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  • 99
    Qiagen rnase inhibitor
    (a) SDS-PAGE of <t>NLP.</t> Lane 1, protein molecular mass marker; lane 2, untreated VSV NLP; lane 3, empty NLP after <t>RNase</t> A treatment; lane 4, reconstituted VSV NLP containing poly(rA) RNA. (b) Image of crystals of NLP with encapsidated poly(rG) RNA.
    Rnase Inhibitor, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 553 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher rnasin
    VSV M protein interacts with the Rae1–Nup98 complex during both interphase and mitosis. ( A , B ) HeLa cell lysates synchronized at the G1/S boundary and at mitosis were incubated with immobilized recombinant GST–M or GST–M(D) proteins. Bound fractions were analysed by SDS–PAGE, and immunoblot (IB) analysis was carried out with Rae1, Nup98, EIB-AP5 or hnRNP U antibodies. Total lysates were subjected to IB analysis with phospho-histone H3 (Ser 28) antibody. ( C , D ) Mitotic and G1/S lysates were subjected to immunoprecipitation (IP) with Rae1 or Nup98 antibodies in the presence of <t>RNasin</t> or <t>RNase</t> A. Samples were subjected to SDS–PAGE and immunoblot analysis was carried out by using E1B-AP5 antibodies. ( E ) Cells in mitosis were subjected to immunofluorescence with E1B-AP5 and α-tubulin antibodies followed by Apotome microscopy. GST, glutathione- S -transferase; hnRNP, heterogeneous nuclear ribonucleoprotein; M, matrix; SDS–PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; VSV, vesicular stomatitis virus.
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    99
    Promega rnase inhibitor rnasin
    Telomerase activity in kinetoplastid parasites. Lanes 1–6, T. brucei DEAE eluate; lanes 8–13, L. tarentolae DEAE eluate; lanes 15–20, L. major DEAE eluate. Telomerase products were fractionated in 10% sequencing gels to reveal the periodicity of banding pattern. In lanes 2, 9, and 16, extracts were pretreated with <t>RNase</t> A; lanes 3, 10, and 17, <t>RNasin</t> incubated with extract before addition of RNase; lanes 4, 11, and 18, RNase A was added after telomerase step incubation (+). nTS, reaction performed without the forward primer; nC, reaction performed in the absence of CX-ext; nE, reaction in which the extracts were omitted. ( A ) One-tube TRAP using primer CX-ext as reverse primer and semipurified extracts. ( B ) Two-tube modified TRAP using CX-ext reverse primer. The assays were performed with half the amount of DEAE fractions used in A .
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    84
    GE Healthcare rnasin rnase inhibitor
    Telomerase activity in kinetoplastid parasites. Lanes 1–6, T. brucei DEAE eluate; lanes 8–13, L. tarentolae DEAE eluate; lanes 15–20, L. major DEAE eluate. Telomerase products were fractionated in 10% sequencing gels to reveal the periodicity of banding pattern. In lanes 2, 9, and 16, extracts were pretreated with <t>RNase</t> A; lanes 3, 10, and 17, <t>RNasin</t> incubated with extract before addition of RNase; lanes 4, 11, and 18, RNase A was added after telomerase step incubation (+). nTS, reaction performed without the forward primer; nC, reaction performed in the absence of CX-ext; nE, reaction in which the extracts were omitted. ( A ) One-tube TRAP using primer CX-ext as reverse primer and semipurified extracts. ( B ) Two-tube modified TRAP using CX-ext reverse primer. The assays were performed with half the amount of DEAE fractions used in A .
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    94
    TaKaRa rnasin plus rnase inhibitor
    Telomerase activity in kinetoplastid parasites. Lanes 1–6, T. brucei DEAE eluate; lanes 8–13, L. tarentolae DEAE eluate; lanes 15–20, L. major DEAE eluate. Telomerase products were fractionated in 10% sequencing gels to reveal the periodicity of banding pattern. In lanes 2, 9, and 16, extracts were pretreated with <t>RNase</t> A; lanes 3, 10, and 17, <t>RNasin</t> incubated with extract before addition of RNase; lanes 4, 11, and 18, RNase A was added after telomerase step incubation (+). nTS, reaction performed without the forward primer; nC, reaction performed in the absence of CX-ext; nE, reaction in which the extracts were omitted. ( A ) One-tube TRAP using primer CX-ext as reverse primer and semipurified extracts. ( B ) Two-tube modified TRAP using CX-ext reverse primer. The assays were performed with half the amount of DEAE fractions used in A .
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    85
    Millipore recombinant rnasin rnase inhibitor
    Telomerase activity in kinetoplastid parasites. Lanes 1–6, T. brucei DEAE eluate; lanes 8–13, L. tarentolae DEAE eluate; lanes 15–20, L. major DEAE eluate. Telomerase products were fractionated in 10% sequencing gels to reveal the periodicity of banding pattern. In lanes 2, 9, and 16, extracts were pretreated with <t>RNase</t> A; lanes 3, 10, and 17, <t>RNasin</t> incubated with extract before addition of RNase; lanes 4, 11, and 18, RNase A was added after telomerase step incubation (+). nTS, reaction performed without the forward primer; nC, reaction performed in the absence of CX-ext; nE, reaction in which the extracts were omitted. ( A ) One-tube TRAP using primer CX-ext as reverse primer and semipurified extracts. ( B ) Two-tube modified TRAP using CX-ext reverse primer. The assays were performed with half the amount of DEAE fractions used in A .
    Recombinant Rnasin Rnase Inhibitor, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore rnase inhibitor rnasin
    Telomerase activity in kinetoplastid parasites. Lanes 1–6, T. brucei DEAE eluate; lanes 8–13, L. tarentolae DEAE eluate; lanes 15–20, L. major DEAE eluate. Telomerase products were fractionated in 10% sequencing gels to reveal the periodicity of banding pattern. In lanes 2, 9, and 16, extracts were pretreated with <t>RNase</t> A; lanes 3, 10, and 17, <t>RNasin</t> incubated with extract before addition of RNase; lanes 4, 11, and 18, RNase A was added after telomerase step incubation (+). nTS, reaction performed without the forward primer; nC, reaction performed in the absence of CX-ext; nE, reaction in which the extracts were omitted. ( A ) One-tube TRAP using primer CX-ext as reverse primer and semipurified extracts. ( B ) Two-tube modified TRAP using CX-ext reverse primer. The assays were performed with half the amount of DEAE fractions used in A .
    Rnase Inhibitor Rnasin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GenBiotech rnase inhibitor rnasin
    Telomerase activity in kinetoplastid parasites. Lanes 1–6, T. brucei DEAE eluate; lanes 8–13, L. tarentolae DEAE eluate; lanes 15–20, L. major DEAE eluate. Telomerase products were fractionated in 10% sequencing gels to reveal the periodicity of banding pattern. In lanes 2, 9, and 16, extracts were pretreated with <t>RNase</t> A; lanes 3, 10, and 17, <t>RNasin</t> incubated with extract before addition of RNase; lanes 4, 11, and 18, RNase A was added after telomerase step incubation (+). nTS, reaction performed without the forward primer; nC, reaction performed in the absence of CX-ext; nE, reaction in which the extracts were omitted. ( A ) One-tube TRAP using primer CX-ext as reverse primer and semipurified extracts. ( B ) Two-tube modified TRAP using CX-ext reverse primer. The assays were performed with half the amount of DEAE fractions used in A .
    Rnase Inhibitor Rnasin, supplied by GenBiotech, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher u rnase inhibitor
    <t>RNase</t> protection assay (nondenaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes (N/P 5) as a function of the RNase A concentration. Dendriplexes incubated in presence (+) or absence (−) of the treatments: RNase A (0.35, 0.7, 1.0, 1.5, and 3.5 μg per 1 μg siRNA, in lanes 4–7 , 8–11 , 12–15 , 16–19 , 20–23 , respectively) for 6 h at 37 °C, followed by 1 μL (40 U) <t>RiboLock</t> RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (250 ng) in lane 1 , after incubation with heparin ( lane 2 ) and 0.35 μg RNase A per 1 μg siRNA ( lane 3 ).
    U Rnase Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 109 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega super rnasein
    <t>RNase</t> protection assay (nondenaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes (N/P 5) as a function of the RNase A concentration. Dendriplexes incubated in presence (+) or absence (−) of the treatments: RNase A (0.35, 0.7, 1.0, 1.5, and 3.5 μg per 1 μg siRNA, in lanes 4–7 , 8–11 , 12–15 , 16–19 , 20–23 , respectively) for 6 h at 37 °C, followed by 1 μL (40 U) <t>RiboLock</t> RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (250 ng) in lane 1 , after incubation with heparin ( lane 2 ) and 0.35 μg RNase A per 1 μg siRNA ( lane 3 ).
    Super Rnasein, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher superase• in rnase inhibitor
    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total <t>RNA</t> was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by <t>RT-PCR.</t> Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p
    Superase• In Rnase Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 901 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Jena Bioscience rnase inhibitor
    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total <t>RNA</t> was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by <t>RT-PCR.</t> Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p
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    90
    New England Biolabs rnase inhibitor
    Summary of the different steps performed in the nextPARS protocol. From the cells or tissue of interest ( A ), total <t>RNA</t> is extracted ( B ) and then poly(A) + RNA is selected ( C ) to initially prepare the samples for nextPARS analyses. Once the quality and quantity of poly(A) + RNA samples is confirmed, RNA samples are denatured and in vitro folded to perform the enzymatic probing of the molecules with the corresponding concentrations of <t>RNase</t> V1 and S1 nuclease ( D ). For the library preparation using the Illumina TruSeq Small RNA Sample Preparation Kit, an initial phosphatase treatment of the 3′ends and a kinase treatment of the 5′ ends are required ( E ) to then ligate the corresponding 5′ and 3′ adapters at the ends of the RNA fragments ( F ). Then a reverse transcription of the RNA fragments and a PCR amplification are performed to obtain the library ( G ). The library is size-selected to get rid of primers and adapters dimers using an acrylamide gel and a final quality control is performed ( H ). Libraries are sequenced in single-reads with read lengths of 50 nucleotides (nt) using Illumina sequencing platforms ( I ) and computational analyses are done as described in the Materials and Methods section in order to map Illumina reads and determine the enzymatic cleavage points, using the first nucleotide in the 5′ end of the reads (which correspond to the 5′end of original RNA fragments) ( J ).
    Rnase Inhibitor, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 1649 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    (a) SDS-PAGE of NLP. Lane 1, protein molecular mass marker; lane 2, untreated VSV NLP; lane 3, empty NLP after RNase A treatment; lane 4, reconstituted VSV NLP containing poly(rA) RNA. (b) Image of crystals of NLP with encapsidated poly(rG) RNA.

    Journal: Journal of Virology

    Article Title: Access to RNA Encapsidated in the Nucleocapsid of Vesicular Stomatitis Virus ▿

    doi: 10.1128/JVI.01927-10

    Figure Lengend Snippet: (a) SDS-PAGE of NLP. Lane 1, protein molecular mass marker; lane 2, untreated VSV NLP; lane 3, empty NLP after RNase A treatment; lane 4, reconstituted VSV NLP containing poly(rA) RNA. (b) Image of crystals of NLP with encapsidated poly(rG) RNA.

    Article Snippet: In the presence of RNase inhibitor (Qiagen), the empty NLP was incubated with poly(rA) (Midland) at a molar ratio of 1:5 for 15 min at 42°C.

    Techniques: SDS Page, Marker

    RNA analysis and electron microscopy of VSV nucleocapsid-like particles (NLP) and viral nucleocapsids digested with RNase A. (a) RNA electrophoresis. Purified VSV NLP treated with RNase A (1 mg/ml, final concentration) at room temperature (lane 3), 37°C

    Journal: Journal of Virology

    Article Title: Access to RNA Encapsidated in the Nucleocapsid of Vesicular Stomatitis Virus ▿

    doi: 10.1128/JVI.01927-10

    Figure Lengend Snippet: RNA analysis and electron microscopy of VSV nucleocapsid-like particles (NLP) and viral nucleocapsids digested with RNase A. (a) RNA electrophoresis. Purified VSV NLP treated with RNase A (1 mg/ml, final concentration) at room temperature (lane 3), 37°C

    Article Snippet: In the presence of RNase inhibitor (Qiagen), the empty NLP was incubated with poly(rA) (Midland) at a molar ratio of 1:5 for 15 min at 42°C.

    Techniques: Electron Microscopy, Electrophoresis, Purification, Concentration Assay

    VSV M protein interacts with the Rae1–Nup98 complex during both interphase and mitosis. ( A , B ) HeLa cell lysates synchronized at the G1/S boundary and at mitosis were incubated with immobilized recombinant GST–M or GST–M(D) proteins. Bound fractions were analysed by SDS–PAGE, and immunoblot (IB) analysis was carried out with Rae1, Nup98, EIB-AP5 or hnRNP U antibodies. Total lysates were subjected to IB analysis with phospho-histone H3 (Ser 28) antibody. ( C , D ) Mitotic and G1/S lysates were subjected to immunoprecipitation (IP) with Rae1 or Nup98 antibodies in the presence of RNasin or RNase A. Samples were subjected to SDS–PAGE and immunoblot analysis was carried out by using E1B-AP5 antibodies. ( E ) Cells in mitosis were subjected to immunofluorescence with E1B-AP5 and α-tubulin antibodies followed by Apotome microscopy. GST, glutathione- S -transferase; hnRNP, heterogeneous nuclear ribonucleoprotein; M, matrix; SDS–PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; VSV, vesicular stomatitis virus.

    Journal: EMBO Reports

    Article Title: Vesicular stomatitis virus inhibits mitotic progression and triggers cell death

    doi: 10.1038/embor.2009.179

    Figure Lengend Snippet: VSV M protein interacts with the Rae1–Nup98 complex during both interphase and mitosis. ( A , B ) HeLa cell lysates synchronized at the G1/S boundary and at mitosis were incubated with immobilized recombinant GST–M or GST–M(D) proteins. Bound fractions were analysed by SDS–PAGE, and immunoblot (IB) analysis was carried out with Rae1, Nup98, EIB-AP5 or hnRNP U antibodies. Total lysates were subjected to IB analysis with phospho-histone H3 (Ser 28) antibody. ( C , D ) Mitotic and G1/S lysates were subjected to immunoprecipitation (IP) with Rae1 or Nup98 antibodies in the presence of RNasin or RNase A. Samples were subjected to SDS–PAGE and immunoblot analysis was carried out by using E1B-AP5 antibodies. ( E ) Cells in mitosis were subjected to immunofluorescence with E1B-AP5 and α-tubulin antibodies followed by Apotome microscopy. GST, glutathione- S -transferase; hnRNP, heterogeneous nuclear ribonucleoprotein; M, matrix; SDS–PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis; VSV, vesicular stomatitis virus.

    Article Snippet: For RNase A or RNAsin pre-treatments, cell lysates were pre-incubated with RNAsin (1,000 units/ml) or RNase A (50 μg/ml; Ambion, Austin, TX, USA) for 15 min at 37°C, followed by incubation on ice for 20 min.

    Techniques: Incubation, Recombinant, SDS Page, Immunoprecipitation, Immunofluorescence, Microscopy, Polyacrylamide Gel Electrophoresis

    Complementary-template reverse-transcription (CT-RT) of single-stranded DNA primers reverse-transcribed from FFPE-RNA. ( a ) RNA extracted from FFPE tissue is reverse-transcribed, the mRNA/DNA duplex is filtered on an YM-50 column and the DNA is single-stranded with RNase-H and column purified. The 5′-NB-Oligo-dA (24) -cT7-3′ (complementary to the T7 promoter) is annealed to the FFPE-cDNA primers. ( b ) Total RNA from universal human reference (UHR, Stratagene) is amplified using the Sense-Amp cRNA amplification kit from Genisphere to provide RNA with the same orientation as messenger RNA ( 32 ). ( c ) Single-stranded DNA primers are hybridized to their sense-RNA template between 70 and 42°C for 90 min. The hybridized products are reverse-transcribed by a process described as CT-RT. The restored FFPE-cDNAs are doubled stranded and transcribed in vitro using T7 polymerase. (See Supplementary Data for technical description of points 1 through 6.)

    Journal: Nucleic Acids Research

    Article Title: Molecular restoration of archived transcriptional profiles by complementary-template reverse-transcription (CT-RT)

    doi: 10.1093/nar/gkm510

    Figure Lengend Snippet: Complementary-template reverse-transcription (CT-RT) of single-stranded DNA primers reverse-transcribed from FFPE-RNA. ( a ) RNA extracted from FFPE tissue is reverse-transcribed, the mRNA/DNA duplex is filtered on an YM-50 column and the DNA is single-stranded with RNase-H and column purified. The 5′-NB-Oligo-dA (24) -cT7-3′ (complementary to the T7 promoter) is annealed to the FFPE-cDNA primers. ( b ) Total RNA from universal human reference (UHR, Stratagene) is amplified using the Sense-Amp cRNA amplification kit from Genisphere to provide RNA with the same orientation as messenger RNA ( 32 ). ( c ) Single-stranded DNA primers are hybridized to their sense-RNA template between 70 and 42°C for 90 min. The hybridized products are reverse-transcribed by a process described as CT-RT. The restored FFPE-cDNAs are doubled stranded and transcribed in vitro using T7 polymerase. (See Supplementary Data for technical description of points 1 through 6.)

    Article Snippet: Each reaction contained 5 μg of RNA, 2 μl of 10× arrayscript buffer, 1 μl of RNase inhibitor mix, 4 μl dNTP mix 2.5 mM each (Ambion), 1 μl (100 ng) of T7-Oligo dT(24) -VN(5′-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGGdT(24) (A/C/G)(A/C/G/T)-3′) and 1 μl of arrayscript reverse-transcriptase.

    Techniques: Formalin-fixed Paraffin-Embedded, Purification, Amplification, In Vitro

    Telomerase activity in kinetoplastid parasites. Lanes 1–6, T. brucei DEAE eluate; lanes 8–13, L. tarentolae DEAE eluate; lanes 15–20, L. major DEAE eluate. Telomerase products were fractionated in 10% sequencing gels to reveal the periodicity of banding pattern. In lanes 2, 9, and 16, extracts were pretreated with RNase A; lanes 3, 10, and 17, RNasin incubated with extract before addition of RNase; lanes 4, 11, and 18, RNase A was added after telomerase step incubation (+). nTS, reaction performed without the forward primer; nC, reaction performed in the absence of CX-ext; nE, reaction in which the extracts were omitted. ( A ) One-tube TRAP using primer CX-ext as reverse primer and semipurified extracts. ( B ) Two-tube modified TRAP using CX-ext reverse primer. The assays were performed with half the amount of DEAE fractions used in A .

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Telomerase in kinetoplastid parasitic protozoa

    doi:

    Figure Lengend Snippet: Telomerase activity in kinetoplastid parasites. Lanes 1–6, T. brucei DEAE eluate; lanes 8–13, L. tarentolae DEAE eluate; lanes 15–20, L. major DEAE eluate. Telomerase products were fractionated in 10% sequencing gels to reveal the periodicity of banding pattern. In lanes 2, 9, and 16, extracts were pretreated with RNase A; lanes 3, 10, and 17, RNasin incubated with extract before addition of RNase; lanes 4, 11, and 18, RNase A was added after telomerase step incubation (+). nTS, reaction performed without the forward primer; nC, reaction performed in the absence of CX-ext; nE, reaction in which the extracts were omitted. ( A ) One-tube TRAP using primer CX-ext as reverse primer and semipurified extracts. ( B ) Two-tube modified TRAP using CX-ext reverse primer. The assays were performed with half the amount of DEAE fractions used in A .

    Article Snippet: Activity in the extracts was tested for RNase A sensitivity by incubation with 100 ng of RNase A (Sigma) for 5 min at 37°C before or after the telomerase reaction step and with or without 1 unit of RNase inhibitor RNasin (Promega) before addition of RNase A.

    Techniques: Activity Assay, Sequencing, Incubation, Modification

    T. brucei activity monitored directly by telomerase primer-extension assay. Reactions were performed with DEAE fraction and primer tel 2. Lane 1, standard reaction; lane 2, extract pretreated with 100 ng of RNase A; lane 3, extract incubated with RNasin before addition of RNase A; lane 4, RNase A treatment after telomerase reaction (+); lane 5, nP, no input primer, lane 6, nE, extract substituted by reaction buffer; lane M, terminal deoxynucleotidyltransferase used to label tel 6 with [α- 32 P]dCTP (19 indicates the position of the primer plus 1-nt molecular weight marker).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Telomerase in kinetoplastid parasitic protozoa

    doi:

    Figure Lengend Snippet: T. brucei activity monitored directly by telomerase primer-extension assay. Reactions were performed with DEAE fraction and primer tel 2. Lane 1, standard reaction; lane 2, extract pretreated with 100 ng of RNase A; lane 3, extract incubated with RNasin before addition of RNase A; lane 4, RNase A treatment after telomerase reaction (+); lane 5, nP, no input primer, lane 6, nE, extract substituted by reaction buffer; lane M, terminal deoxynucleotidyltransferase used to label tel 6 with [α- 32 P]dCTP (19 indicates the position of the primer plus 1-nt molecular weight marker).

    Article Snippet: Activity in the extracts was tested for RNase A sensitivity by incubation with 100 ng of RNase A (Sigma) for 5 min at 37°C before or after the telomerase reaction step and with or without 1 unit of RNase inhibitor RNasin (Promega) before addition of RNase A.

    Techniques: Activity Assay, Primer Extension Assay, Incubation, Molecular Weight, Marker

    FUS forms a complex with itself, PABP and RNA. ( A ) Co-immunoprecipitation of HA-FUS, Myc-FUS and PABP from HEK293T cells. Immunoprecipitation with a Myc (mouse anti-Myc, CST) antibody pulled down PABP (mouse anti-PABP, Sigma) along with anti-HA-FUS (rabbit HA, CST). RNasin was added to block all RNase activity. ( B ) The interaction between HA-FUS and Myc-FUS was not altered by the addition of RNase, while the co-immunoprecipitation of PABP was completely abolished. ( C ) Immunoprecipitation of FUS (mouse anti-FUS, Santa Cruz) from untransfected cells shows that endogenous FUS (rabbit anti-FUS, Novus Biologicals) also interacts with PABP (mouse anti-PABP, Sigma) and treatment with RNase shows that this interaction is also dependent on RNA. ( D ) Mock immunoprecipitation experiments with either untransfected cells or a single transfection of either HA-FUS WT or Myc-FUS WT with no antibody showed that neither endogenous nor tagged FUS binds to the beads. ( E ) Immunoprecipitation of single transfections with an antibody to the wrong tag showed that a Myc antibody does not pull down HA-FUS and a HA antibody does not precipitate Myc-FUS. FT indicates flow-through of proteins that did not bind to the beads.

    Journal: Human Molecular Genetics

    Article Title: ALS mutant FUS disrupts nuclear localization and sequesters wild-type FUS within cytoplasmic stress granules

    doi: 10.1093/hmg/ddt117

    Figure Lengend Snippet: FUS forms a complex with itself, PABP and RNA. ( A ) Co-immunoprecipitation of HA-FUS, Myc-FUS and PABP from HEK293T cells. Immunoprecipitation with a Myc (mouse anti-Myc, CST) antibody pulled down PABP (mouse anti-PABP, Sigma) along with anti-HA-FUS (rabbit HA, CST). RNasin was added to block all RNase activity. ( B ) The interaction between HA-FUS and Myc-FUS was not altered by the addition of RNase, while the co-immunoprecipitation of PABP was completely abolished. ( C ) Immunoprecipitation of FUS (mouse anti-FUS, Santa Cruz) from untransfected cells shows that endogenous FUS (rabbit anti-FUS, Novus Biologicals) also interacts with PABP (mouse anti-PABP, Sigma) and treatment with RNase shows that this interaction is also dependent on RNA. ( D ) Mock immunoprecipitation experiments with either untransfected cells or a single transfection of either HA-FUS WT or Myc-FUS WT with no antibody showed that neither endogenous nor tagged FUS binds to the beads. ( E ) Immunoprecipitation of single transfections with an antibody to the wrong tag showed that a Myc antibody does not pull down HA-FUS and a HA antibody does not precipitate Myc-FUS. FT indicates flow-through of proteins that did not bind to the beads.

    Article Snippet: 5 µl of an RNase A/T1 mix (Fermentas, Yorkshire, UK) or RNasin Plus RNase inhibitor (Promega, Southampton, UK) was added to the tubes and incubated at 37°C for 5 min, then placed back on ice.

    Techniques: Immunoprecipitation, Blocking Assay, Activity Assay, Transfection, Flow Cytometry

    Coligo topology is necessary but not sufficient to template the synthesis of stable released sRNA transcripts in human WCE. ( A ) Circularization stabilizes oligonucleotides in human WCE. Circular (C) or linear (L) templates (Input) were recovered (Post) from HEK293T WCE IVT, digested with RNase cocktail to reduce cellular RNA, and stained after DPAGE. Linear forms were degraded during IVT; coligos were stable. Coligo 19aRL sequence is shown in Supplementary Figure S2 . ( B ) DPAGE separation of HEK293T WCE IVT of the three coligos and linear precursors from the reactions shown in panel A. ( C ) Transcripts are released from the coligo template during IVT. RNase H (RH) was added to (+) or withheld from (−) the indicated coligo IVT reactions at the end of a typical 90-min incubation period. Following additional incubation, the RNA products were separated by DPAGE. Lanes 1 and 2, validation of exhaustive RNase H activity on a 32 P-RNA:DNA hybrid. Reaction in lane 2 was supplemented with total HEK293T cellular RNA to normalize non-specific competing RNAs among all RNase H reactions. The result shows that the coligo 19aTAR ’s transcripts do not remain hybridized to the coligo template, while ∼20% of coligo 122 ’s transcripts do remain bound to the coligo template.

    Journal: Nucleic Acids Research

    Article Title: Circularized synthetic oligodeoxynucleotides serve as promoterless RNA polymerase III templates for small RNA generation in human cells

    doi: 10.1093/nar/gks1334

    Figure Lengend Snippet: Coligo topology is necessary but not sufficient to template the synthesis of stable released sRNA transcripts in human WCE. ( A ) Circularization stabilizes oligonucleotides in human WCE. Circular (C) or linear (L) templates (Input) were recovered (Post) from HEK293T WCE IVT, digested with RNase cocktail to reduce cellular RNA, and stained after DPAGE. Linear forms were degraded during IVT; coligos were stable. Coligo 19aRL sequence is shown in Supplementary Figure S2 . ( B ) DPAGE separation of HEK293T WCE IVT of the three coligos and linear precursors from the reactions shown in panel A. ( C ) Transcripts are released from the coligo template during IVT. RNase H (RH) was added to (+) or withheld from (−) the indicated coligo IVT reactions at the end of a typical 90-min incubation period. Following additional incubation, the RNA products were separated by DPAGE. Lanes 1 and 2, validation of exhaustive RNase H activity on a 32 P-RNA:DNA hybrid. Reaction in lane 2 was supplemented with total HEK293T cellular RNA to normalize non-specific competing RNAs among all RNase H reactions. The result shows that the coligo 19aTAR ’s transcripts do not remain hybridized to the coligo template, while ∼20% of coligo 122 ’s transcripts do remain bound to the coligo template.

    Article Snippet: A typical 20 µl reaction mixture contained 25 µg total WCE protein, 20 units RNase inhibitor (Promega), 1.25 mM each adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), 0.2 mM uridine triphosphate (UTP), [except B, which contained 1.25 mM each nucleotide triphosphate (NTP)], ∼2 μCi [α-32 P]-UTP, 40 mM Tris–HCl pH 7.9, 6 mM MgCl2 , 10 mM dithiothreitol (DTT), 2 mM spermidine, 100 µM NaCl, 100 nM coligo template unless otherwise indicated.

    Techniques: Staining, Sequencing, Incubation, Activity Assay

    Characterization of MRPP3 ΔMTS alone and in complex with MRPP1 ΔMTS and MRPP2. A , domain organization for MRPP3 ΔMTS indicating the N extension (residues 50–206), PPR domain (residues 207–329, red ), core domain (residues 330–361 and 542–583, yellow ), NYN domain (residues 362–541, brown ), and structural zinc ion bound in a Cys-Cys-His-Cys motif in the core domain. B , top , analytical SEC profiles for the mixture of MRPP1 ΔMTS , MRPP2, and MRPP3 ΔMTS ( green line ) and the mixture of MRPP1 ΔMTS , MRPP2, MRPP3 ΔMTS , and pre-(mt)tRNA Ile ( black line ) applied to a Superdex S200 column. A replicate of this experiment is shown in Fig. S5 . The elution volumes for individual MRPP1 ΔMTS (from Fig. 3 A ), MRPP2 (from Fig. 3 A ), and MRPP3 ΔMTS (from Fig. S4 F ) proteins, as well as the mixture of MRPP1 ΔMTS and MRPP2 (from Fig. 3 A ), are indicated with arrows above the chromatogram ( blue , orange , red , and pink , respectively). Bottom , SDS-PAGE ( first two gel panels ) and urea-PAGE ( last two gel panels ) analysis of eluted fractions to visualize proteins and pre-(mt)tRNA Ile , respectively. The black dashed box indicates lanes where the complex containing MRPP1 ΔMTS , MRPP2, MRPP3, and pre-(mt)tRNA Ile would be found on the SDS-PAGE. C , urea-polyacrylamide denaturing gels showing the RNase P reaction of pre-(mt)tRNA Ile set up, for mixtures of MRPP2 and MRPP3 ΔMTS with different MRPP1 truncated proteins (MRPP1 ΔMTS , MRPP1 MT+C(Δ202) , MRPP1 N ). All lanes shown are taken from one experimental gel only. A downward shift in band location of processed (mt)tRNA Ile relative to that of pre-(mt)tRNA Ile and the concomitant appearance of a band corresponding to the removed 5′-leader indicate RNase P activity. D , urea-polyacrylamide denaturing gels showing the RNase P reaction of pre-(mt)tRNA Ile set up for the complex of MRPP1 ΔMTS and MRPP2 without MRPP3 ( lane 2 ) and in the presence of different truncated MRPP3 proteins ( lanes 3–6 ). A negative control of MRPP1 ΔMTS , MRPP2, and MRPP3 ΔMTS mixed with EDTA ( lane 1 ) is included. M r RNA markers are shown ( lane M ). All lanes shown are taken from one experimental gel only. Reactions in C and D were run for 30 min at protein concentrations of 300 n m MRPP1–MRPP2 and 150 n m MRPP3.

    Journal: The Journal of Biological Chemistry

    Article Title: Structural insight into the human mitochondrial tRNA purine N1-methyltransferase and ribonuclease P complexes

    doi: 10.1074/jbc.RA117.001286

    Figure Lengend Snippet: Characterization of MRPP3 ΔMTS alone and in complex with MRPP1 ΔMTS and MRPP2. A , domain organization for MRPP3 ΔMTS indicating the N extension (residues 50–206), PPR domain (residues 207–329, red ), core domain (residues 330–361 and 542–583, yellow ), NYN domain (residues 362–541, brown ), and structural zinc ion bound in a Cys-Cys-His-Cys motif in the core domain. B , top , analytical SEC profiles for the mixture of MRPP1 ΔMTS , MRPP2, and MRPP3 ΔMTS ( green line ) and the mixture of MRPP1 ΔMTS , MRPP2, MRPP3 ΔMTS , and pre-(mt)tRNA Ile ( black line ) applied to a Superdex S200 column. A replicate of this experiment is shown in Fig. S5 . The elution volumes for individual MRPP1 ΔMTS (from Fig. 3 A ), MRPP2 (from Fig. 3 A ), and MRPP3 ΔMTS (from Fig. S4 F ) proteins, as well as the mixture of MRPP1 ΔMTS and MRPP2 (from Fig. 3 A ), are indicated with arrows above the chromatogram ( blue , orange , red , and pink , respectively). Bottom , SDS-PAGE ( first two gel panels ) and urea-PAGE ( last two gel panels ) analysis of eluted fractions to visualize proteins and pre-(mt)tRNA Ile , respectively. The black dashed box indicates lanes where the complex containing MRPP1 ΔMTS , MRPP2, MRPP3, and pre-(mt)tRNA Ile would be found on the SDS-PAGE. C , urea-polyacrylamide denaturing gels showing the RNase P reaction of pre-(mt)tRNA Ile set up, for mixtures of MRPP2 and MRPP3 ΔMTS with different MRPP1 truncated proteins (MRPP1 ΔMTS , MRPP1 MT+C(Δ202) , MRPP1 N ). All lanes shown are taken from one experimental gel only. A downward shift in band location of processed (mt)tRNA Ile relative to that of pre-(mt)tRNA Ile and the concomitant appearance of a band corresponding to the removed 5′-leader indicate RNase P activity. D , urea-polyacrylamide denaturing gels showing the RNase P reaction of pre-(mt)tRNA Ile set up for the complex of MRPP1 ΔMTS and MRPP2 without MRPP3 ( lane 2 ) and in the presence of different truncated MRPP3 proteins ( lanes 3–6 ). A negative control of MRPP1 ΔMTS , MRPP2, and MRPP3 ΔMTS mixed with EDTA ( lane 1 ) is included. M r RNA markers are shown ( lane M ). All lanes shown are taken from one experimental gel only. Reactions in C and D were run for 30 min at protein concentrations of 300 n m MRPP1–MRPP2 and 150 n m MRPP3.

    Article Snippet: RNase P activity assay RNase P cleavage was performed by mixing 300 nm MRPP1–MRPP2, 150 nm MRPP3, 10 units of RNase inhibitors (RNasin from Promega), and 400 nm in vitro transcribed pre-(mt) tRNAIle in a buffer of 30 mm Tris-HCl, pH 8, 40 mm NaCl, 4.5 mm MgCl2 , and 2 mm DTT to a total reaction volume of 8.25 μl.

    Techniques: Size-exclusion Chromatography, SDS Page, Polyacrylamide Gel Electrophoresis, Activity Assay, Negative Control

    RNase protection assay (nondenaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes (N/P 5) as a function of the RNase A concentration. Dendriplexes incubated in presence (+) or absence (−) of the treatments: RNase A (0.35, 0.7, 1.0, 1.5, and 3.5 μg per 1 μg siRNA, in lanes 4–7 , 8–11 , 12–15 , 16–19 , 20–23 , respectively) for 6 h at 37 °C, followed by 1 μL (40 U) RiboLock RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (250 ng) in lane 1 , after incubation with heparin ( lane 2 ) and 0.35 μg RNase A per 1 μg siRNA ( lane 3 ).

    Journal: Molecular Pharmaceutics

    Article Title: Poly(amidoamine) Dendrimer Nanocarriers and Their Aerosol Formulations for siRNA Delivery to the Lung Epithelium

    doi: 10.1021/mp4006358

    Figure Lengend Snippet: RNase protection assay (nondenaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes (N/P 5) as a function of the RNase A concentration. Dendriplexes incubated in presence (+) or absence (−) of the treatments: RNase A (0.35, 0.7, 1.0, 1.5, and 3.5 μg per 1 μg siRNA, in lanes 4–7 , 8–11 , 12–15 , 16–19 , 20–23 , respectively) for 6 h at 37 °C, followed by 1 μL (40 U) RiboLock RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (250 ng) in lane 1 , after incubation with heparin ( lane 2 ) and 0.35 μg RNase A per 1 μg siRNA ( lane 3 ).

    Article Snippet: RiboLock RNase Inhibitor (RI, EO0381, 40 U × μL–1 ) was purchased from Thermo Scientific (part of Thermo Fisher Scientific, Waltham, MA, U.S.A.).

    Techniques: Rnase Protection Assay, Agarose Gel Electrophoresis, Concentration Assay, Incubation, Blocking Assay, Activity Assay

    RNase protection assay (non-denaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes as a function of the N/P ratio. Dendriplexes incubated in the absence (−) or presence (+) of the treatments: RNase A (0.162 μg per 1 μg siRNA) for 6 h at 37 °C, followed by 1 μL (40 U) RiboLock RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (300 ng) before ( lane 1 ) and after ( lane 2 ) incubation with RNase A.

    Journal: Molecular Pharmaceutics

    Article Title: Poly(amidoamine) Dendrimer Nanocarriers and Their Aerosol Formulations for siRNA Delivery to the Lung Epithelium

    doi: 10.1021/mp4006358

    Figure Lengend Snippet: RNase protection assay (non-denaturing agarose gel electrophoresis) of the siRNA–G4NH2 dendriplexes as a function of the N/P ratio. Dendriplexes incubated in the absence (−) or presence (+) of the treatments: RNase A (0.162 μg per 1 μg siRNA) for 6 h at 37 °C, followed by 1 μL (40 U) RiboLock RNase inhibitor for 30 min at 37 °C to block RNase activity, and heparin (455 U per 1 μg siRNA) for 30 min at 37 °C to dissociate the siRNA from the dendrimer. Aqueous medium: TE buffer 1X pH 8. Untreated siRNA control (300 ng) before ( lane 1 ) and after ( lane 2 ) incubation with RNase A.

    Article Snippet: RiboLock RNase Inhibitor (RI, EO0381, 40 U × μL–1 ) was purchased from Thermo Scientific (part of Thermo Fisher Scientific, Waltham, MA, U.S.A.).

    Techniques: Rnase Protection Assay, Agarose Gel Electrophoresis, Incubation, Blocking Assay, Activity Assay

    A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total RNA was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by RT-PCR. Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p

    Journal: PLoS Pathogens

    Article Title: KSHV induces immunoglobulin rearrangements in mature B lymphocytes

    doi: 10.1371/journal.ppat.1006967

    Figure Lengend Snippet: A variety of B lymphocyte lineages from human tonsil are susceptible to infection with BAC16 KSHV. Magnetically sorted total B lymphocytes from four tonsil specimens were infected with KSHV or mock-infected and analyzed by FCM at indicated timepoints for (A) GFP expression and (B) immunophenotypic markers for lineage. In both cases, cells were gated for singlet/viable/CD19+. Memory B cells were further defined as CD38low/IgD-/CD27+, naïve B cells were CD38low/IgD+/CD27-, natural effector (Nat Effector) cells were CD38low/IgD+/CD27+ and germinal center (GC) cells were CD38hi/IgD-. (C) In similar infection experiments with four tonsil specimens, total RNA was extracted at 2, 4 and 6 days post-infection and viral gene transcription was verified in two technical replicates by RT-PCR. Replicate RT negative cDNA reactions for KSHV infected samples at 6 days post-infection were included as a control for DNA contamination and mean NRT Cq values (n = 8) for each target were as follows: 39.44 for LANA, 40.52 for ORF59 and > 40 (not detectable) for K8.1. For a 40-cycle reaction, non-amplifying samples were set to Cq = 41 for the purposes of calculation. The lowest Cq value obtained in a mock infected sample was assigned as the limit of detection for each target, and data points that fall below this threshold are denoted with red shading. Yellow shading highlights values between 1.7 and 3.3 cycles lower than the limit of detection and corresponds to 5–10 fold increases in gene expression. Green shading highlights values more than 3.3 cycles lower than the limit of detection and corresponds to gene expression levels greater than 10-fold above the limit of detection. ANOVA analysis of raw Cq values revealed a statistically significant effect of KSHV infection for all target genes when comparing aggregate trends for mock vs KSHV samples over time: LANA p = 0.0006; K8.1 p = 0.02, ORF59 p

    Article Snippet: Single cell RT-PCR for immunoglobulin light chains Single cells were harvested by flow sorting into 96-well PCR plates containing 4μl of RNA lysis buffer (0.5x PBS+10mM DTT+4U SUPERas-In (Thermo Cat #AM2694)).

    Techniques: Infection, Expressing, Reverse Transcription Polymerase Chain Reaction

    Unique binding motifs in the N-terminus of SLBP interact with FEM1A, FEM1B, and FEM1C. (A) Diagram representing the domain structure of SLBP. The amino acid sequence and substrate receptor binding motifs representing the “degron hotspot” are shown. TAD, translational activation domain; NLS, nuclear localization sequence; RBD, RNA binding domain. (B) FEM1A, FEM1B, and FEM1C interact with amino acids 1–99 of SLBP. C-E Mapping the FEM1A, FEM1B and FEM1C binding regions in SLBP. HEK293T cells were transfected with either empty vector (EV) or FS-tagged SLBP constructs. MLN4924 was added to the cells for 4 hours before collection. Cell lysates were affinity precipitated with anti-STREP resin, and affinity precipitations were probed with the indicated antibodies. (F) The ligase-deficient SLBP(ABCdegron) mutant is unable to bind to CTIF. HEK293T cells were transfected with FLAG-tagged SLBP constructs. Cell lysates were supplemented with SUPERase-In™ RNase Inhibitor and immunoprecipitated with anti-FLAG resin. The immunoprecipitations were probed with the indicated antibodies.

    Journal: Cell Cycle

    Article Title: FEM1 proteins are ancient regulators of SLBP degradation

    doi: 10.1080/15384101.2017.1284715

    Figure Lengend Snippet: Unique binding motifs in the N-terminus of SLBP interact with FEM1A, FEM1B, and FEM1C. (A) Diagram representing the domain structure of SLBP. The amino acid sequence and substrate receptor binding motifs representing the “degron hotspot” are shown. TAD, translational activation domain; NLS, nuclear localization sequence; RBD, RNA binding domain. (B) FEM1A, FEM1B, and FEM1C interact with amino acids 1–99 of SLBP. C-E Mapping the FEM1A, FEM1B and FEM1C binding regions in SLBP. HEK293T cells were transfected with either empty vector (EV) or FS-tagged SLBP constructs. MLN4924 was added to the cells for 4 hours before collection. Cell lysates were affinity precipitated with anti-STREP resin, and affinity precipitations were probed with the indicated antibodies. (F) The ligase-deficient SLBP(ABCdegron) mutant is unable to bind to CTIF. HEK293T cells were transfected with FLAG-tagged SLBP constructs. Cell lysates were supplemented with SUPERase-In™ RNase Inhibitor and immunoprecipitated with anti-FLAG resin. The immunoprecipitations were probed with the indicated antibodies.

    Article Snippet: SUPERase-In™ RNase Inhibitor (Thermo Fisher Scientific) was used at 1U/μL where indicated.

    Techniques: Binding Assay, Sequencing, Activation Assay, RNA Binding Assay, Transfection, Plasmid Preparation, Construct, Mutagenesis, Immunoprecipitation

    Summary of the different steps performed in the nextPARS protocol. From the cells or tissue of interest ( A ), total RNA is extracted ( B ) and then poly(A) + RNA is selected ( C ) to initially prepare the samples for nextPARS analyses. Once the quality and quantity of poly(A) + RNA samples is confirmed, RNA samples are denatured and in vitro folded to perform the enzymatic probing of the molecules with the corresponding concentrations of RNase V1 and S1 nuclease ( D ). For the library preparation using the Illumina TruSeq Small RNA Sample Preparation Kit, an initial phosphatase treatment of the 3′ends and a kinase treatment of the 5′ ends are required ( E ) to then ligate the corresponding 5′ and 3′ adapters at the ends of the RNA fragments ( F ). Then a reverse transcription of the RNA fragments and a PCR amplification are performed to obtain the library ( G ). The library is size-selected to get rid of primers and adapters dimers using an acrylamide gel and a final quality control is performed ( H ). Libraries are sequenced in single-reads with read lengths of 50 nucleotides (nt) using Illumina sequencing platforms ( I ) and computational analyses are done as described in the Materials and Methods section in order to map Illumina reads and determine the enzymatic cleavage points, using the first nucleotide in the 5′ end of the reads (which correspond to the 5′end of original RNA fragments) ( J ).

    Journal: RNA

    Article Title: nextPARS: parallel probing of RNA structures in Illumina

    doi: 10.1261/rna.063073.117

    Figure Lengend Snippet: Summary of the different steps performed in the nextPARS protocol. From the cells or tissue of interest ( A ), total RNA is extracted ( B ) and then poly(A) + RNA is selected ( C ) to initially prepare the samples for nextPARS analyses. Once the quality and quantity of poly(A) + RNA samples is confirmed, RNA samples are denatured and in vitro folded to perform the enzymatic probing of the molecules with the corresponding concentrations of RNase V1 and S1 nuclease ( D ). For the library preparation using the Illumina TruSeq Small RNA Sample Preparation Kit, an initial phosphatase treatment of the 3′ends and a kinase treatment of the 5′ ends are required ( E ) to then ligate the corresponding 5′ and 3′ adapters at the ends of the RNA fragments ( F ). Then a reverse transcription of the RNA fragments and a PCR amplification are performed to obtain the library ( G ). The library is size-selected to get rid of primers and adapters dimers using an acrylamide gel and a final quality control is performed ( H ). Libraries are sequenced in single-reads with read lengths of 50 nucleotides (nt) using Illumina sequencing platforms ( I ) and computational analyses are done as described in the Materials and Methods section in order to map Illumina reads and determine the enzymatic cleavage points, using the first nucleotide in the 5′ end of the reads (which correspond to the 5′end of original RNA fragments) ( J ).

    Article Snippet: Samples were then put on ice, and 2 µL of 5× HM Ligation Buffer, 1 µL of RNase inhibitor and 1 µL of T4 RNA Ligase 2, truncated (New England BioLabs) were added.

    Techniques: In Vitro, Sample Prep, Polymerase Chain Reaction, Amplification, Acrylamide Gel Assay, Sequencing

    Probing of RNA molecules with RNase A enzyme. Examples of the signals obtained in some RNA molecules when performing nextPARS using RNase A, an enzyme that cuts specifically in single-stranded cytosines (C) and uracils (U). Scores were calculated for each site by first capping all read counts for a given transcript at the 95th percentile and then normalizing to have a maximum of 1 (as done in the “Computation of nextPARS scores” of the Materials and Methods, but since Rnase A is the only enzyme in this case, there will be no subtraction performed, so all values will then fall in the range of 0 to 1). Cuts are considered for signals above a threshold of 0.8. ( A ]). In green, nucleotides with a cut signal above 0.8; green crosses (+) show cuts obtained in a C or U; pink asterisks (*) show cuts obtained in a G or A; and blue arrows (→) show cuts obtained in double-stranded positions. ( B ) Table summarizing the total number (N) and percentages (%) of cuts with a signal above 0.8 threshold obtained in five different RNA fragments with known secondary structure (TETp4p6, TETp9-9.1, SRA, B2, U1): first column, N and % of cuts with a signal above 0.8 in the molecules; second column, N and % of these cuts in C or U nucleotides; and third column, N and % of cuts in G or A nucleotides.

    Journal: RNA

    Article Title: nextPARS: parallel probing of RNA structures in Illumina

    doi: 10.1261/rna.063073.117

    Figure Lengend Snippet: Probing of RNA molecules with RNase A enzyme. Examples of the signals obtained in some RNA molecules when performing nextPARS using RNase A, an enzyme that cuts specifically in single-stranded cytosines (C) and uracils (U). Scores were calculated for each site by first capping all read counts for a given transcript at the 95th percentile and then normalizing to have a maximum of 1 (as done in the “Computation of nextPARS scores” of the Materials and Methods, but since Rnase A is the only enzyme in this case, there will be no subtraction performed, so all values will then fall in the range of 0 to 1). Cuts are considered for signals above a threshold of 0.8. ( A ]). In green, nucleotides with a cut signal above 0.8; green crosses (+) show cuts obtained in a C or U; pink asterisks (*) show cuts obtained in a G or A; and blue arrows (→) show cuts obtained in double-stranded positions. ( B ) Table summarizing the total number (N) and percentages (%) of cuts with a signal above 0.8 threshold obtained in five different RNA fragments with known secondary structure (TETp4p6, TETp9-9.1, SRA, B2, U1): first column, N and % of cuts with a signal above 0.8 in the molecules; second column, N and % of these cuts in C or U nucleotides; and third column, N and % of cuts in G or A nucleotides.

    Article Snippet: Samples were then put on ice, and 2 µL of 5× HM Ligation Buffer, 1 µL of RNase inhibitor and 1 µL of T4 RNA Ligase 2, truncated (New England BioLabs) were added.

    Techniques:

    Biochemical characterization of LwaCas13a RNA cleavage activity a, LwaCas13a has more active RNAse activity than LshCas13a. b, Gel electrophoresis of ssRNA1 after incubation with LwaCas13a and with and without crRNA 1 for varying amounts of times. c, Gel electrophoresis of ssRNA1 after incubation with varying amounts of LwaCas13a-crRNA complex. d, Sequence and structure of ssRNA 4 and ssRNA 5. crRNA spacer sequence is highlighted in blue. e, Gel electrophoresis of ssRNA 4 and ssRNA 5 after incubation with LwaCas13a and crRNA 1. f, Sequence and structure of ssRNA 4 with sites of poly-x modifications highlighted in red. crRNA spacer sequence is highlighted in blue. g, Gel electrophoresis of ssRNA 4 with each of 4 possible poly-x modifications incubated with LwaCas13a and crRNA 1. h, LwaCas13a can process pre-crRNA from the L. wadei CRISPR-Cas locus. i, Cleavage efficiency of ssRNA 1 for crRNA spacer truncations after incubation with LwaCas13a.

    Journal: Nature

    Article Title: RNA targeting with CRISPR-Cas13a

    doi: 10.1038/nature24049

    Figure Lengend Snippet: Biochemical characterization of LwaCas13a RNA cleavage activity a, LwaCas13a has more active RNAse activity than LshCas13a. b, Gel electrophoresis of ssRNA1 after incubation with LwaCas13a and with and without crRNA 1 for varying amounts of times. c, Gel electrophoresis of ssRNA1 after incubation with varying amounts of LwaCas13a-crRNA complex. d, Sequence and structure of ssRNA 4 and ssRNA 5. crRNA spacer sequence is highlighted in blue. e, Gel electrophoresis of ssRNA 4 and ssRNA 5 after incubation with LwaCas13a and crRNA 1. f, Sequence and structure of ssRNA 4 with sites of poly-x modifications highlighted in red. crRNA spacer sequence is highlighted in blue. g, Gel electrophoresis of ssRNA 4 with each of 4 possible poly-x modifications incubated with LwaCas13a and crRNA 1. h, LwaCas13a can process pre-crRNA from the L. wadei CRISPR-Cas locus. i, Cleavage efficiency of ssRNA 1 for crRNA spacer truncations after incubation with LwaCas13a.

    Article Snippet: Briefly, reactions consisted of 45 nM purified LwaCas13a, 22.5 nM crRNA, 125 nM quenched fluorescent RNA reporter (RNAse Alert v2, Thermo Scientific), 2 μL murine RNase inhibitor (New England Biolabs), 100 ng of background total human RNA (purified from HEK293FT culture), and varying amounts of input nucleic acid target, unless otherwise indicated, in nuclease assay buffer (40 mM Tris-HCl, 60 mM NaCl, 6 mM MgCl2, pH 7.3).

    Techniques: Activity Assay, Nucleic Acid Electrophoresis, Incubation, Sequencing, CRISPR