rnase inhibitor  (Thermo Fisher)


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    Name:
    RNase Inhibitor
    Description:
    RNase Inhibitor (ribonuclease inhibitor) is a 50 kDa recombinant enzyme used to inhibit RNase activity. It does not contain DNase or endonuclease activity. Features of this enzyme:• Inhibits RNase activity—preventing degradation of RNA template• Lacks DNA endonuclease activity—for better product yield
    Catalog Number:
    N8080119
    Price:
    None
    Applications:
    PCR & Real-Time PCR|Reverse Transcription
    Size:
    2 000 units|2000 units
    Category:
    Proteins, Enzymes, & Peptides, Enzyme Inhibitors, Molecular Biology Enzyme Inhibitors
    Score:
    85
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    Structured Review

    Thermo Fisher rnase inhibitor
    Ribonuclease activity of the recombinant <t>TcPR-4b</t> on tomato ( Solanum lycopersicum var. Micro-Tom) total RNA (5 μg). The incubation with TcPR-4b was carried out for 30 min at 25°C. The boiling conditions were 10 min at 95°C. The <t>RNase</t> inhibitor was the RiboLock (40 U; Thermo Scientific). The incubation conditions of the RNase A (Thermo Scientific) were 10 min at 25°C.
    RNase Inhibitor (ribonuclease inhibitor) is a 50 kDa recombinant enzyme used to inhibit RNase activity. It does not contain DNase or endonuclease activity. Features of this enzyme:• Inhibits RNase activity—preventing degradation of RNA template• Lacks DNA endonuclease activity—for better product yield
    https://www.bioz.com/result/rnase inhibitor/product/Thermo Fisher
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    rnase inhibitor - by Bioz Stars, 2019-10
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    Images

    1) Product Images from "The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca2+ and Mg2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa"

    Article Title: The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca2+ and Mg2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa

    Journal: BMC Plant Biology

    doi: 10.1186/1471-2229-14-161

    Ribonuclease activity of the recombinant TcPR-4b on tomato ( Solanum lycopersicum var. Micro-Tom) total RNA (5 μg). The incubation with TcPR-4b was carried out for 30 min at 25°C. The boiling conditions were 10 min at 95°C. The RNase inhibitor was the RiboLock (40 U; Thermo Scientific). The incubation conditions of the RNase A (Thermo Scientific) were 10 min at 25°C.
    Figure Legend Snippet: Ribonuclease activity of the recombinant TcPR-4b on tomato ( Solanum lycopersicum var. Micro-Tom) total RNA (5 μg). The incubation with TcPR-4b was carried out for 30 min at 25°C. The boiling conditions were 10 min at 95°C. The RNase inhibitor was the RiboLock (40 U; Thermo Scientific). The incubation conditions of the RNase A (Thermo Scientific) were 10 min at 25°C.

    Techniques Used: Activity Assay, Recombinant, Incubation

    Action of TcPR-4b on dikaryotic M. perniciosa survival in relation to RNase and DNase activity. A . Action of TcPR-4b on dikaryotic M. perniciosa survival in presence of RNase inhibitor. The following concentrations were used: 40 μg/ml of TcPR-4b and 800 U of RNase inhibitor. B . Action of TcPR-4b on dikaryotic M. perniciosa survival in presence of MgCl 2 . The following concentrations were used: 40 μg/ml of TcPR-4b and 10 mM of MgCl 2 .
    Figure Legend Snippet: Action of TcPR-4b on dikaryotic M. perniciosa survival in relation to RNase and DNase activity. A . Action of TcPR-4b on dikaryotic M. perniciosa survival in presence of RNase inhibitor. The following concentrations were used: 40 μg/ml of TcPR-4b and 800 U of RNase inhibitor. B . Action of TcPR-4b on dikaryotic M. perniciosa survival in presence of MgCl 2 . The following concentrations were used: 40 μg/ml of TcPR-4b and 10 mM of MgCl 2 .

    Techniques Used: Activity Assay

    2) Product Images from "Obesity reduces the pro-angiogenic potential of adipose tissue stem cell-derived extracellular vesicles (EVs) by impairing miR-126 content: impact on clinical applications"

    Article Title: Obesity reduces the pro-angiogenic potential of adipose tissue stem cell-derived extracellular vesicles (EVs) by impairing miR-126 content: impact on clinical applications

    Journal: International Journal of Obesity (2005)

    doi: 10.1038/ijo.2015.123

    EV-mediated miR-126 transfer is required for EV functional activities. ( a ) Migration assays performed on ECs treated with EVs recovered from nASCs that had either been treated with anti-miR-126 antagomir or nEVs+5U RNAse or not at all. The results are representative of five different experiments performed in triplicate ( n =5) (*** P
    Figure Legend Snippet: EV-mediated miR-126 transfer is required for EV functional activities. ( a ) Migration assays performed on ECs treated with EVs recovered from nASCs that had either been treated with anti-miR-126 antagomir or nEVs+5U RNAse or not at all. The results are representative of five different experiments performed in triplicate ( n =5) (*** P

    Techniques Used: Functional Assay, Migration

    3) Product Images from "The Not5 Subunit of the Ccr4-Not Complex Connects Transcription and Translation"

    Article Title: The Not5 Subunit of the Ccr4-Not Complex Connects Transcription and Translation

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1004569

    Polymerase subunits are present in polysome fractions and interact with Rpl25. A and B . Cells expressing Tap-tagged polymerase subunits were analyzed on sucrose gradients as in Fig. 1C . The polysome profiles and protein loading for these experiments are available in Fig. S15 . Extracts were treated either with CHX to preserve polysomes or with EDTA to disrupt them or C . with RNase to disrupt them. D . Rpb2-TT was immunoprecipitated from extracts of cells expressing HA-tagged Rpl25. Cells expressing untagged Rpb2 were used as a control. The immunoprecipitates were incubated with antibodies against HA or CBP to reveal Rpl25 and Rpb2, respectively. The total extract (Input) or immunoprecipitates (IP) were analyzed.
    Figure Legend Snippet: Polymerase subunits are present in polysome fractions and interact with Rpl25. A and B . Cells expressing Tap-tagged polymerase subunits were analyzed on sucrose gradients as in Fig. 1C . The polysome profiles and protein loading for these experiments are available in Fig. S15 . Extracts were treated either with CHX to preserve polysomes or with EDTA to disrupt them or C . with RNase to disrupt them. D . Rpb2-TT was immunoprecipitated from extracts of cells expressing HA-tagged Rpl25. Cells expressing untagged Rpb2 were used as a control. The immunoprecipitates were incubated with antibodies against HA or CBP to reveal Rpl25 and Rpb2, respectively. The total extract (Input) or immunoprecipitates (IP) were analyzed.

    Techniques Used: Expressing, Immunoprecipitation, Hemagglutination Assay, Incubation

    4) Product Images from "Synthesis and Labeling of RNA In Vitro"

    Article Title: Synthesis and Labeling of RNA In Vitro

    Journal:

    doi: 10.1002/0471142727.mb0415s102

    Schematic representation of site-specific internal radiolabeling of RNA. The procedure includes four steps as described in the text. (1) The RNA substrate hybridizes with the complementary 2′-O-methyl RNA-DNA chimera, which, in turn, directs RNase
    Figure Legend Snippet: Schematic representation of site-specific internal radiolabeling of RNA. The procedure includes four steps as described in the text. (1) The RNA substrate hybridizes with the complementary 2′-O-methyl RNA-DNA chimera, which, in turn, directs RNase

    Techniques Used: Radioactivity

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr085

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

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

    6) Product Images from "Bi-directional processing of pri-miRNAs with branched terminal loops by Arabidopsis Dicer-like1"

    Article Title: Bi-directional processing of pri-miRNAs with branched terminal loops by Arabidopsis Dicer-like1

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.2646

    Terminal loop affects steady-state abundance but not processing pattern of pre-miRNAs. (a) In vitro DCL1 reconstitution assays with the 5′ end labeled transcripts with pre-miR166c and pre-miR166f. Protein immunoprecipitation, cleavage assays, and processing of RNA products were performed as described in Figure 2b . The positions of intact substrates, cleavage products, and RNA markers are shown. Schematic illustration of the cleavage products by DCL1 is shown on right. Red asterisks on the transcripts indicate 32 P-labelling positions. Black arrows show expected processing sites. (b) RNA blot analysis of pre-miR166 in the stable transformants expressing 35S-MIR166c mutants. RNA blot was probed using 32 P-labelled oligo probes complementary to junction regions of miR166 and upper stem in pre-miR166c. Black triangles indicate pre-miRNAs. miR164 was probed as a loading control. (c) A model for bi-directional processing of pri-miRNA by DCL1. BTLs regulate miRNA biogenesis by triggering abortive processing of pri-miRNAs and destabilizing pre-miRNAs. Exo- or endo-ribonucleases are shown in yellow.
    Figure Legend Snippet: Terminal loop affects steady-state abundance but not processing pattern of pre-miRNAs. (a) In vitro DCL1 reconstitution assays with the 5′ end labeled transcripts with pre-miR166c and pre-miR166f. Protein immunoprecipitation, cleavage assays, and processing of RNA products were performed as described in Figure 2b . The positions of intact substrates, cleavage products, and RNA markers are shown. Schematic illustration of the cleavage products by DCL1 is shown on right. Red asterisks on the transcripts indicate 32 P-labelling positions. Black arrows show expected processing sites. (b) RNA blot analysis of pre-miR166 in the stable transformants expressing 35S-MIR166c mutants. RNA blot was probed using 32 P-labelled oligo probes complementary to junction regions of miR166 and upper stem in pre-miR166c. Black triangles indicate pre-miRNAs. miR164 was probed as a loading control. (c) A model for bi-directional processing of pri-miRNA by DCL1. BTLs regulate miRNA biogenesis by triggering abortive processing of pri-miRNAs and destabilizing pre-miRNAs. Exo- or endo-ribonucleases are shown in yellow.

    Techniques Used: In Vitro, Labeling, Immunoprecipitation, Northern blot, Expressing

    7) Product Images from "Human IFIT proteins inhibit lytic replication of KSHV: A new feed-forward loop in the innate immune system"

    Article Title: Human IFIT proteins inhibit lytic replication of KSHV: A new feed-forward loop in the innate immune system

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007609

    Activation of RNase L in cells depleted of IFIT2, IFIT1 and IFIT3. iSLK cells were depleted of IFIT1, IFIT2 and IFIT3 (IFITs) with siRNAs or mock depleted with negative control siRNA (NC Si). Cells were induced to permit lytic KSHV replication by treatment with doxycycline or mock-induced. RNA was harvested at 48 hr from each sample. U6 was used for normalization. (A) Diagram of site-specific qPCR for detection of RNase L site-specific cleavage. RNAs cleaved by RNase L and containing a 2′,3′-cyclic phosphate (waved lines, black vertical bar) were ligated using RtcB to an RNA-DNA adaptor (green-brown) containing a 5’OH RNA (green). The EDTA-quenched ligation reaction was used as a template for reverse transcription with Multiscribe RT. Reverse transcription was carried out using a primer with a 3′-end complementary to the adaptor and a 5′-overhang that serves as a universal priming site (yellow). SYBR-green based qPCR was conducted using a universal reverse primer (blue) that binds to the cDNA overhang and cleavage site-specific forward primers designed for each RNA target complementary to the RtcB ligation junction (red-green). (B-D) Quantitation of RNase L activity. Three specific RNase L cleavage site in His-tRNA and Y RNA were measured. (E) Total RNA yield from cells induced to permit lytic replication and transfected with either IFITs siRNA or NC siRNA. Results are shown as the ratio of each RNA amount to the yield from uninduced, NC transfected cells. Error bars show SEM of qPCR or ratio of RNA yield from three biological replicates; *P
    Figure Legend Snippet: Activation of RNase L in cells depleted of IFIT2, IFIT1 and IFIT3. iSLK cells were depleted of IFIT1, IFIT2 and IFIT3 (IFITs) with siRNAs or mock depleted with negative control siRNA (NC Si). Cells were induced to permit lytic KSHV replication by treatment with doxycycline or mock-induced. RNA was harvested at 48 hr from each sample. U6 was used for normalization. (A) Diagram of site-specific qPCR for detection of RNase L site-specific cleavage. RNAs cleaved by RNase L and containing a 2′,3′-cyclic phosphate (waved lines, black vertical bar) were ligated using RtcB to an RNA-DNA adaptor (green-brown) containing a 5’OH RNA (green). The EDTA-quenched ligation reaction was used as a template for reverse transcription with Multiscribe RT. Reverse transcription was carried out using a primer with a 3′-end complementary to the adaptor and a 5′-overhang that serves as a universal priming site (yellow). SYBR-green based qPCR was conducted using a universal reverse primer (blue) that binds to the cDNA overhang and cleavage site-specific forward primers designed for each RNA target complementary to the RtcB ligation junction (red-green). (B-D) Quantitation of RNase L activity. Three specific RNase L cleavage site in His-tRNA and Y RNA were measured. (E) Total RNA yield from cells induced to permit lytic replication and transfected with either IFITs siRNA or NC siRNA. Results are shown as the ratio of each RNA amount to the yield from uninduced, NC transfected cells. Error bars show SEM of qPCR or ratio of RNA yield from three biological replicates; *P

    Techniques Used: Activation Assay, Negative Control, Real-time Polymerase Chain Reaction, Ligation, SYBR Green Assay, Quantitation Assay, Activity Assay, Transfection

    8) Product Images from "Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor"

    Article Title: Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor

    Journal: Nature

    doi: 10.1038/nature17978

    RocA clamps eIF4A on polypurine motif even after ATP hydrolysis (a, b) Direct measurement of the eIF4A/RNA affinity by fluorescence polarization for eIF4A and 5′ FAM-labeled RNAs in the presence or absence of RocA. Data represent mean and S.D. (n = 3). (c) Motif enrichments along entire 4-mer motifs in Bind-n-Seq with ADP + Pi and highest-scoring elements (inset). (d) Competition assay with unlabeled RNA. Data represent mean (n = 3). (e) Ribosome toeprinting assay performed in RRL in the presence of GMP-PNP in the presence or absence of 3 μM RocA treatment. (f) Relative RNase I cleavage protected by eIF4A/RocA complex on mRNA containg one AGAGAG at the middle in footprinting assay. See the original data in Extended Data Figure 9f .
    Figure Legend Snippet: RocA clamps eIF4A on polypurine motif even after ATP hydrolysis (a, b) Direct measurement of the eIF4A/RNA affinity by fluorescence polarization for eIF4A and 5′ FAM-labeled RNAs in the presence or absence of RocA. Data represent mean and S.D. (n = 3). (c) Motif enrichments along entire 4-mer motifs in Bind-n-Seq with ADP + Pi and highest-scoring elements (inset). (d) Competition assay with unlabeled RNA. Data represent mean (n = 3). (e) Ribosome toeprinting assay performed in RRL in the presence of GMP-PNP in the presence or absence of 3 μM RocA treatment. (f) Relative RNase I cleavage protected by eIF4A/RocA complex on mRNA containg one AGAGAG at the middle in footprinting assay. See the original data in Extended Data Figure 9f .

    Techniques Used: Fluorescence, Labeling, Competitive Binding Assay, Toeprinting Assay, Footprinting

    Characterization of toeprinting assay (a) Diagram of the reporters used in this study. (b, and c) In vitro translation in RRL with mRNAs containing seven polypurine motif (AGAGAG) insertions (b) and qPCR from the samples (c). (d) Dideoxy terminated sequencing of RNA by reverse transcription verified the toeprinting product length terminated by 48S ribosomes. (e) Ribosome toeprinting assay performed in RRL in the presence of m7-GTP in the presence or absence of 3 μM RocA treatment. (f) Toeprinting assay using 10 μM recombinant eIF4A in the presence or absence of 10 μM RocA treatment. (g) Toeprinting assay (top) and RNase I footprinting assay (bottom) using 10 μM recombinant eIF4A with mRNA containing one AGAGAG motif at the middle in the presence or absence of 10 μM RocA treatment. (h and i) Toeprinting assay using 10 μM recombinant eIF4A (VX 4 GKT) or (D296A-T298K) with mRNA containing seven AGAGAG motifs in the presence or absence of 10 μM RocA treatment. (j) Pre-formation of the complex with RocA and eIF4A (VX 4 GKT) or (D296A-T298K) on the mRNA bearing seven polypurine motifs represses the translation from the mRNA in RRL. (k) Basal translation level from mRNA containing seven AGAGAG with the supplementation of recombinant eIF4A. (l) In vitro translation in RRL with mRNAs with single polypurine motif (AGAGAG) insertion at the different positions in 5′ UTR (m) Basal translation level from mRNAs bearing PV IRES and PV IRES with three AGAGAG. In b-c and h-j, data represent mean and S.D. (n = 3).
    Figure Legend Snippet: Characterization of toeprinting assay (a) Diagram of the reporters used in this study. (b, and c) In vitro translation in RRL with mRNAs containing seven polypurine motif (AGAGAG) insertions (b) and qPCR from the samples (c). (d) Dideoxy terminated sequencing of RNA by reverse transcription verified the toeprinting product length terminated by 48S ribosomes. (e) Ribosome toeprinting assay performed in RRL in the presence of m7-GTP in the presence or absence of 3 μM RocA treatment. (f) Toeprinting assay using 10 μM recombinant eIF4A in the presence or absence of 10 μM RocA treatment. (g) Toeprinting assay (top) and RNase I footprinting assay (bottom) using 10 μM recombinant eIF4A with mRNA containing one AGAGAG motif at the middle in the presence or absence of 10 μM RocA treatment. (h and i) Toeprinting assay using 10 μM recombinant eIF4A (VX 4 GKT) or (D296A-T298K) with mRNA containing seven AGAGAG motifs in the presence or absence of 10 μM RocA treatment. (j) Pre-formation of the complex with RocA and eIF4A (VX 4 GKT) or (D296A-T298K) on the mRNA bearing seven polypurine motifs represses the translation from the mRNA in RRL. (k) Basal translation level from mRNA containing seven AGAGAG with the supplementation of recombinant eIF4A. (l) In vitro translation in RRL with mRNAs with single polypurine motif (AGAGAG) insertion at the different positions in 5′ UTR (m) Basal translation level from mRNAs bearing PV IRES and PV IRES with three AGAGAG. In b-c and h-j, data represent mean and S.D. (n = 3).

    Techniques Used: Toeprinting Assay, In Vitro, Real-time Polymerase Chain Reaction, Sequencing, Recombinant, Footprinting

    9) Product Images from "Mesenchymal Stem Cell-Derived Microvesicles Protect Against Acute Tubular Injury"

    Article Title: Mesenchymal Stem Cell-Derived Microvesicles Protect Against Acute Tubular Injury

    Journal:

    doi: 10.1681/ASN.2008070798

    Cytofluorimetric characterization of mesenchymal stem cell (MSC)-derived microvesicles (MVs). Representative FACS analyses of MVs (A) and MVs treated with RNase (B) showing the size (with 1-, 2- and −4-μm beads used as internal size standards) and the expression of CD44, CD29, α-4 integrin, α-5 integrin, CD73, α-6 integrin, and HLA-class I (thick lines) surface molecules. Dot lines indicate the isotypic controls. Ten different MV preparations were analyzed with similar results. In the CD44, CD29, α-4 integrin, α-5 integrin, and CD73 experiments, the Kolmogrov-Smirnov statistical analyses between relevant antibodies and the isotypic control was significant ( P < 0.001). No significant expression of α-5-integrin and HLA class I was observed.
    Figure Legend Snippet: Cytofluorimetric characterization of mesenchymal stem cell (MSC)-derived microvesicles (MVs). Representative FACS analyses of MVs (A) and MVs treated with RNase (B) showing the size (with 1-, 2- and −4-μm beads used as internal size standards) and the expression of CD44, CD29, α-4 integrin, α-5 integrin, CD73, α-6 integrin, and HLA-class I (thick lines) surface molecules. Dot lines indicate the isotypic controls. Ten different MV preparations were analyzed with similar results. In the CD44, CD29, α-4 integrin, α-5 integrin, and CD73 experiments, the Kolmogrov-Smirnov statistical analyses between relevant antibodies and the isotypic control was significant ( P < 0.001). No significant expression of α-5-integrin and HLA class I was observed.

    Techniques Used: Derivative Assay, FACS, Expressing

    10) Product Images from "Increased sensitivity and accuracy of a single-stranded DNA splint-mediated ligation assay (sPAT) reveals poly(A) tail length dynamics of developmentally regulated mRNAs"

    Article Title: Increased sensitivity and accuracy of a single-stranded DNA splint-mediated ligation assay (sPAT) reveals poly(A) tail length dynamics of developmentally regulated mRNAs

    Journal:

    doi: 10.4161/rna.27992

    Figure 1. The splint-mediated poly(A) tail measurement (sPAT) assay. ( A ) A graphical representation of the sPAT assay. The starting point is total RNA. To establish a reference for mRNAs without a poly(A) tail, a fraction of the total RNA is hybridized to oligo-dT12-18 and RNase H treated. mRNAs are denoted with a filled circle at the mRNA’s 5′end, a filled box for the open reading frame, and a poly(A) tail of varying sizes in its 3′ end. Both RNA fractions are then separately hybridized to a DNA splint-mediated RNA anchor. The DNA splint positions the mRNA 3′ end and the 5′ phosphate of the RNA anchor for T4 RNA ligase-mediated ligation. The length of the DNA splint’s 5′ overhang increases the likelihood of annealing to mRNA and prevents non-favorable mRNA-to-RNA ligation artifacts. A DNase I treatment removes the splint and the RNA is cleaned up for reverse transcription, using a primer that preferentially hybridizes to the ligated RNA anchor. PCR amplifications are performed to detect the variation of the poly(A) tail on a specific mRNA, using a gene-specific forward primer and an RNA anchor reverse primer, which is identical to the RT primer. To enhance specificity and sensitivity, an additional PCR reaction can be performed using nested primer sets. ( B ) The RNA anchor and DNA splint combination is designed to support high annealing temperatures. The RNA anchor carries two synthesized modifications: a 5′ phosphorylation to aid ligation and a 3′ amino group to prevent further ligations by T4 RNA ligase 2 (Rnl2). The first 16 nts of the DNA splint are complementary to the last 16 nts of RNA anchor (sequence in capital case). Its remaining nucleotides are nine consecutive thymidines, which anneal to the end of the poly(A) tail forming a nicked double strand duplex of mRNA-3′OH: 5-P-Anchor RNA bridged by the DNA splint. Shorter overhangs as few as five nucleotides have been successfully used in testing analogous combinations.
    Figure Legend Snippet: Figure 1. The splint-mediated poly(A) tail measurement (sPAT) assay. ( A ) A graphical representation of the sPAT assay. The starting point is total RNA. To establish a reference for mRNAs without a poly(A) tail, a fraction of the total RNA is hybridized to oligo-dT12-18 and RNase H treated. mRNAs are denoted with a filled circle at the mRNA’s 5′end, a filled box for the open reading frame, and a poly(A) tail of varying sizes in its 3′ end. Both RNA fractions are then separately hybridized to a DNA splint-mediated RNA anchor. The DNA splint positions the mRNA 3′ end and the 5′ phosphate of the RNA anchor for T4 RNA ligase-mediated ligation. The length of the DNA splint’s 5′ overhang increases the likelihood of annealing to mRNA and prevents non-favorable mRNA-to-RNA ligation artifacts. A DNase I treatment removes the splint and the RNA is cleaned up for reverse transcription, using a primer that preferentially hybridizes to the ligated RNA anchor. PCR amplifications are performed to detect the variation of the poly(A) tail on a specific mRNA, using a gene-specific forward primer and an RNA anchor reverse primer, which is identical to the RT primer. To enhance specificity and sensitivity, an additional PCR reaction can be performed using nested primer sets. ( B ) The RNA anchor and DNA splint combination is designed to support high annealing temperatures. The RNA anchor carries two synthesized modifications: a 5′ phosphorylation to aid ligation and a 3′ amino group to prevent further ligations by T4 RNA ligase 2 (Rnl2). The first 16 nts of the DNA splint are complementary to the last 16 nts of RNA anchor (sequence in capital case). Its remaining nucleotides are nine consecutive thymidines, which anneal to the end of the poly(A) tail forming a nicked double strand duplex of mRNA-3′OH: 5-P-Anchor RNA bridged by the DNA splint. Shorter overhangs as few as five nucleotides have been successfully used in testing analogous combinations.

    Techniques Used: Ligation, Polymerase Chain Reaction, Synthesized, Sequencing

    11) Product Images from "Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury"

    Article Title: Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0033115

    RNase treatment does not modify MV size, but reduces RNA content of MVs. A) Representative MV size analyses by direct measurement with NTA, showing no difference among MVs treated or not with RNase. B) Representative Bioanalyzer profile, showing the size distribution of total RNA extracted from MVs treated or not with RNAse. The first peak (left side of each panel) represents an internal standard. The two peaks in Sample 1 (black arrows) represent 18 S (left) and 28 S (right) ribosomal RNA, only partially detectable in MVs. The red arrows showed the reduction of 18 and 28 S fragment inside RNAse-treated MVs. C) Histogram showing the expression level of SUMO-1 , POLR2 and Act B transcripts in MVs treated or not with RNase, express as 2 -δCt , as described in material and methods.
    Figure Legend Snippet: RNase treatment does not modify MV size, but reduces RNA content of MVs. A) Representative MV size analyses by direct measurement with NTA, showing no difference among MVs treated or not with RNase. B) Representative Bioanalyzer profile, showing the size distribution of total RNA extracted from MVs treated or not with RNAse. The first peak (left side of each panel) represents an internal standard. The two peaks in Sample 1 (black arrows) represent 18 S (left) and 28 S (right) ribosomal RNA, only partially detectable in MVs. The red arrows showed the reduction of 18 and 28 S fragment inside RNAse-treated MVs. C) Histogram showing the expression level of SUMO-1 , POLR2 and Act B transcripts in MVs treated or not with RNase, express as 2 -δCt , as described in material and methods.

    Techniques Used: Expressing, Activated Clotting Time Assay

    MV infusion protects SCID mice with cisplatin-induced AKI from tubular injury. Representative micrographs of renal histology of healthy SCID mice and of SCID mice treated with cisplatin and injected with vehicle alone or with MV pre-treated with RNase or with different regiments of MVs (single or multiple injections) and sacrificed at different time points (day 4, 14 and 21). Original Magnification: ×200. The typical aspect of intra-tubular casts, tubular necrosis and tubular atrophy are respectively shown by asterisks, arrows and head arrows.
    Figure Legend Snippet: MV infusion protects SCID mice with cisplatin-induced AKI from tubular injury. Representative micrographs of renal histology of healthy SCID mice and of SCID mice treated with cisplatin and injected with vehicle alone or with MV pre-treated with RNase or with different regiments of MVs (single or multiple injections) and sacrificed at different time points (day 4, 14 and 21). Original Magnification: ×200. The typical aspect of intra-tubular casts, tubular necrosis and tubular atrophy are respectively shown by asterisks, arrows and head arrows.

    Techniques Used: Mouse Assay, Injection

    12) Product Images from "Substrate recognition by ribonucleoprotein ribonuclease MRP"

    Article Title: Substrate recognition by ribonucleoprotein ribonuclease MRP

    Journal:

    doi: 10.1261/rna.2393711

    A putative hairpin located 5′ to the A3 site in the internal transcribed spacer 1 of pre-rRNA () is not essential for RNase MRP cleavage. (Lanes 1–6 ) RNase MRP cleavage of an ITS1 fragment encompassing nucleotides −81 to
    Figure Legend Snippet: A putative hairpin located 5′ to the A3 site in the internal transcribed spacer 1 of pre-rRNA () is not essential for RNase MRP cleavage. (Lanes 1–6 ) RNase MRP cleavage of an ITS1 fragment encompassing nucleotides −81 to

    Techniques Used:

    Secondary structure diagrams. ( A ) S. cerevisiae RNase P. The catalytic (C-) and specificity (S-) domains are separated by a solid line. ( B ) S. cerevisiae RNase MRP. Domains 1 and 2 are separated by a solid line. The phylogenetically conserved sequence
    Figure Legend Snippet: Secondary structure diagrams. ( A ) S. cerevisiae RNase P. The catalytic (C-) and specificity (S-) domains are separated by a solid line. ( B ) S. cerevisiae RNase MRP. Domains 1 and 2 are separated by a solid line. The phylogenetically conserved sequence

    Techniques Used: Sequencing

    Cytosine in the position +4 [C(+4)] is required for RNase MRP cleavage (shown by an arrow); a replacement of C(+4) for any other base eliminates RNase MRP cleavage. (Crossed arrows) Positions of expected cleavage. (Lanes 1 , 6 , 11 , 16 ) Digest with RNase T1
    Figure Legend Snippet: Cytosine in the position +4 [C(+4)] is required for RNase MRP cleavage (shown by an arrow); a replacement of C(+4) for any other base eliminates RNase MRP cleavage. (Crossed arrows) Positions of expected cleavage. (Lanes 1 , 6 , 11 , 16 ) Digest with RNase T1

    Techniques Used:

    Guanines flanking RNase MRP cleavage site reduce the efficiency of cleavage. (Lanes 1 , 6 , 11 , 16 ) Digest with RNase T1 (markers); (lanes 2 , 7 , 12 , 17 ) alkaline hydrolysis (markers); (lanes 3 , 8 , 13 , 18 ) untreated RNA substrates; (lanes 4 , 5 ) RNase MRP digest of
    Figure Legend Snippet: Guanines flanking RNase MRP cleavage site reduce the efficiency of cleavage. (Lanes 1 , 6 , 11 , 16 ) Digest with RNase T1 (markers); (lanes 2 , 7 , 12 , 17 ) alkaline hydrolysis (markers); (lanes 3 , 8 , 13 , 18 ) untreated RNA substrates; (lanes 4 , 5 ) RNase MRP digest of

    Techniques Used:

    Sequences of in vitro selected RNase MRP substrates. ( A ) Consensus sequence logo. (Arrow) RNase MRP cleavage site. ( B ) Frequencies of the occurrence of different sequences at positions +2 to +4 in all RNase MRP substrates. ( C ) Frequencies of the occurrence
    Figure Legend Snippet: Sequences of in vitro selected RNase MRP substrates. ( A ) Consensus sequence logo. (Arrow) RNase MRP cleavage site. ( B ) Frequencies of the occurrence of different sequences at positions +2 to +4 in all RNase MRP substrates. ( C ) Frequencies of the occurrence

    Techniques Used: In Vitro, Sequencing

    The insertion of the affinity tag does not affect 5.8S rRNA processing by RNase MRP in vivo. Ethidium bromide–stained total RNA separated on an 8% denaturing (8 M urea) polyacrylamide gel. (Lane 1 ) Wild-type yeast; (lane 2 ) the affinity tag fused
    Figure Legend Snippet: The insertion of the affinity tag does not affect 5.8S rRNA processing by RNase MRP in vivo. Ethidium bromide–stained total RNA separated on an 8% denaturing (8 M urea) polyacrylamide gel. (Lane 1 ) Wild-type yeast; (lane 2 ) the affinity tag fused

    Techniques Used: In Vivo, Staining

    Cytosine in the position +4 [C(+4)] is required for RNase MRP cleavage at the A3 site. ( A ) Cleavage of a 56-nt-long substrate containing the A3 site. (Arrow) The location of the cleavage site. ( B ) Cleavage of the same substrate as in A , but with C(+4)
    Figure Legend Snippet: Cytosine in the position +4 [C(+4)] is required for RNase MRP cleavage at the A3 site. ( A ) Cleavage of a 56-nt-long substrate containing the A3 site. (Arrow) The location of the cleavage site. ( B ) Cleavage of the same substrate as in A , but with C(+4)

    Techniques Used:

    RNase MRP cleavage of partially randomized sequences. Random sequences were used to eliminate the potential influence of the upstream and downstream sequences or secondary structures on the results. ( A ) 5′-N 19 -UUUU-H*H-AUC-N 13 -3′. ( B
    Figure Legend Snippet: RNase MRP cleavage of partially randomized sequences. Random sequences were used to eliminate the potential influence of the upstream and downstream sequences or secondary structures on the results. ( A ) 5′-N 19 -UUUU-H*H-AUC-N 13 -3′. ( B

    Techniques Used:

    13) Product Images from "IKKα-mediated biogenesis of miR-196a through interaction with Drosha regulates the sensitivity of cancer cells to radiotherapy"

    Article Title: IKKα-mediated biogenesis of miR-196a through interaction with Drosha regulates the sensitivity of cancer cells to radiotherapy

    Journal:

    doi: 10.1038/cdd.2016.32

    IKK α T23 phosphorylation status determines its association with pri-miR196a. ( a ) RNA dependence of interactions of IKK α with Drosha. Immunoprecipitates were treated with RNase A and subjected to immunoblot analysis. ( b ) RIP analysis of
    Figure Legend Snippet: IKK α T23 phosphorylation status determines its association with pri-miR196a. ( a ) RNA dependence of interactions of IKK α with Drosha. Immunoprecipitates were treated with RNase A and subjected to immunoblot analysis. ( b ) RIP analysis of

    Techniques Used:

    14) Product Images from "Hairpin structure within the 3?UTR of DNA polymerase ? mRNA acts as a post-transcriptional regulatory element and interacts with Hax-1"

    Article Title: Hairpin structure within the 3?UTR of DNA polymerase ? mRNA acts as a post-transcriptional regulatory element and interacts with Hax-1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm502

    The evolutionarily conserved part of the 3′UTR of the DNA polymerase β transcript forms a stable hairpin structure. ( A ) Structural analysis of 5′-end labeled 3′UTR of the Pol β transcript (208 nt) with use of the following probes: Pb ions (0.25, 0.5 and 1 mM); S1 nuclease (0.5, 1 and 2 units/μl (1 mM ZnCl 2 was present in each reaction); RNase T1 (0.5, 1 and 1.5 units/μl); RNase T2 (0.1, 0.2 and 0.4 unit/μl); RNase Cl3 (0.2, 0.4 and 0.6 unit/μl) and RNase A (0.4, 0.8 and 1.6 unit/μl). Lane Ci, control incubation (no probe added); lane F, formamide, statistical ladder; lane T, guanine-specific ladder obtained with T1 ribonuclease digestion. The positions of selected G residues are shown along the T1 ladder. Fragments of autoradiograms corresponding to sequences forming the structure modules M1, M2 and M3 are indicated. ( B ) Proposed secondary structure model of the 3′UTR of the Pol β transcript. Cleavage sites are indicated for each probe used (see figure inset for probe designations and cleavage intensity classification). Three RNA structure modules (M1, M2 and M3), names of loop regions (a–k) and Poly-A signal (122–127 nt) are also marked.
    Figure Legend Snippet: The evolutionarily conserved part of the 3′UTR of the DNA polymerase β transcript forms a stable hairpin structure. ( A ) Structural analysis of 5′-end labeled 3′UTR of the Pol β transcript (208 nt) with use of the following probes: Pb ions (0.25, 0.5 and 1 mM); S1 nuclease (0.5, 1 and 2 units/μl (1 mM ZnCl 2 was present in each reaction); RNase T1 (0.5, 1 and 1.5 units/μl); RNase T2 (0.1, 0.2 and 0.4 unit/μl); RNase Cl3 (0.2, 0.4 and 0.6 unit/μl) and RNase A (0.4, 0.8 and 1.6 unit/μl). Lane Ci, control incubation (no probe added); lane F, formamide, statistical ladder; lane T, guanine-specific ladder obtained with T1 ribonuclease digestion. The positions of selected G residues are shown along the T1 ladder. Fragments of autoradiograms corresponding to sequences forming the structure modules M1, M2 and M3 are indicated. ( B ) Proposed secondary structure model of the 3′UTR of the Pol β transcript. Cleavage sites are indicated for each probe used (see figure inset for probe designations and cleavage intensity classification). Three RNA structure modules (M1, M2 and M3), names of loop regions (a–k) and Poly-A signal (122–127 nt) are also marked.

    Techniques Used: Labeling, Incubation

    15) Product Images from "The adenovirus L4-22K protein regulates transcription and RNA splicing via a sequence-specific single-stranded RNA binding"

    Article Title: The adenovirus L4-22K protein regulates transcription and RNA splicing via a sequence-specific single-stranded RNA binding

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1145

    L4-22K recruits U1 snRNP to the major late leader 1 exon. ( A ) RNA from HeLa–NE pretreated with RNase H and a complementary oligonucleotide directed against the 5΄-end of U1 snRNA (U1 oligo) or the 5S rRNA (5S oligo) was separated on an 8% denaturing polyacrylamide gel. The position of the full length (U1) and shortened (U1*) U1 snRNA are indicated. ( B ) 0.2 μg recombinant L4-22K protein was incubated with 0.4 pmol radiolabeled sense-strand major late first leader-R1 ssRNA (depicted in panel E) and 1.5 μg U1 snRNA-depleted HeLa–NE (U1 oligo) or 5S rRNA-depleted HeLa–NE (5S oligo) in a gel shift assay. ( C and D ) 0.4 μg recombinant L4-22K protein was incubated with 2.6 μg HeLa–NE and 0.4 pmol radiolabeled sense-strand major late first leader-R1 ssRNA (WT in panel C and D), the sense-strand major late first leader-R1 5΄splice site mutant ssRNA (5΄ssM in panel C) or the sense-strand major late first leader-R1 5΄-CAAA-3΄ motif mutant ssRNA (MR4 in panel D). ‘–’ denotes no recombinant L4-22K protein added. ( E ) Schematic drawing depicting the structure of the major late first leader-R1 ssRNA used in the gel shift assays. ( F ) The sequence of the R1 wild type, mutant MR4 and the 5΄ splice site mutant sense-strand ssRNAs used as probes in the gel shift assays. ( G ) HEK293 cells were co-transfected with a Flag-L4-22K expressing plasmid and an MLP reporter plasmid lacking the DE element. Following a 36 hours incubation extracts were UV crosslinked and immunoprecipitated with an anti-Flag or anti-IgG antibodies, RNA was extracted and quantitated by RT-qPCR. Results are shown as the fold change of the major late first leader-R1 ssRNA (MLT) or U1 snRNA in Flag-IP versus IgG-IP. ( H ) Immunoblotting of the same samples used in panel G using antibodies directed against Flag-L4-22K and Actin. The experiments shown in panels B and D have been conducted three times and in panel C four times. The data shown in panel G is the result from three independent experiments with error bars indicating the standard deviation.
    Figure Legend Snippet: L4-22K recruits U1 snRNP to the major late leader 1 exon. ( A ) RNA from HeLa–NE pretreated with RNase H and a complementary oligonucleotide directed against the 5΄-end of U1 snRNA (U1 oligo) or the 5S rRNA (5S oligo) was separated on an 8% denaturing polyacrylamide gel. The position of the full length (U1) and shortened (U1*) U1 snRNA are indicated. ( B ) 0.2 μg recombinant L4-22K protein was incubated with 0.4 pmol radiolabeled sense-strand major late first leader-R1 ssRNA (depicted in panel E) and 1.5 μg U1 snRNA-depleted HeLa–NE (U1 oligo) or 5S rRNA-depleted HeLa–NE (5S oligo) in a gel shift assay. ( C and D ) 0.4 μg recombinant L4-22K protein was incubated with 2.6 μg HeLa–NE and 0.4 pmol radiolabeled sense-strand major late first leader-R1 ssRNA (WT in panel C and D), the sense-strand major late first leader-R1 5΄splice site mutant ssRNA (5΄ssM in panel C) or the sense-strand major late first leader-R1 5΄-CAAA-3΄ motif mutant ssRNA (MR4 in panel D). ‘–’ denotes no recombinant L4-22K protein added. ( E ) Schematic drawing depicting the structure of the major late first leader-R1 ssRNA used in the gel shift assays. ( F ) The sequence of the R1 wild type, mutant MR4 and the 5΄ splice site mutant sense-strand ssRNAs used as probes in the gel shift assays. ( G ) HEK293 cells were co-transfected with a Flag-L4-22K expressing plasmid and an MLP reporter plasmid lacking the DE element. Following a 36 hours incubation extracts were UV crosslinked and immunoprecipitated with an anti-Flag or anti-IgG antibodies, RNA was extracted and quantitated by RT-qPCR. Results are shown as the fold change of the major late first leader-R1 ssRNA (MLT) or U1 snRNA in Flag-IP versus IgG-IP. ( H ) Immunoblotting of the same samples used in panel G using antibodies directed against Flag-L4-22K and Actin. The experiments shown in panels B and D have been conducted three times and in panel C four times. The data shown in panel G is the result from three independent experiments with error bars indicating the standard deviation.

    Techniques Used: Recombinant, Incubation, Electrophoretic Mobility Shift Assay, Mutagenesis, Sequencing, Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Quantitative RT-PCR, Standard Deviation

    16) Product Images from "Exosomes from Drug-Resistant Breast Cancer Cells Transmit Chemoresistance by a Horizontal Transfer of MicroRNAs"

    Article Title: Exosomes from Drug-Resistant Breast Cancer Cells Transmit Chemoresistance by a Horizontal Transfer of MicroRNAs

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0095240

    Effects of different exosomes. (A) Flow cytometry of cell cycle distribution was analyzed after MCF-7/S was incubated with vehicle, S/exo, D/exo, RNase S/exo, and RNase D/exo for 72 h. Data are expressed as the mean ± SD, n = 3: * P
    Figure Legend Snippet: Effects of different exosomes. (A) Flow cytometry of cell cycle distribution was analyzed after MCF-7/S was incubated with vehicle, S/exo, D/exo, RNase S/exo, and RNase D/exo for 72 h. Data are expressed as the mean ± SD, n = 3: * P

    Techniques Used: Flow Cytometry, Cytometry, Incubation

    17) Product Images from ""

    Article Title:

    Journal:

    doi: 10.1074/jbc.M112.354001

    The 7SL RNA mediates the interaction between A3G and MOV10. A , 293T cells transfected with A3G-HA- (or A3G-W127L-HA-) and MOV10-FLAG-expressing plasmids were collected and lysed. B , 293T cells transfected with A3G-HA- (or A3G-W127L-HA-) expressing plasmids were collected and lysed. A and B , after immunoprecipitation with anti-HA agarose beads, the samples were treated with or without RNase mixture and then analyzed by immunoblotting using anti-FLAG ( A ), anti-MOV10 ( B ), or anti-HA antibody ( A and B ). C , SRP14 mRNA in 293T cells expressing control-shRNA and SRP14 -shRNA was quantified using real-time RT-PCR at 72 h post-transfection. The SRP14 mRNA in cells expressing control-shRNA was defined as 100%. D , 7SL RNA in 293T cells expressing control-shRNA and SRP14 -shRNA was quantified by real-time RT-PCR at 6 days post-transfection. The 7SL RNA in cells expressing control-shRNA was defined as 100%. Data in C and D represent mean ± S.D. E , 293T cells expressing control-shRNA or SRP14 -shRNA were co-transfected with MOV10-FLAG- and A3G-HA- (or A3G-W127L-HA-) expressing plasmids. F , 293T cells expressing control-shRNA or SRP14 -shRNA were transfected with A3G-HA- (or A3G-W127L-HA-) expressing plasmids. E and F , after 24 h, cells were lysed and co-immunoprecipitated with anti-HA agarose beads. The precipitated samples were analyzed by immunoblotting using anti-FLAG ( E ), anti-MOV10 ( F ) or anti-HA antibody ( E and F ). Values in A , B , E , and F represent portions of MOV10-FLAG (or MOV10) normalized against A3G-HA (or A3G-W127L-HA) relative to control values.
    Figure Legend Snippet: The 7SL RNA mediates the interaction between A3G and MOV10. A , 293T cells transfected with A3G-HA- (or A3G-W127L-HA-) and MOV10-FLAG-expressing plasmids were collected and lysed. B , 293T cells transfected with A3G-HA- (or A3G-W127L-HA-) expressing plasmids were collected and lysed. A and B , after immunoprecipitation with anti-HA agarose beads, the samples were treated with or without RNase mixture and then analyzed by immunoblotting using anti-FLAG ( A ), anti-MOV10 ( B ), or anti-HA antibody ( A and B ). C , SRP14 mRNA in 293T cells expressing control-shRNA and SRP14 -shRNA was quantified using real-time RT-PCR at 72 h post-transfection. The SRP14 mRNA in cells expressing control-shRNA was defined as 100%. D , 7SL RNA in 293T cells expressing control-shRNA and SRP14 -shRNA was quantified by real-time RT-PCR at 6 days post-transfection. The 7SL RNA in cells expressing control-shRNA was defined as 100%. Data in C and D represent mean ± S.D. E , 293T cells expressing control-shRNA or SRP14 -shRNA were co-transfected with MOV10-FLAG- and A3G-HA- (or A3G-W127L-HA-) expressing plasmids. F , 293T cells expressing control-shRNA or SRP14 -shRNA were transfected with A3G-HA- (or A3G-W127L-HA-) expressing plasmids. E and F , after 24 h, cells were lysed and co-immunoprecipitated with anti-HA agarose beads. The precipitated samples were analyzed by immunoblotting using anti-FLAG ( E ), anti-MOV10 ( F ) or anti-HA antibody ( E and F ). Values in A , B , E , and F represent portions of MOV10-FLAG (or MOV10) normalized against A3G-HA (or A3G-W127L-HA) relative to control values.

    Techniques Used: Transfection, Hemagglutination Assay, Expressing, Immunoprecipitation, shRNA, Quantitative RT-PCR

    18) Product Images from "Bi-directional processing of pri-miRNAs with branched terminal loops by Arabidopsis Dicer-like1"

    Article Title: Bi-directional processing of pri-miRNAs with branched terminal loops by Arabidopsis Dicer-like1

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.2646

    Terminal loop affects steady-state abundance but not processing pattern of pre-miRNAs. (a) In vitro DCL1 reconstitution assays with the 5′ end labeled transcripts with pre-miR166c and pre-miR166f. Protein immunoprecipitation, cleavage assays, and processing of RNA products were performed as described in Figure 2b . The positions of intact substrates, cleavage products, and RNA markers are shown. Schematic illustration of the cleavage products by DCL1 is shown on right. Red asterisks on the transcripts indicate 32 P-labelling positions. Black arrows show expected processing sites. (b) RNA blot analysis of pre-miR166 in the stable transformants expressing 35S-MIR166c mutants. RNA blot was probed using 32 P-labelled oligo probes complementary to junction regions of miR166 and upper stem in pre-miR166c. Black triangles indicate pre-miRNAs. miR164 was probed as a loading control. (c) A model for bi-directional processing of pri-miRNA by DCL1. BTLs regulate miRNA biogenesis by triggering abortive processing of pri-miRNAs and destabilizing pre-miRNAs. Exo- or endo-ribonucleases are shown in yellow.
    Figure Legend Snippet: Terminal loop affects steady-state abundance but not processing pattern of pre-miRNAs. (a) In vitro DCL1 reconstitution assays with the 5′ end labeled transcripts with pre-miR166c and pre-miR166f. Protein immunoprecipitation, cleavage assays, and processing of RNA products were performed as described in Figure 2b . The positions of intact substrates, cleavage products, and RNA markers are shown. Schematic illustration of the cleavage products by DCL1 is shown on right. Red asterisks on the transcripts indicate 32 P-labelling positions. Black arrows show expected processing sites. (b) RNA blot analysis of pre-miR166 in the stable transformants expressing 35S-MIR166c mutants. RNA blot was probed using 32 P-labelled oligo probes complementary to junction regions of miR166 and upper stem in pre-miR166c. Black triangles indicate pre-miRNAs. miR164 was probed as a loading control. (c) A model for bi-directional processing of pri-miRNA by DCL1. BTLs regulate miRNA biogenesis by triggering abortive processing of pri-miRNAs and destabilizing pre-miRNAs. Exo- or endo-ribonucleases are shown in yellow.

    Techniques Used: In Vitro, Labeling, Immunoprecipitation, Northern blot, Expressing

    19) Product Images from "Conserved regions of ribonucleoprotein ribonuclease MRP are involved in interactions with its substrate"

    Article Title: Conserved regions of ribonucleoprotein ribonuclease MRP are involved in interactions with its substrate

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt432

    Using the crystal structure of bacterial RNase P in a complex with a product ( 42 ) as a proxy to position cross-links S 1 and S 2 . The RNA component of the enzyme is shown in gray; the nucleotides corresponding to cross-links S 1 and S 2 are shown is red; the 5′ part of the cleaved substrate is shown in green; the 3′ part of the cleaved substrate in shown in blue. Magnesium ions are shown as yellow spheres. The location of the cleavage site is indicated by a black arrow.
    Figure Legend Snippet: Using the crystal structure of bacterial RNase P in a complex with a product ( 42 ) as a proxy to position cross-links S 1 and S 2 . The RNA component of the enzyme is shown in gray; the nucleotides corresponding to cross-links S 1 and S 2 are shown is red; the 5′ part of the cleaved substrate is shown in green; the 3′ part of the cleaved substrate in shown in blue. Magnesium ions are shown as yellow spheres. The location of the cleavage site is indicated by a black arrow.

    Techniques Used:

    Cross-linking of a photoreactive substrate to the isolated RNase MRP holoenzyme: primer extension analysis. Lanes 1, 2, 8 and 9: sequence ladder; lanes 3 and 10: no substrate and no UV irradiation (control); lanes 4 and 11: UV irradiation in the absence of the substrate (control); lanes 5 and 12: substrate was present at a 10 to 1 substrate to enzyme molar ratio, but the sample was not UV-irradiated (control); lanes 6 and 13: UV cross-linking in the presence of the substrate (at a 1 to 1 substrate to enzyme molar ratio); lanes 7 and 14: UV cross-linking in the presence of the substrate (at a 10 to 1 substrate to enzyme molar ratio). The locations of secondary structure elements ( Figure 1 ) are shown on the left. The locations of the identified substrate–RNase MRP RNA cross-links are indicated by arrows and marked S 1 to S 4 according to the labeling in Figure 1 .
    Figure Legend Snippet: Cross-linking of a photoreactive substrate to the isolated RNase MRP holoenzyme: primer extension analysis. Lanes 1, 2, 8 and 9: sequence ladder; lanes 3 and 10: no substrate and no UV irradiation (control); lanes 4 and 11: UV irradiation in the absence of the substrate (control); lanes 5 and 12: substrate was present at a 10 to 1 substrate to enzyme molar ratio, but the sample was not UV-irradiated (control); lanes 6 and 13: UV cross-linking in the presence of the substrate (at a 1 to 1 substrate to enzyme molar ratio); lanes 7 and 14: UV cross-linking in the presence of the substrate (at a 10 to 1 substrate to enzyme molar ratio). The locations of secondary structure elements ( Figure 1 ) are shown on the left. The locations of the identified substrate–RNase MRP RNA cross-links are indicated by arrows and marked S 1 to S 4 according to the labeling in Figure 1 .

    Techniques Used: Isolation, Sequencing, Irradiation, Labeling

    Secondary structure of the S. cerevisiae RNase MRP RNA (NME1). Phylogenetically conserved nucleotides ( 33 ), including the 5′-GARAR-3′ element ( 4 ) (where ‘R’ designates purines), are highlighted in black. Substrate cross-linking sites are shown by solid arrows and marked by S 1 , S 2, S 3, S 4. Cross-linking sites for protein components Pop1, Pop4 and Pop5 [as determined in ( 34 , 35 )] are shown by dashed lines. The secondary structure and the nomenclature of elements are based on previously published data ( 36–38 ).
    Figure Legend Snippet: Secondary structure of the S. cerevisiae RNase MRP RNA (NME1). Phylogenetically conserved nucleotides ( 33 ), including the 5′-GARAR-3′ element ( 4 ) (where ‘R’ designates purines), are highlighted in black. Substrate cross-linking sites are shown by solid arrows and marked by S 1 , S 2, S 3, S 4. Cross-linking sites for protein components Pop1, Pop4 and Pop5 [as determined in ( 34 , 35 )] are shown by dashed lines. The secondary structure and the nomenclature of elements are based on previously published data ( 36–38 ).

    Techniques Used:

    UV-induced cross-linking of a radiolabeled photoreactive RNase MRP substrate to the isolated RNase MRP holoenzyme. ( A ) Lane 1: control (no UV irradiation); lane 2: UV-induced cross-links. The identities of the RNase MRP components that are involved in the cross-links are shown on the right; see ‘Results’ section for explanation. Fifteen per cent denaturing (SDS) polyacrylamide gel. ( B ) Low-mobility radioactive bands resolved on a 6% denaturing (SDS) polyacrylamide gel. Lane 1: UV-induced cross-links; lane 2: same as lane 1, but the sample was treated with proteinase K to differentiate between protein and RNA bands; lane 3: control (no UV irradiation). ( C ) Identification of the band corresponding to the cross-link to the protein component Rpp1. Lane 3: UV-induced cross-links for the isolated ‘wild-type’ RNase MRP holoenzyme; lane 4: UV-induced cross-links for the isolated RNase MRP holoenzyme that had an elongated version of its protein component Rpp1 (denoted ‘Rpp1 + Δ’). The single band that had changed its mobility in lane 4 was identified as the one associated with Rpp1. Lanes 1 and 2: controls (same as lanes 3, 4, respectively, but without UV irradiation). Twelve per cent denaturing (SDS) polyacrylamide gel.
    Figure Legend Snippet: UV-induced cross-linking of a radiolabeled photoreactive RNase MRP substrate to the isolated RNase MRP holoenzyme. ( A ) Lane 1: control (no UV irradiation); lane 2: UV-induced cross-links. The identities of the RNase MRP components that are involved in the cross-links are shown on the right; see ‘Results’ section for explanation. Fifteen per cent denaturing (SDS) polyacrylamide gel. ( B ) Low-mobility radioactive bands resolved on a 6% denaturing (SDS) polyacrylamide gel. Lane 1: UV-induced cross-links; lane 2: same as lane 1, but the sample was treated with proteinase K to differentiate between protein and RNA bands; lane 3: control (no UV irradiation). ( C ) Identification of the band corresponding to the cross-link to the protein component Rpp1. Lane 3: UV-induced cross-links for the isolated ‘wild-type’ RNase MRP holoenzyme; lane 4: UV-induced cross-links for the isolated RNase MRP holoenzyme that had an elongated version of its protein component Rpp1 (denoted ‘Rpp1 + Δ’). The single band that had changed its mobility in lane 4 was identified as the one associated with Rpp1. Lanes 1 and 2: controls (same as lanes 3, 4, respectively, but without UV irradiation). Twelve per cent denaturing (SDS) polyacrylamide gel.

    Techniques Used: Isolation, Irradiation

    20) Product Images from "Reverse Genetics for Type I Feline Coronavirus Field Isolate To Study the Molecular Pathogenesis of Feline Infectious Peritonitis"

    Article Title: Reverse Genetics for Type I Feline Coronavirus Field Isolate To Study the Molecular Pathogenesis of Feline Infectious Peritonitis

    Journal: mBio

    doi: 10.1128/mBio.01422-18

    Generation and characterization of recFECV. (A) The genome organization of recombinant vaccinia virus vrecFECV and recovered recFECV is shown. (B) Electron micrographs of purified recFECV and recFECV-S 79 originating from supernatant of electroporated cells are depicted. Negative staining was performed with 1% uranylacetate. (C) Detection of M protein in purified recFECVs using Western blot analysis with anti-M monoclonal antibody (α-M mAb). Cells were electroporated either with recFECV RNA or with PBS (mock). Supernatants were harvested 24 h after electroporation and purified by two rounds of ultracentrifugation. A 5-μl volume of purified particles was separated by SDS-PAGE (10%) under reducing conditions and analyzed by Western blotting. (D and E) Comparison of the amounts of FCoV M protein in recFECV-S 79 versus recFECV. Ten-microliter volumes of purified viruses (stock) as well as their 1:2 and 1:10 dilutions were separated by SDS-PAGE (10%) under reducing conditions, and the results were analyzed by Western blotting using anti-M monoclonal antibody (α-M MAb) (D) and quantified (E). The intensity of the bands was analyzed using a ChemiDoc imaging system and Image Lab software. The intensity of each band was compared to that of the recFECV-S 79 stock. (F) Ten-microliter volumes of purified viruses (recFECV and recFECV-S 79 ) as well as their 1:2 and 1:10 dilutions were subjected to RNase A digestion to remove RNA outside viral particles. Upon RNA extraction, genome copy numbers in each of the fractions (stock; 1:2 and 1:10 dilutions) were determined (by qRT-PCR) and compared. GE, genome equivalents.
    Figure Legend Snippet: Generation and characterization of recFECV. (A) The genome organization of recombinant vaccinia virus vrecFECV and recovered recFECV is shown. (B) Electron micrographs of purified recFECV and recFECV-S 79 originating from supernatant of electroporated cells are depicted. Negative staining was performed with 1% uranylacetate. (C) Detection of M protein in purified recFECVs using Western blot analysis with anti-M monoclonal antibody (α-M mAb). Cells were electroporated either with recFECV RNA or with PBS (mock). Supernatants were harvested 24 h after electroporation and purified by two rounds of ultracentrifugation. A 5-μl volume of purified particles was separated by SDS-PAGE (10%) under reducing conditions and analyzed by Western blotting. (D and E) Comparison of the amounts of FCoV M protein in recFECV-S 79 versus recFECV. Ten-microliter volumes of purified viruses (stock) as well as their 1:2 and 1:10 dilutions were separated by SDS-PAGE (10%) under reducing conditions, and the results were analyzed by Western blotting using anti-M monoclonal antibody (α-M MAb) (D) and quantified (E). The intensity of the bands was analyzed using a ChemiDoc imaging system and Image Lab software. The intensity of each band was compared to that of the recFECV-S 79 stock. (F) Ten-microliter volumes of purified viruses (recFECV and recFECV-S 79 ) as well as their 1:2 and 1:10 dilutions were subjected to RNase A digestion to remove RNA outside viral particles. Upon RNA extraction, genome copy numbers in each of the fractions (stock; 1:2 and 1:10 dilutions) were determined (by qRT-PCR) and compared. GE, genome equivalents.

    Techniques Used: Recombinant, Purification, Negative Staining, Western Blot, Electroporation, SDS Page, Imaging, Software, RNA Extraction, Quantitative RT-PCR

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    Article Snippet: DNase I (TaKaRa Biotechnology, Japan) and RNase inhibitor (Thermo Scientific, USA) were added at a final concentration of 0.5 U/ml to wipe off contaminating DNAs. .. DNase I (TaKaRa Biotechnology, Japan) and RNase inhibitor (Thermo Scientific, USA) were added at a final concentration of 0.5 U/ml to wipe off contaminating DNAs.

    Article Title: The RNA-binding protein TTP is a global post-transcriptional regulator of feedback control in inflammation
    Article Snippet: After washing with ice-cold PBS, intact cells were irradiated with UV-C light (300 mJ/cm2 , Stratalinker 2400) and total cell extracts were obtained using lysis buffer (50 mM Tris/HCl [pH 8.0], 150 mM NaCl, 0.5% (v/v) Triton X-100 and 1 mM EDTA) and sonication (3 times for 20 s in ice). .. Extract were cleared by centrifugation (15 min at 4°C) and treated with RNase A (0.9375 × 10−3 Units/ml, Roche) and DNase I (20 Units/ml, Ambion) for 3 min at 37°C and cooled on ice for 5 min after adding urea to 0.5 M and RNase inhibitors (266.8 Units of RiboLock, Thermo/Fermentas; and 133.4 U of RNaseOUT, Life Technologies). .. Immunoprecipitation of GFP–TTP-RNA complexes was performed using 25 μl of Epoxy Dynabeads (M-270, Life Technologies) coupled GFP-nanobody slurry per 1 ml of total cell extracts for 1 h at 4°C.

    Amplification:

    Article Title: Brain microbiota disruption within inflammatory demyelinating lesions in multiple sclerosis
    Article Snippet: All data in which any steps showed evidence of reagent contamination were excluded and the previous steps were performed with new lots of reagents. .. All amplification steps were set up in areas and with equipment treated with DNase and RNase inhibitor (Molecular BioProducts, San Diego CA, USA). .. All post-amplification steps were performed in a discrete space with separate equipment.

    Mass Spectrometry:

    Article Title: Brain microbiota disruption within inflammatory demyelinating lesions in multiple sclerosis
    Article Snippet: Brain tissues from MS and nonMS patients was obtained by aseptic collection at autopsy into sterile vessels with immediate flash freezing. .. All amplification steps were set up in areas and with equipment treated with DNase and RNase inhibitor (Molecular BioProducts, San Diego CA, USA).

    Synthesized:

    Article Title: Identifying and avoiding off-target effects of RNase H-dependent antisense oligonucleotides in mice
    Article Snippet: RNA was extracted using the PureLink Pro 96 RNA Purification kit according to the manufacturer's instructions (Ambion). .. From this, cDNA was synthesized using M-MLT Reverse Transcriptase, random decamers RETROscript and RNase inhibitor (Ambion) with 100 mM dNTP set PCR Grade (Invitrogen) and DNase/RNase free water (Gibco) according the manufacturers’ instructions. .. For gene expression analysis, qPCR was performed using TaqMan Fast Advanced Master Mix (2×) (Ambion) in a doublex set-up.

    Article Title: LTBP2 is secreted from lung myofibroblasts and is a potential biomarker for idiopathic pulmonary fibrosis
    Article Snippet: Extracted RNA was treated with RNase free DNase 1 (Ambion, U.S.A.) in the presence of RNase Inhibitor (Invitrogen, U.S.A.) for 10 min at 37°C. .. RNA was purified using the RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions.

    Article Title: Increased sensitivity and accuracy of a single-stranded DNA splint-mediated ligation assay (sPAT) reveals poly(A) tail length dynamics of developmentally regulated mRNAs
    Article Snippet: To allow ligation to the mRNA, but block RNA anchor self-ligation, the anchor was synthesized with the typical 5′ phosphate group and an alternative 3′ amino group in comparison to a hydroxyl group (Metabion or biomers.net). .. The ligation was performed in a reaction volume of 20 μl with 50 mM TRIS-HCl pH 7.5, 2 mM MgCl2, 1 mM DTT, 400 μM ATP, 10 U RNA ligase 2 (Rnl2, NEB), and 40 U RNase inhibitor (Ribolock, Fermentas) at 15 °C for 16 h. The DNA splint was removed by the addition of a 20 μl master mix that contains 80 mM TRIS-HCl pH7.9, 20 mM NaCl, 12 mM MgCl2, 2 mM CaCl2, 20 U DNase I (Roche), and 40 U Ribolock (Fermentas) at 37 °C for 3 h. The ligated RNA was phenol/chloroform extracted, ethanol precipitated, and dissolved in 20 μl water.

    Autoradiography:

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: 5 × buffer for transcription (see recipe) 3 NTP mix (see recipe) 100 µM CTP (Thermo Scientific) 10 µCi/µl [α32 P]CTP (sp. act. .. 800 Ci/mmol; PerkinElmer) 1 µg/µl DNA template (from linearized plasmid, PCR, or oligodeoxynucleotides) 40 U/µl RNase inhibitor (Thermo Scientific) 20 U/µl T7 RNA polymerase (Thermo Scientific) DNase I (RNase-free; Thermo Scientific) G50 buffer (see recipe) 25:24:1 phenol/chloroform/isoamyl alcohol 100% ethanol Additional reagents and equipment for urea-PAGE ( ) and autoradiography ( APPENDIX 3A ) Prepare the following reaction mixture at room temperature in a 1.5-ml microcen-trifuge tube with the components added in the indicated order (total reaction volume, 20 µl): 4 µl 5 × buffer for transcription 4.6 µl distilled deionized H2 O 1 µl 3 NTP mix, 10 mM each of ATP, GTP, UTP (0.5 mM each NTP final concentration) 2.4 µl 100 µM CTP (12 µM final concentration) 5 µl 10 µCi/µl [α32 P]CTP (sp. act. .. 800 Ci/mmol) 1 µl 1 µg/µl linear DNA template 1 µl 40 U/µl RNase inhibitor 1 µl 20 U/µl T7 RNA polymerase.

    Quantitative RT-PCR:

    Article Title: Identifying and avoiding off-target effects of RNase H-dependent antisense oligonucleotides in mice
    Article Snippet: Paragraph title: RNA isolation from human cells and analysis by qRT-PCR ... From this, cDNA was synthesized using M-MLT Reverse Transcriptase, random decamers RETROscript and RNase inhibitor (Ambion) with 100 mM dNTP set PCR Grade (Invitrogen) and DNase/RNase free water (Gibco) according the manufacturers’ instructions.

    Article Title: LTBP2 is secreted from lung myofibroblasts and is a potential biomarker for idiopathic pulmonary fibrosis
    Article Snippet: Paragraph title: Quantitative RT-PCR for lung fibroblasts and myofibroblasts ... Extracted RNA was treated with RNase free DNase 1 (Ambion, U.S.A.) in the presence of RNase Inhibitor (Invitrogen, U.S.A.) for 10 min at 37°C.

    Article Title: The chromatin scaffold protein SAFB1 localizes SUMO-1 to the promoters of ribosomal protein genes to facilitate transcription initiation and splicing
    Article Snippet: Paragraph title: Isolation of nuclear and cytoplasmic RNA, RT-qPCR ... The nuclear pellet was resuspended in 100 μl of DNAse I buffer (50 mM Tris–HCl, pH 7.5, 1 mM EDTA, 10 mM MgCl2 and 0.5 U RNase inhibitor) and treated 20 U of DNase I (Invitrogen) for 60 min at 37°C.

    SYBR Green Assay:

    Article Title: LTBP2 is secreted from lung myofibroblasts and is a potential biomarker for idiopathic pulmonary fibrosis
    Article Snippet: Extracted RNA was treated with RNase free DNase 1 (Ambion, U.S.A.) in the presence of RNase Inhibitor (Invitrogen, U.S.A.) for 10 min at 37°C. .. Gene-specific primers were designed using Primer 3 software (v.0.4.0) to generate short amplicons (100–150 bp).

    Article Title: Scavenger receptor BI promotes cytoplasmic accumulation of lipoproteins in clear-cell renal cell carcinoma
    Article Snippet: Genomic DNA was removed by digestion using DNase (Roche) and RNase inhibitor (Ribolock; Thermo Scientific). .. Genomic DNA was removed by digestion using DNase (Roche) and RNase inhibitor (Ribolock; Thermo Scientific).

    Article Title: Preserved DNA Damage Checkpoint Pathway Protects against Complications in Long-Standing Type 1 Diabetes
    Article Snippet: For miR200 expression analyses, total RNA was extracted using either fibroblasts, iPSCs, or serum samples obtained from three clinical groups (Control, Medalist −C, and Medalist +C) using standard TRIzol-based RNA extraction protocols in the presence of RNase inhibitors (Life Technologies) and DNase treatment (QIAGEN). .. Samples with a 260/280 optical densitometry ratios of 2 were used for further analyses that included cDNA conversion using miRCURY LNA Universal RT microRNA PCR cDNA synthesis kit (Exiqon).

    Incubation:

    Article Title: Elucidation of the Photorhabdus temperata Genome and Generation of a Transposon Mutant Library To Identify Motility Mutants Altered in Pathogenesis
    Article Snippet: The RNA was extracted by the use of the Qiagen RNeasy minikit (Qiagen Sciences, Valencia, CA), and the manufacturer's protocol was followed except that in the final elution step, 50 μl of nuclease-free water was added directly to the column membrane and incubated at room temperature for 10 min before elution. .. To remove residual DNA, RNA samples were treated with RNase-free DNase as follows: 50 μl RNA, 10 μl 10× DNase I buffer (New England BioLabs, Ipswich, MA), 5 U DNase I (2 U/μl; New England BioLabs, Ipswich, MA), 5 μl RNase Out recombinant RNase inhibitor (40 U/μl; Invitrogen, Carlsbad, CA) and RNase-free water to reach a total reaction volume of 100 μl.

    Article Title: Cell differentiation defines acute and chronic infection cell types in Staphylococcus aureus
    Article Snippet: After washing, the total RNA was resuspended in 42 μl of RNAse-free water (Qiagen) were added and the sample was incubated at 65°C at 1000 rpm for 5 min prior storage in ice. .. To remove any DNA traces, the isolated RNA was treated (one to three times, depending on the sample) with 4 Units of RNase-free DNase I (Thermo), 10 Units of SUPERase In RNase Inhibitor (Life Technologies) and incubated for 45 min at 37°C. .. To remove the DNase I, 50 μl of RNAse-free water and 100 μl of Roti-Aqua-P/C/I (Phenol, Chloroform, Isoamyl alcohol 25:24:1 pH 4.5–5) (Carl Roth) were added to the reaction tube, mixed, transferred to a 2 mL PLGH tube and centrifuged for 12 min at 13000 rpm and 15°C.

    Article Title: Identifying and avoiding off-target effects of RNase H-dependent antisense oligonucleotides in mice
    Article Snippet: Cells were incubated for 30 min before addition of oligonucleotides dissolved in PBS and harvested 3 days later. .. From this, cDNA was synthesized using M-MLT Reverse Transcriptase, random decamers RETROscript and RNase inhibitor (Ambion) with 100 mM dNTP set PCR Grade (Invitrogen) and DNase/RNase free water (Gibco) according the manufacturers’ instructions.

    Article Title: Increased sensitivity and accuracy of a single-stranded DNA splint-mediated ligation assay (sPAT) reveals poly(A) tail length dynamics of developmentally regulated mRNAs
    Article Snippet: In a 10 μl reaction volume, a mixture of total RNA, 2 μM DNA splint, and 3 μM RNA anchor were annealed in a thermo cycler (MJ Research) incubated at 70 °C for 5 min, 60 °C for 5 min, 42 °C for 5 min, 25 °C for 5 min, before storage at 15 °C. .. The ligation was performed in a reaction volume of 20 μl with 50 mM TRIS-HCl pH 7.5, 2 mM MgCl2, 1 mM DTT, 400 μM ATP, 10 U RNA ligase 2 (Rnl2, NEB), and 40 U RNase inhibitor (Ribolock, Fermentas) at 15 °C for 16 h. The DNA splint was removed by the addition of a 20 μl master mix that contains 80 mM TRIS-HCl pH7.9, 20 mM NaCl, 12 mM MgCl2, 2 mM CaCl2, 20 U DNase I (Roche), and 40 U Ribolock (Fermentas) at 37 °C for 3 h. The ligated RNA was phenol/chloroform extracted, ethanol precipitated, and dissolved in 20 μl water.

    Article Title: Gene Expression Response of Salmonella enterica Serotype Enteritidis Phage Type 8 to Subinhibitory Concentrations of the Plant-Derived Compounds Trans-Cinnamaldehyde and Eugenol
    Article Snippet: The pellet was resuspended in 2 ml of RNase-free DEPC-treated water. .. The sample was split into two and each was provided with 500 U of RNase inhibitor (Ribolock®, Fermentas), 250 U of DNase (Fermentas), 20 μl of 1 M Tris (pH 8.3) and 10 μl of 1 M MgCl, followed by mixing and incubation at 37°C for 30 min. .. The RNA sample was then extracted once each with phenol and phenol–chloroform and twice with chloroform.

    Article Title: Role of sph2 Gene Regulation in Hemolytic and Sphingomyelinase Activities Produced by Leptospira interrogans
    Article Snippet: RNA was precipitated by the addition of equal volumes of isopropanol, mixed and incubated for 10 min and subjected to centrifugation at 16,000 x g for 15 min at 4°C. .. The dissolved RNA was protected from degradation by addition of 1μL of 20 U/μL SUPERase In RNase inhibitor (Life Technologies).

    Activity Assay:

    Article Title: The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca2+ and Mg2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa
    Article Snippet: On the contrary, the RNase activity of TcPR-4b was independent of bivalent ions, suggesting probable different catalytic sites for the two nuclease actions. .. As observed in other works [ , , ], the RNase activity of TcPR-4b was inhibited by heating and in the presence of RNase inhibitor (RiboLock, Thermo Scientific) which is able to annul the activity of type A, B and C RNases. .. However, some exceptions were reported, for example, the CcPR-4 seemed to have a RNase activity mechanism different from those of the type A, B and C RNases [ ].

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: 800 Ci/mmol; PerkinElmer) 1 µg/µl DNA template (from linearized plasmid, PCR, or oligodeoxynucleotides) 40 U/µl RNase inhibitor (Thermo Scientific) 20 U/µl T7 RNA polymerase (Thermo Scientific) DNase I (RNase-free; Thermo Scientific) G50 buffer (see recipe) 25:24:1 phenol/chloroform/isoamyl alcohol 100% ethanol Additional reagents and equipment for urea-PAGE ( ) and autoradiography ( APPENDIX 3A ) Prepare the following reaction mixture at room temperature in a 1.5-ml microcen-trifuge tube with the components added in the indicated order (total reaction volume, 20 µl): 4 µl 5 × buffer for transcription 4.6 µl distilled deionized H2 O 1 µl 3 NTP mix, 10 mM each of ATP, GTP, UTP (0.5 mM each NTP final concentration) 2.4 µl 100 µM CTP (12 µM final concentration) 5 µl 10 µCi/µl [α32 P]CTP (sp. act. .. 800 Ci/mmol; PerkinElmer) 1 µg/µl DNA template (from linearized plasmid, PCR, or oligodeoxynucleotides) 40 U/µl RNase inhibitor (Thermo Scientific) 20 U/µl T7 RNA polymerase (Thermo Scientific) DNase I (RNase-free; Thermo Scientific) G50 buffer (see recipe) 25:24:1 phenol/chloroform/isoamyl alcohol 100% ethanol Additional reagents and equipment for urea-PAGE ( ) and autoradiography ( APPENDIX 3A ) Prepare the following reaction mixture at room temperature in a 1.5-ml microcen-trifuge tube with the components added in the indicated order (total reaction volume, 20 µl): 4 µl 5 × buffer for transcription 4.6 µl distilled deionized H2 O 1 µl 3 NTP mix, 10 mM each of ATP, GTP, UTP (0.5 mM each NTP final concentration) 2.4 µl 100 µM CTP (12 µM final concentration) 5 µl 10 µCi/µl [α32 P]CTP (sp. act.

    Infection:

    Article Title: Identification of two novel HSP90 proteins in Babesia orientalis: molecular characterization, and computational analyses of their structure, function, antigenicity and inhibitor interaction
    Article Snippet: The blood samples from the jugular veins of experimentally infected buffaloes with 3% parasitaemia were collected in BD Vacutainer® tubes containing EDTA (Qingdao Pharmacypro Co., Ltd.) for the extraction of DNA and RNA. .. RNA samples were treated with DNase I (Invitrogen, USA); RNase inhibitor (RNaseOUT™ Recombinant Ribonuclease Inhibitor, Invitrogen, USA) was added and then the RNA was reverse transcribed using FastQuant® RT Kit (TIANGEN Biotech (Beijing) Co., Ltd.) according to the manufacturer’s instruction.

    Expressing:

    Article Title: Preserved DNA Damage Checkpoint Pathway Protects against Complications in Long-Standing Type 1 Diabetes
    Article Snippet: All virus infections used 5 µg/ml Polybrene reagent (EMD Millipore). .. For miR200 expression analyses, total RNA was extracted using either fibroblasts, iPSCs, or serum samples obtained from three clinical groups (Control, Medalist −C, and Medalist +C) using standard TRIzol-based RNA extraction protocols in the presence of RNase inhibitors (Life Technologies) and DNase treatment (QIAGEN). .. RNA measurements used ND-1000 spectrophotometer V3.5 (NanoDrop Technologies, Inc.).

    Article Title: Transcriptional profile and Epstein-Barr virus infection status of laser-cut immune infiltrates from the brain of patients with progressive multiple sclerosis
    Article Snippet: Total RNA was extracted from the microdissected samples using Picopure RNA isolation kit (Arcturus, Life Technologies) following the manufacturer’s instructions, including Qiagen DNase treatment, and immediately reverse transcribed using the High Capacity Reverse Transcription kit with RNase inhibitor (Life Technologies). cDNA was diluted to a final volume of 50 μl and split into four 12.5 μl aliquots. .. To increase the number of targeted copies, cDNA was preamplified for a total of 75 cellular and 6 viral transcripts (maximum of 22–26 transcripts/aliquot) using a pool of 100-fold diluted 20× Taqman Gene Expression Assays and the TaqMan PreAmp Master Mix (Life Technologies) at the following thermal conditions: 50 °C for 2 min and 95 °C for 10 min, followed by 95 °C for 15 s and 1 min at 60 °C for 14 cycles.

    Real-time Polymerase Chain Reaction:

    Article Title: Identifying and avoiding off-target effects of RNase H-dependent antisense oligonucleotides in mice
    Article Snippet: From this, cDNA was synthesized using M-MLT Reverse Transcriptase, random decamers RETROscript and RNase inhibitor (Ambion) with 100 mM dNTP set PCR Grade (Invitrogen) and DNase/RNase free water (Gibco) according the manufacturers’ instructions. .. For gene expression analysis, qPCR was performed using TaqMan Fast Advanced Master Mix (2×) (Ambion) in a doublex set-up.

    Article Title: The chromatin scaffold protein SAFB1 localizes SUMO-1 to the promoters of ribosomal protein genes to facilitate transcription initiation and splicing
    Article Snippet: The nuclear pellet was resuspended in 100 μl of DNAse I buffer (50 mM Tris–HCl, pH 7.5, 1 mM EDTA, 10 mM MgCl2 and 0.5 U RNase inhibitor) and treated 20 U of DNase I (Invitrogen) for 60 min at 37°C. .. The nuclear pellet was resuspended in 100 μl of DNAse I buffer (50 mM Tris–HCl, pH 7.5, 1 mM EDTA, 10 mM MgCl2 and 0.5 U RNase inhibitor) and treated 20 U of DNase I (Invitrogen) for 60 min at 37°C.

    Article Title: Scavenger receptor BI promotes cytoplasmic accumulation of lipoproteins in clear-cell renal cell carcinoma
    Article Snippet: Paragraph title: Real-time PCR ... Genomic DNA was removed by digestion using DNase (Roche) and RNase inhibitor (Ribolock; Thermo Scientific).

    Article Title: Preserved DNA Damage Checkpoint Pathway Protects against Complications in Long-Standing Type 1 Diabetes
    Article Snippet: For miR200 expression analyses, total RNA was extracted using either fibroblasts, iPSCs, or serum samples obtained from three clinical groups (Control, Medalist −C, and Medalist +C) using standard TRIzol-based RNA extraction protocols in the presence of RNase inhibitors (Life Technologies) and DNase treatment (QIAGEN). .. Samples with a 260/280 optical densitometry ratios of 2 were used for further analyses that included cDNA conversion using miRCURY LNA Universal RT microRNA PCR cDNA synthesis kit (Exiqon).

    Article Title: Transcriptional profile and Epstein-Barr virus infection status of laser-cut immune infiltrates from the brain of patients with progressive multiple sclerosis
    Article Snippet: Total RNA was extracted from the microdissected samples using Picopure RNA isolation kit (Arcturus, Life Technologies) following the manufacturer’s instructions, including Qiagen DNase treatment, and immediately reverse transcribed using the High Capacity Reverse Transcription kit with RNase inhibitor (Life Technologies). cDNA was diluted to a final volume of 50 μl and split into four 12.5 μl aliquots. .. The final preamplification product was diluted 1:5 and used as template for downstream PCR analysis.

    Ligation:

    Article Title: Increased sensitivity and accuracy of a single-stranded DNA splint-mediated ligation assay (sPAT) reveals poly(A) tail length dynamics of developmentally regulated mRNAs
    Article Snippet: In a 10 μl reaction volume, a mixture of total RNA, 2 μM DNA splint, and 3 μM RNA anchor were annealed in a thermo cycler (MJ Research) incubated at 70 °C for 5 min, 60 °C for 5 min, 42 °C for 5 min, 25 °C for 5 min, before storage at 15 °C. .. The ligation was performed in a reaction volume of 20 μl with 50 mM TRIS-HCl pH 7.5, 2 mM MgCl2, 1 mM DTT, 400 μM ATP, 10 U RNA ligase 2 (Rnl2, NEB), and 40 U RNase inhibitor (Ribolock, Fermentas) at 15 °C for 16 h. The DNA splint was removed by the addition of a 20 μl master mix that contains 80 mM TRIS-HCl pH7.9, 20 mM NaCl, 12 mM MgCl2, 2 mM CaCl2, 20 U DNase I (Roche), and 40 U Ribolock (Fermentas) at 37 °C for 3 h. The ligated RNA was phenol/chloroform extracted, ethanol precipitated, and dissolved in 20 μl water. .. The RNA anchor and DNA splint sequences are listed in .

    Cell Culture:

    Article Title: LTBP2 is secreted from lung myofibroblasts and is a potential biomarker for idiopathic pulmonary fibrosis
    Article Snippet: Total RNA was extracted from freshly isolated mouse cells or cultured human cells using TRIzol (Invitrogen, U.S.A.) with glycogen as a carrier. .. Extracted RNA was treated with RNase free DNase 1 (Ambion, U.S.A.) in the presence of RNase Inhibitor (Invitrogen, U.S.A.) for 10 min at 37°C.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Variations of Growth and Toxin Yield in Cylindrospermopsis raciborskii under Different Phosphorus Concentrations
    Article Snippet: DNA digestion was performed after RNA isolation with RNase-free DNase (Promega, Madison, WI, USA) and RNase Inhibitor (Thermo, Waltham, MA, USA). .. DNA digestion was performed after RNA isolation with RNase-free DNase (Promega, Madison, WI, USA) and RNase Inhibitor (Thermo, Waltham, MA, USA).

    Digital PCR:

    Article Title: Transcriptional profile and Epstein-Barr virus infection status of laser-cut immune infiltrates from the brain of patients with progressive multiple sclerosis
    Article Snippet: Paragraph title: Preamplification real-time reverse transcription PCR and droplet digital PCR ... Total RNA was extracted from the microdissected samples using Picopure RNA isolation kit (Arcturus, Life Technologies) following the manufacturer’s instructions, including Qiagen DNase treatment, and immediately reverse transcribed using the High Capacity Reverse Transcription kit with RNase inhibitor (Life Technologies). cDNA was diluted to a final volume of 50 μl and split into four 12.5 μl aliquots.

    Inhibition:

    Article Title: The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca2+ and Mg2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa
    Article Snippet: As observed in other works [ , , ], the RNase activity of TcPR-4b was inhibited by heating and in the presence of RNase inhibitor (RiboLock, Thermo Scientific) which is able to annul the activity of type A, B and C RNases. .. The antifungal activity of TcPR-4b was verified in vitro on M. perniciosa hyphae (Figure A) and was associated to the increase of mitochondrial O2 - production detected by DHE (Figure C and E).

    Sequencing:

    Article Title: Brain microbiota disruption within inflammatory demyelinating lesions in multiple sclerosis
    Article Snippet: All assays including RNA and DNA extractions contained water controls that were carried forward through all subsequent steps and each new step added an additional water control including cloning and Sanger sequencing. .. All amplification steps were set up in areas and with equipment treated with DNase and RNase inhibitor (Molecular BioProducts, San Diego CA, USA).

    Sonication:

    Article Title: The RNA-binding protein TTP is a global post-transcriptional regulator of feedback control in inflammation
    Article Snippet: After washing with ice-cold PBS, intact cells were irradiated with UV-C light (300 mJ/cm2 , Stratalinker 2400) and total cell extracts were obtained using lysis buffer (50 mM Tris/HCl [pH 8.0], 150 mM NaCl, 0.5% (v/v) Triton X-100 and 1 mM EDTA) and sonication (3 times for 20 s in ice). .. Extract were cleared by centrifugation (15 min at 4°C) and treated with RNase A (0.9375 × 10−3 Units/ml, Roche) and DNase I (20 Units/ml, Ambion) for 3 min at 37°C and cooled on ice for 5 min after adding urea to 0.5 M and RNase inhibitors (266.8 Units of RiboLock, Thermo/Fermentas; and 133.4 U of RNaseOUT, Life Technologies).

    Recombinant:

    Article Title: Elucidation of the Photorhabdus temperata Genome and Generation of a Transposon Mutant Library To Identify Motility Mutants Altered in Pathogenesis
    Article Snippet: The RNA was extracted by the use of the Qiagen RNeasy minikit (Qiagen Sciences, Valencia, CA), and the manufacturer's protocol was followed except that in the final elution step, 50 μl of nuclease-free water was added directly to the column membrane and incubated at room temperature for 10 min before elution. .. To remove residual DNA, RNA samples were treated with RNase-free DNase as follows: 50 μl RNA, 10 μl 10× DNase I buffer (New England BioLabs, Ipswich, MA), 5 U DNase I (2 U/μl; New England BioLabs, Ipswich, MA), 5 μl RNase Out recombinant RNase inhibitor (40 U/μl; Invitrogen, Carlsbad, CA) and RNase-free water to reach a total reaction volume of 100 μl. .. Samples were incubated at 37°C for 30 min. DNase activity was inactivated with the addition of 1 μl 0.5 M EDTA (pH 8.0) (Ambion, Inc., Austin, TX) and a 10-min incubation at 75°C.

    Article Title: The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca2+ and Mg2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa
    Article Snippet: The enzymatic tests carried out with the recombinant TcPR-4b revealed that this protein presented both DNase and RNase activities (Figures and ). .. As observed in other works [ , , ], the RNase activity of TcPR-4b was inhibited by heating and in the presence of RNase inhibitor (RiboLock, Thermo Scientific) which is able to annul the activity of type A, B and C RNases.

    Article Title: Identification of two novel HSP90 proteins in Babesia orientalis: molecular characterization, and computational analyses of their structure, function, antigenicity and inhibitor interaction
    Article Snippet: The leukocytes were removed from the blood using Plasmodipur filters (EuroProxima, Arnhem, the Netherlands) and total RNA was extracted from 250 μl of RBCs using TRIzol® RNA extraction kit (Invitrogen, USA) according to the manufacturer’s instruction. .. RNA samples were treated with DNase I (Invitrogen, USA); RNase inhibitor (RNaseOUT™ Recombinant Ribonuclease Inhibitor, Invitrogen, USA) was added and then the RNA was reverse transcribed using FastQuant® RT Kit (TIANGEN Biotech (Beijing) Co., Ltd.) according to the manufacturer’s instruction. .. The genomic DNA was extracted from 200 μl of infected blood using QIAamp® DNA mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions and stored at -20 °C till further use.

    Isolation:

    Article Title: Hepatic expression profiles in retroviral infection: relevance to drug hypersensitivity risk. Hepatic expression profiles in retroviral infection: relevance to drug hypersensitivity risk
    Article Snippet: Paragraph title: Liver sample collection and RNA isolation ... RNA pellets were resuspended in RNase free water and were treated with 2U DNase I (Ambion®) at 37°C for 30 min, followed by inactivation with EDTA (5 mmol/L) and heating at 75°C for 10 min. RNase inhibitor (Applied Biosystems, Foster City, CA) was added to a final concentration of 1U/μ L. RNA integrity (Schroeder et al. ) was assessed by Agilent 2100 BioAnalyzer, and RNA was quantified by NanoDrop ND‐1000 (ThermoFisher, Madison, WI).

    Article Title: Variations of Growth and Toxin Yield in Cylindrospermopsis raciborskii under Different Phosphorus Concentrations
    Article Snippet: Cells were collected via centrifugation of 9.0–40 mL of culture at 8000 rpm, 4 °C for 2 min, and total RNA was extracted using the RNAiso Plus Kit (Takara, Otsu, Japan) according to the manufacturer’s instructions. .. DNA digestion was performed after RNA isolation with RNase-free DNase (Promega, Madison, WI, USA) and RNase Inhibitor (Thermo, Waltham, MA, USA). .. RNA quality and purity was assessed using a Nanodrop spectrophotometer (Thermo, Waltham, MA, USA) and a total of 900 ng of RNA was taken for reverse transcription (RT) reaction using the PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Otsu, Japan) according to the manufacturer’s instructions.

    Article Title: Cell differentiation defines acute and chronic infection cell types in Staphylococcus aureus
    Article Snippet: After washing, the total RNA was resuspended in 42 μl of RNAse-free water (Qiagen) were added and the sample was incubated at 65°C at 1000 rpm for 5 min prior storage in ice. .. To remove any DNA traces, the isolated RNA was treated (one to three times, depending on the sample) with 4 Units of RNase-free DNase I (Thermo), 10 Units of SUPERase In RNase Inhibitor (Life Technologies) and incubated for 45 min at 37°C. .. To remove the DNase I, 50 μl of RNAse-free water and 100 μl of Roti-Aqua-P/C/I (Phenol, Chloroform, Isoamyl alcohol 25:24:1 pH 4.5–5) (Carl Roth) were added to the reaction tube, mixed, transferred to a 2 mL PLGH tube and centrifuged for 12 min at 13000 rpm and 15°C.

    Article Title: Identifying and avoiding off-target effects of RNase H-dependent antisense oligonucleotides in mice
    Article Snippet: Paragraph title: RNA isolation from human cells and analysis by qRT-PCR ... From this, cDNA was synthesized using M-MLT Reverse Transcriptase, random decamers RETROscript and RNase inhibitor (Ambion) with 100 mM dNTP set PCR Grade (Invitrogen) and DNase/RNase free water (Gibco) according the manufacturers’ instructions.

    Article Title: LTBP2 is secreted from lung myofibroblasts and is a potential biomarker for idiopathic pulmonary fibrosis
    Article Snippet: Total RNA was extracted from freshly isolated mouse cells or cultured human cells using TRIzol (Invitrogen, U.S.A.) with glycogen as a carrier. .. Extracted RNA was treated with RNase free DNase 1 (Ambion, U.S.A.) in the presence of RNase Inhibitor (Invitrogen, U.S.A.) for 10 min at 37°C.

    Article Title: The chromatin scaffold protein SAFB1 localizes SUMO-1 to the promoters of ribosomal protein genes to facilitate transcription initiation and splicing
    Article Snippet: Paragraph title: Isolation of nuclear and cytoplasmic RNA, RT-qPCR ... The nuclear pellet was resuspended in 100 μl of DNAse I buffer (50 mM Tris–HCl, pH 7.5, 1 mM EDTA, 10 mM MgCl2 and 0.5 U RNase inhibitor) and treated 20 U of DNase I (Invitrogen) for 60 min at 37°C.

    Article Title: Scavenger receptor BI promotes cytoplasmic accumulation of lipoproteins in clear-cell renal cell carcinoma
    Article Snippet: Total RNA was isolated using TRI reagent (Sigma T9424) according to the manufacturer’s instructions. .. Genomic DNA was removed by digestion using DNase (Roche) and RNase inhibitor (Ribolock; Thermo Scientific).

    Article Title: Role of sph2 Gene Regulation in Hemolytic and Sphingomyelinase Activities Produced by Leptospira interrogans
    Article Snippet: RNA was isolated from leptospires as follows: 1 mL Trizol (Life Technologies; Grand Island, New York, USA) was added to the cell pellet, resuspended thoroughly by pipetting followed by incubation at room temperature for 5 min. 200 μL of chloroform (Sigma-Aldrich) was added, mixed vigorously for 15 s followed by incubation at room temperature for 3 min. .. The dissolved RNA was protected from degradation by addition of 1μL of 20 U/μL SUPERase In RNase inhibitor (Life Technologies).

    Article Title: Transcriptional profile and Epstein-Barr virus infection status of laser-cut immune infiltrates from the brain of patients with progressive multiple sclerosis
    Article Snippet: After microdissection, the pooled tissue fragments of each series were incubated immediately in 50 μl of RNA stabilizing, extraction buffer (PicoPure RNA isolation kit, Arcturus, Life Technologies) at 42 °C for 30 min and centrifuged at 800×g for 2 min. Lysates were stored at − 80 °C until use. .. Total RNA was extracted from the microdissected samples using Picopure RNA isolation kit (Arcturus, Life Technologies) following the manufacturer’s instructions, including Qiagen DNase treatment, and immediately reverse transcribed using the High Capacity Reverse Transcription kit with RNase inhibitor (Life Technologies). cDNA was diluted to a final volume of 50 μl and split into four 12.5 μl aliquots. .. To increase the number of targeted copies, cDNA was preamplified for a total of 75 cellular and 6 viral transcripts (maximum of 22–26 transcripts/aliquot) using a pool of 100-fold diluted 20× Taqman Gene Expression Assays and the TaqMan PreAmp Master Mix (Life Technologies) at the following thermal conditions: 50 °C for 2 min and 95 °C for 10 min, followed by 95 °C for 15 s and 1 min at 60 °C for 14 cycles.

    Labeling:

    Article Title: Identifying and avoiding off-target effects of RNase H-dependent antisense oligonucleotides in mice
    Article Snippet: From this, cDNA was synthesized using M-MLT Reverse Transcriptase, random decamers RETROscript and RNase inhibitor (Ambion) with 100 mM dNTP set PCR Grade (Invitrogen) and DNase/RNase free water (Gibco) according the manufacturers’ instructions. .. For gene expression analysis, qPCR was performed using TaqMan Fast Advanced Master Mix (2×) (Ambion) in a doublex set-up.

    Purification:

    Article Title: Identifying and avoiding off-target effects of RNase H-dependent antisense oligonucleotides in mice
    Article Snippet: RNA was extracted using the PureLink Pro 96 RNA Purification kit according to the manufacturer's instructions (Ambion). .. From this, cDNA was synthesized using M-MLT Reverse Transcriptase, random decamers RETROscript and RNase inhibitor (Ambion) with 100 mM dNTP set PCR Grade (Invitrogen) and DNase/RNase free water (Gibco) according the manufacturers’ instructions.

    Article Title: The chromatin scaffold protein SAFB1 localizes SUMO-1 to the promoters of ribosomal protein genes to facilitate transcription initiation and splicing
    Article Snippet: Nuclear and cytoplasmic RNAs were purified by lysis of HeLa-SUMO1 cells in 200 μl of lysis buffer (10 mM Tris–HCl, pH 8, 1.5 mM MgCl2 , 0.5% NP-40, 140 mM NaCl and 0.5 U of RNase inhibitor) and loaded onto 200 μl of cushion buffer (10 mM Tris–HCl, pH 8, 1.5 mM MgCl2 , 1% NP-40, 140 mM NaCl and 0.4 M sucrose). .. The nuclear pellet was resuspended in 100 μl of DNAse I buffer (50 mM Tris–HCl, pH 7.5, 1 mM EDTA, 10 mM MgCl2 and 0.5 U RNase inhibitor) and treated 20 U of DNase I (Invitrogen) for 60 min at 37°C.

    Polymerase Chain Reaction:

    Article Title: Identifying and avoiding off-target effects of RNase H-dependent antisense oligonucleotides in mice
    Article Snippet: RNA was extracted using the PureLink Pro 96 RNA Purification kit according to the manufacturer's instructions (Ambion). .. From this, cDNA was synthesized using M-MLT Reverse Transcriptase, random decamers RETROscript and RNase inhibitor (Ambion) with 100 mM dNTP set PCR Grade (Invitrogen) and DNase/RNase free water (Gibco) according the manufacturers’ instructions. .. For gene expression analysis, qPCR was performed using TaqMan Fast Advanced Master Mix (2×) (Ambion) in a doublex set-up.

    Article Title: LTBP2 is secreted from lung myofibroblasts and is a potential biomarker for idiopathic pulmonary fibrosis
    Article Snippet: Extracted RNA was treated with RNase free DNase 1 (Ambion, U.S.A.) in the presence of RNase Inhibitor (Invitrogen, U.S.A.) for 10 min at 37°C. .. Extracted RNA was treated with RNase free DNase 1 (Ambion, U.S.A.) in the presence of RNase Inhibitor (Invitrogen, U.S.A.) for 10 min at 37°C.

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: 5 × buffer for transcription (see recipe) 3 NTP mix (see recipe) 100 µM CTP (Thermo Scientific) 10 µCi/µl [α32 P]CTP (sp. act. .. 800 Ci/mmol; PerkinElmer) 1 µg/µl DNA template (from linearized plasmid, PCR, or oligodeoxynucleotides) 40 U/µl RNase inhibitor (Thermo Scientific) 20 U/µl T7 RNA polymerase (Thermo Scientific) DNase I (RNase-free; Thermo Scientific) G50 buffer (see recipe) 25:24:1 phenol/chloroform/isoamyl alcohol 100% ethanol Additional reagents and equipment for urea-PAGE ( ) and autoradiography ( APPENDIX 3A ) Prepare the following reaction mixture at room temperature in a 1.5-ml microcen-trifuge tube with the components added in the indicated order (total reaction volume, 20 µl): 4 µl 5 × buffer for transcription 4.6 µl distilled deionized H2 O 1 µl 3 NTP mix, 10 mM each of ATP, GTP, UTP (0.5 mM each NTP final concentration) 2.4 µl 100 µM CTP (12 µM final concentration) 5 µl 10 µCi/µl [α32 P]CTP (sp. act. .. 800 Ci/mmol) 1 µl 1 µg/µl linear DNA template 1 µl 40 U/µl RNase inhibitor 1 µl 20 U/µl T7 RNA polymerase.

    Article Title: Preserved DNA Damage Checkpoint Pathway Protects against Complications in Long-Standing Type 1 Diabetes
    Article Snippet: For miR200 expression analyses, total RNA was extracted using either fibroblasts, iPSCs, or serum samples obtained from three clinical groups (Control, Medalist −C, and Medalist +C) using standard TRIzol-based RNA extraction protocols in the presence of RNase inhibitors (Life Technologies) and DNase treatment (QIAGEN). .. RNA measurements used ND-1000 spectrophotometer V3.5 (NanoDrop Technologies, Inc.).

    Article Title: Transcriptional profile and Epstein-Barr virus infection status of laser-cut immune infiltrates from the brain of patients with progressive multiple sclerosis
    Article Snippet: Paragraph title: Preamplification real-time reverse transcription PCR and droplet digital PCR ... Total RNA was extracted from the microdissected samples using Picopure RNA isolation kit (Arcturus, Life Technologies) following the manufacturer’s instructions, including Qiagen DNase treatment, and immediately reverse transcribed using the High Capacity Reverse Transcription kit with RNase inhibitor (Life Technologies). cDNA was diluted to a final volume of 50 μl and split into four 12.5 μl aliquots.

    Blocking Assay:

    Article Title: Increased sensitivity and accuracy of a single-stranded DNA splint-mediated ligation assay (sPAT) reveals poly(A) tail length dynamics of developmentally regulated mRNAs
    Article Snippet: To allow ligation to the mRNA, but block RNA anchor self-ligation, the anchor was synthesized with the typical 5′ phosphate group and an alternative 3′ amino group in comparison to a hydroxyl group (Metabion or biomers.net). .. The ligation was performed in a reaction volume of 20 μl with 50 mM TRIS-HCl pH 7.5, 2 mM MgCl2, 1 mM DTT, 400 μM ATP, 10 U RNA ligase 2 (Rnl2, NEB), and 40 U RNase inhibitor (Ribolock, Fermentas) at 15 °C for 16 h. The DNA splint was removed by the addition of a 20 μl master mix that contains 80 mM TRIS-HCl pH7.9, 20 mM NaCl, 12 mM MgCl2, 2 mM CaCl2, 20 U DNase I (Roche), and 40 U Ribolock (Fermentas) at 37 °C for 3 h. The ligated RNA was phenol/chloroform extracted, ethanol precipitated, and dissolved in 20 μl water.

    De-Phosphorylation Assay:

    Article Title: The RNA-binding protein TTP is a global post-transcriptional regulator of feedback control in inflammation
    Article Snippet: Extract were cleared by centrifugation (15 min at 4°C) and treated with RNase A (0.9375 × 10−3 Units/ml, Roche) and DNase I (20 Units/ml, Ambion) for 3 min at 37°C and cooled on ice for 5 min after adding urea to 0.5 M and RNase inhibitors (266.8 Units of RiboLock, Thermo/Fermentas; and 133.4 U of RNaseOUT, Life Technologies). .. Extract were cleared by centrifugation (15 min at 4°C) and treated with RNase A (0.9375 × 10−3 Units/ml, Roche) and DNase I (20 Units/ml, Ambion) for 3 min at 37°C and cooled on ice for 5 min after adding urea to 0.5 M and RNase inhibitors (266.8 Units of RiboLock, Thermo/Fermentas; and 133.4 U of RNaseOUT, Life Technologies).

    Concentration Assay:

    Article Title: Hepatic expression profiles in retroviral infection: relevance to drug hypersensitivity risk. Hepatic expression profiles in retroviral infection: relevance to drug hypersensitivity risk
    Article Snippet: Total RNA was extracted by homogenizing liver samples in TRIzol (Ambion®) according to the manufacturer's protocol. .. RNA pellets were resuspended in RNase free water and were treated with 2U DNase I (Ambion®) at 37°C for 30 min, followed by inactivation with EDTA (5 mmol/L) and heating at 75°C for 10 min. RNase inhibitor (Applied Biosystems, Foster City, CA) was added to a final concentration of 1U/μ L. RNA integrity (Schroeder et al. ) was assessed by Agilent 2100 BioAnalyzer, and RNA was quantified by NanoDrop ND‐1000 (ThermoFisher, Madison, WI). .. RNA was stored at −80°C until preparation for arrays.

    Article Title: The expression and construction of engineering Escherichia coli producing humanized AluY RNAs
    Article Snippet: The precipitate was washed three times with 75% ethanol to gain RNAs. .. DNase I (TaKaRa Biotechnology, Japan) and RNase inhibitor (Thermo Scientific, USA) were added at a final concentration of 0.5 U/ml to wipe off contaminating DNAs. .. Then RNAs without contaminated DNAs were used to detect AluY RNAs using northern blotting method.

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: 5 × buffer for transcription (see recipe) 3 NTP mix (see recipe) 100 µM CTP (Thermo Scientific) 10 µCi/µl [α32 P]CTP (sp. act. .. 800 Ci/mmol; PerkinElmer) 1 µg/µl DNA template (from linearized plasmid, PCR, or oligodeoxynucleotides) 40 U/µl RNase inhibitor (Thermo Scientific) 20 U/µl T7 RNA polymerase (Thermo Scientific) DNase I (RNase-free; Thermo Scientific) G50 buffer (see recipe) 25:24:1 phenol/chloroform/isoamyl alcohol 100% ethanol Additional reagents and equipment for urea-PAGE ( ) and autoradiography ( APPENDIX 3A ) Prepare the following reaction mixture at room temperature in a 1.5-ml microcen-trifuge tube with the components added in the indicated order (total reaction volume, 20 µl): 4 µl 5 × buffer for transcription 4.6 µl distilled deionized H2 O 1 µl 3 NTP mix, 10 mM each of ATP, GTP, UTP (0.5 mM each NTP final concentration) 2.4 µl 100 µM CTP (12 µM final concentration) 5 µl 10 µCi/µl [α32 P]CTP (sp. act. .. 800 Ci/mmol) 1 µl 1 µg/µl linear DNA template 1 µl 40 U/µl RNase inhibitor 1 µl 20 U/µl T7 RNA polymerase.

    Article Title: Gene Expression Response of Salmonella enterica Serotype Enteritidis Phage Type 8 to Subinhibitory Concentrations of the Plant-Derived Compounds Trans-Cinnamaldehyde and Eugenol
    Article Snippet: The sample was split into two and each was provided with 500 U of RNase inhibitor (Ribolock®, Fermentas), 250 U of DNase (Fermentas), 20 μl of 1 M Tris (pH 8.3) and 10 μl of 1 M MgCl, followed by mixing and incubation at 37°C for 30 min. .. The sample was split into two and each was provided with 500 U of RNase inhibitor (Ribolock®, Fermentas), 250 U of DNase (Fermentas), 20 μl of 1 M Tris (pH 8.3) and 10 μl of 1 M MgCl, followed by mixing and incubation at 37°C for 30 min.

    Article Title: Role of sph2 Gene Regulation in Hemolytic and Sphingomyelinase Activities Produced by Leptospira interrogans
    Article Snippet: The dissolved RNA was protected from degradation by addition of 1μL of 20 U/μL SUPERase In RNase inhibitor (Life Technologies). .. DNA was digested by addition of 10 μL of 10X Turbo DNase buffer and 5 μL of Turbo DNase followed by incubation in 37°C water bath for 2 h. The RNA samples were subjected to clean-up using the RNeasy Mini kit (Qiagen; USA) as described in the manufacturer’s instructions.

    Activated Clotting Time Assay:

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: 5 × buffer for transcription (see recipe) 3 NTP mix (see recipe) 100 µM CTP (Thermo Scientific) 10 µCi/µl [α32 P]CTP (sp. act. .. 800 Ci/mmol; PerkinElmer) 1 µg/µl DNA template (from linearized plasmid, PCR, or oligodeoxynucleotides) 40 U/µl RNase inhibitor (Thermo Scientific) 20 U/µl T7 RNA polymerase (Thermo Scientific) DNase I (RNase-free; Thermo Scientific) G50 buffer (see recipe) 25:24:1 phenol/chloroform/isoamyl alcohol 100% ethanol Additional reagents and equipment for urea-PAGE ( ) and autoradiography ( APPENDIX 3A ) Prepare the following reaction mixture at room temperature in a 1.5-ml microcen-trifuge tube with the components added in the indicated order (total reaction volume, 20 µl): 4 µl 5 × buffer for transcription 4.6 µl distilled deionized H2 O 1 µl 3 NTP mix, 10 mM each of ATP, GTP, UTP (0.5 mM each NTP final concentration) 2.4 µl 100 µM CTP (12 µM final concentration) 5 µl 10 µCi/µl [α32 P]CTP (sp. act. .. 800 Ci/mmol) 1 µl 1 µg/µl linear DNA template 1 µl 40 U/µl RNase inhibitor 1 µl 20 U/µl T7 RNA polymerase.

    Plasmid Preparation:

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: 5 × buffer for transcription (see recipe) 3 NTP mix (see recipe) 100 µM CTP (Thermo Scientific) 10 µCi/µl [α32 P]CTP (sp. act. .. 800 Ci/mmol; PerkinElmer) 1 µg/µl DNA template (from linearized plasmid, PCR, or oligodeoxynucleotides) 40 U/µl RNase inhibitor (Thermo Scientific) 20 U/µl T7 RNA polymerase (Thermo Scientific) DNase I (RNase-free; Thermo Scientific) G50 buffer (see recipe) 25:24:1 phenol/chloroform/isoamyl alcohol 100% ethanol Additional reagents and equipment for urea-PAGE ( ) and autoradiography ( APPENDIX 3A ) Prepare the following reaction mixture at room temperature in a 1.5-ml microcen-trifuge tube with the components added in the indicated order (total reaction volume, 20 µl): 4 µl 5 × buffer for transcription 4.6 µl distilled deionized H2 O 1 µl 3 NTP mix, 10 mM each of ATP, GTP, UTP (0.5 mM each NTP final concentration) 2.4 µl 100 µM CTP (12 µM final concentration) 5 µl 10 µCi/µl [α32 P]CTP (sp. act. .. 800 Ci/mmol) 1 µl 1 µg/µl linear DNA template 1 µl 40 U/µl RNase inhibitor 1 µl 20 U/µl T7 RNA polymerase.

    Software:

    Article Title: LTBP2 is secreted from lung myofibroblasts and is a potential biomarker for idiopathic pulmonary fibrosis
    Article Snippet: Extracted RNA was treated with RNase free DNase 1 (Ambion, U.S.A.) in the presence of RNase Inhibitor (Invitrogen, U.S.A.) for 10 min at 37°C. .. After first-strand cDNA was synthesized using SuperScript Reverse Transcriptase (Invitrogen, U.S.A.) with random primers (Invitrogen, U.S.A.), cDNA equivalent to 100 cells was used for each PCR.

    Irradiation:

    Article Title: The RNA-binding protein TTP is a global post-transcriptional regulator of feedback control in inflammation
    Article Snippet: After washing with ice-cold PBS, intact cells were irradiated with UV-C light (300 mJ/cm2 , Stratalinker 2400) and total cell extracts were obtained using lysis buffer (50 mM Tris/HCl [pH 8.0], 150 mM NaCl, 0.5% (v/v) Triton X-100 and 1 mM EDTA) and sonication (3 times for 20 s in ice). .. Extract were cleared by centrifugation (15 min at 4°C) and treated with RNase A (0.9375 × 10−3 Units/ml, Roche) and DNase I (20 Units/ml, Ambion) for 3 min at 37°C and cooled on ice for 5 min after adding urea to 0.5 M and RNase inhibitors (266.8 Units of RiboLock, Thermo/Fermentas; and 133.4 U of RNaseOUT, Life Technologies).

    RNA Extraction:

    Article Title: Elucidation of the Photorhabdus temperata Genome and Generation of a Transposon Mutant Library To Identify Motility Mutants Altered in Pathogenesis
    Article Snippet: Paragraph title: RNA extraction and DNase treatment. ... To remove residual DNA, RNA samples were treated with RNase-free DNase as follows: 50 μl RNA, 10 μl 10× DNase I buffer (New England BioLabs, Ipswich, MA), 5 U DNase I (2 U/μl; New England BioLabs, Ipswich, MA), 5 μl RNase Out recombinant RNase inhibitor (40 U/μl; Invitrogen, Carlsbad, CA) and RNase-free water to reach a total reaction volume of 100 μl.

    Article Title: Identification of two novel HSP90 proteins in Babesia orientalis: molecular characterization, and computational analyses of their structure, function, antigenicity and inhibitor interaction
    Article Snippet: The leukocytes were removed from the blood using Plasmodipur filters (EuroProxima, Arnhem, the Netherlands) and total RNA was extracted from 250 μl of RBCs using TRIzol® RNA extraction kit (Invitrogen, USA) according to the manufacturer’s instruction. .. RNA samples were treated with DNase I (Invitrogen, USA); RNase inhibitor (RNaseOUT™ Recombinant Ribonuclease Inhibitor, Invitrogen, USA) was added and then the RNA was reverse transcribed using FastQuant® RT Kit (TIANGEN Biotech (Beijing) Co., Ltd.) according to the manufacturer’s instruction.

    Article Title: Preserved DNA Damage Checkpoint Pathway Protects against Complications in Long-Standing Type 1 Diabetes
    Article Snippet: All virus infections used 5 µg/ml Polybrene reagent (EMD Millipore). .. For miR200 expression analyses, total RNA was extracted using either fibroblasts, iPSCs, or serum samples obtained from three clinical groups (Control, Medalist −C, and Medalist +C) using standard TRIzol-based RNA extraction protocols in the presence of RNase inhibitors (Life Technologies) and DNase treatment (QIAGEN). .. RNA measurements used ND-1000 spectrophotometer V3.5 (NanoDrop Technologies, Inc.).

    Article Title: Gene Expression Response of Salmonella enterica Serotype Enteritidis Phage Type 8 to Subinhibitory Concentrations of the Plant-Derived Compounds Trans-Cinnamaldehyde and Eugenol
    Article Snippet: Paragraph title: Total RNA extraction ... The sample was split into two and each was provided with 500 U of RNase inhibitor (Ribolock®, Fermentas), 250 U of DNase (Fermentas), 20 μl of 1 M Tris (pH 8.3) and 10 μl of 1 M MgCl, followed by mixing and incubation at 37°C for 30 min.

    Article Title: Role of sph2 Gene Regulation in Hemolytic and Sphingomyelinase Activities Produced by Leptospira interrogans
    Article Snippet: Paragraph title: RNA extraction and DNase treatment ... The dissolved RNA was protected from degradation by addition of 1μL of 20 U/μL SUPERase In RNase inhibitor (Life Technologies).

    Agarose Gel Electrophoresis:

    Article Title: Role of sph2 Gene Regulation in Hemolytic and Sphingomyelinase Activities Produced by Leptospira interrogans
    Article Snippet: The dissolved RNA was protected from degradation by addition of 1μL of 20 U/μL SUPERase In RNase inhibitor (Life Technologies). .. DNA was digested by addition of 10 μL of 10X Turbo DNase buffer and 5 μL of Turbo DNase followed by incubation in 37°C water bath for 2 h. The RNA samples were subjected to clean-up using the RNeasy Mini kit (Qiagen; USA) as described in the manufacturer’s instructions.

    In Vitro:

    Article Title: The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca2+ and Mg2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa
    Article Snippet: As observed in other works [ , , ], the RNase activity of TcPR-4b was inhibited by heating and in the presence of RNase inhibitor (RiboLock, Thermo Scientific) which is able to annul the activity of type A, B and C RNases. .. Moreover, for class I PR-4 s, as FaPR4, the presence of the chitin-binding domain may contribute to the thermal stability of the protein avoiding the lack of RNase activity when the protein was submitted to heating [ ].

    Electrophoresis:

    Article Title: Role of sph2 Gene Regulation in Hemolytic and Sphingomyelinase Activities Produced by Leptospira interrogans
    Article Snippet: The dissolved RNA was protected from degradation by addition of 1μL of 20 U/μL SUPERase In RNase inhibitor (Life Technologies). .. DNA was digested by addition of 10 μL of 10X Turbo DNase buffer and 5 μL of Turbo DNase followed by incubation in 37°C water bath for 2 h. The RNA samples were subjected to clean-up using the RNeasy Mini kit (Qiagen; USA) as described in the manufacturer’s instructions.

    Spectrophotometry:

    Article Title: Elucidation of the Photorhabdus temperata Genome and Generation of a Transposon Mutant Library To Identify Motility Mutants Altered in Pathogenesis
    Article Snippet: To remove residual DNA, RNA samples were treated with RNase-free DNase as follows: 50 μl RNA, 10 μl 10× DNase I buffer (New England BioLabs, Ipswich, MA), 5 U DNase I (2 U/μl; New England BioLabs, Ipswich, MA), 5 μl RNase Out recombinant RNase inhibitor (40 U/μl; Invitrogen, Carlsbad, CA) and RNase-free water to reach a total reaction volume of 100 μl. .. Samples were incubated at 37°C for 30 min. DNase activity was inactivated with the addition of 1 μl 0.5 M EDTA (pH 8.0) (Ambion, Inc., Austin, TX) and a 10-min incubation at 75°C.

    Article Title: Role of sph2 Gene Regulation in Hemolytic and Sphingomyelinase Activities Produced by Leptospira interrogans
    Article Snippet: The dissolved RNA was protected from degradation by addition of 1μL of 20 U/μL SUPERase In RNase inhibitor (Life Technologies). .. DNA was digested by addition of 10 μL of 10X Turbo DNase buffer and 5 μL of Turbo DNase followed by incubation in 37°C water bath for 2 h. The RNA samples were subjected to clean-up using the RNeasy Mini kit (Qiagen; USA) as described in the manufacturer’s instructions.

    Sampling:

    Article Title: Brain microbiota disruption within inflammatory demyelinating lesions in multiple sclerosis
    Article Snippet: All post-collection tissue manipulation was performed in decontaminated biosafety hoods with autoclaved or chemically decontaminated tools and no sampling was performed from the exposed surface of tissues. .. All amplification steps were set up in areas and with equipment treated with DNase and RNase inhibitor (Molecular BioProducts, San Diego CA, USA).

    Immunoprecipitation:

    Article Title: The RNA-binding protein TTP is a global post-transcriptional regulator of feedback control in inflammation
    Article Snippet: Paragraph title: Individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP) ... Extract were cleared by centrifugation (15 min at 4°C) and treated with RNase A (0.9375 × 10−3 Units/ml, Roche) and DNase I (20 Units/ml, Ambion) for 3 min at 37°C and cooled on ice for 5 min after adding urea to 0.5 M and RNase inhibitors (266.8 Units of RiboLock, Thermo/Fermentas; and 133.4 U of RNaseOUT, Life Technologies).

    Lysis:

    Article Title: Elucidation of the Photorhabdus temperata Genome and Generation of a Transposon Mutant Library To Identify Motility Mutants Altered in Pathogenesis
    Article Snippet: To remove residual DNA, RNA samples were treated with RNase-free DNase as follows: 50 μl RNA, 10 μl 10× DNase I buffer (New England BioLabs, Ipswich, MA), 5 U DNase I (2 U/μl; New England BioLabs, Ipswich, MA), 5 μl RNase Out recombinant RNase inhibitor (40 U/μl; Invitrogen, Carlsbad, CA) and RNase-free water to reach a total reaction volume of 100 μl. .. To remove residual DNA, RNA samples were treated with RNase-free DNase as follows: 50 μl RNA, 10 μl 10× DNase I buffer (New England BioLabs, Ipswich, MA), 5 U DNase I (2 U/μl; New England BioLabs, Ipswich, MA), 5 μl RNase Out recombinant RNase inhibitor (40 U/μl; Invitrogen, Carlsbad, CA) and RNase-free water to reach a total reaction volume of 100 μl.

    Article Title: The chromatin scaffold protein SAFB1 localizes SUMO-1 to the promoters of ribosomal protein genes to facilitate transcription initiation and splicing
    Article Snippet: Nuclear and cytoplasmic RNAs were purified by lysis of HeLa-SUMO1 cells in 200 μl of lysis buffer (10 mM Tris–HCl, pH 8, 1.5 mM MgCl2 , 0.5% NP-40, 140 mM NaCl and 0.5 U of RNase inhibitor) and loaded onto 200 μl of cushion buffer (10 mM Tris–HCl, pH 8, 1.5 mM MgCl2 , 1% NP-40, 140 mM NaCl and 0.4 M sucrose). .. The nuclear pellet was resuspended in 100 μl of DNAse I buffer (50 mM Tris–HCl, pH 7.5, 1 mM EDTA, 10 mM MgCl2 and 0.5 U RNase inhibitor) and treated 20 U of DNase I (Invitrogen) for 60 min at 37°C.

    Article Title: The RNA-binding protein TTP is a global post-transcriptional regulator of feedback control in inflammation
    Article Snippet: After washing with ice-cold PBS, intact cells were irradiated with UV-C light (300 mJ/cm2 , Stratalinker 2400) and total cell extracts were obtained using lysis buffer (50 mM Tris/HCl [pH 8.0], 150 mM NaCl, 0.5% (v/v) Triton X-100 and 1 mM EDTA) and sonication (3 times for 20 s in ice). .. Extract were cleared by centrifugation (15 min at 4°C) and treated with RNase A (0.9375 × 10−3 Units/ml, Roche) and DNase I (20 Units/ml, Ambion) for 3 min at 37°C and cooled on ice for 5 min after adding urea to 0.5 M and RNase inhibitors (266.8 Units of RiboLock, Thermo/Fermentas; and 133.4 U of RNaseOUT, Life Technologies).

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    Thermo Fisher v5 tagged rnase h1
    Both RNase H1 and P32 interact with mitochondrial DNA and pre-rRNA. ( A ) The positions of Probes and PCR primers for the human mitochondrial DNA. The DNA map was derived from published review   [65] . Two oligonucleotide probes specific to 12 S and 16 S mitochondria rRNA regions are shown in  Blue bars . Three sets of PCR probes corresponding to the A, B and C regions are indicated in  Green arrows . ( B ) RNase H1 and P32 bind mitochondrial DNA. Cell extracts were prepared from an HA-H1 stably expressing cell line (RNase H1), control HEK cells or HEK cells transfected with the HA-P32 expression plasmid (P32). Equal amounts of each extract were used for immunoprecipitation with anti-HA beads. Nucleic acids were extracted from the precipitated samples using phenol/chloroform and subjected to PCR analysis. The probe sets for PCR were shown in   Figure 6A . Genomic DNA from HEK cells that was used as a positive control. The PCR products were analyzed on 2% Agarose gels. ( C ) RNase H1 may interact with the mitochondrial rDNA region. The extracts from HA-H1 cell and control HEK cells were used for immunoprecipitation with HA-antibody. The precipitates were digested on beads with (+) or without (−) DNase I. The DNA associated with beads was then extracted and subjected to PCR analysis. The PCR products were separated in 2% agarose gel. ( D ) RNase H1 and P32 also co-immunoprecipitated with mitochondrial pre-rRNA. The same extracted nucleic acids from panel B were digested with DNase I. The RNA is used for reverse transcription with (+) or without (−) reverse transcriptase, followed by PCR amplification using different primer sets as indicated below the panels. PCR reaction using primers specific to U16 snoRNA was used as control.
    V5 Tagged Rnase H1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rnase inhibitor
    Ribonuclease activity of the recombinant <t>TcPR-4b</t> on tomato ( Solanum lycopersicum var. Micro-Tom) total RNA (5 μg). The incubation with TcPR-4b was carried out for 30 min at 25°C. The boiling conditions were 10 min at 95°C. The <t>RNase</t> inhibitor was the RiboLock (40 U; Thermo Scientific). The incubation conditions of the RNase A (Thermo Scientific) were 10 min at 25°C.
    Rnase Inhibitor, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase inhibitor/product/Thermo Fisher
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    Thermo Fisher gene exp drosha hs01095033 m1
    <t>Drosha</t> expression in endometrial cancer samples and in the control group (values presented in the log scale); * p = 0.008
    Gene Exp Drosha Hs01095033 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 83/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Both RNase H1 and P32 interact with mitochondrial DNA and pre-rRNA. ( A ) The positions of Probes and PCR primers for the human mitochondrial DNA. The DNA map was derived from published review   [65] . Two oligonucleotide probes specific to 12 S and 16 S mitochondria rRNA regions are shown in  Blue bars . Three sets of PCR probes corresponding to the A, B and C regions are indicated in  Green arrows . ( B ) RNase H1 and P32 bind mitochondrial DNA. Cell extracts were prepared from an HA-H1 stably expressing cell line (RNase H1), control HEK cells or HEK cells transfected with the HA-P32 expression plasmid (P32). Equal amounts of each extract were used for immunoprecipitation with anti-HA beads. Nucleic acids were extracted from the precipitated samples using phenol/chloroform and subjected to PCR analysis. The probe sets for PCR were shown in   Figure 6A . Genomic DNA from HEK cells that was used as a positive control. The PCR products were analyzed on 2% Agarose gels. ( C ) RNase H1 may interact with the mitochondrial rDNA region. The extracts from HA-H1 cell and control HEK cells were used for immunoprecipitation with HA-antibody. The precipitates were digested on beads with (+) or without (−) DNase I. The DNA associated with beads was then extracted and subjected to PCR analysis. The PCR products were separated in 2% agarose gel. ( D ) RNase H1 and P32 also co-immunoprecipitated with mitochondrial pre-rRNA. The same extracted nucleic acids from panel B were digested with DNase I. The RNA is used for reverse transcription with (+) or without (−) reverse transcriptase, followed by PCR amplification using different primer sets as indicated below the panels. PCR reaction using primers specific to U16 snoRNA was used as control.

    Journal: PLoS ONE

    Article Title: Human RNase H1 Is Associated with Protein P32 and Is Involved in Mitochondrial Pre-rRNA Processing

    doi: 10.1371/journal.pone.0071006

    Figure Lengend Snippet: Both RNase H1 and P32 interact with mitochondrial DNA and pre-rRNA. ( A ) The positions of Probes and PCR primers for the human mitochondrial DNA. The DNA map was derived from published review [65] . Two oligonucleotide probes specific to 12 S and 16 S mitochondria rRNA regions are shown in Blue bars . Three sets of PCR probes corresponding to the A, B and C regions are indicated in Green arrows . ( B ) RNase H1 and P32 bind mitochondrial DNA. Cell extracts were prepared from an HA-H1 stably expressing cell line (RNase H1), control HEK cells or HEK cells transfected with the HA-P32 expression plasmid (P32). Equal amounts of each extract were used for immunoprecipitation with anti-HA beads. Nucleic acids were extracted from the precipitated samples using phenol/chloroform and subjected to PCR analysis. The probe sets for PCR were shown in Figure 6A . Genomic DNA from HEK cells that was used as a positive control. The PCR products were analyzed on 2% Agarose gels. ( C ) RNase H1 may interact with the mitochondrial rDNA region. The extracts from HA-H1 cell and control HEK cells were used for immunoprecipitation with HA-antibody. The precipitates were digested on beads with (+) or without (−) DNase I. The DNA associated with beads was then extracted and subjected to PCR analysis. The PCR products were separated in 2% agarose gel. ( D ) RNase H1 and P32 also co-immunoprecipitated with mitochondrial pre-rRNA. The same extracted nucleic acids from panel B were digested with DNase I. The RNA is used for reverse transcription with (+) or without (−) reverse transcriptase, followed by PCR amplification using different primer sets as indicated below the panels. PCR reaction using primers specific to U16 snoRNA was used as control.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: Polymerase Chain Reaction, Derivative Assay, Hemagglutination Assay, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Positive Control, Agarose Gel Electrophoresis, Amplification

    Depletion of RNase H1 or P32 resulted in accumulation of mitochondrial pre-12S/16S rRNA. HeLa cells were treated with 2 nM or 20 nM of RNase H1-siRNA or P32 –siRNA for 24 or 48 hours. ( A ) The mRNA levels of RNase H1 and P32 were determined by qRT-PCR 24 hrs after siRNA treatment. ( B ) Protein levels of RNase H1 and P32 were analyzed by western analysis 24 hours post siRNA treatment. ( C ) Reduction of RNase H1 or P32 significantly increased the level of mitochondrial pre-rRNA. HeLa cells were treated with either RNase H1-siRNA (2 nM) or P32-siRNA (2 nM) for 24 hours. Total RNA was prepared and subjected to Northern analysis with  32 P labeled probes specific to 12S or 16S rRNAs. U3 snoRNA was detected and served as a control. The relative levels of pre-rRNA were measured from the results obtained with 12 S probe, normalized to U3, and plotted in the right panel. The error bars indicate standard error of the three replicates. (D) RT-PCR assay for the levels of pre-16 S and pre-ND3 RNAs. Total RNA prepared from HeLa cells treated for 24 hrs with corresponding siRNAs was analyzed by qRT-PCR, using primer probe sets specific to the tRNA Val -16 S rRNA junction (pre-16 S) or to the tRNA Gly -ND3 junction (pre-ND3). The error bars represent standard deviation of three replicates.

    Journal: PLoS ONE

    Article Title: Human RNase H1 Is Associated with Protein P32 and Is Involved in Mitochondrial Pre-rRNA Processing

    doi: 10.1371/journal.pone.0071006

    Figure Lengend Snippet: Depletion of RNase H1 or P32 resulted in accumulation of mitochondrial pre-12S/16S rRNA. HeLa cells were treated with 2 nM or 20 nM of RNase H1-siRNA or P32 –siRNA for 24 or 48 hours. ( A ) The mRNA levels of RNase H1 and P32 were determined by qRT-PCR 24 hrs after siRNA treatment. ( B ) Protein levels of RNase H1 and P32 were analyzed by western analysis 24 hours post siRNA treatment. ( C ) Reduction of RNase H1 or P32 significantly increased the level of mitochondrial pre-rRNA. HeLa cells were treated with either RNase H1-siRNA (2 nM) or P32-siRNA (2 nM) for 24 hours. Total RNA was prepared and subjected to Northern analysis with 32 P labeled probes specific to 12S or 16S rRNAs. U3 snoRNA was detected and served as a control. The relative levels of pre-rRNA were measured from the results obtained with 12 S probe, normalized to U3, and plotted in the right panel. The error bars indicate standard error of the three replicates. (D) RT-PCR assay for the levels of pre-16 S and pre-ND3 RNAs. Total RNA prepared from HeLa cells treated for 24 hrs with corresponding siRNAs was analyzed by qRT-PCR, using primer probe sets specific to the tRNA Val -16 S rRNA junction (pre-16 S) or to the tRNA Gly -ND3 junction (pre-ND3). The error bars represent standard deviation of three replicates.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: Quantitative RT-PCR, Western Blot, Northern Blot, Labeling, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

    Co-localization of P32 and RNase H1. ( A ) Immunofluorescence Staining of P32 and RNase H1. Upper panel: HeLa cells were stained for endogenous P32 and RNase H1 using mouse monoclonal anti-P32 antibody and rabbit anti-RNase H1 antibody, respectively, followed by FITC conjugated donkey anti-mouse ( green ) and TRITC conjugated anti-rabbit secondary antibodies ( red ). Nuclei were stained with DAP1 ( Blue ) and Mitochondria were stained with mitotracker ( white ). Lower panel: HeLa cells were infected with adenovirus expressing RNase H1. Cells were stained as described in upper panel. ( B ) Subcellular fractionation of P32 protein. The proteins from sub-cellular compartments (cytosol, mitochondrial and ER membranes, nucleus and cytoskeleton) were prepared from HEK cells using proteome cell compartment kit (Qiagen). About 10 µg protein samples from each fraction were analyzed by western for P32. The same blot was stripped and tubulin-γ was detected to serve as a control.

    Journal: PLoS ONE

    Article Title: Human RNase H1 Is Associated with Protein P32 and Is Involved in Mitochondrial Pre-rRNA Processing

    doi: 10.1371/journal.pone.0071006

    Figure Lengend Snippet: Co-localization of P32 and RNase H1. ( A ) Immunofluorescence Staining of P32 and RNase H1. Upper panel: HeLa cells were stained for endogenous P32 and RNase H1 using mouse monoclonal anti-P32 antibody and rabbit anti-RNase H1 antibody, respectively, followed by FITC conjugated donkey anti-mouse ( green ) and TRITC conjugated anti-rabbit secondary antibodies ( red ). Nuclei were stained with DAP1 ( Blue ) and Mitochondria were stained with mitotracker ( white ). Lower panel: HeLa cells were infected with adenovirus expressing RNase H1. Cells were stained as described in upper panel. ( B ) Subcellular fractionation of P32 protein. The proteins from sub-cellular compartments (cytosol, mitochondrial and ER membranes, nucleus and cytoskeleton) were prepared from HEK cells using proteome cell compartment kit (Qiagen). About 10 µg protein samples from each fraction were analyzed by western for P32. The same blot was stripped and tubulin-γ was detected to serve as a control.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: Immunofluorescence, Staining, Infection, Expressing, Fractionation, Western Blot

    Recombinant P32 binds to recombinant RNase H1, enhances its turnover rate, and reduces the binding affinity of the enzyme for the heteroduplex substrate. ( A ) Coomassie blue staining of the purified human His-H1, GST protein, and GST-P32 proteins separated by SDS-PAGE. The sizes for the standard protein markers are indicated. ( B ) RNase H1 but not P32 appears to bind the heteroduplex substrate. Gel shift assay was performed using 0.4 ug purified RNase H1, GST-P32, or GST proteins incubated at 4°C for 30 min with a non-cleavable heteroduplex containing  32 P labeled uniformly modified 2′-fluoro RNA annealed to DNA and subjected to native gel electrophoresis. ( C ) The interaction between RNase H1 and P32 appears to be equal molar. A fixed amount of GST-P32 was bound to GST affinity beads and then incubated with increasing amounts of RNase H1. Glutathione (GSH) eluted RNase H1 and P32 were quantified by Western blot as described in the Material and Methods. The amounts of bead-bound P32 and P32-associated RNase H1 were determined by loading known amounts of the respective proteins (left panel). The molecular ratio of bound RNase H1 relative to P32 was calculated and plotted in the right panel. ( D ) The effects of ionic strength on RNase H1/P32 interaction. Left panel: RNase H1 binds GST-P32 but not GST protein. GST or GST-P32 bound to anti-GST beads was incubated with RNase H1 in NaCl concentrations ranging from 0-950 mM as described in the Material and Methods. Middle panel: increasing NaCl concentration inhibits binding of RNase H1 to P32. Both unbound (flow through) and bound (affinity eluted) fractions were collected and the levels of RNase H1 and P32 evaluated by western blot. Right panel: Increasing pH reduced binding of RNase H1 to P32. ( E ) Michaelis-Menten kinetics and binding constants for RNase H1 cleavage of an RNA/DNA duplex in the presence or absence of P32. The K m , V max , and K d  were determined by incubating the Apo B RNA/DNA duplex with RNase H1 plus GST (as control) or RNase H1 plus different amounts of P32 resulting in an H1:P32 ratio = 1∶1 or 1∶5. An uncleavable competitive inhibitor (2′-fluororibonucleotide/DNA) was used to determine the binding to the RNA/DNA duplex, as described in the Material and Methods. The calculated constants are indicated in the right panel. The error bars indicate the standard error from three parallel experiments.

    Journal: PLoS ONE

    Article Title: Human RNase H1 Is Associated with Protein P32 and Is Involved in Mitochondrial Pre-rRNA Processing

    doi: 10.1371/journal.pone.0071006

    Figure Lengend Snippet: Recombinant P32 binds to recombinant RNase H1, enhances its turnover rate, and reduces the binding affinity of the enzyme for the heteroduplex substrate. ( A ) Coomassie blue staining of the purified human His-H1, GST protein, and GST-P32 proteins separated by SDS-PAGE. The sizes for the standard protein markers are indicated. ( B ) RNase H1 but not P32 appears to bind the heteroduplex substrate. Gel shift assay was performed using 0.4 ug purified RNase H1, GST-P32, or GST proteins incubated at 4°C for 30 min with a non-cleavable heteroduplex containing 32 P labeled uniformly modified 2′-fluoro RNA annealed to DNA and subjected to native gel electrophoresis. ( C ) The interaction between RNase H1 and P32 appears to be equal molar. A fixed amount of GST-P32 was bound to GST affinity beads and then incubated with increasing amounts of RNase H1. Glutathione (GSH) eluted RNase H1 and P32 were quantified by Western blot as described in the Material and Methods. The amounts of bead-bound P32 and P32-associated RNase H1 were determined by loading known amounts of the respective proteins (left panel). The molecular ratio of bound RNase H1 relative to P32 was calculated and plotted in the right panel. ( D ) The effects of ionic strength on RNase H1/P32 interaction. Left panel: RNase H1 binds GST-P32 but not GST protein. GST or GST-P32 bound to anti-GST beads was incubated with RNase H1 in NaCl concentrations ranging from 0-950 mM as described in the Material and Methods. Middle panel: increasing NaCl concentration inhibits binding of RNase H1 to P32. Both unbound (flow through) and bound (affinity eluted) fractions were collected and the levels of RNase H1 and P32 evaluated by western blot. Right panel: Increasing pH reduced binding of RNase H1 to P32. ( E ) Michaelis-Menten kinetics and binding constants for RNase H1 cleavage of an RNA/DNA duplex in the presence or absence of P32. The K m , V max , and K d were determined by incubating the Apo B RNA/DNA duplex with RNase H1 plus GST (as control) or RNase H1 plus different amounts of P32 resulting in an H1:P32 ratio = 1∶1 or 1∶5. An uncleavable competitive inhibitor (2′-fluororibonucleotide/DNA) was used to determine the binding to the RNA/DNA duplex, as described in the Material and Methods. The calculated constants are indicated in the right panel. The error bars indicate the standard error from three parallel experiments.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: Recombinant, Binding Assay, Staining, Purification, SDS Page, Electrophoretic Mobility Shift Assay, Incubation, Labeling, Modification, Nucleic Acid Electrophoresis, Western Blot, Concentration Assay, Flow Cytometry

    P32 appears to interact with the N-terminal duplex binding domain of RNase H1. ( A ) Expression and purification of RNase H1 deletion mutants. Left panel: Schematic depiction of the different human RNase H1 deletion mutants. DL1 deletes the hybrid binding domain (amino acid positions 1–73); DL2 deletes both the hybrid binding domain and the spacer domain (amino acid 1–129). The black bars at the N-terminus of each mutant represent a His tag. Right panel: Coomassie blue staining of the purified RNase H1 deletion mutants. The sizes of the standard markers are given. ( B ) Interaction of full length RNase H1 and its deletion mutants with P32. The full length or truncated RNase H1 proteins were incubated with GST-P32 bound to GST-beads under different NaCl concentrations ranging from 150–450 mM in both the binding and washing solutions. The P32 and RNase H1 or deletion mutants were eluted and analyzed by Western blot, using P32 or RNase H1 antibodies, respectively (right panel). Western blot to RNase H1 and deletion mutants DL1 and DL2 demonstrates that the mutant proteins are recognized by the RNase H1 antibody (left panel). ( C ) Michaelis-Menten Kinetics of DL-1 mutant in the presence or absence of P32. K m , V max , and k cat  for DL-1 plus GST or GST-P32 (DL-1:P32 = 1:5 in molecular ratio) were determined in 50 and 150 mM NaCl concentration with the Apo B RNA/DNA duplex as described in the Material and Methods.

    Journal: PLoS ONE

    Article Title: Human RNase H1 Is Associated with Protein P32 and Is Involved in Mitochondrial Pre-rRNA Processing

    doi: 10.1371/journal.pone.0071006

    Figure Lengend Snippet: P32 appears to interact with the N-terminal duplex binding domain of RNase H1. ( A ) Expression and purification of RNase H1 deletion mutants. Left panel: Schematic depiction of the different human RNase H1 deletion mutants. DL1 deletes the hybrid binding domain (amino acid positions 1–73); DL2 deletes both the hybrid binding domain and the spacer domain (amino acid 1–129). The black bars at the N-terminus of each mutant represent a His tag. Right panel: Coomassie blue staining of the purified RNase H1 deletion mutants. The sizes of the standard markers are given. ( B ) Interaction of full length RNase H1 and its deletion mutants with P32. The full length or truncated RNase H1 proteins were incubated with GST-P32 bound to GST-beads under different NaCl concentrations ranging from 150–450 mM in both the binding and washing solutions. The P32 and RNase H1 or deletion mutants were eluted and analyzed by Western blot, using P32 or RNase H1 antibodies, respectively (right panel). Western blot to RNase H1 and deletion mutants DL1 and DL2 demonstrates that the mutant proteins are recognized by the RNase H1 antibody (left panel). ( C ) Michaelis-Menten Kinetics of DL-1 mutant in the presence or absence of P32. K m , V max , and k cat for DL-1 plus GST or GST-P32 (DL-1:P32 = 1:5 in molecular ratio) were determined in 50 and 150 mM NaCl concentration with the Apo B RNA/DNA duplex as described in the Material and Methods.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: Binding Assay, Expressing, Purification, Mutagenesis, Staining, Incubation, Western Blot, Concentration Assay

    Human RNase H1 is associated with P32. ( A ) Western blot analysis of cell lysates and immunoprecipitated samples show Flag-tagged RNase H1 and H2 expression from cells stably transformed with RNase H1 (H1) or H2 (H2) or wild type (control) HEK cell lines. ( B ) Co-selection of RNase H1 binding proteins by immunoprecipitation. Extracts from cells expressing the Flag-H1, Flag-H2, or HA-H1 cell lines were immunoprecipitated with either anti-Flag or anti-HA antibody. Co-precipitated proteins were resolved by SDS-PAGE, and visualized by silver staining. Protein bands that were different from the co-precipitated proteins from control cells were subjected to mass spectrometry. The protein bands corresponding to the tagged RNase H1, H2 and the co-precipitated P32 proteins are indicated. The size marker was SeeBlue Plus2 Pre-Stained Standard (Invitrogen). ( C ) 2D gel electrophoresis of proteins co-precipitated with Flag-H1 or Flag-H2. About 5 mg cell lysates were prepared for immunoprecipitation with anti-flag beads from cell lines which stably express Flag-H1 or Flag-H2. The immunoprecipitates were washed four times with RIPA buffer and directly sent to Applied Biomics Inc. (San Francisco, CA) for 2D gel electrophoresis coupled with MS analysis. In brief, the co-precipitated proteins from Flag-H1 or Flag-H2 cells were labeled by fluorescent DIGE CyDyers, respectively, followed by 2D gel electrophoresis. The protein image was scanned with a fluorescence detector. The figure illustrates the proteins differentially associated with RNase H1 (green) or H2 (red). The P32 protein was confirmed with mass spectrum from the extracted gel sample. Circled spots were identified as RNase H1, H2 or P32 by mass spectrometric analysis. ( D ) Both endogenous and expressed RNase H1 are co-precipitated with the expressed P32. Left panel: western blots with P32, RNase H1, or H2 antibodies for proteins co-precipitated using anti-HA antibody from extracts of control HeLa cells or cells transfected with HA-P32 expression plasmid. Right panel: western blots for proteins co-selected using anti-HA antibody from extracts of Flag-H1, Flag-H2 stable cell lines and control cells, all of which were transfected with HA-P32 expression plasmid. ( E ) Confirmation of the specific interaction between RNase H1 and P32. RNase H cleavage activity indicates that the P32 co-immunoprecipitated material contains only RNase H1 enzyme activity. Upper panel: Cleavage patterns of human RNase H1 and H2 from IP-coupled enzyme activity assays. Immunoprecipitations were performed with either anti-flag, anti-RNase H1 or anti-H2 antibodies from extracts of Flag-H1, Flag-H2 expressing cells or control cells. The co-precipitated samples were incubated for the indicated times with a  32 P-labeled RNA/DNA-methoxyethyl (MOE) gapmer duplex and the cleavage products were separated using denaturing gel electrophoresis. The preferred cleavage sites of RNase H1 and H2 are indicated with * or #, respectively. The positions of the preferred cleavage sites in the heteroduplex are shown in the middle panel with the sequences of the RNA substrate (upper strand) and the oligonucleotide (lower strand). The bold nucleotides in the oligonucleotide strand indicate the position of the MOE substitutions. Lower panel: only the RNase H1 enzyme activity was detected in the co-precipitated material from lysates containing tagged P32. Immunoprecipitations were performed with anti-HA antibody from extracts of Flag-H1 or Flag-H2 stable cell lines or control HEK cells, which were all transfected or not transfected with HA-P32 expression plasmid. The precipitated samples were analyzed for cleavage patterns as described above. The position of the cleavage bands relative to the sequence of the cleavage products is shown on the left. A partial alkaline digestion of the same labeled RNA was used as a sequence ladder. The cleavage pattern of purified human RNase H1 is shown at the far right of the lower panel.

    Journal: PLoS ONE

    Article Title: Human RNase H1 Is Associated with Protein P32 and Is Involved in Mitochondrial Pre-rRNA Processing

    doi: 10.1371/journal.pone.0071006

    Figure Lengend Snippet: Human RNase H1 is associated with P32. ( A ) Western blot analysis of cell lysates and immunoprecipitated samples show Flag-tagged RNase H1 and H2 expression from cells stably transformed with RNase H1 (H1) or H2 (H2) or wild type (control) HEK cell lines. ( B ) Co-selection of RNase H1 binding proteins by immunoprecipitation. Extracts from cells expressing the Flag-H1, Flag-H2, or HA-H1 cell lines were immunoprecipitated with either anti-Flag or anti-HA antibody. Co-precipitated proteins were resolved by SDS-PAGE, and visualized by silver staining. Protein bands that were different from the co-precipitated proteins from control cells were subjected to mass spectrometry. The protein bands corresponding to the tagged RNase H1, H2 and the co-precipitated P32 proteins are indicated. The size marker was SeeBlue Plus2 Pre-Stained Standard (Invitrogen). ( C ) 2D gel electrophoresis of proteins co-precipitated with Flag-H1 or Flag-H2. About 5 mg cell lysates were prepared for immunoprecipitation with anti-flag beads from cell lines which stably express Flag-H1 or Flag-H2. The immunoprecipitates were washed four times with RIPA buffer and directly sent to Applied Biomics Inc. (San Francisco, CA) for 2D gel electrophoresis coupled with MS analysis. In brief, the co-precipitated proteins from Flag-H1 or Flag-H2 cells were labeled by fluorescent DIGE CyDyers, respectively, followed by 2D gel electrophoresis. The protein image was scanned with a fluorescence detector. The figure illustrates the proteins differentially associated with RNase H1 (green) or H2 (red). The P32 protein was confirmed with mass spectrum from the extracted gel sample. Circled spots were identified as RNase H1, H2 or P32 by mass spectrometric analysis. ( D ) Both endogenous and expressed RNase H1 are co-precipitated with the expressed P32. Left panel: western blots with P32, RNase H1, or H2 antibodies for proteins co-precipitated using anti-HA antibody from extracts of control HeLa cells or cells transfected with HA-P32 expression plasmid. Right panel: western blots for proteins co-selected using anti-HA antibody from extracts of Flag-H1, Flag-H2 stable cell lines and control cells, all of which were transfected with HA-P32 expression plasmid. ( E ) Confirmation of the specific interaction between RNase H1 and P32. RNase H cleavage activity indicates that the P32 co-immunoprecipitated material contains only RNase H1 enzyme activity. Upper panel: Cleavage patterns of human RNase H1 and H2 from IP-coupled enzyme activity assays. Immunoprecipitations were performed with either anti-flag, anti-RNase H1 or anti-H2 antibodies from extracts of Flag-H1, Flag-H2 expressing cells or control cells. The co-precipitated samples were incubated for the indicated times with a 32 P-labeled RNA/DNA-methoxyethyl (MOE) gapmer duplex and the cleavage products were separated using denaturing gel electrophoresis. The preferred cleavage sites of RNase H1 and H2 are indicated with * or #, respectively. The positions of the preferred cleavage sites in the heteroduplex are shown in the middle panel with the sequences of the RNA substrate (upper strand) and the oligonucleotide (lower strand). The bold nucleotides in the oligonucleotide strand indicate the position of the MOE substitutions. Lower panel: only the RNase H1 enzyme activity was detected in the co-precipitated material from lysates containing tagged P32. Immunoprecipitations were performed with anti-HA antibody from extracts of Flag-H1 or Flag-H2 stable cell lines or control HEK cells, which were all transfected or not transfected with HA-P32 expression plasmid. The precipitated samples were analyzed for cleavage patterns as described above. The position of the cleavage bands relative to the sequence of the cleavage products is shown on the left. A partial alkaline digestion of the same labeled RNA was used as a sequence ladder. The cleavage pattern of purified human RNase H1 is shown at the far right of the lower panel.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: Western Blot, Immunoprecipitation, Expressing, Stable Transfection, Transformation Assay, Selection, Binding Assay, Hemagglutination Assay, SDS Page, Silver Staining, Mass Spectrometry, Marker, Staining, Two-Dimensional Gel Electrophoresis, Electrophoresis, Labeling, Fluorescence, Transfection, Plasmid Preparation, Activity Assay, Incubation, Nucleic Acid Electrophoresis, Sequencing, Purification

    Overexpression of rnh1 relieves replication pausing. A–D , 2DNAGE of four restriction fragments of Drosophila S2 cells mtDNA, probed as indicated, in material from control cells and cells overexpressing RNase H1 in the form of epitope-tagged RNase H1-V5 (denoted OE ), both treated with 500 μ m CuSO 4 for 48 h to induce expression. E , schematic map of Drosophila mtDNA, as also shown in Fig. 8 , indicating the location of relevant restriction sites ( open circles ), mTTF-binding sites (bs1 and bs2; filled circles ), the noncoding region ( bold ), and the probes used. The open arrowhead marks the location and direction of replication initiation (see Ref. 40 ). The directions of first- and second-dimension electrophoresis in all gels are as indicated by the arrows . The images show relatively low exposures to reveal fine details of the arcs of RIs.

    Journal: The Journal of Biological Chemistry

    Article Title: RNase H1 promotes replication fork progression through oppositely transcribed regions of Drosophila mitochondrial DNA

    doi: 10.1074/jbc.RA118.007015

    Figure Lengend Snippet: Overexpression of rnh1 relieves replication pausing. A–D , 2DNAGE of four restriction fragments of Drosophila S2 cells mtDNA, probed as indicated, in material from control cells and cells overexpressing RNase H1 in the form of epitope-tagged RNase H1-V5 (denoted OE ), both treated with 500 μ m CuSO 4 for 48 h to induce expression. E , schematic map of Drosophila mtDNA, as also shown in Fig. 8 , indicating the location of relevant restriction sites ( open circles ), mTTF-binding sites (bs1 and bs2; filled circles ), the noncoding region ( bold ), and the probes used. The open arrowhead marks the location and direction of replication initiation (see Ref. 40 ). The directions of first- and second-dimension electrophoresis in all gels are as indicated by the arrows . The images show relatively low exposures to reveal fine details of the arcs of RIs.

    Article Snippet: To establish cell clones stably expressing V5-tagged RNase H1 and variants, pCoBlast (Thermo Fisher Scientific) was included in transfections.

    Techniques: Over Expression, Expressing, Binding Assay, Electrophoresis

    Subcellular localization of epitope-tagged RNase H1. A , immunocytochemistry of cells transiently transfected with RNase H1-V5, probed for the V5 epitope tag ( red ), Cox4 ( green ), and DAPI ( blue ), showing examples of the three types of intracellular distribution of V5-tagged RNase H1: nucleus and mitochondria ( i ), mitochondria only ( ii ), and nucleus only ( iii ). B , subcellular distribution of RNase H1-V5 in 100 transfected cells as indicated (mean of three experiments, error bars denote S.D.). C , Western blots of subcellular fractions from cells transfected with RNase H1-V5, highly enriched for nuclei ( nuc ) or mitochondria ( mt ) as indicated, probed simultaneously for V5 and for the markers indicated. M , molecular mass markers.

    Journal: The Journal of Biological Chemistry

    Article Title: RNase H1 promotes replication fork progression through oppositely transcribed regions of Drosophila mitochondrial DNA

    doi: 10.1074/jbc.RA118.007015

    Figure Lengend Snippet: Subcellular localization of epitope-tagged RNase H1. A , immunocytochemistry of cells transiently transfected with RNase H1-V5, probed for the V5 epitope tag ( red ), Cox4 ( green ), and DAPI ( blue ), showing examples of the three types of intracellular distribution of V5-tagged RNase H1: nucleus and mitochondria ( i ), mitochondria only ( ii ), and nucleus only ( iii ). B , subcellular distribution of RNase H1-V5 in 100 transfected cells as indicated (mean of three experiments, error bars denote S.D.). C , Western blots of subcellular fractions from cells transfected with RNase H1-V5, highly enriched for nuclei ( nuc ) or mitochondria ( mt ) as indicated, probed simultaneously for V5 and for the markers indicated. M , molecular mass markers.

    Article Snippet: To establish cell clones stably expressing V5-tagged RNase H1 and variants, pCoBlast (Thermo Fisher Scientific) was included in transfections.

    Techniques: Immunocytochemistry, Transfection, Western Blot

    Subcellular targeting of RNase H1 variants. A , intracellular localization of RNase H1-V5 variants in cultures of stably transfected cells exemplified in B . M1V and M16V, N-terminal methionine variants (see Fig. S2 A ); ΔNLS, with the putative nuclear localization signal deleted (see Fig. S2 C ). C , intracellular localization of RNase H1-V5 in cells synchronized in G1 and G2 (see FACS profiles in Fig. S2 E ). All plotted values are means of three experiments. Error bars denote S.D. nuc , nuclei; mt , mitochondria.

    Journal: The Journal of Biological Chemistry

    Article Title: RNase H1 promotes replication fork progression through oppositely transcribed regions of Drosophila mitochondrial DNA

    doi: 10.1074/jbc.RA118.007015

    Figure Lengend Snippet: Subcellular targeting of RNase H1 variants. A , intracellular localization of RNase H1-V5 variants in cultures of stably transfected cells exemplified in B . M1V and M16V, N-terminal methionine variants (see Fig. S2 A ); ΔNLS, with the putative nuclear localization signal deleted (see Fig. S2 C ). C , intracellular localization of RNase H1-V5 in cells synchronized in G1 and G2 (see FACS profiles in Fig. S2 E ). All plotted values are means of three experiments. Error bars denote S.D. nuc , nuclei; mt , mitochondria.

    Article Snippet: To establish cell clones stably expressing V5-tagged RNase H1 and variants, pCoBlast (Thermo Fisher Scientific) was included in transfections.

    Techniques: Stable Transfection, Transfection, FACS

    Ribonuclease activity of the recombinant TcPR-4b on tomato ( Solanum lycopersicum var. Micro-Tom) total RNA (5 μg). The incubation with TcPR-4b was carried out for 30 min at 25°C. The boiling conditions were 10 min at 95°C. The RNase inhibitor was the RiboLock (40 U; Thermo Scientific). The incubation conditions of the RNase A (Thermo Scientific) were 10 min at 25°C.

    Journal: BMC Plant Biology

    Article Title: The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca2+ and Mg2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa

    doi: 10.1186/1471-2229-14-161

    Figure Lengend Snippet: Ribonuclease activity of the recombinant TcPR-4b on tomato ( Solanum lycopersicum var. Micro-Tom) total RNA (5 μg). The incubation with TcPR-4b was carried out for 30 min at 25°C. The boiling conditions were 10 min at 95°C. The RNase inhibitor was the RiboLock (40 U; Thermo Scientific). The incubation conditions of the RNase A (Thermo Scientific) were 10 min at 25°C.

    Article Snippet: As observed in other works [ , , ], the RNase activity of TcPR-4b was inhibited by heating and in the presence of RNase inhibitor (RiboLock, Thermo Scientific) which is able to annul the activity of type A, B and C RNases.

    Techniques: Activity Assay, Recombinant, Incubation

    Action of TcPR-4b on dikaryotic M. perniciosa survival in relation to RNase and DNase activity. A . Action of TcPR-4b on dikaryotic M. perniciosa survival in presence of RNase inhibitor. The following concentrations were used: 40 μg/ml of TcPR-4b and 800 U of RNase inhibitor. B . Action of TcPR-4b on dikaryotic M. perniciosa survival in presence of MgCl 2 . The following concentrations were used: 40 μg/ml of TcPR-4b and 10 mM of MgCl 2 .

    Journal: BMC Plant Biology

    Article Title: The pathogenesis-related protein PR-4b from Theobroma cacao presents RNase activity, Ca2+ and Mg2+ dependent-DNase activity and antifungal action on Moniliophthora perniciosa

    doi: 10.1186/1471-2229-14-161

    Figure Lengend Snippet: Action of TcPR-4b on dikaryotic M. perniciosa survival in relation to RNase and DNase activity. A . Action of TcPR-4b on dikaryotic M. perniciosa survival in presence of RNase inhibitor. The following concentrations were used: 40 μg/ml of TcPR-4b and 800 U of RNase inhibitor. B . Action of TcPR-4b on dikaryotic M. perniciosa survival in presence of MgCl 2 . The following concentrations were used: 40 μg/ml of TcPR-4b and 10 mM of MgCl 2 .

    Article Snippet: As observed in other works [ , , ], the RNase activity of TcPR-4b was inhibited by heating and in the presence of RNase inhibitor (RiboLock, Thermo Scientific) which is able to annul the activity of type A, B and C RNases.

    Techniques: Activity Assay

    Drosha expression in endometrial cancer samples and in the control group (values presented in the log scale); * p = 0.008

    Journal: Tumour Biology

    Article Title: Major regulators of microRNAs biogenesis Dicer and Drosha are down-regulated in endometrial cancer

    doi: 10.1007/s13277-011-0179-0

    Figure Lengend Snippet: Drosha expression in endometrial cancer samples and in the control group (values presented in the log scale); * p = 0.008

    Article Snippet: Amplifications of Dicer and Drosha were performed utilizing specific TaqMan® probes and primers purchased from Applied Biosystems, USA (assays IDs Hs00229023_ml and Hs01095033_m1, respectively).

    Techniques: Expressing

    The influence of histological grading on Dicer and Drosha expressions (values are presented in the log scale): one-way ANOVA with the Bonferroni post hoc test revealed the influence of histological grading on Drosha expression, with the significantly higher difference detected between controls and higher-grade cancers; * p = 0.038

    Journal: Tumour Biology

    Article Title: Major regulators of microRNAs biogenesis Dicer and Drosha are down-regulated in endometrial cancer

    doi: 10.1007/s13277-011-0179-0

    Figure Lengend Snippet: The influence of histological grading on Dicer and Drosha expressions (values are presented in the log scale): one-way ANOVA with the Bonferroni post hoc test revealed the influence of histological grading on Drosha expression, with the significantly higher difference detected between controls and higher-grade cancers; * p = 0.038

    Article Snippet: Amplifications of Dicer and Drosha were performed utilizing specific TaqMan® probes and primers purchased from Applied Biosystems, USA (assays IDs Hs00229023_ml and Hs01095033_m1, respectively).

    Techniques: Expressing

    Dicer and Drosha expression in endometrial cancer (red) and normal endometrial (green) samples

    Journal: Tumour Biology

    Article Title: Major regulators of microRNAs biogenesis Dicer and Drosha are down-regulated in endometrial cancer

    doi: 10.1007/s13277-011-0179-0

    Figure Lengend Snippet: Dicer and Drosha expression in endometrial cancer (red) and normal endometrial (green) samples

    Article Snippet: Amplifications of Dicer and Drosha were performed utilizing specific TaqMan® probes and primers purchased from Applied Biosystems, USA (assays IDs Hs00229023_ml and Hs01095033_m1, respectively).

    Techniques: Expressing