rna  (Worthington Biochemical)

Journal: Nature Communications

Article Title: Activating frataxin expression by repeat-targeted nucleic acids

doi: 10.1038/ncomms10606

Figure Lengend Snippet: Mechanism of FXN activation by repeat-targeted duplex RNAs. ( a ) RIP examining the association of Ago2 with FXN pre-mRNA after treatment with 50 nM duplex RNA and analysis by real-time PCR. An arrow marks the PCR product of FXN pre-mRNA, which was confirmed by sequencing ( Supplementary Fig. 7 ) ( b ) Anti-GAA duplex RNA with central mismatches (siGAA 9,10 mm with mismatches on both strands; 25 nM) activates FXN expression at a level similar to the analogous fully complementary duplex RNA ( n =3). siExon3 is a positive control for transfection efficiency targeting exon 3 of FXN . ( c ) ChIP for RNAP2 using four different primer sets ( n =4). ( d ) ChIP for transcription-associated histone modification markers H3K4me3, H3K9me2, H3K9me3, H3K9Ac, H3K27me3 and H4Ac ( n =4–8). ( e ) FXN mRNA stability assay. Cells were transfected with duplex RNAs siGAA or CM at 25 nM ( n =3). Actinomycin D (5 μg ml −1 ) was added with fresh media 3 days after transfection and cells were collected at the indicated time points. HPRT expression was measured for normalization. All experiments were performed in GM03816 patient-derived cells. All data are presented as mean±STDEV. * P

Article Snippet: Quantitative PCR Identical volumes of RNA (representing approximately the same number of cells and ranging from 1 to 2 μg of RNA) were treated with 2 units of DNase I (Worthington) in DNase I buffer (10 mM Tris-HCl, pH 7.0, 10 mM NaCl, 2 mM MgCl2 and 0.5 mM CaCl2 ) for 15 min at room temperature to degrade any genomic DNA contamination.

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Sequencing, Expressing, Positive Control, Transfection, Chromatin Immunoprecipitation, Modification, Stability Assay, Derivative Assay

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

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

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

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

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

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

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

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

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

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

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

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

Journal: Nature Communications

Article Title: Functionally distinct disease-associated fibroblast subsets in rheumatoid arthritis

doi: 10.1038/s41467-018-02892-y

Figure Lengend Snippet: Distinct protein and mRNA expression between fibroblast subsets. a Gating strategy for synovial fibroblasts with heterogeneous expression of surface proteins. b Analysis of variance (ANOVA) reveals 436 genes with significant (1% FDR) variation across seven gated populations that are measured and statistically significant in both microarray and RNA-seq datasets. Each column in the heatmap corresponds to the average of multiple samples of a cell sorting gate. Each square beneath a column represents a donor from which this sample was taken. c Principal component analysis (PCA) with 2,986 genes (1% FDR, ANOVA) in microarray data separates the 32 microarray samples into three subsets: CD34 – THY1 – , CD34 – THY1 + , and CD34 + . d Pairwise Pearson's correlation of microarrays also suggests three major subsets of fibroblasts

Article Snippet: For microarray analysis, low input bulk RNA-seq and in vitro assays, we digested tissues with 4 mg/mL collagenase type 4 (Worthington, NJ, USA), 0.8 mg/mL dispase II, 0.1 mg/mL DNaseI (Roche) in Dulbecco’s modified Eagle’s medium (DMEM) at 37 °C.

Techniques: Expressing, Microarray, RNA Sequencing Assay, FACS