superscriptiii transcriptase Thermo Fisher Search Results


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  • 99
    Thermo Fisher superscriptiii reverse transcriptase
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    Single HV + Cells in 2i Can Contribute to Both Epiblast and Extraembryonic Lineages Single HV − or HV + cells from serum/LIF or 2i were sorted by FACS and analyzed by <t>qRT-PCR</t> using the Biomark HD system (Fluidigm). (A) Heat map for each condition and cell type showing the expression of genes with reliable primer melting curves. Adjacent squares show technical replicates for each cell. (B) Single cells were sorted by flow cytometry into LIF withdrawal conditions. After 7 days, differentiated colonies were immunostained for GATA6, CDX2, and BRACHYURY. (C) Single-sorted HV + cells (sorted from the top 10% of HV expression), previously cultured in 2i/LIF, constitutively expressing H2B-Tomato were injected into morulae and dissected at E6.5 and chimeras (n = 23/64) were assessed for lineage contribution. Examples of different contribution patterns of single cells are shown. Some cells showed even contribution across the whole epiblast while others showed a contribution that was biased toward a particular area of the epiblast. Red arrowheads indicate trophoblast contribution, green arrowheads indicate visceral endoderm contribution, and black arrowheads indicate parietal endoderm contribution. Epiblast contribution alone was observed in nine chimeras, extraembryonic contribution alone in one chimera, and 13 chimeras showed both epiblast and extraembryonic contribution. See also Figure S5 .
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    Image Search Results


    Single HV + Cells in 2i Can Contribute to Both Epiblast and Extraembryonic Lineages Single HV − or HV + cells from serum/LIF or 2i were sorted by FACS and analyzed by qRT-PCR using the Biomark HD system (Fluidigm). (A) Heat map for each condition and cell type showing the expression of genes with reliable primer melting curves. Adjacent squares show technical replicates for each cell. (B) Single cells were sorted by flow cytometry into LIF withdrawal conditions. After 7 days, differentiated colonies were immunostained for GATA6, CDX2, and BRACHYURY. (C) Single-sorted HV + cells (sorted from the top 10% of HV expression), previously cultured in 2i/LIF, constitutively expressing H2B-Tomato were injected into morulae and dissected at E6.5 and chimeras (n = 23/64) were assessed for lineage contribution. Examples of different contribution patterns of single cells are shown. Some cells showed even contribution across the whole epiblast while others showed a contribution that was biased toward a particular area of the epiblast. Red arrowheads indicate trophoblast contribution, green arrowheads indicate visceral endoderm contribution, and black arrowheads indicate parietal endoderm contribution. Epiblast contribution alone was observed in nine chimeras, extraembryonic contribution alone in one chimera, and 13 chimeras showed both epiblast and extraembryonic contribution. See also Figure S5 .

    Journal: Cell Reports

    Article Title: Totipotent Embryonic Stem Cells Arise in Ground-State Culture Conditions

    doi: 10.1016/j.celrep.2013.04.034

    Figure Lengend Snippet: Single HV + Cells in 2i Can Contribute to Both Epiblast and Extraembryonic Lineages Single HV − or HV + cells from serum/LIF or 2i were sorted by FACS and analyzed by qRT-PCR using the Biomark HD system (Fluidigm). (A) Heat map for each condition and cell type showing the expression of genes with reliable primer melting curves. Adjacent squares show technical replicates for each cell. (B) Single cells were sorted by flow cytometry into LIF withdrawal conditions. After 7 days, differentiated colonies were immunostained for GATA6, CDX2, and BRACHYURY. (C) Single-sorted HV + cells (sorted from the top 10% of HV expression), previously cultured in 2i/LIF, constitutively expressing H2B-Tomato were injected into morulae and dissected at E6.5 and chimeras (n = 23/64) were assessed for lineage contribution. Examples of different contribution patterns of single cells are shown. Some cells showed even contribution across the whole epiblast while others showed a contribution that was biased toward a particular area of the epiblast. Red arrowheads indicate trophoblast contribution, green arrowheads indicate visceral endoderm contribution, and black arrowheads indicate parietal endoderm contribution. Epiblast contribution alone was observed in nine chimeras, extraembryonic contribution alone in one chimera, and 13 chimeras showed both epiblast and extraembryonic contribution. See also Figure S5 .

    Article Snippet: Single-Cell qRT-PCR Single cells were sorted into 96-well PCR plates containing 5 μl CellsDirect reaction mix, 0.2 μl SuperScriptIII/Platinum Taq mix (CellsDirect One-Step qRT-PCR kit, Invitrogen), 2.8 μl DNA suspension buffer (TEKnova), and 1 μl 500 nM primer mix containing a mix of 48 DELTAgene Assays (Fluidigm) (sequences in ).

    Techniques: FACS, Quantitative RT-PCR, Expressing, Flow Cytometry, Cytometry, Cell Culture, Injection

    LIF Supports an Extraembryonically Primed Population of Cells (A) Flow cytometry of HV or TNGB Nanog -GFP cells cultured in serum or 2i medium, with increasing LIF concentrations (n = 3). Raw data and controls are in Figures S6 A–S6C. Error bars indicate mean ± SD of two biological replicates. (B) qRT-PCR showing relative gene expression of extraembryonic markers in ESCs cultured in 2i or 2i/LIF. Data were normalized to the housekeeping gene TBP and are shown relative to 2i samples. Error bars indicate mean ± SD of biological replicates. (C) Confocal optical section showing immunostaining of HV cells for NANOG after culture of cells in serum/LIF, 2i, or 2i/LIF. (D) qRT-PCR showing relative gene expression in Stat3 +/+ or −/− ESCs. Data are shown relative to wild-type expression levels. All qRT-PCR transcript levels in this figure were normalized to TBP and are shown as mean ± SD. (E) Statistical significance of correlation of gene expression differences between ESCs cultured in the presence and absence of LIF and tissue-specific gene expression in the GNF Ver.3 database. Correlation was estimated from data on 5,085 genes, which represent the overlap between 10,000 most informative genes in our RNA-seq data and 10,000 most informative genes in the GNF database. Organs/tissues sorted by decreasing correlation. (F) Confocal optical section of HV cells immunostained for the proliferation marker KI67. White arrowheads indicate KI67-negative cells. (G) Quantification of KI67 immunostaining in HV + and HV − ESCs. The absolute number of proliferating cells (expressing KI67, red bar) and quiescent cells (not expressing KI67, black bar) were counted. Error bars indicate SD of the mean from five colonies in each condition, analyzed by confocal microscopy. See also Figure S5 .

    Journal: Cell Reports

    Article Title: Totipotent Embryonic Stem Cells Arise in Ground-State Culture Conditions

    doi: 10.1016/j.celrep.2013.04.034

    Figure Lengend Snippet: LIF Supports an Extraembryonically Primed Population of Cells (A) Flow cytometry of HV or TNGB Nanog -GFP cells cultured in serum or 2i medium, with increasing LIF concentrations (n = 3). Raw data and controls are in Figures S6 A–S6C. Error bars indicate mean ± SD of two biological replicates. (B) qRT-PCR showing relative gene expression of extraembryonic markers in ESCs cultured in 2i or 2i/LIF. Data were normalized to the housekeeping gene TBP and are shown relative to 2i samples. Error bars indicate mean ± SD of biological replicates. (C) Confocal optical section showing immunostaining of HV cells for NANOG after culture of cells in serum/LIF, 2i, or 2i/LIF. (D) qRT-PCR showing relative gene expression in Stat3 +/+ or −/− ESCs. Data are shown relative to wild-type expression levels. All qRT-PCR transcript levels in this figure were normalized to TBP and are shown as mean ± SD. (E) Statistical significance of correlation of gene expression differences between ESCs cultured in the presence and absence of LIF and tissue-specific gene expression in the GNF Ver.3 database. Correlation was estimated from data on 5,085 genes, which represent the overlap between 10,000 most informative genes in our RNA-seq data and 10,000 most informative genes in the GNF database. Organs/tissues sorted by decreasing correlation. (F) Confocal optical section of HV cells immunostained for the proliferation marker KI67. White arrowheads indicate KI67-negative cells. (G) Quantification of KI67 immunostaining in HV + and HV − ESCs. The absolute number of proliferating cells (expressing KI67, red bar) and quiescent cells (not expressing KI67, black bar) were counted. Error bars indicate SD of the mean from five colonies in each condition, analyzed by confocal microscopy. See also Figure S5 .

    Article Snippet: Single-Cell qRT-PCR Single cells were sorted into 96-well PCR plates containing 5 μl CellsDirect reaction mix, 0.2 μl SuperScriptIII/Platinum Taq mix (CellsDirect One-Step qRT-PCR kit, Invitrogen), 2.8 μl DNA suspension buffer (TEKnova), and 1 μl 500 nM primer mix containing a mix of 48 DELTAgene Assays (Fluidigm) (sequences in ).

    Techniques: Flow Cytometry, Cytometry, Cell Culture, Quantitative RT-PCR, Expressing, Immunostaining, RNA Sequencing Assay, Marker, Confocal Microscopy

    Single HV + Cells Are Totipotent in the Ground State, Related to Figure 4 Single cell qRT-PCR was carried out on a BioMark HD System (Fluidigm) using a 96 × 96 chip. DELTAgene Assay custom designed primers (Fluidigm) were used for amplification. Technical replicates were generated for each single cell. (A) Histograms showing the distribution of expression of 4 genes in each population. Data shown unnormalized. (B) Single cells were sorted by flow cytometry into LIF withdrawal conditions. After 7 days differentiated colonies were immunostained for GATA6, CDX2 and BRACHYURY. Quantification of marker expression patterns. 2 and 3 marker exclusive expression indicates colonies that contain independent cells positive for different lineage markers. (C) Whole-mount immunostaining of an E6.5 chimeric embryo generated by injection of a single HV+ ES cell constitutively expressed H2B-Tomato, previously cultured in 2i/LIF, into a morula stage wild-type embryo. Upper whole-mount image shows an extended focus view of the entire embryo. Panels below show optical sections, generated by confocal microscopy, with ES cells contributing to trophoblast and visceral and parietal endoderm (VE and PE, respectively). (D) E6.5 chimeras generated by injection of a single HV- cell that had been cultured in 2i/LIF. The majority of single cells contributed only to epiblast (11/13), with only 2 showing extraembryonic contribution.

    Journal: Cell Reports

    Article Title: Totipotent Embryonic Stem Cells Arise in Ground-State Culture Conditions

    doi: 10.1016/j.celrep.2013.04.034

    Figure Lengend Snippet: Single HV + Cells Are Totipotent in the Ground State, Related to Figure 4 Single cell qRT-PCR was carried out on a BioMark HD System (Fluidigm) using a 96 × 96 chip. DELTAgene Assay custom designed primers (Fluidigm) were used for amplification. Technical replicates were generated for each single cell. (A) Histograms showing the distribution of expression of 4 genes in each population. Data shown unnormalized. (B) Single cells were sorted by flow cytometry into LIF withdrawal conditions. After 7 days differentiated colonies were immunostained for GATA6, CDX2 and BRACHYURY. Quantification of marker expression patterns. 2 and 3 marker exclusive expression indicates colonies that contain independent cells positive for different lineage markers. (C) Whole-mount immunostaining of an E6.5 chimeric embryo generated by injection of a single HV+ ES cell constitutively expressed H2B-Tomato, previously cultured in 2i/LIF, into a morula stage wild-type embryo. Upper whole-mount image shows an extended focus view of the entire embryo. Panels below show optical sections, generated by confocal microscopy, with ES cells contributing to trophoblast and visceral and parietal endoderm (VE and PE, respectively). (D) E6.5 chimeras generated by injection of a single HV- cell that had been cultured in 2i/LIF. The majority of single cells contributed only to epiblast (11/13), with only 2 showing extraembryonic contribution.

    Article Snippet: Single-Cell qRT-PCR Single cells were sorted into 96-well PCR plates containing 5 μl CellsDirect reaction mix, 0.2 μl SuperScriptIII/Platinum Taq mix (CellsDirect One-Step qRT-PCR kit, Invitrogen), 2.8 μl DNA suspension buffer (TEKnova), and 1 μl 500 nM primer mix containing a mix of 48 DELTAgene Assays (Fluidigm) (sequences in ).

    Techniques: Quantitative RT-PCR, Chromatin Immunoprecipitation, Amplification, Generated, Expressing, Flow Cytometry, Cytometry, Marker, Immunostaining, Injection, Cell Culture, Confocal Microscopy

    Transcriptomics Suggest the Presence of a Population Primed toward Trophoblast as well as Endoderm, Related to Figure 2 (A–D) Common RNA-seq signatures in specific gene classes. The behavior of the individual genes belonging to the classes described in the text is shown. Plots compare mean log intensity values for individual genes among four sorted populations; serum/LIF HV-, serum/LIF HV+, 2i/LIF HV-, 2i/LIF HV+. Error bars represent s.d. between 2 biological replicates. (A) Markers of undifferentiated ESCs, (B) imprinted genes ( ∗ = member of Dlk1-Dio3 cluster), (C) trophoblast markers, (D) 2-cell stage embryo retroviral genes. (E) High-magnification image of GATA6 and CDX2 immunostaining of ESCs, after 7 days differentiation in TSC conditions, demonstrating that there was no coexpression of these 2 markers. (F) qRT-PCR of HV- and HV+ populations cultured in serum/LIF or 2i after LIF withdrawal for 4 days. Values were normalized to the housekeeping gene TBP and are shown relative to serum/LIF HV+. Error bars indicate mean ± SD.

    Journal: Cell Reports

    Article Title: Totipotent Embryonic Stem Cells Arise in Ground-State Culture Conditions

    doi: 10.1016/j.celrep.2013.04.034

    Figure Lengend Snippet: Transcriptomics Suggest the Presence of a Population Primed toward Trophoblast as well as Endoderm, Related to Figure 2 (A–D) Common RNA-seq signatures in specific gene classes. The behavior of the individual genes belonging to the classes described in the text is shown. Plots compare mean log intensity values for individual genes among four sorted populations; serum/LIF HV-, serum/LIF HV+, 2i/LIF HV-, 2i/LIF HV+. Error bars represent s.d. between 2 biological replicates. (A) Markers of undifferentiated ESCs, (B) imprinted genes ( ∗ = member of Dlk1-Dio3 cluster), (C) trophoblast markers, (D) 2-cell stage embryo retroviral genes. (E) High-magnification image of GATA6 and CDX2 immunostaining of ESCs, after 7 days differentiation in TSC conditions, demonstrating that there was no coexpression of these 2 markers. (F) qRT-PCR of HV- and HV+ populations cultured in serum/LIF or 2i after LIF withdrawal for 4 days. Values were normalized to the housekeeping gene TBP and are shown relative to serum/LIF HV+. Error bars indicate mean ± SD.

    Article Snippet: Single-Cell qRT-PCR Single cells were sorted into 96-well PCR plates containing 5 μl CellsDirect reaction mix, 0.2 μl SuperScriptIII/Platinum Taq mix (CellsDirect One-Step qRT-PCR kit, Invitrogen), 2.8 μl DNA suspension buffer (TEKnova), and 1 μl 500 nM primer mix containing a mix of 48 DELTAgene Assays (Fluidigm) (sequences in ).

    Techniques: RNA Sequencing Assay, Immunostaining, Quantitative RT-PCR, Cell Culture

    Quantification of Lineage Priming In Vitro, Related to Figure 2 (A) A typical GATA6+ endodermal colony, also expressing HV, as scored in differentiation assays. (B) Quantification of number of GATA6+ cells per endodermal cluster. (C and D) Immunostaining of ESCs differentiated by LIF withdrawal (C) or neural differentiation (D) after previous culture in serum/LIF or 2i and then sorting into HV- and HV+, PECAM-1+ ESCs. Cells were plated immediately into differentiation conditions after sorting. Endoderm levels were quantified by GATA6 immunostaining (n = 3) (values relative to serum/LIF HV+), (D) and neural differentiation by TUJ1 immunostaining (n = 5) (Values relative to serum/LIF HV-) (E and F) qRT-PCR showing relative gene expression of sorted cells differentiated by LIF withdrawal (E) or neural differentiation (F). Data were normalized to the housekeeping gene TBP and is shown relative to serum/LIF HV sample. Values indicate mean ± SD of biological replicates. (G) Flow cytometry of sorted HV- and HV+ populations following LIF withdrawal and staining for the endoderm marker PDGFR-alpha.

    Journal: Cell Reports

    Article Title: Totipotent Embryonic Stem Cells Arise in Ground-State Culture Conditions

    doi: 10.1016/j.celrep.2013.04.034

    Figure Lengend Snippet: Quantification of Lineage Priming In Vitro, Related to Figure 2 (A) A typical GATA6+ endodermal colony, also expressing HV, as scored in differentiation assays. (B) Quantification of number of GATA6+ cells per endodermal cluster. (C and D) Immunostaining of ESCs differentiated by LIF withdrawal (C) or neural differentiation (D) after previous culture in serum/LIF or 2i and then sorting into HV- and HV+, PECAM-1+ ESCs. Cells were plated immediately into differentiation conditions after sorting. Endoderm levels were quantified by GATA6 immunostaining (n = 3) (values relative to serum/LIF HV+), (D) and neural differentiation by TUJ1 immunostaining (n = 5) (Values relative to serum/LIF HV-) (E and F) qRT-PCR showing relative gene expression of sorted cells differentiated by LIF withdrawal (E) or neural differentiation (F). Data were normalized to the housekeeping gene TBP and is shown relative to serum/LIF HV sample. Values indicate mean ± SD of biological replicates. (G) Flow cytometry of sorted HV- and HV+ populations following LIF withdrawal and staining for the endoderm marker PDGFR-alpha.

    Article Snippet: Single-Cell qRT-PCR Single cells were sorted into 96-well PCR plates containing 5 μl CellsDirect reaction mix, 0.2 μl SuperScriptIII/Platinum Taq mix (CellsDirect One-Step qRT-PCR kit, Invitrogen), 2.8 μl DNA suspension buffer (TEKnova), and 1 μl 500 nM primer mix containing a mix of 48 DELTAgene Assays (Fluidigm) (sequences in ).

    Techniques: In Vitro, Expressing, Immunostaining, Quantitative RT-PCR, Flow Cytometry, Cytometry, Staining, Marker