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  • 99
    Integrated DNA Technologies rna template
    Rna Template, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna template/product/Integrated DNA Technologies
    Average 99 stars, based on 12 article reviews
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    94
    Worthington Biochemical rna template
    In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- <t>RNA,</t> which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. bRT is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and dNTPs, including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with <t>RNase</t> (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.
    Rna Template, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna template/product/Worthington Biochemical
    Average 94 stars, based on 4 article reviews
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    99
    Millipore template rna
    <t>CT-RT-LAMP</t> assays for YMV detection in yam. Amplification products were visualised by agarose gel electrophoresis (A) and with SYBR Green (B). Samples in lanes 1, 2 and 3 are YMV-infected samples. B, non-template control; M, 100-bp DNA ladder. (C) CT-RT-LAMP with loop primers. (D) CT-RT-LAMP, without loop primers. Samples in lanes 1 to 4: YMV-infected samples, 5 – 6, healthy yam samples. ( E and F ) Comparison between RT-PCR (E) and CT-RT-LAMP (F) for detecting YMV in infected yam leaf sample. Tenfold dilutions of total <t>RNA</t> from 100 ng/µl were tested. RT-PCR products were analyzed by agarose gel electrophoresis, and visual detection of RT-LAMP was done using SYBR Green dye. M, 100-bp DNA ladder; H, healthy yam RNA sample; NTC, no-template control
    Template Rna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/template rna/product/Millipore
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    99
    Millipore standard rna templates
    Stage and tissue expression profiles of Ixodes ricinus cysteine and aspartic peptidases . Messenger <t>RNA</t> levels of individual enzymes were determined by semi-quantitative two-step RT <t>PCR.</t> Panel A : Expression of peptidase mRNAs in whole body homogenates of I. ricinus eggs, unfed larvae, unfed nymphs, males, unfed females and females attached for 1 day on the guinea pigs. Panel B : Expression of peptidase mRNAs in tissues dissected from partially engorged females (the 5-th day of feeding). The abbreviations used are as in the text. IrFer shows the mRNA amplification of tick ferritin used as template loading control. (For details, see Methods).
    Standard Rna Templates, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/standard rna templates/product/Millipore
    Average 99 stars, based on 18 article reviews
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    92
    PerkinElmer rna template
    Stage and tissue expression profiles of Ixodes ricinus cysteine and aspartic peptidases . Messenger <t>RNA</t> levels of individual enzymes were determined by semi-quantitative two-step RT <t>PCR.</t> Panel A : Expression of peptidase mRNAs in whole body homogenates of I. ricinus eggs, unfed larvae, unfed nymphs, males, unfed females and females attached for 1 day on the guinea pigs. Panel B : Expression of peptidase mRNAs in tissues dissected from partially engorged females (the 5-th day of feeding). The abbreviations used are as in the text. IrFer shows the mRNA amplification of tick ferritin used as template loading control. (For details, see Methods).
    Rna Template, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 92/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna template/product/PerkinElmer
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    88
    Thermo Fisher rna century marker templates
    Detection of DHPR α 1S gene expression in C2C12 cells and mouse skeletal muscle A 140 bp protected fragment obtained by specific antisense <t>RNA</t> probe hybridized to the same amount of total RNA (25 μg) from mouse skeletal muscle, C2C12 cell myoblasts in growth medium and C2C12 cell myotubes after 5 days in differentiation medium. The relative amount of total RNA loaded for each sample is indicated by the signal resulting from the hybridization of the 115 bp protected fragment for <t>28S</t> rRNA in the same reaction.
    Rna Century Marker Templates, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna century marker templates/product/Thermo Fisher
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    88
    Roche ms2 rna template
    Detection of DHPR α 1S gene expression in C2C12 cells and mouse skeletal muscle A 140 bp protected fragment obtained by specific antisense <t>RNA</t> probe hybridized to the same amount of total RNA (25 μg) from mouse skeletal muscle, C2C12 cell myoblasts in growth medium and C2C12 cell myotubes after 5 days in differentiation medium. The relative amount of total RNA loaded for each sample is indicated by the signal resulting from the hybridization of the 115 bp protected fragment for <t>28S</t> rRNA in the same reaction.
    Ms2 Rna Template, supplied by Roche, used in various techniques. Bioz Stars score: 88/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher rna template
    The size distribution of both total small <t>RNA</t> and <t>miRNA</t> sequences that were identified in Healthy (H) and Phytoplasma-infected (I) plants. Whereas most of the small RNAs were either 21 nt or 24 nt, most of the miRNAs were either 21 nt or 22 nt.
    Rna Template, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 3288 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Horizon Discovery rna oligonucleotide template
    Metabolism and mechanism of antiviral activity of GS-5734. a , Chemical structures of GS-5734 and metabolic conversion to NTP. b , NTP formation in human monocyte-derived macrophages following 72-h incubation with 1 μM GS-5734 (black) or Nuc (red); mean ± SD, from 3 donors. c , Antiviral activity of GS-5734 in HeLa cells against EBOV-Makona (black symbols), EBOV-Kikwit (open symbols), MARV (red), BDBV (orange), SUDV (blue); mean ± SD from triplicates. d , Amino acid sequence homology of EBOV and <t>RSV</t> RdRp active site. *, residues predicted to contact NTP. e , Inhibition of RSV RdRp (blue), but not human <t>RNA</t> Pol II (black) or mitochondrial (mt) RNA (red) polymerases by NTP ; mean ± SD, n=3 biological replicates. f , NTP-induced RNA chain termination in RSV RdRp primer-extension assay. For gel source data, see Supplementary Figure 1 .
    Rna Oligonucleotide Template, supplied by Horizon Discovery, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna oligonucleotide template/product/Horizon Discovery
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    93
    Thermo Fisher rna century plus marker template
    The requirement of the response regulator RisA for rgtA expression. ( A ) Schematic representation of RisA binding motifs in rgtA promoter region. ( Left ) Three plausible RisA binding sites (gray circles 1 – 3 ) are present in a 60-bp region (−277 to −217) upstream of the rgtA transcriptional start site. This 60-bp region has been deleted, resulting in the rgtA Δ277/217 mutant. ( Right ) Sequences of canonical RisA-binding motif as well of three RisA-binding motifs found upstream of the rgtA promoter are depicted; nucleotides different from consensus are in gray. ( B ) B. pertussis Tohama I strain (lane 1 ) and its isogenic Δ risA (lane 2 ) and rgtA Δ277/217 (lane 3 ) mutants were grown in SS medium to exponential phase. <t>RNA</t> isolated from harvested cells was probed with <t>biotinylated</t> RgtA-specific probe and subjected to northern blot analysis ( upper panel). Signals obtained upon rehybridization of the membrane with SsrA-specific probe ( lower panel) served as loading control. Only relevant parts of the membranes are shown. The result is a representative of two experiments.
    Rna Century Plus Marker Template, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. bRT is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and dNTPs, including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. bRT is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and dNTPs, including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.

    Article Snippet: Reverse transcription reactions with HIV-1 RT were carried out as above, except in 10 μl and containing 10 units RNase inhibitor (NEB), 0.1 μCi/μl [α-32 P]dCTP, 30 ng/μl RNA template, 1 μM PG117 primer ( ) and 2 units of HIV-1 RT (Worthington Biochemical), and the reaction was carried out for 30 min.

    Techniques: In Vitro, Binding Assay, Sequencing, Incubation, Polyacrylamide Gel Electrophoresis, Labeling, Marker, Activity Assay, Produced

    Branched RNA–cDNA. ( A ) The 580 nt DGR RNA was biotinylated at its 3′ end and used as a template for reverse-transcription by bRT-Avd, after which biotinylated RNA was captured with streptavidin beads, and the presence of TR- cDNA was detected by PCR using the indicated primers. ( B ) The 580 nt DGR RNA was biotinylated at its 3′ end (RNA-Bio), and either reacted with no protein or used as a template for reverse transcription with bRT-Avd. The 580 nt DGR RNA in its unbiotinylated form (RNA) was also used as a template for reverse transcription with bRT-Avd. Samples were then purified using streptavidin beads, and the presence of TR -cDNA in the purified samples was assessed by PCR. Products from the PCR reaction were resolved on an agarose gel. ( C ) Hybrid dA56 580 nt DGR RNA containing deoxyadenosine at Sp 56 (indicated with H at 2′ position) and hybrid d56 580 nt DGR RNA containing adenosine at Sp 56 (indicated with OH at 2′). Both molecules terminate at Sp 140 and have a dideoxynucleotide at the 3′ end (indicated with H at 3′). ( D ) Radiolabeled products resulting from bRT-Avd activity for 12 h with 580 nt DGR RNA, hybrid 580 nt dA56, or hybrid 580 nt A56 DGR RNA as template. Products were untreated (U) or RNase-treated (+R), and resolved by denaturing PAGE. Separate samples of dA56 and A56 were 5′ 32 P-labeled for visualization of input templates (I). The positions of the 120 and 90 nt cDNAs are indicated.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: Branched RNA–cDNA. ( A ) The 580 nt DGR RNA was biotinylated at its 3′ end and used as a template for reverse-transcription by bRT-Avd, after which biotinylated RNA was captured with streptavidin beads, and the presence of TR- cDNA was detected by PCR using the indicated primers. ( B ) The 580 nt DGR RNA was biotinylated at its 3′ end (RNA-Bio), and either reacted with no protein or used as a template for reverse transcription with bRT-Avd. The 580 nt DGR RNA in its unbiotinylated form (RNA) was also used as a template for reverse transcription with bRT-Avd. Samples were then purified using streptavidin beads, and the presence of TR -cDNA in the purified samples was assessed by PCR. Products from the PCR reaction were resolved on an agarose gel. ( C ) Hybrid dA56 580 nt DGR RNA containing deoxyadenosine at Sp 56 (indicated with H at 2′ position) and hybrid d56 580 nt DGR RNA containing adenosine at Sp 56 (indicated with OH at 2′). Both molecules terminate at Sp 140 and have a dideoxynucleotide at the 3′ end (indicated with H at 3′). ( D ) Radiolabeled products resulting from bRT-Avd activity for 12 h with 580 nt DGR RNA, hybrid 580 nt dA56, or hybrid 580 nt A56 DGR RNA as template. Products were untreated (U) or RNase-treated (+R), and resolved by denaturing PAGE. Separate samples of dA56 and A56 were 5′ 32 P-labeled for visualization of input templates (I). The positions of the 120 and 90 nt cDNAs are indicated.

    Article Snippet: Reverse transcription reactions with HIV-1 RT were carried out as above, except in 10 μl and containing 10 units RNase inhibitor (NEB), 0.1 μCi/μl [α-32 P]dCTP, 30 ng/μl RNA template, 1 μM PG117 primer ( ) and 2 units of HIV-1 RT (Worthington Biochemical), and the reaction was carried out for 30 min.

    Techniques: Polymerase Chain Reaction, Purification, Agarose Gel Electrophoresis, Activity Assay, Polyacrylamide Gel Electrophoresis, Labeling

    Core DGR RNA. ( A ) Schematic of core DGR RNA. ( B ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the core DGR RNA as template. Prior to the reverse transcription reaction, the RNA template was untreated (-Per) or treated with periodate (+Per). Products from the reaction were untreated (U) or treated with RNase (+R), and resolved by 6% denaturing PAGE. Lane T corresponds to internally-labeled core DGR RNA as a marker for the size of the template. Red arrowheads indicate radiolabeled product bands that migrate at the same position or slower than the core DGR RNA, and green arrowheads ones that migrate faster. The positions of the 120 and 90 nt cDNA bands are indicated. The two panels are from the same gel, with the black line indicating that intermediate lanes were removed. ( C ) Internally-labeled core DGR RNA was not incubated (–), or incubated with bRT-Avd alone or bRT-Avd with 100 μM standard dNTPs (+dNTP), 100 μM dCTP (+CTP), 100 μM dNTPs excluding dCTP (+d(A,T,G)TP), or 100 μM nonhydrolyzeable analog of dCTP (+N-dCTP) for 2 h. Incubation products were resolved by denaturing PAGE. The band corresponding to the 5′ fragment of the cleaved core RNA containing either a deoxycytidine alone (5′+dC) or cDNA (5′+cDNA), and the band corresponding to the 3′ fragment of the RNA are indicated. ( D ) The core DGR RNA was biotinylated at its 3′ end (RNA-Bio), and either reacted with no protein or used as a template for reverse transcription with bRT-Avd. The core DGR RNA in its unbiotinylated form (RNA) was also used as a template for reverse transcription with bRT-Avd. Samples were then purified using streptavidin beads, and the presence of TR -cDNA in the purified samples was assessed by PCR. Products from the PCR reaction were resolved on an agarose gel. ( E ) Radiolabeled products resulting from bRT-Avd activity for 12 h with core, hybrid core dA56, or hybrid core A56 DGR RNA as template. Products were untreated (U) or treated with RNase (+R), and resolved by denaturing PAGE. Separate samples of core dA56 and A56 were 5′ 32 P-labeled for visualization of inputs (I). The positions of the 120 and 90 nt cDNAs are indicated.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: Core DGR RNA. ( A ) Schematic of core DGR RNA. ( B ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the core DGR RNA as template. Prior to the reverse transcription reaction, the RNA template was untreated (-Per) or treated with periodate (+Per). Products from the reaction were untreated (U) or treated with RNase (+R), and resolved by 6% denaturing PAGE. Lane T corresponds to internally-labeled core DGR RNA as a marker for the size of the template. Red arrowheads indicate radiolabeled product bands that migrate at the same position or slower than the core DGR RNA, and green arrowheads ones that migrate faster. The positions of the 120 and 90 nt cDNA bands are indicated. The two panels are from the same gel, with the black line indicating that intermediate lanes were removed. ( C ) Internally-labeled core DGR RNA was not incubated (–), or incubated with bRT-Avd alone or bRT-Avd with 100 μM standard dNTPs (+dNTP), 100 μM dCTP (+CTP), 100 μM dNTPs excluding dCTP (+d(A,T,G)TP), or 100 μM nonhydrolyzeable analog of dCTP (+N-dCTP) for 2 h. Incubation products were resolved by denaturing PAGE. The band corresponding to the 5′ fragment of the cleaved core RNA containing either a deoxycytidine alone (5′+dC) or cDNA (5′+cDNA), and the band corresponding to the 3′ fragment of the RNA are indicated. ( D ) The core DGR RNA was biotinylated at its 3′ end (RNA-Bio), and either reacted with no protein or used as a template for reverse transcription with bRT-Avd. The core DGR RNA in its unbiotinylated form (RNA) was also used as a template for reverse transcription with bRT-Avd. Samples were then purified using streptavidin beads, and the presence of TR -cDNA in the purified samples was assessed by PCR. Products from the PCR reaction were resolved on an agarose gel. ( E ) Radiolabeled products resulting from bRT-Avd activity for 12 h with core, hybrid core dA56, or hybrid core A56 DGR RNA as template. Products were untreated (U) or treated with RNase (+R), and resolved by denaturing PAGE. Separate samples of core dA56 and A56 were 5′ 32 P-labeled for visualization of inputs (I). The positions of the 120 and 90 nt cDNAs are indicated.

    Article Snippet: Reverse transcription reactions with HIV-1 RT were carried out as above, except in 10 μl and containing 10 units RNase inhibitor (NEB), 0.1 μCi/μl [α-32 P]dCTP, 30 ng/μl RNA template, 1 μM PG117 primer ( ) and 2 units of HIV-1 RT (Worthington Biochemical), and the reaction was carried out for 30 min.

    Techniques: Activity Assay, Polyacrylamide Gel Electrophoresis, Labeling, Marker, Incubation, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    TR -Sp interactions. ( A ) Complementarity between TR (blue) and Sp (purple) segments. Potential basepairs are numbered (wobble in red). ( B ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2h with the WT or mutated 580 nt DGR RNA as template. Products were treated with RNase, and resolved by denaturing PAGE. Numbers over lane labels correspond to the basepair tested by the mutation, with ‘R’ referring to restoration of the basepair. Sp Mut4 corresponds to Sp 55-CAGC substituted with 55-GUCG.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: TR -Sp interactions. ( A ) Complementarity between TR (blue) and Sp (purple) segments. Potential basepairs are numbered (wobble in red). ( B ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2h with the WT or mutated 580 nt DGR RNA as template. Products were treated with RNase, and resolved by denaturing PAGE. Numbers over lane labels correspond to the basepair tested by the mutation, with ‘R’ referring to restoration of the basepair. Sp Mut4 corresponds to Sp 55-CAGC substituted with 55-GUCG.

    Article Snippet: Reverse transcription reactions with HIV-1 RT were carried out as above, except in 10 μl and containing 10 units RNase inhibitor (NEB), 0.1 μCi/μl [α-32 P]dCTP, 30 ng/μl RNA template, 1 μM PG117 primer ( ) and 2 units of HIV-1 RT (Worthington Biochemical), and the reaction was carried out for 30 min.

    Techniques: Activity Assay, Polyacrylamide Gel Electrophoresis, Mutagenesis

    Adenine mutagenesis and template-priming. ( A ) Covalently-linked RNA–cDNA molecule. The linkage is to Sp A56 of the RNA, and the first nucleotide reverse transcribed is TR G117. The RT-PCR product resulting from primers 1 and 2 (blue arrows) is indicated by the dashed red line. ( B ) RT-PCR amplicons from 580 nt DGR RNA reacted with no protein (–), bRT, Avd, or bRT-Avd, separated on a 2% agarose gel and ethidium bromide-stained. The specific amplicon produced from reaction with bRT-Avd shown by the red arrowhead. ( C ) Percentage of substitutions in TR -cDNA determined by sequencing. ( D ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity with the 580 nt DGR RNA as template for 2 h (left) or 12 h (right). Either standard dNTPs (dATP, dGTP, dCTP, TTP), as indicated by ‘+’,were present in the reaction, or standard dNTPs excluding dATP (-A), dGTP (–G), or TTP (-T) were present. Products were treated with RNase, and resolved by denaturing PAGE. ( E ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying TTP (top) or dUTP (bottom) concentrations. Products were treated with RNase, and resolved by denaturing PAGE. ( F ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying dUTP concentrations. Products were either RNase-treated (top), or both RNase- and UDG-treated (bottom), and resolved by denaturing PAGE.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: Adenine mutagenesis and template-priming. ( A ) Covalently-linked RNA–cDNA molecule. The linkage is to Sp A56 of the RNA, and the first nucleotide reverse transcribed is TR G117. The RT-PCR product resulting from primers 1 and 2 (blue arrows) is indicated by the dashed red line. ( B ) RT-PCR amplicons from 580 nt DGR RNA reacted with no protein (–), bRT, Avd, or bRT-Avd, separated on a 2% agarose gel and ethidium bromide-stained. The specific amplicon produced from reaction with bRT-Avd shown by the red arrowhead. ( C ) Percentage of substitutions in TR -cDNA determined by sequencing. ( D ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity with the 580 nt DGR RNA as template for 2 h (left) or 12 h (right). Either standard dNTPs (dATP, dGTP, dCTP, TTP), as indicated by ‘+’,were present in the reaction, or standard dNTPs excluding dATP (-A), dGTP (–G), or TTP (-T) were present. Products were treated with RNase, and resolved by denaturing PAGE. ( E ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying TTP (top) or dUTP (bottom) concentrations. Products were treated with RNase, and resolved by denaturing PAGE. ( F ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying dUTP concentrations. Products were either RNase-treated (top), or both RNase- and UDG-treated (bottom), and resolved by denaturing PAGE.

    Article Snippet: Reverse transcription reactions with HIV-1 RT were carried out as above, except in 10 μl and containing 10 units RNase inhibitor (NEB), 0.1 μCi/μl [α-32 P]dCTP, 30 ng/μl RNA template, 1 μM PG117 primer ( ) and 2 units of HIV-1 RT (Worthington Biochemical), and the reaction was carried out for 30 min.

    Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Amplification, Produced, Sequencing, Activity Assay, Polyacrylamide Gel Electrophoresis

    Specificity to DGR RNA. ( A ) Top, schematic of DGR RNA template and primer P G117 . Bottom, radiolabeled products resulting from bRT-Avd activity for 2 h with intact 580 nt DGR RNA or DGR RNA truncated at Sp A56 as template. Reverse transcription reactions were carried out in the absence (-P) or presence of primer P G117 . Reaction products were untreated (U), treated with RNase (+R), or treated with DNase (+D), and resolved by denaturing PAGE. The blue line indicates ODN-primed cDNA products. The red dot indicates ODN-primed 120 nt cDNA (cDNA + 20 nt primer for a 140 nt band). ( B ) Protection of internally-labeled 580 nt DGR RNA from RNase by bRT, Avd, or bRT-Avd, with products resolved by 15% denaturing PAGE. The protected band (P) is indicated. ( C ) RNase protection by Avd, as in panel B, carried out on internally-labeled wild-type 580 nt DGR RNA or 580 nt DGR RNA with scrambled (Sc) Sp sequences, with the first lane in each pair untreated and the second RNase-treated. Products were resolved by denaturing PAGE. The protected band (P) is indicated. ( D ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the WT 580 nt DGR RNA or DGR RNA containing scrambled (Sc) Sp sequences as template. The last lane shows the activity of Avd alone for 2 h with the WT 580 nt DGR RNA as template. Products were treated with RNase, and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs indicated. ( E ) Model of processive polymerization of adenine-mutagenized cDNA by bRT-Avd/RNA ribonucleoprotein particle. The 2′-OH of Sp 56 serves as the priming site and forms a 2′-5′ phosphodiester bond with the cDNA. The first nucleotide reverse transcribed is TR 117. Adenines in TR are unfaithfully reverse transcribed by bRT-Avd (represented by ‘N’). The RNP promotes processive polymerization, which terminates at one of two stops in the DGR RNA.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: Specificity to DGR RNA. ( A ) Top, schematic of DGR RNA template and primer P G117 . Bottom, radiolabeled products resulting from bRT-Avd activity for 2 h with intact 580 nt DGR RNA or DGR RNA truncated at Sp A56 as template. Reverse transcription reactions were carried out in the absence (-P) or presence of primer P G117 . Reaction products were untreated (U), treated with RNase (+R), or treated with DNase (+D), and resolved by denaturing PAGE. The blue line indicates ODN-primed cDNA products. The red dot indicates ODN-primed 120 nt cDNA (cDNA + 20 nt primer for a 140 nt band). ( B ) Protection of internally-labeled 580 nt DGR RNA from RNase by bRT, Avd, or bRT-Avd, with products resolved by 15% denaturing PAGE. The protected band (P) is indicated. ( C ) RNase protection by Avd, as in panel B, carried out on internally-labeled wild-type 580 nt DGR RNA or 580 nt DGR RNA with scrambled (Sc) Sp sequences, with the first lane in each pair untreated and the second RNase-treated. Products were resolved by denaturing PAGE. The protected band (P) is indicated. ( D ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the WT 580 nt DGR RNA or DGR RNA containing scrambled (Sc) Sp sequences as template. The last lane shows the activity of Avd alone for 2 h with the WT 580 nt DGR RNA as template. Products were treated with RNase, and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs indicated. ( E ) Model of processive polymerization of adenine-mutagenized cDNA by bRT-Avd/RNA ribonucleoprotein particle. The 2′-OH of Sp 56 serves as the priming site and forms a 2′-5′ phosphodiester bond with the cDNA. The first nucleotide reverse transcribed is TR 117. Adenines in TR are unfaithfully reverse transcribed by bRT-Avd (represented by ‘N’). The RNP promotes processive polymerization, which terminates at one of two stops in the DGR RNA.

    Article Snippet: Reverse transcription reactions with HIV-1 RT were carried out as above, except in 10 μl and containing 10 units RNase inhibitor (NEB), 0.1 μCi/μl [α-32 P]dCTP, 30 ng/μl RNA template, 1 μM PG117 primer ( ) and 2 units of HIV-1 RT (Worthington Biochemical), and the reaction was carried out for 30 min.

    Techniques: Activity Assay, Polyacrylamide Gel Electrophoresis, Labeling

    CT-RT-LAMP assays for YMV detection in yam. Amplification products were visualised by agarose gel electrophoresis (A) and with SYBR Green (B). Samples in lanes 1, 2 and 3 are YMV-infected samples. B, non-template control; M, 100-bp DNA ladder. (C) CT-RT-LAMP with loop primers. (D) CT-RT-LAMP, without loop primers. Samples in lanes 1 to 4: YMV-infected samples, 5 – 6, healthy yam samples. ( E and F ) Comparison between RT-PCR (E) and CT-RT-LAMP (F) for detecting YMV in infected yam leaf sample. Tenfold dilutions of total RNA from 100 ng/µl were tested. RT-PCR products were analyzed by agarose gel electrophoresis, and visual detection of RT-LAMP was done using SYBR Green dye. M, 100-bp DNA ladder; H, healthy yam RNA sample; NTC, no-template control

    Journal: Archives of Virology

    Article Title: Chromogenic detection of yam mosaic virus by closed-tube reverse transcription loop-mediated isothermal amplification (CT-RT-LAMP)

    doi: 10.1007/s00705-018-3706-0

    Figure Lengend Snippet: CT-RT-LAMP assays for YMV detection in yam. Amplification products were visualised by agarose gel electrophoresis (A) and with SYBR Green (B). Samples in lanes 1, 2 and 3 are YMV-infected samples. B, non-template control; M, 100-bp DNA ladder. (C) CT-RT-LAMP with loop primers. (D) CT-RT-LAMP, without loop primers. Samples in lanes 1 to 4: YMV-infected samples, 5 – 6, healthy yam samples. ( E and F ) Comparison between RT-PCR (E) and CT-RT-LAMP (F) for detecting YMV in infected yam leaf sample. Tenfold dilutions of total RNA from 100 ng/µl were tested. RT-PCR products were analyzed by agarose gel electrophoresis, and visual detection of RT-LAMP was done using SYBR Green dye. M, 100-bp DNA ladder; H, healthy yam RNA sample; NTC, no-template control

    Article Snippet: The final optimized RT-LAMP reaction conditions consisted of 13-μl reactions including 2 μl of RNA template, 0.16 μM each of primers YMV-F3 and YMV-B3, 1.2 μM each of primers YMV-FIP and YMV-BIP, 0.5 μM each of primers YMV-LF and YMV-LB, 1.2 mM each dNTP, 0.6 M betaine (Sigma-Aldrich, USA), 4 mM MgSO4 , 8 U of Bst 2.0 polymerase (New England Biolabs, UK), 14 U of M-MLV reverse transcriptase (Promega, USA), and 1× isothermal amplification buffer (New England Biolabs, UK).

    Techniques: Amplification, Agarose Gel Electrophoresis, SYBR Green Assay, Infection, Reverse Transcription Polymerase Chain Reaction

    Stage and tissue expression profiles of Ixodes ricinus cysteine and aspartic peptidases . Messenger RNA levels of individual enzymes were determined by semi-quantitative two-step RT PCR. Panel A : Expression of peptidase mRNAs in whole body homogenates of I. ricinus eggs, unfed larvae, unfed nymphs, males, unfed females and females attached for 1 day on the guinea pigs. Panel B : Expression of peptidase mRNAs in tissues dissected from partially engorged females (the 5-th day of feeding). The abbreviations used are as in the text. IrFer shows the mRNA amplification of tick ferritin used as template loading control. (For details, see Methods).

    Journal: Parasites & Vectors

    Article Title: Profiling of proteolytic enzymes in the gut of the tick Ixodes ricinus reveals an evolutionarily conserved network of aspartic and cysteine peptidases

    doi: 10.1186/1756-3305-1-7

    Figure Lengend Snippet: Stage and tissue expression profiles of Ixodes ricinus cysteine and aspartic peptidases . Messenger RNA levels of individual enzymes were determined by semi-quantitative two-step RT PCR. Panel A : Expression of peptidase mRNAs in whole body homogenates of I. ricinus eggs, unfed larvae, unfed nymphs, males, unfed females and females attached for 1 day on the guinea pigs. Panel B : Expression of peptidase mRNAs in tissues dissected from partially engorged females (the 5-th day of feeding). The abbreviations used are as in the text. IrFer shows the mRNA amplification of tick ferritin used as template loading control. (For details, see Methods).

    Article Snippet: Two-step RT-PCR was performed using total RNA templates (prepared as described above; 50 ng/μl final concentration) and the Enhanced Avian HS RT-PCR Kit (Sigma) according to the protocol provided by the manufacturer.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification

    Detection of DHPR α 1S gene expression in C2C12 cells and mouse skeletal muscle A 140 bp protected fragment obtained by specific antisense RNA probe hybridized to the same amount of total RNA (25 μg) from mouse skeletal muscle, C2C12 cell myoblasts in growth medium and C2C12 cell myotubes after 5 days in differentiation medium. The relative amount of total RNA loaded for each sample is indicated by the signal resulting from the hybridization of the 115 bp protected fragment for 28S rRNA in the same reaction.

    Journal: The Journal of Physiology

    Article Title: Charge movement and transcription regulation of L-type calcium channel ?1S in skeletal muscle cells

    doi: 10.1113/jphysiol.2001.013464

    Figure Lengend Snippet: Detection of DHPR α 1S gene expression in C2C12 cells and mouse skeletal muscle A 140 bp protected fragment obtained by specific antisense RNA probe hybridized to the same amount of total RNA (25 μg) from mouse skeletal muscle, C2C12 cell myoblasts in growth medium and C2C12 cell myotubes after 5 days in differentiation medium. The relative amount of total RNA loaded for each sample is indicated by the signal resulting from the hybridization of the 115 bp protected fragment for 28S rRNA in the same reaction.

    Article Snippet: Both the pTRI-RNA-28S antisense control template and the RNA century marker template set (Ambion) were also labelled by the in vitro transcription method using a different dilution of 32 P-UTP.

    Techniques: Expressing, Hybridization

    Determination of the mouse DHPR α 1S gene transcription start site by RPA A protected fragment was obtained using antisense probe to hybridize to total RNA from skeletal muscle (lane RPA Mu), yeast tRNA (lane RPA Yt) as negative RNA control, and without RNA (lane RPA Pb). Lanes G, A, T and C in the DNA sequence show the control M13mp18 vector sequenced with control primer (Sequenase Sequence Kit, Amersham).

    Journal: The Journal of Physiology

    Article Title: Charge movement and transcription regulation of L-type calcium channel ?1S in skeletal muscle cells

    doi: 10.1113/jphysiol.2001.013464

    Figure Lengend Snippet: Determination of the mouse DHPR α 1S gene transcription start site by RPA A protected fragment was obtained using antisense probe to hybridize to total RNA from skeletal muscle (lane RPA Mu), yeast tRNA (lane RPA Yt) as negative RNA control, and without RNA (lane RPA Pb). Lanes G, A, T and C in the DNA sequence show the control M13mp18 vector sequenced with control primer (Sequenase Sequence Kit, Amersham).

    Article Snippet: Both the pTRI-RNA-28S antisense control template and the RNA century marker template set (Ambion) were also labelled by the in vitro transcription method using a different dilution of 32 P-UTP.

    Techniques: Recombinase Polymerase Amplification, Sequencing, Plasmid Preparation

    In vitro splicing assays. a In vitro splicing assay with the Arabidopsis LHCB3 pre-mRNA substrate. Radioactive LHCB3 pre-mRNA substrate was synthesized in vitro with a DNA template using SP6 RNA polymerase (see Fig. 1 b) as described in materials and methods. [ 32 P]-labeled LHCB3 pre-mRNA substrate (25,000 cpm) was incubated with nuclear extract from Arabidopsis etiolated seedlings at 30 °C as described in materials and methods. Samples were withdrawn at intervals (0, 90 and 180 min), [ 32 P]-RNA was extracted and analyzed by electrophoresis on a 6% polyacrylamide gel containing 7 M urea. The gel was dried and exposed to a phosphor-imaging screen. b Heat-inactivation of Arabidopsis NE abolished the production of a spliced product. NE from Arabidopsis etiolated seedlings was incubated at 90 °C for 3 min or kept on ice (as a control) were used for splicing assays at 30 °C with the LHCB3 [ 32 P]-pre-mRNA. Samples were withdrawn at different time points (0, 90, and 180 min), [ 32 P]-RNA was extracted and analyzed as described above. c The spliced product is increased with increasing nuclear extract concentration. In vitro splicing of [ 32 P]-labeled LHCB3 pre-mRNA substrate (25,000 cpm) was carried out at 30 °C in 25 μl reaction volume containing different concentrations 0–50% (v/v) of nuclear extract as described in materials and methods. All reactions were stopped after three hours; [ 32 P]-RNA was extracted and analyzed as described above. M indicates [ 32 P]-labeled RNA markers synthesized in vitro using RNA Century™-Plus Marker Templates (Applied Biosystems, AM7782). M* lane contains [ 32 P]-labeled LHCB3 pre-mRNA, spliced mRNA, and exon1. Schematic diagrams on the right show pre-mRNA, spliced mRNA and exon 1 and their sizes. One of the [ 32 P]-RNA products formed in in vitro splicing assay corresponds to the size of spliced [ 32 P]-mRNA marker, suggesting that it could be a spliced product. The asterisks indicate the potential splicing intermediates. Other [ 32 P]-RNA products could be another pre-mRNA splicing intermediates and/or degradation products

    Journal: Plant Methods

    Article Title: Development of an in vitro pre-mRNA splicing assay using plant nuclear extract

    doi: 10.1186/s13007-017-0271-6

    Figure Lengend Snippet: In vitro splicing assays. a In vitro splicing assay with the Arabidopsis LHCB3 pre-mRNA substrate. Radioactive LHCB3 pre-mRNA substrate was synthesized in vitro with a DNA template using SP6 RNA polymerase (see Fig. 1 b) as described in materials and methods. [ 32 P]-labeled LHCB3 pre-mRNA substrate (25,000 cpm) was incubated with nuclear extract from Arabidopsis etiolated seedlings at 30 °C as described in materials and methods. Samples were withdrawn at intervals (0, 90 and 180 min), [ 32 P]-RNA was extracted and analyzed by electrophoresis on a 6% polyacrylamide gel containing 7 M urea. The gel was dried and exposed to a phosphor-imaging screen. b Heat-inactivation of Arabidopsis NE abolished the production of a spliced product. NE from Arabidopsis etiolated seedlings was incubated at 90 °C for 3 min or kept on ice (as a control) were used for splicing assays at 30 °C with the LHCB3 [ 32 P]-pre-mRNA. Samples were withdrawn at different time points (0, 90, and 180 min), [ 32 P]-RNA was extracted and analyzed as described above. c The spliced product is increased with increasing nuclear extract concentration. In vitro splicing of [ 32 P]-labeled LHCB3 pre-mRNA substrate (25,000 cpm) was carried out at 30 °C in 25 μl reaction volume containing different concentrations 0–50% (v/v) of nuclear extract as described in materials and methods. All reactions were stopped after three hours; [ 32 P]-RNA was extracted and analyzed as described above. M indicates [ 32 P]-labeled RNA markers synthesized in vitro using RNA Century™-Plus Marker Templates (Applied Biosystems, AM7782). M* lane contains [ 32 P]-labeled LHCB3 pre-mRNA, spliced mRNA, and exon1. Schematic diagrams on the right show pre-mRNA, spliced mRNA and exon 1 and their sizes. One of the [ 32 P]-RNA products formed in in vitro splicing assay corresponds to the size of spliced [ 32 P]-mRNA marker, suggesting that it could be a spliced product. The asterisks indicate the potential splicing intermediates. Other [ 32 P]-RNA products could be another pre-mRNA splicing intermediates and/or degradation products

    Article Snippet: M indicates [32 P]-labeled RNA markers synthesized in vitro using RNA Century™-Plus Marker Templates (Applied Biosystems, AM7782).

    Techniques: In Vitro, Splicing Assay, Synthesized, Labeling, Incubation, Electrophoresis, Imaging, Concentration Assay, Marker

    The size distribution of both total small RNA and miRNA sequences that were identified in Healthy (H) and Phytoplasma-infected (I) plants. Whereas most of the small RNAs were either 21 nt or 24 nt, most of the miRNAs were either 21 nt or 22 nt.

    Journal: PLoS ONE

    Article Title: Phytoplasma-Responsive microRNAs Modulate Hormonal, Nutritional, and Stress Signalling Pathways in Mexican Lime Trees

    doi: 10.1371/journal.pone.0066372

    Figure Lengend Snippet: The size distribution of both total small RNA and miRNA sequences that were identified in Healthy (H) and Phytoplasma-infected (I) plants. Whereas most of the small RNAs were either 21 nt or 24 nt, most of the miRNAs were either 21 nt or 22 nt.

    Article Snippet: Stem-loop Real-time PCR Analysis to Validate miRNA Expression Patterns All cDNAs were synthesized using an RNA template and reverse transcriptase (Invitrogen, USA).

    Techniques: Infection

    Metabolism and mechanism of antiviral activity of GS-5734. a , Chemical structures of GS-5734 and metabolic conversion to NTP. b , NTP formation in human monocyte-derived macrophages following 72-h incubation with 1 μM GS-5734 (black) or Nuc (red); mean ± SD, from 3 donors. c , Antiviral activity of GS-5734 in HeLa cells against EBOV-Makona (black symbols), EBOV-Kikwit (open symbols), MARV (red), BDBV (orange), SUDV (blue); mean ± SD from triplicates. d , Amino acid sequence homology of EBOV and RSV RdRp active site. *, residues predicted to contact NTP. e , Inhibition of RSV RdRp (blue), but not human RNA Pol II (black) or mitochondrial (mt) RNA (red) polymerases by NTP ; mean ± SD, n=3 biological replicates. f , NTP-induced RNA chain termination in RSV RdRp primer-extension assay. For gel source data, see Supplementary Figure 1 .

    Journal: Nature

    Article Title: Therapeutic Efficacy of the Small Molecule GS-5734 against Ebola Virus in Rhesus Monkeys

    doi: 10.1038/nature17180

    Figure Lengend Snippet: Metabolism and mechanism of antiviral activity of GS-5734. a , Chemical structures of GS-5734 and metabolic conversion to NTP. b , NTP formation in human monocyte-derived macrophages following 72-h incubation with 1 μM GS-5734 (black) or Nuc (red); mean ± SD, from 3 donors. c , Antiviral activity of GS-5734 in HeLa cells against EBOV-Makona (black symbols), EBOV-Kikwit (open symbols), MARV (red), BDBV (orange), SUDV (blue); mean ± SD from triplicates. d , Amino acid sequence homology of EBOV and RSV RdRp active site. *, residues predicted to contact NTP. e , Inhibition of RSV RdRp (blue), but not human RNA Pol II (black) or mitochondrial (mt) RNA (red) polymerases by NTP ; mean ± SD, n=3 biological replicates. f , NTP-induced RNA chain termination in RSV RdRp primer-extension assay. For gel source data, see Supplementary Figure 1 .

    Article Snippet: In vitro RSV RNA synthesis assay RNA synthesis by the RSV polymerase was reconstituted in vitro using purified RSV L/P complexes and an RNA oligonucleotide template (Dharmacon), representing nucleotides 1–14 of the RSV leader promoter (3′-UGCGCUUUUUUACG-5′) – .

    Techniques: Activity Assay, Derivative Assay, Incubation, Sequencing, Inhibition, Primer Extension Assay

    The requirement of the response regulator RisA for rgtA expression. ( A ) Schematic representation of RisA binding motifs in rgtA promoter region. ( Left ) Three plausible RisA binding sites (gray circles 1 – 3 ) are present in a 60-bp region (−277 to −217) upstream of the rgtA transcriptional start site. This 60-bp region has been deleted, resulting in the rgtA Δ277/217 mutant. ( Right ) Sequences of canonical RisA-binding motif as well of three RisA-binding motifs found upstream of the rgtA promoter are depicted; nucleotides different from consensus are in gray. ( B ) B. pertussis Tohama I strain (lane 1 ) and its isogenic Δ risA (lane 2 ) and rgtA Δ277/217 (lane 3 ) mutants were grown in SS medium to exponential phase. RNA isolated from harvested cells was probed with biotinylated RgtA-specific probe and subjected to northern blot analysis ( upper panel). Signals obtained upon rehybridization of the membrane with SsrA-specific probe ( lower panel) served as loading control. Only relevant parts of the membranes are shown. The result is a representative of two experiments.

    Journal: RNA

    Article Title: Signal transduction-dependent small regulatory RNA is involved in glutamate metabolism of the human pathogen Bordetella pertussis

    doi: 10.1261/rna.067306.118

    Figure Lengend Snippet: The requirement of the response regulator RisA for rgtA expression. ( A ) Schematic representation of RisA binding motifs in rgtA promoter region. ( Left ) Three plausible RisA binding sites (gray circles 1 – 3 ) are present in a 60-bp region (−277 to −217) upstream of the rgtA transcriptional start site. This 60-bp region has been deleted, resulting in the rgtA Δ277/217 mutant. ( Right ) Sequences of canonical RisA-binding motif as well of three RisA-binding motifs found upstream of the rgtA promoter are depicted; nucleotides different from consensus are in gray. ( B ) B. pertussis Tohama I strain (lane 1 ) and its isogenic Δ risA (lane 2 ) and rgtA Δ277/217 (lane 3 ) mutants were grown in SS medium to exponential phase. RNA isolated from harvested cells was probed with biotinylated RgtA-specific probe and subjected to northern blot analysis ( upper panel). Signals obtained upon rehybridization of the membrane with SsrA-specific probe ( lower panel) served as loading control. Only relevant parts of the membranes are shown. The result is a representative of two experiments.

    Article Snippet: Biotinylated RNAs transcribed in vitro using RNA Century-Plus Marker Template (Invitrogen) served as size markers.

    Techniques: Ribosomal Intergenic Spacer Analysis, Expressing, Binding Assay, Mutagenesis, Isolation, Northern Blot

    Abundance of RgtA is affected by the Hfq protein and phenotypic modulation. ( A ) Northern blot analysis was performed using total RNA isolated from Tohama I strain (lane 1 ) and its isogenic Δ hfq mutant (lane 2 ) probed with biotinylated probes specific to RgtA ( upper panel) and to SsrA RNAs ( lower panel, loading control). Only relevant parts of the membranes are shown. The result is a representative of three independent experiments. ( B ) B. pertussis Tohama I strain was grown in SS medium in the absence or presence of magnesium sulfate (lanes 1 , 2 ) and nicotinic acid (lanes 3,4 ) for 2 h. In another experiment, Tohama I cells were grown in parallel at 37°C and 22°C for 48 h in SS medium (lanes 5 , 6 ). At these time points cells were harvested and isolated RNA was probed with biotinylated RgtA-specific probe and subjected to northern blot analysis ( upper panels). Signals obtained upon rehybridization of the membrane with SsrA-specific probe ( lower panels) served as loading control. Only relevant parts of the membranes are shown. The result is a representative of four experiments. ( C ) B. pertussis Tohama I strain (lane 1 ) and its isogenic mutants carrying deletions of bvgA (lane 2 ), bvgS (lane 3 ), bvgR (lane 4 ), Δ risA (lane 5 ), and Δ risA Δ bvgR (lane 6 ) genes were grown in SS medium to exponential phase. RNA isolated from harvested cells was probed with biotinylated RgtA-specific probe and subjected to northern blot analysis. Signals obtained upon rehybridization of the membrane with SsrA-specific probe ( lower panels) served as loading control. Only relevant parts of the membranes are shown. The result is a representative of three experiments.

    Journal: RNA

    Article Title: Signal transduction-dependent small regulatory RNA is involved in glutamate metabolism of the human pathogen Bordetella pertussis

    doi: 10.1261/rna.067306.118

    Figure Lengend Snippet: Abundance of RgtA is affected by the Hfq protein and phenotypic modulation. ( A ) Northern blot analysis was performed using total RNA isolated from Tohama I strain (lane 1 ) and its isogenic Δ hfq mutant (lane 2 ) probed with biotinylated probes specific to RgtA ( upper panel) and to SsrA RNAs ( lower panel, loading control). Only relevant parts of the membranes are shown. The result is a representative of three independent experiments. ( B ) B. pertussis Tohama I strain was grown in SS medium in the absence or presence of magnesium sulfate (lanes 1 , 2 ) and nicotinic acid (lanes 3,4 ) for 2 h. In another experiment, Tohama I cells were grown in parallel at 37°C and 22°C for 48 h in SS medium (lanes 5 , 6 ). At these time points cells were harvested and isolated RNA was probed with biotinylated RgtA-specific probe and subjected to northern blot analysis ( upper panels). Signals obtained upon rehybridization of the membrane with SsrA-specific probe ( lower panels) served as loading control. Only relevant parts of the membranes are shown. The result is a representative of four experiments. ( C ) B. pertussis Tohama I strain (lane 1 ) and its isogenic mutants carrying deletions of bvgA (lane 2 ), bvgS (lane 3 ), bvgR (lane 4 ), Δ risA (lane 5 ), and Δ risA Δ bvgR (lane 6 ) genes were grown in SS medium to exponential phase. RNA isolated from harvested cells was probed with biotinylated RgtA-specific probe and subjected to northern blot analysis. Signals obtained upon rehybridization of the membrane with SsrA-specific probe ( lower panels) served as loading control. Only relevant parts of the membranes are shown. The result is a representative of three experiments.

    Article Snippet: Biotinylated RNAs transcribed in vitro using RNA Century-Plus Marker Template (Invitrogen) served as size markers.

    Techniques: Northern Blot, Isolation, Mutagenesis, Ribosomal Intergenic Spacer Analysis

    Identification and characterization of RgtA sRNA in B. pertussis . ( A ) Visualization of the dRNA-seq normalized data set (±treatment with terminal exonuclease) showing the region between the BP2735 and BP2736 genes. Graphs display sequencing depth of the positive (dark red) and negative (light red) strands. (The different color intensities depict the different library replicates.) Gene annotations are depicted as green arrows. Red bar above the picture denotes the genomic position of RgtA RNA. ( B ) Detection of RgtA by northern blot analysis. Total RNA isolated from B. pertussis Tohama I (lane 2 ) and Δ rgtA (lane 3 ) strains was separated on 8% PAA-8M urea gel, transferred to membrane, and probed with biotinylated oligo specific to RgtA. Biotinylated Century-Plus RNA ladder was loaded as a molecular size marker (lane 1 ). ( C ) Determination of the transcriptional start site of RgtA by primer extension analysis. The product of the primer extension reaction (PE, lane 5 ) was separated together with sequencing reactions performed with the primer used for primer extension (lanes 1 – 4 ) on a 6% polyacrylamide–8 M urea gel. Only the relevant part of the autoradiograph is presented. The sequence of the coding strand is shown on the left with the transcriptional start site (asterisk) and the plausible −10 sequence ( vertical line). ( D ) Nucleotide sequence map of the rgtA locus. The transcriptional start site identified by primer extension and dRNA-seq analysis (rectangular arrow, +1), plausible −35 and −10 promoter sequences (underlined), putative RisA binding sites (gray boxes), and two possible terminators (convergent arrows) are depicted.

    Journal: RNA

    Article Title: Signal transduction-dependent small regulatory RNA is involved in glutamate metabolism of the human pathogen Bordetella pertussis

    doi: 10.1261/rna.067306.118

    Figure Lengend Snippet: Identification and characterization of RgtA sRNA in B. pertussis . ( A ) Visualization of the dRNA-seq normalized data set (±treatment with terminal exonuclease) showing the region between the BP2735 and BP2736 genes. Graphs display sequencing depth of the positive (dark red) and negative (light red) strands. (The different color intensities depict the different library replicates.) Gene annotations are depicted as green arrows. Red bar above the picture denotes the genomic position of RgtA RNA. ( B ) Detection of RgtA by northern blot analysis. Total RNA isolated from B. pertussis Tohama I (lane 2 ) and Δ rgtA (lane 3 ) strains was separated on 8% PAA-8M urea gel, transferred to membrane, and probed with biotinylated oligo specific to RgtA. Biotinylated Century-Plus RNA ladder was loaded as a molecular size marker (lane 1 ). ( C ) Determination of the transcriptional start site of RgtA by primer extension analysis. The product of the primer extension reaction (PE, lane 5 ) was separated together with sequencing reactions performed with the primer used for primer extension (lanes 1 – 4 ) on a 6% polyacrylamide–8 M urea gel. Only the relevant part of the autoradiograph is presented. The sequence of the coding strand is shown on the left with the transcriptional start site (asterisk) and the plausible −10 sequence ( vertical line). ( D ) Nucleotide sequence map of the rgtA locus. The transcriptional start site identified by primer extension and dRNA-seq analysis (rectangular arrow, +1), plausible −35 and −10 promoter sequences (underlined), putative RisA binding sites (gray boxes), and two possible terminators (convergent arrows) are depicted.

    Article Snippet: Biotinylated RNAs transcribed in vitro using RNA Century-Plus Marker Template (Invitrogen) served as size markers.

    Techniques: Sequencing, Northern Blot, Isolation, Marker, Autoradiography, Ribosomal Intergenic Spacer Analysis, Binding Assay