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  • 99
    Thermo Fisher ffpe dna pellet
    MicroRNA expression analysis of matched fresh and <t>FFPE</t> RNA from MCF10A cells using different RNA extraction methods. The upper panel displays a graphic representation of quantitative RT-PCR (Taqman® miRNA assays). Measurements obtain for miR-10a, miR-196b, miR-135b, miR-32a and miR-21 using matched fresh and FFPE RNA from MCF10A cells. MiRNAs were quantified using FFPE RNA extracted with TRIzol (TRI), Qiagen AllPrep <t>DNA/RNA</t> FFPE (QDR), AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB) kits and compared to control RNA extracted from fresh cells with TRIzol (TRI-Fr). Results are represented as ΔδC t (δC t target miRNA - δC t miR-10a (least expressed miRNA)). The lower panels show the comparison of global miRNA quantification obtained between fresh and FFPE RNA samples using the Illumina miRNA platform. Comparisons were performed between triplicate RNA extractions obtained from matched fresh (TRI-Fr1, TRI-Fr2, TRI-Fr3) and FFPE (TRI1-3, QDR1-3, and AMB1-3) cells. The correlation coefficient (r) between matched fresh and FFPE RNAs is displayed in each graph.
    Ffpe Dna Pellet, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ffpe dna pellet/product/Thermo Fisher
    Average 99 stars, based on 15 article reviews
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    ffpe dna pellet - by Bioz Stars, 2020-08
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    99
    Millipore dna pellet
    MM cells cultured in <t>3D</t> acquire STAT3 activity. ( A ) The activity of various signaling pathways (STAT3, Erk/MAPK, PI3K/Akt, NF-κB and Notch) in U266 and RPMI8226 cells cultured conventionally (2D) or in 3D was examined by Western blot analysis after 48 h; ( B ) The STAT3 activity of U266 and RPMI8226 cells in 2D or 3D culture from day 1 to day 4 were examined by Western blot analysis of pSTAT3 levels. SupM2 cells were included as a positive control for the pSTAT3 level; ( C ) The <t>DNA</t> binding ability of STAT3 in U266 and RPMI8226 cells cultured in 2D or 3D was examined by DNA pulldown immunoblotting assay. The cells were harvested and lysed after 48 h in culture. STAT3 in cell lysate was pulled down by a STAT3 DNA probe (described in Materials and Methods); ( D ) Immunocytochemical analysis of pSTAT3 level in U266, U266-3D, Karpas 299 and U266 xenograft cells. The cells were fixed after 48 h in culture. The procedure of processing, embedding and sectioning was described in Materials and Methods. Two representative pictures were shown. Karpas 299 cells were included as a positive control for pSTAT3 staining; ( E ) Western blot analysis of pSTAT3 and STAT3 levels of primary MM bone marrow cells in 2D or 3D culture from day 1 to day 3. SupM2 cells were included as a positive control for pSTAT3 level. β-actin was probed as a loading control in each blot.
    Dna Pellet, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 327 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 327 article reviews
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    dna pellet - by Bioz Stars, 2020-08
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    99
    Millipore dry dna pellets
    MM cells cultured in <t>3D</t> acquire STAT3 activity. ( A ) The activity of various signaling pathways (STAT3, Erk/MAPK, PI3K/Akt, NF-κB and Notch) in U266 and RPMI8226 cells cultured conventionally (2D) or in 3D was examined by Western blot analysis after 48 h; ( B ) The STAT3 activity of U266 and RPMI8226 cells in 2D or 3D culture from day 1 to day 4 were examined by Western blot analysis of pSTAT3 levels. SupM2 cells were included as a positive control for the pSTAT3 level; ( C ) The <t>DNA</t> binding ability of STAT3 in U266 and RPMI8226 cells cultured in 2D or 3D was examined by DNA pulldown immunoblotting assay. The cells were harvested and lysed after 48 h in culture. STAT3 in cell lysate was pulled down by a STAT3 DNA probe (described in Materials and Methods); ( D ) Immunocytochemical analysis of pSTAT3 level in U266, U266-3D, Karpas 299 and U266 xenograft cells. The cells were fixed after 48 h in culture. The procedure of processing, embedding and sectioning was described in Materials and Methods. Two representative pictures were shown. Karpas 299 cells were included as a positive control for pSTAT3 staining; ( E ) Western blot analysis of pSTAT3 and STAT3 levels of primary MM bone marrow cells in 2D or 3D culture from day 1 to day 3. SupM2 cells were included as a positive control for pSTAT3 level. β-actin was probed as a loading control in each blot.
    Dry Dna Pellets, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dry dna pellets/product/Millipore
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    dry dna pellets - by Bioz Stars, 2020-08
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    92
    Eppendorf AG dna pellet
    MM cells cultured in <t>3D</t> acquire STAT3 activity. ( A ) The activity of various signaling pathways (STAT3, Erk/MAPK, PI3K/Akt, NF-κB and Notch) in U266 and RPMI8226 cells cultured conventionally (2D) or in 3D was examined by Western blot analysis after 48 h; ( B ) The STAT3 activity of U266 and RPMI8226 cells in 2D or 3D culture from day 1 to day 4 were examined by Western blot analysis of pSTAT3 levels. SupM2 cells were included as a positive control for the pSTAT3 level; ( C ) The <t>DNA</t> binding ability of STAT3 in U266 and RPMI8226 cells cultured in 2D or 3D was examined by DNA pulldown immunoblotting assay. The cells were harvested and lysed after 48 h in culture. STAT3 in cell lysate was pulled down by a STAT3 DNA probe (described in Materials and Methods); ( D ) Immunocytochemical analysis of pSTAT3 level in U266, U266-3D, Karpas 299 and U266 xenograft cells. The cells were fixed after 48 h in culture. The procedure of processing, embedding and sectioning was described in Materials and Methods. Two representative pictures were shown. Karpas 299 cells were included as a positive control for pSTAT3 staining; ( E ) Western blot analysis of pSTAT3 and STAT3 levels of primary MM bone marrow cells in 2D or 3D culture from day 1 to day 3. SupM2 cells were included as a positive control for pSTAT3 level. β-actin was probed as a loading control in each blot.
    Dna Pellet, supplied by Eppendorf AG, used in various techniques. Bioz Stars score: 92/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna pellet/product/Eppendorf AG
    Average 92 stars, based on 52 article reviews
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    dna pellet - by Bioz Stars, 2020-08
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    92
    Thermo Fisher dna pellet
    MM cells cultured in <t>3D</t> acquire STAT3 activity. ( A ) The activity of various signaling pathways (STAT3, Erk/MAPK, PI3K/Akt, NF-κB and Notch) in U266 and RPMI8226 cells cultured conventionally (2D) or in 3D was examined by Western blot analysis after 48 h; ( B ) The STAT3 activity of U266 and RPMI8226 cells in 2D or 3D culture from day 1 to day 4 were examined by Western blot analysis of pSTAT3 levels. SupM2 cells were included as a positive control for the pSTAT3 level; ( C ) The <t>DNA</t> binding ability of STAT3 in U266 and RPMI8226 cells cultured in 2D or 3D was examined by DNA pulldown immunoblotting assay. The cells were harvested and lysed after 48 h in culture. STAT3 in cell lysate was pulled down by a STAT3 DNA probe (described in Materials and Methods); ( D ) Immunocytochemical analysis of pSTAT3 level in U266, U266-3D, Karpas 299 and U266 xenograft cells. The cells were fixed after 48 h in culture. The procedure of processing, embedding and sectioning was described in Materials and Methods. Two representative pictures were shown. Karpas 299 cells were included as a positive control for pSTAT3 staining; ( E ) Western blot analysis of pSTAT3 and STAT3 levels of primary MM bone marrow cells in 2D or 3D culture from day 1 to day 3. SupM2 cells were included as a positive control for pSTAT3 level. β-actin was probed as a loading control in each blot.
    Dna Pellet, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 994 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna pellet/product/Thermo Fisher
    Average 92 stars, based on 994 article reviews
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    dna pellet - by Bioz Stars, 2020-08
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    92
    Avantor dna pellet
    MM cells cultured in <t>3D</t> acquire STAT3 activity. ( A ) The activity of various signaling pathways (STAT3, Erk/MAPK, PI3K/Akt, NF-κB and Notch) in U266 and RPMI8226 cells cultured conventionally (2D) or in 3D was examined by Western blot analysis after 48 h; ( B ) The STAT3 activity of U266 and RPMI8226 cells in 2D or 3D culture from day 1 to day 4 were examined by Western blot analysis of pSTAT3 levels. SupM2 cells were included as a positive control for the pSTAT3 level; ( C ) The <t>DNA</t> binding ability of STAT3 in U266 and RPMI8226 cells cultured in 2D or 3D was examined by DNA pulldown immunoblotting assay. The cells were harvested and lysed after 48 h in culture. STAT3 in cell lysate was pulled down by a STAT3 DNA probe (described in Materials and Methods); ( D ) Immunocytochemical analysis of pSTAT3 level in U266, U266-3D, Karpas 299 and U266 xenograft cells. The cells were fixed after 48 h in culture. The procedure of processing, embedding and sectioning was described in Materials and Methods. Two representative pictures were shown. Karpas 299 cells were included as a positive control for pSTAT3 staining; ( E ) Western blot analysis of pSTAT3 and STAT3 levels of primary MM bone marrow cells in 2D or 3D culture from day 1 to day 3. SupM2 cells were included as a positive control for pSTAT3 level. β-actin was probed as a loading control in each blot.
    Dna Pellet, supplied by Avantor, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna pellet/product/Avantor
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    dna pellet - by Bioz Stars, 2020-08
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    91
    Merck KGaA dna pellets
    MM cells cultured in <t>3D</t> acquire STAT3 activity. ( A ) The activity of various signaling pathways (STAT3, Erk/MAPK, PI3K/Akt, NF-κB and Notch) in U266 and RPMI8226 cells cultured conventionally (2D) or in 3D was examined by Western blot analysis after 48 h; ( B ) The STAT3 activity of U266 and RPMI8226 cells in 2D or 3D culture from day 1 to day 4 were examined by Western blot analysis of pSTAT3 levels. SupM2 cells were included as a positive control for the pSTAT3 level; ( C ) The <t>DNA</t> binding ability of STAT3 in U266 and RPMI8226 cells cultured in 2D or 3D was examined by DNA pulldown immunoblotting assay. The cells were harvested and lysed after 48 h in culture. STAT3 in cell lysate was pulled down by a STAT3 DNA probe (described in Materials and Methods); ( D ) Immunocytochemical analysis of pSTAT3 level in U266, U266-3D, Karpas 299 and U266 xenograft cells. The cells were fixed after 48 h in culture. The procedure of processing, embedding and sectioning was described in Materials and Methods. Two representative pictures were shown. Karpas 299 cells were included as a positive control for pSTAT3 staining; ( E ) Western blot analysis of pSTAT3 and STAT3 levels of primary MM bone marrow cells in 2D or 3D culture from day 1 to day 3. SupM2 cells were included as a positive control for pSTAT3 level. β-actin was probed as a loading control in each blot.
    Dna Pellets, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Illumina Inc dna pellets
    Distribution of <t>Illumina</t> tags and ChIP–Seq (U+M) scores over main features of the genome. (A) Distribution of ChIP-Seq (U+M) scores over CDS, 5′ UTR, 3′ UTR, intronic, transposon/repetitive and intergenic regions within euchromatic genome and all sequenced genome. (B) Total ChIP-Seq (U+M) scores of epigenetic marks in all transposons within the genome. (C) Pearson correlations between ChIP-Seq (U+M) scores of epigenetic marks over 185 transposon classes. (D) A heat map of ChIP-Seq (U+M) scores of epigenetic marks over 185 transposon classes, which are arranged into 5 major types: <t>DNA,</t> LINE, LTR, Satellite and unknown.
    Dna Pellets, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 91/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna pellets/product/Illumina Inc
    Average 91 stars, based on 17 article reviews
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    91
    WILEY-VCH Verlag dna pellet
    Distribution of <t>Illumina</t> tags and ChIP–Seq (U+M) scores over main features of the genome. (A) Distribution of ChIP-Seq (U+M) scores over CDS, 5′ UTR, 3′ UTR, intronic, transposon/repetitive and intergenic regions within euchromatic genome and all sequenced genome. (B) Total ChIP-Seq (U+M) scores of epigenetic marks in all transposons within the genome. (C) Pearson correlations between ChIP-Seq (U+M) scores of epigenetic marks over 185 transposon classes. (D) A heat map of ChIP-Seq (U+M) scores of epigenetic marks over 185 transposon classes, which are arranged into 5 major types: <t>DNA,</t> LINE, LTR, Satellite and unknown.
    Dna Pellet, supplied by WILEY-VCH Verlag, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna pellet/product/WILEY-VCH Verlag
    Average 91 stars, based on 1 article reviews
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    91
    Beckman Coulter dna pellet
    Distribution of <t>Illumina</t> tags and ChIP–Seq (U+M) scores over main features of the genome. (A) Distribution of ChIP-Seq (U+M) scores over CDS, 5′ UTR, 3′ UTR, intronic, transposon/repetitive and intergenic regions within euchromatic genome and all sequenced genome. (B) Total ChIP-Seq (U+M) scores of epigenetic marks in all transposons within the genome. (C) Pearson correlations between ChIP-Seq (U+M) scores of epigenetic marks over 185 transposon classes. (D) A heat map of ChIP-Seq (U+M) scores of epigenetic marks over 185 transposon classes, which are arranged into 5 major types: <t>DNA,</t> LINE, LTR, Satellite and unknown.
    Dna Pellet, supplied by Beckman Coulter, used in various techniques. Bioz Stars score: 91/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GE Healthcare dna pellet
    Distribution of <t>Illumina</t> tags and ChIP–Seq (U+M) scores over main features of the genome. (A) Distribution of ChIP-Seq (U+M) scores over CDS, 5′ UTR, 3′ UTR, intronic, transposon/repetitive and intergenic regions within euchromatic genome and all sequenced genome. (B) Total ChIP-Seq (U+M) scores of epigenetic marks in all transposons within the genome. (C) Pearson correlations between ChIP-Seq (U+M) scores of epigenetic marks over 185 transposon classes. (D) A heat map of ChIP-Seq (U+M) scores of epigenetic marks over 185 transposon classes, which are arranged into 5 major types: <t>DNA,</t> LINE, LTR, Satellite and unknown.
    Dna Pellet, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    5 PRIME dna pellets
    Distribution of <t>Illumina</t> tags and ChIP–Seq (U+M) scores over main features of the genome. (A) Distribution of ChIP-Seq (U+M) scores over CDS, 5′ UTR, 3′ UTR, intronic, transposon/repetitive and intergenic regions within euchromatic genome and all sequenced genome. (B) Total ChIP-Seq (U+M) scores of epigenetic marks in all transposons within the genome. (C) Pearson correlations between ChIP-Seq (U+M) scores of epigenetic marks over 185 transposon classes. (D) A heat map of ChIP-Seq (U+M) scores of epigenetic marks over 185 transposon classes, which are arranged into 5 major types: <t>DNA,</t> LINE, LTR, Satellite and unknown.
    Dna Pellets, supplied by 5 PRIME, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Applichem dna pellets
    Distribution of <t>Illumina</t> tags and ChIP–Seq (U+M) scores over main features of the genome. (A) Distribution of ChIP-Seq (U+M) scores over CDS, 5′ UTR, 3′ UTR, intronic, transposon/repetitive and intergenic regions within euchromatic genome and all sequenced genome. (B) Total ChIP-Seq (U+M) scores of epigenetic marks in all transposons within the genome. (C) Pearson correlations between ChIP-Seq (U+M) scores of epigenetic marks over 185 transposon classes. (D) A heat map of ChIP-Seq (U+M) scores of epigenetic marks over 185 transposon classes, which are arranged into 5 major types: <t>DNA,</t> LINE, LTR, Satellite and unknown.
    Dna Pellets, supplied by Applichem, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna pellets/product/Applichem
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    dna pellets - by Bioz Stars, 2020-08
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    99
    Millipore plasmid dna pellets
    Distribution of <t>Illumina</t> tags and ChIP–Seq (U+M) scores over main features of the genome. (A) Distribution of ChIP-Seq (U+M) scores over CDS, 5′ UTR, 3′ UTR, intronic, transposon/repetitive and intergenic regions within euchromatic genome and all sequenced genome. (B) Total ChIP-Seq (U+M) scores of epigenetic marks in all transposons within the genome. (C) Pearson correlations between ChIP-Seq (U+M) scores of epigenetic marks over 185 transposon classes. (D) A heat map of ChIP-Seq (U+M) scores of epigenetic marks over 185 transposon classes, which are arranged into 5 major types: <t>DNA,</t> LINE, LTR, Satellite and unknown.
    Plasmid Dna Pellets, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen cell pellets one kit
    Distribution of <t>Illumina</t> tags and ChIP–Seq (U+M) scores over main features of the genome. (A) Distribution of ChIP-Seq (U+M) scores over CDS, 5′ UTR, 3′ UTR, intronic, transposon/repetitive and intergenic regions within euchromatic genome and all sequenced genome. (B) Total ChIP-Seq (U+M) scores of epigenetic marks in all transposons within the genome. (C) Pearson correlations between ChIP-Seq (U+M) scores of epigenetic marks over 185 transposon classes. (D) A heat map of ChIP-Seq (U+M) scores of epigenetic marks over 185 transposon classes, which are arranged into 5 major types: <t>DNA,</t> LINE, LTR, Satellite and unknown.
    Cell Pellets One Kit, supplied by Qiagen, 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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    Image Search Results


    MicroRNA expression analysis of matched fresh and FFPE RNA from MCF10A cells using different RNA extraction methods. The upper panel displays a graphic representation of quantitative RT-PCR (Taqman® miRNA assays). Measurements obtain for miR-10a, miR-196b, miR-135b, miR-32a and miR-21 using matched fresh and FFPE RNA from MCF10A cells. MiRNAs were quantified using FFPE RNA extracted with TRIzol (TRI), Qiagen AllPrep DNA/RNA FFPE (QDR), AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB) kits and compared to control RNA extracted from fresh cells with TRIzol (TRI-Fr). Results are represented as ΔδC t (δC t target miRNA - δC t miR-10a (least expressed miRNA)). The lower panels show the comparison of global miRNA quantification obtained between fresh and FFPE RNA samples using the Illumina miRNA platform. Comparisons were performed between triplicate RNA extractions obtained from matched fresh (TRI-Fr1, TRI-Fr2, TRI-Fr3) and FFPE (TRI1-3, QDR1-3, and AMB1-3) cells. The correlation coefficient (r) between matched fresh and FFPE RNAs is displayed in each graph.

    Journal: PLoS ONE

    Article Title: Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens

    doi: 10.1371/journal.pone.0034683

    Figure Lengend Snippet: MicroRNA expression analysis of matched fresh and FFPE RNA from MCF10A cells using different RNA extraction methods. The upper panel displays a graphic representation of quantitative RT-PCR (Taqman® miRNA assays). Measurements obtain for miR-10a, miR-196b, miR-135b, miR-32a and miR-21 using matched fresh and FFPE RNA from MCF10A cells. MiRNAs were quantified using FFPE RNA extracted with TRIzol (TRI), Qiagen AllPrep DNA/RNA FFPE (QDR), AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB) kits and compared to control RNA extracted from fresh cells with TRIzol (TRI-Fr). Results are represented as ΔδC t (δC t target miRNA - δC t miR-10a (least expressed miRNA)). The lower panels show the comparison of global miRNA quantification obtained between fresh and FFPE RNA samples using the Illumina miRNA platform. Comparisons were performed between triplicate RNA extractions obtained from matched fresh (TRI-Fr1, TRI-Fr2, TRI-Fr3) and FFPE (TRI1-3, QDR1-3, and AMB1-3) cells. The correlation coefficient (r) between matched fresh and FFPE RNAs is displayed in each graph.

    Article Snippet: While an FFPE-DNA pellet was observable and DNA readable on a NanoDrop ND-1000, it could not be observed on an agarose gel and did not produce PCR amplicons (data not shown).

    Techniques: Expressing, Formalin-fixed Paraffin-Embedded, RNA Extraction, Quantitative RT-PCR, Isolation

    Summary of sequential recovery of DNA and RNA from MCF10A Fresh and FFPE samples using different extraction methods. (A) Schematic representation of cell culture and DNA/RNA extraction methods used with matched fresh and 1 month-old formalin-fixed paraffin-embedded (FFPE) human mammary epithelial MCF10A cells. FFPE DNA and RNA extractions (QD, TRI, QDR, AMB) were performed in triplicate using three 10 µm sections for each replicate. (B) Analysis of RNA extracted from matched fresh and FFPE MCF10A cells. Total RNA extracted from fresh cells using TRIzol (TRI-Fr; Lane 2), and total RNA extracted from FFPE cells using TRIzol (TRI; lane 3), Qiagen QIAamp DNA/RNA extraction kit (QDR; lane 4), and AMBion RecoverAll™ Total Nucleic Acid Isolation kit (AMB; lane 5) was analyzed and quantified using an Agilent 2100 Bioanalyzer 6000 Nanochip (size ladder in lane 1). The bar graph placed above the Bioanalyzer image displays total amounts of RNA recovered from three consecutive 10 µm sections, in triplicate experiments, using the three different methods (TRI, QDR, AMB). (C) Analysis of genomic DNA extracted from matched fresh and FFPE MCF10A cells. DNA was extracted from fresh cells using a phenol/chloroform based method (PC-Fr; lane 2), and TRIzol (TRI-Fr lane 3); and from FFPE cells using Qiagen QIAamp DNA FFPE kit (QD; lane 4), TRIzol DNA/RNA extraction method (TRI; lane 5), Qiagen AllPrep DNA/RNA FFPE kit (QDR; lane 6), and AMBion RecoverAll™ Total Nucleic Acid Isolation kit (AMB; lane 7) was analyzed on a 1% agarose gel (size ladder in lane 1). The bar graph placed above the agarose gel displays total amounts of DNA recovered alone (QD), simultaneously with RNA (TRI, QDR), or separately from RNA (AMB), using three consecutive 10 µm sections, in triplicate experiments for each method.

    Journal: PLoS ONE

    Article Title: Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens

    doi: 10.1371/journal.pone.0034683

    Figure Lengend Snippet: Summary of sequential recovery of DNA and RNA from MCF10A Fresh and FFPE samples using different extraction methods. (A) Schematic representation of cell culture and DNA/RNA extraction methods used with matched fresh and 1 month-old formalin-fixed paraffin-embedded (FFPE) human mammary epithelial MCF10A cells. FFPE DNA and RNA extractions (QD, TRI, QDR, AMB) were performed in triplicate using three 10 µm sections for each replicate. (B) Analysis of RNA extracted from matched fresh and FFPE MCF10A cells. Total RNA extracted from fresh cells using TRIzol (TRI-Fr; Lane 2), and total RNA extracted from FFPE cells using TRIzol (TRI; lane 3), Qiagen QIAamp DNA/RNA extraction kit (QDR; lane 4), and AMBion RecoverAll™ Total Nucleic Acid Isolation kit (AMB; lane 5) was analyzed and quantified using an Agilent 2100 Bioanalyzer 6000 Nanochip (size ladder in lane 1). The bar graph placed above the Bioanalyzer image displays total amounts of RNA recovered from three consecutive 10 µm sections, in triplicate experiments, using the three different methods (TRI, QDR, AMB). (C) Analysis of genomic DNA extracted from matched fresh and FFPE MCF10A cells. DNA was extracted from fresh cells using a phenol/chloroform based method (PC-Fr; lane 2), and TRIzol (TRI-Fr lane 3); and from FFPE cells using Qiagen QIAamp DNA FFPE kit (QD; lane 4), TRIzol DNA/RNA extraction method (TRI; lane 5), Qiagen AllPrep DNA/RNA FFPE kit (QDR; lane 6), and AMBion RecoverAll™ Total Nucleic Acid Isolation kit (AMB; lane 7) was analyzed on a 1% agarose gel (size ladder in lane 1). The bar graph placed above the agarose gel displays total amounts of DNA recovered alone (QD), simultaneously with RNA (TRI, QDR), or separately from RNA (AMB), using three consecutive 10 µm sections, in triplicate experiments for each method.

    Article Snippet: While an FFPE-DNA pellet was observable and DNA readable on a NanoDrop ND-1000, it could not be observed on an agarose gel and did not produce PCR amplicons (data not shown).

    Techniques: Formalin-fixed Paraffin-Embedded, Cell Culture, RNA Extraction, Isolation, Agarose Gel Electrophoresis

    DNA/RNA extractions using archived human specimens. Four different methods were tested on seven different archived tissues: (A) Qiagen QIAamp DNA FFPE kit for DNA (QD), (B) TRIzol DNA/RNA extraction method for DNA and RNA (TRI), (C) Qiagen AllPrep DNA/RNA FFPE kit for DNA and RNA (QDR), and (D) Ambion RecoverAll™ Total Nucleic Acid Isolation (AMB) for DNA and for RNA. Each nucleic acid extraction was done in triplicate to determine technical reproducibility.

    Journal: PLoS ONE

    Article Title: Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens

    doi: 10.1371/journal.pone.0034683

    Figure Lengend Snippet: DNA/RNA extractions using archived human specimens. Four different methods were tested on seven different archived tissues: (A) Qiagen QIAamp DNA FFPE kit for DNA (QD), (B) TRIzol DNA/RNA extraction method for DNA and RNA (TRI), (C) Qiagen AllPrep DNA/RNA FFPE kit for DNA and RNA (QDR), and (D) Ambion RecoverAll™ Total Nucleic Acid Isolation (AMB) for DNA and for RNA. Each nucleic acid extraction was done in triplicate to determine technical reproducibility.

    Article Snippet: While an FFPE-DNA pellet was observable and DNA readable on a NanoDrop ND-1000, it could not be observed on an agarose gel and did not produce PCR amplicons (data not shown).

    Techniques: Formalin-fixed Paraffin-Embedded, RNA Extraction, Isolation

    Methylation analysis of CpG regions in genes of interest using matched fresh and FFPE genomic DNA obtained by different extraction methods. Representative 2% agarose gel electrophoresis images of PCR products for (A) ESR1 and (B) CCND2 genes. Graphs depict methylation values as a percentage for CpG dinucleotide rich regions in (C) ESR1, (D) CCND2, (E) GHSR, and (F) ARID3A as assayed via the MassARRAY system (Sequenom). Data were analyzed and confirmed using the MassArray R script statistical package. Methylation values for fresh MCF10A DNA isolated with control methods (DNA from fresh cells recovered by phenol/chloroform (PC-Fr) and from FFPE cells using the Qiagen QIAamp DNA FFPE kit (QD)) are compared against methods used for matched FFPE DNA (TRIzol extraction (TRI), Qiagen AllPrep DNA/RNA FFPE (QDR), and AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB)). The bar graphs display the correlation between DNA methylation measurements obtained from fresh genomic DNA and each FFPE genomic DNA recovered by the different extraction methods.

    Journal: PLoS ONE

    Article Title: Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens

    doi: 10.1371/journal.pone.0034683

    Figure Lengend Snippet: Methylation analysis of CpG regions in genes of interest using matched fresh and FFPE genomic DNA obtained by different extraction methods. Representative 2% agarose gel electrophoresis images of PCR products for (A) ESR1 and (B) CCND2 genes. Graphs depict methylation values as a percentage for CpG dinucleotide rich regions in (C) ESR1, (D) CCND2, (E) GHSR, and (F) ARID3A as assayed via the MassARRAY system (Sequenom). Data were analyzed and confirmed using the MassArray R script statistical package. Methylation values for fresh MCF10A DNA isolated with control methods (DNA from fresh cells recovered by phenol/chloroform (PC-Fr) and from FFPE cells using the Qiagen QIAamp DNA FFPE kit (QD)) are compared against methods used for matched FFPE DNA (TRIzol extraction (TRI), Qiagen AllPrep DNA/RNA FFPE (QDR), and AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB)). The bar graphs display the correlation between DNA methylation measurements obtained from fresh genomic DNA and each FFPE genomic DNA recovered by the different extraction methods.

    Article Snippet: While an FFPE-DNA pellet was observable and DNA readable on a NanoDrop ND-1000, it could not be observed on an agarose gel and did not produce PCR amplicons (data not shown).

    Techniques: Methylation, Formalin-fixed Paraffin-Embedded, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Isolation, DNA Methylation Assay

    Optimized TRIzol extraction of DNA from archived specimens. (A) Schematic representation of DNA recovery from the lower phase of TRIzol (upper phase yields RNA). In step 1 (yellow bullet), tissue digestion is performed following the procedure described in Loudig et al. 2007. In step 2 (yellow bullet), using TRIzol RNA and DNA are separated into the upper and lower phases, respectively. The DNA is recovered from the lower phase, using our optimized approach described in the materials and methods . The four steps describing optimization of DNA recovery from the lower phase of TRIzol include: a. Precipitate DNA; b. Process DNA pellet (using reagents from Qiagen DNA FFPE kit for steps b to d); c. Purify DNA; d. Bind, wash, and elute DNA. (B) Analysis of DNA from FFPE tissue recovered from the lower phase of TRIzol. The upper panel shows the histogram of DNA recovery. The lower panel shows a 1.5% agarose gel electrophoresis image of fresh DNA recovered from a TRIzol treatment lower phase (lane 1), FFPE DNA recovered from a TRIzol lower phase (lanes 2–6), and the size ladder (lane 7). For DNA, precipitation was tested for 600 µl (lane 2 and lane 4), 1000 µl (lane 3 and lane 5), and 1200 µl of Ethanol (lane 6). Proteinase K (PK) treatment was performed for 24 (lanes 2–3) or 48 hours (lanes 4–6). Electrophoresis reveals integrity of the extracted DNA samples. The histogram and agarose gel show that precipitation with a combination of 1200 µl ethanol and 48 hours of PK treatment gives the best quality and quantity of DNA. 500 ng of DNA was loaded per well of the gel.

    Journal: PLoS ONE

    Article Title: Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens

    doi: 10.1371/journal.pone.0034683

    Figure Lengend Snippet: Optimized TRIzol extraction of DNA from archived specimens. (A) Schematic representation of DNA recovery from the lower phase of TRIzol (upper phase yields RNA). In step 1 (yellow bullet), tissue digestion is performed following the procedure described in Loudig et al. 2007. In step 2 (yellow bullet), using TRIzol RNA and DNA are separated into the upper and lower phases, respectively. The DNA is recovered from the lower phase, using our optimized approach described in the materials and methods . The four steps describing optimization of DNA recovery from the lower phase of TRIzol include: a. Precipitate DNA; b. Process DNA pellet (using reagents from Qiagen DNA FFPE kit for steps b to d); c. Purify DNA; d. Bind, wash, and elute DNA. (B) Analysis of DNA from FFPE tissue recovered from the lower phase of TRIzol. The upper panel shows the histogram of DNA recovery. The lower panel shows a 1.5% agarose gel electrophoresis image of fresh DNA recovered from a TRIzol treatment lower phase (lane 1), FFPE DNA recovered from a TRIzol lower phase (lanes 2–6), and the size ladder (lane 7). For DNA, precipitation was tested for 600 µl (lane 2 and lane 4), 1000 µl (lane 3 and lane 5), and 1200 µl of Ethanol (lane 6). Proteinase K (PK) treatment was performed for 24 (lanes 2–3) or 48 hours (lanes 4–6). Electrophoresis reveals integrity of the extracted DNA samples. The histogram and agarose gel show that precipitation with a combination of 1200 µl ethanol and 48 hours of PK treatment gives the best quality and quantity of DNA. 500 ng of DNA was loaded per well of the gel.

    Article Snippet: While an FFPE-DNA pellet was observable and DNA readable on a NanoDrop ND-1000, it could not be observed on an agarose gel and did not produce PCR amplicons (data not shown).

    Techniques: Formalin-fixed Paraffin-Embedded, Agarose Gel Electrophoresis, Electrophoresis

    Messenger RNA expression analysis of matched fresh and FFPE RNA using different RNA extraction methods. The upper panel displays a graphic representation of quantitative RT-PCR (Taqman® mRNA assays) Measurements obtained for ESR1, CCND2 and KRT14 genes using matched fresh and FFPE RNA from MCF10A cells. The three genes were quantified using matched fresh RNA recovered with TRIzol (TRI-Fr), and FFPE RNA recovered with TRIzol (TRI), with Qiagen AllPrep DNA/RNA FFPE (QDR), with AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB) and with the Roche RNA FFPE (Roche) kits. The results are represented as fold changes. The lower panels show the comparison of global mRNA quantifications obtained between fresh and FFPE RNA samples using the Illumina whole-Genome DASL platform. The different panels display comparison between triplicate RNA extractions from matched fresh (TRI-Fr1, TRI-Fr2, TRI-Fr3 (bottom to top panel)) and FFPE (TRI1-3, QDR1-3, AMB1-3 and Roche1-3 (from left to right panel)) cells. The correlation coefficient (r) between matched fresh and FFPE RNAs is displayed in each graph.

    Journal: PLoS ONE

    Article Title: Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens

    doi: 10.1371/journal.pone.0034683

    Figure Lengend Snippet: Messenger RNA expression analysis of matched fresh and FFPE RNA using different RNA extraction methods. The upper panel displays a graphic representation of quantitative RT-PCR (Taqman® mRNA assays) Measurements obtained for ESR1, CCND2 and KRT14 genes using matched fresh and FFPE RNA from MCF10A cells. The three genes were quantified using matched fresh RNA recovered with TRIzol (TRI-Fr), and FFPE RNA recovered with TRIzol (TRI), with Qiagen AllPrep DNA/RNA FFPE (QDR), with AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB) and with the Roche RNA FFPE (Roche) kits. The results are represented as fold changes. The lower panels show the comparison of global mRNA quantifications obtained between fresh and FFPE RNA samples using the Illumina whole-Genome DASL platform. The different panels display comparison between triplicate RNA extractions from matched fresh (TRI-Fr1, TRI-Fr2, TRI-Fr3 (bottom to top panel)) and FFPE (TRI1-3, QDR1-3, AMB1-3 and Roche1-3 (from left to right panel)) cells. The correlation coefficient (r) between matched fresh and FFPE RNAs is displayed in each graph.

    Article Snippet: While an FFPE-DNA pellet was observable and DNA readable on a NanoDrop ND-1000, it could not be observed on an agarose gel and did not produce PCR amplicons (data not shown).

    Techniques: RNA Expression, Formalin-fixed Paraffin-Embedded, RNA Extraction, Quantitative RT-PCR, Isolation

    MM cells cultured in 3D acquire STAT3 activity. ( A ) The activity of various signaling pathways (STAT3, Erk/MAPK, PI3K/Akt, NF-κB and Notch) in U266 and RPMI8226 cells cultured conventionally (2D) or in 3D was examined by Western blot analysis after 48 h; ( B ) The STAT3 activity of U266 and RPMI8226 cells in 2D or 3D culture from day 1 to day 4 were examined by Western blot analysis of pSTAT3 levels. SupM2 cells were included as a positive control for the pSTAT3 level; ( C ) The DNA binding ability of STAT3 in U266 and RPMI8226 cells cultured in 2D or 3D was examined by DNA pulldown immunoblotting assay. The cells were harvested and lysed after 48 h in culture. STAT3 in cell lysate was pulled down by a STAT3 DNA probe (described in Materials and Methods); ( D ) Immunocytochemical analysis of pSTAT3 level in U266, U266-3D, Karpas 299 and U266 xenograft cells. The cells were fixed after 48 h in culture. The procedure of processing, embedding and sectioning was described in Materials and Methods. Two representative pictures were shown. Karpas 299 cells were included as a positive control for pSTAT3 staining; ( E ) Western blot analysis of pSTAT3 and STAT3 levels of primary MM bone marrow cells in 2D or 3D culture from day 1 to day 3. SupM2 cells were included as a positive control for pSTAT3 level. β-actin was probed as a loading control in each blot.

    Journal: Cancers

    Article Title: Constitutive Activation of STAT3 in Myeloma Cells Cultured in a Three-Dimensional, Reconstructed Bone Marrow Model

    doi: 10.3390/cancers10060206

    Figure Lengend Snippet: MM cells cultured in 3D acquire STAT3 activity. ( A ) The activity of various signaling pathways (STAT3, Erk/MAPK, PI3K/Akt, NF-κB and Notch) in U266 and RPMI8226 cells cultured conventionally (2D) or in 3D was examined by Western blot analysis after 48 h; ( B ) The STAT3 activity of U266 and RPMI8226 cells in 2D or 3D culture from day 1 to day 4 were examined by Western blot analysis of pSTAT3 levels. SupM2 cells were included as a positive control for the pSTAT3 level; ( C ) The DNA binding ability of STAT3 in U266 and RPMI8226 cells cultured in 2D or 3D was examined by DNA pulldown immunoblotting assay. The cells were harvested and lysed after 48 h in culture. STAT3 in cell lysate was pulled down by a STAT3 DNA probe (described in Materials and Methods); ( D ) Immunocytochemical analysis of pSTAT3 level in U266, U266-3D, Karpas 299 and U266 xenograft cells. The cells were fixed after 48 h in culture. The procedure of processing, embedding and sectioning was described in Materials and Methods. Two representative pictures were shown. Karpas 299 cells were included as a positive control for pSTAT3 staining; ( E ) Western blot analysis of pSTAT3 and STAT3 levels of primary MM bone marrow cells in 2D or 3D culture from day 1 to day 3. SupM2 cells were included as a positive control for pSTAT3 level. β-actin was probed as a loading control in each blot.

    Article Snippet: DNA Pulldown Assay Cell pellets from MM or MM-3D cells were lysed with CellLytic M (Sigma) with protease inhibitor and phosphatase inhibitor cocktails (Millipore, Etobicoke, ON, Canada) on ice for 30 min. 300 µg of total cell lysate was mixed with 3 pmol of STAT3 DNA probe (Biotin-5′-GATCTAGGAATTCCCAGAAGG-3′) for 30 min on a rotator at room temperature.

    Techniques: Cell Culture, Activity Assay, Western Blot, Positive Control, Binding Assay, Staining

    Distribution of Illumina tags and ChIP–Seq (U+M) scores over main features of the genome. (A) Distribution of ChIP-Seq (U+M) scores over CDS, 5′ UTR, 3′ UTR, intronic, transposon/repetitive and intergenic regions within euchromatic genome and all sequenced genome. (B) Total ChIP-Seq (U+M) scores of epigenetic marks in all transposons within the genome. (C) Pearson correlations between ChIP-Seq (U+M) scores of epigenetic marks over 185 transposon classes. (D) A heat map of ChIP-Seq (U+M) scores of epigenetic marks over 185 transposon classes, which are arranged into 5 major types: DNA, LINE, LTR, Satellite and unknown.

    Journal: PLoS Genetics

    Article Title: A High-Resolution Whole-Genome Map of Key Chromatin Modifications in the Adult Drosophila melanogaster

    doi: 10.1371/journal.pgen.1002380

    Figure Lengend Snippet: Distribution of Illumina tags and ChIP–Seq (U+M) scores over main features of the genome. (A) Distribution of ChIP-Seq (U+M) scores over CDS, 5′ UTR, 3′ UTR, intronic, transposon/repetitive and intergenic regions within euchromatic genome and all sequenced genome. (B) Total ChIP-Seq (U+M) scores of epigenetic marks in all transposons within the genome. (C) Pearson correlations between ChIP-Seq (U+M) scores of epigenetic marks over 185 transposon classes. (D) A heat map of ChIP-Seq (U+M) scores of epigenetic marks over 185 transposon classes, which are arranged into 5 major types: DNA, LINE, LTR, Satellite and unknown.

    Article Snippet: The precipitated DNA pellets were submitted for Illumina library construction and sequencing.

    Techniques: Chromatin Immunoprecipitation