luna universal one step rt qpcr kit  (New England Biolabs)


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    Name:
    Luna Universal One Step RT qPCR Kit
    Description:
    Luna Universal One Step RT qPCR Kit 2 500 rxns
    Catalog Number:
    E3005E
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    2074
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    RT PCR Kits
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    2 500 rxns
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    New England Biolabs luna universal one step rt qpcr kit
    Luna Universal One Step RT qPCR Kit
    Luna Universal One Step RT qPCR Kit 2 500 rxns
    https://www.bioz.com/result/luna universal one step rt qpcr kit/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
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    luna universal one step rt qpcr kit - by Bioz Stars, 2021-06
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    1) Product Images from "Characterization and functional interrogation of SARS-CoV-2 RNA interactome"

    Article Title: Characterization and functional interrogation of SARS-CoV-2 RNA interactome

    Journal: bioRxiv

    doi: 10.1101/2021.03.23.436611

    Screening of compounds with antiviral activity targeting SARS-CoV-2 host RBP. Related to Figure 4 . (A) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous presence of compounds (10 and 1 μM). Virus released in supernatant was quantified 24 hpi by RT-qPCR (top panel). Cell viability was assessed in parallel (bottom panel). Data shown are mean +/- SD of three independent experiments in duplicate. Significance was calculated using two-way ANOVA statistical test with Dunnett’s multiple comparisons test. (ns not significant, ** p
    Figure Legend Snippet: Screening of compounds with antiviral activity targeting SARS-CoV-2 host RBP. Related to Figure 4 . (A) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous presence of compounds (10 and 1 μM). Virus released in supernatant was quantified 24 hpi by RT-qPCR (top panel). Cell viability was assessed in parallel (bottom panel). Data shown are mean +/- SD of three independent experiments in duplicate. Significance was calculated using two-way ANOVA statistical test with Dunnett’s multiple comparisons test. (ns not significant, ** p

    Techniques Used: Activity Assay, Infection, Quantitative RT-PCR

    Functional interrogation of the SARS-CoV-2 RNA interactome and compounds screening. (A) Schematic illustrating the loss-of-function screen procedure. (B and C) A549-ACE2 cells were transfected with an arrayed siRNA library and challenged with SARS-CoV-2 (MOI 0.05) for 24h hours. (B) Yield of viral particles released in the supernatant of infected cells was quantified by RT-qPCR and normalized to the siNT-transfected cells. (C) Viral replication was assessed by flow cytometry using anti-N protein mAb, and normalized to the siNT-transfected cells. Data shown are means of two independent experiments. Adjusted p-values were calculated by one-way ANOVA with Benjamini and Hochberg correction. Host dependency factors are marked in blue and host restriction factors are marked in red. Positive controls (CTSL and ATP6V1B2) are highlighted in yellow. (D) Intersection of the data obtained from N protein quantification by flow cytometry and virus release in supernatant of infected cells by RT-qPCR. Data shown are means of two independent experiments. Host dependency factors are marked in blue and host restriction factors are marked in red. (E) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous presence of increased concentrations of remdesivir or sunitinib malate. Virus released in supernatant was quantified 24 hpi by RT-qPCR (red lane). Cell viability was assessed in parallel (black lane). Data shown are mean +/- SD of three independent experiments in duplicate.
    Figure Legend Snippet: Functional interrogation of the SARS-CoV-2 RNA interactome and compounds screening. (A) Schematic illustrating the loss-of-function screen procedure. (B and C) A549-ACE2 cells were transfected with an arrayed siRNA library and challenged with SARS-CoV-2 (MOI 0.05) for 24h hours. (B) Yield of viral particles released in the supernatant of infected cells was quantified by RT-qPCR and normalized to the siNT-transfected cells. (C) Viral replication was assessed by flow cytometry using anti-N protein mAb, and normalized to the siNT-transfected cells. Data shown are means of two independent experiments. Adjusted p-values were calculated by one-way ANOVA with Benjamini and Hochberg correction. Host dependency factors are marked in blue and host restriction factors are marked in red. Positive controls (CTSL and ATP6V1B2) are highlighted in yellow. (D) Intersection of the data obtained from N protein quantification by flow cytometry and virus release in supernatant of infected cells by RT-qPCR. Data shown are means of two independent experiments. Host dependency factors are marked in blue and host restriction factors are marked in red. (E) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous presence of increased concentrations of remdesivir or sunitinib malate. Virus released in supernatant was quantified 24 hpi by RT-qPCR (red lane). Cell viability was assessed in parallel (black lane). Data shown are mean +/- SD of three independent experiments in duplicate.

    Techniques Used: Functional Assay, Transfection, Infection, Quantitative RT-PCR, Flow Cytometry

    2) Product Images from "LncRNA TUG1 was upregulated in osteoporosis and regulates the proliferation and apoptosis of osteoclasts"

    Article Title: LncRNA TUG1 was upregulated in osteoporosis and regulates the proliferation and apoptosis of osteoclasts

    Journal: Journal of Orthopaedic Surgery and Research

    doi: 10.1186/s13018-019-1430-4

    Plasma lncRNA TUG1 was upregulated in osteoporosis patients than in healthy participants. RT-qPCR results showed that plasma levels of lncRNA TUG1 were significantly higher in osteoporosis patients than in healthy participants (* p
    Figure Legend Snippet: Plasma lncRNA TUG1 was upregulated in osteoporosis patients than in healthy participants. RT-qPCR results showed that plasma levels of lncRNA TUG1 were significantly higher in osteoporosis patients than in healthy participants (* p

    Techniques Used: Quantitative RT-PCR

    3) Product Images from "Globoside Is Dispensable for Parvovirus B19 Entry but Essential at a Postentry Step for Productive Infection"

    Article Title: Globoside Is Dispensable for Parvovirus B19 Entry but Essential at a Postentry Step for Productive Infection

    Journal: Journal of Virology

    doi: 10.1128/JVI.00972-19

    B3GalNT1 KO UT7/Epo cells lack B3GalNT1 transcripts, do not express Gb4, and proliferate normally. (A) Detection of B3GalNT1 mRNA. Total mRNA was isolated from WT cells and from two single cell-derived RFP-expressing clones (KO1 and KO2) and used to detect B3GalNT1 transcripts by RT-qPCR. The amplicons were used in a nested PCR to ensure sufficient sensitivity. Dilutions (1% and 10%) of the WT amplicons were loaded as a reference. GAPDH mRNA was used as a loading control. (B) Detection of Gb4 by immunofluorescence. WT and KO cells were stained with anti-Gb4 antibody, fixed, and visualized by confocal microscopy. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). (C) Phase-contrast images of WT and KO cells showing no morphological differences. (D) Cell proliferation of WT and KO cells. Cells were incubated at 37°C and counted at the indicated days.
    Figure Legend Snippet: B3GalNT1 KO UT7/Epo cells lack B3GalNT1 transcripts, do not express Gb4, and proliferate normally. (A) Detection of B3GalNT1 mRNA. Total mRNA was isolated from WT cells and from two single cell-derived RFP-expressing clones (KO1 and KO2) and used to detect B3GalNT1 transcripts by RT-qPCR. The amplicons were used in a nested PCR to ensure sufficient sensitivity. Dilutions (1% and 10%) of the WT amplicons were loaded as a reference. GAPDH mRNA was used as a loading control. (B) Detection of Gb4 by immunofluorescence. WT and KO cells were stained with anti-Gb4 antibody, fixed, and visualized by confocal microscopy. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). (C) Phase-contrast images of WT and KO cells showing no morphological differences. (D) Cell proliferation of WT and KO cells. Cells were incubated at 37°C and counted at the indicated days.

    Techniques Used: Isolation, Derivative Assay, Expressing, Clone Assay, Quantitative RT-PCR, Nested PCR, Immunofluorescence, Staining, Confocal Microscopy, Incubation

    Gb4 is dispensable for B19V cell attachment, internalization, and VP1u exposure. (A) Detection of B19V attachment by immunofluorescence. B19V was incubated with cells at 4°C for 1 h, followed by four washes with cold PBS. Cells were fixed, stained with antibody 860-55D against capsids, and visualized by confocal microscopy. (B) Detection of B19V internalization by immunofluorescence. B19V was incubated with cells at 37°C for 1 h, washed four times with PBS, and trypsinized to remove noninternalized viruses. Cells were fixed, stained with antibody 860-55D, and visualized by confocal microscopy. (C) Quantification of B19V attachment. B19V was incubated with cells at 4°C for 1 h, followed by four washes with cold PBS. The number of virions bound to the cells was quantified by PCR. (D) Quantification of B19V internalization. B19V was incubated with cells at 37°C for 1 h, washed four times with PBS, trypsinized to remove noninternalized viruses, and quantified by PCR. WT cells incubated at 4°C serve as negative controls (no internalization). (E) Quantification of VP1u exposure from free virus or bound to cells. Virions were immunoprecipitated with antibody 860-55D against capsids (total capsids) and a rabbit antibody against the PLA 2 region (α-VP1u), followed by qPCR. Normal rabbit IgG was used as a negative control. P values were calculated according to Student’s t test. *, P
    Figure Legend Snippet: Gb4 is dispensable for B19V cell attachment, internalization, and VP1u exposure. (A) Detection of B19V attachment by immunofluorescence. B19V was incubated with cells at 4°C for 1 h, followed by four washes with cold PBS. Cells were fixed, stained with antibody 860-55D against capsids, and visualized by confocal microscopy. (B) Detection of B19V internalization by immunofluorescence. B19V was incubated with cells at 37°C for 1 h, washed four times with PBS, and trypsinized to remove noninternalized viruses. Cells were fixed, stained with antibody 860-55D, and visualized by confocal microscopy. (C) Quantification of B19V attachment. B19V was incubated with cells at 4°C for 1 h, followed by four washes with cold PBS. The number of virions bound to the cells was quantified by PCR. (D) Quantification of B19V internalization. B19V was incubated with cells at 37°C for 1 h, washed four times with PBS, trypsinized to remove noninternalized viruses, and quantified by PCR. WT cells incubated at 4°C serve as negative controls (no internalization). (E) Quantification of VP1u exposure from free virus or bound to cells. Virions were immunoprecipitated with antibody 860-55D against capsids (total capsids) and a rabbit antibody against the PLA 2 region (α-VP1u), followed by qPCR. Normal rabbit IgG was used as a negative control. P values were calculated according to Student’s t test. *, P

    Techniques Used: Cell Attachment Assay, Immunofluorescence, Incubation, Staining, Confocal Microscopy, Polymerase Chain Reaction, Immunoprecipitation, Proximity Ligation Assay, Real-time Polymerase Chain Reaction, Negative Control

    4) Product Images from "Histone methyltransferases EHMT1 and EHMT2 (GLP/G9A) maintain PARP inhibitor resistance in high-grade serous ovarian carcinoma"

    Article Title: Histone methyltransferases EHMT1 and EHMT2 (GLP/G9A) maintain PARP inhibitor resistance in high-grade serous ovarian carcinoma

    Journal: Clinical Epigenetics

    doi: 10.1186/s13148-019-0758-2

    Histone methyltransferases EHMT1 and EHMT2 are upregulated in olaparib-resistant HGSOC. a Four olaparib resistant clones of PEO1-OR were analyzed by RNA-Seq. Of all enzymes involved in H3K9 methylation, EHMT1 (red squares), and KDM1B (green circles) were significantly changed in all four populations of PEO1-OR relative to PEO1. b – d RT-qPCR analysis of histone methyltransferases EHMT1 and EHMT2 , and zinc-finger gene ZNF644 in PEO1 and PEO1-OR cells (mean ± SD, n = 3, unpaired t test). e Protein lysates from PEO1 and PEO1-OR cells were analyzed by immunoblot for EHMT1, EHMT2, and Actin loading control. f , g Patient-derived HGSOC ascites cells were injected intraperitoneally into NSG mice. After 21-day treatment with olaparib or vehicle control, mice were sacrificed and ascites cells were collected and analyzed by RT-qPCR for EHMT1 and EHMT2 (mRNA expression is normalized to GAPDH and plotted as mean ± SD, n = 3 technical PCR replicates, unpaired test; numbers below bars are mouse ear-tag numbers). h Protein lysates from ascites cells were analyzed by immunoblot for EHMT1, EHMT2, and Actin loading control (ear tag numbers correspond with mRNA data)
    Figure Legend Snippet: Histone methyltransferases EHMT1 and EHMT2 are upregulated in olaparib-resistant HGSOC. a Four olaparib resistant clones of PEO1-OR were analyzed by RNA-Seq. Of all enzymes involved in H3K9 methylation, EHMT1 (red squares), and KDM1B (green circles) were significantly changed in all four populations of PEO1-OR relative to PEO1. b – d RT-qPCR analysis of histone methyltransferases EHMT1 and EHMT2 , and zinc-finger gene ZNF644 in PEO1 and PEO1-OR cells (mean ± SD, n = 3, unpaired t test). e Protein lysates from PEO1 and PEO1-OR cells were analyzed by immunoblot for EHMT1, EHMT2, and Actin loading control. f , g Patient-derived HGSOC ascites cells were injected intraperitoneally into NSG mice. After 21-day treatment with olaparib or vehicle control, mice were sacrificed and ascites cells were collected and analyzed by RT-qPCR for EHMT1 and EHMT2 (mRNA expression is normalized to GAPDH and plotted as mean ± SD, n = 3 technical PCR replicates, unpaired test; numbers below bars are mouse ear-tag numbers). h Protein lysates from ascites cells were analyzed by immunoblot for EHMT1, EHMT2, and Actin loading control (ear tag numbers correspond with mRNA data)

    Techniques Used: Clone Assay, RNA Sequencing Assay, Methylation, Quantitative RT-PCR, Derivative Assay, Injection, Mouse Assay, Expressing, Polymerase Chain Reaction

    5) Product Images from "Hfq CLASH uncovers sRNA-target interaction networks linked to nutrient availability adaptation"

    Article Title: Hfq CLASH uncovers sRNA-target interaction networks linked to nutrient availability adaptation

    Journal: eLife

    doi: 10.7554/eLife.54655

    ArcZ can influence CyaR levels. ( A ) Base-pairing interactions predicted from the ArcZ-CyaR chimeras using RNACofold. The nucleotide substitutions for experimental validation of direct base-pairing are shown as red or green residues. ( B ) Northern blot analysis of ArcZ and CyaR. The cells containing both the empty pZA and pJV300 plasmids (lanes 1, 5, 7) do not express ArcZ and CyaR at detectable levels. ( C ) Validation of ArcZ-CyaR interaction by over-expression analyses. ArcZ (panel I) orCyaR (panel II) was over-expressed and the levels of their targets were monitored by RT-qPCR. The tpx and sdaC mRNAs are ArcZ targets (panel I). The nadE and yqaE mRNAs are CyaR targets (panel II). The dashed horizontal line indicates the level in the control plasmid (pJV300) that expresses a ~50 nt randomly generated RNA sequence. Panel III: The sRNAs and mutants (as in (A)) were ectopically co-expressed in E. coli and CyaR and CyaR 38–39 levels were quantified by RT-qPCR. Experiments were performed in biological and technical triplicates; Error bars indicate the standard error of the mean (SEM) of the three biological replicates. ( D ) ArcZ and CyaR were overexpressed from a plasmid-borne IPTG inducible promoter (pZE-ArcZ and pZE-CyaR) and the data were compared to data from cells carrying plasmid pJV300. The co-expressed candidate target sRNAs (expressed from pZA-derived backbone) were induced with anhydrotetracycline hydrochloride (panels I and II). The bars indicate the mean fold-change in expression relative to the level of 5S rRNA ( rrfD ) in cells with the indicated vector. In panel III endogenous ArcZ levels were measured upon over-expression of CyaR. Error bars indicate the standard error of the mean from three biological replicates and three technical replicates per experiment. Source data are provided as a Source Data file. Source data for Figure 7B . Source data for Figure 7C . Source data for Figure 7D .
    Figure Legend Snippet: ArcZ can influence CyaR levels. ( A ) Base-pairing interactions predicted from the ArcZ-CyaR chimeras using RNACofold. The nucleotide substitutions for experimental validation of direct base-pairing are shown as red or green residues. ( B ) Northern blot analysis of ArcZ and CyaR. The cells containing both the empty pZA and pJV300 plasmids (lanes 1, 5, 7) do not express ArcZ and CyaR at detectable levels. ( C ) Validation of ArcZ-CyaR interaction by over-expression analyses. ArcZ (panel I) orCyaR (panel II) was over-expressed and the levels of their targets were monitored by RT-qPCR. The tpx and sdaC mRNAs are ArcZ targets (panel I). The nadE and yqaE mRNAs are CyaR targets (panel II). The dashed horizontal line indicates the level in the control plasmid (pJV300) that expresses a ~50 nt randomly generated RNA sequence. Panel III: The sRNAs and mutants (as in (A)) were ectopically co-expressed in E. coli and CyaR and CyaR 38–39 levels were quantified by RT-qPCR. Experiments were performed in biological and technical triplicates; Error bars indicate the standard error of the mean (SEM) of the three biological replicates. ( D ) ArcZ and CyaR were overexpressed from a plasmid-borne IPTG inducible promoter (pZE-ArcZ and pZE-CyaR) and the data were compared to data from cells carrying plasmid pJV300. The co-expressed candidate target sRNAs (expressed from pZA-derived backbone) were induced with anhydrotetracycline hydrochloride (panels I and II). The bars indicate the mean fold-change in expression relative to the level of 5S rRNA ( rrfD ) in cells with the indicated vector. In panel III endogenous ArcZ levels were measured upon over-expression of CyaR. Error bars indicate the standard error of the mean from three biological replicates and three technical replicates per experiment. Source data are provided as a Source Data file. Source data for Figure 7B . Source data for Figure 7C . Source data for Figure 7D .

    Techniques Used: Northern Blot, Over Expression, Quantitative RT-PCR, Plasmid Preparation, Generated, Sequencing, Derivative Assay, Expressing

    6) Product Images from "SARS-CoV-2 and SARS-CoV differ in their cell tropism and drug sensitivity profiles"

    Article Title: SARS-CoV-2 and SARS-CoV differ in their cell tropism and drug sensitivity profiles

    Journal: bioRxiv

    doi: 10.1101/2020.04.03.024257

    N A) Western blots indicating cellular ACE2 and TMPRSS2 protein levels. B) CPE formation in SARS-CoV and SARS-CoV-2 (MOI 0.01)-infected ACE2-negative 293 cells and 293 cells stably expressing ACE2 cells (293/ACE2) 48h post infection. C) Immunostaining for double-stranded RNA in SARS-CoV-2 and SARS-CoV (MOI 0.01)-infected 293/ACE2 cells 48h post infection. D) Quantification of virus genomes by qPCR in SARS-CoV-2 and SARS-CoV (MOI 0.01)-infected 293/ACE2 cells 48h post infection. E) Cytopathogenic effect (CPE) formation in SARS-CoV-2 and SARS-CoV (MOI 0.01)-infected Caco2 cells in the presence of antibodies directed against ACE2 or DPP4 (MERS-CoV receptor) 48h post infection.
    Figure Legend Snippet: N A) Western blots indicating cellular ACE2 and TMPRSS2 protein levels. B) CPE formation in SARS-CoV and SARS-CoV-2 (MOI 0.01)-infected ACE2-negative 293 cells and 293 cells stably expressing ACE2 cells (293/ACE2) 48h post infection. C) Immunostaining for double-stranded RNA in SARS-CoV-2 and SARS-CoV (MOI 0.01)-infected 293/ACE2 cells 48h post infection. D) Quantification of virus genomes by qPCR in SARS-CoV-2 and SARS-CoV (MOI 0.01)-infected 293/ACE2 cells 48h post infection. E) Cytopathogenic effect (CPE) formation in SARS-CoV-2 and SARS-CoV (MOI 0.01)-infected Caco2 cells in the presence of antibodies directed against ACE2 or DPP4 (MERS-CoV receptor) 48h post infection.

    Techniques Used: Western Blot, Infection, Stable Transfection, Expressing, Immunostaining, Real-time Polymerase Chain Reaction

    SARS-CoV-2 and SARS-CoV susceptibility of colorectal cancer cell lines. A) Cytopathogenic effect (CPE) formation 48h post infection in MOI 0.01-infected cells. B) Representative images showing MOI 0.01-infected cells immunostained for double-stranded RNA 48h post infection. C) Quantification of virus genomes by qPCR at different time points post infection (p.i.).
    Figure Legend Snippet: SARS-CoV-2 and SARS-CoV susceptibility of colorectal cancer cell lines. A) Cytopathogenic effect (CPE) formation 48h post infection in MOI 0.01-infected cells. B) Representative images showing MOI 0.01-infected cells immunostained for double-stranded RNA 48h post infection. C) Quantification of virus genomes by qPCR at different time points post infection (p.i.).

    Techniques Used: Infection, Real-time Polymerase Chain Reaction

    7) Product Images from "VELCRO-IP RNA-seq explores ribosome expansion segment function in translation genome-wide"

    Article Title: VELCRO-IP RNA-seq explores ribosome expansion segment function in translation genome-wide

    Journal: bioRxiv

    doi: 10.1101/2020.07.01.179515

    Development of VELCRO-IP RNA-seq to identify global ES-mRNA interactions. Schematic representation of the VELCRO-IP approach. 40S ribosomes are tagged by endogenously Flag-tagging Rps2 at the C-terminus and by generating WT or hES9S rDNA containing Rps2-Flag yeast strains. Lysates are generated by cryo-milling, and 40S ribosomal subunits from each strain are coupled to Flag agarose beads and washed. For a proof-of-principle VELCRO-IP RT-qPCR experiment, in vitro transcripts (IVT) described in Figure 3 are incubated with washed ribosome beads. Upon 3xFlag peptide-elution of 40S-RNA complexes, total RNA is eluted with TRIzol, and IVT enrichment is determined by RT-qPCR using primers specific for Fluc and the 18S rRNA tag to normalize for 40S-IP efficiency. For the genome-wide VELCRO-IP RNA-seq experiment, total RNA from E11.5 FVB mouse embryos is extracted, mRNAs are purified, and the mRNA is fragmented to 100-200 nt long fragments. Refolded RNA fragments are used as input for IP and Flag elution. After yeast rRNA depletion from eluted RNAs, Illumina sequencing libraries of the ribosome-bound mRNA fragments are generated to identify hES9S-specific mRNA elements. IVT, in vitro transcript.
    Figure Legend Snippet: Development of VELCRO-IP RNA-seq to identify global ES-mRNA interactions. Schematic representation of the VELCRO-IP approach. 40S ribosomes are tagged by endogenously Flag-tagging Rps2 at the C-terminus and by generating WT or hES9S rDNA containing Rps2-Flag yeast strains. Lysates are generated by cryo-milling, and 40S ribosomal subunits from each strain are coupled to Flag agarose beads and washed. For a proof-of-principle VELCRO-IP RT-qPCR experiment, in vitro transcripts (IVT) described in Figure 3 are incubated with washed ribosome beads. Upon 3xFlag peptide-elution of 40S-RNA complexes, total RNA is eluted with TRIzol, and IVT enrichment is determined by RT-qPCR using primers specific for Fluc and the 18S rRNA tag to normalize for 40S-IP efficiency. For the genome-wide VELCRO-IP RNA-seq experiment, total RNA from E11.5 FVB mouse embryos is extracted, mRNAs are purified, and the mRNA is fragmented to 100-200 nt long fragments. Refolded RNA fragments are used as input for IP and Flag elution. After yeast rRNA depletion from eluted RNAs, Illumina sequencing libraries of the ribosome-bound mRNA fragments are generated to identify hES9S-specific mRNA elements. IVT, in vitro transcript.

    Techniques Used: RNA Sequencing Assay, Generated, Quantitative RT-PCR, In Vitro, Incubation, Genome Wide, Purification, Sequencing

    VELCRO-IP RT-qPCR serves as a proof-of-principle to identify novel hES9S-interacting 5’ UTRs and mRNA fragmentation. Related to Figure 3. (A) Schematic of in vitro transcripts used for the proof-of-principle experiment of the VELCRO-IP RT-qPCR. Reproduced from Figure 3B . (B) For qualitative analysis of the integrity of in vitro transcripts, RNAs were subjected to 4-20% polyacrylamide/TBE/native PAGE and visualized by SYBR Gold staining. (C) Analysis of total RNA in the 3xFlag peptide elution by RT-qPCR using same volumes of RNA per sample for the RT. Normalization of Ct values for Fluc to the 18S rRNA tag internally controls for ribosome-IP efficiency per sample. The native/WT sample was used to normalize for fold enrichment of RNA binding (set to 1). Representation of the raw data in Figure 3D . Average RNA fold enrichment, SEM, n = 5; ns, not significant. (D) Full view of the Bioanalyzer (Agilent) quantification and electronic gel analysis in Figure 3G, H is shown for optimization of mouse mRNA fragmentation from C3H/10T1/2 cells and stage E11.5 mouse embryos. The marker (M, grey) is overlaid for reference. (E) Full view of the Bioanalyzer (Agilent) quantification and electronic gel analysis in Figure 4B is shown for the eluted and yeast rRNA-depleted mouse embryo RNA from three independent replicates of WT and hES9S VELCRO-IP experiments. The marker (M, grey) is overlaid for reference.
    Figure Legend Snippet: VELCRO-IP RT-qPCR serves as a proof-of-principle to identify novel hES9S-interacting 5’ UTRs and mRNA fragmentation. Related to Figure 3. (A) Schematic of in vitro transcripts used for the proof-of-principle experiment of the VELCRO-IP RT-qPCR. Reproduced from Figure 3B . (B) For qualitative analysis of the integrity of in vitro transcripts, RNAs were subjected to 4-20% polyacrylamide/TBE/native PAGE and visualized by SYBR Gold staining. (C) Analysis of total RNA in the 3xFlag peptide elution by RT-qPCR using same volumes of RNA per sample for the RT. Normalization of Ct values for Fluc to the 18S rRNA tag internally controls for ribosome-IP efficiency per sample. The native/WT sample was used to normalize for fold enrichment of RNA binding (set to 1). Representation of the raw data in Figure 3D . Average RNA fold enrichment, SEM, n = 5; ns, not significant. (D) Full view of the Bioanalyzer (Agilent) quantification and electronic gel analysis in Figure 3G, H is shown for optimization of mouse mRNA fragmentation from C3H/10T1/2 cells and stage E11.5 mouse embryos. The marker (M, grey) is overlaid for reference. (E) Full view of the Bioanalyzer (Agilent) quantification and electronic gel analysis in Figure 4B is shown for the eluted and yeast rRNA-depleted mouse embryo RNA from three independent replicates of WT and hES9S VELCRO-IP experiments. The marker (M, grey) is overlaid for reference.

    Techniques Used: Quantitative RT-PCR, In Vitro, Clear Native PAGE, Staining, RNA Binding Assay, Marker

    VELCRO-IP RT-qPCR serves as a proof-of-principle and mouse embryo mRNA fragmentation. (A) VELCRO-IP RT-qPCR: A zoomed-in view on the potential interactions between mRNAs and ESs, here hES9S binding to the Hoxa9 P4 stem-loop ( Leppek et al., 2020 ) or other target 5’ UTRs, that can be identified by the VELCRO-IP approach. The 4-nt inactive P4 mutant M5 (P4(M5)) serves as a negative control. (B) In vitro transcripts are generated using reporter mRNA plasmids as templates (see ( Leppek et al., 2020 )). The IVT RNAs contain the native spacer (-, negative control), P4-native (P4) or P4(M5)-native (P4(M5), P4-specific negative control) embedded in flanking constant regions (5’ TIE and 3’ Fluc ORF sequence). IVT RNAs have a total length of 475-510 nt and the Fluc ORF portion can be used for qPCR amplification across the three IVT RNA constructs. (C) WB analysis of same volumes of lysate (input), unbound fraction, and Flag peptide-eluted protein from beads to monitor ribosome enrichment of tagged (Rps2-Flag) and untagged (Rps5) 40S and 60S (Rpl10a) components in IVT RNA samples in combination with WT and hES9S yeast ribosomes. Cytoplasmic enzyme Pgk1 served as a negative control. The fraction loaded of input, unbound, and elution samples is expressed as percentage of the original lysate volume. Representative of n = 5 is shown. (D) Analysis of total RNA in the Flag peptide-elution by RT-qPCR using the same volumes of RNA per sample for the RT. Fluc transcript enrichment was assessed by internally normalizing Ct values to that of the respective 18S rRNA tag which controls for ribosome-IP efficiency per sample. We then compared respective hES9S to WT samples to assess specific RNA fold-enrichment of IVT RNAs. Average RNA fold enrichment ± SEM, n = 5. See also Figure S3A-C . (E) Schematic representation of embryo mRNA fragmentation for VELCRO-IP RNA-seq. In brief, total RNA extraction of stage E11.5 mouse embryos yields 2-3% of mRNA isolated on oligo(dT) beads, which is fragmented with magnesium ions to a length of 100-200 nt, and overall recovers
    Figure Legend Snippet: VELCRO-IP RT-qPCR serves as a proof-of-principle and mouse embryo mRNA fragmentation. (A) VELCRO-IP RT-qPCR: A zoomed-in view on the potential interactions between mRNAs and ESs, here hES9S binding to the Hoxa9 P4 stem-loop ( Leppek et al., 2020 ) or other target 5’ UTRs, that can be identified by the VELCRO-IP approach. The 4-nt inactive P4 mutant M5 (P4(M5)) serves as a negative control. (B) In vitro transcripts are generated using reporter mRNA plasmids as templates (see ( Leppek et al., 2020 )). The IVT RNAs contain the native spacer (-, negative control), P4-native (P4) or P4(M5)-native (P4(M5), P4-specific negative control) embedded in flanking constant regions (5’ TIE and 3’ Fluc ORF sequence). IVT RNAs have a total length of 475-510 nt and the Fluc ORF portion can be used for qPCR amplification across the three IVT RNA constructs. (C) WB analysis of same volumes of lysate (input), unbound fraction, and Flag peptide-eluted protein from beads to monitor ribosome enrichment of tagged (Rps2-Flag) and untagged (Rps5) 40S and 60S (Rpl10a) components in IVT RNA samples in combination with WT and hES9S yeast ribosomes. Cytoplasmic enzyme Pgk1 served as a negative control. The fraction loaded of input, unbound, and elution samples is expressed as percentage of the original lysate volume. Representative of n = 5 is shown. (D) Analysis of total RNA in the Flag peptide-elution by RT-qPCR using the same volumes of RNA per sample for the RT. Fluc transcript enrichment was assessed by internally normalizing Ct values to that of the respective 18S rRNA tag which controls for ribosome-IP efficiency per sample. We then compared respective hES9S to WT samples to assess specific RNA fold-enrichment of IVT RNAs. Average RNA fold enrichment ± SEM, n = 5. See also Figure S3A-C . (E) Schematic representation of embryo mRNA fragmentation for VELCRO-IP RNA-seq. In brief, total RNA extraction of stage E11.5 mouse embryos yields 2-3% of mRNA isolated on oligo(dT) beads, which is fragmented with magnesium ions to a length of 100-200 nt, and overall recovers

    Techniques Used: Quantitative RT-PCR, Binding Assay, Mutagenesis, Negative Control, In Vitro, Generated, Sequencing, Real-time Polymerase Chain Reaction, Amplification, Construct, Western Blot, RNA Sequencing Assay, RNA Extraction, Isolation

    Plasmid shuffling in yeast and strain characterization. Related to Figure 1, 2. (A) A yeast strain containing the plasmid-encoded chimeric 18S rRNA is generated by plasmid shuffling. Schematic of the plasmid shuffling approach to generate yeast strains (NOY890, RPS2-Flag) that contain a homozygous knock-out of the rDNA locus (NOY890) and generate ribosomes exclusively from plasmids. All rDNA plasmids contain unique 18S and 25S rRNA sequence tags. 5-FOA-based selection of transformed yeast cells allows for isolation of clones that retain a transformed LEU2 -plasmid (pNOY373) and lost the original URA3 -plasmid (pNOY373). Successful plasmid exchange from URA3 (WT) to LEU2 (tagged WT or hES9S)-plasmids in isolates is achieved by growth on SD- LEU2 , and SD+5-FOA but not on SD- LEU/URA . (B) RT-PCR analysis using ES9S-specific primers that span ES9S allow analysis of expression of WT or hES9S 18S rRNA due to a PCR product of 7 nt difference in length between WT and hES9S (ES span PCR). Similarly, the presence of the 18S tag can be distinguished from WT rRNA (18S tag PCR). Total RNA for cDNA synthesis or plasmid DNA was extracted from clones and used for RT-PCR. Plasmid-derived PCR products serve as controls. PCR products were resolved by 12% native PAGE and stained with SYBR Gold. Two independent isolates of tagged-WT and tagged- hES9S strains (NOY890/RPS2-Flag background) used in this study are presented. RT-PCR specific for the 18S rRNA tag confirms presence of the tag in transformed plasmid and derived mature 18S rRNA. A 10 bp DNA ladder (Invitrogen) was loaded as reference. (C) For yeast strain characterization after plasmid shuffling and isolation of clones, RT-qPCR analysis using specific primers for rRNA tags and endogenous rRNA is used to derive a tag/endogenous rRNA level that assesses the substitution rate of WT with tagged-WT or tagged- hES9S ribosomes present in isolated strains. For NOY890/RPS2-Flag strains, for 44 and 22 tagged WT and hES9S ribosomes, respectively, one endogenous plasmid-derived WT ribosome is left in the cell. (D) Sucrose gradient fractionation analysis of yeast lysates derived from WT and hES9S-stains in the background of NOY890 and NOY890/RPS2-Flag, containing scarless C-terminal RPS2-Flag ( Jan et al., 2014 ), on 10-45% sucrose gradients (n = 3). In comparison to WT rRNA-containing cells, humanized ribosome-containing cells show a slight growth defect. Polysome traces demonstrate proper ribosomal assembly. Incorporation of the Flag tag into polysomes demonstrates its non-perturbative nature. (E) Mapping of the components of the ES engineering system onto the cryo-EM structure of the yeast 80S and 40S ribosome (PDB: 4V6I). The sites of rRNA tag insertion, the last 10 amino acids of the C-terminus of Rps2, and ES9S are highlighted according to the schematic representation.
    Figure Legend Snippet: Plasmid shuffling in yeast and strain characterization. Related to Figure 1, 2. (A) A yeast strain containing the plasmid-encoded chimeric 18S rRNA is generated by plasmid shuffling. Schematic of the plasmid shuffling approach to generate yeast strains (NOY890, RPS2-Flag) that contain a homozygous knock-out of the rDNA locus (NOY890) and generate ribosomes exclusively from plasmids. All rDNA plasmids contain unique 18S and 25S rRNA sequence tags. 5-FOA-based selection of transformed yeast cells allows for isolation of clones that retain a transformed LEU2 -plasmid (pNOY373) and lost the original URA3 -plasmid (pNOY373). Successful plasmid exchange from URA3 (WT) to LEU2 (tagged WT or hES9S)-plasmids in isolates is achieved by growth on SD- LEU2 , and SD+5-FOA but not on SD- LEU/URA . (B) RT-PCR analysis using ES9S-specific primers that span ES9S allow analysis of expression of WT or hES9S 18S rRNA due to a PCR product of 7 nt difference in length between WT and hES9S (ES span PCR). Similarly, the presence of the 18S tag can be distinguished from WT rRNA (18S tag PCR). Total RNA for cDNA synthesis or plasmid DNA was extracted from clones and used for RT-PCR. Plasmid-derived PCR products serve as controls. PCR products were resolved by 12% native PAGE and stained with SYBR Gold. Two independent isolates of tagged-WT and tagged- hES9S strains (NOY890/RPS2-Flag background) used in this study are presented. RT-PCR specific for the 18S rRNA tag confirms presence of the tag in transformed plasmid and derived mature 18S rRNA. A 10 bp DNA ladder (Invitrogen) was loaded as reference. (C) For yeast strain characterization after plasmid shuffling and isolation of clones, RT-qPCR analysis using specific primers for rRNA tags and endogenous rRNA is used to derive a tag/endogenous rRNA level that assesses the substitution rate of WT with tagged-WT or tagged- hES9S ribosomes present in isolated strains. For NOY890/RPS2-Flag strains, for 44 and 22 tagged WT and hES9S ribosomes, respectively, one endogenous plasmid-derived WT ribosome is left in the cell. (D) Sucrose gradient fractionation analysis of yeast lysates derived from WT and hES9S-stains in the background of NOY890 and NOY890/RPS2-Flag, containing scarless C-terminal RPS2-Flag ( Jan et al., 2014 ), on 10-45% sucrose gradients (n = 3). In comparison to WT rRNA-containing cells, humanized ribosome-containing cells show a slight growth defect. Polysome traces demonstrate proper ribosomal assembly. Incorporation of the Flag tag into polysomes demonstrates its non-perturbative nature. (E) Mapping of the components of the ES engineering system onto the cryo-EM structure of the yeast 80S and 40S ribosome (PDB: 4V6I). The sites of rRNA tag insertion, the last 10 amino acids of the C-terminus of Rps2, and ES9S are highlighted according to the schematic representation.

    Techniques Used: Plasmid Preparation, Generated, Knock-Out, Sequencing, Selection, Transformation Assay, Isolation, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Polymerase Chain Reaction, Derivative Assay, Clear Native PAGE, Staining, Quantitative RT-PCR, Fractionation, FLAG-tag

    VELCRO-IP RNA-seq identifies hES9S-interacting 5’ UTRs with cap-independent initiation activity. (A) Based on the analysis in Figure 4 , full 5’ UTRs as annotated in the ENSEMBL database were extracted for experimental validation. Bicistronic mRNA reporter genes containing no insert in the intergenic region (pRF, vector), candidate or control 5’ UTR sequences in the intergenic region were transiently transfected into mouse C3H/10T1/2 cells. Cells were harvested after 24 hours and cells from the same transfection were split in half for protein lysates and total RNA extraction, and subjected to luciferase activity measurement and RT-qPCR analysis, respectively. Relative luciferase activity is expressed as a Fluc(IRES)/Rluc(cap-initiation) ratio normalized to respective Fluc/Rluc mRNA levels, for the integrity of the bicistronic reporter mRNA to support cap-independent initiation activity of candidate 5’ UTRs, and expressed as average cap-independent initiation activity ± standard error of the mean (SEM), n = 3-8. pRF serves as negative control, the EMCV and HCV IRESs as IRES controls, and the full-length Hoxa9 IRES-like element and P4-native as Hoxa9 IRES-like references, respectively. EMCV IRES activity was used as a cutoff to assess candidate 5’ UTR cap-independent initiation activity. a9 IRES FL: FL, full-length. (B) Schematic of the 4xS1m pulldown to probe the interactions of control and candidate 5’ UTR-4xS1m in vitro transcribed RNAs with WT and hES9S yeast ribosomes. Pre-coupled 5’ UTR-4xS1m RNA on SA-sepharose beads are incubated with lysates of WT and hES9S yeast strains (NOY890). Ribosome-RNA RNP enrichment in vitro is monitored by RT-qPCR for tagged rRNA and other RNA classes normalized to the input, and WB analysis for RPs. (C) 4xS1m pulldown to probe the interactions of candidate 5’ UTR-4xS1m in vitro transcribed RNA with WT and hES9S yeast ribosomes. Three control 5’ UTRs served as negative controls and the four candidate 5’ UTRs that display highest cap-independent initiation activity in (A) were tested using Hoxa9 P4 as a reference. Pre-coupled 5’ UTR-4xS1m RNA on SA-sepharose beads are incubated with yeast lysates to form ribonucleoproteins (RNPs) in vitro and protein and RNA are specifically eluted. After the formation of ribosome-RNA RNPs in vitro , beads are split in half: total RNA is eluted with TRIzol, and protein is eluted with RNase A. rRNA bound to the 4xS1m- fused RNA is quantified with primers specific for 18S and 25S rRNA tags (RNA on beads). The P4 serve as a positive control. To indicate specific enrichment of RNA, fold enrichment of RNAs was determined by RT-qPCR using same volumes of eluted RNA and normalizing Ct values of each sample to their respective RNA input (WT or hES9S). Yeast actin ( act1 ) and yeast UsnRNA1 serve as negative controls for an mRNA and a non-coding RNA, respectively. Ribosome enrichment, particularly ribosomal proteins of the 40S and 60S subunit, was assessed by WB analysis of same volumes of protein released from beads by RNase A. The fraction loaded of input and elution samples is expressed as percentage of the original lysate volume. The P4-4xS1m/WT sample was used to normalize for RNA fold enrichment (set to 1). Average RNA fold enrichment, SEM, n = 3; ns, not significant; long exp., long exposure. See also Figure S7C .
    Figure Legend Snippet: VELCRO-IP RNA-seq identifies hES9S-interacting 5’ UTRs with cap-independent initiation activity. (A) Based on the analysis in Figure 4 , full 5’ UTRs as annotated in the ENSEMBL database were extracted for experimental validation. Bicistronic mRNA reporter genes containing no insert in the intergenic region (pRF, vector), candidate or control 5’ UTR sequences in the intergenic region were transiently transfected into mouse C3H/10T1/2 cells. Cells were harvested after 24 hours and cells from the same transfection were split in half for protein lysates and total RNA extraction, and subjected to luciferase activity measurement and RT-qPCR analysis, respectively. Relative luciferase activity is expressed as a Fluc(IRES)/Rluc(cap-initiation) ratio normalized to respective Fluc/Rluc mRNA levels, for the integrity of the bicistronic reporter mRNA to support cap-independent initiation activity of candidate 5’ UTRs, and expressed as average cap-independent initiation activity ± standard error of the mean (SEM), n = 3-8. pRF serves as negative control, the EMCV and HCV IRESs as IRES controls, and the full-length Hoxa9 IRES-like element and P4-native as Hoxa9 IRES-like references, respectively. EMCV IRES activity was used as a cutoff to assess candidate 5’ UTR cap-independent initiation activity. a9 IRES FL: FL, full-length. (B) Schematic of the 4xS1m pulldown to probe the interactions of control and candidate 5’ UTR-4xS1m in vitro transcribed RNAs with WT and hES9S yeast ribosomes. Pre-coupled 5’ UTR-4xS1m RNA on SA-sepharose beads are incubated with lysates of WT and hES9S yeast strains (NOY890). Ribosome-RNA RNP enrichment in vitro is monitored by RT-qPCR for tagged rRNA and other RNA classes normalized to the input, and WB analysis for RPs. (C) 4xS1m pulldown to probe the interactions of candidate 5’ UTR-4xS1m in vitro transcribed RNA with WT and hES9S yeast ribosomes. Three control 5’ UTRs served as negative controls and the four candidate 5’ UTRs that display highest cap-independent initiation activity in (A) were tested using Hoxa9 P4 as a reference. Pre-coupled 5’ UTR-4xS1m RNA on SA-sepharose beads are incubated with yeast lysates to form ribonucleoproteins (RNPs) in vitro and protein and RNA are specifically eluted. After the formation of ribosome-RNA RNPs in vitro , beads are split in half: total RNA is eluted with TRIzol, and protein is eluted with RNase A. rRNA bound to the 4xS1m- fused RNA is quantified with primers specific for 18S and 25S rRNA tags (RNA on beads). The P4 serve as a positive control. To indicate specific enrichment of RNA, fold enrichment of RNAs was determined by RT-qPCR using same volumes of eluted RNA and normalizing Ct values of each sample to their respective RNA input (WT or hES9S). Yeast actin ( act1 ) and yeast UsnRNA1 serve as negative controls for an mRNA and a non-coding RNA, respectively. Ribosome enrichment, particularly ribosomal proteins of the 40S and 60S subunit, was assessed by WB analysis of same volumes of protein released from beads by RNase A. The fraction loaded of input and elution samples is expressed as percentage of the original lysate volume. The P4-4xS1m/WT sample was used to normalize for RNA fold enrichment (set to 1). Average RNA fold enrichment, SEM, n = 3; ns, not significant; long exp., long exposure. See also Figure S7C .

    Techniques Used: RNA Sequencing Assay, Activity Assay, Plasmid Preparation, Transfection, RNA Extraction, Luciferase, Quantitative RT-PCR, Negative Control, In Vitro, Incubation, Western Blot, Positive Control

    8) Product Images from "Dual-level autoregulation of the E. coli DeaD RNA helicase via mRNA stability and Rho-dependent transcription termination"

    Article Title: Dual-level autoregulation of the E. coli DeaD RNA helicase via mRNA stability and Rho-dependent transcription termination

    Journal: bioRxiv

    doi: 10.1101/2020.04.19.049098

    Autoregulation of deaD is mediated via mRNA stability and transcription termination. (A) Northern-blot analysis. WT or DM strains were treated with rifampicin and aliquots of the cell cultures were harvested at different times after rifampicin addition, as indicated at the top. Total RNA was prepared from these cultures and analyzed by northern blot using a probe for deaD mRNA. (B) Half-life analysis. The levels of deaD mRNA from (A) were normalized and plotted as a function of time after rifampicin addition on a semi-logarithmic scale. Each data point represents an average value from three experiments. The calculated half-life of the deaD mRNA in each strain is indicated. (C) Inactivation of RNase E increases deaD mRNA levels. WT and DM strains, as well as their rne-3071 derivatives, were transferred to 42°C for 30 min after growth to midlog phase at 30°C, followed by harvesting of cultures, RNA preparation and northern blot analysis. (D) Effects of Rho inactivation on deaD mRNA. Total RNA was isolated from WT or DM strains containing either wild-type rho or a rho ts allele. The strains were grown to mid-log phase at 30°C and then transferred to 42°C for 30 min before harvesting. RNA was isolated from the strains and analyzed by northern blotting. (E) Effect of BCM addition on deaD mRNA. Cultures of WT or DM strains were grown at 37°C and were either untreated or treated with 20 or 40 μg/ml of BCM for 20 min before harvesting. RNA was isolated from the strains and analyzed by northern blotting. (F) RNA was isolated from WT, rho ts or DM strains after growth at 42°C for 30 min and the levels of RNA transcription from 538 to 638 bps downstream of the guaC coding region were quantified by qRT-PCR. The convergently oriented guaC and hofC coding regions are shown in blue and yellow, respectively. The arrow depicts transcription from the guaC promoter with dotted lines indicating readthrough antisense transcription into the hofC coding region. The region analyzed by qRT-PCR is indicated by a red line.
    Figure Legend Snippet: Autoregulation of deaD is mediated via mRNA stability and transcription termination. (A) Northern-blot analysis. WT or DM strains were treated with rifampicin and aliquots of the cell cultures were harvested at different times after rifampicin addition, as indicated at the top. Total RNA was prepared from these cultures and analyzed by northern blot using a probe for deaD mRNA. (B) Half-life analysis. The levels of deaD mRNA from (A) were normalized and plotted as a function of time after rifampicin addition on a semi-logarithmic scale. Each data point represents an average value from three experiments. The calculated half-life of the deaD mRNA in each strain is indicated. (C) Inactivation of RNase E increases deaD mRNA levels. WT and DM strains, as well as their rne-3071 derivatives, were transferred to 42°C for 30 min after growth to midlog phase at 30°C, followed by harvesting of cultures, RNA preparation and northern blot analysis. (D) Effects of Rho inactivation on deaD mRNA. Total RNA was isolated from WT or DM strains containing either wild-type rho or a rho ts allele. The strains were grown to mid-log phase at 30°C and then transferred to 42°C for 30 min before harvesting. RNA was isolated from the strains and analyzed by northern blotting. (E) Effect of BCM addition on deaD mRNA. Cultures of WT or DM strains were grown at 37°C and were either untreated or treated with 20 or 40 μg/ml of BCM for 20 min before harvesting. RNA was isolated from the strains and analyzed by northern blotting. (F) RNA was isolated from WT, rho ts or DM strains after growth at 42°C for 30 min and the levels of RNA transcription from 538 to 638 bps downstream of the guaC coding region were quantified by qRT-PCR. The convergently oriented guaC and hofC coding regions are shown in blue and yellow, respectively. The arrow depicts transcription from the guaC promoter with dotted lines indicating readthrough antisense transcription into the hofC coding region. The region analyzed by qRT-PCR is indicated by a red line.

    Techniques Used: Northern Blot, Isolation, Quantitative RT-PCR

    (A) Deletion mapping of regions important for autoregulation. A wild-type single-copy deaD-lacZ fusion or variants containing the indicated deletions within the deaD 5’ UTR were incorporated into DlacZ derivatives of the WT or DdeaD strain and the β-galactosidase activity of the resulting strains was measured. (B) RNA was isolated from WT or rho ts strains containing a WT deaD-lacZ fusion or a fusion containing a 720 nt deletion in the deaD 5’ UTR. The RNA samples were analyzed by qRT-PCR, with amplification performed either over the junction region of the deaD-lacZ transcript (100 nts of deaD and 24 nts of lacZ sequence) or a 123 nt region near the 5’ end of the transcript. n. d., not determined.
    Figure Legend Snippet: (A) Deletion mapping of regions important for autoregulation. A wild-type single-copy deaD-lacZ fusion or variants containing the indicated deletions within the deaD 5’ UTR were incorporated into DlacZ derivatives of the WT or DdeaD strain and the β-galactosidase activity of the resulting strains was measured. (B) RNA was isolated from WT or rho ts strains containing a WT deaD-lacZ fusion or a fusion containing a 720 nt deletion in the deaD 5’ UTR. The RNA samples were analyzed by qRT-PCR, with amplification performed either over the junction region of the deaD-lacZ transcript (100 nts of deaD and 24 nts of lacZ sequence) or a 123 nt region near the 5’ end of the transcript. n. d., not determined.

    Techniques Used: Activity Assay, Isolation, Quantitative RT-PCR, Amplification, Sequencing

    9) Product Images from "Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry"

    Article Title: Glyoxal fixation facilitates transcriptome analysis after antigen staining and cell sorting by flow cytometry

    Journal: bioRxiv

    doi: 10.1101/2020.10.05.326082

    Determination of RNA-compatible fixation and permeabilisation conditions A : 1×10 6 COLO205 cells were either unfixed (lane 1), fixed with 70% ethanol on ice for 15 minutes (lane 2) or fixed with 4% formaldehyde on ice for 15 minutes (lane 3). Unfixed cells were dissolved immediately in TRI Reagent, fixed cells were washed once in PBS by centrifugation at 2000 x g for 3 minutes at 4°C before RNA extraction with TRI Reagent. 20% of RNA obtained was separated on a 1.2% glyoxal gel and imaged by ethidium bromide staining. B : COLO205 cells were either unfixed (lane 1), fixed with 70% ethanol on ice for 15 minutes (lane 2) or fixed with 100% methanol on ice for 15 minutes (lane 3). Unfixed cells were dissolved immediately in TRI Reagent, fixed cells were washed once in PBS by centrifugation at 2000 x g for 3 minutes at 4°C before RNA extraction with TRI Reagent. RNA was analysed as in A . C : COLO205 cells were fixed with glyoxal fixation mix (pH4) either without or with 20% ethanol and incubated on ice for 15 minutes and washed once in PBS by centrifugation at 2000 x g for 3 minutes at 4°C. RNA was extracted and analysed as in A . D : COLO205 cells were either unfixed (lane 1), fixed with glyoxal fixation mix (pH4) with 20% ethanol (lane 2) or with 4% formaldehyde on ice for 15 minutes (lane 3). Cells were washed once in PBS by centrifugation at 2000 x g for 3 minutes at 4°C and incubated on ice for 1 hour in 100 μl PBS followed by centrifugation at 2000 x g for 3 minutes at 4°C before RNA extraction with an RNeasy mini kit. E : 100 ng RNA per reaction from D was subjected to one-step combined reverse transcription and quantitative PCR reactions for ACT1B, GAPDH and PGK1. Ct is the cycle number at which the fluorescence exceeded threshold. 3 technical replicates for each RT-qPCR reaction were performed. F : COLO205 cells were either unfixed (lane 1) or fixed with glyoxal fixation mix (pH4) with 20% ethanol on ice for 15 minutes and permeabilised in 100% methanol on ice for 30 minutes (lane 2), or 0.5% saponin on ice for 30 minutes (lane 3) or 0.3% Triton X-100 on ice for 30 minutes (lane 4). RNA was analysed as in A . G : COLO205 cells were either unfixed (lane 1) or fixed with glyoxal fixation mix (pH4) with 20% ethanol on ice for 15 minutes, permeabilised in 100% methanol on ice for 30 minutes followed by incubation in primary antibody for 1 hour on ice and secondary antibody for 30 minutes on ice in dark (lane 2). RNA was analysed as in A .
    Figure Legend Snippet: Determination of RNA-compatible fixation and permeabilisation conditions A : 1×10 6 COLO205 cells were either unfixed (lane 1), fixed with 70% ethanol on ice for 15 minutes (lane 2) or fixed with 4% formaldehyde on ice for 15 minutes (lane 3). Unfixed cells were dissolved immediately in TRI Reagent, fixed cells were washed once in PBS by centrifugation at 2000 x g for 3 minutes at 4°C before RNA extraction with TRI Reagent. 20% of RNA obtained was separated on a 1.2% glyoxal gel and imaged by ethidium bromide staining. B : COLO205 cells were either unfixed (lane 1), fixed with 70% ethanol on ice for 15 minutes (lane 2) or fixed with 100% methanol on ice for 15 minutes (lane 3). Unfixed cells were dissolved immediately in TRI Reagent, fixed cells were washed once in PBS by centrifugation at 2000 x g for 3 minutes at 4°C before RNA extraction with TRI Reagent. RNA was analysed as in A . C : COLO205 cells were fixed with glyoxal fixation mix (pH4) either without or with 20% ethanol and incubated on ice for 15 minutes and washed once in PBS by centrifugation at 2000 x g for 3 minutes at 4°C. RNA was extracted and analysed as in A . D : COLO205 cells were either unfixed (lane 1), fixed with glyoxal fixation mix (pH4) with 20% ethanol (lane 2) or with 4% formaldehyde on ice for 15 minutes (lane 3). Cells were washed once in PBS by centrifugation at 2000 x g for 3 minutes at 4°C and incubated on ice for 1 hour in 100 μl PBS followed by centrifugation at 2000 x g for 3 minutes at 4°C before RNA extraction with an RNeasy mini kit. E : 100 ng RNA per reaction from D was subjected to one-step combined reverse transcription and quantitative PCR reactions for ACT1B, GAPDH and PGK1. Ct is the cycle number at which the fluorescence exceeded threshold. 3 technical replicates for each RT-qPCR reaction were performed. F : COLO205 cells were either unfixed (lane 1) or fixed with glyoxal fixation mix (pH4) with 20% ethanol on ice for 15 minutes and permeabilised in 100% methanol on ice for 30 minutes (lane 2), or 0.5% saponin on ice for 30 minutes (lane 3) or 0.3% Triton X-100 on ice for 30 minutes (lane 4). RNA was analysed as in A . G : COLO205 cells were either unfixed (lane 1) or fixed with glyoxal fixation mix (pH4) with 20% ethanol on ice for 15 minutes, permeabilised in 100% methanol on ice for 30 minutes followed by incubation in primary antibody for 1 hour on ice and secondary antibody for 30 minutes on ice in dark (lane 2). RNA was analysed as in A .

    Techniques Used: Centrifugation, RNA Extraction, Staining, Incubation, Real-time Polymerase Chain Reaction, Fluorescence, Quantitative RT-PCR

    10) Product Images from "A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing"

    Article Title: A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing

    Journal: bioRxiv

    doi: 10.1101/2020.06.23.166397

    A sensitive, robust RT-LAMP assay compatible with crude patient samples. A) Schematic illustrating loop-mediated amplification (LAMP) of SARS-CoV-2 RNA and the regions targeted in this study (Orf1ab, E and N genes; depicted above). Each target region is recognized by a defined set of primers (B3, LB, BIP, LF, FIP, F3). The RNA template (red) is reverse transcribed and displaced after first-strand synthesis; the outer primer binding sites are added in the subsequent amplification step. The resulting dumbbell DNA structure acts as template for further rounds of amplification, ultimately leading to high molecular weight amplicons. B) Readout of a real-time fluorescence RT-LAMP reaction using 500 copies of synthetic SARS-CoV-2 (red) or water as non-targeting control (NTC, black) as input. ‘Time to threshold’ indicates the time at which the fluorescence value reaches threshold level (equivalent to Cq value in RT-qPCR assays), ‘end-point RFU’ indicates the fluorescence value (FAM filter set, absorption/emission at 494 nm/518 nm) after 35 minutes reaction time (used throughout this study unless indicated otherwise); RFU: relative fluorescence units. C) Performance of the three top primer sets for RT-LAMP-based SARS-CoV-2 detection. End-point relative fluorescence units (RFUs) of RT-LAMP reactions (in duplicates) using the indicated primer sets and serially diluted synthetic SARS-CoV-2 RNA standard as input. Water was used as no-target control (NTC). D) Cartoon indicating the workflow for SARS-CoV-2 detection by either RT-LAMP or 1-step RT-qPCR from patient samples (nasopharyngeal swab or gargle) with prior RNA isolation. E) Comparison of RT-LAMP and RT-qPCR performance. Plotted are RT-LAMP end-point fluorescence values after 35 minutes versus the respective RT-qPCR Cq values. RNA was derived from gargle (green) or nasopharyngeal swabs (black); two no-target controls were included (black cross). Reactions in which no amplification was recorded are labelled as qPCR negative. F) Predictive agreement between RT-LAMP and 1-step RT-qPCR assays. Shown are percentages of positive (detected in RT-LAMP and RT-qPCR, black bars) and negative (not detected in either RT-LAMP or RT-qPCR, purple bars) predictive agreement for sample groups (defined by RT-qPCR-derived Cq values) between RT-LAMP (using E- and/or N-gene primers) and 1-step RT-qPCR. G) Performance of different crude sample preparation methods in RT-LAMP. Shown are end-point relative fluorescence units (RFUs) for RT-LAMP reactions targeting human RNAseP on sample inputs derived from defined numbers of HEK293 cells mixed 1:1 with indicated 2x buffers (extracted RNsA served as a positive control). H) Cartoon indicating the workflow for RT-LAMP using QuickExtract crude lysate as sample input. I) Comparison of QuickExtract crude sample input versus extracted RNA as input using 1-step RT-qPCR. Covid-19 patient nasopharyngeal swabs or gargle samples (color coded according to the indicated collection medium) were either processed with the QuickExtract workflow (crude sample input) or RNA was extracted using an automated King Fisher RNA bead purification protocol. Reactions in which no amplification was recorded are labelled as qPCR negative. J) Performance of RT-LAMP with QuickExtract treated crude Covid-19 patient sample input (same samples as in I). Depicted is the correlation of Cq values from RT-qPCR performed on QuickExtract treated samples versus corresponding end-point relative fluorescence units (RFUs) from RT-LAMP reactions.
    Figure Legend Snippet: A sensitive, robust RT-LAMP assay compatible with crude patient samples. A) Schematic illustrating loop-mediated amplification (LAMP) of SARS-CoV-2 RNA and the regions targeted in this study (Orf1ab, E and N genes; depicted above). Each target region is recognized by a defined set of primers (B3, LB, BIP, LF, FIP, F3). The RNA template (red) is reverse transcribed and displaced after first-strand synthesis; the outer primer binding sites are added in the subsequent amplification step. The resulting dumbbell DNA structure acts as template for further rounds of amplification, ultimately leading to high molecular weight amplicons. B) Readout of a real-time fluorescence RT-LAMP reaction using 500 copies of synthetic SARS-CoV-2 (red) or water as non-targeting control (NTC, black) as input. ‘Time to threshold’ indicates the time at which the fluorescence value reaches threshold level (equivalent to Cq value in RT-qPCR assays), ‘end-point RFU’ indicates the fluorescence value (FAM filter set, absorption/emission at 494 nm/518 nm) after 35 minutes reaction time (used throughout this study unless indicated otherwise); RFU: relative fluorescence units. C) Performance of the three top primer sets for RT-LAMP-based SARS-CoV-2 detection. End-point relative fluorescence units (RFUs) of RT-LAMP reactions (in duplicates) using the indicated primer sets and serially diluted synthetic SARS-CoV-2 RNA standard as input. Water was used as no-target control (NTC). D) Cartoon indicating the workflow for SARS-CoV-2 detection by either RT-LAMP or 1-step RT-qPCR from patient samples (nasopharyngeal swab or gargle) with prior RNA isolation. E) Comparison of RT-LAMP and RT-qPCR performance. Plotted are RT-LAMP end-point fluorescence values after 35 minutes versus the respective RT-qPCR Cq values. RNA was derived from gargle (green) or nasopharyngeal swabs (black); two no-target controls were included (black cross). Reactions in which no amplification was recorded are labelled as qPCR negative. F) Predictive agreement between RT-LAMP and 1-step RT-qPCR assays. Shown are percentages of positive (detected in RT-LAMP and RT-qPCR, black bars) and negative (not detected in either RT-LAMP or RT-qPCR, purple bars) predictive agreement for sample groups (defined by RT-qPCR-derived Cq values) between RT-LAMP (using E- and/or N-gene primers) and 1-step RT-qPCR. G) Performance of different crude sample preparation methods in RT-LAMP. Shown are end-point relative fluorescence units (RFUs) for RT-LAMP reactions targeting human RNAseP on sample inputs derived from defined numbers of HEK293 cells mixed 1:1 with indicated 2x buffers (extracted RNsA served as a positive control). H) Cartoon indicating the workflow for RT-LAMP using QuickExtract crude lysate as sample input. I) Comparison of QuickExtract crude sample input versus extracted RNA as input using 1-step RT-qPCR. Covid-19 patient nasopharyngeal swabs or gargle samples (color coded according to the indicated collection medium) were either processed with the QuickExtract workflow (crude sample input) or RNA was extracted using an automated King Fisher RNA bead purification protocol. Reactions in which no amplification was recorded are labelled as qPCR negative. J) Performance of RT-LAMP with QuickExtract treated crude Covid-19 patient sample input (same samples as in I). Depicted is the correlation of Cq values from RT-qPCR performed on QuickExtract treated samples versus corresponding end-point relative fluorescence units (RFUs) from RT-LAMP reactions.

    Techniques Used: RT Lamp Assay, Amplification, Binding Assay, Molecular Weight, Fluorescence, Quantitative RT-PCR, Isolation, Derivative Assay, Real-time Polymerase Chain Reaction, Sample Prep, Positive Control, Purification

    11) Product Images from "A streamlined, cost-effective, and specific method to deplete transcripts for RNA-seq"

    Article Title: A streamlined, cost-effective, and specific method to deplete transcripts for RNA-seq

    Journal: bioRxiv

    doi: 10.1101/2020.05.21.109033

    RNaseH-mediated rRNA and non-specific mRNA depletion. A) Nondenaturing 1.2% agarose gel depicting the following lanes from left to right: 1) ssRNA ladder; 2) total RNA input; 3) input with mock incubations; 4) RNaseH treatment and DNaseI treatment without oligos; 5) DNaseI and oligos only; and 6-8) increasing oligo:RNA mass ratio (1:1, 2:1, and 4:1; total RNA fixed at 1 µg) with RNaseH and DNaseI treatment. B) Fold depletion of 18S rRNA, ACTB, and GAPDH transcripts normalized to input total RNA by RT-qPCR performed on the RNA samples in panel A. Total RNA samples incubated with RNaseH and DNaseI without oligos (grey bar) or increasing oligo:RNA ratio (red bars) with RNaseH and DNaseI treatment. NEB E.coli RNaseH treatment for 1 hour at 37°C and NEB E.coli DNaseI treatment for 30 minutes at 37°C used for samples in this experiment.
    Figure Legend Snippet: RNaseH-mediated rRNA and non-specific mRNA depletion. A) Nondenaturing 1.2% agarose gel depicting the following lanes from left to right: 1) ssRNA ladder; 2) total RNA input; 3) input with mock incubations; 4) RNaseH treatment and DNaseI treatment without oligos; 5) DNaseI and oligos only; and 6-8) increasing oligo:RNA mass ratio (1:1, 2:1, and 4:1; total RNA fixed at 1 µg) with RNaseH and DNaseI treatment. B) Fold depletion of 18S rRNA, ACTB, and GAPDH transcripts normalized to input total RNA by RT-qPCR performed on the RNA samples in panel A. Total RNA samples incubated with RNaseH and DNaseI without oligos (grey bar) or increasing oligo:RNA ratio (red bars) with RNaseH and DNaseI treatment. NEB E.coli RNaseH treatment for 1 hour at 37°C and NEB E.coli DNaseI treatment for 30 minutes at 37°C used for samples in this experiment.

    Techniques Used: Agarose Gel Electrophoresis, Quantitative RT-PCR, Incubation

    12) Product Images from "Attenuation of a very virulent Marek's disease herpesvirus (MDV) by codon pair bias deoptimization"

    Article Title: Attenuation of a very virulent Marek's disease herpesvirus (MDV) by codon pair bias deoptimization

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006857

    Quantification of RNA expression and protein production from the recoded UL30 genes. HEK 293T cells were transfected with dual expression plasmids pVITRO2-TagBFP-UL30-EGFP that carried differently recoded UL30 genes fused in frame with EGFP gene. 24 h post transfection RNA expression (A) from the recoded genes was quantified by qPCR, and protein production by flow cytometry (B). The UL30 RNA levels were normalized against the TagBFP levels. We used EGFP fluorescence as a reporter to quantify protein production of the fusion UL30-EGFP genes. The EGFP fluorescence was normalized against the TagBFP fluorescence. P-values were calculated using Kruskal-Wallis H test, * indicates P
    Figure Legend Snippet: Quantification of RNA expression and protein production from the recoded UL30 genes. HEK 293T cells were transfected with dual expression plasmids pVITRO2-TagBFP-UL30-EGFP that carried differently recoded UL30 genes fused in frame with EGFP gene. 24 h post transfection RNA expression (A) from the recoded genes was quantified by qPCR, and protein production by flow cytometry (B). The UL30 RNA levels were normalized against the TagBFP levels. We used EGFP fluorescence as a reporter to quantify protein production of the fusion UL30-EGFP genes. The EGFP fluorescence was normalized against the TagBFP fluorescence. P-values were calculated using Kruskal-Wallis H test, * indicates P

    Techniques Used: RNA Expression, Transfection, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Fluorescence

    Characterization of recoded MDV UL30 mutants. (A) Effect of recoding on UL30 expression from the virus background. CEC were transfected with the parental or mutant BAC clones that carried differently recoded UL30 genes. 24 h post transfection RNA levels of UL29, UL30 and UL42 genes were quantified by qPCR. P-values were calculated using Kruskal-Wallis H test, * indicates P
    Figure Legend Snippet: Characterization of recoded MDV UL30 mutants. (A) Effect of recoding on UL30 expression from the virus background. CEC were transfected with the parental or mutant BAC clones that carried differently recoded UL30 genes. 24 h post transfection RNA levels of UL29, UL30 and UL42 genes were quantified by qPCR. P-values were calculated using Kruskal-Wallis H test, * indicates P

    Techniques Used: Expressing, Capillary Electrochromatography, Transfection, Mutagenesis, BAC Assay, Clone Assay, Real-time Polymerase Chain Reaction

    Related Articles

    Plasmid Preparation:

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    Article Snippet: Derivatives of the plasmid pSP73-4xS1m(MCS) ( ) were generated by PCR-amplifying 5’ UTR sequences from pRF plasmids using AccuPrime Pfx DNA Polymerase (Thermo, Invitrogen, 12344024). pSP73-4xS1m(MCS) and derivatives can then be linearized at the EcoRI site downstream of the 4xS1m aptamers for run-off in vitro transcription. .. For pNOY373-18S/25S-tag, into the yeast plasmid derivatives of pNOY373, we inserted rRNA tag sequences , a 16-nt tag into 18S rRNA ( ) and a 24-nt tag into 25S rRNA , for RT-PCR and RT-qPCR analysis. .. In a second step, the yeast ES9S was exchanged for the human ES9S in pNOY373-18S/25S-tag, which were generated by overlap extension PCR and were subsequently introduced into SacII-MluI-sites of pNOY373-18S/25S-tag, respectively.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: VELCRO-IP RNA-seq explores ribosome expansion segment function in translation genome-wide
    Article Snippet: Derivatives of the plasmid pSP73-4xS1m(MCS) ( ) were generated by PCR-amplifying 5’ UTR sequences from pRF plasmids using AccuPrime Pfx DNA Polymerase (Thermo, Invitrogen, 12344024). pSP73-4xS1m(MCS) and derivatives can then be linearized at the EcoRI site downstream of the 4xS1m aptamers for run-off in vitro transcription. .. For pNOY373-18S/25S-tag, into the yeast plasmid derivatives of pNOY373, we inserted rRNA tag sequences , a 16-nt tag into 18S rRNA ( ) and a 24-nt tag into 25S rRNA , for RT-PCR and RT-qPCR analysis. .. In a second step, the yeast ES9S was exchanged for the human ES9S in pNOY373-18S/25S-tag, which were generated by overlap extension PCR and were subsequently introduced into SacII-MluI-sites of pNOY373-18S/25S-tag, respectively.

    Quantitative RT-PCR:

    Article Title: VELCRO-IP RNA-seq explores ribosome expansion segment function in translation genome-wide
    Article Snippet: Derivatives of the plasmid pSP73-4xS1m(MCS) ( ) were generated by PCR-amplifying 5’ UTR sequences from pRF plasmids using AccuPrime Pfx DNA Polymerase (Thermo, Invitrogen, 12344024). pSP73-4xS1m(MCS) and derivatives can then be linearized at the EcoRI site downstream of the 4xS1m aptamers for run-off in vitro transcription. .. For pNOY373-18S/25S-tag, into the yeast plasmid derivatives of pNOY373, we inserted rRNA tag sequences , a 16-nt tag into 18S rRNA ( ) and a 24-nt tag into 25S rRNA , for RT-PCR and RT-qPCR analysis. .. In a second step, the yeast ES9S was exchanged for the human ES9S in pNOY373-18S/25S-tag, which were generated by overlap extension PCR and were subsequently introduced into SacII-MluI-sites of pNOY373-18S/25S-tag, respectively.

    Article Title: Dual-level autoregulation of the E. coli DeaD RNA helicase via mRNA stability and Rho-dependent transcription termination
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    Article Title: Globoside Is Dispensable for Parvovirus B19 Entry but Essential at a Postentry Step for Productive Infection
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    Article Title: Histone methyltransferases EHMT1 and EHMT2 (GLP/G9A) maintain PARP inhibitor resistance in high-grade serous ovarian carcinoma
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    Polymerase Chain Reaction:

    Article Title: Globoside Is Dispensable for Parvovirus B19 Entry but Essential at a Postentry Step for Productive Infection
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    Article Title: LncRNA TUG1 was upregulated in osteoporosis and regulates the proliferation and apoptosis of osteoclasts
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    Real-time Polymerase Chain Reaction:

    Article Title: Hfq CLASH uncovers sRNA-target interaction networks linked to nutrient availability adaptation
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    Article Title: Characterization and functional interrogation of SARS-CoV-2 RNA interactome
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    Article Title: Histone methyltransferases EHMT1 and EHMT2 (GLP/G9A) maintain PARP inhibitor resistance in high-grade serous ovarian carcinoma
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    Expressing:

    Article Title: LncRNA TUG1 was upregulated in osteoporosis and regulates the proliferation and apoptosis of osteoclasts
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    Article Title: Histone methyltransferases EHMT1 and EHMT2 (GLP/G9A) maintain PARP inhibitor resistance in high-grade serous ovarian carcinoma
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    Isolation:

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    SYBR Green Assay:

    Article Title: Histone methyltransferases EHMT1 and EHMT2 (GLP/G9A) maintain PARP inhibitor resistance in high-grade serous ovarian carcinoma
    Article Snippet: .. Reverse-transcriptase quantitative PCR RNA was isolated from cells using the RNeasy Plus Mini Kit (Qiagen). mRNA expression was determined using SYBR green Luna One Step reverse-transcriptase quantitative PCR (RT-qPCR) Kit (New England BioLabs) on a C1000 Touch (Bio-Rad) or QuantStudio 6 (Applied Biosystems) thermocycler. .. Expression was quantified by the ΔΔCt method using target-specific and control primers. β-2-microglobulin (B2M ) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH ) were used as internal controls. mRNA-specific primers were designed to span exon-exon junctions to avoid detection of genomic DNA.

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    New England Biolabs luna universal one step rt qpcr kit
    Screening of compounds with antiviral activity targeting <t>SARS-CoV-2</t> host RBP. Related to Figure 4 . (A) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous presence of compounds (10 and 1 μM). Virus released in supernatant was quantified 24 hpi by <t>RT-qPCR</t> (top panel). Cell viability was assessed in parallel (bottom panel). Data shown are mean +/- SD of three independent experiments in duplicate. Significance was calculated using two-way ANOVA statistical test with Dunnett’s multiple comparisons test. (ns not significant, ** p
    Luna Universal One Step Rt Qpcr Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs luna universal probe one step rt qpcr kit
    Mitoprotein-induced stress triggers the UPR ER . A , Fusion of DHFR to the N-terminus of Cytochrome b 2 generates a mitochondrial ‘clogger’ that jams the protein import machinery. B , The mitochondrial clogger b 2 -DHFR or cytosolic DHFR were expressed for 4.5 h. The precursor form of the mitochondrial proteins Mdj1 and Rip1 were detected by Western Blotting. C , Expression of b 2 -DHFR leads to attenuated growth. D , The mitoprotein-induced stress response encompasses an early transcriptional induction of chaperones and the proteasome and a downregulation of cytosolic ribosomes and OXPHOS components. E , Protein levels in clogger-expressing versus control cells after 18 h of induction were measured by quantitative mass spectrometry 28 . Highlighted are proteins which are reported targets of the UPR ER 21 . Data from n =3 independent biological replicates are shown. F, G , The cellular transcriptome and translatome after 4.5 h of clogger induction were measured by <t>RNA-Seq</t> ( n =4) 28 and ribosome profiling ( n =3), respectively. Shown are log 2 fold changes of b 2 -DHFR versus cytosolic DHFR. HAC1 transcripts are slightly reduced, but its translation is upregulated. H , Ribosome footprints along the HAC1 gene from cells expressing b 2 -DHFR or cytosolic DHFR for 4.5 h are shown. I , Levels of spliced HAC1 mRNA in cells expressing b 2 -DHFR or cytosolic DHFR were measured by <t>RT-qPCR</t> over time ( n =3).
    Luna Universal Probe One Step Rt Qpcr Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Screening of compounds with antiviral activity targeting SARS-CoV-2 host RBP. Related to Figure 4 . (A) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous presence of compounds (10 and 1 μM). Virus released in supernatant was quantified 24 hpi by RT-qPCR (top panel). Cell viability was assessed in parallel (bottom panel). Data shown are mean +/- SD of three independent experiments in duplicate. Significance was calculated using two-way ANOVA statistical test with Dunnett’s multiple comparisons test. (ns not significant, ** p

    Journal: bioRxiv

    Article Title: Characterization and functional interrogation of SARS-CoV-2 RNA interactome

    doi: 10.1101/2021.03.23.436611

    Figure Lengend Snippet: Screening of compounds with antiviral activity targeting SARS-CoV-2 host RBP. Related to Figure 4 . (A) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous presence of compounds (10 and 1 μM). Virus released in supernatant was quantified 24 hpi by RT-qPCR (top panel). Cell viability was assessed in parallel (bottom panel). Data shown are mean +/- SD of three independent experiments in duplicate. Significance was calculated using two-way ANOVA statistical test with Dunnett’s multiple comparisons test. (ns not significant, ** p

    Article Snippet: Yields of viral RNA were quantified by real-time qPCR by using SARS-CoV-2 specific primers targeting the E gene with the Luna®Universal One-Step RT-qPCR Kit (New England Biolabs) in a LightCycler 480 thermocycler (Roche) according to the manufacturer’s protocol.

    Techniques: Activity Assay, Infection, Quantitative RT-PCR

    Functional interrogation of the SARS-CoV-2 RNA interactome and compounds screening. (A) Schematic illustrating the loss-of-function screen procedure. (B and C) A549-ACE2 cells were transfected with an arrayed siRNA library and challenged with SARS-CoV-2 (MOI 0.05) for 24h hours. (B) Yield of viral particles released in the supernatant of infected cells was quantified by RT-qPCR and normalized to the siNT-transfected cells. (C) Viral replication was assessed by flow cytometry using anti-N protein mAb, and normalized to the siNT-transfected cells. Data shown are means of two independent experiments. Adjusted p-values were calculated by one-way ANOVA with Benjamini and Hochberg correction. Host dependency factors are marked in blue and host restriction factors are marked in red. Positive controls (CTSL and ATP6V1B2) are highlighted in yellow. (D) Intersection of the data obtained from N protein quantification by flow cytometry and virus release in supernatant of infected cells by RT-qPCR. Data shown are means of two independent experiments. Host dependency factors are marked in blue and host restriction factors are marked in red. (E) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous presence of increased concentrations of remdesivir or sunitinib malate. Virus released in supernatant was quantified 24 hpi by RT-qPCR (red lane). Cell viability was assessed in parallel (black lane). Data shown are mean +/- SD of three independent experiments in duplicate.

    Journal: bioRxiv

    Article Title: Characterization and functional interrogation of SARS-CoV-2 RNA interactome

    doi: 10.1101/2021.03.23.436611

    Figure Lengend Snippet: Functional interrogation of the SARS-CoV-2 RNA interactome and compounds screening. (A) Schematic illustrating the loss-of-function screen procedure. (B and C) A549-ACE2 cells were transfected with an arrayed siRNA library and challenged with SARS-CoV-2 (MOI 0.05) for 24h hours. (B) Yield of viral particles released in the supernatant of infected cells was quantified by RT-qPCR and normalized to the siNT-transfected cells. (C) Viral replication was assessed by flow cytometry using anti-N protein mAb, and normalized to the siNT-transfected cells. Data shown are means of two independent experiments. Adjusted p-values were calculated by one-way ANOVA with Benjamini and Hochberg correction. Host dependency factors are marked in blue and host restriction factors are marked in red. Positive controls (CTSL and ATP6V1B2) are highlighted in yellow. (D) Intersection of the data obtained from N protein quantification by flow cytometry and virus release in supernatant of infected cells by RT-qPCR. Data shown are means of two independent experiments. Host dependency factors are marked in blue and host restriction factors are marked in red. (E) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous presence of increased concentrations of remdesivir or sunitinib malate. Virus released in supernatant was quantified 24 hpi by RT-qPCR (red lane). Cell viability was assessed in parallel (black lane). Data shown are mean +/- SD of three independent experiments in duplicate.

    Article Snippet: Yields of viral RNA were quantified by real-time qPCR by using SARS-CoV-2 specific primers targeting the E gene with the Luna®Universal One-Step RT-qPCR Kit (New England Biolabs) in a LightCycler 480 thermocycler (Roche) according to the manufacturer’s protocol.

    Techniques: Functional Assay, Transfection, Infection, Quantitative RT-PCR, Flow Cytometry

    Plasma lncRNA TUG1 was upregulated in osteoporosis patients than in healthy participants. RT-qPCR results showed that plasma levels of lncRNA TUG1 were significantly higher in osteoporosis patients than in healthy participants (* p

    Journal: Journal of Orthopaedic Surgery and Research

    Article Title: LncRNA TUG1 was upregulated in osteoporosis and regulates the proliferation and apoptosis of osteoclasts

    doi: 10.1186/s13018-019-1430-4

    Figure Lengend Snippet: Plasma lncRNA TUG1 was upregulated in osteoporosis patients than in healthy participants. RT-qPCR results showed that plasma levels of lncRNA TUG1 were significantly higher in osteoporosis patients than in healthy participants (* p

    Article Snippet: To detect the expression of lncRNA TUG1 and PTEN mRNA, Luna® Universal One-Step RT-qPCR Kit (NEB) was used to prepare PCR reaction systems.

    Techniques: Quantitative RT-PCR

    B3GalNT1 KO UT7/Epo cells lack B3GalNT1 transcripts, do not express Gb4, and proliferate normally. (A) Detection of B3GalNT1 mRNA. Total mRNA was isolated from WT cells and from two single cell-derived RFP-expressing clones (KO1 and KO2) and used to detect B3GalNT1 transcripts by RT-qPCR. The amplicons were used in a nested PCR to ensure sufficient sensitivity. Dilutions (1% and 10%) of the WT amplicons were loaded as a reference. GAPDH mRNA was used as a loading control. (B) Detection of Gb4 by immunofluorescence. WT and KO cells were stained with anti-Gb4 antibody, fixed, and visualized by confocal microscopy. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). (C) Phase-contrast images of WT and KO cells showing no morphological differences. (D) Cell proliferation of WT and KO cells. Cells were incubated at 37°C and counted at the indicated days.

    Journal: Journal of Virology

    Article Title: Globoside Is Dispensable for Parvovirus B19 Entry but Essential at a Postentry Step for Productive Infection

    doi: 10.1128/JVI.00972-19

    Figure Lengend Snippet: B3GalNT1 KO UT7/Epo cells lack B3GalNT1 transcripts, do not express Gb4, and proliferate normally. (A) Detection of B3GalNT1 mRNA. Total mRNA was isolated from WT cells and from two single cell-derived RFP-expressing clones (KO1 and KO2) and used to detect B3GalNT1 transcripts by RT-qPCR. The amplicons were used in a nested PCR to ensure sufficient sensitivity. Dilutions (1% and 10%) of the WT amplicons were loaded as a reference. GAPDH mRNA was used as a loading control. (B) Detection of Gb4 by immunofluorescence. WT and KO cells were stained with anti-Gb4 antibody, fixed, and visualized by confocal microscopy. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). (C) Phase-contrast images of WT and KO cells showing no morphological differences. (D) Cell proliferation of WT and KO cells. Cells were incubated at 37°C and counted at the indicated days.

    Article Snippet: For detection of the B3GalNT1 mRNA, the Luna Universal one-step reverse transcription-quantitative PCR (RT-qPCR) kit (New England BioLabs, Ipswich, MA) was used.

    Techniques: Isolation, Derivative Assay, Expressing, Clone Assay, Quantitative RT-PCR, Nested PCR, Immunofluorescence, Staining, Confocal Microscopy, Incubation

    Gb4 is dispensable for B19V cell attachment, internalization, and VP1u exposure. (A) Detection of B19V attachment by immunofluorescence. B19V was incubated with cells at 4°C for 1 h, followed by four washes with cold PBS. Cells were fixed, stained with antibody 860-55D against capsids, and visualized by confocal microscopy. (B) Detection of B19V internalization by immunofluorescence. B19V was incubated with cells at 37°C for 1 h, washed four times with PBS, and trypsinized to remove noninternalized viruses. Cells were fixed, stained with antibody 860-55D, and visualized by confocal microscopy. (C) Quantification of B19V attachment. B19V was incubated with cells at 4°C for 1 h, followed by four washes with cold PBS. The number of virions bound to the cells was quantified by PCR. (D) Quantification of B19V internalization. B19V was incubated with cells at 37°C for 1 h, washed four times with PBS, trypsinized to remove noninternalized viruses, and quantified by PCR. WT cells incubated at 4°C serve as negative controls (no internalization). (E) Quantification of VP1u exposure from free virus or bound to cells. Virions were immunoprecipitated with antibody 860-55D against capsids (total capsids) and a rabbit antibody against the PLA 2 region (α-VP1u), followed by qPCR. Normal rabbit IgG was used as a negative control. P values were calculated according to Student’s t test. *, P

    Journal: Journal of Virology

    Article Title: Globoside Is Dispensable for Parvovirus B19 Entry but Essential at a Postentry Step for Productive Infection

    doi: 10.1128/JVI.00972-19

    Figure Lengend Snippet: Gb4 is dispensable for B19V cell attachment, internalization, and VP1u exposure. (A) Detection of B19V attachment by immunofluorescence. B19V was incubated with cells at 4°C for 1 h, followed by four washes with cold PBS. Cells were fixed, stained with antibody 860-55D against capsids, and visualized by confocal microscopy. (B) Detection of B19V internalization by immunofluorescence. B19V was incubated with cells at 37°C for 1 h, washed four times with PBS, and trypsinized to remove noninternalized viruses. Cells were fixed, stained with antibody 860-55D, and visualized by confocal microscopy. (C) Quantification of B19V attachment. B19V was incubated with cells at 4°C for 1 h, followed by four washes with cold PBS. The number of virions bound to the cells was quantified by PCR. (D) Quantification of B19V internalization. B19V was incubated with cells at 37°C for 1 h, washed four times with PBS, trypsinized to remove noninternalized viruses, and quantified by PCR. WT cells incubated at 4°C serve as negative controls (no internalization). (E) Quantification of VP1u exposure from free virus or bound to cells. Virions were immunoprecipitated with antibody 860-55D against capsids (total capsids) and a rabbit antibody against the PLA 2 region (α-VP1u), followed by qPCR. Normal rabbit IgG was used as a negative control. P values were calculated according to Student’s t test. *, P

    Article Snippet: For detection of the B3GalNT1 mRNA, the Luna Universal one-step reverse transcription-quantitative PCR (RT-qPCR) kit (New England BioLabs, Ipswich, MA) was used.

    Techniques: Cell Attachment Assay, Immunofluorescence, Incubation, Staining, Confocal Microscopy, Polymerase Chain Reaction, Immunoprecipitation, Proximity Ligation Assay, Real-time Polymerase Chain Reaction, Negative Control

    Mitoprotein-induced stress triggers the UPR ER . A , Fusion of DHFR to the N-terminus of Cytochrome b 2 generates a mitochondrial ‘clogger’ that jams the protein import machinery. B , The mitochondrial clogger b 2 -DHFR or cytosolic DHFR were expressed for 4.5 h. The precursor form of the mitochondrial proteins Mdj1 and Rip1 were detected by Western Blotting. C , Expression of b 2 -DHFR leads to attenuated growth. D , The mitoprotein-induced stress response encompasses an early transcriptional induction of chaperones and the proteasome and a downregulation of cytosolic ribosomes and OXPHOS components. E , Protein levels in clogger-expressing versus control cells after 18 h of induction were measured by quantitative mass spectrometry 28 . Highlighted are proteins which are reported targets of the UPR ER 21 . Data from n =3 independent biological replicates are shown. F, G , The cellular transcriptome and translatome after 4.5 h of clogger induction were measured by RNA-Seq ( n =4) 28 and ribosome profiling ( n =3), respectively. Shown are log 2 fold changes of b 2 -DHFR versus cytosolic DHFR. HAC1 transcripts are slightly reduced, but its translation is upregulated. H , Ribosome footprints along the HAC1 gene from cells expressing b 2 -DHFR or cytosolic DHFR for 4.5 h are shown. I , Levels of spliced HAC1 mRNA in cells expressing b 2 -DHFR or cytosolic DHFR were measured by RT-qPCR over time ( n =3).

    Journal: bioRxiv

    Article Title: The unfolded protein response of the endoplasmic reticulum supports mitochondrial biogenesis by buffering non-imported proteins

    doi: 10.1101/2021.05.19.444788

    Figure Lengend Snippet: Mitoprotein-induced stress triggers the UPR ER . A , Fusion of DHFR to the N-terminus of Cytochrome b 2 generates a mitochondrial ‘clogger’ that jams the protein import machinery. B , The mitochondrial clogger b 2 -DHFR or cytosolic DHFR were expressed for 4.5 h. The precursor form of the mitochondrial proteins Mdj1 and Rip1 were detected by Western Blotting. C , Expression of b 2 -DHFR leads to attenuated growth. D , The mitoprotein-induced stress response encompasses an early transcriptional induction of chaperones and the proteasome and a downregulation of cytosolic ribosomes and OXPHOS components. E , Protein levels in clogger-expressing versus control cells after 18 h of induction were measured by quantitative mass spectrometry 28 . Highlighted are proteins which are reported targets of the UPR ER 21 . Data from n =3 independent biological replicates are shown. F, G , The cellular transcriptome and translatome after 4.5 h of clogger induction were measured by RNA-Seq ( n =4) 28 and ribosome profiling ( n =3), respectively. Shown are log 2 fold changes of b 2 -DHFR versus cytosolic DHFR. HAC1 transcripts are slightly reduced, but its translation is upregulated. H , Ribosome footprints along the HAC1 gene from cells expressing b 2 -DHFR or cytosolic DHFR for 4.5 h are shown. I , Levels of spliced HAC1 mRNA in cells expressing b 2 -DHFR or cytosolic DHFR were measured by RT-qPCR over time ( n =3).

    Article Snippet: 100 ng total RNA per 20 µl reaction were analyzed using the Luna Universal Probe One-Step RT-qPCR Kit (NEB, # E3006) in technical triplicates. cDNA was generated by reverse transcription for 10 min at 55°C.

    Techniques: Western Blot, Expressing, Mass Spectrometry, RNA Sequencing Assay, Quantitative RT-PCR

    Detection of UPR ER induction with mass spectrometry and RT-qPCR. A , Protein levels in clogger-expressing versus control cells after different times of induction were measured by quantitative mass spectrometry 28 . Highlighted are proteins which are reported targets of the UPR ER 21 . Data from n =3 independent biological replicates are shown. The data for 18 h are the same as shown in Fig. 1E . B , The change in translational efficiency after 4.5 h clogger expression was calculated for all genes measured in both the RNA-seq 28 and Ribo-Seq on clogger-expressing cells by dividing the translatome fold change by the transcriptome fold change. C , Schematic depiction of the primer-probe combinations used to quantify total HAC1 as well as spliced HAC1 i mRNA levels via RT-qPCR. D , Wild type, Δ ire1 and Δ hac1 cells were grown in presence or absence of 1 µg/ml tunicamycin and HAC1 and HAC1 i levels were analyzed with the primer-probe assay shown in C. As expected, HAC1 i levels increased in wild type cells treated with tunicamyin, but no HAC1 i was detected in cells lacking HAC1 or IRE1 , confirming the specificity of the RT-qPCR assay.

    Journal: bioRxiv

    Article Title: The unfolded protein response of the endoplasmic reticulum supports mitochondrial biogenesis by buffering non-imported proteins

    doi: 10.1101/2021.05.19.444788

    Figure Lengend Snippet: Detection of UPR ER induction with mass spectrometry and RT-qPCR. A , Protein levels in clogger-expressing versus control cells after different times of induction were measured by quantitative mass spectrometry 28 . Highlighted are proteins which are reported targets of the UPR ER 21 . Data from n =3 independent biological replicates are shown. The data for 18 h are the same as shown in Fig. 1E . B , The change in translational efficiency after 4.5 h clogger expression was calculated for all genes measured in both the RNA-seq 28 and Ribo-Seq on clogger-expressing cells by dividing the translatome fold change by the transcriptome fold change. C , Schematic depiction of the primer-probe combinations used to quantify total HAC1 as well as spliced HAC1 i mRNA levels via RT-qPCR. D , Wild type, Δ ire1 and Δ hac1 cells were grown in presence or absence of 1 µg/ml tunicamycin and HAC1 and HAC1 i levels were analyzed with the primer-probe assay shown in C. As expected, HAC1 i levels increased in wild type cells treated with tunicamyin, but no HAC1 i was detected in cells lacking HAC1 or IRE1 , confirming the specificity of the RT-qPCR assay.

    Article Snippet: 100 ng total RNA per 20 µl reaction were analyzed using the Luna Universal Probe One-Step RT-qPCR Kit (NEB, # E3006) in technical triplicates. cDNA was generated by reverse transcription for 10 min at 55°C.

    Techniques: Mass Spectrometry, Quantitative RT-PCR, Expressing, RNA Sequencing Assay