrows and corresponding to transcripts 2 to 4 or polyadenylation signals A and B, were gel-purified and Sanger sequenced. Other RACE products mentioned in the text are indicated by white arrows.
    Figure Legend Snippet: Map of the MKPV genome. (A-B) Maps of the MKPV/MuCPV strains from Centenary Institute (CI, accession MH670587), Memorial Sloan Kettering Cancer Center (MSKCC, accession MH670588) and New York City basements (wild-NY, MF175078). “Bowties” indicate terminal repeats (TR). (A) Single nucleotide variations (SNV) between the CI, MSKCC and wild-NY accessions. Vertical lines—differences between accessions. Half height vertical lines—polymorphisms within an accession. ▼; 2 bp insertion in the CI strain. ▲; 1 bp insertion in a CI sub-strain. Dashed lines—missing extremities in MSKCC and wild-NY accessions, which consist of the exterior inverted repeats in the full-length CI sequence. (B-C) Alternative splicing allows production of the polypeptides p10, p15, NS1, NS2, NP and VP. Black, brown or blue shading indicate the relative reading frames of ORFs. p15, p10 and NP could theoretically be produced from multiple transcripts. Orange or red indicate peptides present in LC-MS/MS datasets PXD014938 (this paper) or PXD010540 [ 9 ], respectively. Exon or intron sequences flanking splice sites are shown in black or red text, respectively. (C) Quantitation of spliced MKPV reads in RNAseq data pooled from two MKPV-infected kidneys. Columns indicate splice site usage (left y-axis); heights of arcs (right y-axis) indicate the abundance of specific splice combinations. See S4 Table for more information. (D-E) Detection of spliced transcripts by RT-dependent PCR, using primers mapped in A-B. Input templates were MKPV-infected (D) kidney DNA or (E) DNAse/ExoI-treated kidney RNA, converted (+RT) or mock-converted (-RT) to cDNA. RT-PCR products corresponding to transcripts 1 to 4 are indicated by white numbers. (F) Mapping of transcription start and stop sites by RACE. See S1 Fig for RACE details. Major 5’ and 3’ RACE products, indicated by black arrows and corresponding to transcripts 2 to 4 or polyadenylation signals A and B, were gel-purified and Sanger sequenced. Other RACE products mentioned in the text are indicated by white arrows.

    Techniques Used: Sequencing, Produced, Liquid Chromatography with Mass Spectroscopy, Quantitation Assay, Infection, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Purification

    17) Product Images from "Addressing fluorogenic real-time qPCR inhibition using the novel custom Excel file system 'FocusField2-6GallupqPCRSet-upTool-001' to attain consistently high fidelity qPCR reactions"

    Article Title: Addressing fluorogenic real-time qPCR inhibition using the novel custom Excel file system 'FocusField2-6GallupqPCRSet-upTool-001' to attain consistently high fidelity qPCR reactions

    Journal: Biological Procedures Online

    doi: 10.1251/bpo122

    Demonstration of typical inhibitory qPCR profiles exhibited on qPCR Test Plates by the more concentrated RNA samples (on the right hand side of each graph) in a progressive dilution series. Targets here were Gallus gallus Gallinacin 1, Gallinacin 2 and Gallus gallus 18S ribosomal RNA (the single housekeeper). Stock I here was an equivolumetric mixture of the 26 total tissue RNA samples used in this study: just after their isolation by Trizol method, each RNA pellet was resolubilized in 150 ?l of 0.1 mM EDTA pH 6.75, warmed to 65°C for 5 minutes, and their 260 nm and 260 nm /280 nm measurements at 1:50 were taken. 70 ?l of each resolubilized RNA was then Turbo-DNAse treated [70 ?l RNA isolate + 10 ?l 10X Turbo DNase Buffer + 20 ?l Turbo DNase enzyme (40 Units); and finally 10 ?l Inactivation Reagent] and 80 ?l of each was then diluted 1:10 with nuclease-free water. Subsequently, 50 ?l of each of these 1:10 RNA isolates was mixed together into a single tube attaining a final volume of 1,300 ?l. This was the Stock I RNA solution from which all standards and inter-plate calibrators were prepared. It was also the mixture which served as the source of the serially-diluted template samples for the Test Plate which we ran early on to identify the best RNA dilution ranges for each of the 3 targets. All calculations for this study were quickly performed by the FF2-6-001 qPCR set-up tool.
    Figure Legend Snippet: Demonstration of typical inhibitory qPCR profiles exhibited on qPCR Test Plates by the more concentrated RNA samples (on the right hand side of each graph) in a progressive dilution series. Targets here were Gallus gallus Gallinacin 1, Gallinacin 2 and Gallus gallus 18S ribosomal RNA (the single housekeeper). Stock I here was an equivolumetric mixture of the 26 total tissue RNA samples used in this study: just after their isolation by Trizol method, each RNA pellet was resolubilized in 150 ?l of 0.1 mM EDTA pH 6.75, warmed to 65°C for 5 minutes, and their 260 nm and 260 nm /280 nm measurements at 1:50 were taken. 70 ?l of each resolubilized RNA was then Turbo-DNAse treated [70 ?l RNA isolate + 10 ?l 10X Turbo DNase Buffer + 20 ?l Turbo DNase enzyme (40 Units); and finally 10 ?l Inactivation Reagent] and 80 ?l of each was then diluted 1:10 with nuclease-free water. Subsequently, 50 ?l of each of these 1:10 RNA isolates was mixed together into a single tube attaining a final volume of 1,300 ?l. This was the Stock I RNA solution from which all standards and inter-plate calibrators were prepared. It was also the mixture which served as the source of the serially-diluted template samples for the Test Plate which we ran early on to identify the best RNA dilution ranges for each of the 3 targets. All calculations for this study were quickly performed by the FF2-6-001 qPCR set-up tool.

    Techniques Used: Real-time Polymerase Chain Reaction, Isolation

    Different approaches to fluorogenic qPCR; we use the highlighted TaqMan® hydrolysis probe-based real-time qPCR method. All primers and probes are optimized and validated according to ABI procedural guidelines ( 31 ) using all-target-inclusive ( Stock I ) cDNA prepared from Turbo DNase-treated total RNA isolated (using Trizol®) from whole tissue homogenates as described previously ( 17 - 20 ). Our optimization approach is a very common/well-known procedure whereby one first studies different combinations of primer concentrations in the range of 50 nM-900 nM while keeping the probe at a constant 200 or 225 nM, after which the probe is studied by challenging it from 25 nM to 225 nM while primers are used at their optimal concentrations. All samples are performed in triplicate or quadruplicate during these evaluations to bolster significance of final evaluations. After optimization, a 'validation plate' or Test Plate is performed on up to eleven serial dilutions of the same cDNA or RNA (starting with full-strength cDNA or RNA which is assigned a relative dilution strength value of
    Figure Legend Snippet: Different approaches to fluorogenic qPCR; we use the highlighted TaqMan® hydrolysis probe-based real-time qPCR method. All primers and probes are optimized and validated according to ABI procedural guidelines ( 31 ) using all-target-inclusive ( Stock I ) cDNA prepared from Turbo DNase-treated total RNA isolated (using Trizol®) from whole tissue homogenates as described previously ( 17 - 20 ). Our optimization approach is a very common/well-known procedure whereby one first studies different combinations of primer concentrations in the range of 50 nM-900 nM while keeping the probe at a constant 200 or 225 nM, after which the probe is studied by challenging it from 25 nM to 225 nM while primers are used at their optimal concentrations. All samples are performed in triplicate or quadruplicate during these evaluations to bolster significance of final evaluations. After optimization, a 'validation plate' or Test Plate is performed on up to eleven serial dilutions of the same cDNA or RNA (starting with full-strength cDNA or RNA which is assigned a relative dilution strength value of "1") using the optimal primer and probe concentrations established during optimization for each target. The highest Rn (normalized reporter fluorescence) value achieved using the lowest primer concentrations is the indicator by which one selects the appropriate optimal primer concentrations in each case; the higher the Rn, the higher the magnitude of real-time fluorescent signal. Once the Rn value no longer increases with increasing primer concentrations, one has effectively attained the useful optimal primer concentrations. C T values (not Rn values) are evaluated during probe optimizations, and the lowest C T (threshold cycle) value with the lowest probe concentration is the criteria by which one chooses optimal probe concentrations. Once C T values no longer decrease with increasing probe concentration, one has effectively attained the useful optimal probe concentration. Little known is the fact that most real-time target signals can be found with greater than 75% amplification efficiency simply using 'saturating concentrations' of primers (1 ?M) and probes (150 nM) in most experimental situations if optimal RNA dilution ranges are established for each target and inhibition is entirely avoided (unpublished multiple observations from our lab, 2001-2006).

    Techniques Used: Real-time Polymerase Chain Reaction, Isolation, Fluorescence, Concentration Assay, Amplification, Inhibition

    18) Product Images from "Probing Oral Microbial Functionality - Expression of spxB in Plaque Samples"

    Article Title: Probing Oral Microbial Functionality - Expression of spxB in Plaque Samples

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0086685

    RNA integrity and concentration. A) Gel-eletrophoretic separation of isolated total RNA after DNase digest and clean-up on 1% agarose. B) Gel images of RNA samples generated by the Agilent Bioanalyzer using RNA 6000 Nano Lab Chip. C) RNA concentration and RIN as determined by the Agilent Bioanalyzer. RIN = RNA Integrity Number; L = RNA Ladder. Green line in Figure 4B: Bioanalyzer internal marker.
    Figure Legend Snippet: RNA integrity and concentration. A) Gel-eletrophoretic separation of isolated total RNA after DNase digest and clean-up on 1% agarose. B) Gel images of RNA samples generated by the Agilent Bioanalyzer using RNA 6000 Nano Lab Chip. C) RNA concentration and RIN as determined by the Agilent Bioanalyzer. RIN = RNA Integrity Number; L = RNA Ladder. Green line in Figure 4B: Bioanalyzer internal marker.

    Techniques Used: Concentration Assay, Isolation, Generated, Chromatin Immunoprecipitation, Marker

    19) Product Images from "Strand-specific deep sequencing of the transcriptome"

    Article Title: Strand-specific deep sequencing of the transcriptome

    Journal: Genome Research

    doi: 10.1101/gr.094318.109

    Comparative evaluation of deep sequencing. ( A ) Cumulative coverage of read data set, expressed as the ratio of sequenced bases located in annotated genes and the total number of bases in annotated genes. Exhaustive coverage is reached after 14 lanes (Supplemental Fig. 5). ( B ) Comparison of dynamic range of expression measurements (protein coding genes only) on tiling array and DSSS expressed on a log 2 scale. ( C ) Validation of DSSS results using qPCR. DNase-treated RNA was reverse transcribed and subjected to SYBR green real-time PCR. Non-reverse-transcribed controls were included for each gene, as well as a genomic DNA dilution series. DSSS signal corresponds to the log 2 transformed mean number of counts along the gene.
    Figure Legend Snippet: Comparative evaluation of deep sequencing. ( A ) Cumulative coverage of read data set, expressed as the ratio of sequenced bases located in annotated genes and the total number of bases in annotated genes. Exhaustive coverage is reached after 14 lanes (Supplemental Fig. 5). ( B ) Comparison of dynamic range of expression measurements (protein coding genes only) on tiling array and DSSS expressed on a log 2 scale. ( C ) Validation of DSSS results using qPCR. DNase-treated RNA was reverse transcribed and subjected to SYBR green real-time PCR. Non-reverse-transcribed controls were included for each gene, as well as a genomic DNA dilution series. DSSS signal corresponds to the log 2 transformed mean number of counts along the gene.

    Techniques Used: Sequencing, Expressing, Real-time Polymerase Chain Reaction, SYBR Green Assay, Transformation Assay

    20) Product Images from "Circular RNA Profiling by Illumina Sequencing via Template-Dependent Multiple Displacement Amplification"

    Article Title: Circular RNA Profiling by Illumina Sequencing via Template-Dependent Multiple Displacement Amplification

    Journal: BioMed Research International

    doi: 10.1155/2019/2756516

    Amplification of cDNA by Phi29 DNA polymerase . Total RNA from N. benthamiana (a) and O. sativa (b) was treated with DNase and RNase R to enrich circRNAs. The enriched circRNAs were converted into cDNA using random hexamer and subjected to amplification by Phi29 DNA polymerase.
    Figure Legend Snippet: Amplification of cDNA by Phi29 DNA polymerase . Total RNA from N. benthamiana (a) and O. sativa (b) was treated with DNase and RNase R to enrich circRNAs. The enriched circRNAs were converted into cDNA using random hexamer and subjected to amplification by Phi29 DNA polymerase.

    Techniques Used: Amplification, Random Hexamer Labeling

    21) Product Images from "Hepatitis B Virus Capsid Assembly Modulators, but Not Nucleoside Analogs, Inhibit the Production of Extracellular Pregenomic RNA and Spliced RNA Variants"

    Article Title: Hepatitis B Virus Capsid Assembly Modulators, but Not Nucleoside Analogs, Inhibit the Production of Extracellular Pregenomic RNA and Spliced RNA Variants

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00680-17

    Effect of nuclease treatment on extracted HBV DNA and RNA from the supernatant of infected HepaRG cells treated with HBV inhibitors. Cells were incubated with DMSO or 30 μM NVR 3-1983 or LMV for 6 days. HBV DNA and RNA from culture supernatants were extracted and treated with DNase or RNase, prior to quantitative assays. Extracellular HBV DNA (A) and HBV RNA (B) levels were normalized to those for DMSO-treated samples without nuclease treatment. Results and error bars represented means and standard deviations from at least three independent experiments, respectively.
    Figure Legend Snippet: Effect of nuclease treatment on extracted HBV DNA and RNA from the supernatant of infected HepaRG cells treated with HBV inhibitors. Cells were incubated with DMSO or 30 μM NVR 3-1983 or LMV for 6 days. HBV DNA and RNA from culture supernatants were extracted and treated with DNase or RNase, prior to quantitative assays. Extracellular HBV DNA (A) and HBV RNA (B) levels were normalized to those for DMSO-treated samples without nuclease treatment. Results and error bars represented means and standard deviations from at least three independent experiments, respectively.

    Techniques Used: Infection, Incubation

    22) Product Images from "Thiol reductive stress induces cellulose-anchored biofilm formation in Mycobacterium tuberculosis"

    Article Title: Thiol reductive stress induces cellulose-anchored biofilm formation in Mycobacterium tuberculosis

    Journal: Nature Communications

    doi: 10.1038/ncomms11392

    Mtb biofilms are disrupted by cellulase and protease treatments. Mature Mtb biofilms were treated with cellulase (5 mg ml −1 ), alpha-amylase, proteinase K (100 μg ml −1 ), lipase (1 mg ml −1 ) and DNase (2 U). ( a ) CLSM of mature Mtb biofilms overexpressing GFP following treatment with cellulase, lipase, proteinase K, DNase or their respective controls. EPS of Mtb biofilm was destroyed after treatment with cellulase and proteinase K, as depicted by significantly small cross-section compared with the control. ( b ) CV assays of Mtb biofilms after treatment with cellulase, lipase, proteinase K or DNase. Cellulase and proteinase K disrupted biofilm, whereas lipase, DNase and α-amylase had little effect on the biofilms. ( c ) Concentration of glucose released on treating Mtb biofilms with cellulase as measured by a glucose assay kit (Sigma). ( d ) Cellulase treatment of biofilms resulted in significantly higher reduction of 3,5-dinitrosalicylic acid (DNS) by the reducing sugars released on the cellulase treatment. ( e ) The biomass in the untreated biofilms and the biofilms independently treated with the cellulase, lipase, proteinase K or DNase was estimated using COMSTAT. The data presented in b – e are expressed as the mean (±s.e.m.). Statistical significance was determined using Student's t -test. * P
    Figure Legend Snippet: Mtb biofilms are disrupted by cellulase and protease treatments. Mature Mtb biofilms were treated with cellulase (5 mg ml −1 ), alpha-amylase, proteinase K (100 μg ml −1 ), lipase (1 mg ml −1 ) and DNase (2 U). ( a ) CLSM of mature Mtb biofilms overexpressing GFP following treatment with cellulase, lipase, proteinase K, DNase or their respective controls. EPS of Mtb biofilm was destroyed after treatment with cellulase and proteinase K, as depicted by significantly small cross-section compared with the control. ( b ) CV assays of Mtb biofilms after treatment with cellulase, lipase, proteinase K or DNase. Cellulase and proteinase K disrupted biofilm, whereas lipase, DNase and α-amylase had little effect on the biofilms. ( c ) Concentration of glucose released on treating Mtb biofilms with cellulase as measured by a glucose assay kit (Sigma). ( d ) Cellulase treatment of biofilms resulted in significantly higher reduction of 3,5-dinitrosalicylic acid (DNS) by the reducing sugars released on the cellulase treatment. ( e ) The biomass in the untreated biofilms and the biofilms independently treated with the cellulase, lipase, proteinase K or DNase was estimated using COMSTAT. The data presented in b – e are expressed as the mean (±s.e.m.). Statistical significance was determined using Student's t -test. * P

    Techniques Used: Confocal Laser Scanning Microscopy, Concentration Assay, Glucose Assay

    Effect of ECM-degrading enzymes on the development of Mtb biofilms. ( a ) Mtb cells overexpressing GFP were treated with 6 mM DTT to induce TRS. After 3 h of DTT exposure, the cultures were treated with proteinase K, cellulase, α-amylase, lipase or DNase. After 29 h, the effects of various enzymes on the formation of Mtb biofilms were observed through CLSM. Cellulase and proteinase K inhibited the Mtb biofilm formation, suggesting that cellulose fibres and unidentified structural proteins play a critical role in the early stages of biofilm attachment. In contrast, amylase, lipase and DNase had no effect on the structural integrity of biofilm initiation and maturation. ( b ) The biomass of biofilms developed in the presence of enzymes capable of degrading the ECM was estimated using COMSTAT. ( c ) CV assays of Mtb biofilms developed despite the presence of cellulase, lipase, proteinase K and DNase. Cellulase and proteinase K inhibited biofilm development, whereas lipase, DNase and α-amylase had no effect on the biofilm formation and hence on the CV staining. The data presented in b , c are expressed as the mean (±s.e.m.). Statistical significance was determined using Student's t -test. * P
    Figure Legend Snippet: Effect of ECM-degrading enzymes on the development of Mtb biofilms. ( a ) Mtb cells overexpressing GFP were treated with 6 mM DTT to induce TRS. After 3 h of DTT exposure, the cultures were treated with proteinase K, cellulase, α-amylase, lipase or DNase. After 29 h, the effects of various enzymes on the formation of Mtb biofilms were observed through CLSM. Cellulase and proteinase K inhibited the Mtb biofilm formation, suggesting that cellulose fibres and unidentified structural proteins play a critical role in the early stages of biofilm attachment. In contrast, amylase, lipase and DNase had no effect on the structural integrity of biofilm initiation and maturation. ( b ) The biomass of biofilms developed in the presence of enzymes capable of degrading the ECM was estimated using COMSTAT. ( c ) CV assays of Mtb biofilms developed despite the presence of cellulase, lipase, proteinase K and DNase. Cellulase and proteinase K inhibited biofilm development, whereas lipase, DNase and α-amylase had no effect on the biofilm formation and hence on the CV staining. The data presented in b , c are expressed as the mean (±s.e.m.). Statistical significance was determined using Student's t -test. * P

    Techniques Used: Confocal Laser Scanning Microscopy, Staining

    23) Product Images from "Hepatitis B Virus Capsid Assembly Modulators, but Not Nucleoside Analogs, Inhibit the Production of Extracellular Pregenomic RNA and Spliced RNA Variants"

    Article Title: Hepatitis B Virus Capsid Assembly Modulators, but Not Nucleoside Analogs, Inhibit the Production of Extracellular Pregenomic RNA and Spliced RNA Variants

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00680-17

    Effect of nuclease treatment on extracted HBV DNA and RNA from the supernatant of infected HepaRG cells treated with HBV inhibitors. Cells were incubated with DMSO or 30 μM NVR 3-1983 or LMV for 6 days. HBV DNA and RNA from culture supernatants were extracted and treated with DNase or RNase, prior to quantitative assays. Extracellular HBV DNA (A) and HBV RNA (B) levels were normalized to those for DMSO-treated samples without nuclease treatment. Results and error bars represented means and standard deviations from at least three independent experiments, respectively.
    Figure Legend Snippet: Effect of nuclease treatment on extracted HBV DNA and RNA from the supernatant of infected HepaRG cells treated with HBV inhibitors. Cells were incubated with DMSO or 30 μM NVR 3-1983 or LMV for 6 days. HBV DNA and RNA from culture supernatants were extracted and treated with DNase or RNase, prior to quantitative assays. Extracellular HBV DNA (A) and HBV RNA (B) levels were normalized to those for DMSO-treated samples without nuclease treatment. Results and error bars represented means and standard deviations from at least three independent experiments, respectively.

    Techniques Used: Infection, Incubation

    24) Product Images from "G-Quadruplex DNA Motifs in the Malaria Parasite Plasmodium falciparum and Their Potential as Novel Antimalarial Drug Targets"

    Article Title: G-Quadruplex DNA Motifs in the Malaria Parasite Plasmodium falciparum and Their Potential as Novel Antimalarial Drug Targets

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.01828-17

    G-quadruplexes can be detected in P. falciparum parasites. (A) Schematic of a G-quadruplex DNA motif. Guanine tetrads are shown as green squares, and guanine backbones are shown as dashed black lines. G-quartets stack on top of one another to form the quadruplex, which is stabilized by cations, such as potassium (K + ). An example of a PQS that could fold into such a structure is shown below. (B) Immunofluorescence images showing G-quadruplexes detected with the structure-specific antibody IH6 in P. falciparum intraerythrocytic stages (3D7 parasite strain). Images are representative of those from 3 independent experiments examining mixed-stage cultures. Troph, trophozoite. (C) DNase treatment abolishes G-quadruplex (IH6) staining but has no effect on the distribution of a protein, ERD2, used as a control (middle). RNase treatment has no discernible effect on G-quadruplex (IH6) staining (bottom). Images are representative of those from 3 independent experiments.
    Figure Legend Snippet: G-quadruplexes can be detected in P. falciparum parasites. (A) Schematic of a G-quadruplex DNA motif. Guanine tetrads are shown as green squares, and guanine backbones are shown as dashed black lines. G-quartets stack on top of one another to form the quadruplex, which is stabilized by cations, such as potassium (K + ). An example of a PQS that could fold into such a structure is shown below. (B) Immunofluorescence images showing G-quadruplexes detected with the structure-specific antibody IH6 in P. falciparum intraerythrocytic stages (3D7 parasite strain). Images are representative of those from 3 independent experiments examining mixed-stage cultures. Troph, trophozoite. (C) DNase treatment abolishes G-quadruplex (IH6) staining but has no effect on the distribution of a protein, ERD2, used as a control (middle). RNase treatment has no discernible effect on G-quadruplex (IH6) staining (bottom). Images are representative of those from 3 independent experiments.

    Techniques Used: Immunofluorescence, Staining

    25) Product Images from "A Re-Examination of Global Suppression of RNA Interference by HIV-1"

    Article Title: A Re-Examination of Global Suppression of RNA Interference by HIV-1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017246

    The effect of Tat over-expression on the silencing potency of miEGFP. (A) Immunoblot analysis of protein extracts obtained from P4R5 cells 2 days post-transfection with indicated plasmids. The blot was analyzed using antibodies specific to EGFP and β-actin, which serves as a loading control. (B) Spot-densitometry analysis of two individual experiments, as described in (A). The data are shown as the ratio of EGFP to β-actin and presented as the percentage of control (no miEGFP, no Tat). Error bars represents standard deviation from 3 replicates. (C) Total RNA was extracted from cells transfected in (A) and, following DNase treatment, qRT-PCR was performed to quantitate EGFP mRNA. The data are normalized to β-actin and presented as fold change over control (no miEGFP). Error bars represent standard deviation from 3 replicates. (D) Lower panel: the RNA preparation from (C) was subjected to primer extension reaction to detect mature miEGFP. Upper panel: U6 RNA was detected by northern blotting to confirm RNA integrity and quantification.
    Figure Legend Snippet: The effect of Tat over-expression on the silencing potency of miEGFP. (A) Immunoblot analysis of protein extracts obtained from P4R5 cells 2 days post-transfection with indicated plasmids. The blot was analyzed using antibodies specific to EGFP and β-actin, which serves as a loading control. (B) Spot-densitometry analysis of two individual experiments, as described in (A). The data are shown as the ratio of EGFP to β-actin and presented as the percentage of control (no miEGFP, no Tat). Error bars represents standard deviation from 3 replicates. (C) Total RNA was extracted from cells transfected in (A) and, following DNase treatment, qRT-PCR was performed to quantitate EGFP mRNA. The data are normalized to β-actin and presented as fold change over control (no miEGFP). Error bars represent standard deviation from 3 replicates. (D) Lower panel: the RNA preparation from (C) was subjected to primer extension reaction to detect mature miEGFP. Upper panel: U6 RNA was detected by northern blotting to confirm RNA integrity and quantification.

    Techniques Used: Over Expression, Transfection, Standard Deviation, Quantitative RT-PCR, Northern Blot

    Efficacy of EGFP silencing by miEGFP in the presence of HIV-1 replication. (A) Protein extracts were prepared 2d post-transfection from 293T cells transfected with pCMV-dsEGFP and the indicated plasmids, and then analyzed by immunoblotting with antibodies specific to EGFP and β-actin. Results show two replicates from the same experiment. (B) Total RNA was isolated from cells transfected in (A) and, following DNase treatment, qRT-PCR was performed to determine the level of EGFP mRNA. The data are normalized to β-actin mRNA and presented as fold change over control. Two individual replicates from the same experiment are shown. Error bars represent standard deviation from three qPCR replicates of the same sample.
    Figure Legend Snippet: Efficacy of EGFP silencing by miEGFP in the presence of HIV-1 replication. (A) Protein extracts were prepared 2d post-transfection from 293T cells transfected with pCMV-dsEGFP and the indicated plasmids, and then analyzed by immunoblotting with antibodies specific to EGFP and β-actin. Results show two replicates from the same experiment. (B) Total RNA was isolated from cells transfected in (A) and, following DNase treatment, qRT-PCR was performed to determine the level of EGFP mRNA. The data are normalized to β-actin mRNA and presented as fold change over control. Two individual replicates from the same experiment are shown. Error bars represent standard deviation from three qPCR replicates of the same sample.

    Techniques Used: Transfection, Isolation, Quantitative RT-PCR, Standard Deviation, Real-time Polymerase Chain Reaction

    The impact of Dicer depletion on HIV-1 replication in 293T cells. (A) Dicer knockdown was confirmed by immunoblotting total protein extracts prepared 2d post-transfection from 293T cells transfected with indicated amounts of pU6-miDicer. The immunoblot was incubated with antibody specific to Dicer and β-actin that serves as a loading control. (B) 293T cells were transfected with pLAI, together with pmiEGFP or pU6-miDicer, as indicated. 2d post transfection infectious virus released into the supernatant was assayed using P4R5 indicator cells (see Materials and Methods ). Error bars represent standard deviation from 6 replicates. (C) Total RNA isolated from cells in (B) was subjected to semi-quantitative RT-PCR to determine the mRNA levels of Dicer, HIV-1 Tat, and β-actin. Following PCR, products were analyzed by electrophoresis in 2% agarose and ethidium bromide staining. PCR products are resolved on the same gel and irrelevant samples are cropped out. D) Total RNA isolated in (C) was treated with DNase, and following cDNA synthesis with a Gag mRNA specific primer, semi-quantitative PCR was performed and products were analyzed as in (C). PCR for β-actin following oligo(dT)-primed cDNA synthesis serves as a loading control.
    Figure Legend Snippet: The impact of Dicer depletion on HIV-1 replication in 293T cells. (A) Dicer knockdown was confirmed by immunoblotting total protein extracts prepared 2d post-transfection from 293T cells transfected with indicated amounts of pU6-miDicer. The immunoblot was incubated with antibody specific to Dicer and β-actin that serves as a loading control. (B) 293T cells were transfected with pLAI, together with pmiEGFP or pU6-miDicer, as indicated. 2d post transfection infectious virus released into the supernatant was assayed using P4R5 indicator cells (see Materials and Methods ). Error bars represent standard deviation from 6 replicates. (C) Total RNA isolated from cells in (B) was subjected to semi-quantitative RT-PCR to determine the mRNA levels of Dicer, HIV-1 Tat, and β-actin. Following PCR, products were analyzed by electrophoresis in 2% agarose and ethidium bromide staining. PCR products are resolved on the same gel and irrelevant samples are cropped out. D) Total RNA isolated in (C) was treated with DNase, and following cDNA synthesis with a Gag mRNA specific primer, semi-quantitative PCR was performed and products were analyzed as in (C). PCR for β-actin following oligo(dT)-primed cDNA synthesis serves as a loading control.

    Techniques Used: Transfection, Incubation, Standard Deviation, Isolation, Quantitative RT-PCR, Polymerase Chain Reaction, Electrophoresis, Staining, Real-time Polymerase Chain Reaction

    26) Product Images from "Plasmodium yoelii Macrophage Migration Inhibitory Factor Is Necessary for Efficient Liver-Stage Development"

    Article Title: Plasmodium yoelii Macrophage Migration Inhibitory Factor Is Necessary for Efficient Liver-Stage Development

    Journal: Infection and Immunity

    doi: 10.1128/IAI.05861-11

    Py-MIF transcript is expressed in liver-stage (LS) and blood-stage (BS) parasites. The expression of Py- mif was determined by RT-PCR on RNA derived from P. yoelii 17XNL. RNA was isolated from salivary gland sporozoites, mixed blood-stage parasites, or mouse livers infected with 1 × 10 6 sporozoites (isolated at 24 or 44 hpi). Genomic DNA was removed by DNase treatment, and cDNA was generated by reverse transcriptase single-strand DNA synthesis. Either Py- mif or 18S rRNA was amplified from cDNAs via 35 cycles of PCR. Non-RT controls (−RT) were included to confirm that mif amplification was due to cDNA and not to residual genomic DNA.
    Figure Legend Snippet: Py-MIF transcript is expressed in liver-stage (LS) and blood-stage (BS) parasites. The expression of Py- mif was determined by RT-PCR on RNA derived from P. yoelii 17XNL. RNA was isolated from salivary gland sporozoites, mixed blood-stage parasites, or mouse livers infected with 1 × 10 6 sporozoites (isolated at 24 or 44 hpi). Genomic DNA was removed by DNase treatment, and cDNA was generated by reverse transcriptase single-strand DNA synthesis. Either Py- mif or 18S rRNA was amplified from cDNAs via 35 cycles of PCR. Non-RT controls (−RT) were included to confirm that mif amplification was due to cDNA and not to residual genomic DNA.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Isolation, Infection, Generated, DNA Synthesis, Amplification, Polymerase Chain Reaction

    27) Product Images from "TLR9 Ligands Induce S100A8 in Macrophages via a STAT3-Dependent Pathway which Requires IL-10 and PGE2"

    Article Title: TLR9 Ligands Induce S100A8 in Macrophages via a STAT3-Dependent Pathway which Requires IL-10 and PGE2

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0103629

    Induction of S100A8 by CpG-DNA is dose-dependent. (A) RAW 264.7 macrophages were incubated with E. coli DNA at the indicated concentrations ±0.3 ng/ml LPS for 24 h before harvesting and (A) mRNAs quantitated by qRT-PCR with different doses of DNase-treated E. coli DNA ± LPS (0.3 ng/ml) as control. (B) S100A8 levels in culture supernates from (A) were quantitated by ELISA. Other controls included various concentrations of placental DNA ± LPS (0.3 ng/ml) or DNase-treated E. coli DNA, but mRNA levels were same as control and proteins were below the detectable level (not shown). (C) S100A8 in supernates from RAW cells treated with synthetic nCpG-1982 or CpG-1826 at the indicated concentrations was quantitated. QRT-PCR data represent means (relative to the γ-actin mRNA levels) ± SD of duplicates and are representative of 3 experiments. ELISA data are means ± SD of duplicate samples from 2 separate experiments.
    Figure Legend Snippet: Induction of S100A8 by CpG-DNA is dose-dependent. (A) RAW 264.7 macrophages were incubated with E. coli DNA at the indicated concentrations ±0.3 ng/ml LPS for 24 h before harvesting and (A) mRNAs quantitated by qRT-PCR with different doses of DNase-treated E. coli DNA ± LPS (0.3 ng/ml) as control. (B) S100A8 levels in culture supernates from (A) were quantitated by ELISA. Other controls included various concentrations of placental DNA ± LPS (0.3 ng/ml) or DNase-treated E. coli DNA, but mRNA levels were same as control and proteins were below the detectable level (not shown). (C) S100A8 in supernates from RAW cells treated with synthetic nCpG-1982 or CpG-1826 at the indicated concentrations was quantitated. QRT-PCR data represent means (relative to the γ-actin mRNA levels) ± SD of duplicates and are representative of 3 experiments. ELISA data are means ± SD of duplicate samples from 2 separate experiments.

    Techniques Used: Incubation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    Induction of S100A8 mRNA in macrophages by E. coli DNA, M. luteus DNA, and synthetic CpG-ONDs. (A) RAW 264.7 macrophages were incubated with E. coli DNA (3 µg/ml) or M. luteus DNA (3 µg/ml) ±0.3 ng/ml LPS. Controls include placental DNA (3 µg/ml) ± LPS (0.3 ng/ml), Turbo DNase-treated E. coli DNA or M. luteus DNA ± LPS (0.3 ng/ml). RAW cells were also stimulated with two doses of LPS alone as positive controls (0.3 or 100 ng/ml). (B) RAW cells were untreated (medium) or treated with a synthetic non-CpG-containing OND 1982 (nCpG), a murine-oriented CpG-containing ODN (CpG-1826), or a human-oriented CpG-containing ODN (CpG-2006) (all 3 µM final concentration) ± LPS (0.3 ng/ml). (C) medium control, CpG-1826 (3 µM) or LPS (20 ng/ml) were co-stimulated with inhibitory CpG ODN 2088 (iCpG) 2 or 6 µM. (D) Elicited murine macrophages were untreated or stimulated with human placental DNA or E. coli DNA at the indicated concentrations. (E) Human monocyte-derived macrophages stimulated with 3 µM of nCpG or CpG-2216; LPS (20 ng/ml) was positive control. Combined data from 3 independent experiments are presented as the mean ± SD (*, p
    Figure Legend Snippet: Induction of S100A8 mRNA in macrophages by E. coli DNA, M. luteus DNA, and synthetic CpG-ONDs. (A) RAW 264.7 macrophages were incubated with E. coli DNA (3 µg/ml) or M. luteus DNA (3 µg/ml) ±0.3 ng/ml LPS. Controls include placental DNA (3 µg/ml) ± LPS (0.3 ng/ml), Turbo DNase-treated E. coli DNA or M. luteus DNA ± LPS (0.3 ng/ml). RAW cells were also stimulated with two doses of LPS alone as positive controls (0.3 or 100 ng/ml). (B) RAW cells were untreated (medium) or treated with a synthetic non-CpG-containing OND 1982 (nCpG), a murine-oriented CpG-containing ODN (CpG-1826), or a human-oriented CpG-containing ODN (CpG-2006) (all 3 µM final concentration) ± LPS (0.3 ng/ml). (C) medium control, CpG-1826 (3 µM) or LPS (20 ng/ml) were co-stimulated with inhibitory CpG ODN 2088 (iCpG) 2 or 6 µM. (D) Elicited murine macrophages were untreated or stimulated with human placental DNA or E. coli DNA at the indicated concentrations. (E) Human monocyte-derived macrophages stimulated with 3 µM of nCpG or CpG-2216; LPS (20 ng/ml) was positive control. Combined data from 3 independent experiments are presented as the mean ± SD (*, p

    Techniques Used: Incubation, Concentration Assay, Derivative Assay, Positive Control

    28) Product Images from "N6-methyladenosine modification and the YTHDF2 reader protein play cell type specific roles in lytic viral gene expression during Kaposi's sarcoma-associated herpesvirus infection"

    Article Title: N6-methyladenosine modification and the YTHDF2 reader protein play cell type specific roles in lytic viral gene expression during Kaposi's sarcoma-associated herpesvirus infection

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006995

    KSHV mRNA contains m 6 A modifications. (A) Two independent replicates of iSLK.219 cells containing latent KSHV were treated with dox for 5 days to induce the viral lytic cycle (induced) or left untreated to preserve viral latency (uninduced). DNase-treated RNA was isolated and subjected to m 6 Aseq. Displayed are peaks with a fold change of four or higher, comparing reads in the m 6 A-IP to the corresponding input. Numbers above peaks correspond to the base position within the KSHV genome. (B) Overview of sequencing reads from induced and uninduced m 6 A IP samples, aligned to the ORF50 transcript and the annotated GG(m 6 A)C consensus motifs found in exon 2 of ORF50. Numbers to the left of sequencing reads indicate the scale of the read count. (C) Cells were induced as in (A), and total RNA was subjected to m 6 A RIP, followed by RT-qPCR using primers for the indicated viral and cellular genes. Values are displayed as fold change over input, normalized to GAPDH. (D) Quantification of cellular m 6 A peaks from m 6 Aseq analysis.
    Figure Legend Snippet: KSHV mRNA contains m 6 A modifications. (A) Two independent replicates of iSLK.219 cells containing latent KSHV were treated with dox for 5 days to induce the viral lytic cycle (induced) or left untreated to preserve viral latency (uninduced). DNase-treated RNA was isolated and subjected to m 6 Aseq. Displayed are peaks with a fold change of four or higher, comparing reads in the m 6 A-IP to the corresponding input. Numbers above peaks correspond to the base position within the KSHV genome. (B) Overview of sequencing reads from induced and uninduced m 6 A IP samples, aligned to the ORF50 transcript and the annotated GG(m 6 A)C consensus motifs found in exon 2 of ORF50. Numbers to the left of sequencing reads indicate the scale of the read count. (C) Cells were induced as in (A), and total RNA was subjected to m 6 A RIP, followed by RT-qPCR using primers for the indicated viral and cellular genes. Values are displayed as fold change over input, normalized to GAPDH. (D) Quantification of cellular m 6 A peaks from m 6 Aseq analysis.

    Techniques Used: Isolation, Sequencing, Quantitative RT-PCR

    29) Product Images from "In Vivo T-Box Transcription Factor Profiling Reveals Joint Regulation of Embryonic Neuromesodermal Bipotency"

    Article Title: In Vivo T-Box Transcription Factor Profiling Reveals Joint Regulation of Embryonic Neuromesodermal Bipotency

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2013.08.012

    Combined Loss of T-box TFs Causes Embryos to Produce Excess Neural Tissue at the Expense of Axial and Paraxial Mesoderm and in the Absence of Apoptosis, Related to Figure 6 (A) WMISH on control, Xbra/Xbra3, Xbra/Xbra3/Eomes, Xbra/Xbra3/zVegT and Xbra/Xbra3/Eomes/zVegT KD embryos for neural differentiation marker N-tubulin at early tailbud stage (stage 20-21, lateral view). 1, 2 and 3 mark the position of cross-sections through control, Xbra/Xbra3 KD and Xbra/Xbra3/zVegT KD embryos. Abbreviations: no, notochord; nt, neural tube (d, dorsal; v, ventral); pm, paraxial mesoderm. (B) WMISH for actc1 , hoxd8 and tal1 at late tailbud stage illustrates the loss of mesodermal derivatives such as skeletal muscle (sm), heart (he), pronephros (pn) and ventral blood island (vbi) upon Xbra/Xbra3/Eomes/zVegT KD. Statistics in bottom right corner indicates the number of embryos observed with the depicted WMISH pattern versus the number of embryos analyzed in total. (C) TUNEL staining on control, Xbra/Xbra3 and Xbra/Xbra3/Eomes/zVegT KD embryos (cleared with Murray’s clear) at early tailbud stage (stage 20). Positive controls, embryos treated for 4 hr in 35 μM cycloheximide (chx) and fixed embryos incubated with DNase I. Arrowheads mark apoptotic cells in the brain region (where apoptosis can occasionally be observed in embryos even under normal conditions) and the posterior nervous system induced by cycloheximide. Scale bar, 0.2 mm.
    Figure Legend Snippet: Combined Loss of T-box TFs Causes Embryos to Produce Excess Neural Tissue at the Expense of Axial and Paraxial Mesoderm and in the Absence of Apoptosis, Related to Figure 6 (A) WMISH on control, Xbra/Xbra3, Xbra/Xbra3/Eomes, Xbra/Xbra3/zVegT and Xbra/Xbra3/Eomes/zVegT KD embryos for neural differentiation marker N-tubulin at early tailbud stage (stage 20-21, lateral view). 1, 2 and 3 mark the position of cross-sections through control, Xbra/Xbra3 KD and Xbra/Xbra3/zVegT KD embryos. Abbreviations: no, notochord; nt, neural tube (d, dorsal; v, ventral); pm, paraxial mesoderm. (B) WMISH for actc1 , hoxd8 and tal1 at late tailbud stage illustrates the loss of mesodermal derivatives such as skeletal muscle (sm), heart (he), pronephros (pn) and ventral blood island (vbi) upon Xbra/Xbra3/Eomes/zVegT KD. Statistics in bottom right corner indicates the number of embryos observed with the depicted WMISH pattern versus the number of embryos analyzed in total. (C) TUNEL staining on control, Xbra/Xbra3 and Xbra/Xbra3/Eomes/zVegT KD embryos (cleared with Murray’s clear) at early tailbud stage (stage 20). Positive controls, embryos treated for 4 hr in 35 μM cycloheximide (chx) and fixed embryos incubated with DNase I. Arrowheads mark apoptotic cells in the brain region (where apoptosis can occasionally be observed in embryos even under normal conditions) and the posterior nervous system induced by cycloheximide. Scale bar, 0.2 mm.

    Techniques Used: Marker, TUNEL Assay, Staining, Incubation

    30) Product Images from "In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase"

    Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

    Journal: Journal of Virology

    doi: 10.1128/JVI.07137-11

    Detection of in vitro protein priming by purified HP. Priming reactions were performed by incubating immunoaffinity-purified HP with TMgNK buffer and [α- 32 P]dGTP (A to C ) or another labeled nucleotide as indicated (D and E). After priming, the beads were washed, and the labeled HP was resolved on an SDS–12.5% polyacrylamide gel. A priming reaction was also performed with the DHBV MiniRT2 (DP) in TMnNK buffer and resolved on the same gel for comparison (A, lane 1). Labeled HP and DP priming products were detected by autoradiography after SDS-PAGE. (A) In vitro priming reactions with WT (lanes 3 and 4) or mutant (lanes 5 and 6) HP with (lanes 4 to 6) or without Hε (lane 3) coexpression in cells. GFP + Hε (lane 2) represents priming using the control purification product from cells cotransfected with GFP and the Hε-expressing plasmid. (B) After protein priming, primed HP was untreated (−; lane 1) or treated with DNase I (D; lane 2) or pronase (P; lane 3) before analysis by SDS-PAGE. (C) The purified HP was mock treated (lane 1) or RNase treated (lane 2) before being used in protein priming. Labeled HP was detected by autoradiography after SDS-PAGE (top), and HP protein levels were measured by Western blotting using the anti-FLAG (α-Flag) antibody (bottom). (D) HP purified either with (lanes 5 to 8) or without (lanes 1 to 4) the coexpressed Hε was assayed for priming activity in the presence of [α- 32 P]dGTP (G; lanes 2 and 6), [α- 32 P]TTP (T; lanes 1 and 5), [α- 32 P]dCTP (C; lanes 3 and 7), or [α- 32 P]dATP (A; lanes 4 and 8). Priming signals were quantified via phosphorimaging, normalized to the highest signal (dGTP priming, set as 100%), and denoted below the lane numbers (as a percentage of dGTP signal). The labeled HP and DP priming products are indicated. (E) Shown on the top is a schematic diagram of the mutant Hε RNAs, with the last 4 nucleotides of the internal bulge and part of the upper stem, including its bottom A-U base pair. In Hε-B6G (left), the last (6th) bulge residue (i.e., B6) was changed (from rC in the WT) to rG and in Hε-B6A (right), the same residue was changed to rA. The mutated residues are highlighted in bold. Shown at the bottom are priming products obtained with the mutant Hε RNAs. The Hε-B6G (lanes 1 and 2) or -B6A (lanes 3 and 4) mutant was coexpressed with HP, and the purified HP-Hε complex was assayed for protein priming in vitro in the presence of the indicated 32 P-labeled nucleotide. The labeled HP priming products are indicated, as is the position of the protein molecular mass marker (in kDa).
    Figure Legend Snippet: Detection of in vitro protein priming by purified HP. Priming reactions were performed by incubating immunoaffinity-purified HP with TMgNK buffer and [α- 32 P]dGTP (A to C ) or another labeled nucleotide as indicated (D and E). After priming, the beads were washed, and the labeled HP was resolved on an SDS–12.5% polyacrylamide gel. A priming reaction was also performed with the DHBV MiniRT2 (DP) in TMnNK buffer and resolved on the same gel for comparison (A, lane 1). Labeled HP and DP priming products were detected by autoradiography after SDS-PAGE. (A) In vitro priming reactions with WT (lanes 3 and 4) or mutant (lanes 5 and 6) HP with (lanes 4 to 6) or without Hε (lane 3) coexpression in cells. GFP + Hε (lane 2) represents priming using the control purification product from cells cotransfected with GFP and the Hε-expressing plasmid. (B) After protein priming, primed HP was untreated (−; lane 1) or treated with DNase I (D; lane 2) or pronase (P; lane 3) before analysis by SDS-PAGE. (C) The purified HP was mock treated (lane 1) or RNase treated (lane 2) before being used in protein priming. Labeled HP was detected by autoradiography after SDS-PAGE (top), and HP protein levels were measured by Western blotting using the anti-FLAG (α-Flag) antibody (bottom). (D) HP purified either with (lanes 5 to 8) or without (lanes 1 to 4) the coexpressed Hε was assayed for priming activity in the presence of [α- 32 P]dGTP (G; lanes 2 and 6), [α- 32 P]TTP (T; lanes 1 and 5), [α- 32 P]dCTP (C; lanes 3 and 7), or [α- 32 P]dATP (A; lanes 4 and 8). Priming signals were quantified via phosphorimaging, normalized to the highest signal (dGTP priming, set as 100%), and denoted below the lane numbers (as a percentage of dGTP signal). The labeled HP and DP priming products are indicated. (E) Shown on the top is a schematic diagram of the mutant Hε RNAs, with the last 4 nucleotides of the internal bulge and part of the upper stem, including its bottom A-U base pair. In Hε-B6G (left), the last (6th) bulge residue (i.e., B6) was changed (from rC in the WT) to rG and in Hε-B6A (right), the same residue was changed to rA. The mutated residues are highlighted in bold. Shown at the bottom are priming products obtained with the mutant Hε RNAs. The Hε-B6G (lanes 1 and 2) or -B6A (lanes 3 and 4) mutant was coexpressed with HP, and the purified HP-Hε complex was assayed for protein priming in vitro in the presence of the indicated 32 P-labeled nucleotide. The labeled HP priming products are indicated, as is the position of the protein molecular mass marker (in kDa).

    Techniques Used: In Vitro, Purification, Labeling, Autoradiography, SDS Page, Mutagenesis, Expressing, Plasmid Preparation, Western Blot, Activity Assay, Marker

    31) Product Images from "Heat Shock Affects Mitotic Segregation of Human Chromosomes Bound to Stress-Induced Satellite III RNAs"

    Article Title: Heat Shock Affects Mitotic Segregation of Human Chromosomes Bound to Stress-Induced Satellite III RNAs

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21082812

    Characterization of short SatIII RNAs. ( A ) Short RNAs were prepared from heat-shocked HeLa cells allowed to recover at 37 °C for 42 h. RNAs were digested with DNase I or with RNase A and then analyzed using Northern blotting with the LNA oligo specific for SatIII repeats. ( B ) Short RNAs were prepared from heat-shocked hamster B14-150 cells and from the hamster > human somatic cell hybrid GM-10611A containing human chromosome 9. RNAs were then analyzed using Northern blotting as in panel A. Hybridization to Mir16 was used as a loading control. The histogram shows the quantitation of long SatIII RNAs in unstressed (C) and in heat-shocked GM10611A cells allowed to recover at 37 °C for the indicated time periods. Data are represented as mean fold increases ± standard deviation of three independent experiments. ( C ) Short RNAs were prepared from unstressed (C) and from heat-shocked (1 h at 42 °C) HeLa cells allowed to recover for 30 h at 37 °C in the presence or in the absence of Actinomycin D (5 μg/mL) during the last 6 h of recovery. The same membrane was hybridized with the LNA probe against SatIII RNAs and with a probe against Mir16 (loading control). M: molecular size marker. Total RNAs prepared from the same cells were analyzed using RT-PCR to assess the level of c-myc RNAs to control the efficacy of Act D treatment. RT-PCR analysis of hP0 RNA was used as a loading control. ( D ) Unstressed (37) and heat-shocked HeLa cells were collected after 40 h of recovery at 37 °C and fractionated in nuclear pellet (N-P), nucleoplasm (Nu) and cytoplasm (Cy) fractions. Total and short RNAs were prepared from each fraction. Short RNAs were analyzed using Northern blotting with an LNA probe against SatIII repeats. As a control of loading and fractionation quality, the same filter was hybridized to a probe against Mir16 (mainly cytoplasmic) and U6 RNAs. Total RNAs from nuclear pellet, nucleoplasm and cytoplasm fractions were prepared from heat-shocked HeLa cells allowed to recover for 40 h at 37 °C. RNAs were analyzed using quantitative RT-PCR. The histogram shows the relative abundance of SatIII RNAs in the three factions as determined in three independent experiments. Data are mean ± standard deviation.
    Figure Legend Snippet: Characterization of short SatIII RNAs. ( A ) Short RNAs were prepared from heat-shocked HeLa cells allowed to recover at 37 °C for 42 h. RNAs were digested with DNase I or with RNase A and then analyzed using Northern blotting with the LNA oligo specific for SatIII repeats. ( B ) Short RNAs were prepared from heat-shocked hamster B14-150 cells and from the hamster > human somatic cell hybrid GM-10611A containing human chromosome 9. RNAs were then analyzed using Northern blotting as in panel A. Hybridization to Mir16 was used as a loading control. The histogram shows the quantitation of long SatIII RNAs in unstressed (C) and in heat-shocked GM10611A cells allowed to recover at 37 °C for the indicated time periods. Data are represented as mean fold increases ± standard deviation of three independent experiments. ( C ) Short RNAs were prepared from unstressed (C) and from heat-shocked (1 h at 42 °C) HeLa cells allowed to recover for 30 h at 37 °C in the presence or in the absence of Actinomycin D (5 μg/mL) during the last 6 h of recovery. The same membrane was hybridized with the LNA probe against SatIII RNAs and with a probe against Mir16 (loading control). M: molecular size marker. Total RNAs prepared from the same cells were analyzed using RT-PCR to assess the level of c-myc RNAs to control the efficacy of Act D treatment. RT-PCR analysis of hP0 RNA was used as a loading control. ( D ) Unstressed (37) and heat-shocked HeLa cells were collected after 40 h of recovery at 37 °C and fractionated in nuclear pellet (N-P), nucleoplasm (Nu) and cytoplasm (Cy) fractions. Total and short RNAs were prepared from each fraction. Short RNAs were analyzed using Northern blotting with an LNA probe against SatIII repeats. As a control of loading and fractionation quality, the same filter was hybridized to a probe against Mir16 (mainly cytoplasmic) and U6 RNAs. Total RNAs from nuclear pellet, nucleoplasm and cytoplasm fractions were prepared from heat-shocked HeLa cells allowed to recover for 40 h at 37 °C. RNAs were analyzed using quantitative RT-PCR. The histogram shows the relative abundance of SatIII RNAs in the three factions as determined in three independent experiments. Data are mean ± standard deviation.

    Techniques Used: Northern Blot, Hybridization, Quantitation Assay, Standard Deviation, Marker, Reverse Transcription Polymerase Chain Reaction, Fractionation, Quantitative RT-PCR

    32) Product Images from "Life without tRNAArg-adenosine deaminase TadA: evolutionary consequences of decoding the four CGN codons as arginine in Mycoplasmas and other Mollicutes"

    Article Title: Life without tRNAArg-adenosine deaminase TadA: evolutionary consequences of decoding the four CGN codons as arginine in Mycoplasmas and other Mollicutes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt356

    Reverse transcriptase–PCR of tRNA Arg I CG from M. capricolum and B. subtilis . ( A ) Comparison of the nucleotide sequences of M. capricolum (Mca) and B. subtilis (Bsu) tRNA Arg I CG , obtained from ( 15 ). The cloverleaf structures are shown. I, 4, D, K, P, 7 and T represent inosine, 4-thio-uridine, dihydrouridine, 1-methylguanosine, pseudouridine, 7-methylguanosine and 5-methyluridine (ribosylthymine), respectively. Regions of primers for reverse transcription of the first strand (and first primers for PCR) are shown with black arrows. Regions of the second primers for PCR are shown with gray arrows. ( B ) Summary of sequences of cDNA clones for M. capricolum and B. subtilis tRNA Arg I CG . The DNA sequences of the cDNA clones, except for the PCR primer regions, are shown in brackets. The RNA sequences corresponding to the obtained DNA sequences are shown in parentheses. I (inosine) in the RNA sequence corresponds to G in the DNA sequence obtained by reverse transcription. ( C ) Agarose gel electrophoresis of reverse transcriptase–PCR products. Lane M: size marker (100-bp ladder, the position of 100 bp is shown with an arrow). Lanes 1–10: PCR products of various templates. Lane 1: reverse-transcribed McatRNA Arg I CG solution treated with DNase before reverse transcription. Lane 2: total McatRNA solution with DNase treatment. Lane 3: Reverse-transcribed McatRNA Arg I CG solution without DNase treatment before reverse transcription. Lane 4: total McatRNA solution without DNase treatment. Lane 6: reverse-transcribed BsutRNA Arg I CG solution with DNase treatment before reverse transcription. Lane 7: total BsutRNA solution with DNase treatment. Lane 8: reverse-transcribed BsutRNA Arg I CG solution without DNase treatment before reverse transcription. Lane 9: total BsutRNA solution without DNase treatment. Lanes 5 and 10: control (no RNA/DNA).
    Figure Legend Snippet: Reverse transcriptase–PCR of tRNA Arg I CG from M. capricolum and B. subtilis . ( A ) Comparison of the nucleotide sequences of M. capricolum (Mca) and B. subtilis (Bsu) tRNA Arg I CG , obtained from ( 15 ). The cloverleaf structures are shown. I, 4, D, K, P, 7 and T represent inosine, 4-thio-uridine, dihydrouridine, 1-methylguanosine, pseudouridine, 7-methylguanosine and 5-methyluridine (ribosylthymine), respectively. Regions of primers for reverse transcription of the first strand (and first primers for PCR) are shown with black arrows. Regions of the second primers for PCR are shown with gray arrows. ( B ) Summary of sequences of cDNA clones for M. capricolum and B. subtilis tRNA Arg I CG . The DNA sequences of the cDNA clones, except for the PCR primer regions, are shown in brackets. The RNA sequences corresponding to the obtained DNA sequences are shown in parentheses. I (inosine) in the RNA sequence corresponds to G in the DNA sequence obtained by reverse transcription. ( C ) Agarose gel electrophoresis of reverse transcriptase–PCR products. Lane M: size marker (100-bp ladder, the position of 100 bp is shown with an arrow). Lanes 1–10: PCR products of various templates. Lane 1: reverse-transcribed McatRNA Arg I CG solution treated with DNase before reverse transcription. Lane 2: total McatRNA solution with DNase treatment. Lane 3: Reverse-transcribed McatRNA Arg I CG solution without DNase treatment before reverse transcription. Lane 4: total McatRNA solution without DNase treatment. Lane 6: reverse-transcribed BsutRNA Arg I CG solution with DNase treatment before reverse transcription. Lane 7: total BsutRNA solution with DNase treatment. Lane 8: reverse-transcribed BsutRNA Arg I CG solution without DNase treatment before reverse transcription. Lane 9: total BsutRNA solution without DNase treatment. Lanes 5 and 10: control (no RNA/DNA).

    Techniques Used: Polymerase Chain Reaction, Clone Assay, Sequencing, Agarose Gel Electrophoresis, Marker

    33) Product Images from "An Rrp6-like Protein Positively Regulates Non-coding RNA Levels and DNA Methylation in Arabidopsis"

    Article Title: An Rrp6-like Protein Positively Regulates Non-coding RNA Levels and DNA Methylation in Arabidopsis

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2014.03.019

    AtRRP6L1 associates with scaffold RNAs and partially co-localizes with Pol V in subnuclear foci [A] AtRRP6L1 binds to RNA in vitro as shown by EMSA. The N-terminal half (N) and C-terminal half (C) of GST-fused AtRRP6L1 proteins were used. [B] AtRRP6L1 associates with scaffold RNAs in vivo as shown by RNA IP using atrrp6l1-2/AtRRP6L1-HA and Col-0 plants. Transcripts were detected by gene-specific RT-PCR after DNase treatment. Total RNA controls, assayed from input samples without IP, show that the RNAs are present in equivalent amounts in Col-0 and atrrp6l1-2/AtRRP6L1-HA plants. No RT controls used SN1C primers. Background signals of Actin2 RNA show that equal RNA amounts from the IP fractions were tested. [C] AtRR6L1 co-localizes with NRPE1 in the perinucleolar dot in 38% of the nuclei that show a colocalization or partial colocalization of the two proteins (upper panel). Partial colocalization was also observed in discrete nucleoplasmic foci in 62% of the nuclei that show a colocalization or partial colocalization of the two proteins (lower panel).. The majority (55%) of 158 nuclei examined in 4 biological replicates showed a colocalization or partial colocalization of the two proteins. NRPE1 (red) is localized by its specific antibody in cells expressing HA-tagged AtRRP6L1 (green). The yellow signals due to the overlap of red and green channels in merged images indicate protein co-localization. DNA (blue) was stained with DAPI.
    Figure Legend Snippet: AtRRP6L1 associates with scaffold RNAs and partially co-localizes with Pol V in subnuclear foci [A] AtRRP6L1 binds to RNA in vitro as shown by EMSA. The N-terminal half (N) and C-terminal half (C) of GST-fused AtRRP6L1 proteins were used. [B] AtRRP6L1 associates with scaffold RNAs in vivo as shown by RNA IP using atrrp6l1-2/AtRRP6L1-HA and Col-0 plants. Transcripts were detected by gene-specific RT-PCR after DNase treatment. Total RNA controls, assayed from input samples without IP, show that the RNAs are present in equivalent amounts in Col-0 and atrrp6l1-2/AtRRP6L1-HA plants. No RT controls used SN1C primers. Background signals of Actin2 RNA show that equal RNA amounts from the IP fractions were tested. [C] AtRR6L1 co-localizes with NRPE1 in the perinucleolar dot in 38% of the nuclei that show a colocalization or partial colocalization of the two proteins (upper panel). Partial colocalization was also observed in discrete nucleoplasmic foci in 62% of the nuclei that show a colocalization or partial colocalization of the two proteins (lower panel).. The majority (55%) of 158 nuclei examined in 4 biological replicates showed a colocalization or partial colocalization of the two proteins. NRPE1 (red) is localized by its specific antibody in cells expressing HA-tagged AtRRP6L1 (green). The yellow signals due to the overlap of red and green channels in merged images indicate protein co-localization. DNA (blue) was stained with DAPI.

    Techniques Used: In Vitro, In Vivo, Reverse Transcription Polymerase Chain Reaction, Expressing, Staining

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    Article Snippet: .. One microgram of RNA from each sample was treated with Turbo DNA-free DNase (Ambion) before RT-PCR, to exclude DNA contamination. .. First strand cDNA was synthesized using SuperScriptIII reverse transcriptase (Invitrogen) and random hexamer primers.

    Concentration Assay:

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    Incubation:

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    Characterization of short SatIII RNAs. ( A ) Short RNAs were prepared from heat-shocked HeLa cells allowed to recover at 37 °C for 42 h. RNAs were digested with <t>DNase</t> I or with RNase A and then analyzed using Northern blotting with the LNA oligo specific for SatIII repeats. ( B ) Short RNAs were prepared from heat-shocked hamster B14-150 cells and from the hamster > human somatic cell hybrid GM-10611A containing human chromosome 9. RNAs were then analyzed using Northern blotting as in panel A. Hybridization to Mir16 was used as a loading control. The histogram shows the quantitation of long SatIII RNAs in unstressed (C) and in heat-shocked GM10611A cells allowed to recover at 37 °C for the indicated time periods. Data are represented as mean fold increases ± standard deviation of three independent experiments. ( C ) Short RNAs were prepared from unstressed (C) and from heat-shocked (1 h at 42 °C) HeLa cells allowed to recover for 30 h at 37 °C in the presence or in the absence of Actinomycin D (5 μg/mL) during the last 6 h of recovery. The same membrane was hybridized with the LNA probe against SatIII RNAs and with a probe against Mir16 (loading control). M: molecular size marker. Total RNAs prepared from the same cells were analyzed using RT-PCR to assess the level of c-myc RNAs to control the efficacy of Act D treatment. RT-PCR analysis of hP0 <t>RNA</t> was used as a loading control. ( D ) Unstressed (37) and heat-shocked HeLa cells were collected after 40 h of recovery at 37 °C and fractionated in nuclear pellet (N-P), nucleoplasm (Nu) and cytoplasm (Cy) fractions. Total and short RNAs were prepared from each fraction. Short RNAs were analyzed using Northern blotting with an LNA probe against SatIII repeats. As a control of loading and fractionation quality, the same filter was hybridized to a probe against Mir16 (mainly cytoplasmic) and U6 RNAs. Total RNAs from nuclear pellet, nucleoplasm and cytoplasm fractions were prepared from heat-shocked HeLa cells allowed to recover for 40 h at 37 °C. RNAs were analyzed using quantitative RT-PCR. The histogram shows the relative abundance of SatIII RNAs in the three factions as determined in three independent experiments. Data are mean ± standard deviation.
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    Characterization of short SatIII RNAs. ( A ) Short RNAs were prepared from heat-shocked HeLa cells allowed to recover at 37 °C for 42 h. RNAs were digested with DNase I or with RNase A and then analyzed using Northern blotting with the LNA oligo specific for SatIII repeats. ( B ) Short RNAs were prepared from heat-shocked hamster B14-150 cells and from the hamster > human somatic cell hybrid GM-10611A containing human chromosome 9. RNAs were then analyzed using Northern blotting as in panel A. Hybridization to Mir16 was used as a loading control. The histogram shows the quantitation of long SatIII RNAs in unstressed (C) and in heat-shocked GM10611A cells allowed to recover at 37 °C for the indicated time periods. Data are represented as mean fold increases ± standard deviation of three independent experiments. ( C ) Short RNAs were prepared from unstressed (C) and from heat-shocked (1 h at 42 °C) HeLa cells allowed to recover for 30 h at 37 °C in the presence or in the absence of Actinomycin D (5 μg/mL) during the last 6 h of recovery. The same membrane was hybridized with the LNA probe against SatIII RNAs and with a probe against Mir16 (loading control). M: molecular size marker. Total RNAs prepared from the same cells were analyzed using RT-PCR to assess the level of c-myc RNAs to control the efficacy of Act D treatment. RT-PCR analysis of hP0 RNA was used as a loading control. ( D ) Unstressed (37) and heat-shocked HeLa cells were collected after 40 h of recovery at 37 °C and fractionated in nuclear pellet (N-P), nucleoplasm (Nu) and cytoplasm (Cy) fractions. Total and short RNAs were prepared from each fraction. Short RNAs were analyzed using Northern blotting with an LNA probe against SatIII repeats. As a control of loading and fractionation quality, the same filter was hybridized to a probe against Mir16 (mainly cytoplasmic) and U6 RNAs. Total RNAs from nuclear pellet, nucleoplasm and cytoplasm fractions were prepared from heat-shocked HeLa cells allowed to recover for 40 h at 37 °C. RNAs were analyzed using quantitative RT-PCR. The histogram shows the relative abundance of SatIII RNAs in the three factions as determined in three independent experiments. Data are mean ± standard deviation.

    Journal: International Journal of Molecular Sciences

    Article Title: Heat Shock Affects Mitotic Segregation of Human Chromosomes Bound to Stress-Induced Satellite III RNAs

    doi: 10.3390/ijms21082812

    Figure Lengend Snippet: Characterization of short SatIII RNAs. ( A ) Short RNAs were prepared from heat-shocked HeLa cells allowed to recover at 37 °C for 42 h. RNAs were digested with DNase I or with RNase A and then analyzed using Northern blotting with the LNA oligo specific for SatIII repeats. ( B ) Short RNAs were prepared from heat-shocked hamster B14-150 cells and from the hamster > human somatic cell hybrid GM-10611A containing human chromosome 9. RNAs were then analyzed using Northern blotting as in panel A. Hybridization to Mir16 was used as a loading control. The histogram shows the quantitation of long SatIII RNAs in unstressed (C) and in heat-shocked GM10611A cells allowed to recover at 37 °C for the indicated time periods. Data are represented as mean fold increases ± standard deviation of three independent experiments. ( C ) Short RNAs were prepared from unstressed (C) and from heat-shocked (1 h at 42 °C) HeLa cells allowed to recover for 30 h at 37 °C in the presence or in the absence of Actinomycin D (5 μg/mL) during the last 6 h of recovery. The same membrane was hybridized with the LNA probe against SatIII RNAs and with a probe against Mir16 (loading control). M: molecular size marker. Total RNAs prepared from the same cells were analyzed using RT-PCR to assess the level of c-myc RNAs to control the efficacy of Act D treatment. RT-PCR analysis of hP0 RNA was used as a loading control. ( D ) Unstressed (37) and heat-shocked HeLa cells were collected after 40 h of recovery at 37 °C and fractionated in nuclear pellet (N-P), nucleoplasm (Nu) and cytoplasm (Cy) fractions. Total and short RNAs were prepared from each fraction. Short RNAs were analyzed using Northern blotting with an LNA probe against SatIII repeats. As a control of loading and fractionation quality, the same filter was hybridized to a probe against Mir16 (mainly cytoplasmic) and U6 RNAs. Total RNAs from nuclear pellet, nucleoplasm and cytoplasm fractions were prepared from heat-shocked HeLa cells allowed to recover for 40 h at 37 °C. RNAs were analyzed using quantitative RT-PCR. The histogram shows the relative abundance of SatIII RNAs in the three factions as determined in three independent experiments. Data are mean ± standard deviation.

    Article Snippet: Subsequently, the RNA was treated with Turbo DNase (Thermo Fisher Scientific) for 30 min at 37 °C, to remove any contaminating DNA.

    Techniques: Northern Blot, Hybridization, Quantitation Assay, Standard Deviation, Marker, Reverse Transcription Polymerase Chain Reaction, Fractionation, Quantitative RT-PCR

    Characterization of anti-dsRNA J2 antibody and mtDNA depletion results in loss of mtdsRNA formation. a , RT–qPCR analysis of L-mRNA expression in encephalomyocarditis virus (EMCV) infected HeLa cells at MOI 1 at the indicated time points after infection. Data are from two independent experiments. b , Confocal microscopy images of uninfected or EMCV-infected HeLa cells at multiplicity of infection (MOI) of 1, 8 h after infection stained with anti-dsRNA (J2) antibody (green) and DAPI (nuclei stained blue). Images are representative of two experiments. Scale bars, 10 μm. c , Immunostaining of dsRNA (green) and DNA (red) in HeLa cells treated with indicated nucleases before staining. Signal from J2 antibody is specific for RNA but not for DNA and is sensitive only to RNase III treatment. Images are representative of three experiments. Scale bars, 10 μm. d , Quantification of fluorescence signal from HeLa cells treated as in c . Data are mean ± s.e.m. from 4,095, 1,755, 4,766 and 5,585 cells for the untreated, RNase T1, RNase III and DNase Turbo groups, respectively. e , Autoradiogram showing substrate specificity of J2 on the basis of immunoprecipitation efficiency for uniformly 32 . f , Chromosome-wise coverage plot of dsRNA-seq reads. Inset, read distribution of dsRNA-seq on the basis of RNA class biotypes. g , Left, dsRNA and DNA staining of HeLa cells transfected with constructs encoding the indicated proteins, the expression of which results in mtDNA depletion. Plasmids encoding mtDNA-depletion factors co-express EGFP from an independent promoter, which enables identification of transfected cells. Mitochondria were stained using anti-OXA1L antibody. Scale bars, 10 μm. Right, quantitative analysis of fluorescence signal from HeLa cells. Data are mean ± s.e.m. from ten cells.

    Journal: Nature

    Article Title: Mitochondrial double-stranded RNA triggers antiviral signalling in humans

    doi: 10.1038/s41586-018-0363-0

    Figure Lengend Snippet: Characterization of anti-dsRNA J2 antibody and mtDNA depletion results in loss of mtdsRNA formation. a , RT–qPCR analysis of L-mRNA expression in encephalomyocarditis virus (EMCV) infected HeLa cells at MOI 1 at the indicated time points after infection. Data are from two independent experiments. b , Confocal microscopy images of uninfected or EMCV-infected HeLa cells at multiplicity of infection (MOI) of 1, 8 h after infection stained with anti-dsRNA (J2) antibody (green) and DAPI (nuclei stained blue). Images are representative of two experiments. Scale bars, 10 μm. c , Immunostaining of dsRNA (green) and DNA (red) in HeLa cells treated with indicated nucleases before staining. Signal from J2 antibody is specific for RNA but not for DNA and is sensitive only to RNase III treatment. Images are representative of three experiments. Scale bars, 10 μm. d , Quantification of fluorescence signal from HeLa cells treated as in c . Data are mean ± s.e.m. from 4,095, 1,755, 4,766 and 5,585 cells for the untreated, RNase T1, RNase III and DNase Turbo groups, respectively. e , Autoradiogram showing substrate specificity of J2 on the basis of immunoprecipitation efficiency for uniformly 32 . f , Chromosome-wise coverage plot of dsRNA-seq reads. Inset, read distribution of dsRNA-seq on the basis of RNA class biotypes. g , Left, dsRNA and DNA staining of HeLa cells transfected with constructs encoding the indicated proteins, the expression of which results in mtDNA depletion. Plasmids encoding mtDNA-depletion factors co-express EGFP from an independent promoter, which enables identification of transfected cells. Mitochondria were stained using anti-OXA1L antibody. Scale bars, 10 μm. Right, quantitative analysis of fluorescence signal from HeLa cells. Data are mean ± s.e.m. from ten cells.

    Article Snippet: Following enzymes were used: RNase T1 (EN0541, Thermo Fisher Scientific, concentration 100 U ml−1 ), RNase III (M0245S, NEB, concentration 40 U ml−1 ), TURBO DNase (AM2238, Thermo Fisher Scientific, concentration 40 U ml−1 ).

    Techniques: Quantitative RT-PCR, Expressing, Infection, Confocal Microscopy, Staining, Immunostaining, Fluorescence, Immunoprecipitation, Transfection, Construct

    Workflow for bacterial RNase H based rRNA depletion. a) Probes used for depletion can be either designed and chemically synthesized from known rRNA sequences (oligo-based) or generated by PCR from genomic DNA with 5’-phosphorylated forward primers and subsequent lambda exonuclease digestion (amplicon-based). b) Probes are then hybridized to total RNA and the rRNA bound by the ssDNA probes is degraded by RNase H. Finally, all remaining probes are degraded by DNase I or removed by SPRI beads-based size selection, resulting in enriched mRNAs.

    Journal: bioRxiv

    Article Title: Scalable and cost-effective ribonuclease-based rRNA depletion for transcriptomics

    doi: 10.1101/645895

    Figure Lengend Snippet: Workflow for bacterial RNase H based rRNA depletion. a) Probes used for depletion can be either designed and chemically synthesized from known rRNA sequences (oligo-based) or generated by PCR from genomic DNA with 5’-phosphorylated forward primers and subsequent lambda exonuclease digestion (amplicon-based). b) Probes are then hybridized to total RNA and the rRNA bound by the ssDNA probes is degraded by RNase H. Finally, all remaining probes are degraded by DNase I or removed by SPRI beads-based size selection, resulting in enriched mRNAs.

    Article Snippet: After RNase H digestion, 30μL DNase I reaction mix (6U TURBO DNase [ThermoFisher AM2239] and 5μL 10X TURBO DNase Buffer) was added, and the mixture was incubated at 37°C for 30 minutes to degrade ssDNA probes.

    Techniques: Synthesized, Generated, Polymerase Chain Reaction, Amplification, Selection