bisulfite treated dna  (Thermo Fisher)


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    Structured Review

    Thermo Fisher bisulfite treated dna
    Discrimination of <t>DNA</t> hypermethylation at maternal and paternal GSTP1 alleles using a <t>PCR</t> strategy. DNA from matched normal (normal) and neoplastic (tumor) prostate tissues was left untreated (U; lanes 1 , 4 , 7 , and 10 ), or was treated with Hpa II (H; lanes 2 , 5 , 8 , and 11 ), which cuts CCGG but not C 5-m CGG, or treated with Msp I (M; lanes 3 , 6 , 9 , and 12 ), which cuts CCGG and C 5-m CGG, before being subjected to PCR amplification using oligonucleotide primers targeting a polymorphic [ATAAA] n repeat sequence near the GSTP1 regulatory region. For primer set B, the amplification of polymorphic GSTP1 promoter sequences after Hpa II digestion, but not after Msp I digestion, indicated the presence of CpG dinucleotide methylation at the Hpa II/ Msp I sites in the DNA analyzed.
    Bisulfite Treated Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 39794 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "GSTP1 CpG Island Hypermethylation Is Responsible for the Absence of GSTP1 Expression in Human Prostate Cancer Cells "

    Article Title: GSTP1 CpG Island Hypermethylation Is Responsible for the Absence of GSTP1 Expression in Human Prostate Cancer Cells

    Journal: The American Journal of Pathology

    doi:

    Discrimination of DNA hypermethylation at maternal and paternal GSTP1 alleles using a PCR strategy. DNA from matched normal (normal) and neoplastic (tumor) prostate tissues was left untreated (U; lanes 1 , 4 , 7 , and 10 ), or was treated with Hpa II (H; lanes 2 , 5 , 8 , and 11 ), which cuts CCGG but not C 5-m CGG, or treated with Msp I (M; lanes 3 , 6 , 9 , and 12 ), which cuts CCGG and C 5-m CGG, before being subjected to PCR amplification using oligonucleotide primers targeting a polymorphic [ATAAA] n repeat sequence near the GSTP1 regulatory region. For primer set B, the amplification of polymorphic GSTP1 promoter sequences after Hpa II digestion, but not after Msp I digestion, indicated the presence of CpG dinucleotide methylation at the Hpa II/ Msp I sites in the DNA analyzed.
    Figure Legend Snippet: Discrimination of DNA hypermethylation at maternal and paternal GSTP1 alleles using a PCR strategy. DNA from matched normal (normal) and neoplastic (tumor) prostate tissues was left untreated (U; lanes 1 , 4 , 7 , and 10 ), or was treated with Hpa II (H; lanes 2 , 5 , 8 , and 11 ), which cuts CCGG but not C 5-m CGG, or treated with Msp I (M; lanes 3 , 6 , 9 , and 12 ), which cuts CCGG and C 5-m CGG, before being subjected to PCR amplification using oligonucleotide primers targeting a polymorphic [ATAAA] n repeat sequence near the GSTP1 regulatory region. For primer set B, the amplification of polymorphic GSTP1 promoter sequences after Hpa II digestion, but not after Msp I digestion, indicated the presence of CpG dinucleotide methylation at the Hpa II/ Msp I sites in the DNA analyzed.

    Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing, Methylation

    2) Product Images from "Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gni064

    Agarose gel electrophoresis of RDA products ( A ) and its hybridized autoradiogram ( B ). In vitro transcribed RNA and total cellular RNA were mixed as described in the legend to Figure 2 . Double-stranded cDNAs were synthesized and subjected to RDA as described in Materials and Methods. One-twentieth of the volume of the amplified products was separated on 3% agarose gel and stained with ethidium bromide ( A ), blotted onto a nylon membrane and hybridized with 32 P-labelled pCIneo ( B ). Positions and sizes (bp) of markers are present on the left.
    Figure Legend Snippet: Agarose gel electrophoresis of RDA products ( A ) and its hybridized autoradiogram ( B ). In vitro transcribed RNA and total cellular RNA were mixed as described in the legend to Figure 2 . Double-stranded cDNAs were synthesized and subjected to RDA as described in Materials and Methods. One-twentieth of the volume of the amplified products was separated on 3% agarose gel and stained with ethidium bromide ( A ), blotted onto a nylon membrane and hybridized with 32 P-labelled pCIneo ( B ). Positions and sizes (bp) of markers are present on the left.

    Techniques Used: Agarose Gel Electrophoresis, In Vitro, Synthesized, Amplification, Staining

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

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

    Agarose gel electrophoresis of RDA products with PCR products used for probes for hybridization ( A ) and a hybridized fluorogram ( B ). RNA was extracted from SARS-CoV-infected cells and subjected to RDA according to the method described in Materials and Methods. Mock-infected cells were used for the synthesis of driver amplicons for RDA. One-twentieth of the volume of the amplified products was separated on 3% agarose gels and blotted on a Nylon membrane. The membrane was then cut into slits that contained the lane showing the presence of DNA. On the other hand, the PCR fragments predicted to be amplified in the RDA reaction were amplified and subsequently ascertained by agarose gel electrophoresis (A). The amplified genomic fragments of SARS-CoV were Dig-labelled and used as probes for hybridization to each slit of the Nylon membrane containing the RDA product. Hybridization was performed in separate hybridization bags. After washing with 1× SSC and 0.1% SDS solution, the hybridized probes were detected on a fluorogram (B). Positions and sizes (bp) of markers are present on the left.
    Figure Legend Snippet: Agarose gel electrophoresis of RDA products with PCR products used for probes for hybridization ( A ) and a hybridized fluorogram ( B ). RNA was extracted from SARS-CoV-infected cells and subjected to RDA according to the method described in Materials and Methods. Mock-infected cells were used for the synthesis of driver amplicons for RDA. One-twentieth of the volume of the amplified products was separated on 3% agarose gels and blotted on a Nylon membrane. The membrane was then cut into slits that contained the lane showing the presence of DNA. On the other hand, the PCR fragments predicted to be amplified in the RDA reaction were amplified and subsequently ascertained by agarose gel electrophoresis (A). The amplified genomic fragments of SARS-CoV were Dig-labelled and used as probes for hybridization to each slit of the Nylon membrane containing the RDA product. Hybridization was performed in separate hybridization bags. After washing with 1× SSC and 0.1% SDS solution, the hybridized probes were detected on a fluorogram (B). Positions and sizes (bp) of markers are present on the left.

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Hybridization, Infection, Amplification

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

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

    3) Product Images from "Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gni064

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

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

    4) Product Images from "Calcium/calmodulin‐dependent kinase 2 mediates Epac‐induced spontaneous transient outward currents in rat vascular smooth muscle"

    Article Title: Calcium/calmodulin‐dependent kinase 2 mediates Epac‐induced spontaneous transient outward currents in rat vascular smooth muscle

    Journal: The Journal of Physiology

    doi: 10.1113/JP274754

    Selective activation of Epac causes autophosphorylation of specific CaMKII isoforms in rat mesenteric artery A , primers designed to amplify γ and δ isoforms ( Aa ) and γ and δ CaMKII splice variants ( Ab ) were used to probe rat mesenteric artery cDNA. PCR products were separated on a 3% agarose gel. M indicates DNA markers. Ba , Incubation of first‐order branches of rat mesenteric artery (left) or renal artery (right) with 8‐pCPT‐AM (10 μ m ) induces phosphorylation of specific CaMKII isoforms at Thr 286/7 (pCaMKII (T 286/7 ); upper). Arteries were incubated with vehicle control (DMSO) or 8‐pCPT‐AM (10 μ m ) for 5 min prior to homogenization. As a positive control, arteries were incubated in the Ca 2+ ionophore, A23187, (12.5 μ m ) for 15 min prior to homogenization. Proteins within the arterial homogenates were separated on 10% polyacrylamide‐Tris gels and immunoblotted with an antibody directed against phospho‐CaMKII Thr 286/7 . The membrane was then stripped and re‐blotted with pan‐specific CaMKII antibodies (total CaMKII; lower; blots shown representative of three similar experiments). Film exposure time for all blots ∼5 min ( Bb ) Densitometry analysis of three similar blots. C , immunoblots of lysates from wild‐type mice mesenteric ( Ca ) or renal artery ( Cb ) using CaMKII antibodies showed a pattern of immunoreactive bands (arrowheads) similar to that of rat arterial lysates. In arterial lysates obtained from CaMKIIδ knockout animals, the lowest molecular weight band was absent. By contrast, in CaMKIIγ knockout lysates, the lower band remained but the higher molecular weight bands were missing. Film exposure time for all blots ∼10 min.
    Figure Legend Snippet: Selective activation of Epac causes autophosphorylation of specific CaMKII isoforms in rat mesenteric artery A , primers designed to amplify γ and δ isoforms ( Aa ) and γ and δ CaMKII splice variants ( Ab ) were used to probe rat mesenteric artery cDNA. PCR products were separated on a 3% agarose gel. M indicates DNA markers. Ba , Incubation of first‐order branches of rat mesenteric artery (left) or renal artery (right) with 8‐pCPT‐AM (10 μ m ) induces phosphorylation of specific CaMKII isoforms at Thr 286/7 (pCaMKII (T 286/7 ); upper). Arteries were incubated with vehicle control (DMSO) or 8‐pCPT‐AM (10 μ m ) for 5 min prior to homogenization. As a positive control, arteries were incubated in the Ca 2+ ionophore, A23187, (12.5 μ m ) for 15 min prior to homogenization. Proteins within the arterial homogenates were separated on 10% polyacrylamide‐Tris gels and immunoblotted with an antibody directed against phospho‐CaMKII Thr 286/7 . The membrane was then stripped and re‐blotted with pan‐specific CaMKII antibodies (total CaMKII; lower; blots shown representative of three similar experiments). Film exposure time for all blots ∼5 min ( Bb ) Densitometry analysis of three similar blots. C , immunoblots of lysates from wild‐type mice mesenteric ( Ca ) or renal artery ( Cb ) using CaMKII antibodies showed a pattern of immunoreactive bands (arrowheads) similar to that of rat arterial lysates. In arterial lysates obtained from CaMKIIδ knockout animals, the lowest molecular weight band was absent. By contrast, in CaMKIIγ knockout lysates, the lower band remained but the higher molecular weight bands were missing. Film exposure time for all blots ∼10 min.

    Techniques Used: Activation Assay, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Incubation, Homogenization, Positive Control, Western Blot, Mouse Assay, Knock-Out, Molecular Weight

    5) Product Images from "Differentially expressed non-coding RNAs induced by transmissible gastroenteritis virus potentially regulate inflammation and NF-κB pathway in porcine intestinal epithelial cell line"

    Article Title: Differentially expressed non-coding RNAs induced by transmissible gastroenteritis virus potentially regulate inflammation and NF-κB pathway in porcine intestinal epithelial cell line

    Journal: BMC Genomics

    doi: 10.1186/s12864-018-5128-5

    qRT-PCR validation of ncRNAs and mRNAs. a Both divergent primers ( ) and convergent primers ( ) were designed to detect the circular and linear form. The divergent primers were used to amplify circRNAs using cDNA but not gDNA as template. b The relative levels of differentially expressed mRNAs, miRNAs, and circRNAs. The fold change was determined normalized to U6 using the 2-ΔΔCt method. The data from real-time PCR are shown as mean ± standard deviation (S.D.)
    Figure Legend Snippet: qRT-PCR validation of ncRNAs and mRNAs. a Both divergent primers ( ) and convergent primers ( ) were designed to detect the circular and linear form. The divergent primers were used to amplify circRNAs using cDNA but not gDNA as template. b The relative levels of differentially expressed mRNAs, miRNAs, and circRNAs. The fold change was determined normalized to U6 using the 2-ΔΔCt method. The data from real-time PCR are shown as mean ± standard deviation (S.D.)

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

    KEGG analysis of differentially expressed mRNAs and ncRNAs. a KEGG enrichment analysis of differentially expressed mRNAs. b Target genes of differentially expressed miRNAs. c Source genes of differentially expressed circRNAs. The degree of KEGG enrichment is assessed by the Rich Factor, P -value, and Gene Number. The closer the P -value is to zero, the greater the Rich factor is. The greater the Gene Number is, the more the enrichment is significant
    Figure Legend Snippet: KEGG analysis of differentially expressed mRNAs and ncRNAs. a KEGG enrichment analysis of differentially expressed mRNAs. b Target genes of differentially expressed miRNAs. c Source genes of differentially expressed circRNAs. The degree of KEGG enrichment is assessed by the Rich Factor, P -value, and Gene Number. The closer the P -value is to zero, the greater the Rich factor is. The greater the Gene Number is, the more the enrichment is significant

    Techniques Used:

    6) Product Images from "Differentially expressed non-coding RNAs induced by transmissible gastroenteritis virus potentially regulate inflammation and NF-κB pathway in porcine intestinal epithelial cell line"

    Article Title: Differentially expressed non-coding RNAs induced by transmissible gastroenteritis virus potentially regulate inflammation and NF-κB pathway in porcine intestinal epithelial cell line

    Journal: BMC Genomics

    doi: 10.1186/s12864-018-5128-5

    qRT-PCR validation of ncRNAs and mRNAs. a Both divergent primers ( ) and convergent primers ( ) were designed to detect the circular and linear form. The divergent primers were used to amplify circRNAs using cDNA but not gDNA as template. b The relative levels of differentially expressed mRNAs, miRNAs, and circRNAs. The fold change was determined normalized to U6 using the 2-ΔΔCt method. The data from real-time PCR are shown as mean ± standard deviation (S.D.)
    Figure Legend Snippet: qRT-PCR validation of ncRNAs and mRNAs. a Both divergent primers ( ) and convergent primers ( ) were designed to detect the circular and linear form. The divergent primers were used to amplify circRNAs using cDNA but not gDNA as template. b The relative levels of differentially expressed mRNAs, miRNAs, and circRNAs. The fold change was determined normalized to U6 using the 2-ΔΔCt method. The data from real-time PCR are shown as mean ± standard deviation (S.D.)

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

    KEGG analysis of differentially expressed mRNAs and ncRNAs. a KEGG enrichment analysis of differentially expressed mRNAs. b Target genes of differentially expressed miRNAs. c Source genes of differentially expressed circRNAs. The degree of KEGG enrichment is assessed by the Rich Factor, P -value, and Gene Number. The closer the P -value is to zero, the greater the Rich factor is. The greater the Gene Number is, the more the enrichment is significant
    Figure Legend Snippet: KEGG analysis of differentially expressed mRNAs and ncRNAs. a KEGG enrichment analysis of differentially expressed mRNAs. b Target genes of differentially expressed miRNAs. c Source genes of differentially expressed circRNAs. The degree of KEGG enrichment is assessed by the Rich Factor, P -value, and Gene Number. The closer the P -value is to zero, the greater the Rich factor is. The greater the Gene Number is, the more the enrichment is significant

    Techniques Used:

    7) Product Images from "An RNA-Seq Strategy to Detect the Complete Coding and Non-Coding Transcriptome Including Full-Length Imprinted Macro ncRNAs"

    Article Title: An RNA-Seq Strategy to Detect the Complete Coding and Non-Coding Transcriptome Including Full-Length Imprinted Macro ncRNAs

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0027288

    Optimisation and reproducibility of ribo-depleted RNA-Seq. ( A ) Distribution of different sequence tag types from RNA prepared from CCE differentiated ES cells subject to ribosomal RNA depletion using either the RiboMinus or the Ribo-Zero Kit and fragmented either by RNA-hydrolysis or by cDNA-shearing. Sequencing was performed in two different sequencing locations (Vienna-IMP, Nijmegen, RiboMinus) or in one sequencing location (Vienna-CeMM, Ribo-Zero). The percentage of tags in each category is shown for two technical sequencing replicates (CCE1, CCE2) of material prepared by RiboMinus and cDNA-shearing (sheared, lanes nr. 1,2,5,6) or RiboMinus and RNA-hydrolysis (hydrolysed, lanes nr. 3,4,7,8), for the combination of three technical sequencing replicates of RiboMinus and RNA-hydrolysis (lane nr. 9) and for one sequencing of Ribo-Zero and RNA-hydrolysis (lane nr. 10). green: unique tags matching only once in the genome; blue: rRNA+mitoRNA tags matching to ribosomal (RiboMinus and Ribo-Zero) or mitochondrial (RiboMinus) RNAs; red: repeat tags matching more than once in the genome; purple: nomatch tags do not match to the genome. ( B ) Scatter plots comparing the RPKM ( R eads P er K ilobase of exon model per M illion of reads) transcription levels of RefSeq protein-coding genes between combined tags from RiboMinus and RNA-hydrolysis (H) and RiboMinus and cDNA-shearing (S) from CCE within the same location: Vienna-IMP (left) and Nijmegen (right). ( C ) Scatter plots as in B comparing RPKM transcript levels of all combined tags from the two sequencing locations (Vienna-IMP and Nijmegen, left) or between the combined RiboMinus data and the Ribo-Zero data (right). R: Pearson's correlation, note that a perfect correlation is R = 1.
    Figure Legend Snippet: Optimisation and reproducibility of ribo-depleted RNA-Seq. ( A ) Distribution of different sequence tag types from RNA prepared from CCE differentiated ES cells subject to ribosomal RNA depletion using either the RiboMinus or the Ribo-Zero Kit and fragmented either by RNA-hydrolysis or by cDNA-shearing. Sequencing was performed in two different sequencing locations (Vienna-IMP, Nijmegen, RiboMinus) or in one sequencing location (Vienna-CeMM, Ribo-Zero). The percentage of tags in each category is shown for two technical sequencing replicates (CCE1, CCE2) of material prepared by RiboMinus and cDNA-shearing (sheared, lanes nr. 1,2,5,6) or RiboMinus and RNA-hydrolysis (hydrolysed, lanes nr. 3,4,7,8), for the combination of three technical sequencing replicates of RiboMinus and RNA-hydrolysis (lane nr. 9) and for one sequencing of Ribo-Zero and RNA-hydrolysis (lane nr. 10). green: unique tags matching only once in the genome; blue: rRNA+mitoRNA tags matching to ribosomal (RiboMinus and Ribo-Zero) or mitochondrial (RiboMinus) RNAs; red: repeat tags matching more than once in the genome; purple: nomatch tags do not match to the genome. ( B ) Scatter plots comparing the RPKM ( R eads P er K ilobase of exon model per M illion of reads) transcription levels of RefSeq protein-coding genes between combined tags from RiboMinus and RNA-hydrolysis (H) and RiboMinus and cDNA-shearing (S) from CCE within the same location: Vienna-IMP (left) and Nijmegen (right). ( C ) Scatter plots as in B comparing RPKM transcript levels of all combined tags from the two sequencing locations (Vienna-IMP and Nijmegen, left) or between the combined RiboMinus data and the Ribo-Zero data (right). R: Pearson's correlation, note that a perfect correlation is R = 1.

    Techniques Used: RNA Sequencing Assay, Sequencing

    The template preparation protocol determines the comparability of ribo-depleted RNA-Seq to polyA RNA-Seq. The cDNA size distribution of genes showing more than 8× expression difference ( Figure S2 ), in the comparison of ( A ) FH RiboMinus - FH-RiboZero (left) and CCE RiboMinus - CCE Ribo-Zero (right). ( B ) as in A for the comparisons of CCE RiboMinus-Cloonan et al. EB (left), FH RiboMinus-Cui et al. adult mouse brain polyA (middle) and FH RiboMinus-Mortazavi et al. adult mouse brain polyA (right). ( C ) as in A for the comparisons of CCE Ribo-Zero-Cloonan et al. EB (left), FH Ribo-Zero-Cui et al. adult mouse brain polyA (middle) and FH Ribo-Zero-Mortazavi et al. adult mouse brain polyA (right). For Cloonan et al. EB both the gene expression data from the published alignment (shown in B, C, see Materials and methods ) and from an alignment done with the pipeline used here (data not shown) were used and produced the same highly significant differences. Two different size classes are shown with different bin sizes (0–2 kb, 100 bp bins and > 2 kb, 500 bp bins). Genes bigger than 11.5 kb are grouped in the last bin (arrow).
    Figure Legend Snippet: The template preparation protocol determines the comparability of ribo-depleted RNA-Seq to polyA RNA-Seq. The cDNA size distribution of genes showing more than 8× expression difference ( Figure S2 ), in the comparison of ( A ) FH RiboMinus - FH-RiboZero (left) and CCE RiboMinus - CCE Ribo-Zero (right). ( B ) as in A for the comparisons of CCE RiboMinus-Cloonan et al. EB (left), FH RiboMinus-Cui et al. adult mouse brain polyA (middle) and FH RiboMinus-Mortazavi et al. adult mouse brain polyA (right). ( C ) as in A for the comparisons of CCE Ribo-Zero-Cloonan et al. EB (left), FH Ribo-Zero-Cui et al. adult mouse brain polyA (middle) and FH Ribo-Zero-Mortazavi et al. adult mouse brain polyA (right). For Cloonan et al. EB both the gene expression data from the published alignment (shown in B, C, see Materials and methods ) and from an alignment done with the pipeline used here (data not shown) were used and produced the same highly significant differences. Two different size classes are shown with different bin sizes (0–2 kb, 100 bp bins and > 2 kb, 500 bp bins). Genes bigger than 11.5 kb are grouped in the last bin (arrow).

    Techniques Used: RNA Sequencing Assay, Expressing, Produced

    Tag coverage of genes differs between fragmentation methods and ribosomal RNA depletion methods. The coverage of genes with sequence tags is shown as the normalized number of tags at relative positions throughout the gene length. UTRs and coding exons were analysed separately and are plotted as 10 bins for 5′UTRs and 3′UTRs and 100bins for the coding exons (separated by vertical dotted line). ( A ) Comparison of the coverage in the RiboMinus dataset for the combined tags of CCE and FH from RNA-hydrolysis (black) and cDNA-shearing (grey). ( B ) Comparison of the coverage in the RNA-hydrolysis RiboMinus dataset (dotted line, same as in A) and in Ribo-Zero dataset plotted separately for CCE (black) and FH (grey). For all analyses the genes were separated into three groups according to their cDNA length (coding exons and 5′ and 3′ UTRs) as indicated.
    Figure Legend Snippet: Tag coverage of genes differs between fragmentation methods and ribosomal RNA depletion methods. The coverage of genes with sequence tags is shown as the normalized number of tags at relative positions throughout the gene length. UTRs and coding exons were analysed separately and are plotted as 10 bins for 5′UTRs and 3′UTRs and 100bins for the coding exons (separated by vertical dotted line). ( A ) Comparison of the coverage in the RiboMinus dataset for the combined tags of CCE and FH from RNA-hydrolysis (black) and cDNA-shearing (grey). ( B ) Comparison of the coverage in the RNA-hydrolysis RiboMinus dataset (dotted line, same as in A) and in Ribo-Zero dataset plotted separately for CCE (black) and FH (grey). For all analyses the genes were separated into three groups according to their cDNA length (coding exons and 5′ and 3′ UTRs) as indicated.

    Techniques Used: Sequencing

    8) Product Images from "Multi-tissue transcriptomics of the black widow spider reveals expansions, co-options, and functional processes of the silk gland gene toolkit"

    Article Title: Multi-tissue transcriptomics of the black widow spider reveals expansions, co-options, and functional processes of the silk gland gene toolkit

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-15-365

    Flowchart of the de novo transcript assembly process. The Western black widow transcriptome was assembled in three major steps. First, high-quality 75 or 100 base paired-end cDNA sequence reads were generated for each of three tissues (dark blue boxes). Second, transcripts were de novo assembled for each tissue separately (light blue boxes). Finally, the high quality non-redundant transcriptome was generated (yellow boxes). Relevant programs are shown parenthetically in the boxes. The number of sequence reads, Trinity de novo assembled sequences, and final assembled transcripts generated in each step are shown in bold below the arrows.
    Figure Legend Snippet: Flowchart of the de novo transcript assembly process. The Western black widow transcriptome was assembled in three major steps. First, high-quality 75 or 100 base paired-end cDNA sequence reads were generated for each of three tissues (dark blue boxes). Second, transcripts were de novo assembled for each tissue separately (light blue boxes). Finally, the high quality non-redundant transcriptome was generated (yellow boxes). Relevant programs are shown parenthetically in the boxes. The number of sequence reads, Trinity de novo assembled sequences, and final assembled transcripts generated in each step are shown in bold below the arrows.

    Techniques Used: Western Blot, Sequencing, Generated

    9) Product Images from "Species-independent detection of RNA virus by representational difference analysis using non-ribosomal hexanucleotides for reverse transcription"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gni064

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

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

    10) Product Images from "Differential ratio amplicons (Ramp) for the evaluation of RNA integrity extracted from complex environmental samples"

    Article Title: Differential ratio amplicons (Ramp) for the evaluation of RNA integrity extracted from complex environmental samples

    Journal: Environmental Microbiology

    doi: 10.1111/1462-2920.14516

    Effect of UV degradation on RNA integrity measured via the RIN (A), with RT‐Q‐PCR (B) and RIN versus R amp (C). For RIN, RNA integrity visualized in virtual gels (A; left) and electropherogram (A; right) are displayed against incubation period under UV. RNA ladder shows size in nucleotides (nt). B. Effect of degradation on transcript quantification; Amp 1–3: average Ct ( n = 3) of one of the three possible glnA amplicons; amoA : average amoA Ct ( n = 3) of the Bacterial amoA transcript; 16S rRNA : average 16S rRNA Ct ( n = 3) of the bacterial 16S rRNA transcript. Effect of RNA degradation on R amp index is presented in figure C; for comparison, RIN values were also plotted. Greek Letters indicate the result of TukeyHSD tests (points with different letters had values significantly different from each other using 0.05 as threshold for the p value).
    Figure Legend Snippet: Effect of UV degradation on RNA integrity measured via the RIN (A), with RT‐Q‐PCR (B) and RIN versus R amp (C). For RIN, RNA integrity visualized in virtual gels (A; left) and electropherogram (A; right) are displayed against incubation period under UV. RNA ladder shows size in nucleotides (nt). B. Effect of degradation on transcript quantification; Amp 1–3: average Ct ( n = 3) of one of the three possible glnA amplicons; amoA : average amoA Ct ( n = 3) of the Bacterial amoA transcript; 16S rRNA : average 16S rRNA Ct ( n = 3) of the bacterial 16S rRNA transcript. Effect of RNA degradation on R amp index is presented in figure C; for comparison, RIN values were also plotted. Greek Letters indicate the result of TukeyHSD tests (points with different letters had values significantly different from each other using 0.05 as threshold for the p value).

    Techniques Used: Polymerase Chain Reaction, Incubation

    Effect of heat degradation on RNA integrity measured via the RIN (A), with RT‐Q‐PCR (B) and RIN versus R amp (C). For RIN, RNA integrity visualized in virtual gels (A; left) and electropherogram (A; right) are displayed against incubation period at 90°C. RNA ladder shows size in nucleotides (nt). B. Effect of degradation on transcript quantification; Amp 1–3: average Ct ( n = 3) of one of the three possible glnA amplicons; amoA : average amoA Ct ( n = 3) of the Bacterial amoA transcript; 16S rRNA : average 16S rRNA Ct ( n = 3) of the bacterial 16S rRNA transcript. Effect of RNA degradation on R amp index is presented in figure C; for comparison, RIN values were also plotted. Greek Letters indicate the result of TukeyHSD tests (points with different letters had values significantly different from each other using 0.05 as threshold for the p value).
    Figure Legend Snippet: Effect of heat degradation on RNA integrity measured via the RIN (A), with RT‐Q‐PCR (B) and RIN versus R amp (C). For RIN, RNA integrity visualized in virtual gels (A; left) and electropherogram (A; right) are displayed against incubation period at 90°C. RNA ladder shows size in nucleotides (nt). B. Effect of degradation on transcript quantification; Amp 1–3: average Ct ( n = 3) of one of the three possible glnA amplicons; amoA : average amoA Ct ( n = 3) of the Bacterial amoA transcript; 16S rRNA : average 16S rRNA Ct ( n = 3) of the bacterial 16S rRNA transcript. Effect of RNA degradation on R amp index is presented in figure C; for comparison, RIN values were also plotted. Greek Letters indicate the result of TukeyHSD tests (points with different letters had values significantly different from each other using 0.05 as threshold for the p value).

    Techniques Used: Polymerase Chain Reaction, Incubation

    Effect of RNase I degradation on RNA integrity measured via the RIN (A), with RT‐Q‐PCR (B) and RIN versus R amp (C). For RIN, RNA integrity visualized in virtual gels (A; left) and electropherogram (A; right) are displayed against incubation period with RNase I . RNA ladder shows size in nucleotides (nt). B. Effect of degradation on transcript quantification; Amp 1–3: average Ct ( n = 3) of one of the three possible glnA amplicons; amoA : average amoA Ct ( n = 3) of the Bacterial amoA transcript; 16S rRNA : average 16S rRNA Ct ( n = 3) of the bacterial 16S rRNA transcript. Effect of RNA degradation on R amp index is presented in figure C; for comparison, RIN values were also plotted. Greek letters indicate the result of TukeyHSD tests (points with different letters had values significantly different from each other using 0.05 as threshold for the p value).
    Figure Legend Snippet: Effect of RNase I degradation on RNA integrity measured via the RIN (A), with RT‐Q‐PCR (B) and RIN versus R amp (C). For RIN, RNA integrity visualized in virtual gels (A; left) and electropherogram (A; right) are displayed against incubation period with RNase I . RNA ladder shows size in nucleotides (nt). B. Effect of degradation on transcript quantification; Amp 1–3: average Ct ( n = 3) of one of the three possible glnA amplicons; amoA : average amoA Ct ( n = 3) of the Bacterial amoA transcript; 16S rRNA : average 16S rRNA Ct ( n = 3) of the bacterial 16S rRNA transcript. Effect of RNA degradation on R amp index is presented in figure C; for comparison, RIN values were also plotted. Greek letters indicate the result of TukeyHSD tests (points with different letters had values significantly different from each other using 0.05 as threshold for the p value).

    Techniques Used: Polymerase Chain Reaction, Incubation

    Effect of RNase I treatment on 16S rRNA transcript composition. Bar charts (A) represent changes in community composition of the 50 most abundant taxa. Scatterplots (B) represent log2 changes of individual taxa along the degradation gradient relative to control experiments (no treatment control (NT) or buffer only control (0U RNase I μl −1 )) as indicated by black arrows. Taxa with a significant difference ( p value
    Figure Legend Snippet: Effect of RNase I treatment on 16S rRNA transcript composition. Bar charts (A) represent changes in community composition of the 50 most abundant taxa. Scatterplots (B) represent log2 changes of individual taxa along the degradation gradient relative to control experiments (no treatment control (NT) or buffer only control (0U RNase I μl −1 )) as indicated by black arrows. Taxa with a significant difference ( p value

    Techniques Used:

    11) Product Images from "Transcriptional read-through of the long non-coding RNA SVALKA governs plant cold acclimation"

    Article Title: Transcriptional read-through of the long non-coding RNA SVALKA governs plant cold acclimation

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07010-6

    SVALKA transcription mediates transcription activity antisense to CBF1 . a Representative Northern blot of a cold exposure time series of Col-0 (WT) and hen2-2 . The probe used for asCBF1 is shown in b . Blots were repeated with three biological replicates with similar results. UBI is used as loading control. Uncropped blots can be found in the Source Data file. b Graphical representation of the CBF1-SVK genomic region. The probe for asCBF1 is shown in red. RT was done with an oligo-linked primer to ensure strand-specificity and generated cDNA was used in c . c RT-qPCR of asCBF1 after cold exposure in hen2-2 and the double mutants, hen2-2uns-1, and hen2-2svk-1 . The RT-primer and the qPCR primers are shown in the graphical representation above the graph. Bars represent mean (white: 4 h 4 °C, black: 8 h 4 °C, ±SEM) from three biological replicates (rings). The relative level of asCBF1 was normalized to the level in hen2-2 after 4 h of 4 °C. Statistically significant differences were determined with Student’s t -test (*** p
    Figure Legend Snippet: SVALKA transcription mediates transcription activity antisense to CBF1 . a Representative Northern blot of a cold exposure time series of Col-0 (WT) and hen2-2 . The probe used for asCBF1 is shown in b . Blots were repeated with three biological replicates with similar results. UBI is used as loading control. Uncropped blots can be found in the Source Data file. b Graphical representation of the CBF1-SVK genomic region. The probe for asCBF1 is shown in red. RT was done with an oligo-linked primer to ensure strand-specificity and generated cDNA was used in c . c RT-qPCR of asCBF1 after cold exposure in hen2-2 and the double mutants, hen2-2uns-1, and hen2-2svk-1 . The RT-primer and the qPCR primers are shown in the graphical representation above the graph. Bars represent mean (white: 4 h 4 °C, black: 8 h 4 °C, ±SEM) from three biological replicates (rings). The relative level of asCBF1 was normalized to the level in hen2-2 after 4 h of 4 °C. Statistically significant differences were determined with Student’s t -test (*** p

    Techniques Used: Activity Assay, Northern Blot, Generated, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    12) Product Images from "Variable Suites of Non-effector Genes Are Co-regulated in the Type III Secretion Virulence Regulon across the Pseudomonas syringae Phylogeny"

    Article Title: Variable Suites of Non-effector Genes Are Co-regulated in the Type III Secretion Virulence Regulon across the Pseudomonas syringae Phylogeny

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003807

    A novel HrpL-regulated virulence operon linked to avrD in Pph 1448A identified by RNA-seq analysis. ( A ) Color coded genomic context of avrD and downstream genes from PSPPH_A0106 to A0112 . Grey arrows represent transposases. White arrows represent additional ORFs that are not necessary related. ( B ) Graphical representation of avrD operon according to IMG/ER conserved neighborhood region search with the 45 Pseudomonas strains presented in Figure 5 and S5 . Grey, white, black, and colored arrows as in A. Brackets represent breaks and end of contigs or scaffolds. Not to scale. ( C ) PCR was performed using primers spanning intragenic regions between PSPPH_A0109 and A0110 (top panels), or PSPPH_A0112 and avrD (middle panels) on cDNA prepared from Pph 1448A ( Pph ) or an isogenic Pph 1448A Δ hrpL mutant (Δ hrpL ) grown in MM media. Total RNA was subject to reverse transcriptase (+RT), or without reverse transcriptase (−RT, as negative control). gDNA, DW indicate respectively that genomic DNA or distilled water were used as template for positive and negative controls of amplification. Equal loading was controlled by monitoring gap-1 amplification across samples (bottom panels). ( D ) Two week old bean cv. Tendergreen beans were dip inoculated with wild type Pph 1448A (Pph) , two independent clean deletion avrD mutants (Δa vrD #1, Δa vrD #2), and two independent clean deletion PSPPH_A0107 mutants (Δ PSPPH_A0107 #1, Δ PSPPH_A0107 #2) at OD 600 = 0.001. Bacterial growth of each strain was determined after 3.5 dpi. Letters represent significant differences with P
    Figure Legend Snippet: A novel HrpL-regulated virulence operon linked to avrD in Pph 1448A identified by RNA-seq analysis. ( A ) Color coded genomic context of avrD and downstream genes from PSPPH_A0106 to A0112 . Grey arrows represent transposases. White arrows represent additional ORFs that are not necessary related. ( B ) Graphical representation of avrD operon according to IMG/ER conserved neighborhood region search with the 45 Pseudomonas strains presented in Figure 5 and S5 . Grey, white, black, and colored arrows as in A. Brackets represent breaks and end of contigs or scaffolds. Not to scale. ( C ) PCR was performed using primers spanning intragenic regions between PSPPH_A0109 and A0110 (top panels), or PSPPH_A0112 and avrD (middle panels) on cDNA prepared from Pph 1448A ( Pph ) or an isogenic Pph 1448A Δ hrpL mutant (Δ hrpL ) grown in MM media. Total RNA was subject to reverse transcriptase (+RT), or without reverse transcriptase (−RT, as negative control). gDNA, DW indicate respectively that genomic DNA or distilled water were used as template for positive and negative controls of amplification. Equal loading was controlled by monitoring gap-1 amplification across samples (bottom panels). ( D ) Two week old bean cv. Tendergreen beans were dip inoculated with wild type Pph 1448A (Pph) , two independent clean deletion avrD mutants (Δa vrD #1, Δa vrD #2), and two independent clean deletion PSPPH_A0107 mutants (Δ PSPPH_A0107 #1, Δ PSPPH_A0107 #2) at OD 600 = 0.001. Bacterial growth of each strain was determined after 3.5 dpi. Letters represent significant differences with P

    Techniques Used: RNA Sequencing Assay, Polymerase Chain Reaction, Mutagenesis, Negative Control, Amplification

    13) Product Images from "HLA genotyping by next-generation sequencing of complementary DNA"

    Article Title: HLA genotyping by next-generation sequencing of complementary DNA

    Journal: BMC Genomics

    doi: 10.1186/s12864-017-4300-7

    Complete sequencing of individual cDNA molecules with molecular barcodes. a , cDNA synthesis and PCR. b , Fragmentation and circularization. c , Template preparation through PCR using a circularized molecule as the template
    Figure Legend Snippet: Complete sequencing of individual cDNA molecules with molecular barcodes. a , cDNA synthesis and PCR. b , Fragmentation and circularization. c , Template preparation through PCR using a circularized molecule as the template

    Techniques Used: Sequencing, Polymerase Chain Reaction

    14) Product Images from "Dynamics of gene silencing during X inactivation using allele-specific RNA-seq"

    Article Title: Dynamics of gene silencing during X inactivation using allele-specific RNA-seq

    Journal: Genome Biology

    doi: 10.1186/s13059-015-0698-x

    Allele-specific RNA-seq on three NPC lines identifies three distal regions of genes that escape XCI. a Ratio of Xi/Xa ( y-axis ; for each of the three NPC lines sorted from highest to lowest) for genes showing a log2 ratio of at least −5. We set the cutoff for escape on 10 % relative expression from the Xi versus the Xa (log 2 ratio of > −3.32; similar to Yang et al. [ 37 ]). b Xi/Xa ratio of genes that escape XCI in all three NPC lines. c Distribution of the escape genes identified in *NPC_129-Xi over the four clusters as characterized in Fig. 4a . d Localization of the escape genes within each NPC line over the linear X chromosome (see also Table 1 ). The black dots on the fourth row represent all X-linked genes for which high-confidence allele-specific ratios were obtained in NPCs. e Validation of the escape genes within the three escape regions by Sanger sequencing of cDNA. See Additional file 1 : Figure S13 for the full panel of 13 genes that we validated, and for further details
    Figure Legend Snippet: Allele-specific RNA-seq on three NPC lines identifies three distal regions of genes that escape XCI. a Ratio of Xi/Xa ( y-axis ; for each of the three NPC lines sorted from highest to lowest) for genes showing a log2 ratio of at least −5. We set the cutoff for escape on 10 % relative expression from the Xi versus the Xa (log 2 ratio of > −3.32; similar to Yang et al. [ 37 ]). b Xi/Xa ratio of genes that escape XCI in all three NPC lines. c Distribution of the escape genes identified in *NPC_129-Xi over the four clusters as characterized in Fig. 4a . d Localization of the escape genes within each NPC line over the linear X chromosome (see also Table 1 ). The black dots on the fourth row represent all X-linked genes for which high-confidence allele-specific ratios were obtained in NPCs. e Validation of the escape genes within the three escape regions by Sanger sequencing of cDNA. See Additional file 1 : Figure S13 for the full panel of 13 genes that we validated, and for further details

    Techniques Used: RNA Sequencing Assay, Expressing, Sequencing

    15) Product Images from "Allele-specific regulation of mutant Huntingtin by Wig1, a downstream target of p53"

    Article Title: Allele-specific regulation of mutant Huntingtin by Wig1, a downstream target of p53

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddw115

    Role for Wig1 in mHtt-elicited pathology in HD models. (A) Suppression of mutant Htt-elicited cell toxicity by knockdown of Wig1 in rat cortical primary neurons. Green, GFP. Inset shows DAPI-stained nucleus. The scale bar represents 20 µm. (B) Amelioration of mutant Htt-elicited aggregate formation by knockdown of Wig1 in rat cortical primary neurons. Red, Htt aggregates; blue, DAPI nuclear staining. The scale bar represents 10 µm. (C) RNA-binding region in Wig1 is critical for its facilitation of mutant Htt-elicited cytotoxicity and aggregate formation. Expression of wt Wig1 in rat cortical primary neurons expressing mutant Htt fragment (N171-Htt-Q82) and RNAi against Wig1 completely suppresses Wig1 RNAi-mediated amelioration of cell death (the left panel) and aggregate formation (the right panel), whereas RNA binding-defective mutant Wig1 (Wig1-H88A) does not. All the graphs represent at least three independent experiments. (D) Reduction of the Htt-positive aggregate formation by knockdown of Wig1 in the striatum of R6/2 mice. Htt-positive aggregates were labeled with an antibody against Htt MAB5374 (EM48; green) at 9 weeks of age. White arrowhead indicates the AAV-infected striatal cells that do not contain Htt-positive aggregates. Con, control AAV; KD, Wig1 KD AAV. Scale bars represent 20 μm. High magnification (High mag.): Scale bars represent 10 μm. Data are represented as mean ± s.e.m. (* P
    Figure Legend Snippet: Role for Wig1 in mHtt-elicited pathology in HD models. (A) Suppression of mutant Htt-elicited cell toxicity by knockdown of Wig1 in rat cortical primary neurons. Green, GFP. Inset shows DAPI-stained nucleus. The scale bar represents 20 µm. (B) Amelioration of mutant Htt-elicited aggregate formation by knockdown of Wig1 in rat cortical primary neurons. Red, Htt aggregates; blue, DAPI nuclear staining. The scale bar represents 10 µm. (C) RNA-binding region in Wig1 is critical for its facilitation of mutant Htt-elicited cytotoxicity and aggregate formation. Expression of wt Wig1 in rat cortical primary neurons expressing mutant Htt fragment (N171-Htt-Q82) and RNAi against Wig1 completely suppresses Wig1 RNAi-mediated amelioration of cell death (the left panel) and aggregate formation (the right panel), whereas RNA binding-defective mutant Wig1 (Wig1-H88A) does not. All the graphs represent at least three independent experiments. (D) Reduction of the Htt-positive aggregate formation by knockdown of Wig1 in the striatum of R6/2 mice. Htt-positive aggregates were labeled with an antibody against Htt MAB5374 (EM48; green) at 9 weeks of age. White arrowhead indicates the AAV-infected striatal cells that do not contain Htt-positive aggregates. Con, control AAV; KD, Wig1 KD AAV. Scale bars represent 20 μm. High magnification (High mag.): Scale bars represent 10 μm. Data are represented as mean ± s.e.m. (* P

    Techniques Used: Mutagenesis, Staining, RNA Binding Assay, Expressing, Mouse Assay, Labeling, Infection

    16) Product Images from "Mycobacterium avium Genes MAV_5138 and MAV_3679 Are Transcriptional Regulators That Play a Role in Invasion of Epithelial Cells, in Part by Their Regulation of CipA, a Putative Surface Protein Interacting with Host Cell Signaling Pathways ▿"

    Article Title: Mycobacterium avium Genes MAV_5138 and MAV_3679 Are Transcriptional Regulators That Play a Role in Invasion of Epithelial Cells, in Part by Their Regulation of CipA, a Putative Surface Protein Interacting with Host Cell Signaling Pathways ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01359-07

    Real-time PCR results comparing the fold change in gene expression upon 15 min of exposure to HEp-2 cells between the MAC109 and MAC109 Δ fadD2 strains. The y axis represents the fold change between broth grown bacteria and bacteria incubated for 15 min with HEp-2 cells, using the 16S rDNA as an internal control. An asterisk (*) indicates significant differences between the two strains, based on three independent experiments ( P
    Figure Legend Snippet: Real-time PCR results comparing the fold change in gene expression upon 15 min of exposure to HEp-2 cells between the MAC109 and MAC109 Δ fadD2 strains. The y axis represents the fold change between broth grown bacteria and bacteria incubated for 15 min with HEp-2 cells, using the 16S rDNA as an internal control. An asterisk (*) indicates significant differences between the two strains, based on three independent experiments ( P

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Incubation

    17) Product Images from "Lariat capping as a tool to manipulate the 5′ end of individual yeast mRNA species in vivo"

    Article Title: Lariat capping as a tool to manipulate the 5′ end of individual yeast mRNA species in vivo

    Journal: RNA

    doi: 10.1261/rna.059337.116

    Transcripts are lariat-capped cotranscriptionally and processed to have oligo(A) tails. ( A ) Outline of the RNase H assay applied to measure poly(A) tail lengths of GFP mRNAs. Relative positions of the oligo used to cleave the GFP mRNA, the d(T)NN oligo used to trim the poly(A) tail, and the Northern hybridization probe oligo are indicated. ( B ) RNase H/Northern assay of whole cell RNA extracted from wt cells expressing m 7 G GFP-, LC GFP-, and LCmutGFP-mRNA as indicated. The mobility shift between samples treated without (−) and with (+) oligo d(T)NN indicates the length of the poly(A) tail. ( C ) RNase H/Northern assay of whole cell RNA from cells expressing m 7 G GFP- or LC GFP-mRNA in the ccr4 Δ and ccr4 Δ pan2 Δ mutant cell backgrounds as indicated. The gel was run at identical conditions and to the same length as in B as judged by the xylene cyanol and bromophenol blue dye markers. ( D ) Box-plot showing lengths of poly(A) tails from individual clones of PCR products derived from 3′ RACE experiments of RNA from B and C . ( E ) Map of amplicons used for nascent RNA analysis by qRT-PCR. Three primer sets were used to generate amplicon a located upstream of the IPS (the LCrz processing site), amplicon b spanning the IPS, and amplicon c targeting the GFP part of the transcript downstream from the IPS. ( F ) qRT-PCR of nascent RNA. All amplicon signals were normalized to that of amplicon c and plotted as the ratio between LC- and LCmut-RNA (uncleaved). The error bars indicate the standard error of the mean (SEM), n = 3.
    Figure Legend Snippet: Transcripts are lariat-capped cotranscriptionally and processed to have oligo(A) tails. ( A ) Outline of the RNase H assay applied to measure poly(A) tail lengths of GFP mRNAs. Relative positions of the oligo used to cleave the GFP mRNA, the d(T)NN oligo used to trim the poly(A) tail, and the Northern hybridization probe oligo are indicated. ( B ) RNase H/Northern assay of whole cell RNA extracted from wt cells expressing m 7 G GFP-, LC GFP-, and LCmutGFP-mRNA as indicated. The mobility shift between samples treated without (−) and with (+) oligo d(T)NN indicates the length of the poly(A) tail. ( C ) RNase H/Northern assay of whole cell RNA from cells expressing m 7 G GFP- or LC GFP-mRNA in the ccr4 Δ and ccr4 Δ pan2 Δ mutant cell backgrounds as indicated. The gel was run at identical conditions and to the same length as in B as judged by the xylene cyanol and bromophenol blue dye markers. ( D ) Box-plot showing lengths of poly(A) tails from individual clones of PCR products derived from 3′ RACE experiments of RNA from B and C . ( E ) Map of amplicons used for nascent RNA analysis by qRT-PCR. Three primer sets were used to generate amplicon a located upstream of the IPS (the LCrz processing site), amplicon b spanning the IPS, and amplicon c targeting the GFP part of the transcript downstream from the IPS. ( F ) qRT-PCR of nascent RNA. All amplicon signals were normalized to that of amplicon c and plotted as the ratio between LC- and LCmut-RNA (uncleaved). The error bars indicate the standard error of the mean (SEM), n = 3.

    Techniques Used: Rnase H Assay, Northern Blot, Hybridization, Expressing, Mobility Shift, Mutagenesis, Clone Assay, Polymerase Chain Reaction, Derivative Assay, Quantitative RT-PCR, Amplification

    18) Product Images from "Transcription, Processing, and Function of CRISPR Cassettes in Escherichia coli"

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

    Journal: Molecular microbiology

    doi: 10.1111/j.1365-2958.2010.07265.x

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

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

    19) Product Images from "The High Level of Aberrant Splicing of ISCU in Slow-Twitch Muscle May Involve the Splicing Factor SRSF3"

    Article Title: The High Level of Aberrant Splicing of ISCU in Slow-Twitch Muscle May Involve the Splicing Factor SRSF3

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0165453

    Incorrect splicing of ISCU in myoblasts with decreased SRSF3 expression. A lentivirus-mediated expression vector for SRSF3 shRNA (sh3) was introduced into myoblasts from HML patients (P1, P2) and a healthy control (C). A) SRSF3 qRTPCR using cDNA from uninfected myoblasts (-) and myoblasts infected with a shSRSF3 lentivirus-mediated expression vector. The graph present the mean fold change ± SD for the SRSF3 expression from at least three independent experiments. β-actin was used as an internal control. B) Western blot of SRSF3 in non-transduced and transduced myoblasts. ACTIN was used as a loading reference. C) Semi-qRTPCR of human ISCU with incorrect (MUT) and correct (WT) splice variants from uninfected myoblasts, (-) or myoblasts infected with lentivirus-mediated vectors expressing shSRSF3 (sh3). D) Quantification of incorrectly spliced ISCU by qRTPCR in in non-transduced and transduced myoblasts. The graph presents the mean percentage of incorrectly spliced ISCU ± SD from at least three independent experiments (* p
    Figure Legend Snippet: Incorrect splicing of ISCU in myoblasts with decreased SRSF3 expression. A lentivirus-mediated expression vector for SRSF3 shRNA (sh3) was introduced into myoblasts from HML patients (P1, P2) and a healthy control (C). A) SRSF3 qRTPCR using cDNA from uninfected myoblasts (-) and myoblasts infected with a shSRSF3 lentivirus-mediated expression vector. The graph present the mean fold change ± SD for the SRSF3 expression from at least three independent experiments. β-actin was used as an internal control. B) Western blot of SRSF3 in non-transduced and transduced myoblasts. ACTIN was used as a loading reference. C) Semi-qRTPCR of human ISCU with incorrect (MUT) and correct (WT) splice variants from uninfected myoblasts, (-) or myoblasts infected with lentivirus-mediated vectors expressing shSRSF3 (sh3). D) Quantification of incorrectly spliced ISCU by qRTPCR in in non-transduced and transduced myoblasts. The graph presents the mean percentage of incorrectly spliced ISCU ± SD from at least three independent experiments (* p

    Techniques Used: Expressing, Plasmid Preparation, shRNA, Infection, Western Blot

    20) Product Images from "Unstructured 5′-tails act through ribosome standby to override inhibitory structure at ribosome binding sites"

    Article Title: Unstructured 5′-tails act through ribosome standby to override inhibitory structure at ribosome binding sites

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky073

    The 5′ standby tails need to be single-stranded to promote translation and initiation complex formation. ( A ) The effect of blocking of the (CA)6 and (CA)8 standby tail by an antisense oligo (at 6.7 times molar excess), or a control oligo, was assayed by in vitro translation. The mRNAs used are indicated, and GFP was detected by Western blot. ( B ) Formation of a heteroduplex between the antisense oligo and the standby tail of (CA)8 mRNA was tested by RNase H cleavage (Materials and Methods). Cleavage was observed after reverse transcription using a 5′-labeled oligo annealed downstream. Cleavage near the base of the stem is observed only in the presence of the antisense oligo and RNase H (far right lane). ( C ) The toeprint experiment was conducted on several mRNA variants, with 30S and tRNA fMet , with or without antisense or control oligo (Materials and Methods), as indicated. The position of the characteristic toeprint at +15 is shown. UAGC shows a sequencing ladder for reference. The gel is representative of three technical replicates.
    Figure Legend Snippet: The 5′ standby tails need to be single-stranded to promote translation and initiation complex formation. ( A ) The effect of blocking of the (CA)6 and (CA)8 standby tail by an antisense oligo (at 6.7 times molar excess), or a control oligo, was assayed by in vitro translation. The mRNAs used are indicated, and GFP was detected by Western blot. ( B ) Formation of a heteroduplex between the antisense oligo and the standby tail of (CA)8 mRNA was tested by RNase H cleavage (Materials and Methods). Cleavage was observed after reverse transcription using a 5′-labeled oligo annealed downstream. Cleavage near the base of the stem is observed only in the presence of the antisense oligo and RNase H (far right lane). ( C ) The toeprint experiment was conducted on several mRNA variants, with 30S and tRNA fMet , with or without antisense or control oligo (Materials and Methods), as indicated. The position of the characteristic toeprint at +15 is shown. UAGC shows a sequencing ladder for reference. The gel is representative of three technical replicates.

    Techniques Used: Blocking Assay, In Vitro, Western Blot, Labeling, Sequencing

    21) Product Images from "Two-dimensional intact mitochondrial DNA agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome"

    Article Title: Two-dimensional intact mitochondrial DNA agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks1324

    Mitochondrial DNA topoisomers are associated with RNA in 2D-IMAGE profiles. ( A ) 2D-IMAGE profile of wild-type MEF DNA without RNAse treatment. New topoisomers relative to those in Figure 3 are assigned letters. ( B ) RNase H treatment, which digests RNA:DNA hybrids, reveals the vertical spikes of DNA that are sensitive to S1 nuclease ( 23–25 ). Molecules that decrease on digestion are indicated with dashed arrows, whereas those that increase are indicated with solid arrows. Numbers correspond to topoisomers in Figure 3 . ( C ) RNase A treatment, which digests heterogeneous RNA after U and C bases, reveals the typical 2D-IMAGE pattern shown in Figure 2 . Dashed arrows indicate a reduction in signal, and solid arrows indicate increased signal. ( D ) Quantitation of change in abundance of topoisomers shown in panels B and C relative to untreated.
    Figure Legend Snippet: Mitochondrial DNA topoisomers are associated with RNA in 2D-IMAGE profiles. ( A ) 2D-IMAGE profile of wild-type MEF DNA without RNAse treatment. New topoisomers relative to those in Figure 3 are assigned letters. ( B ) RNase H treatment, which digests RNA:DNA hybrids, reveals the vertical spikes of DNA that are sensitive to S1 nuclease ( 23–25 ). Molecules that decrease on digestion are indicated with dashed arrows, whereas those that increase are indicated with solid arrows. Numbers correspond to topoisomers in Figure 3 . ( C ) RNase A treatment, which digests heterogeneous RNA after U and C bases, reveals the typical 2D-IMAGE pattern shown in Figure 2 . Dashed arrows indicate a reduction in signal, and solid arrows indicate increased signal. ( D ) Quantitation of change in abundance of topoisomers shown in panels B and C relative to untreated.

    Techniques Used: Quantitation Assay

    22) Product Images from "Differential ratio amplicons (Ramp) for the evaluation of RNA integrity extracted from complex environmental samples"

    Article Title: Differential ratio amplicons (Ramp) for the evaluation of RNA integrity extracted from complex environmental samples

    Journal: Environmental Microbiology

    doi: 10.1111/1462-2920.14516

    Effect of UV degradation on RNA integrity measured via the RIN (A), with RT‐Q‐PCR (B) and RIN versus R amp (C). For RIN, RNA integrity visualized in virtual gels (A; left) and electropherogram (A; right) are displayed against incubation period under UV. RNA ladder shows size in nucleotides (nt). B. Effect of degradation on transcript quantification; Amp 1–3: average Ct ( n = 3) of one of the three possible glnA amplicons; amoA : average amoA Ct ( n = 3) of the Bacterial amoA transcript; 16S rRNA : average 16S rRNA Ct ( n = 3) of the bacterial 16S rRNA transcript. Effect of RNA degradation on R amp index is presented in figure C; for comparison, RIN values were also plotted. Greek Letters indicate the result of TukeyHSD tests (points with different letters had values significantly different from each other using 0.05 as threshold for the p value).
    Figure Legend Snippet: Effect of UV degradation on RNA integrity measured via the RIN (A), with RT‐Q‐PCR (B) and RIN versus R amp (C). For RIN, RNA integrity visualized in virtual gels (A; left) and electropherogram (A; right) are displayed against incubation period under UV. RNA ladder shows size in nucleotides (nt). B. Effect of degradation on transcript quantification; Amp 1–3: average Ct ( n = 3) of one of the three possible glnA amplicons; amoA : average amoA Ct ( n = 3) of the Bacterial amoA transcript; 16S rRNA : average 16S rRNA Ct ( n = 3) of the bacterial 16S rRNA transcript. Effect of RNA degradation on R amp index is presented in figure C; for comparison, RIN values were also plotted. Greek Letters indicate the result of TukeyHSD tests (points with different letters had values significantly different from each other using 0.05 as threshold for the p value).

    Techniques Used: Polymerase Chain Reaction, Incubation

    Effect of heat degradation on RNA integrity measured via the RIN (A), with RT‐Q‐PCR (B) and RIN versus R amp (C). For RIN, RNA integrity visualized in virtual gels (A; left) and electropherogram (A; right) are displayed against incubation period at 90°C. RNA ladder shows size in nucleotides (nt). B. Effect of degradation on transcript quantification; Amp 1–3: average Ct ( n = 3) of one of the three possible glnA amplicons; amoA : average amoA Ct ( n = 3) of the Bacterial amoA transcript; 16S rRNA : average 16S rRNA Ct ( n = 3) of the bacterial 16S rRNA transcript. Effect of RNA degradation on R amp index is presented in figure C; for comparison, RIN values were also plotted. Greek Letters indicate the result of TukeyHSD tests (points with different letters had values significantly different from each other using 0.05 as threshold for the p value).
    Figure Legend Snippet: Effect of heat degradation on RNA integrity measured via the RIN (A), with RT‐Q‐PCR (B) and RIN versus R amp (C). For RIN, RNA integrity visualized in virtual gels (A; left) and electropherogram (A; right) are displayed against incubation period at 90°C. RNA ladder shows size in nucleotides (nt). B. Effect of degradation on transcript quantification; Amp 1–3: average Ct ( n = 3) of one of the three possible glnA amplicons; amoA : average amoA Ct ( n = 3) of the Bacterial amoA transcript; 16S rRNA : average 16S rRNA Ct ( n = 3) of the bacterial 16S rRNA transcript. Effect of RNA degradation on R amp index is presented in figure C; for comparison, RIN values were also plotted. Greek Letters indicate the result of TukeyHSD tests (points with different letters had values significantly different from each other using 0.05 as threshold for the p value).

    Techniques Used: Polymerase Chain Reaction, Incubation

    Effect of RNase I degradation on RNA integrity measured via the RIN (A), with RT‐Q‐PCR (B) and RIN versus R amp (C). For RIN, RNA integrity visualized in virtual gels (A; left) and electropherogram (A; right) are displayed against incubation period with RNase I . RNA ladder shows size in nucleotides (nt). B. Effect of degradation on transcript quantification; Amp 1–3: average Ct ( n = 3) of one of the three possible glnA amplicons; amoA : average amoA Ct ( n = 3) of the Bacterial amoA transcript; 16S rRNA : average 16S rRNA Ct ( n = 3) of the bacterial 16S rRNA transcript. Effect of RNA degradation on R amp index is presented in figure C; for comparison, RIN values were also plotted. Greek letters indicate the result of TukeyHSD tests (points with different letters had values significantly different from each other using 0.05 as threshold for the p value).
    Figure Legend Snippet: Effect of RNase I degradation on RNA integrity measured via the RIN (A), with RT‐Q‐PCR (B) and RIN versus R amp (C). For RIN, RNA integrity visualized in virtual gels (A; left) and electropherogram (A; right) are displayed against incubation period with RNase I . RNA ladder shows size in nucleotides (nt). B. Effect of degradation on transcript quantification; Amp 1–3: average Ct ( n = 3) of one of the three possible glnA amplicons; amoA : average amoA Ct ( n = 3) of the Bacterial amoA transcript; 16S rRNA : average 16S rRNA Ct ( n = 3) of the bacterial 16S rRNA transcript. Effect of RNA degradation on R amp index is presented in figure C; for comparison, RIN values were also plotted. Greek letters indicate the result of TukeyHSD tests (points with different letters had values significantly different from each other using 0.05 as threshold for the p value).

    Techniques Used: Polymerase Chain Reaction, Incubation

    23) Product Images from "Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells"

    Article Title: Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells

    Journal: Nature biotechnology

    doi: 10.1038/nbt.2720

    Microwell displacement amplification system. (a) Each slide contains 16 arrays of 255 microwells each. Cells, lysis solution, denaturing buffer, neutralization buffer and MDA master mix were each added to the microwells with a single pipette pump. Amplicon growth was then visualized with a fluorescent microscope using a real-time MDA system. Microwells showing increasing fluorescence over time were positive amplicons. The amplicons were extracted with fine glass pipettes attached to a micromanipulation system. (b) Scanning electron microscopy of a single E. coli cell displayed at different magnifications. This particular well contains only one cell, and most wells observed also contained no more than one cell. (c) A custom microscope incubation chamber was used for real time MDA. The chamber was temperature and humidity controlled to mitigate evaporation of reagents. Additionally, it prevented contamination during amplicon extraction by self-containing the micromanipulation system. An image of the entire microwell array is also shown, as well as a micropipette probing a well. (d ) Complex three-dimensional MDA amplicons were reduced to linear DNA using DNA polymerase I and Ampligase. This process substantially improved the complexity of the library during sequencing.
    Figure Legend Snippet: Microwell displacement amplification system. (a) Each slide contains 16 arrays of 255 microwells each. Cells, lysis solution, denaturing buffer, neutralization buffer and MDA master mix were each added to the microwells with a single pipette pump. Amplicon growth was then visualized with a fluorescent microscope using a real-time MDA system. Microwells showing increasing fluorescence over time were positive amplicons. The amplicons were extracted with fine glass pipettes attached to a micromanipulation system. (b) Scanning electron microscopy of a single E. coli cell displayed at different magnifications. This particular well contains only one cell, and most wells observed also contained no more than one cell. (c) A custom microscope incubation chamber was used for real time MDA. The chamber was temperature and humidity controlled to mitigate evaporation of reagents. Additionally, it prevented contamination during amplicon extraction by self-containing the micromanipulation system. An image of the entire microwell array is also shown, as well as a micropipette probing a well. (d ) Complex three-dimensional MDA amplicons were reduced to linear DNA using DNA polymerase I and Ampligase. This process substantially improved the complexity of the library during sequencing.

    Techniques Used: Amplification, Lysis, Neutralization, Multiple Displacement Amplification, Transferring, Microscopy, Fluorescence, Micromanipulation, Electron Microscopy, Incubation, Evaporation, Sequencing

    24) Product Images from "Scalable and cost-effective ribonuclease-based rRNA depletion for transcriptomics"

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

    Journal: bioRxiv

    doi: 10.1101/645895

    Oligo probes can be applied to closely related species. a) Proportion of rRNA-aligning reads of total mapped reads (% rRNA reads), for un-depleted and RNase H depleted samples using an oligo probe library designed for B. dorei across closely related species ( B. uniformis and B. vulgatus ) and distantly related species ( C. aerofaciens and D. longicatena ). b) Scatter plot between probe-to-target sequence similarity and fold enrichment of non-rRNA reads for five species. Probe-to-target sequence similarity was calculated as the average of percentages of base with mismatches in 16S and 23S rRNA alignments.
    Figure Legend Snippet: Oligo probes can be applied to closely related species. a) Proportion of rRNA-aligning reads of total mapped reads (% rRNA reads), for un-depleted and RNase H depleted samples using an oligo probe library designed for B. dorei across closely related species ( B. uniformis and B. vulgatus ) and distantly related species ( C. aerofaciens and D. longicatena ). b) Scatter plot between probe-to-target sequence similarity and fold enrichment of non-rRNA reads for five species. Probe-to-target sequence similarity was calculated as the average of percentages of base with mismatches in 16S and 23S rRNA alignments.

    Techniques Used: Sequencing

    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.
    Figure Legend 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.

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

    RNase H based rRNA depletion with amplicons. a) Proportion of rRNA-aligning reads of total mapped reads (% rRNA reads), for un-depleted, Ribo-Zero depleted, and RNase H depleted samples with chemically synthesized oligonucleotides (oligo) or amplicon-based ssDNA (amplicon) probes. b, c) Scatter plot (b) and Quantile-Quantile (Q-Q) plot (c) for depleted sample using amplicon probes with 5:1 probe-to-RNA ratio and un-depleted sample.
    Figure Legend Snippet: RNase H based rRNA depletion with amplicons. a) Proportion of rRNA-aligning reads of total mapped reads (% rRNA reads), for un-depleted, Ribo-Zero depleted, and RNase H depleted samples with chemically synthesized oligonucleotides (oligo) or amplicon-based ssDNA (amplicon) probes. b, c) Scatter plot (b) and Quantile-Quantile (Q-Q) plot (c) for depleted sample using amplicon probes with 5:1 probe-to-RNA ratio and un-depleted sample.

    Techniques Used: Synthesized, Amplification

    High-throughput microbial RNA-seq screening of B. dorei on different substrates. a) The 5 short-chain fatty acids (SCFAs) and 14 carbohydrates tested. B. dorei cultures were treated with each substrate at 5 mg/mL final concentration during exponential growth phase. b) The RNase H based method showed efficient and consistent rRNA depletion on pooled RNA samples from different conditions. Three biological replicates were performed for each condition. c) Multidimensional scaling ordination of 20 conditions based on overall transcriptomes. Pairwise Spearman correlations between conditions were calculated using expression profiles and multidimensional scaling was then performed on normalized Spearman correlations to visualize the impact of substrates on bacterial transcriptome.
    Figure Legend Snippet: High-throughput microbial RNA-seq screening of B. dorei on different substrates. a) The 5 short-chain fatty acids (SCFAs) and 14 carbohydrates tested. B. dorei cultures were treated with each substrate at 5 mg/mL final concentration during exponential growth phase. b) The RNase H based method showed efficient and consistent rRNA depletion on pooled RNA samples from different conditions. Three biological replicates were performed for each condition. c) Multidimensional scaling ordination of 20 conditions based on overall transcriptomes. Pairwise Spearman correlations between conditions were calculated using expression profiles and multidimensional scaling was then performed on normalized Spearman correlations to visualize the impact of substrates on bacterial transcriptome.

    Techniques Used: High Throughput Screening Assay, RNA Sequencing Assay, Concentration Assay, Expressing

    Application of RNase H based rRNA depletion with oligos to three diverse gut microbiota species. a) Proportion of rRNA-aligning reads of total mapped reads (% rRNA reads), for un-depleted, Ribo-Zero depleted, and RNase H depleted samples under optimized reaction condition across three microbiota species from different phyla. The same RNA samples used for Ribo-Zero depletion were pooled together to yield the RNA used for RNase H reaction. b, c) Consistency in transcriptome between rRNA depleted samples and un-depleted samples in terms of expression correlation (b) and expression distribution (c) . TPM indicates transcripts per million for each CDS. c) Points lie on a straight line in Quantile-Quantile (Q-Q) plots if there is no global shift in the distribution of expression profile between depleted samples and un-depleted samples.
    Figure Legend Snippet: Application of RNase H based rRNA depletion with oligos to three diverse gut microbiota species. a) Proportion of rRNA-aligning reads of total mapped reads (% rRNA reads), for un-depleted, Ribo-Zero depleted, and RNase H depleted samples under optimized reaction condition across three microbiota species from different phyla. The same RNA samples used for Ribo-Zero depletion were pooled together to yield the RNA used for RNase H reaction. b, c) Consistency in transcriptome between rRNA depleted samples and un-depleted samples in terms of expression correlation (b) and expression distribution (c) . TPM indicates transcripts per million for each CDS. c) Points lie on a straight line in Quantile-Quantile (Q-Q) plots if there is no global shift in the distribution of expression profile between depleted samples and un-depleted samples.

    Techniques Used: Expressing

    Optimization of RNase H reaction conditions. a) Proportion of rRNA-aligning reads of total mapped reads (% rRNA reads), for un-depleted and RNase H depleted samples for two RNase H enzymes and various reaction times with probe-to-RNA fixed to 1:1, or b) various probe-to-RNA ratios with reaction time fixed to 30 minutes. The same RNA sample isolated from Bacteroides dorei was split for rRNA depletion across different reaction conditions.
    Figure Legend Snippet: Optimization of RNase H reaction conditions. a) Proportion of rRNA-aligning reads of total mapped reads (% rRNA reads), for un-depleted and RNase H depleted samples for two RNase H enzymes and various reaction times with probe-to-RNA fixed to 1:1, or b) various probe-to-RNA ratios with reaction time fixed to 30 minutes. The same RNA sample isolated from Bacteroides dorei was split for rRNA depletion across different reaction conditions.

    Techniques Used: Isolation

    25) Product Images from "Angiogenin/ribonuclease 5 is an EGFR ligand and a serum biomarker for erlotinib sensitivity in pancreatic cancer"

    Article Title: Angiogenin/ribonuclease 5 is an EGFR ligand and a serum biomarker for erlotinib sensitivity in pancreatic cancer

    Journal: Cancer cell

    doi: 10.1016/j.ccell.2018.02.012

    Catalytic activity of RNase is not required for activation of and binding to EGFR (A) IB of secreted proteins from conditioned media (CM) in HeLa cells transiently transfected with the indicated plasmids. Signals were quantified using ImageJ. Relative density of Flag-bRNaseA-WT was set as 1. (B) Detection of RNase enzyme activity in CM-secreted proteins collected from (A). Following the addition of fluorescent substrate, the mixtures were subjected to RNaseAlert ® Lab Test kit. Signals were monitored by a BioTek Synergy™ Neo real-time fluorometer. (C) HeLa cells were treated with secreted proteins from CM as described in (A) for 5 min and subjected to IB with the indicated antibodies. (D) HeLa cells were treated with CM-secreted proteins as described in (A) for 15 min, fixed and stained with EGFR and bRNaseA antibodies, and subjected to Duolink in situ PLA. Bar, 10 μm. Right, the number of interactions per cell normalized to the amount of the secreted proteins. (E) IB of CM-secreted proteins in HeLa stable transfectants as indicated. Signals were quantified using ImageJ. Relative density of HeLa-Flag-bRNaseA-WT was set as 1. (F) Detection of RNase enzyme activity in CM collected from (E). (G) HeLa cells were treated with CM-secreted proteins as described in (E) for 15 min, fixed and stained with EGFR and Flag antibodies, and subjected to Duolink in situ PLA. Bar, 10 μm. Right, the number of interactions per cell normalized to the amount of the secreted proteins. (H) Cell morphological changes of HeLa cells treated with the indicated proteins for 3 days. Bar, 50 μm. (I) IB of HeLa cells lysates extracted from (H). (J) IB of HeLa cells treated with the indicated proteins or water (−) for 5 min. (K) IB of CM-secreted proteins in HeLa stable transfectants expressing the indicated plasmids. Signals were quantified using ImageJ. Relative density of HeLa-Flag-ANG-WT was set as 1. (L) HeLa cells were treated with CM-secreted proteins as indicated in (K) for 5 min and subjected to IB with the indicated antibodies. These experiments were performed in duplicate. (M) HeLa cells were treated with CM-secreted proteins as indicated in (K) for 15 min, fixed and stained with EGFR and Flag antibodies and subjected to Duolink in situ PLA. Bar, 10 μm. Bottom, the number of interactions per cell normalized to the amount of the secreted proteins. All error bars represent mean ± SD. *p
    Figure Legend Snippet: Catalytic activity of RNase is not required for activation of and binding to EGFR (A) IB of secreted proteins from conditioned media (CM) in HeLa cells transiently transfected with the indicated plasmids. Signals were quantified using ImageJ. Relative density of Flag-bRNaseA-WT was set as 1. (B) Detection of RNase enzyme activity in CM-secreted proteins collected from (A). Following the addition of fluorescent substrate, the mixtures were subjected to RNaseAlert ® Lab Test kit. Signals were monitored by a BioTek Synergy™ Neo real-time fluorometer. (C) HeLa cells were treated with secreted proteins from CM as described in (A) for 5 min and subjected to IB with the indicated antibodies. (D) HeLa cells were treated with CM-secreted proteins as described in (A) for 15 min, fixed and stained with EGFR and bRNaseA antibodies, and subjected to Duolink in situ PLA. Bar, 10 μm. Right, the number of interactions per cell normalized to the amount of the secreted proteins. (E) IB of CM-secreted proteins in HeLa stable transfectants as indicated. Signals were quantified using ImageJ. Relative density of HeLa-Flag-bRNaseA-WT was set as 1. (F) Detection of RNase enzyme activity in CM collected from (E). (G) HeLa cells were treated with CM-secreted proteins as described in (E) for 15 min, fixed and stained with EGFR and Flag antibodies, and subjected to Duolink in situ PLA. Bar, 10 μm. Right, the number of interactions per cell normalized to the amount of the secreted proteins. (H) Cell morphological changes of HeLa cells treated with the indicated proteins for 3 days. Bar, 50 μm. (I) IB of HeLa cells lysates extracted from (H). (J) IB of HeLa cells treated with the indicated proteins or water (−) for 5 min. (K) IB of CM-secreted proteins in HeLa stable transfectants expressing the indicated plasmids. Signals were quantified using ImageJ. Relative density of HeLa-Flag-ANG-WT was set as 1. (L) HeLa cells were treated with CM-secreted proteins as indicated in (K) for 5 min and subjected to IB with the indicated antibodies. These experiments were performed in duplicate. (M) HeLa cells were treated with CM-secreted proteins as indicated in (K) for 15 min, fixed and stained with EGFR and Flag antibodies and subjected to Duolink in situ PLA. Bar, 10 μm. Bottom, the number of interactions per cell normalized to the amount of the secreted proteins. All error bars represent mean ± SD. *p

    Techniques Used: Activity Assay, Activation Assay, Binding Assay, Transfection, Staining, In Situ, Proximity Ligation Assay, Expressing

    26) Product Images from "Cloning, expression, purification and preliminary crystallographic analysis of the RNase HI domain of the Mycobacterium tuberculosis protein Rv2228c as a maltose-binding protein fusion"

    Article Title: Cloning, expression, purification and preliminary crystallographic analysis of the RNase HI domain of the Mycobacterium tuberculosis protein Rv2228c as a maltose-binding protein fusion

    Journal: Acta Crystallographica Section F: Structural Biology and Crystallization Communications

    doi: 10.1107/S1744309108021118

    Crystals of the RNase HI domain-MBP fusion protein formed in 20% PEG 2000 and 0.2 M diammonium tartrate.
    Figure Legend Snippet: Crystals of the RNase HI domain-MBP fusion protein formed in 20% PEG 2000 and 0.2 M diammonium tartrate.

    Techniques Used:

    SDS–PAGE gel showing total cell extract (lane A ), soluble fraction (lane B ) and insoluble fraction (lane C ) from IPTG-based induction of the RNase HI-MBP fusion protein in pMAL-C2.
    Figure Legend Snippet: SDS–PAGE gel showing total cell extract (lane A ), soluble fraction (lane B ) and insoluble fraction (lane C ) from IPTG-based induction of the RNase HI-MBP fusion protein in pMAL-C2.

    Techniques Used: SDS Page

    27) Product Images from "Genome-wide analysis of Musashi-2 targets reveals novel functions in governing epithelial cell migration"

    Article Title: Genome-wide analysis of Musashi-2 targets reveals novel functions in governing epithelial cell migration

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw207

    Msi2-HITS-CLIP identifies direct Msi2 targets in keratinocytes. ( A ) Autoradiogram of 32 P-labelled Msi2-RNA complexes treated with different RNase concentration resolved on a 10% Bis-Tris gel. ( B ) Pie chart of the genomic locations of the aligned reads and filtered peaks before (left panel) and after (right panel) the filtering processes, respectively. ( C ) Metagene of exonic coverage along a scaled mRNA for aligned reads (top panel) and filtered peaks (bottom panel). Reads densities are normalized for library sizes. Peak and reads densities are averaged along all detectable transcripts based on RNA-seq (see ‘Materials and Methods’). ( D ) Msi2 recognized motifs are identified from de novo motif search for 3–9 mers in the 3′UTR peaks. The top motif identified for each N-mer search is displayed and positioned to highlight the shared UAG motif. ( E ) Motif occurrences are tabulated in a +/- 100-nucleotide window surrounding the peak summits for all 3–9mer motifs displayed in panel D (top panel) or UAG (bottom panel). ( F ) Number of UAG motif ( 1 – 4 ) occurrences in a +/- 225-nucleotide window around the peak summit in CLIP sites, flanking region or random 3′UTR background (left panel), with Fisher Exact Test showing motif enrichment in CLIP sites over the flanking region or 3′UTR background (right panel). ( G ) Gene tracks of Msi2 HITS-CLIP reads for Msi2 bound transcripts. Reads from all libraries were combined and positive strand reads are coloured blue whereas the negative strand reads are coloured green. The UAG motifs and its reverse complement, CUA, in a window around the peak summits are highlighted in red.
    Figure Legend Snippet: Msi2-HITS-CLIP identifies direct Msi2 targets in keratinocytes. ( A ) Autoradiogram of 32 P-labelled Msi2-RNA complexes treated with different RNase concentration resolved on a 10% Bis-Tris gel. ( B ) Pie chart of the genomic locations of the aligned reads and filtered peaks before (left panel) and after (right panel) the filtering processes, respectively. ( C ) Metagene of exonic coverage along a scaled mRNA for aligned reads (top panel) and filtered peaks (bottom panel). Reads densities are normalized for library sizes. Peak and reads densities are averaged along all detectable transcripts based on RNA-seq (see ‘Materials and Methods’). ( D ) Msi2 recognized motifs are identified from de novo motif search for 3–9 mers in the 3′UTR peaks. The top motif identified for each N-mer search is displayed and positioned to highlight the shared UAG motif. ( E ) Motif occurrences are tabulated in a +/- 100-nucleotide window surrounding the peak summits for all 3–9mer motifs displayed in panel D (top panel) or UAG (bottom panel). ( F ) Number of UAG motif ( 1 – 4 ) occurrences in a +/- 225-nucleotide window around the peak summit in CLIP sites, flanking region or random 3′UTR background (left panel), with Fisher Exact Test showing motif enrichment in CLIP sites over the flanking region or 3′UTR background (right panel). ( G ) Gene tracks of Msi2 HITS-CLIP reads for Msi2 bound transcripts. Reads from all libraries were combined and positive strand reads are coloured blue whereas the negative strand reads are coloured green. The UAG motifs and its reverse complement, CUA, in a window around the peak summits are highlighted in red.

    Techniques Used: Cross-linking Immunoprecipitation, Concentration Assay, RNA Sequencing Assay

    28) Product Images from "Alkaloids of fascaplysin are effective conventional chemotherapeutic drugs, inhibiting the proliferation of C6 glioma cells and causing their death in vitro"

    Article Title: Alkaloids of fascaplysin are effective conventional chemotherapeutic drugs, inhibiting the proliferation of C6 glioma cells and causing their death in vitro

    Journal: Oncology Letters

    doi: 10.3892/ol.2016.5478

    Staining by terminal deoxynucleotidyl transferase dUTP nick end labeling method following 6 h exposure to 0.5 µM fascaplysin in C6 cell culture. (A) Initial first signs of nuclear fragmentation of neoplastic cells, with a large number of fluorescent apoptotic cells, which indicated oligonucleosomic DNA degradation (green). (B) Control (positive) cells. Prior to staining, cells were pretreated with DNAse to induce apoptosis. (C) Control (negative) glioma C6 cells not treated by fascaplysin.
    Figure Legend Snippet: Staining by terminal deoxynucleotidyl transferase dUTP nick end labeling method following 6 h exposure to 0.5 µM fascaplysin in C6 cell culture. (A) Initial first signs of nuclear fragmentation of neoplastic cells, with a large number of fluorescent apoptotic cells, which indicated oligonucleosomic DNA degradation (green). (B) Control (positive) cells. Prior to staining, cells were pretreated with DNAse to induce apoptosis. (C) Control (negative) glioma C6 cells not treated by fascaplysin.

    Techniques Used: Staining, TUNEL Assay, Cell Culture

    29) Product Images from "A Novel Microplate 3D Bioprinting Platform for the Engineering of Muscle and Tendon Tissues"

    Article Title: A Novel Microplate 3D Bioprinting Platform for the Engineering of Muscle and Tendon Tissues

    Journal: Slas Technology

    doi: 10.1177/2472630318776594

    Calcium signaling of printed muscle models. Ca 2+ imaging of a two-channel muscle model with G5 that was differentiated for 22 days and was loaded with Fluo-4 AM calcium dye. Ca 2+ signal curve after single electrical pulse stimulation (EPS) of 1 ms duration (300 ms, 50 Hz). Top right: Ca 2+ signal imaging, while electrically stimulated. The time on the x-axis is in arbitrary units. The length of 1 s is indicated in the figure. The inset demonstrates fluorescent Ca 2+ .
    Figure Legend Snippet: Calcium signaling of printed muscle models. Ca 2+ imaging of a two-channel muscle model with G5 that was differentiated for 22 days and was loaded with Fluo-4 AM calcium dye. Ca 2+ signal curve after single electrical pulse stimulation (EPS) of 1 ms duration (300 ms, 50 Hz). Top right: Ca 2+ signal imaging, while electrically stimulated. The time on the x-axis is in arbitrary units. The length of 1 s is indicated in the figure. The inset demonstrates fluorescent Ca 2+ .

    Techniques Used: Imaging, Mass Spectrometry

    30) Product Images from "A Heterogeneous Nuclear Ribonucleoprotein A/B-Related Protein Binds to Single-Stranded DNA near the 5? End or within the Genome of Feline Parvovirus and Can Modify Virus Replication"

    Article Title: A Heterogeneous Nuclear Ribonucleoprotein A/B-Related Protein Binds to Single-Stranded DNA near the 5? End or within the Genome of Feline Parvovirus and Can Modify Virus Replication

    Journal: Journal of Virology

    doi:

    Effect of DBP40 on the in vitro DNA filled-in of FPV ssDNA by the Klenow fragment of DNA polymerase I. Viral DNA recovered from purified virions was incubated with the polymerase in the presence of deoxynucleoside triphosphates, [ 32 P]dCTP, and either 0 or 50 ng of DBP40. (A) The product generated was electrophoresed in a 1% agarose gel, with the number of disintegrations per minute (in phosphorimager units) in the total DNA product shown. (B) dsDNA produced was digested with Sna I (nt 289) or Bse RI (nt 469) and electrophoresed in a 5% nondenaturing acrylamide gel, which was exposed to X-ray film. Incorporation into the lower band is shown below each lane; size markers are indicated in base pairs. (C) Positions of Sna I and Bse RI sites relative to the 3′-end palindrome of the FPV ssDNA genome.
    Figure Legend Snippet: Effect of DBP40 on the in vitro DNA filled-in of FPV ssDNA by the Klenow fragment of DNA polymerase I. Viral DNA recovered from purified virions was incubated with the polymerase in the presence of deoxynucleoside triphosphates, [ 32 P]dCTP, and either 0 or 50 ng of DBP40. (A) The product generated was electrophoresed in a 1% agarose gel, with the number of disintegrations per minute (in phosphorimager units) in the total DNA product shown. (B) dsDNA produced was digested with Sna I (nt 289) or Bse RI (nt 469) and electrophoresed in a 5% nondenaturing acrylamide gel, which was exposed to X-ray film. Incorporation into the lower band is shown below each lane; size markers are indicated in base pairs. (C) Positions of Sna I and Bse RI sites relative to the 3′-end palindrome of the FPV ssDNA genome.

    Techniques Used: In Vitro, Purification, Incubation, Generated, Agarose Gel Electrophoresis, Produced, Acrylamide Gel Assay

    31) 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

    32) Product Images from "Sulfotransferases of Two Specificities Function in the Reconstitution of High Endothelial Cell Ligands for L-selectin "

    Article Title: Sulfotransferases of Two Specificities Function in the Reconstitution of High Endothelial Cell Ligands for L-selectin

    Journal: The Journal of Cell Biology

    doi:

    Expression of HEC-GlcNAc6ST transcripts in HECs. (A) Semiquantitative RT-PCR analysis. Fragments of the HEC-GlcNAc6ST and HPRT sequences were amplified by PCR from serial dilutions of cDNA prepared from purified HECs, HUVECs, and tonsillar lymphocytes. The reaction products (456 and 300 bp, respectively) were analyzed by agarose electrophoresis and ethidium bromide staining. −RT, PCR reactions in which the template was generated by omission of RT. (B) Northern blotting. Northern blots containing poly(A) + RNA from various human tissues (left and center) and from HECs and HUVECs (right) were probed with a 500-bp fragment from the HEC-GlcNAc6ST cDNA (top panels). The blots were stripped and reprobed with a 300-bp probe for β-act in (bottom panels).
    Figure Legend Snippet: Expression of HEC-GlcNAc6ST transcripts in HECs. (A) Semiquantitative RT-PCR analysis. Fragments of the HEC-GlcNAc6ST and HPRT sequences were amplified by PCR from serial dilutions of cDNA prepared from purified HECs, HUVECs, and tonsillar lymphocytes. The reaction products (456 and 300 bp, respectively) were analyzed by agarose electrophoresis and ethidium bromide staining. −RT, PCR reactions in which the template was generated by omission of RT. (B) Northern blotting. Northern blots containing poly(A) + RNA from various human tissues (left and center) and from HECs and HUVECs (right) were probed with a 500-bp fragment from the HEC-GlcNAc6ST cDNA (top panels). The blots were stripped and reprobed with a 300-bp probe for β-act in (bottom panels).

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Purification, Electrophoresis, Staining, Generated, Northern Blot, Activated Clotting Time Assay

    33) Product Images from "RNase HII saves rnhA mutant Escherichia coli from R-loop-associated chromosomal fragmentation"

    Article Title: RNase HII saves rnhA mutant Escherichia coli from R-loop-associated chromosomal fragmentation

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2017.08.004

    Chromosome fragmentation analysis by RNase HI, RNase HII and RNase A treatment in vitro A. A scheme of various hypothetical R-lesions (R-tract, two types of R-gaps) with positions of cleavage by RNase HI, HII and A (in low salt (LS) and high salt (HS) conditions) shown with arrows of the corresponding color. Small blue “d” letters, dNs; small orange “r” letters, rNs. The strand polarity in a duplex is identified on the left. B. A representative pulsed-field gel detecting chromosomal fragmentation after RNase HII treatment. The lanes are marked either with “b” (buffer treatment control) or “H2” (RNase HII treatment). Strains: WT, AB1157; rnhA , L-413; rnhB , L-415; rnhAB , L-416; uvrA rnhAB , L-417. C. Quantification of the RNase treatment-induced fragmentation. The plotted values are means ± SEM from 3-6 independent measurements from gels like in “B”. For RNase A treatment, both low salt (LS) and high salt (HS) conditions are plotted. Since individual fragmentation values are differences between the enzyme and the buffer treatments, some values are negative.
    Figure Legend Snippet: Chromosome fragmentation analysis by RNase HI, RNase HII and RNase A treatment in vitro A. A scheme of various hypothetical R-lesions (R-tract, two types of R-gaps) with positions of cleavage by RNase HI, HII and A (in low salt (LS) and high salt (HS) conditions) shown with arrows of the corresponding color. Small blue “d” letters, dNs; small orange “r” letters, rNs. The strand polarity in a duplex is identified on the left. B. A representative pulsed-field gel detecting chromosomal fragmentation after RNase HII treatment. The lanes are marked either with “b” (buffer treatment control) or “H2” (RNase HII treatment). Strains: WT, AB1157; rnhA , L-413; rnhB , L-415; rnhAB , L-416; uvrA rnhAB , L-417. C. Quantification of the RNase treatment-induced fragmentation. The plotted values are means ± SEM from 3-6 independent measurements from gels like in “B”. For RNase A treatment, both low salt (LS) and high salt (HS) conditions are plotted. Since individual fragmentation values are differences between the enzyme and the buffer treatments, some values are negative.

    Techniques Used: In Vitro, Pulsed-Field Gel

    Growth, morphology and viability of the double rnhAB mutants A. A scheme of in vivo substrates of the two RNase H enzymes. The common substrate, framed in bright green, is the RNA-run with at least four contiguous rNs, which we call “R-tract”. HI and H1, HII and H2 refer to RNase H enzymes of prokaryotes and eukaryotes accordingly. B. Colony size on LB agar, 37°C, 24 hours. Strains: WT, AB1157; Δ rnhA , L-413; Δ rnhB , L-415; Δ rnhAB , L-416. C. Images of rnh and wild type strains stained with DAPI and observed by Hiraga's fluo-phase combined method. Cells were grown at 37°C in LB. The strains are like in “B”. D. Viability of the strains, determined as the ratio of the colony forming units (CFUs) to the microscopic counts in the same volume of the culture. Overnight cultures grown at 30°C were diluted and grown at the temperature (indicated by the first number) to OD 0.2-0.3 (about 2 hours), then cultures were serially diluted and plated on LB plates developed for 16 hours at the temperature indicated by the second number in pairs. Average viability (± SEM) of the eight WT measurements and six measurements for the rnhAB mutant cells is shown (the low titers of the two MG1655 Δ rnhAB cultures at 42°C were not used in the calculation). Strains: AB1157, L-416, MG1655, L-419. E. An enlarged image of the rnhAB mutant cells (processed as in panel C), to show nucleoids of both filamenting and normal-looking cells in some detail. F. Anaerobic growth inhibition of the rnhA and anaerobic lethality of rnhAB strains. Dilution-spotting of strains (like in “B”) was done in an anaerobic chamber on LB plates. Plates were incubated at room temperature in the chamber for 24 hours, then shifted to 28°C aerobic conditions for another 48 hours. G. The uvrA defect further reduces the colony size of the rnhAB double mutant. Strains: rnhAB , L-416; uvrA rnhA , L-414; uvrA rnhAB , L-417.
    Figure Legend Snippet: Growth, morphology and viability of the double rnhAB mutants A. A scheme of in vivo substrates of the two RNase H enzymes. The common substrate, framed in bright green, is the RNA-run with at least four contiguous rNs, which we call “R-tract”. HI and H1, HII and H2 refer to RNase H enzymes of prokaryotes and eukaryotes accordingly. B. Colony size on LB agar, 37°C, 24 hours. Strains: WT, AB1157; Δ rnhA , L-413; Δ rnhB , L-415; Δ rnhAB , L-416. C. Images of rnh and wild type strains stained with DAPI and observed by Hiraga's fluo-phase combined method. Cells were grown at 37°C in LB. The strains are like in “B”. D. Viability of the strains, determined as the ratio of the colony forming units (CFUs) to the microscopic counts in the same volume of the culture. Overnight cultures grown at 30°C were diluted and grown at the temperature (indicated by the first number) to OD 0.2-0.3 (about 2 hours), then cultures were serially diluted and plated on LB plates developed for 16 hours at the temperature indicated by the second number in pairs. Average viability (± SEM) of the eight WT measurements and six measurements for the rnhAB mutant cells is shown (the low titers of the two MG1655 Δ rnhAB cultures at 42°C were not used in the calculation). Strains: AB1157, L-416, MG1655, L-419. E. An enlarged image of the rnhAB mutant cells (processed as in panel C), to show nucleoids of both filamenting and normal-looking cells in some detail. F. Anaerobic growth inhibition of the rnhA and anaerobic lethality of rnhAB strains. Dilution-spotting of strains (like in “B”) was done in an anaerobic chamber on LB plates. Plates were incubated at room temperature in the chamber for 24 hours, then shifted to 28°C aerobic conditions for another 48 hours. G. The uvrA defect further reduces the colony size of the rnhAB double mutant. Strains: rnhAB , L-416; uvrA rnhA , L-414; uvrA rnhAB , L-417.

    Techniques Used: In Vivo, Staining, Mutagenesis, Inhibition, Incubation

    Verification of RNase HI and RNase HII rN-DNA substrate specificity in vitro and the rN-density in DNA of the RNase H + cells and rnh mutants A. A scheme of the two double stranded oligo substrates: 38R1 (single rN) and 34R5 (five consecutive rN). The 32 P label at the 5′ end is shown as a red asterisk. DNA nucleotides are shown as blue lower case “d”, ribonucleotides are orange uppercase “R”. B. Products of the rN-DNA substrate hydrolysis by E. coli RNase HI and RNase HII enzymes. The radiolabelled rN-containing dsDNA oligos (shown in A) were incubated with the RNase HI or RNase HII enzymes. “0.1 M NaOH” and “Na Carb. pH 9.3” refer to alkali conditions in which rN hydrolysis produces reference size products. Numbers “1” or “5” refer to 38R1 or 34R5 oligos (A); ss/ds refers to whether the substrate used in the reaction was single-stranded or double-stranded. RNase H1 and RNase H2 were the E. coli enzymes RNase HI and RNase HII. RNase H1-1 and RNase H1-2 were RNase HI enzymes from different producers. The numbers on the side of the gel represent the sizes of the substrate and cleavage products. The reaction products were analyzed in 18% urea-PAGE gel. C. Only 34R5 oligo was used as either ss or ds substrate. All designations are like in “B”. D. Treatment with RNase HII of the plasmid isolated by alkaline lysis protocol. SCM, supercoiled monomer; b, buffer; H2, RNase HII. Plasmid: pEAK86, plasmid isolation was done at 0°C. Strains for results shown in panels D-I were: WT, AB1157; rnhA , L-413; rnhB , L-415; rnhAB , L-416; uvrA rnhAB L-417. Product of the reactions were run in 1.1% agarose gel; autoradiogram of the representative Southern blot with the radiolabelled pEAK86 DNA as a probe is shown here and also in E and G. E. Treatment with either RNase HI or RNase HII enzymes of the plasmid isolated by the total genomic DNA protocol. SC, supercoiled plasmid; relaxed, relaxed plasmid; chrom., chromosomal DNA. Plasmid: pEAK86. Analysis of plasmid species was carried out as in D. F. Summary of quantification of the RNaseHII-revealed density of rNs in plasmid DNA isolated by various methods from the rnhAB double mutant. The density calculations are described in Methods. “Form.”, formamide. G. Alkali treatment analysis of rN-density. The plasmid DNA isolated by alkaline lysis at 0°C, was linearized and treated with NaOH. Treatment: “—”, no treatment; 0°, 0.2 M NaOH, 20 mM EDTA treatment on ice for 20 min; 45°, 0.3 M NaOH, 20 mM EDTA treatment at 45°C for 90 minutes. ds, linearized plasmid DNA, ss -single stranded plasmid. The samples were run in 1.1% agarose in TAE buffer, at 4°C. H. Summary of quantification of the rN-density determined by either RNase HII or by alkali treatments (from gels like in “G”). Various mutant comparison data are shown, pEAK86 was purified by alkaline lysis only, values are means of three independent measurements ± SEM. The star identifies the value already reported in panel “F”. I. R-loop removal by RNase HI or by RNase A. pAM34 isolated from rnhA (strain L-413) by the total genomic DNA protocol.
    Figure Legend Snippet: Verification of RNase HI and RNase HII rN-DNA substrate specificity in vitro and the rN-density in DNA of the RNase H + cells and rnh mutants A. A scheme of the two double stranded oligo substrates: 38R1 (single rN) and 34R5 (five consecutive rN). The 32 P label at the 5′ end is shown as a red asterisk. DNA nucleotides are shown as blue lower case “d”, ribonucleotides are orange uppercase “R”. B. Products of the rN-DNA substrate hydrolysis by E. coli RNase HI and RNase HII enzymes. The radiolabelled rN-containing dsDNA oligos (shown in A) were incubated with the RNase HI or RNase HII enzymes. “0.1 M NaOH” and “Na Carb. pH 9.3” refer to alkali conditions in which rN hydrolysis produces reference size products. Numbers “1” or “5” refer to 38R1 or 34R5 oligos (A); ss/ds refers to whether the substrate used in the reaction was single-stranded or double-stranded. RNase H1 and RNase H2 were the E. coli enzymes RNase HI and RNase HII. RNase H1-1 and RNase H1-2 were RNase HI enzymes from different producers. The numbers on the side of the gel represent the sizes of the substrate and cleavage products. The reaction products were analyzed in 18% urea-PAGE gel. C. Only 34R5 oligo was used as either ss or ds substrate. All designations are like in “B”. D. Treatment with RNase HII of the plasmid isolated by alkaline lysis protocol. SCM, supercoiled monomer; b, buffer; H2, RNase HII. Plasmid: pEAK86, plasmid isolation was done at 0°C. Strains for results shown in panels D-I were: WT, AB1157; rnhA , L-413; rnhB , L-415; rnhAB , L-416; uvrA rnhAB L-417. Product of the reactions were run in 1.1% agarose gel; autoradiogram of the representative Southern blot with the radiolabelled pEAK86 DNA as a probe is shown here and also in E and G. E. Treatment with either RNase HI or RNase HII enzymes of the plasmid isolated by the total genomic DNA protocol. SC, supercoiled plasmid; relaxed, relaxed plasmid; chrom., chromosomal DNA. Plasmid: pEAK86. Analysis of plasmid species was carried out as in D. F. Summary of quantification of the RNaseHII-revealed density of rNs in plasmid DNA isolated by various methods from the rnhAB double mutant. The density calculations are described in Methods. “Form.”, formamide. G. Alkali treatment analysis of rN-density. The plasmid DNA isolated by alkaline lysis at 0°C, was linearized and treated with NaOH. Treatment: “—”, no treatment; 0°, 0.2 M NaOH, 20 mM EDTA treatment on ice for 20 min; 45°, 0.3 M NaOH, 20 mM EDTA treatment at 45°C for 90 minutes. ds, linearized plasmid DNA, ss -single stranded plasmid. The samples were run in 1.1% agarose in TAE buffer, at 4°C. H. Summary of quantification of the rN-density determined by either RNase HII or by alkali treatments (from gels like in “G”). Various mutant comparison data are shown, pEAK86 was purified by alkaline lysis only, values are means of three independent measurements ± SEM. The star identifies the value already reported in panel “F”. I. R-loop removal by RNase HI or by RNase A. pAM34 isolated from rnhA (strain L-413) by the total genomic DNA protocol.

    Techniques Used: In Vitro, Incubation, Polyacrylamide Gel Electrophoresis, Plasmid Preparation, Isolation, Alkaline Lysis, Agarose Gel Electrophoresis, Southern Blot, Mutagenesis, Purification

    34) Product Images from "Transcript assembly and abundance estimation from RNA-Seq reveals thousands of new transcripts and switching among isoforms"

    Article Title: Transcript assembly and abundance estimation from RNA-Seq reveals thousands of new transcripts and switching among isoforms

    Journal: Nature biotechnology

    doi: 10.1038/nbt.1621

    Overview of Cufflinks. The algorithm takes as input cDNA fragment sequences that have been ( a ) aligned to the genome by software capable of producing spliced alignments, such as TopHat. With paired-end RNA-Seq, Cufflinks treats each pair of fragment reads as a single alignment. The algorithm assembles overlapping ‘bundles’ of fragment alignments ( b-c ) separately, which reduces running time and memory use because each bundle typically contains the fragments from no more than a few genes. Cufflinks then estimates the abundances of the assembled transcripts ( d-e ). ( b ) The first step in fragment assembly is to identify pairs of ‘incompatible’ fragments that must have originated from distinct spliced mRNA isoforms. Fragments are connected in an ‘overlap graph’ when they are compatible and their alignments overlap in the genome. Each fragment has one node in the graph, and an edge, directed from left to right along the genome, is placed between each pair of compatible fragments. In this example, the yellow, blue, and red fragments must have originated from separate isoforms, but any other fragment could have come from the same transcript as one of these three. ( c ) Assembling isoforms from the overlap graph. Paths through the graph correspond to sets of mutually compatible fragments that could be merged into complete isoforms. The overlap graph here can be minimally ‘covered’ by three paths, each representing a different isoform. Dilworth's Theorem states that the number of mutually incompatible reads is the same as the minimum number of transcripts needed to “explain” all the fragments. Cufflinks implements a proof of Dilworth's Theorem that produces a minimal set of paths that cover all the fragments in the overlap graph by finding the largest set of reads with the property that no two could have originated from the same isoform. ( d ) Estimating transcript abundance. Fragments are matched (denoted here using color) to the transcripts from which they could have originated. The violet fragment could have originated from the blue or red isoform. Gray fragments could have come from any of the three shown. Cufflinks estimates transcript abundances using a statistical model in which the probability of observing each fragment is a linear function of the abundances of the transcripts from which it could have originated. Because only the ends of each fragment are sequenced, the length of each may be unknown. Assigning a fragment to different isoforms often implies a different length for it. Cufflinks can incorporate the distribution of fragment lengths to help assign fragments to isoforms. For example, the violet fragment would be much longer, and very improbable according to Cufflinks' model, if it were to come from the red isoform instead of the blue isoform. ( e ) The program then numerically maximizes a function that assigns a likelihood to all possible sets of relative abundances of the yellow, red and blue isoforms (γ 1 ,γ 2 ,γ 3 ), producing the abundances that best explain the observed fragments, shown as a pie chart.
    Figure Legend Snippet: Overview of Cufflinks. The algorithm takes as input cDNA fragment sequences that have been ( a ) aligned to the genome by software capable of producing spliced alignments, such as TopHat. With paired-end RNA-Seq, Cufflinks treats each pair of fragment reads as a single alignment. The algorithm assembles overlapping ‘bundles’ of fragment alignments ( b-c ) separately, which reduces running time and memory use because each bundle typically contains the fragments from no more than a few genes. Cufflinks then estimates the abundances of the assembled transcripts ( d-e ). ( b ) The first step in fragment assembly is to identify pairs of ‘incompatible’ fragments that must have originated from distinct spliced mRNA isoforms. Fragments are connected in an ‘overlap graph’ when they are compatible and their alignments overlap in the genome. Each fragment has one node in the graph, and an edge, directed from left to right along the genome, is placed between each pair of compatible fragments. In this example, the yellow, blue, and red fragments must have originated from separate isoforms, but any other fragment could have come from the same transcript as one of these three. ( c ) Assembling isoforms from the overlap graph. Paths through the graph correspond to sets of mutually compatible fragments that could be merged into complete isoforms. The overlap graph here can be minimally ‘covered’ by three paths, each representing a different isoform. Dilworth's Theorem states that the number of mutually incompatible reads is the same as the minimum number of transcripts needed to “explain” all the fragments. Cufflinks implements a proof of Dilworth's Theorem that produces a minimal set of paths that cover all the fragments in the overlap graph by finding the largest set of reads with the property that no two could have originated from the same isoform. ( d ) Estimating transcript abundance. Fragments are matched (denoted here using color) to the transcripts from which they could have originated. The violet fragment could have originated from the blue or red isoform. Gray fragments could have come from any of the three shown. Cufflinks estimates transcript abundances using a statistical model in which the probability of observing each fragment is a linear function of the abundances of the transcripts from which it could have originated. Because only the ends of each fragment are sequenced, the length of each may be unknown. Assigning a fragment to different isoforms often implies a different length for it. Cufflinks can incorporate the distribution of fragment lengths to help assign fragments to isoforms. For example, the violet fragment would be much longer, and very improbable according to Cufflinks' model, if it were to come from the red isoform instead of the blue isoform. ( e ) The program then numerically maximizes a function that assigns a likelihood to all possible sets of relative abundances of the yellow, red and blue isoforms (γ 1 ,γ 2 ,γ 3 ), producing the abundances that best explain the observed fragments, shown as a pie chart.

    Techniques Used: Software, RNA Sequencing Assay

    35) Product Images from "The E2A-HLF Oncoprotein Activates Groucho-Related Genes and Suppresses Runx1"

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

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.21.17.5935-5945.2001

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

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

    36) Product Images from "AICAR-mediated AMPK activation induces protective innate responses in bacterial endophthalmitis"

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

    Journal: Cellular microbiology

    doi: 10.1111/cmi.12625

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

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

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

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

    37) Product Images from "Affinity selection of DNA-binding protein complexes using mRNA display"

    Article Title: Affinity selection of DNA-binding protein complexes using mRNA display

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gnj025

    Schematic representation of the IVV selection procedure. (1) A cDNA library is transcribed and ligated with a Fluoro-PEG Puro spacer. (2) The IVV template RNA library is translated in a wheat germ cell-free translation system. (3) The constructed IVV library is purified with anti-FLAG M2 antibody-immobilized beads to remove untranslated mRNA and impurities contained in the wheat germ mixture. (4) The purified IVV are (5) subjected to affinity selection with mutated bait DNA-immobilized beads to eliminate non-specific binders to DNA. (6) Unbound IVV are then subjected to affinity selection with bait DNA-immobilized beads. (7) After washing, DNA-binding IVV are eluted by DNase I digestion. (8) The mRNA portions of the selected IVV are reverse-transcribed, PCR-amplified and (9) subjected to the next round of selection or (10) identified by cloning and sequencing.
    Figure Legend Snippet: Schematic representation of the IVV selection procedure. (1) A cDNA library is transcribed and ligated with a Fluoro-PEG Puro spacer. (2) The IVV template RNA library is translated in a wheat germ cell-free translation system. (3) The constructed IVV library is purified with anti-FLAG M2 antibody-immobilized beads to remove untranslated mRNA and impurities contained in the wheat germ mixture. (4) The purified IVV are (5) subjected to affinity selection with mutated bait DNA-immobilized beads to eliminate non-specific binders to DNA. (6) Unbound IVV are then subjected to affinity selection with bait DNA-immobilized beads. (7) After washing, DNA-binding IVV are eluted by DNase I digestion. (8) The mRNA portions of the selected IVV are reverse-transcribed, PCR-amplified and (9) subjected to the next round of selection or (10) identified by cloning and sequencing.

    Techniques Used: Selection, cDNA Library Assay, Construct, Purification, Binding Assay, Polymerase Chain Reaction, Amplification, Clone Assay, Sequencing

    38) Product Images from "Conserved Subgroups and Developmental Regulation in the Monocot rop Gene Family 1 Gene Family 1 [w]"

    Article Title: Conserved Subgroups and Developmental Regulation in the Monocot rop Gene Family 1 Gene Family 1 [w]

    Journal: Plant Physiology

    doi: 10.1104/pp.103.029900

    Agarose gel electrophoresis of MTRP amplification products shows a high degree of overlap in individual maize rop expression patterns in vegetative tissues. PCR templates are 4-fold serial dilutions of cDNA made from root tip (RT), root shank (RS), shoot apex (SA), mature leaf (ML), and pollen (P) RNA samples. Lanes correspond to the dilution steps taken from the original cDNA (i.e. the template used in the lane 1 reaction was the initial 4-fold dilution of cDNA). Amplification of the actin1 gene is an internal control for the vegetative tissue samples; Elongation Factor1 -α is the internal control for the pollen samples. The left-most lane in the RT panel shows the M r standard (100-bp markers; the 500 bp marker is the brightest band). Similar results for the relative expression levels of each rop were observed in three independent experiments.
    Figure Legend Snippet: Agarose gel electrophoresis of MTRP amplification products shows a high degree of overlap in individual maize rop expression patterns in vegetative tissues. PCR templates are 4-fold serial dilutions of cDNA made from root tip (RT), root shank (RS), shoot apex (SA), mature leaf (ML), and pollen (P) RNA samples. Lanes correspond to the dilution steps taken from the original cDNA (i.e. the template used in the lane 1 reaction was the initial 4-fold dilution of cDNA). Amplification of the actin1 gene is an internal control for the vegetative tissue samples; Elongation Factor1 -α is the internal control for the pollen samples. The left-most lane in the RT panel shows the M r standard (100-bp markers; the 500 bp marker is the brightest band). Similar results for the relative expression levels of each rop were observed in three independent experiments.

    Techniques Used: Agarose Gel Electrophoresis, Amplification, Expressing, Polymerase Chain Reaction, Marker

    39) Product Images from "Differential Gene Expression in CD8+ Cells from HIV-1 Infected Subjects Showing Suppression of HIV Replication"

    Article Title: Differential Gene Expression in CD8+ Cells from HIV-1 Infected Subjects Showing Suppression of HIV Replication

    Journal: Virology

    doi: 10.1016/j.virol.2006.12.007

    Preparation of cRNA for microarray hybridization. Total RNA was isolated and purified from CD8+ cells from HIV+ subjects showing high CNAR and seronegative control subjects with none or low CNAR using a combined Trizol / RNeasy method. Complementary DNA (cDNA) was synthesized with the SuperScript™ Double Stranded cDNA Synthesis Kit. The cDNA was then subjected to an in vitro for details.
    Figure Legend Snippet: Preparation of cRNA for microarray hybridization. Total RNA was isolated and purified from CD8+ cells from HIV+ subjects showing high CNAR and seronegative control subjects with none or low CNAR using a combined Trizol / RNeasy method. Complementary DNA (cDNA) was synthesized with the SuperScript™ Double Stranded cDNA Synthesis Kit. The cDNA was then subjected to an in vitro for details.

    Techniques Used: Microarray, Hybridization, Isolation, Purification, Synthesized, In Vitro

    40) Product Images from "MasABK Proteins Interact with Proteins of the Type IV Pilin System to Affect Social Motility of Myxococcus xanthus"

    Article Title: MasABK Proteins Interact with Proteins of the Type IV Pilin System to Affect Social Motility of Myxococcus xanthus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0054557

    Deletion of pilA does not affect masB transcription. RNA was harvested from 1×10 8 cells and mRNA was used to generate cDNA using a random hexanucleotide primer. Gene specific primers for masB and the 16S gene (endogenous control) were used in the amplification reaction with cDNA. masB expression was amplified from cDNA using 50, 5 and 0.5 ng template, while 16 S expression was amplified using 200, 20 and 2 pg cDNA. Both the WT and pilA strain express masB (black band in lanes 1 and 4 respectively). Hence, deletion of pilA does not adversely affect the expression of masB in M. xanthus . Deletion of masABK abolished masB expression (lanes 7–9).
    Figure Legend Snippet: Deletion of pilA does not affect masB transcription. RNA was harvested from 1×10 8 cells and mRNA was used to generate cDNA using a random hexanucleotide primer. Gene specific primers for masB and the 16S gene (endogenous control) were used in the amplification reaction with cDNA. masB expression was amplified from cDNA using 50, 5 and 0.5 ng template, while 16 S expression was amplified using 200, 20 and 2 pg cDNA. Both the WT and pilA strain express masB (black band in lanes 1 and 4 respectively). Hence, deletion of pilA does not adversely affect the expression of masB in M. xanthus . Deletion of masABK abolished masB expression (lanes 7–9).

    Techniques Used: Amplification, Expressing

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