thermostable rnase h  (New England Biolabs)


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    Thermostable RNase H
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    Thermostable RNase H 250 units
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    M0523S
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    Ribonucleases RNase
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    250 units
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    New England Biolabs thermostable rnase h
    Thermostable RNase H
    Thermostable RNase H 250 units
    https://www.bioz.com/result/thermostable rnase h/product/New England Biolabs
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    thermostable rnase h - by Bioz Stars, 2021-06
    98/100 stars

    Images

    1) Product Images from "N6-methyladenosine in poly(A) tails stabilize VSG transcripts"

    Article Title: N6-methyladenosine in poly(A) tails stabilize VSG transcripts

    Journal: bioRxiv

    doi: 10.1101/2020.01.30.925776

    m 6 A is present in the poly(A) tail of VSG mRNA and other transcripts. a . Immunoblotting with anti-m 6 A antibody. RNA samples (from left to right): total RNA (Total), Poly(A)-enriched (A+) RNA and Poly(A)-depleted (A-) RNA from two life cycle stages (BSF and PCF). The last lane contains total mouse liver RNA (Mouse). 2 µ g of total RNA, 2 µ g of poly(A)-depleted RNA and 100 ng of poly(A)-enriched RNA was loaded per lane. rRNA was detected by staining RNA with methylene blue to confirm equal loading between total and poly(A)-depleted fractions. As expected the rRNA are undetectable in the poly(A)-enriched fraction. b . Intensity of the m 6 A signal in immunoblot, measured by Image J, in the whole lane containing the poly(A)-enriched RNA of bloodstream forms. The intensity of the ∼1.8 kb band was compared with the signal intensity of the entire lane, and averaged from 5 independent samples. c . Diagram displaying the location of the oligonucleotides used in RNase H digestion of VSG mRNA. The digestion products detected in the immunoblot (panel D) after incubation with each oligonucleotide (SL, A, B, C, dT) are also indicated. d . Immunoblotting with anti-m 6 A antibody of mammalian bloodstream forms total RNA pre-incubated with indicated oligonucleotides and digested with RNase H. 2 µ g of total RNA were loaded per lane. Staining of rRNA with Methylene Blue confirmed equal loading. SL: spliced leader, dT: poly deoxi-thymidines. Tub: α-Tubulin. (see also Extended Data Fig. S3)
    Figure Legend Snippet: m 6 A is present in the poly(A) tail of VSG mRNA and other transcripts. a . Immunoblotting with anti-m 6 A antibody. RNA samples (from left to right): total RNA (Total), Poly(A)-enriched (A+) RNA and Poly(A)-depleted (A-) RNA from two life cycle stages (BSF and PCF). The last lane contains total mouse liver RNA (Mouse). 2 µ g of total RNA, 2 µ g of poly(A)-depleted RNA and 100 ng of poly(A)-enriched RNA was loaded per lane. rRNA was detected by staining RNA with methylene blue to confirm equal loading between total and poly(A)-depleted fractions. As expected the rRNA are undetectable in the poly(A)-enriched fraction. b . Intensity of the m 6 A signal in immunoblot, measured by Image J, in the whole lane containing the poly(A)-enriched RNA of bloodstream forms. The intensity of the ∼1.8 kb band was compared with the signal intensity of the entire lane, and averaged from 5 independent samples. c . Diagram displaying the location of the oligonucleotides used in RNase H digestion of VSG mRNA. The digestion products detected in the immunoblot (panel D) after incubation with each oligonucleotide (SL, A, B, C, dT) are also indicated. d . Immunoblotting with anti-m 6 A antibody of mammalian bloodstream forms total RNA pre-incubated with indicated oligonucleotides and digested with RNase H. 2 µ g of total RNA were loaded per lane. Staining of rRNA with Methylene Blue confirmed equal loading. SL: spliced leader, dT: poly deoxi-thymidines. Tub: α-Tubulin. (see also Extended Data Fig. S3)

    Techniques Used: Staining, Incubation

    Conserved VSG 16-mer motif is required for inclusion of m6A in adjacent poly(A) tail. a . Diagram of 16-mer motif VSg double-expressor (DE) cell-lines. VSG117 was inserted immediately downstream of the promoter of the active bloodstream expression site, which naturally contains VSG 2 at the telomeric end. In VSG double expresor 16-mer WT cell-line, VSG117 contains its endogenous 3’UTR with the conserved 16-mer motif (sequence shown in blue). In VSG double expresor 16-mer MUT cell-line, the 16-mer motif was scrambled (sequence shown in orange). b . Transcript levels of VSG117 and VSG2 transcripts measured by qRT-PCR in both reporter cell-lines. Levels were normalized to transcript levels in cell-lines expressing only VSG2 or only VSG117 . c . Immunoblot with anti-m 6 A antibody of mRNA from VSG double-expressor cell-lines. RNase H digestion of VSG2 mRNA was used to resolve VSG2 and VSG117 transcripts. Different quantities of the same VSG double expresor 16-mer WT cell-line was loaded in two separet lanes (50ng and 12.5ng) to show that the VSG117 band is detectable in both conditions. d . m 6 A index calculated as the ratio of m 6 A intensity and mRNA levels, measured in panels c. and b., respectively. und., undetectable. # intensities measured in lane 3 of Figure 5C (see also Extended Data Fig. S5)
    Figure Legend Snippet: Conserved VSG 16-mer motif is required for inclusion of m6A in adjacent poly(A) tail. a . Diagram of 16-mer motif VSg double-expressor (DE) cell-lines. VSG117 was inserted immediately downstream of the promoter of the active bloodstream expression site, which naturally contains VSG 2 at the telomeric end. In VSG double expresor 16-mer WT cell-line, VSG117 contains its endogenous 3’UTR with the conserved 16-mer motif (sequence shown in blue). In VSG double expresor 16-mer MUT cell-line, the 16-mer motif was scrambled (sequence shown in orange). b . Transcript levels of VSG117 and VSG2 transcripts measured by qRT-PCR in both reporter cell-lines. Levels were normalized to transcript levels in cell-lines expressing only VSG2 or only VSG117 . c . Immunoblot with anti-m 6 A antibody of mRNA from VSG double-expressor cell-lines. RNase H digestion of VSG2 mRNA was used to resolve VSG2 and VSG117 transcripts. Different quantities of the same VSG double expresor 16-mer WT cell-line was loaded in two separet lanes (50ng and 12.5ng) to show that the VSG117 band is detectable in both conditions. d . m 6 A index calculated as the ratio of m 6 A intensity and mRNA levels, measured in panels c. and b., respectively. und., undetectable. # intensities measured in lane 3 of Figure 5C (see also Extended Data Fig. S5)

    Techniques Used: Expressing, Sequencing, Quantitative RT-PCR

    2) Product Images from "Control of ribosomal protein synthesis by Microprocessor complex"

    Article Title: Control of ribosomal protein synthesis by Microprocessor complex

    Journal: bioRxiv

    doi: 10.1101/2020.04.24.060236

    The microprocessor binds to the transcription start sites at RP gene loci. a. ChIP-seq profiles of RNAPII, H3K4me3, Drosha and Dgcr8 at the Rps15a, Rps24, Rpl4, and Rpl28 loci in mouse embryonic stem cells (mES). b. ChIP (IP: Drosha)-qPCR analysis of different RP genes as indicated with anti-Flag (M2) antibody or nonspecific IgG (control) in Flag tagged Drosha-expressing MEF (F-Drosha) or control (pBABE-MEF). Tuba1a and Tubb1 (negative control). Fold enrichment of anti-Flag IP over IgG IP is plotted as Mean ± SEM. n=3. c. ChIP (IP: Drosha)-qPCR analysis of different RP genes as indicated with anti-Flag (M2) antibody or nonspecific IgG (negative control) in Flag-Drosha-expressing MEF or control-MEF. Tuba1a and Tubb1 (negative control). Cells were treated with 1μg/ml actinomycin D (ActD) or vehicle (DMSO), followed by ChIP-qPCR analysis. Fold enrichment of anti-Flag IP over IgG IP is plotted as Mean ± SEM. n=3. d. ChIP (IP: Drosha)-qPCR analysis of different RP genes as indicated in MEF treated with 1μg/μl RNase A or vehicle (water) with anti-Drosha antibody or nonspecific IgG (control). Tuba1a and Tubb1 (negative control). Fold enrichment of Drosha IP over IgG IP is plotted as Mean ± SEM.n=3. e. ChIP (anti-Drosha antibody)-qPCR analysis of different RP genes in MEFs treated with 100U/ml RNase H or vehicle (water) with anti-Drosha antibody. Tuba1a and Tubb1 (negative control). Fold enrichment of Drosha IP over IgG IP is plotted as Mean ± SEM.n=3. f. ChIP (IP: Drosha)-qPCR analysis of different RP genes as indicated with anti-Drosha antibody or nonspecific IgG (control) in control MEFs (Ctrl) or MEFs deleted in Dgcr8 gene (Dgcr8-KO). Tuba1a (negative control). Fold enrichment of Drosha IP over IgG IP is plotted as Mean ± SEM.n=3. g. qRT-PCR analysis of different RP mRNAs and control mRNAs (Itgb1 and IL-6) (relative to GAPDH) as indicated in Ctrl or Dgcr8-KO MEFs. Result is plotted as Mean ± SEM. n=3.
    Figure Legend Snippet: The microprocessor binds to the transcription start sites at RP gene loci. a. ChIP-seq profiles of RNAPII, H3K4me3, Drosha and Dgcr8 at the Rps15a, Rps24, Rpl4, and Rpl28 loci in mouse embryonic stem cells (mES). b. ChIP (IP: Drosha)-qPCR analysis of different RP genes as indicated with anti-Flag (M2) antibody or nonspecific IgG (control) in Flag tagged Drosha-expressing MEF (F-Drosha) or control (pBABE-MEF). Tuba1a and Tubb1 (negative control). Fold enrichment of anti-Flag IP over IgG IP is plotted as Mean ± SEM. n=3. c. ChIP (IP: Drosha)-qPCR analysis of different RP genes as indicated with anti-Flag (M2) antibody or nonspecific IgG (negative control) in Flag-Drosha-expressing MEF or control-MEF. Tuba1a and Tubb1 (negative control). Cells were treated with 1μg/ml actinomycin D (ActD) or vehicle (DMSO), followed by ChIP-qPCR analysis. Fold enrichment of anti-Flag IP over IgG IP is plotted as Mean ± SEM. n=3. d. ChIP (IP: Drosha)-qPCR analysis of different RP genes as indicated in MEF treated with 1μg/μl RNase A or vehicle (water) with anti-Drosha antibody or nonspecific IgG (control). Tuba1a and Tubb1 (negative control). Fold enrichment of Drosha IP over IgG IP is plotted as Mean ± SEM.n=3. e. ChIP (anti-Drosha antibody)-qPCR analysis of different RP genes in MEFs treated with 100U/ml RNase H or vehicle (water) with anti-Drosha antibody. Tuba1a and Tubb1 (negative control). Fold enrichment of Drosha IP over IgG IP is plotted as Mean ± SEM.n=3. f. ChIP (IP: Drosha)-qPCR analysis of different RP genes as indicated with anti-Drosha antibody or nonspecific IgG (control) in control MEFs (Ctrl) or MEFs deleted in Dgcr8 gene (Dgcr8-KO). Tuba1a (negative control). Fold enrichment of Drosha IP over IgG IP is plotted as Mean ± SEM.n=3. g. qRT-PCR analysis of different RP mRNAs and control mRNAs (Itgb1 and IL-6) (relative to GAPDH) as indicated in Ctrl or Dgcr8-KO MEFs. Result is plotted as Mean ± SEM. n=3.

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing, Negative Control, Quantitative RT-PCR

    The Ddx5 helicase reduces DNA/RNA hybrid and facilitates transcription elongation. A. DNA/RNA hybrid IP-sequencing (DRIP-seq) data indicate increased R-loops at the RPG loci (Rps2, Rps3A, Rpl28, and Rpl37A) and control loci (GAPDH and Tuba1a) in Drosha KD cells (red) compared to control U-2 OS cells (blue) (top). Quantitation of the DRIP-seq data is shown (bottom). b. DRIP analysis of RPG loci (Rps2, Rps5, Rpl14) and control locus (Tuba1a) locus in the presence or absence of RNase H in HCT116 cells expressing CRISPR/Cas9 against Drosha (KO) or non-specific control (Ctrl). Four sets of primers used for DRIP analysis are indicated as black boxes in the top panel (bottom). Signal relative to input is plotted as Mean ± SEM. n=3 c. ChIP-qPCR analysis using anti-RNAPII antibody was performed in K562 cells cells expressing CRISPR/Cas9 against Drosha (KO) or non-specific control (Ctrl). Primers for RPG loci (Rps2, Rps26, Rpl14, Rpl28) and control locus (Tuba1a) are shown. Mean ± SEM. n=3. d. ChIP-qPCR analysis using anti-Ddx5 antibody of RPG loci (Rps2, Rps5, Rps10, Rps12, Rps26, Rpl4, Rpl14, Rpl19, Rpl28) and control locus(Tuba1a) in control (Ctrl) or Drosha KO HCT116 cells. Fold enrichment of Ddx5 antibody pull-down against IgG pull-down is plotted as Mean ± SEM. n=3. e. Immunoprecipitation of DNA/RNA hybrids with the S9.6 antibody, followed by immunoblot analysis of Ddx5, Drosha, Dgcr8 and Lamin A/C (negative control) in Ctrl or Drosha KO K562 cells. f. DRIP analysis of RPG loci (Rps2, Rps5, Rpl4, Rpl28) and control loci (Tuba1a and Tubb1) locus in the presence or absence of RNase H in K562 cells targeting Ddx5 gene ( Ddx5 KO) by RNAi or non-specific control (Ctrl). Result is plotted as Mean ± SEM. n=3 g. qRT-PCR analysis of various RP mRNAs and Drosha mRNAs (relative to GAPDH) in Ctrl or Drosha KO HCT116 cells transfected with empty plasmid (mock), Ddx5 wild type (WT) or the RNA helicase dead (HD) mutant expression plasmid (left). Result is plotted as Mean ± SEM. n=3 Drosha, Ddx5 and GAPDH proteins were examined by western blot in total cell lysates from HCT116 cells (right). Relative protein amount normalized to GAPDH is shown below each blot.
    Figure Legend Snippet: The Ddx5 helicase reduces DNA/RNA hybrid and facilitates transcription elongation. A. DNA/RNA hybrid IP-sequencing (DRIP-seq) data indicate increased R-loops at the RPG loci (Rps2, Rps3A, Rpl28, and Rpl37A) and control loci (GAPDH and Tuba1a) in Drosha KD cells (red) compared to control U-2 OS cells (blue) (top). Quantitation of the DRIP-seq data is shown (bottom). b. DRIP analysis of RPG loci (Rps2, Rps5, Rpl14) and control locus (Tuba1a) locus in the presence or absence of RNase H in HCT116 cells expressing CRISPR/Cas9 against Drosha (KO) or non-specific control (Ctrl). Four sets of primers used for DRIP analysis are indicated as black boxes in the top panel (bottom). Signal relative to input is plotted as Mean ± SEM. n=3 c. ChIP-qPCR analysis using anti-RNAPII antibody was performed in K562 cells cells expressing CRISPR/Cas9 against Drosha (KO) or non-specific control (Ctrl). Primers for RPG loci (Rps2, Rps26, Rpl14, Rpl28) and control locus (Tuba1a) are shown. Mean ± SEM. n=3. d. ChIP-qPCR analysis using anti-Ddx5 antibody of RPG loci (Rps2, Rps5, Rps10, Rps12, Rps26, Rpl4, Rpl14, Rpl19, Rpl28) and control locus(Tuba1a) in control (Ctrl) or Drosha KO HCT116 cells. Fold enrichment of Ddx5 antibody pull-down against IgG pull-down is plotted as Mean ± SEM. n=3. e. Immunoprecipitation of DNA/RNA hybrids with the S9.6 antibody, followed by immunoblot analysis of Ddx5, Drosha, Dgcr8 and Lamin A/C (negative control) in Ctrl or Drosha KO K562 cells. f. DRIP analysis of RPG loci (Rps2, Rps5, Rpl4, Rpl28) and control loci (Tuba1a and Tubb1) locus in the presence or absence of RNase H in K562 cells targeting Ddx5 gene ( Ddx5 KO) by RNAi or non-specific control (Ctrl). Result is plotted as Mean ± SEM. n=3 g. qRT-PCR analysis of various RP mRNAs and Drosha mRNAs (relative to GAPDH) in Ctrl or Drosha KO HCT116 cells transfected with empty plasmid (mock), Ddx5 wild type (WT) or the RNA helicase dead (HD) mutant expression plasmid (left). Result is plotted as Mean ± SEM. n=3 Drosha, Ddx5 and GAPDH proteins were examined by western blot in total cell lysates from HCT116 cells (right). Relative protein amount normalized to GAPDH is shown below each blot.

    Techniques Used: Sequencing, Quantitation Assay, Expressing, CRISPR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation, Negative Control, Quantitative RT-PCR, Transfection, Plasmid Preparation, Mutagenesis, Western Blot

    3) Product Images from "N6-methyladenosine in poly(A) tails stabilize VSG transcripts"

    Article Title: N6-methyladenosine in poly(A) tails stabilize VSG transcripts

    Journal: bioRxiv

    doi: 10.1101/2020.01.30.925776

    m 6 A is present in the poly(A) tail of VSG mRNA and other transcripts. a . Immunoblotting with anti-m 6 A antibody. RNA samples (from left to right): total RNA (Total), Poly(A)-enriched (A+) RNA and Poly(A)-depleted (A-) RNA from two life cycle stages (BSF and PCF). The last lane contains total mouse liver RNA (Mouse). 2 µ g of total RNA, 2 µ g of poly(A)-depleted RNA and 100 ng of poly(A)-enriched RNA was loaded per lane. rRNA was detected by staining RNA with methylene blue to confirm equal loading between total and poly(A)-depleted fractions. As expected the rRNA are undetectable in the poly(A)-enriched fraction. b . Intensity of the m 6 A signal in immunoblot, measured by Image J, in the whole lane containing the poly(A)-enriched RNA of bloodstream forms. The intensity of the ∼1.8 kb band was compared with the signal intensity of the entire lane, and averaged from 5 independent samples. c . Diagram displaying the location of the oligonucleotides used in RNase H digestion of VSG mRNA. The digestion products detected in the immunoblot (panel D) after incubation with each oligonucleotide (SL, A, B, C, dT) are also indicated. d . Immunoblotting with anti-m 6 A antibody of mammalian bloodstream forms total RNA pre-incubated with indicated oligonucleotides and digested with RNase H. 2 µ g of total RNA were loaded per lane. Staining of rRNA with Methylene Blue confirmed equal loading. SL: spliced leader, dT: poly deoxi-thymidines. Tub: α-Tubulin. (see also Extended Data Fig. S3)
    Figure Legend Snippet: m 6 A is present in the poly(A) tail of VSG mRNA and other transcripts. a . Immunoblotting with anti-m 6 A antibody. RNA samples (from left to right): total RNA (Total), Poly(A)-enriched (A+) RNA and Poly(A)-depleted (A-) RNA from two life cycle stages (BSF and PCF). The last lane contains total mouse liver RNA (Mouse). 2 µ g of total RNA, 2 µ g of poly(A)-depleted RNA and 100 ng of poly(A)-enriched RNA was loaded per lane. rRNA was detected by staining RNA with methylene blue to confirm equal loading between total and poly(A)-depleted fractions. As expected the rRNA are undetectable in the poly(A)-enriched fraction. b . Intensity of the m 6 A signal in immunoblot, measured by Image J, in the whole lane containing the poly(A)-enriched RNA of bloodstream forms. The intensity of the ∼1.8 kb band was compared with the signal intensity of the entire lane, and averaged from 5 independent samples. c . Diagram displaying the location of the oligonucleotides used in RNase H digestion of VSG mRNA. The digestion products detected in the immunoblot (panel D) after incubation with each oligonucleotide (SL, A, B, C, dT) are also indicated. d . Immunoblotting with anti-m 6 A antibody of mammalian bloodstream forms total RNA pre-incubated with indicated oligonucleotides and digested with RNase H. 2 µ g of total RNA were loaded per lane. Staining of rRNA with Methylene Blue confirmed equal loading. SL: spliced leader, dT: poly deoxi-thymidines. Tub: α-Tubulin. (see also Extended Data Fig. S3)

    Techniques Used: Staining, Incubation

    Conserved VSG 16-mer motif is required for inclusion of m6A in adjacent poly(A) tail. a . Diagram of 16-mer motif VSg double-expressor (DE) cell-lines. VSG117 was inserted immediately downstream of the promoter of the active bloodstream expression site, which naturally contains VSG 2 at the telomeric end. In VSG double expresor 16-mer WT cell-line, VSG117 contains its endogenous 3’UTR with the conserved 16-mer motif (sequence shown in blue). In VSG double expresor 16-mer MUT cell-line, the 16-mer motif was scrambled (sequence shown in orange). b . Transcript levels of VSG117 and VSG2 transcripts measured by qRT-PCR in both reporter cell-lines. Levels were normalized to transcript levels in cell-lines expressing only VSG2 or only VSG117 . c . Immunoblot with anti-m 6 A antibody of mRNA from VSG double-expressor cell-lines. RNase H digestion of VSG2 mRNA was used to resolve VSG2 and VSG117 transcripts. Different quantities of the same VSG double expresor 16-mer WT cell-line was loaded in two separet lanes (50ng and 12.5ng) to show that the VSG117 band is detectable in both conditions. d . m 6 A index calculated as the ratio of m 6 A intensity and mRNA levels, measured in panels c. and b., respectively. und., undetectable. # intensities measured in lane 3 of Figure 5C (see also Extended Data Fig. S5)
    Figure Legend Snippet: Conserved VSG 16-mer motif is required for inclusion of m6A in adjacent poly(A) tail. a . Diagram of 16-mer motif VSg double-expressor (DE) cell-lines. VSG117 was inserted immediately downstream of the promoter of the active bloodstream expression site, which naturally contains VSG 2 at the telomeric end. In VSG double expresor 16-mer WT cell-line, VSG117 contains its endogenous 3’UTR with the conserved 16-mer motif (sequence shown in blue). In VSG double expresor 16-mer MUT cell-line, the 16-mer motif was scrambled (sequence shown in orange). b . Transcript levels of VSG117 and VSG2 transcripts measured by qRT-PCR in both reporter cell-lines. Levels were normalized to transcript levels in cell-lines expressing only VSG2 or only VSG117 . c . Immunoblot with anti-m 6 A antibody of mRNA from VSG double-expressor cell-lines. RNase H digestion of VSG2 mRNA was used to resolve VSG2 and VSG117 transcripts. Different quantities of the same VSG double expresor 16-mer WT cell-line was loaded in two separet lanes (50ng and 12.5ng) to show that the VSG117 band is detectable in both conditions. d . m 6 A index calculated as the ratio of m 6 A intensity and mRNA levels, measured in panels c. and b., respectively. und., undetectable. # intensities measured in lane 3 of Figure 5C (see also Extended Data Fig. S5)

    Techniques Used: Expressing, Sequencing, Quantitative RT-PCR

    4) Product Images from "Selective Naked-Eye Detection of SARS-CoV-2 Mediated by N Gene Targeted Antisense Oligonucleotide Capped Plasmonic Nanoparticles"

    Article Title: Selective Naked-Eye Detection of SARS-CoV-2 Mediated by N Gene Targeted Antisense Oligonucleotide Capped Plasmonic Nanoparticles

    Journal: ACS Nano

    doi: 10.1021/acsnano.0c03822

    (a) Comparison of response of the Au-ASO mix nanoparticles toward the RNA (1 ng/μL) isolated from noninfected Vero cells, Vero cells infected with MERS-CoV, and Vero cells infected with SARS-CoV-2 virus. Relative change in absorbance at 660 nm wavelength for the Au-ASO mix nanoparticle treated with SARS-CoV-2 RNA (1 ng/μL) followed by the addition of RNase H has been plotted in (b) when the mixture was incubated at different temperatures for 5 min. The schematic representation for the visual naked-eye detection of SARS-CoV-2 with the treatment of RNase H at 65 °C for 5 min is shown in (c). The error bar indicates the average results obtained from three such independent experiments performed in triplicate.
    Figure Legend Snippet: (a) Comparison of response of the Au-ASO mix nanoparticles toward the RNA (1 ng/μL) isolated from noninfected Vero cells, Vero cells infected with MERS-CoV, and Vero cells infected with SARS-CoV-2 virus. Relative change in absorbance at 660 nm wavelength for the Au-ASO mix nanoparticle treated with SARS-CoV-2 RNA (1 ng/μL) followed by the addition of RNase H has been plotted in (b) when the mixture was incubated at different temperatures for 5 min. The schematic representation for the visual naked-eye detection of SARS-CoV-2 with the treatment of RNase H at 65 °C for 5 min is shown in (c). The error bar indicates the average results obtained from three such independent experiments performed in triplicate.

    Techniques Used: Allele-specific Oligonucleotide, Isolation, Infection, Incubation

    Related Articles

    Incubation:

    Article Title: N6-methyladenosine in poly(A) tails stabilize VSG transcripts
    Article Snippet: Statistical analysis of decay curves Data of the decay of mRNA, length of poly(A) tail and m6A signal were fitted in GraphPad Prism to either “Plateau followed by one phase decay” or “one phase decay”. .. The digestion mixture was prepared on ice with 50 ng of mRNA, (0.2 pmol) complementary DNA oligo, and Thermostable RNase H. The reaction mixture was transferred to the pre-warmed thermoblock at 50°C and incubated for 25 minutes. .. Immunofluorescence assays Parasites were pelleted by centrifugation (800 g, 3 min, room temperature), resuspended in the remaining medium and transferred to an microcentrifuge tube.

    Article Title: R3T (Rapid Research Response Team) One-step RT-qPCR kit for COVID-19 diagnostic using in-house enzymes
    Article Snippet: The final reaction mixtures were incubated at room temperature for 10 minutes, followed by incubation at 42 ̊ C for 50 minutes then by heat inactivation at 70 ̊ C for 15 minutes. .. Lastly, 5 units of thermostable RNase H (New England Biolabs, Cat. No M0523S) were added and incubated at 37 ˚C for 20 minutes to hydrolyze RNA. .. The resulting cDNAs were subjected to RT-PCR assays with 2019-nCoV_N3 primers (IDT, Cat. No 10006770).

    Article Title: Scalable and cost-effective ribonuclease-based rRNA depletion for transcriptomics
    Article Snippet: RNase H reaction optimizationTo optimize RNase H reaction conditions, we added oligo probe mix to 100ng of fragmented RNA after first adapter ligation with various probe-to-RNA ratios (1:1, 5:1 or 10:1), the mixture was incubated in hybridization buffer (200mM NaCl, 100mM Tris-HCl pH7.5) in a final volume of 5μL at 95°C for 2 minutes, the temperature was slowly ramped down (−0.1°C/sec) to 45°C (for Hybridase RNase H) or 22°C (for NEB RNase H), and the mixture was incubated at 45°C (for Hybridase RNase H) or 22°C (for NEB RNase H) for additional 5 minutes. .. After probe hybridization, we added 5μL preheated Hybridase RNase H reaction mix (10U Hybridase Thermostable RNase H [Lucigen H39500], 0.5μmol Tris-HCl pH 7.5, 1μmol NaCl and 0.2μmol MgCl2) or 5μL NEB RNase H reaction mix (10U NEB RNase H [NEB M0297S] and 1uL 10X NEB RNase H reaction buffer) to the RNA-probe mixture and incubated the mixture at 45°C (for Hybridase RNase H) or 37°C (for NEB RNase H) for various depletion times (10 minutes, 30 minutes and 60 minutes). .. After RNase H digestion, 15μL DNase I reaction mix (4U TURBO DNase [ThermoFisher AM2239] and 2.5μL 10X TURBO DNase Buffer) was added, and the mixture was incubated at 37°C for 30 minutes to degrade ssDNA probes.

    other:

    Article Title: Staphylococcus aureus in Intensive Pig Production in South Africa: Antibiotic Resistance, Virulence Determinants, and Clonality
    Article Snippet: Molecular confirmation was performed using S. aureus species-specific primers for the nuc A gene, which codes for a thermostable nuclease [ ].

    Hybridization:

    Article Title: Scalable and cost-effective ribonuclease-based rRNA depletion for transcriptomics
    Article Snippet: RNase H reaction optimizationTo optimize RNase H reaction conditions, we added oligo probe mix to 100ng of fragmented RNA after first adapter ligation with various probe-to-RNA ratios (1:1, 5:1 or 10:1), the mixture was incubated in hybridization buffer (200mM NaCl, 100mM Tris-HCl pH7.5) in a final volume of 5μL at 95°C for 2 minutes, the temperature was slowly ramped down (−0.1°C/sec) to 45°C (for Hybridase RNase H) or 22°C (for NEB RNase H), and the mixture was incubated at 45°C (for Hybridase RNase H) or 22°C (for NEB RNase H) for additional 5 minutes. .. After probe hybridization, we added 5μL preheated Hybridase RNase H reaction mix (10U Hybridase Thermostable RNase H [Lucigen H39500], 0.5μmol Tris-HCl pH 7.5, 1μmol NaCl and 0.2μmol MgCl2) or 5μL NEB RNase H reaction mix (10U NEB RNase H [NEB M0297S] and 1uL 10X NEB RNase H reaction buffer) to the RNA-probe mixture and incubated the mixture at 45°C (for Hybridase RNase H) or 37°C (for NEB RNase H) for various depletion times (10 minutes, 30 minutes and 60 minutes). .. After RNase H digestion, 15μL DNase I reaction mix (4U TURBO DNase [ThermoFisher AM2239] and 2.5μL 10X TURBO DNase Buffer) was added, and the mixture was incubated at 37°C for 30 minutes to degrade ssDNA probes.

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    New England Biolabs thermostable rnase h
    m 6 A is present in the poly(A) tail of VSG mRNA and other transcripts. a . Immunoblotting with anti-m 6 A antibody. RNA samples (from left to right): total RNA (Total), Poly(A)-enriched (A+) RNA and Poly(A)-depleted (A-) RNA from two life cycle stages (BSF and PCF). The last lane contains total mouse liver RNA (Mouse). 2 µ g of total RNA, 2 µ g of poly(A)-depleted RNA and 100 ng of poly(A)-enriched RNA was loaded per lane. rRNA was detected by staining RNA with methylene blue to confirm equal loading between total and poly(A)-depleted fractions. As expected the rRNA are undetectable in the poly(A)-enriched fraction. b . Intensity of the m 6 A signal in immunoblot, measured by Image J, in the whole lane containing the poly(A)-enriched RNA of bloodstream forms. The intensity of the ∼1.8 kb band was compared with the signal intensity of the entire lane, and averaged from 5 independent samples. c . Diagram displaying the location of the oligonucleotides used in <t>RNase</t> H digestion of VSG mRNA. The digestion products detected in the immunoblot (panel D) after incubation with each oligonucleotide (SL, A, B, C, dT) are also indicated. d . Immunoblotting with anti-m 6 A antibody of mammalian bloodstream forms total RNA pre-incubated with indicated oligonucleotides and digested with RNase H. 2 µ g of total RNA were loaded per lane. Staining of rRNA with Methylene Blue confirmed equal loading. SL: spliced leader, dT: poly deoxi-thymidines. Tub: α-Tubulin. (see also Extended Data Fig. S3)
    Thermostable Rnase H, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    m 6 A is present in the poly(A) tail of VSG mRNA and other transcripts. a . Immunoblotting with anti-m 6 A antibody. RNA samples (from left to right): total RNA (Total), Poly(A)-enriched (A+) RNA and Poly(A)-depleted (A-) RNA from two life cycle stages (BSF and PCF). The last lane contains total mouse liver RNA (Mouse). 2 µ g of total RNA, 2 µ g of poly(A)-depleted RNA and 100 ng of poly(A)-enriched RNA was loaded per lane. rRNA was detected by staining RNA with methylene blue to confirm equal loading between total and poly(A)-depleted fractions. As expected the rRNA are undetectable in the poly(A)-enriched fraction. b . Intensity of the m 6 A signal in immunoblot, measured by Image J, in the whole lane containing the poly(A)-enriched RNA of bloodstream forms. The intensity of the ∼1.8 kb band was compared with the signal intensity of the entire lane, and averaged from 5 independent samples. c . Diagram displaying the location of the oligonucleotides used in RNase H digestion of VSG mRNA. The digestion products detected in the immunoblot (panel D) after incubation with each oligonucleotide (SL, A, B, C, dT) are also indicated. d . Immunoblotting with anti-m 6 A antibody of mammalian bloodstream forms total RNA pre-incubated with indicated oligonucleotides and digested with RNase H. 2 µ g of total RNA were loaded per lane. Staining of rRNA with Methylene Blue confirmed equal loading. SL: spliced leader, dT: poly deoxi-thymidines. Tub: α-Tubulin. (see also Extended Data Fig. S3)

    Journal: bioRxiv

    Article Title: N6-methyladenosine in poly(A) tails stabilize VSG transcripts

    doi: 10.1101/2020.01.30.925776

    Figure Lengend Snippet: m 6 A is present in the poly(A) tail of VSG mRNA and other transcripts. a . Immunoblotting with anti-m 6 A antibody. RNA samples (from left to right): total RNA (Total), Poly(A)-enriched (A+) RNA and Poly(A)-depleted (A-) RNA from two life cycle stages (BSF and PCF). The last lane contains total mouse liver RNA (Mouse). 2 µ g of total RNA, 2 µ g of poly(A)-depleted RNA and 100 ng of poly(A)-enriched RNA was loaded per lane. rRNA was detected by staining RNA with methylene blue to confirm equal loading between total and poly(A)-depleted fractions. As expected the rRNA are undetectable in the poly(A)-enriched fraction. b . Intensity of the m 6 A signal in immunoblot, measured by Image J, in the whole lane containing the poly(A)-enriched RNA of bloodstream forms. The intensity of the ∼1.8 kb band was compared with the signal intensity of the entire lane, and averaged from 5 independent samples. c . Diagram displaying the location of the oligonucleotides used in RNase H digestion of VSG mRNA. The digestion products detected in the immunoblot (panel D) after incubation with each oligonucleotide (SL, A, B, C, dT) are also indicated. d . Immunoblotting with anti-m 6 A antibody of mammalian bloodstream forms total RNA pre-incubated with indicated oligonucleotides and digested with RNase H. 2 µ g of total RNA were loaded per lane. Staining of rRNA with Methylene Blue confirmed equal loading. SL: spliced leader, dT: poly deoxi-thymidines. Tub: α-Tubulin. (see also Extended Data Fig. S3)

    Article Snippet: The reaction was stopped by adding formaldehyde loading buffer and incubating at 70°C for 5 min. For RNase H digestions of VSG2 transcript , a thermostable RNase H (NEB M0523) was used to reduce unspecific digestion of the abundant VSG117 transcript.

    Techniques: Staining, Incubation

    Conserved VSG 16-mer motif is required for inclusion of m6A in adjacent poly(A) tail. a . Diagram of 16-mer motif VSg double-expressor (DE) cell-lines. VSG117 was inserted immediately downstream of the promoter of the active bloodstream expression site, which naturally contains VSG 2 at the telomeric end. In VSG double expresor 16-mer WT cell-line, VSG117 contains its endogenous 3’UTR with the conserved 16-mer motif (sequence shown in blue). In VSG double expresor 16-mer MUT cell-line, the 16-mer motif was scrambled (sequence shown in orange). b . Transcript levels of VSG117 and VSG2 transcripts measured by qRT-PCR in both reporter cell-lines. Levels were normalized to transcript levels in cell-lines expressing only VSG2 or only VSG117 . c . Immunoblot with anti-m 6 A antibody of mRNA from VSG double-expressor cell-lines. RNase H digestion of VSG2 mRNA was used to resolve VSG2 and VSG117 transcripts. Different quantities of the same VSG double expresor 16-mer WT cell-line was loaded in two separet lanes (50ng and 12.5ng) to show that the VSG117 band is detectable in both conditions. d . m 6 A index calculated as the ratio of m 6 A intensity and mRNA levels, measured in panels c. and b., respectively. und., undetectable. # intensities measured in lane 3 of Figure 5C (see also Extended Data Fig. S5)

    Journal: bioRxiv

    Article Title: N6-methyladenosine in poly(A) tails stabilize VSG transcripts

    doi: 10.1101/2020.01.30.925776

    Figure Lengend Snippet: Conserved VSG 16-mer motif is required for inclusion of m6A in adjacent poly(A) tail. a . Diagram of 16-mer motif VSg double-expressor (DE) cell-lines. VSG117 was inserted immediately downstream of the promoter of the active bloodstream expression site, which naturally contains VSG 2 at the telomeric end. In VSG double expresor 16-mer WT cell-line, VSG117 contains its endogenous 3’UTR with the conserved 16-mer motif (sequence shown in blue). In VSG double expresor 16-mer MUT cell-line, the 16-mer motif was scrambled (sequence shown in orange). b . Transcript levels of VSG117 and VSG2 transcripts measured by qRT-PCR in both reporter cell-lines. Levels were normalized to transcript levels in cell-lines expressing only VSG2 or only VSG117 . c . Immunoblot with anti-m 6 A antibody of mRNA from VSG double-expressor cell-lines. RNase H digestion of VSG2 mRNA was used to resolve VSG2 and VSG117 transcripts. Different quantities of the same VSG double expresor 16-mer WT cell-line was loaded in two separet lanes (50ng and 12.5ng) to show that the VSG117 band is detectable in both conditions. d . m 6 A index calculated as the ratio of m 6 A intensity and mRNA levels, measured in panels c. and b., respectively. und., undetectable. # intensities measured in lane 3 of Figure 5C (see also Extended Data Fig. S5)

    Article Snippet: The reaction was stopped by adding formaldehyde loading buffer and incubating at 70°C for 5 min. For RNase H digestions of VSG2 transcript , a thermostable RNase H (NEB M0523) was used to reduce unspecific digestion of the abundant VSG117 transcript.

    Techniques: Expressing, Sequencing, Quantitative RT-PCR

    The microprocessor binds to the transcription start sites at RP gene loci. a. ChIP-seq profiles of RNAPII, H3K4me3, Drosha and Dgcr8 at the Rps15a, Rps24, Rpl4, and Rpl28 loci in mouse embryonic stem cells (mES). b. ChIP (IP: Drosha)-qPCR analysis of different RP genes as indicated with anti-Flag (M2) antibody or nonspecific IgG (control) in Flag tagged Drosha-expressing MEF (F-Drosha) or control (pBABE-MEF). Tuba1a and Tubb1 (negative control). Fold enrichment of anti-Flag IP over IgG IP is plotted as Mean ± SEM. n=3. c. ChIP (IP: Drosha)-qPCR analysis of different RP genes as indicated with anti-Flag (M2) antibody or nonspecific IgG (negative control) in Flag-Drosha-expressing MEF or control-MEF. Tuba1a and Tubb1 (negative control). Cells were treated with 1μg/ml actinomycin D (ActD) or vehicle (DMSO), followed by ChIP-qPCR analysis. Fold enrichment of anti-Flag IP over IgG IP is plotted as Mean ± SEM. n=3. d. ChIP (IP: Drosha)-qPCR analysis of different RP genes as indicated in MEF treated with 1μg/μl RNase A or vehicle (water) with anti-Drosha antibody or nonspecific IgG (control). Tuba1a and Tubb1 (negative control). Fold enrichment of Drosha IP over IgG IP is plotted as Mean ± SEM.n=3. e. ChIP (anti-Drosha antibody)-qPCR analysis of different RP genes in MEFs treated with 100U/ml RNase H or vehicle (water) with anti-Drosha antibody. Tuba1a and Tubb1 (negative control). Fold enrichment of Drosha IP over IgG IP is plotted as Mean ± SEM.n=3. f. ChIP (IP: Drosha)-qPCR analysis of different RP genes as indicated with anti-Drosha antibody or nonspecific IgG (control) in control MEFs (Ctrl) or MEFs deleted in Dgcr8 gene (Dgcr8-KO). Tuba1a (negative control). Fold enrichment of Drosha IP over IgG IP is plotted as Mean ± SEM.n=3. g. qRT-PCR analysis of different RP mRNAs and control mRNAs (Itgb1 and IL-6) (relative to GAPDH) as indicated in Ctrl or Dgcr8-KO MEFs. Result is plotted as Mean ± SEM. n=3.

    Journal: bioRxiv

    Article Title: Control of ribosomal protein synthesis by Microprocessor complex

    doi: 10.1101/2020.04.24.060236

    Figure Lengend Snippet: The microprocessor binds to the transcription start sites at RP gene loci. a. ChIP-seq profiles of RNAPII, H3K4me3, Drosha and Dgcr8 at the Rps15a, Rps24, Rpl4, and Rpl28 loci in mouse embryonic stem cells (mES). b. ChIP (IP: Drosha)-qPCR analysis of different RP genes as indicated with anti-Flag (M2) antibody or nonspecific IgG (control) in Flag tagged Drosha-expressing MEF (F-Drosha) or control (pBABE-MEF). Tuba1a and Tubb1 (negative control). Fold enrichment of anti-Flag IP over IgG IP is plotted as Mean ± SEM. n=3. c. ChIP (IP: Drosha)-qPCR analysis of different RP genes as indicated with anti-Flag (M2) antibody or nonspecific IgG (negative control) in Flag-Drosha-expressing MEF or control-MEF. Tuba1a and Tubb1 (negative control). Cells were treated with 1μg/ml actinomycin D (ActD) or vehicle (DMSO), followed by ChIP-qPCR analysis. Fold enrichment of anti-Flag IP over IgG IP is plotted as Mean ± SEM. n=3. d. ChIP (IP: Drosha)-qPCR analysis of different RP genes as indicated in MEF treated with 1μg/μl RNase A or vehicle (water) with anti-Drosha antibody or nonspecific IgG (control). Tuba1a and Tubb1 (negative control). Fold enrichment of Drosha IP over IgG IP is plotted as Mean ± SEM.n=3. e. ChIP (anti-Drosha antibody)-qPCR analysis of different RP genes in MEFs treated with 100U/ml RNase H or vehicle (water) with anti-Drosha antibody. Tuba1a and Tubb1 (negative control). Fold enrichment of Drosha IP over IgG IP is plotted as Mean ± SEM.n=3. f. ChIP (IP: Drosha)-qPCR analysis of different RP genes as indicated with anti-Drosha antibody or nonspecific IgG (control) in control MEFs (Ctrl) or MEFs deleted in Dgcr8 gene (Dgcr8-KO). Tuba1a (negative control). Fold enrichment of Drosha IP over IgG IP is plotted as Mean ± SEM.n=3. g. qRT-PCR analysis of different RP mRNAs and control mRNAs (Itgb1 and IL-6) (relative to GAPDH) as indicated in Ctrl or Dgcr8-KO MEFs. Result is plotted as Mean ± SEM. n=3.

    Article Snippet: For RNase H treatment, RNase H(New England Biolab M0523S) was added into the lysed ChIP sample at a final concentration of 100U/ml.

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing, Negative Control, Quantitative RT-PCR

    The Ddx5 helicase reduces DNA/RNA hybrid and facilitates transcription elongation. A. DNA/RNA hybrid IP-sequencing (DRIP-seq) data indicate increased R-loops at the RPG loci (Rps2, Rps3A, Rpl28, and Rpl37A) and control loci (GAPDH and Tuba1a) in Drosha KD cells (red) compared to control U-2 OS cells (blue) (top). Quantitation of the DRIP-seq data is shown (bottom). b. DRIP analysis of RPG loci (Rps2, Rps5, Rpl14) and control locus (Tuba1a) locus in the presence or absence of RNase H in HCT116 cells expressing CRISPR/Cas9 against Drosha (KO) or non-specific control (Ctrl). Four sets of primers used for DRIP analysis are indicated as black boxes in the top panel (bottom). Signal relative to input is plotted as Mean ± SEM. n=3 c. ChIP-qPCR analysis using anti-RNAPII antibody was performed in K562 cells cells expressing CRISPR/Cas9 against Drosha (KO) or non-specific control (Ctrl). Primers for RPG loci (Rps2, Rps26, Rpl14, Rpl28) and control locus (Tuba1a) are shown. Mean ± SEM. n=3. d. ChIP-qPCR analysis using anti-Ddx5 antibody of RPG loci (Rps2, Rps5, Rps10, Rps12, Rps26, Rpl4, Rpl14, Rpl19, Rpl28) and control locus(Tuba1a) in control (Ctrl) or Drosha KO HCT116 cells. Fold enrichment of Ddx5 antibody pull-down against IgG pull-down is plotted as Mean ± SEM. n=3. e. Immunoprecipitation of DNA/RNA hybrids with the S9.6 antibody, followed by immunoblot analysis of Ddx5, Drosha, Dgcr8 and Lamin A/C (negative control) in Ctrl or Drosha KO K562 cells. f. DRIP analysis of RPG loci (Rps2, Rps5, Rpl4, Rpl28) and control loci (Tuba1a and Tubb1) locus in the presence or absence of RNase H in K562 cells targeting Ddx5 gene ( Ddx5 KO) by RNAi or non-specific control (Ctrl). Result is plotted as Mean ± SEM. n=3 g. qRT-PCR analysis of various RP mRNAs and Drosha mRNAs (relative to GAPDH) in Ctrl or Drosha KO HCT116 cells transfected with empty plasmid (mock), Ddx5 wild type (WT) or the RNA helicase dead (HD) mutant expression plasmid (left). Result is plotted as Mean ± SEM. n=3 Drosha, Ddx5 and GAPDH proteins were examined by western blot in total cell lysates from HCT116 cells (right). Relative protein amount normalized to GAPDH is shown below each blot.

    Journal: bioRxiv

    Article Title: Control of ribosomal protein synthesis by Microprocessor complex

    doi: 10.1101/2020.04.24.060236

    Figure Lengend Snippet: The Ddx5 helicase reduces DNA/RNA hybrid and facilitates transcription elongation. A. DNA/RNA hybrid IP-sequencing (DRIP-seq) data indicate increased R-loops at the RPG loci (Rps2, Rps3A, Rpl28, and Rpl37A) and control loci (GAPDH and Tuba1a) in Drosha KD cells (red) compared to control U-2 OS cells (blue) (top). Quantitation of the DRIP-seq data is shown (bottom). b. DRIP analysis of RPG loci (Rps2, Rps5, Rpl14) and control locus (Tuba1a) locus in the presence or absence of RNase H in HCT116 cells expressing CRISPR/Cas9 against Drosha (KO) or non-specific control (Ctrl). Four sets of primers used for DRIP analysis are indicated as black boxes in the top panel (bottom). Signal relative to input is plotted as Mean ± SEM. n=3 c. ChIP-qPCR analysis using anti-RNAPII antibody was performed in K562 cells cells expressing CRISPR/Cas9 against Drosha (KO) or non-specific control (Ctrl). Primers for RPG loci (Rps2, Rps26, Rpl14, Rpl28) and control locus (Tuba1a) are shown. Mean ± SEM. n=3. d. ChIP-qPCR analysis using anti-Ddx5 antibody of RPG loci (Rps2, Rps5, Rps10, Rps12, Rps26, Rpl4, Rpl14, Rpl19, Rpl28) and control locus(Tuba1a) in control (Ctrl) or Drosha KO HCT116 cells. Fold enrichment of Ddx5 antibody pull-down against IgG pull-down is plotted as Mean ± SEM. n=3. e. Immunoprecipitation of DNA/RNA hybrids with the S9.6 antibody, followed by immunoblot analysis of Ddx5, Drosha, Dgcr8 and Lamin A/C (negative control) in Ctrl or Drosha KO K562 cells. f. DRIP analysis of RPG loci (Rps2, Rps5, Rpl4, Rpl28) and control loci (Tuba1a and Tubb1) locus in the presence or absence of RNase H in K562 cells targeting Ddx5 gene ( Ddx5 KO) by RNAi or non-specific control (Ctrl). Result is plotted as Mean ± SEM. n=3 g. qRT-PCR analysis of various RP mRNAs and Drosha mRNAs (relative to GAPDH) in Ctrl or Drosha KO HCT116 cells transfected with empty plasmid (mock), Ddx5 wild type (WT) or the RNA helicase dead (HD) mutant expression plasmid (left). Result is plotted as Mean ± SEM. n=3 Drosha, Ddx5 and GAPDH proteins were examined by western blot in total cell lysates from HCT116 cells (right). Relative protein amount normalized to GAPDH is shown below each blot.

    Article Snippet: For RNase H treatment, RNase H(New England Biolab M0523S) was added into the lysed ChIP sample at a final concentration of 100U/ml.

    Techniques: Sequencing, Quantitation Assay, Expressing, CRISPR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation, Negative Control, Quantitative RT-PCR, Transfection, Plasmid Preparation, Mutagenesis, Western Blot

    (a) Comparison of response of the Au-ASO mix nanoparticles toward the RNA (1 ng/μL) isolated from noninfected Vero cells, Vero cells infected with MERS-CoV, and Vero cells infected with SARS-CoV-2 virus. Relative change in absorbance at 660 nm wavelength for the Au-ASO mix nanoparticle treated with SARS-CoV-2 RNA (1 ng/μL) followed by the addition of RNase H has been plotted in (b) when the mixture was incubated at different temperatures for 5 min. The schematic representation for the visual naked-eye detection of SARS-CoV-2 with the treatment of RNase H at 65 °C for 5 min is shown in (c). The error bar indicates the average results obtained from three such independent experiments performed in triplicate.

    Journal: ACS Nano

    Article Title: Selective Naked-Eye Detection of SARS-CoV-2 Mediated by N Gene Targeted Antisense Oligonucleotide Capped Plasmonic Nanoparticles

    doi: 10.1021/acsnano.0c03822

    Figure Lengend Snippet: (a) Comparison of response of the Au-ASO mix nanoparticles toward the RNA (1 ng/μL) isolated from noninfected Vero cells, Vero cells infected with MERS-CoV, and Vero cells infected with SARS-CoV-2 virus. Relative change in absorbance at 660 nm wavelength for the Au-ASO mix nanoparticle treated with SARS-CoV-2 RNA (1 ng/μL) followed by the addition of RNase H has been plotted in (b) when the mixture was incubated at different temperatures for 5 min. The schematic representation for the visual naked-eye detection of SARS-CoV-2 with the treatment of RNase H at 65 °C for 5 min is shown in (c). The error bar indicates the average results obtained from three such independent experiments performed in triplicate.

    Article Snippet: Thermostable RNase H was purchased from New England Biolabs.

    Techniques: Allele-specific Oligonucleotide, Isolation, Infection, Incubation