complementary dna synthesis Search Results


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  • 90
    Millipore complementary dna cdna synthesis
    Complementary Dna Cdna Synthesis, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Viogene complementary dna cdna synthesis
    Complementary Dna Cdna Synthesis, supplied by Viogene, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc complementary dna cdna synthesis
    Complementary Dna Cdna Synthesis, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 91/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega complementary dna cdna synthesis kit
    Tissue expression of hMSH6 <t>mRNA.</t> A human multiple-tissue Northern blot (CLONTECH) was probed with the 32 P-labeled complete <t>cDNA</t> clone of hMSH6 according to the manufacturer’s protocol. Colon refers to mucosal lining; p.b. leukocyte refers to peripheral blood leukocyte. The hMSH6 transcript is indicated by the arrow and corresponds to ≈4.5 kb. The amount of RNA loaded in each lane was adjusted to comparable levels as judged spectrophotometrically and by the levels of actin present (CLONTECH).
    Complementary Dna Cdna Synthesis Kit, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bioneer Corporation complementary dna cdna synthesis kit
    Tissue expression of hMSH6 <t>mRNA.</t> A human multiple-tissue Northern blot (CLONTECH) was probed with the 32 P-labeled complete <t>cDNA</t> clone of hMSH6 according to the manufacturer’s protocol. Colon refers to mucosal lining; p.b. leukocyte refers to peripheral blood leukocyte. The hMSH6 transcript is indicated by the arrow and corresponds to ≈4.5 kb. The amount of RNA loaded in each lane was adjusted to comparable levels as judged spectrophotometrically and by the levels of actin present (CLONTECH).
    Complementary Dna Cdna Synthesis Kit, supplied by Bioneer Corporation, used in various techniques. Bioz Stars score: 88/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher complementary dna cdna synthesis
    Expression levels of type I collagen, Smad2/3, and TGF-β1 genes after ES-P treatment in aged mice. (A) The left ears of fourteen-week old mice were treated daily with 30 μg T . spiralis ES-P for 14 days (red color). The right ears of were treated daily with PBS for 14 days as control (blue color). After 14 days, ears were collected. Total <t>RNA</t> was extracted from each ear tissue and <t>cDNA</t> was constructed. (B) The gene expression levels of collagen I , TGF-β1 , and Smad2/3 were analyzed by real-time PCR. The gene expression value of each group was normalized by control value, 14 weeks group. (**; P
    Complementary Dna Cdna Synthesis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 2952 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher complementary dna cdna synthesis kit
    The Total <t>RNA</t> Detection system. A universal RNA adaptor was firstly added to the 3′ ends of all RNA templates. The abundant 18S and 28S ribosome RNAs were then subtracted by using biotinylated ribosomal-specific probes. Small-size RNAs containing the degraded RNA intermediates were removed by size-filtration. The enriched transcripts were converted into double-strand <t>cDNA</t> by using the 3′ end RNA adaptor-based primer. The cDNAs were further digested by NlaIII. The 3′ cDNAs were isolated by using the streptoavidin beads. An adaptor was added to the 5′ ends of the 3′ cDNAs. The 3′ cDNAs were then amplified by PCR using the 5′ adaptor-based sense primer and the 3′ end RNA adaptor-based antisense primer. The amplified 3′ cDNAs were sequenced from the 3′ end by the 454 system. See further details in Materials and Methods .
    Complementary Dna Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 66 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa cdna synthesis
    LIRKO mice show quantitative and qualitative defects in bile acid synthesis on a chow diet (a) Real-time <t>PCR</t> analysis of <t>cDNA</t> prepared from six-week old mice sacrificed during the light cycle following a 24 hour fast; the expression of each gene was normalized to its value in Lox mice (n=5-8, *p=0.05 versus Lox). (b) Since the synthesis of bile acids is equivalent to fecal excretion in the steady state, we assessed bile acid synthesis by collecting feces from 3 month old Lox and LIRKO mice for 72 hours and measuring total bile acid outputs (n=4, p=0.02). Hepatic bile from 5-6 month old Lox and LIRKO mice was subjected to HPLC analysis as described in Methods (n=4-5). (c) Hydrophobicity indices were calculated as described in Methods (*p=0.002). (d) The ratio of muricholates to cholate was calculated using the molar percentages of tauroα- and tauroβ- muricholate and taurocholate (*p=0.001). (e) Three-month old mice were gavaged with an FXR agonist (GW4064) at 100 mg/kg/d or vehicle for 14 days. Mice were fasted overnight, and sacrificed two hours after the last dose of agonist. Bile was expressed from the gallbladder and ratio of muricholates to cholate was determined as above. *p
    Cdna Synthesis, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 15507 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa complementary dna cdna synthesis kit
    Effects of DCA on mRNA and protein levels of OCTN2 in cancer cell lines. (A), analysis of mRNA levels of OCTN2 in DCA-treated cancer cell lines. After treatment with 0.5 and 1 μM DCA, total <t>RNA</t> in cancer cells was purified by TRIzol reagent for <t>cDNA</t> synthesis and the mRNA levels of OCTN2 were analyzed by Quantitative RT-PCR. (B), analysis of protein levels of OCTN2 in DCA-treated cancer cell lines. Total proteins were extracted and protein levels of OCTN2 were analyzed by western blotting. The relative expression of mRNA and protein was normalized to β-actin. Data presented represent the mean ± S.D. of three independent experiments. Significant difference from control group of 0.1% DMSO is denoted with asterisks (*, p
    Complementary Dna Cdna Synthesis Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad complementary dna cdna synthesis kit
    Breast tumor kinase expression in breast cancer cell lines. (a) Total RNA was isolated from the breast cancer cell lines using the RNeasy kit (Qiagen), and the <t>cDNA</t> was generated using <t>iScript</t> cDNA synthesis (Bio-Rad). The cDNA was amplified using primers specifically for breast tumor kinase (Brk) or β-actin. The relative level of Brk mRNA was determined using densitometric analysis and was normalized to the amount of β-actin. The level of Brk mRNA in the MDA-MB-468 cell line was set to 1; numbers indicate the fold increase compared with the 468 cells. (b) Whole-cell lysates were prepared from the breast cancer cell lines. Lysates were immunoblotted with anti-Brk antibody (top) and with anti-Ran antibody (bottom). The relative expression of Brk protein was determined using densitometric analysis and was normalized to the amount of Ran. The expression of Brk protein in the MDA-MB-468 cell line was set to 1; numbers indicate the fold increase compared with the 468 cells.
    Complementary Dna Cdna Synthesis Kit, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher complementary dna cdna
    Breast tumor kinase expression in breast cancer cell lines. (a) Total RNA was isolated from the breast cancer cell lines using the RNeasy kit (Qiagen), and the <t>cDNA</t> was generated using <t>iScript</t> cDNA synthesis (Bio-Rad). The cDNA was amplified using primers specifically for breast tumor kinase (Brk) or β-actin. The relative level of Brk mRNA was determined using densitometric analysis and was normalized to the amount of β-actin. The level of Brk mRNA in the MDA-MB-468 cell line was set to 1; numbers indicate the fold increase compared with the 468 cells. (b) Whole-cell lysates were prepared from the breast cancer cell lines. Lysates were immunoblotted with anti-Brk antibody (top) and with anti-Ran antibody (bottom). The relative expression of Brk protein was determined using densitometric analysis and was normalized to the amount of Ran. The expression of Brk protein in the MDA-MB-468 cell line was set to 1; numbers indicate the fold increase compared with the 468 cells.
    Complementary Dna Cdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 26028 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Quanta Biosciences superscript complementary dna cdna synthesis kit
    Breast tumor kinase expression in breast cancer cell lines. (a) Total RNA was isolated from the breast cancer cell lines using the RNeasy kit (Qiagen), and the <t>cDNA</t> was generated using <t>iScript</t> cDNA synthesis (Bio-Rad). The cDNA was amplified using primers specifically for breast tumor kinase (Brk) or β-actin. The relative level of Brk mRNA was determined using densitometric analysis and was normalized to the amount of β-actin. The level of Brk mRNA in the MDA-MB-468 cell line was set to 1; numbers indicate the fold increase compared with the 468 cells. (b) Whole-cell lysates were prepared from the breast cancer cell lines. Lysates were immunoblotted with anti-Brk antibody (top) and with anti-Ran antibody (bottom). The relative expression of Brk protein was determined using densitometric analysis and was normalized to the amount of Ran. The expression of Brk protein in the MDA-MB-468 cell line was set to 1; numbers indicate the fold increase compared with the 468 cells.
    Superscript Complementary Dna Cdna Synthesis Kit, supplied by Quanta Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Quanta Biosciences cdna synthesis kit
    Breast tumor kinase expression in breast cancer cell lines. (a) Total RNA was isolated from the breast cancer cell lines using the RNeasy kit (Qiagen), and the <t>cDNA</t> was generated using <t>iScript</t> cDNA synthesis (Bio-Rad). The cDNA was amplified using primers specifically for breast tumor kinase (Brk) or β-actin. The relative level of Brk mRNA was determined using densitometric analysis and was normalized to the amount of β-actin. The level of Brk mRNA in the MDA-MB-468 cell line was set to 1; numbers indicate the fold increase compared with the 468 cells. (b) Whole-cell lysates were prepared from the breast cancer cell lines. Lysates were immunoblotted with anti-Brk antibody (top) and with anti-Ran antibody (bottom). The relative expression of Brk protein was determined using densitometric analysis and was normalized to the amount of Ran. The expression of Brk protein in the MDA-MB-468 cell line was set to 1; numbers indicate the fold increase compared with the 468 cells.
    Cdna Synthesis Kit, supplied by Quanta Biosciences, used in various techniques. Bioz Stars score: 93/100, based on 332 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche cdna synthesis kit
    Messenger <t>RNA</t> expression of TNC receptors of MCF-7 cells after TGF-β and TNC treatments. ( a ) mRNA was extracted from cells co-stimulated with TNC (10 μg/ml) and TGF-β1 (5 ng/ml) for synthesis of <t>cDNA</t> and performance of real-time PCR determining the cycle threshold ( C t ) values for each subunit. Differences in C t values of TNC receptor mRNAs from that of GAPDH mRNA in TGF-β1/TNC-treated cells are shown using qPCR. C t values of α7/8/9 and β3 integrin subunits were very high, indicating only limited expression. Calibration curves were obtained using each target gene in the samples. ( b ) Changes of receptor mRNA expression for αv/2 and β1/6 after treatment with TGF-β1 and/or TNC. The results were normalized to GAPDH expression. Integrin αv showed significant increase after TGF-β1/TNC treatment and β6 was dramatically increased after TGF-β1 addition, with or without TNC (mean±s.d.; * P
    Cdna Synthesis Kit, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 1387 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene complementary dna cdna synthesis complementary dna
    Messenger <t>RNA</t> expression of TNC receptors of MCF-7 cells after TGF-β and TNC treatments. ( a ) mRNA was extracted from cells co-stimulated with TNC (10 μg/ml) and TGF-β1 (5 ng/ml) for synthesis of <t>cDNA</t> and performance of real-time PCR determining the cycle threshold ( C t ) values for each subunit. Differences in C t values of TNC receptor mRNAs from that of GAPDH mRNA in TGF-β1/TNC-treated cells are shown using qPCR. C t values of α7/8/9 and β3 integrin subunits were very high, indicating only limited expression. Calibration curves were obtained using each target gene in the samples. ( b ) Changes of receptor mRNA expression for αv/2 and β1/6 after treatment with TGF-β1 and/or TNC. The results were normalized to GAPDH expression. Integrin αv showed significant increase after TGF-β1/TNC treatment and β6 was dramatically increased after TGF-β1 addition, with or without TNC (mean±s.d.; * P
    Complementary Dna Cdna Synthesis Complementary Dna, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher complementary dna cdna synthesis supermix kit
    Messenger <t>RNA</t> expression of TNC receptors of MCF-7 cells after TGF-β and TNC treatments. ( a ) mRNA was extracted from cells co-stimulated with TNC (10 μg/ml) and TGF-β1 (5 ng/ml) for synthesis of <t>cDNA</t> and performance of real-time PCR determining the cycle threshold ( C t ) values for each subunit. Differences in C t values of TNC receptor mRNAs from that of GAPDH mRNA in TGF-β1/TNC-treated cells are shown using qPCR. C t values of α7/8/9 and β3 integrin subunits were very high, indicating only limited expression. Calibration curves were obtained using each target gene in the samples. ( b ) Changes of receptor mRNA expression for αv/2 and β1/6 after treatment with TGF-β1 and/or TNC. The results were normalized to GAPDH expression. Integrin αv showed significant increase after TGF-β1/TNC treatment and β6 was dramatically increased after TGF-β1 addition, with or without TNC (mean±s.d.; * P
    Complementary Dna Cdna Synthesis Supermix Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TransGen biotech co cdna synthesis kit
    Messenger <t>RNA</t> expression of TNC receptors of MCF-7 cells after TGF-β and TNC treatments. ( a ) mRNA was extracted from cells co-stimulated with TNC (10 μg/ml) and TGF-β1 (5 ng/ml) for synthesis of <t>cDNA</t> and performance of real-time PCR determining the cycle threshold ( C t ) values for each subunit. Differences in C t values of TNC receptor mRNAs from that of GAPDH mRNA in TGF-β1/TNC-treated cells are shown using qPCR. C t values of α7/8/9 and β3 integrin subunits were very high, indicating only limited expression. Calibration curves were obtained using each target gene in the samples. ( b ) Changes of receptor mRNA expression for αv/2 and β1/6 after treatment with TGF-β1 and/or TNC. The results were normalized to GAPDH expression. Integrin αv showed significant increase after TGF-β1/TNC treatment and β6 was dramatically increased after TGF-β1 addition, with or without TNC (mean±s.d.; * P
    Cdna Synthesis Kit, supplied by TransGen biotech co, used in various techniques. Bioz Stars score: 93/100, based on 179 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene cdna synthesis kit
    The nucleotide and deduced amino acid sequences of Fus p 4.0101 (GenBank accession no. KF151224). Numbers to the right are the positions of the nucleotides and the deduced residues of the sequences. The stop codon TAA is denoted with an asterisk (*). The potential N-glycosylation site is indicated in bold letters and boxed. Eight conserved amino acid residues that are involved in the catalysis and the substrate binding of transaldolases are shaded and in bold letters. Nucleotide sequences in grey correspond to primer sequences synthesized for <t>PCR</t> experiments in the <t>cDNA</t> cloning of Fus p 4.0101 as described in the Materials and Methods. The sequences corresponding to primers Fu-TAase-f and Fu-TAase-r used in the preparation of rFus p 4.0101 are boxed.
    Cdna Synthesis Kit, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 1002 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies cdna synthesis kit
    Identification of Oplr16 as a pluripotent lncRNA. ( A ) Identification of Oplr16 as a pluripotency-associated lncRNA by integrating RNA-seq and CRIST-seq. RNA-seq was used to identify RNAs that are differentially expressed between fibroblasts and iPSCs collected in the process of reprogramming. In CRIST-seq, the Oct4 promoter was targeted by the expression of the Cas9 Oct4 -gRNA. LncRNAs interacting with Oct4 promoter were reverse transcribed in situ into <t>cDNA</t> with biotin-dCTP. After immunoprecipitation, biotin-cDNAs were purified by streptavidin beads for <t>Illumina</t> sequencing. Both sets of databases were integrated to identify the pluripotency-associated lncRNA Oplr16 . ( B ) Reactivation of Oplr16 in reprogramming. Fibroblasts were transfected with a Oct4-Sox2-Kilf4-c-Myc (OSKM) lentivirus. Cells were collected at various stages of reprogramming and expression of Oplr16 was measured by qPCR. E14: mouse embryonic pluripotent stem cell line used as a positive control; iPSC: induced pluripotent stem cells; non-iPSC: un-reprogrammed cells that express four OSKM factors, but fail to complete reprogramming; FBC: fibroblasts. β-Actin was used as the PCR control. ** P
    Cdna Synthesis Kit, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 732 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Tissue expression of hMSH6 mRNA. A human multiple-tissue Northern blot (CLONTECH) was probed with the 32 P-labeled complete cDNA clone of hMSH6 according to the manufacturer’s protocol. Colon refers to mucosal lining; p.b. leukocyte refers to peripheral blood leukocyte. The hMSH6 transcript is indicated by the arrow and corresponds to ≈4.5 kb. The amount of RNA loaded in each lane was adjusted to comparable levels as judged spectrophotometrically and by the levels of actin present (CLONTECH).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: hMSH2 forms specific mispair-binding complexes with hMSH3 and hMSH6

    doi:

    Figure Lengend Snippet: Tissue expression of hMSH6 mRNA. A human multiple-tissue Northern blot (CLONTECH) was probed with the 32 P-labeled complete cDNA clone of hMSH6 according to the manufacturer’s protocol. Colon refers to mucosal lining; p.b. leukocyte refers to peripheral blood leukocyte. The hMSH6 transcript is indicated by the arrow and corresponds to ≈4.5 kb. The amount of RNA loaded in each lane was adjusted to comparable levels as judged spectrophotometrically and by the levels of actin present (CLONTECH).

    Article Snippet: Single-strand, random-primed cDNA was prepared from human testis poly(A) mRNA (CLONTECH) using a Promega cDNA synthesis kit.

    Techniques: Expressing, Northern Blot, Labeling

    Organization of hMSH6 genomic locus and sequence of the intron region flanking each MSH6 exon. Boxes containing numbers 1 through 10 indicate the individual MSH6 exons and their relative sizes. The size of each exon is given below each exon and the size of each intron is given above the region between each pair of exons. The sizes of exons 1 and 10 are the sizes of the mRNA sequence upstream and downstream of the first and last introns, respectively; these sizes were calculated from the longest MSH6 cDNA sequence available. The first 20 nucleotides of each intron sequence up to the intron-exon junction is given in uppercase letters except for the 3′ side of the last intron, where additional sequence is given. The first 10 nucleotides of each exon sequence up to the intron-exon junction is given in lowercase letters. In addition, the sequence of the 5′ and 3′ ends of the cDNA, minus the poly(A) sequence, is also given in lowercase letters. The numbers in parentheses between intron sequences are the nucleotide coordinates of the exon sequences or cDNA sequences, assuming the A of the ATG is nucleotide 1. (Additional intron sequence including the complete sequence of some introns has been determined in all cases and is available on request from M.F.K. and R.K.)

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: hMSH2 forms specific mispair-binding complexes with hMSH3 and hMSH6

    doi:

    Figure Lengend Snippet: Organization of hMSH6 genomic locus and sequence of the intron region flanking each MSH6 exon. Boxes containing numbers 1 through 10 indicate the individual MSH6 exons and their relative sizes. The size of each exon is given below each exon and the size of each intron is given above the region between each pair of exons. The sizes of exons 1 and 10 are the sizes of the mRNA sequence upstream and downstream of the first and last introns, respectively; these sizes were calculated from the longest MSH6 cDNA sequence available. The first 20 nucleotides of each intron sequence up to the intron-exon junction is given in uppercase letters except for the 3′ side of the last intron, where additional sequence is given. The first 10 nucleotides of each exon sequence up to the intron-exon junction is given in lowercase letters. In addition, the sequence of the 5′ and 3′ ends of the cDNA, minus the poly(A) sequence, is also given in lowercase letters. The numbers in parentheses between intron sequences are the nucleotide coordinates of the exon sequences or cDNA sequences, assuming the A of the ATG is nucleotide 1. (Additional intron sequence including the complete sequence of some introns has been determined in all cases and is available on request from M.F.K. and R.K.)

    Article Snippet: Single-strand, random-primed cDNA was prepared from human testis poly(A) mRNA (CLONTECH) using a Promega cDNA synthesis kit.

    Techniques: Sequencing

    Expression levels of type I collagen, Smad2/3, and TGF-β1 genes after ES-P treatment in aged mice. (A) The left ears of fourteen-week old mice were treated daily with 30 μg T . spiralis ES-P for 14 days (red color). The right ears of were treated daily with PBS for 14 days as control (blue color). After 14 days, ears were collected. Total RNA was extracted from each ear tissue and cDNA was constructed. (B) The gene expression levels of collagen I , TGF-β1 , and Smad2/3 were analyzed by real-time PCR. The gene expression value of each group was normalized by control value, 14 weeks group. (**; P

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Identification of a host collagen inducing factor from the excretory secretory proteins of Trichinella spiralis

    doi: 10.1371/journal.pntd.0006516

    Figure Lengend Snippet: Expression levels of type I collagen, Smad2/3, and TGF-β1 genes after ES-P treatment in aged mice. (A) The left ears of fourteen-week old mice were treated daily with 30 μg T . spiralis ES-P for 14 days (red color). The right ears of were treated daily with PBS for 14 days as control (blue color). After 14 days, ears were collected. Total RNA was extracted from each ear tissue and cDNA was constructed. (B) The gene expression levels of collagen I , TGF-β1 , and Smad2/3 were analyzed by real-time PCR. The gene expression value of each group was normalized by control value, 14 weeks group. (**; P

    Article Snippet: Real-time PCR Homogenized ear tissues were mixed with TRIzol (Invitrogen, Germany), and total RNA extraction and cDNA synthesis (Invitrogen, Germany) was performed in accordance with the manufacturer’s protocols.

    Techniques: Expressing, Mouse Assay, Construct, Real-time Polymerase Chain Reaction

    The Total RNA Detection system. A universal RNA adaptor was firstly added to the 3′ ends of all RNA templates. The abundant 18S and 28S ribosome RNAs were then subtracted by using biotinylated ribosomal-specific probes. Small-size RNAs containing the degraded RNA intermediates were removed by size-filtration. The enriched transcripts were converted into double-strand cDNA by using the 3′ end RNA adaptor-based primer. The cDNAs were further digested by NlaIII. The 3′ cDNAs were isolated by using the streptoavidin beads. An adaptor was added to the 5′ ends of the 3′ cDNAs. The 3′ cDNAs were then amplified by PCR using the 5′ adaptor-based sense primer and the 3′ end RNA adaptor-based antisense primer. The amplified 3′ cDNAs were sequenced from the 3′ end by the 454 system. See further details in Materials and Methods .

    Journal: PLoS ONE

    Article Title: Poly A- Transcripts Expressed in HeLa Cells

    doi: 10.1371/journal.pone.0002803

    Figure Lengend Snippet: The Total RNA Detection system. A universal RNA adaptor was firstly added to the 3′ ends of all RNA templates. The abundant 18S and 28S ribosome RNAs were then subtracted by using biotinylated ribosomal-specific probes. Small-size RNAs containing the degraded RNA intermediates were removed by size-filtration. The enriched transcripts were converted into double-strand cDNA by using the 3′ end RNA adaptor-based primer. The cDNAs were further digested by NlaIII. The 3′ cDNAs were isolated by using the streptoavidin beads. An adaptor was added to the 5′ ends of the 3′ cDNAs. The 3′ cDNAs were then amplified by PCR using the 5′ adaptor-based sense primer and the 3′ end RNA adaptor-based antisense primer. The amplified 3′ cDNAs were sequenced from the 3′ end by the 454 system. See further details in Materials and Methods .

    Article Snippet: cDNA synthesis and 3′ ESTs collection The enriched RNA was converted into double-strand cDNA by using a cDNA synthesis kit (Invitrogen) following the manufacturer's protocol, except for using the biotin-labeled primer based on the 3′ end adaptor sequences for the priming ( 5′ biotin-ATCTAGAGCGGCCGCAATGGCCACTCTGCGTTGATAC ).

    Techniques: RNA Detection, Filtration, Isolation, Amplification, Polymerase Chain Reaction

    LIRKO mice show quantitative and qualitative defects in bile acid synthesis on a chow diet (a) Real-time PCR analysis of cDNA prepared from six-week old mice sacrificed during the light cycle following a 24 hour fast; the expression of each gene was normalized to its value in Lox mice (n=5-8, *p=0.05 versus Lox). (b) Since the synthesis of bile acids is equivalent to fecal excretion in the steady state, we assessed bile acid synthesis by collecting feces from 3 month old Lox and LIRKO mice for 72 hours and measuring total bile acid outputs (n=4, p=0.02). Hepatic bile from 5-6 month old Lox and LIRKO mice was subjected to HPLC analysis as described in Methods (n=4-5). (c) Hydrophobicity indices were calculated as described in Methods (*p=0.002). (d) The ratio of muricholates to cholate was calculated using the molar percentages of tauroα- and tauroβ- muricholate and taurocholate (*p=0.001). (e) Three-month old mice were gavaged with an FXR agonist (GW4064) at 100 mg/kg/d or vehicle for 14 days. Mice were fasted overnight, and sacrificed two hours after the last dose of agonist. Bile was expressed from the gallbladder and ratio of muricholates to cholate was determined as above. *p

    Journal: Nature medicine

    Article Title: Hepatic Insulin Resistance Directly Promotes Formation of Cholesterol Gallstones

    doi: 10.1038/nm1785

    Figure Lengend Snippet: LIRKO mice show quantitative and qualitative defects in bile acid synthesis on a chow diet (a) Real-time PCR analysis of cDNA prepared from six-week old mice sacrificed during the light cycle following a 24 hour fast; the expression of each gene was normalized to its value in Lox mice (n=5-8, *p=0.05 versus Lox). (b) Since the synthesis of bile acids is equivalent to fecal excretion in the steady state, we assessed bile acid synthesis by collecting feces from 3 month old Lox and LIRKO mice for 72 hours and measuring total bile acid outputs (n=4, p=0.02). Hepatic bile from 5-6 month old Lox and LIRKO mice was subjected to HPLC analysis as described in Methods (n=4-5). (c) Hydrophobicity indices were calculated as described in Methods (*p=0.002). (d) The ratio of muricholates to cholate was calculated using the molar percentages of tauroα- and tauroβ- muricholate and taurocholate (*p=0.001). (e) Three-month old mice were gavaged with an FXR agonist (GW4064) at 100 mg/kg/d or vehicle for 14 days. Mice were fasted overnight, and sacrificed two hours after the last dose of agonist. Bile was expressed from the gallbladder and ratio of muricholates to cholate was determined as above. *p

    Article Snippet: In all other experiments, RNA (RNeasy, Qiagen) was isolated from mouse liver and used to direct cDNA synthesis (RT for PCR, Clontech).

    Techniques: Mouse Assay, Real-time Polymerase Chain Reaction, Expressing, High Performance Liquid Chromatography

    Effects of DCA on mRNA and protein levels of OCTN2 in cancer cell lines. (A), analysis of mRNA levels of OCTN2 in DCA-treated cancer cell lines. After treatment with 0.5 and 1 μM DCA, total RNA in cancer cells was purified by TRIzol reagent for cDNA synthesis and the mRNA levels of OCTN2 were analyzed by Quantitative RT-PCR. (B), analysis of protein levels of OCTN2 in DCA-treated cancer cell lines. Total proteins were extracted and protein levels of OCTN2 were analyzed by western blotting. The relative expression of mRNA and protein was normalized to β-actin. Data presented represent the mean ± S.D. of three independent experiments. Significant difference from control group of 0.1% DMSO is denoted with asterisks (*, p

    Journal: PLoS ONE

    Article Title: Different Involvement of Promoter Methylation in the Expression of Organic Cation/Carnitine Transporter 2 (OCTN2) in Cancer Cell Lines

    doi: 10.1371/journal.pone.0076474

    Figure Lengend Snippet: Effects of DCA on mRNA and protein levels of OCTN2 in cancer cell lines. (A), analysis of mRNA levels of OCTN2 in DCA-treated cancer cell lines. After treatment with 0.5 and 1 μM DCA, total RNA in cancer cells was purified by TRIzol reagent for cDNA synthesis and the mRNA levels of OCTN2 were analyzed by Quantitative RT-PCR. (B), analysis of protein levels of OCTN2 in DCA-treated cancer cell lines. Total proteins were extracted and protein levels of OCTN2 were analyzed by western blotting. The relative expression of mRNA and protein was normalized to β-actin. Data presented represent the mean ± S.D. of three independent experiments. Significant difference from control group of 0.1% DMSO is denoted with asterisks (*, p

    Article Snippet: RNA purification, cDNA Synthesis and Quantitative Real-time PCR Total cellular RNA was isolated from the treated cells using TRIzol reagent. cDNA was synthesized from 1 μg of total RNA using the first cDNA synthesis kit (Takara, Dalian).

    Techniques: Purification, Quantitative RT-PCR, Western Blot, Expressing

    Expression of OCTN2 in cancer cell lines. (A), analysis of mRNA levels of OCTN2 in cancer cell lines. Total RNA in cancer cells was purified by TRIzol reagent for cDNA synthesis and the mRNA levels of OCTN2 were analyzed by Quantitative RT-PCR. (B), analysis of protein levels of OCTN2 in cancer cells. Total proteins were extracted and protein levels of OCTN2 were analyzed by western blotting. The relative expression of mRNA and protein was normalized to β-actin. The expression of HepG2 was set as 1. All of mRNA and protein analysis were performed three times. Significant difference from HepG2 or LS174T cell is denoted with asterisk (*, p

    Journal: PLoS ONE

    Article Title: Different Involvement of Promoter Methylation in the Expression of Organic Cation/Carnitine Transporter 2 (OCTN2) in Cancer Cell Lines

    doi: 10.1371/journal.pone.0076474

    Figure Lengend Snippet: Expression of OCTN2 in cancer cell lines. (A), analysis of mRNA levels of OCTN2 in cancer cell lines. Total RNA in cancer cells was purified by TRIzol reagent for cDNA synthesis and the mRNA levels of OCTN2 were analyzed by Quantitative RT-PCR. (B), analysis of protein levels of OCTN2 in cancer cells. Total proteins were extracted and protein levels of OCTN2 were analyzed by western blotting. The relative expression of mRNA and protein was normalized to β-actin. The expression of HepG2 was set as 1. All of mRNA and protein analysis were performed three times. Significant difference from HepG2 or LS174T cell is denoted with asterisk (*, p

    Article Snippet: RNA purification, cDNA Synthesis and Quantitative Real-time PCR Total cellular RNA was isolated from the treated cells using TRIzol reagent. cDNA was synthesized from 1 μg of total RNA using the first cDNA synthesis kit (Takara, Dalian).

    Techniques: Expressing, Purification, Quantitative RT-PCR, Western Blot

    Breast tumor kinase expression in breast cancer cell lines. (a) Total RNA was isolated from the breast cancer cell lines using the RNeasy kit (Qiagen), and the cDNA was generated using iScript cDNA synthesis (Bio-Rad). The cDNA was amplified using primers specifically for breast tumor kinase (Brk) or β-actin. The relative level of Brk mRNA was determined using densitometric analysis and was normalized to the amount of β-actin. The level of Brk mRNA in the MDA-MB-468 cell line was set to 1; numbers indicate the fold increase compared with the 468 cells. (b) Whole-cell lysates were prepared from the breast cancer cell lines. Lysates were immunoblotted with anti-Brk antibody (top) and with anti-Ran antibody (bottom). The relative expression of Brk protein was determined using densitometric analysis and was normalized to the amount of Ran. The expression of Brk protein in the MDA-MB-468 cell line was set to 1; numbers indicate the fold increase compared with the 468 cells.

    Journal: Breast Cancer Research : BCR

    Article Title: Signal transducer and activator of transcription 5b: a new target of breast tumor kinase/protein tyrosine kinase 6

    doi: 10.1186/bcr1794

    Figure Lengend Snippet: Breast tumor kinase expression in breast cancer cell lines. (a) Total RNA was isolated from the breast cancer cell lines using the RNeasy kit (Qiagen), and the cDNA was generated using iScript cDNA synthesis (Bio-Rad). The cDNA was amplified using primers specifically for breast tumor kinase (Brk) or β-actin. The relative level of Brk mRNA was determined using densitometric analysis and was normalized to the amount of β-actin. The level of Brk mRNA in the MDA-MB-468 cell line was set to 1; numbers indicate the fold increase compared with the 468 cells. (b) Whole-cell lysates were prepared from the breast cancer cell lines. Lysates were immunoblotted with anti-Brk antibody (top) and with anti-Ran antibody (bottom). The relative expression of Brk protein was determined using densitometric analysis and was normalized to the amount of Ran. The expression of Brk protein in the MDA-MB-468 cell line was set to 1; numbers indicate the fold increase compared with the 468 cells.

    Article Snippet: The cDNA was generated using iScript cDNA synthesis (Bio-Rad).

    Techniques: Expressing, Isolation, Generated, Amplification, Multiple Displacement Amplification

    Messenger RNA expression of TNC receptors of MCF-7 cells after TGF-β and TNC treatments. ( a ) mRNA was extracted from cells co-stimulated with TNC (10 μg/ml) and TGF-β1 (5 ng/ml) for synthesis of cDNA and performance of real-time PCR determining the cycle threshold ( C t ) values for each subunit. Differences in C t values of TNC receptor mRNAs from that of GAPDH mRNA in TGF-β1/TNC-treated cells are shown using qPCR. C t values of α7/8/9 and β3 integrin subunits were very high, indicating only limited expression. Calibration curves were obtained using each target gene in the samples. ( b ) Changes of receptor mRNA expression for αv/2 and β1/6 after treatment with TGF-β1 and/or TNC. The results were normalized to GAPDH expression. Integrin αv showed significant increase after TGF-β1/TNC treatment and β6 was dramatically increased after TGF-β1 addition, with or without TNC (mean±s.d.; * P

    Journal: Oncogenesis

    Article Title: Binding of ?v?1 and ?v?6 integrins to tenascin-C induces epithelial-mesenchymal transition-like change of breast cancer cells

    doi: 10.1038/oncsis.2013.27

    Figure Lengend Snippet: Messenger RNA expression of TNC receptors of MCF-7 cells after TGF-β and TNC treatments. ( a ) mRNA was extracted from cells co-stimulated with TNC (10 μg/ml) and TGF-β1 (5 ng/ml) for synthesis of cDNA and performance of real-time PCR determining the cycle threshold ( C t ) values for each subunit. Differences in C t values of TNC receptor mRNAs from that of GAPDH mRNA in TGF-β1/TNC-treated cells are shown using qPCR. C t values of α7/8/9 and β3 integrin subunits were very high, indicating only limited expression. Calibration curves were obtained using each target gene in the samples. ( b ) Changes of receptor mRNA expression for αv/2 and β1/6 after treatment with TGF-β1 and/or TNC. The results were normalized to GAPDH expression. Integrin αv showed significant increase after TGF-β1/TNC treatment and β6 was dramatically increased after TGF-β1 addition, with or without TNC (mean±s.d.; * P

    Article Snippet: Total RNA (1 μg) was reverse-transcribed using a cDNA synthesis kit (Roche Diagnostics) with anchored-oligo (dT)18 primers.

    Techniques: RNA Expression, Real-time Polymerase Chain Reaction, Expressing

    The nucleotide and deduced amino acid sequences of Fus p 4.0101 (GenBank accession no. KF151224). Numbers to the right are the positions of the nucleotides and the deduced residues of the sequences. The stop codon TAA is denoted with an asterisk (*). The potential N-glycosylation site is indicated in bold letters and boxed. Eight conserved amino acid residues that are involved in the catalysis and the substrate binding of transaldolases are shaded and in bold letters. Nucleotide sequences in grey correspond to primer sequences synthesized for PCR experiments in the cDNA cloning of Fus p 4.0101 as described in the Materials and Methods. The sequences corresponding to primers Fu-TAase-f and Fu-TAase-r used in the preparation of rFus p 4.0101 are boxed.

    Journal: PLoS ONE

    Article Title: The Transaldolase, a Novel Allergen of Fusarium proliferatum, Demonstrates IgE Cross-Reactivity with Its Human Analogue

    doi: 10.1371/journal.pone.0103488

    Figure Lengend Snippet: The nucleotide and deduced amino acid sequences of Fus p 4.0101 (GenBank accession no. KF151224). Numbers to the right are the positions of the nucleotides and the deduced residues of the sequences. The stop codon TAA is denoted with an asterisk (*). The potential N-glycosylation site is indicated in bold letters and boxed. Eight conserved amino acid residues that are involved in the catalysis and the substrate binding of transaldolases are shaded and in bold letters. Nucleotide sequences in grey correspond to primer sequences synthesized for PCR experiments in the cDNA cloning of Fus p 4.0101 as described in the Materials and Methods. The sequences corresponding to primers Fu-TAase-f and Fu-TAase-r used in the preparation of rFus p 4.0101 are boxed.

    Article Snippet: cDNA Cloning The cDNA encoding the F. proliferatum transaldolase was isolated with polymerase chain reactions (PCR) using an AffinityScript Multiple Temperature cDNA Synthesis kit (Stratagene, La Jolla, Calif., USA) as previously described , .

    Techniques: Binding Assay, Synthesized, Polymerase Chain Reaction, Clone Assay

    Identification of Oplr16 as a pluripotent lncRNA. ( A ) Identification of Oplr16 as a pluripotency-associated lncRNA by integrating RNA-seq and CRIST-seq. RNA-seq was used to identify RNAs that are differentially expressed between fibroblasts and iPSCs collected in the process of reprogramming. In CRIST-seq, the Oct4 promoter was targeted by the expression of the Cas9 Oct4 -gRNA. LncRNAs interacting with Oct4 promoter were reverse transcribed in situ into cDNA with biotin-dCTP. After immunoprecipitation, biotin-cDNAs were purified by streptavidin beads for Illumina sequencing. Both sets of databases were integrated to identify the pluripotency-associated lncRNA Oplr16 . ( B ) Reactivation of Oplr16 in reprogramming. Fibroblasts were transfected with a Oct4-Sox2-Kilf4-c-Myc (OSKM) lentivirus. Cells were collected at various stages of reprogramming and expression of Oplr16 was measured by qPCR. E14: mouse embryonic pluripotent stem cell line used as a positive control; iPSC: induced pluripotent stem cells; non-iPSC: un-reprogrammed cells that express four OSKM factors, but fail to complete reprogramming; FBC: fibroblasts. β-Actin was used as the PCR control. ** P

    Journal: Nucleic Acids Research

    Article Title: Oplr16 serves as a novel chromatin factor to control stem cell fate by modulating pluripotency-specific chromosomal looping and TET2-mediated DNA demethylation

    doi: 10.1093/nar/gkaa097

    Figure Lengend Snippet: Identification of Oplr16 as a pluripotent lncRNA. ( A ) Identification of Oplr16 as a pluripotency-associated lncRNA by integrating RNA-seq and CRIST-seq. RNA-seq was used to identify RNAs that are differentially expressed between fibroblasts and iPSCs collected in the process of reprogramming. In CRIST-seq, the Oct4 promoter was targeted by the expression of the Cas9 Oct4 -gRNA. LncRNAs interacting with Oct4 promoter were reverse transcribed in situ into cDNA with biotin-dCTP. After immunoprecipitation, biotin-cDNAs were purified by streptavidin beads for Illumina sequencing. Both sets of databases were integrated to identify the pluripotency-associated lncRNA Oplr16 . ( B ) Reactivation of Oplr16 in reprogramming. Fibroblasts were transfected with a Oct4-Sox2-Kilf4-c-Myc (OSKM) lentivirus. Cells were collected at various stages of reprogramming and expression of Oplr16 was measured by qPCR. E14: mouse embryonic pluripotent stem cell line used as a positive control; iPSC: induced pluripotent stem cells; non-iPSC: un-reprogrammed cells that express four OSKM factors, but fail to complete reprogramming; FBC: fibroblasts. β-Actin was used as the PCR control. ** P

    Article Snippet: The second strand cDNA was synthesized by using Stratagene cDNA Synthesis kit (Agilent Technologies, CA, USA) for Illumina lnc-cDNA sequencing.

    Techniques: RNA Sequencing Assay, Expressing, In Situ, Immunoprecipitation, Purification, Sequencing, Transfection, Real-time Polymerase Chain Reaction, Positive Control, Polymerase Chain Reaction

    Oplr16 binds to the Oct4 promoter. ( A ) Schematic diagram of the RNA reverse transcription-associated trap sequencing (RAT-seq) assay. Oplr16 lncRNA was in situ reverse transcribed using three Oplr16 -specific complementary primers with biotin-dCTP. The random primers were used as the negative control (RAT-CT). After nuclear lysis, the bitoin- Oplr16 cDNA chromatin complex was isolated by streptavidin beads and the Oplr16 -interacting target DNAs were isolated for Illumina library sequencing. ( B ) The IGV analysis of Oplr16 binding signals in the Oct4 locus. 5′-Enh, 3′-Enh: the Oct4 5′- and 3′-enhancers; pOct4: Oct4 promoter; E1-E5: Oct4 exons. ( C ) Quantitative PCR mapping of Oplr16 binding in the Oct4 locus. The RAT pulldown complex was used to map the Oplr16 binding. 5′-CT, 3′-CT: the RAT control sites in the Oct4 locus. Note the enrichment of the Oplr16 binding signals in the promoter region (Prot-1 and Prot-2) ( N = 9, ** P

    Journal: Nucleic Acids Research

    Article Title: Oplr16 serves as a novel chromatin factor to control stem cell fate by modulating pluripotency-specific chromosomal looping and TET2-mediated DNA demethylation

    doi: 10.1093/nar/gkaa097

    Figure Lengend Snippet: Oplr16 binds to the Oct4 promoter. ( A ) Schematic diagram of the RNA reverse transcription-associated trap sequencing (RAT-seq) assay. Oplr16 lncRNA was in situ reverse transcribed using three Oplr16 -specific complementary primers with biotin-dCTP. The random primers were used as the negative control (RAT-CT). After nuclear lysis, the bitoin- Oplr16 cDNA chromatin complex was isolated by streptavidin beads and the Oplr16 -interacting target DNAs were isolated for Illumina library sequencing. ( B ) The IGV analysis of Oplr16 binding signals in the Oct4 locus. 5′-Enh, 3′-Enh: the Oct4 5′- and 3′-enhancers; pOct4: Oct4 promoter; E1-E5: Oct4 exons. ( C ) Quantitative PCR mapping of Oplr16 binding in the Oct4 locus. The RAT pulldown complex was used to map the Oplr16 binding. 5′-CT, 3′-CT: the RAT control sites in the Oct4 locus. Note the enrichment of the Oplr16 binding signals in the promoter region (Prot-1 and Prot-2) ( N = 9, ** P

    Article Snippet: The second strand cDNA was synthesized by using Stratagene cDNA Synthesis kit (Agilent Technologies, CA, USA) for Illumina lnc-cDNA sequencing.

    Techniques: Sequencing, In Situ, Negative Control, Lysis, Isolation, Binding Assay, Real-time Polymerase Chain Reaction