superscript iii reverse transcriptase  (Thermo Fisher)


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    Name:
    Shandon 10000 Autopsy Saw
    Description:
    Cut safely and reduce operator fatigue with Thermo Scientific Shandon 10000 Autopsy Saw
    Catalog Number:
    10000
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    Thermo Fisher superscript iii reverse transcriptase
    TET proteins facilitate CSR. ( A ) Flowchart of in vivo experiment. f.p., footpad. ( B ) Top: Flow cytometric analysis of CD19 + GL7 + Fas + GC B cells at the draining popliteal lymph nodes from WT and Tet2/3 DKO mice after NP-OVA immunization as in (A). Bottom: Decreased IgG1-switched cells among GC B cells in Tet2/3 DKO (YFP + GC B–gated) compared with WT mice (GC B–gated). ( C to F ) Quantification of experiments shown in (B). Data shown are aggregated results from two independent experiments. Means and SEs are shown. WT, n = 11; DKO, n = 12. ( G ) Flowchart of in vitro IgG1 switching. Cells were labeled with Cell trace violet and activated for 4 days with LPS and IL-4. ( H and I ) Flow cytometry plots (H) and quantification (I) of IgG1-switched B cells in Tet2/3 DKO ( n = 4) and WT ( n = 4) mice. Representative of at least <t>three</t> independent experiments. ( J ) Circular γ1 transcript was quantified by <t>qRT-PCR</t> and normalized to Gapdh and then to the level of WT. Representative experiment of two independent experiments with three technical replicates. ( K ) Flowchart of in vitro IgA switching. Cells were activated for 5 days with anti-CD40, rmIL-4, rmIL-5, and rhTGFβ. ( L to N ) Flow cytometry plots (L) and quantification (M and N) of IgG1-switched (M) and IgA-switched cells (N). Representative of three independent experiments with three technical replicates. Statistical significance was calculated using unpaired two-tailed t test. * P
    Cut safely and reduce operator fatigue with Thermo Scientific Shandon 10000 Autopsy Saw
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    Images

    1) Product Images from "TET enzymes augment activation-induced deaminase (AID) expression via 5-hydroxymethylcytosine modifications at the Aicda superenhancer"

    Article Title: TET enzymes augment activation-induced deaminase (AID) expression via 5-hydroxymethylcytosine modifications at the Aicda superenhancer

    Journal: Science immunology

    doi: 10.1126/sciimmunol.aau7523

    TET proteins facilitate CSR. ( A ) Flowchart of in vivo experiment. f.p., footpad. ( B ) Top: Flow cytometric analysis of CD19 + GL7 + Fas + GC B cells at the draining popliteal lymph nodes from WT and Tet2/3 DKO mice after NP-OVA immunization as in (A). Bottom: Decreased IgG1-switched cells among GC B cells in Tet2/3 DKO (YFP + GC B–gated) compared with WT mice (GC B–gated). ( C to F ) Quantification of experiments shown in (B). Data shown are aggregated results from two independent experiments. Means and SEs are shown. WT, n = 11; DKO, n = 12. ( G ) Flowchart of in vitro IgG1 switching. Cells were labeled with Cell trace violet and activated for 4 days with LPS and IL-4. ( H and I ) Flow cytometry plots (H) and quantification (I) of IgG1-switched B cells in Tet2/3 DKO ( n = 4) and WT ( n = 4) mice. Representative of at least three independent experiments. ( J ) Circular γ1 transcript was quantified by qRT-PCR and normalized to Gapdh and then to the level of WT. Representative experiment of two independent experiments with three technical replicates. ( K ) Flowchart of in vitro IgA switching. Cells were activated for 5 days with anti-CD40, rmIL-4, rmIL-5, and rhTGFβ. ( L to N ) Flow cytometry plots (L) and quantification (M and N) of IgG1-switched (M) and IgA-switched cells (N). Representative of three independent experiments with three technical replicates. Statistical significance was calculated using unpaired two-tailed t test. * P
    Figure Legend Snippet: TET proteins facilitate CSR. ( A ) Flowchart of in vivo experiment. f.p., footpad. ( B ) Top: Flow cytometric analysis of CD19 + GL7 + Fas + GC B cells at the draining popliteal lymph nodes from WT and Tet2/3 DKO mice after NP-OVA immunization as in (A). Bottom: Decreased IgG1-switched cells among GC B cells in Tet2/3 DKO (YFP + GC B–gated) compared with WT mice (GC B–gated). ( C to F ) Quantification of experiments shown in (B). Data shown are aggregated results from two independent experiments. Means and SEs are shown. WT, n = 11; DKO, n = 12. ( G ) Flowchart of in vitro IgG1 switching. Cells were labeled with Cell trace violet and activated for 4 days with LPS and IL-4. ( H and I ) Flow cytometry plots (H) and quantification (I) of IgG1-switched B cells in Tet2/3 DKO ( n = 4) and WT ( n = 4) mice. Representative of at least three independent experiments. ( J ) Circular γ1 transcript was quantified by qRT-PCR and normalized to Gapdh and then to the level of WT. Representative experiment of two independent experiments with three technical replicates. ( K ) Flowchart of in vitro IgA switching. Cells were activated for 5 days with anti-CD40, rmIL-4, rmIL-5, and rhTGFβ. ( L to N ) Flow cytometry plots (L) and quantification (M and N) of IgG1-switched (M) and IgA-switched cells (N). Representative of three independent experiments with three technical replicates. Statistical significance was calculated using unpaired two-tailed t test. * P

    Techniques Used: In Vivo, Flow Cytometry, Mouse Assay, In Vitro, Labeling, Cytometry, Quantitative RT-PCR, Two Tailed Test

    TET2/3 facilitate CSR by regulating expression of the cytidine deaminase AID. ( A ) qRT-PCR analysis of Aicda mRNA expression in WT and Tet2/3 DKO B cells activated 4 days with LPS and IL-4. Aicda expression was normalized to Gapdh and then to the level in WT. Data shown are representative of two independent experiments with three technical replicates. * P
    Figure Legend Snippet: TET2/3 facilitate CSR by regulating expression of the cytidine deaminase AID. ( A ) qRT-PCR analysis of Aicda mRNA expression in WT and Tet2/3 DKO B cells activated 4 days with LPS and IL-4. Aicda expression was normalized to Gapdh and then to the level in WT. Data shown are representative of two independent experiments with three technical replicates. * P

    Techniques Used: Expressing, Quantitative RT-PCR

    2) Product Images from "A comprehensive evaluation of Hippo pathway silencing in sarcomas"

    Article Title: A comprehensive evaluation of Hippo pathway silencing in sarcomas

    Journal: Oncotarget

    doi: 10.18632/oncotarget.25824

    ( A ) Summary of cell lines diagram. Expression of the Hippo kinases was lost at the protein level in 0% (LATS1) to 58% (MST1) of the sarcoma cell lines, indicated by (+). Accumulation of the Hippo kinases with treatment with MG132, indicated by (+), was noted only for MST2, indicating that proteosomal degradation is an important mechanism by which MST2 expression is lost. Loss of expression at the RNA level was identified for MST1 (42%) and MST2 (25%) of sarcoma cell lines. Loss of expression at the RNA level for LATS1 and LATS2 was negligible. Deletions of the Hippo kinases were essentially absent from the sarcoma cell lines with the exception of LATS2 , where 1 of the 12 sarcoma cell lines (8%) demonstrated a deletion. Treatment with 10 μM 5-azacytidine resulted in a modest increase in expression in 8–17% of the sarcoma cell lines. Treatment with 0.5 μM TSA resulted in a reversal of expression in a higher percentage of cell lines, predominantly with MST1 and MST2 . Treatment with TSA and 5-azacytidine showed an additive effect with regards to re-expressing the Hippo kinases in some cell lines. ( B ) Expression of the Hippo kinases is regulated by at least three different mechanisms shown in this model, potentially targetable by different therapeutic interventions. Promoter (CpG island) hypermethylation is one mechanism that appears to modestly regulate the expression of predominantly MST1 and MST2 . Histone deacetylation can also promote silencing the expression of the Hippo kinases, again particularly MST1 and MST2 , and to a lesser degree LATS2 . Proteosomal degradation plays a role in in regulating the expression of MST2 and could be targeted as well.
    Figure Legend Snippet: ( A ) Summary of cell lines diagram. Expression of the Hippo kinases was lost at the protein level in 0% (LATS1) to 58% (MST1) of the sarcoma cell lines, indicated by (+). Accumulation of the Hippo kinases with treatment with MG132, indicated by (+), was noted only for MST2, indicating that proteosomal degradation is an important mechanism by which MST2 expression is lost. Loss of expression at the RNA level was identified for MST1 (42%) and MST2 (25%) of sarcoma cell lines. Loss of expression at the RNA level for LATS1 and LATS2 was negligible. Deletions of the Hippo kinases were essentially absent from the sarcoma cell lines with the exception of LATS2 , where 1 of the 12 sarcoma cell lines (8%) demonstrated a deletion. Treatment with 10 μM 5-azacytidine resulted in a modest increase in expression in 8–17% of the sarcoma cell lines. Treatment with 0.5 μM TSA resulted in a reversal of expression in a higher percentage of cell lines, predominantly with MST1 and MST2 . Treatment with TSA and 5-azacytidine showed an additive effect with regards to re-expressing the Hippo kinases in some cell lines. ( B ) Expression of the Hippo kinases is regulated by at least three different mechanisms shown in this model, potentially targetable by different therapeutic interventions. Promoter (CpG island) hypermethylation is one mechanism that appears to modestly regulate the expression of predominantly MST1 and MST2 . Histone deacetylation can also promote silencing the expression of the Hippo kinases, again particularly MST1 and MST2 , and to a lesser degree LATS2 . Proteosomal degradation plays a role in in regulating the expression of MST2 and could be targeted as well.

    Techniques Used: Expressing

    3) Product Images from "1α,25(OH)2D3-glycosides from Solanum glaucophyllum leaves extract induce myoblasts differentiation through p38 MAPK and AKT activation"

    Article Title: 1α,25(OH)2D3-glycosides from Solanum glaucophyllum leaves extract induce myoblasts differentiation through p38 MAPK and AKT activation

    Journal: Biology Open

    doi: 10.1242/bio.033670

    Genes regulation by SGE or synthetic 1α,25(OH) 2 D 3 during onset of differentiation. C2C12 cells were treated with SGE (10 nM), 1α,25(OH) 2 D 3 (1,25D, 1 nM), or vehicle alone (ctrl) in DM in time course experiments (24–72 h). Total RNA was extracted and reverse transcribed and data analysis was performed for each gene expression by qRT-PCR. Bar graphs show quantitative results from three experiments performed in duplicate and expressed as a ratio between mRNA of each gene (A) myoD1 , (B) myogenin , (C) MHC2b in DM under control conditions (ctrl) or treated (1,25D or SGE) previously normalized to GAPDH mRNA levels and to GM (time 0). The results were then represented in bar graphs. Significant differences between control and treated (SGE or 1,25D) conditions at each time point were analyzed by Student's t -test. ns: not significant. * P
    Figure Legend Snippet: Genes regulation by SGE or synthetic 1α,25(OH) 2 D 3 during onset of differentiation. C2C12 cells were treated with SGE (10 nM), 1α,25(OH) 2 D 3 (1,25D, 1 nM), or vehicle alone (ctrl) in DM in time course experiments (24–72 h). Total RNA was extracted and reverse transcribed and data analysis was performed for each gene expression by qRT-PCR. Bar graphs show quantitative results from three experiments performed in duplicate and expressed as a ratio between mRNA of each gene (A) myoD1 , (B) myogenin , (C) MHC2b in DM under control conditions (ctrl) or treated (1,25D or SGE) previously normalized to GAPDH mRNA levels and to GM (time 0). The results were then represented in bar graphs. Significant differences between control and treated (SGE or 1,25D) conditions at each time point were analyzed by Student's t -test. ns: not significant. * P

    Techniques Used: Expressing, Quantitative RT-PCR

    Related Articles

    Cell Culture:

    Article Title: Bioprinting of stem cell expansion lattices
    Article Snippet: NPCs were cultured following a previously described protocol [ ]. .. Briefly, NPCs were cultured in Stemness Maintenance Medium (Neurobasal-A, 2% B27 Supplement with Vitamin A (Gibco), GlutaMAX (Gibco), 20 ng ml−1 FGF-2 (PeproTech), 20 ng ml−1 EGF (PeproTech), and 1% Pen/Strep (Gibco)) on tissue culture plastic coated with 10 μg mL−1 polyornithine (Sigma-Aldrich) and 5 mg mL−1 laminin (Gibco) at a seeding density of 10,000 NPCs cm−2 . ..

    Mutagenesis:

    Article Title: Specific bone cells produce DLL4 to generate thymus-seeding progenitors from bone marrow
    Article Snippet: .. 35,000 or 10,000 Ly6D- CLPs (Lin− Sca+ cKit+ IL7R+ Thy1.2− Ly6D− ) were sorted from OcnCre;iDTR control and mutant donors (CD45.2-Pacific Blue), labeled with 10 µM CFDA-SE (Invitrogen) and 1 µg/ml VivoTag680 (VT680, VisEn Medical), respectively, mixed in a 1:1 ratio, and transplanted into each congenic SJL recipient (CD45.1-PE) by retro-orbital injection. .. The same number of progenitor cells sorted from CCR7−/− mice served as another control to compete with OcnCre;iDTR mutant donors.

    Labeling:

    Article Title: Specific bone cells produce DLL4 to generate thymus-seeding progenitors from bone marrow
    Article Snippet: .. 35,000 or 10,000 Ly6D- CLPs (Lin− Sca+ cKit+ IL7R+ Thy1.2− Ly6D− ) were sorted from OcnCre;iDTR control and mutant donors (CD45.2-Pacific Blue), labeled with 10 µM CFDA-SE (Invitrogen) and 1 µg/ml VivoTag680 (VT680, VisEn Medical), respectively, mixed in a 1:1 ratio, and transplanted into each congenic SJL recipient (CD45.1-PE) by retro-orbital injection. .. The same number of progenitor cells sorted from CCR7−/− mice served as another control to compete with OcnCre;iDTR mutant donors.

    Injection:

    Article Title: Specific bone cells produce DLL4 to generate thymus-seeding progenitors from bone marrow
    Article Snippet: .. 35,000 or 10,000 Ly6D- CLPs (Lin− Sca+ cKit+ IL7R+ Thy1.2− Ly6D− ) were sorted from OcnCre;iDTR control and mutant donors (CD45.2-Pacific Blue), labeled with 10 µM CFDA-SE (Invitrogen) and 1 µg/ml VivoTag680 (VT680, VisEn Medical), respectively, mixed in a 1:1 ratio, and transplanted into each congenic SJL recipient (CD45.1-PE) by retro-orbital injection. .. The same number of progenitor cells sorted from CCR7−/− mice served as another control to compete with OcnCre;iDTR mutant donors.

    Transfection:

    Article Title: Novel lincRNA SLINKY is a prognostic biomarker in kidney cancer
    Article Snippet: .. siRNA transfections and proliferation assays ccRCC cells were seeded (10,000 cells per well) in 6-well plates and transfected using Lipofectamine 2000 (Life Technologies). .. Two independent small interfering (si)RNAs targeting the first exon of SLINKY (On-TARGETplus, Dharmacon) were transfected at a final siRNA concentration of 50nM for 16 hrs (custom-designed siRNA sequences available in ).

    Multiple Displacement Amplification:

    Article Title: Molecular Imaging of the Translocator Protein (TSPO) in a Pre-Clinical Model of Breast Cancer
    Article Snippet: A Nikon Eclipse TE2000-U fluorescence microscope equipped with a mercury lamp, indocyanine green filter set and a Hamamatsu ORCA II BT 512 camera controlled by Metamorph v6.1 (Molecular Devices Corporation; Downingtown, PA) was used for imaging. .. MDA-MB-231 cells were plated at 10,000 cells per well in parafilm-wrapped 96 MicroWell™ Nunclon™Δ Optical Bottom Plates (Nalge Nunc International; Rochester, NY) and incubated under standard culture conditions for approximately 48 h. Immediately prior to experimentation, the cells were washed once with 37°C FBS-free medium to remove any dead cells and serum. .. The cells were then divided into four populations and evaluated in triplicate: (1) cells incubated with increasing concentrations of NIR-conPK11195 (1, 4, 7, 10, 40, 70, 100, 400, 700 nM, 1 μM) in FBS-free medium (100 μL volume), (2) cells simultaneously incubated with the same concentrations of NIR-conPK11195 and 100 µM PK 11195, (3) cells incubated with the same concentrations of free NIR dye, and (4) undosed cells as blanks.

    Incubation:

    Article Title: Molecular Imaging of the Translocator Protein (TSPO) in a Pre-Clinical Model of Breast Cancer
    Article Snippet: A Nikon Eclipse TE2000-U fluorescence microscope equipped with a mercury lamp, indocyanine green filter set and a Hamamatsu ORCA II BT 512 camera controlled by Metamorph v6.1 (Molecular Devices Corporation; Downingtown, PA) was used for imaging. .. MDA-MB-231 cells were plated at 10,000 cells per well in parafilm-wrapped 96 MicroWell™ Nunclon™Δ Optical Bottom Plates (Nalge Nunc International; Rochester, NY) and incubated under standard culture conditions for approximately 48 h. Immediately prior to experimentation, the cells were washed once with 37°C FBS-free medium to remove any dead cells and serum. .. The cells were then divided into four populations and evaluated in triplicate: (1) cells incubated with increasing concentrations of NIR-conPK11195 (1, 4, 7, 10, 40, 70, 100, 400, 700 nM, 1 μM) in FBS-free medium (100 μL volume), (2) cells simultaneously incubated with the same concentrations of NIR-conPK11195 and 100 µM PK 11195, (3) cells incubated with the same concentrations of free NIR dye, and (4) undosed cells as blanks.

    Real-time Polymerase Chain Reaction:

    Article Title: Seven Novel Probe Systems for Real-Time PCR Provide Absolute Single-Base Discrimination, Higher Signaling, and Generic Components
    Article Snippet: In Universal probe assays, the concentration of linker primer used in the first-step reaction was 20 nmol/L, and Universal probe and antiprobe concentrations were 200 and 400 nmol/L, respectively. .. We typically used 10 ng of viral cDNA or 1000 to 10,000 copies of ultramer template per reaction. qPCR reactions were performed in 25-μL volumes containing 12.5 μL of 2X HotStart-IT Probe qPCR Master Mix (Affymetrix, Inc., Santa Clara, CA), the indicated template, primer, probe, and/or antiprobe concentrations, and additional MgCl2 and dNTPs (added at final concentrations of 5 and 0.1 mmol/L, respectively). .. Typical thermal cycling conditions are 95°C, 5 minutes, and then 40 cycles of either two-step PCR (denaturation at 95°C for 15 seconds, and annealing/extension at 58°C for 30 to 60 seconds), or three-step PCR (denaturation at 95°C, 15 seconds; annealing at 58°C, 45 seconds; and extension at 72°C, 45 seconds).

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    Thermo Fisher superscript iii reverse transcriptase
    The c848+1G > A Intronic Mutation Resulted in Aberrant Splicing of FARSB (A) The mutation of the universal splice site motif is expected to affect the splicing of nearby exons in the FARSB gene, such as skipping of the adjacent exon 9. (B) A splice variant that deleted exon 9 of the FARSB _FL transcript, designated as FARSB -ΔE9, was detected in the proband but not in the mother. (C) Quantitative real-time PCR analysis of mRNA expressions of FARSB _E9-11 (using primers targeting exons 9 and 11 of FARSB , thus primarily detecting the full-length transcript), and FARSB -ΔE9 (using primers specifically amplifying the ΔE9 variant) in primary fibroblasts. Gene expression of FARSB _E9-11 and -ΔE9 in the fibroblasts of the proband, both parents, and <t>three</t> control subjects were calculated based on Ct values normalized to house-keeping genes RPL9 and RPS11 . The fold changes were thus calculated by relative to the FARSB _E9-11 level of CTL-17M. Data were presented as mean ± SEM. The ΔE9 <t>RNA</t> was detected only in the proband and father cells but not in the mother and control subjects (U.D. denotes no detectable amplification within 45 qPCR cycles).
    Superscript Iii Reverse Transcriptase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/superscript iii reverse transcriptase/product/Thermo Fisher
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    The c848+1G > A Intronic Mutation Resulted in Aberrant Splicing of FARSB (A) The mutation of the universal splice site motif is expected to affect the splicing of nearby exons in the FARSB gene, such as skipping of the adjacent exon 9. (B) A splice variant that deleted exon 9 of the FARSB _FL transcript, designated as FARSB -ΔE9, was detected in the proband but not in the mother. (C) Quantitative real-time PCR analysis of mRNA expressions of FARSB _E9-11 (using primers targeting exons 9 and 11 of FARSB , thus primarily detecting the full-length transcript), and FARSB -ΔE9 (using primers specifically amplifying the ΔE9 variant) in primary fibroblasts. Gene expression of FARSB _E9-11 and -ΔE9 in the fibroblasts of the proband, both parents, and three control subjects were calculated based on Ct values normalized to house-keeping genes RPL9 and RPS11 . The fold changes were thus calculated by relative to the FARSB _E9-11 level of CTL-17M. Data were presented as mean ± SEM. The ΔE9 RNA was detected only in the proband and father cells but not in the mother and control subjects (U.D. denotes no detectable amplification within 45 qPCR cycles).

    Journal: American Journal of Human Genetics

    Article Title: Bi-allelic Mutations in Phe-tRNA Synthetase Associated with a Multi-system Pulmonary Disease Support Non-translational Function

    doi: 10.1016/j.ajhg.2018.06.006

    Figure Lengend Snippet: The c848+1G > A Intronic Mutation Resulted in Aberrant Splicing of FARSB (A) The mutation of the universal splice site motif is expected to affect the splicing of nearby exons in the FARSB gene, such as skipping of the adjacent exon 9. (B) A splice variant that deleted exon 9 of the FARSB _FL transcript, designated as FARSB -ΔE9, was detected in the proband but not in the mother. (C) Quantitative real-time PCR analysis of mRNA expressions of FARSB _E9-11 (using primers targeting exons 9 and 11 of FARSB , thus primarily detecting the full-length transcript), and FARSB -ΔE9 (using primers specifically amplifying the ΔE9 variant) in primary fibroblasts. Gene expression of FARSB _E9-11 and -ΔE9 in the fibroblasts of the proband, both parents, and three control subjects were calculated based on Ct values normalized to house-keeping genes RPL9 and RPS11 . The fold changes were thus calculated by relative to the FARSB _E9-11 level of CTL-17M. Data were presented as mean ± SEM. The ΔE9 RNA was detected only in the proband and father cells but not in the mother and control subjects (U.D. denotes no detectable amplification within 45 qPCR cycles).

    Article Snippet: For gene expression assessment by quantitative real-time PCR, messenger RNA was captured by oligo(dT)18 primer and reverse-transcribed into cDNA using SuperScript III Reverse-Transcriptase (Life Technologies).

    Techniques: Mutagenesis, Variant Assay, Real-time Polymerase Chain Reaction, Expressing, CTL Assay, Amplification

    FOXA2-DS-S regulates FOXA2 expression. a ] showing the FOXA2 locus with tracks displaying coverage data for ChIP-Seq experiments for Pol2, FOXA1, FOXA2, HNF4A, HNF6 and CEBPA. The ChIP-Seq tracks were produced by the ENCODE project on HepG2 cells. b Real time PCR data showing the expression of FOXA2 and FOXA2-DS-S in Huh7 cells upon knock-down. Si1 and si2 FOXA2-DS-S indicate two different, non-overlapping siRNAs designed against FOXA2-DS-S . The data are expressed relative to the expression of the control transfected with scrambled siRNAs; the error bars indicate the standard error of the mean across three replicate experiments. c Venn diagram showing the number of significantly differentially expressed genes (adjusted p value

    Journal: Genome Biology

    Article Title: Genomic positional conservation identifies topological anchor point RNAs linked to developmental loci

    doi: 10.1186/s13059-018-1405-5

    Figure Lengend Snippet: FOXA2-DS-S regulates FOXA2 expression. a ] showing the FOXA2 locus with tracks displaying coverage data for ChIP-Seq experiments for Pol2, FOXA1, FOXA2, HNF4A, HNF6 and CEBPA. The ChIP-Seq tracks were produced by the ENCODE project on HepG2 cells. b Real time PCR data showing the expression of FOXA2 and FOXA2-DS-S in Huh7 cells upon knock-down. Si1 and si2 FOXA2-DS-S indicate two different, non-overlapping siRNAs designed against FOXA2-DS-S . The data are expressed relative to the expression of the control transfected with scrambled siRNAs; the error bars indicate the standard error of the mean across three replicate experiments. c Venn diagram showing the number of significantly differentially expressed genes (adjusted p value

    Article Snippet: Briefly, 2 μg total RNA from HepG2 cells were reverse transcribed in 20 μl reaction using Superscript III Reverse Transcriptase (Invitrogen, catalogue number 18080044).

    Techniques: Expressing, Chromatin Immunoprecipitation, Produced, Real-time Polymerase Chain Reaction, Transfection

    CKII Phosphorylation of Spt6 Regulates Chromatin Integrity during Transcription (A) Schematic of the SRG1 and SER3 loci showing their expression patterns in WT and mutant strains, as indicated by red (WT) and green (mutant) arrows. (B) Representative RNA-seq tracks showing an increase in the expression level of the SER3 gene in WT, spt6 S8 → A8 , spt6 S8 → E8 , and spt6–1004 allele. (C) Quantitative real-time PCR detection of SRG1 and SER3 transcripts in the WT and spt6–1004 mutant strain. (D) Quantitative real-time PCR detection of SRG1 and SER3 transcripts in the WT and spt6 mutant ( spt6 S8 → A8 and spt6 S8 → E8 ) strains. (E) ChIP analysis of histone H3 levels across SRG1 and SER3 was performed with WT and spt6 mutant ( spt6 S8 → A8 and spt6 S8 → E8 ) strains. Amplicons are indicated below the schematic diagram of the genes. Quantitative real-time PCR and ChIP data are represented as means ± SDs of three independent biological experiments. Asterisks indicate significance values (**p

    Journal: Cell reports

    Article Title: Casein Kinase II Phosphorylation of Spt6 Enforces Transcriptional Fidelity by Maintaining Spn1-Spt6 Interaction

    doi: 10.1016/j.celrep.2018.11.089

    Figure Lengend Snippet: CKII Phosphorylation of Spt6 Regulates Chromatin Integrity during Transcription (A) Schematic of the SRG1 and SER3 loci showing their expression patterns in WT and mutant strains, as indicated by red (WT) and green (mutant) arrows. (B) Representative RNA-seq tracks showing an increase in the expression level of the SER3 gene in WT, spt6 S8 → A8 , spt6 S8 → E8 , and spt6–1004 allele. (C) Quantitative real-time PCR detection of SRG1 and SER3 transcripts in the WT and spt6–1004 mutant strain. (D) Quantitative real-time PCR detection of SRG1 and SER3 transcripts in the WT and spt6 mutant ( spt6 S8 → A8 and spt6 S8 → E8 ) strains. (E) ChIP analysis of histone H3 levels across SRG1 and SER3 was performed with WT and spt6 mutant ( spt6 S8 → A8 and spt6 S8 → E8 ) strains. Amplicons are indicated below the schematic diagram of the genes. Quantitative real-time PCR and ChIP data are represented as means ± SDs of three independent biological experiments. Asterisks indicate significance values (**p

    Article Snippet: The isolated RNA was treated with 10 U of RNase-free DNase (Promega) for 30 minutes, followed by RNA cleanup (QIAGEN RNeasy Mini Kit, 74106). cDNA was synthesized from one mg of total RNA using random hexamer primers and Superscript Reverse Transcriptase III (Thermo-Fisher Scientific, 108–80044).

    Techniques: Expressing, Mutagenesis, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation

    Replication analysis of HCV JFH1-AM120-LacZ followed RNA transfection. (A) Huh7.5 cells were transfected with the RNA of JFH1-AM120-LacZ and controls of JFH1-AM120, JFH1-AM120-EGFP and JFH1-AM120-Rluc. Three days post-transfection, the cells were lysed for Western blotting using anti-NS5A and anti-β-actin antibodies as described in Methods. Western blot analyses were carried out two times and representative examples are shown. (B) Three days after transfection of Huh7.5 cells in six-well plates with RNAs of JFH-AM120-LacZ and JFH-AM120, JFH-AM120-EGFP and JFH-AM120-Rluc, the total RNA was extracted from cells and qPCR analysis of HCV RNA was performed. Experiments were performed three times and the data presented as the mean ± SD (* P

    Journal: International Journal of Biological Sciences

    Article Title: Adapted HCV JFH1 variant is capable of accommodating a large foreign gene insert and allows lower level HCV replication and viral production

    doi: 10.7150/ijbs.27411

    Figure Lengend Snippet: Replication analysis of HCV JFH1-AM120-LacZ followed RNA transfection. (A) Huh7.5 cells were transfected with the RNA of JFH1-AM120-LacZ and controls of JFH1-AM120, JFH1-AM120-EGFP and JFH1-AM120-Rluc. Three days post-transfection, the cells were lysed for Western blotting using anti-NS5A and anti-β-actin antibodies as described in Methods. Western blot analyses were carried out two times and representative examples are shown. (B) Three days after transfection of Huh7.5 cells in six-well plates with RNAs of JFH-AM120-LacZ and JFH-AM120, JFH-AM120-EGFP and JFH-AM120-Rluc, the total RNA was extracted from cells and qPCR analysis of HCV RNA was performed. Experiments were performed three times and the data presented as the mean ± SD (* P

    Article Snippet: The RNA was reverse transcribed using Superscript III Reverse Transcriptase (Invitrogen) and random primers.

    Techniques: Transfection, Western Blot, Real-time Polymerase Chain Reaction

    Kinetics assay of the JFH1-AM120-LacZ HCV reporter after multiple passages. The cells transfected with the RNA of JFH1-AM120-LacZ were passaged at every three days for a total of 15 days. Supernatants collected were designated P1 to P5. The double titrations were carried out by X-gal staining for β-galactosidase and immunofluoresence staining for NS5A protein (see Materials and Methods). Experiments were performed three times and the data presented as the mean ± SD.

    Journal: International Journal of Biological Sciences

    Article Title: Adapted HCV JFH1 variant is capable of accommodating a large foreign gene insert and allows lower level HCV replication and viral production

    doi: 10.7150/ijbs.27411

    Figure Lengend Snippet: Kinetics assay of the JFH1-AM120-LacZ HCV reporter after multiple passages. The cells transfected with the RNA of JFH1-AM120-LacZ were passaged at every three days for a total of 15 days. Supernatants collected were designated P1 to P5. The double titrations were carried out by X-gal staining for β-galactosidase and immunofluoresence staining for NS5A protein (see Materials and Methods). Experiments were performed three times and the data presented as the mean ± SD.

    Article Snippet: The RNA was reverse transcribed using Superscript III Reverse Transcriptase (Invitrogen) and random primers.

    Techniques: Transfection, Staining

    Analysis of NS5A-LacZ activity followed RNA transfection of JFH1-AM120-LacZ . (A) Huh7.5 cells were transfected with the RNA of JFH1-AM120-LacZ and controls of JFH1-AM120, JFH1-AM120-EGFP and JFH1-AM120-Rluc as described in Methods. Three days post-transfection , the absorption peaks of β-Galactosidase activity were measured using a Bio-Tec plate reader at 405 nm. The values were relative to JFH1-AM120-LacZ. Experiments were performed three times and the data presented as the mean ± SD. (B) Three days post-transfection of Huh7.5 cells in 24-well plates with RNA of JFH1-AM120-LacZ, cells were fixed with 4% paraformaldehyde followed X-gal staining. Cover slips were visualized and images were taken (100x) by bright field microscopy. Experiments were performed two times and representative results are shown. (C) Co-localization of NS5A and β-galactosidase. Three days post-transfection of Huh7.5 cells in 24-well plates with RNA of JFH1-AM120-LacZ, Cells were fixed with 4% paraformaldehyde and were immunostained with anti-NS5A antibody (red) and Nuclei were counterstained using DAPI (blue). Images were taken by immunofluorescence microscopy (200x). Then the coverslip was washed and were processed with X-gal staining followed visualizing and imaging by bright field microscopy (200x). Colocalization assay was performed by Photoshop CC software. Experiments were performed three times and representative results are shown.

    Journal: International Journal of Biological Sciences

    Article Title: Adapted HCV JFH1 variant is capable of accommodating a large foreign gene insert and allows lower level HCV replication and viral production

    doi: 10.7150/ijbs.27411

    Figure Lengend Snippet: Analysis of NS5A-LacZ activity followed RNA transfection of JFH1-AM120-LacZ . (A) Huh7.5 cells were transfected with the RNA of JFH1-AM120-LacZ and controls of JFH1-AM120, JFH1-AM120-EGFP and JFH1-AM120-Rluc as described in Methods. Three days post-transfection , the absorption peaks of β-Galactosidase activity were measured using a Bio-Tec plate reader at 405 nm. The values were relative to JFH1-AM120-LacZ. Experiments were performed three times and the data presented as the mean ± SD. (B) Three days post-transfection of Huh7.5 cells in 24-well plates with RNA of JFH1-AM120-LacZ, cells were fixed with 4% paraformaldehyde followed X-gal staining. Cover slips were visualized and images were taken (100x) by bright field microscopy. Experiments were performed two times and representative results are shown. (C) Co-localization of NS5A and β-galactosidase. Three days post-transfection of Huh7.5 cells in 24-well plates with RNA of JFH1-AM120-LacZ, Cells were fixed with 4% paraformaldehyde and were immunostained with anti-NS5A antibody (red) and Nuclei were counterstained using DAPI (blue). Images were taken by immunofluorescence microscopy (200x). Then the coverslip was washed and were processed with X-gal staining followed visualizing and imaging by bright field microscopy (200x). Colocalization assay was performed by Photoshop CC software. Experiments were performed three times and representative results are shown.

    Article Snippet: The RNA was reverse transcribed using Superscript III Reverse Transcriptase (Invitrogen) and random primers.

    Techniques: Activity Assay, Transfection, Staining, Microscopy, Immunofluorescence, Imaging, Software

    Infectivity assay of virus particles followed RNAs transfection of JFH1-AM120-LacZ and controls . The RNA of JFH1-AM120-LacZ and controls of JFH1-AM120, JFH1-AM120-EGFP and JFH1-AM120-Rluc were electroporated into Huh-7.5 and the infectivity titers in the cultured supernatants at the 6th day were measured (Described in Material and Method). The viral titer is expressed as focus-forming units per ml of supernatant (ffu/ml) as determined by the average number of NS5A-positive foci detected by immunofluorescence for NS5A (Mock, JFH-AM120 and JFH-AM120-Rluc), or directly visualized EGFP positive cells(JFH1-AM120-EGFP) and detected blue color cells after X-gal staining (JFH1-AM120-LacZ). Assays were performed three times and the data are presented as mean ± standard deviation. (** P

    Journal: International Journal of Biological Sciences

    Article Title: Adapted HCV JFH1 variant is capable of accommodating a large foreign gene insert and allows lower level HCV replication and viral production

    doi: 10.7150/ijbs.27411

    Figure Lengend Snippet: Infectivity assay of virus particles followed RNAs transfection of JFH1-AM120-LacZ and controls . The RNA of JFH1-AM120-LacZ and controls of JFH1-AM120, JFH1-AM120-EGFP and JFH1-AM120-Rluc were electroporated into Huh-7.5 and the infectivity titers in the cultured supernatants at the 6th day were measured (Described in Material and Method). The viral titer is expressed as focus-forming units per ml of supernatant (ffu/ml) as determined by the average number of NS5A-positive foci detected by immunofluorescence for NS5A (Mock, JFH-AM120 and JFH-AM120-Rluc), or directly visualized EGFP positive cells(JFH1-AM120-EGFP) and detected blue color cells after X-gal staining (JFH1-AM120-LacZ). Assays were performed three times and the data are presented as mean ± standard deviation. (** P

    Article Snippet: The RNA was reverse transcribed using Superscript III Reverse Transcriptase (Invitrogen) and random primers.

    Techniques: Infection, Transfection, Cell Culture, Immunofluorescence, Staining, Standard Deviation