total rna Thermo Fisher Search Results


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  • 95
    Jena Bioscience total rna extraction
    Total Rna Extraction, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 95/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher control rna
    The expression of PAQR3 partially rescued <t>miR-137-enhanced</t> cell proliferation and invasion. (A) Western blot analysis showed that transfection of small interfering <t>RNA</t> against PAQR3 into T24 cells led to dramatically decreased PAQR3 protein expression. GAPDH was also detected as a loading control. (B) Silencing of PAQR3 significantly enhanced the proliferation of bladder cancer cells. The growth index as assessed at 0, 24, 48 and 72 h. (C) Western blot analysis of PAQR3 in T24 cells co-transfected with either miR-137 mimic or scramble and 2.0 µg pCDNA- PAQR3 or pCDNA empty vector. GAPDH was also detected as a loading control. (D) Cell growth curves in T24 cells transfected with different combinations at 0, 24, 48 and 72 h. (E) Transwell analysis of T24 cells treated with different combinations. The relative ratio of invasive cells per field is shown right. *p
    Control Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 953 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher total rna
    Changes in the <t>RNA</t> and <t>DNA</t> content (fg cell −1 ) of each strain across temperatures, with different symbols for each level of supply C:P. Error bars represent one standard error of the mean.
    Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 477211 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pancreatic total rna
    Changes in the <t>RNA</t> and <t>DNA</t> content (fg cell −1 ) of each strain across temperatures, with different symbols for each level of supply C:P. Error bars represent one standard error of the mean.
    Pancreatic Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher label total rna
    Changes in the <t>RNA</t> and <t>DNA</t> content (fg cell −1 ) of each strain across temperatures, with different symbols for each level of supply C:P. Error bars represent one standard error of the mean.
    Label Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher total rna chemistry
    Changes in the <t>RNA</t> and <t>DNA</t> content (fg cell −1 ) of each strain across temperatures, with different symbols for each level of supply C:P. Error bars represent one standard error of the mean.
    Total Rna Chemistry, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hl60 total rna
    Limit of blank study. ( a ) Summary of the MFI probe signals obtained from repeat testing with the MMA of a no <t>RNA</t> control sample, a total RNA sample purified from the translocation-negative <t>HL60</t> cell line, and eight total RNA samples purified from asymptomatic control donors' white blood cells (WBC control RNA). Minimum (MIN), maximum (MAX), median, mean and s.d. (STDEV) values for the 11 fusion-transcript-specific probes combined are shown for each sample type and overall. ( b ) Results by probe type for all sample types combined. The graph shows the mean, maximum (MAX) and twice the limit of blank (2 × LOB) values for each of the 11 fusion-transcript-specific probes relative to the qualitative 350 MFI cutoff value (dash line). The error bars represent the s.d. of each probe-specific distribution. The complete data set is presented in Supplementary Table 2 .
    Hl60 Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher quality total rna
    Limit of blank study. ( a ) Summary of the MFI probe signals obtained from repeat testing with the MMA of a no <t>RNA</t> control sample, a total RNA sample purified from the translocation-negative <t>HL60</t> cell line, and eight total RNA samples purified from asymptomatic control donors' white blood cells (WBC control RNA). Minimum (MIN), maximum (MAX), median, mean and s.d. (STDEV) values for the 11 fusion-transcript-specific probes combined are shown for each sample type and overall. ( b ) Results by probe type for all sample types combined. The graph shows the mean, maximum (MAX) and twice the limit of blank (2 × LOB) values for each of the 11 fusion-transcript-specific probes relative to the qualitative 350 MFI cutoff value (dash line). The error bars represent the s.d. of each probe-specific distribution. The complete data set is presented in Supplementary Table 2 .
    Quality Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 175 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher total rna kits
    HprK <t>Xcc</t> has a broad regulatory role in Xcc . Functional categories of differential expressed genes (DEGs) in hprK Xcc mutant ΔhprK Xcc . Genome‐scale transcriptome profiling of Xcc strains cultured in nutrition rich medium NYG were investigated by <t>RNA‐sequencing,</t> and 256 genes were found differentially expressed by two‐fold or more in hprK Xcc mutant (Table S2 ). These genes were broadly categorized according to their biological function (He et al ., 2007 ).
    Total Rna Kits, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher hela total rna
    <t>RNA</t> analysis following immunostaining. (A) An agarose gel of 5 µg <t>HeLa</t> total RNA (lane 1) and 5 µg HeLa total RNA incubated with primary antibody for anti–cytokeratin AE1/AE3 for 20 min (lane 2). (B) A BioAnalyzer image of total
    Hela Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher total rna isolation
    <t>RNA</t> analysis following immunostaining. (A) An agarose gel of 5 µg <t>HeLa</t> total RNA (lane 1) and 5 µg HeLa total RNA incubated with primary antibody for anti–cytokeratin AE1/AE3 for 20 min (lane 2). (B) A BioAnalyzer image of total
    Total Rna Isolation, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 145 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher total rna workflow
    <t>RNA</t> analysis following immunostaining. (A) An agarose gel of 5 µg <t>HeLa</t> total RNA (lane 1) and 5 µg HeLa total RNA incubated with primary antibody for anti–cytokeratin AE1/AE3 for 20 min (lane 2). (B) A BioAnalyzer image of total
    Total Rna Workflow, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ng total rna
    <t>RNA</t> analysis following immunostaining. (A) An agarose gel of 5 µg <t>HeLa</t> total RNA (lane 1) and 5 µg HeLa total RNA incubated with primary antibody for anti–cytokeratin AE1/AE3 for 20 min (lane 2). (B) A BioAnalyzer image of total
    Ng Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rats total rna
    Engrafted human umbilical cord-derived mesenchymal stem cells (HUMSCs) differentiate into osteoblasts and inhibit the number of osteoclasts at the distal femur. TRAP staining was performed to detect osteoclasts (arrows) in the metaphysis of the distal femur in the normal + phosphate-buffered saline (PBS; A), normal + HUMSCs (B), ovariectomy (OVX) + PBS (C), and OVX + HUMSCs (D) groups. The number of osteoclasts per trabecular bone area (no./mm 2 ) was quantified. The results indicated no difference in the osteoclast number among the normal + PBS, normal + HUMSCs, and OVX + HUMSCs groups. However, the osteoclast number was significantly higher in the OVX + PBS group than in the other 3 groups (E). Histological sections were immunologically stained with <t>anti-osteocalcin</t> antibody to identify osteocalcin expression near the trabeculae in the metaphysis in each group. The results revealed considerably osteocalcin expression around trabeculae in the normal + HUMSCs group (G). Some osteocalcin expression was detected in the normal + PBS and OVX + HUMSCs groups (F and I). In the OVX + PBS group, only slight expression was detected (H, arrows). Histological sections obtained from the left distal femur were stained with antihuman nuclei antibody to label HUMSCs. Human nuclei were found within the bone marrow cavity (J2) and trabeculae (J1). Double immunofluorescence revealed that numerous HUMSCs (rhodamine, red in K) and abundant osterix (fluorescein, green in L) existed in the rat femur. The merged image showed that many HUMSCs did differentiate into osterix-expressing osteoblasts (M). The results of RT-PCR further demonstrated that the grafted HUMSCs expressed human osteocalcin at the messenger <t>RNA</t> level (N). * Versus normal + PBS group, P
    Rats Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher human islet total rna
    Treatment of human islets with L-WRN+ regulates large numbers of proliferative genes. (A) mRNAs upregulated in response to L-WRN+ by 1.5-fold vs treatment with 8 mM glucose alone, in islets sourced from two different donors. Relative <t>mRNA</t> levels in 5 mM glucose (5), 8 mM glucose (8), 8 mM glucose+LiCl (8+L) and L-WRN+-treated islet aliquots are shown; blue indicates lower abundance, red indicates higher abundance. (B) as for (A), but showing those mRNAs downregulated by 1.5-fold in response to L-WRN+ treatment. (C) qRT-PCR validation of six regulated and four nonregulated mRNAs ( Mafa , Mnx1 , Neurod1 , Pax6 ); fold-changes of L-WRN+ treatment vs 8 mM glucose are shown. Individual gene expression values were normalized to the geometric mean of B2m , Gapdh , Hmbs and Ywhaz [70] . White and black bars, fold-changes observed with <t>RNA-sequencing;</t> red and blue bars, fold-changes observed with qRT-PCR. (D, upper) Classification of upregulated genes into Gene Ontology categories; the most significantly over-represented categories are shown. (D, lower) Classification of upregulated genes into MetaCore custom-defined categories. (E, upper and lower) as for (D), but showing downregulated genes. Further details of Gene Ontology and MetaCore assignments are provided in Tables S2 and S3 .
    Human Islet Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher pure link total rna blood kit
    Treatment of human islets with L-WRN+ regulates large numbers of proliferative genes. (A) mRNAs upregulated in response to L-WRN+ by 1.5-fold vs treatment with 8 mM glucose alone, in islets sourced from two different donors. Relative <t>mRNA</t> levels in 5 mM glucose (5), 8 mM glucose (8), 8 mM glucose+LiCl (8+L) and L-WRN+-treated islet aliquots are shown; blue indicates lower abundance, red indicates higher abundance. (B) as for (A), but showing those mRNAs downregulated by 1.5-fold in response to L-WRN+ treatment. (C) qRT-PCR validation of six regulated and four nonregulated mRNAs ( Mafa , Mnx1 , Neurod1 , Pax6 ); fold-changes of L-WRN+ treatment vs 8 mM glucose are shown. Individual gene expression values were normalized to the geometric mean of B2m , Gapdh , Hmbs and Ywhaz [70] . White and black bars, fold-changes observed with <t>RNA-sequencing;</t> red and blue bars, fold-changes observed with qRT-PCR. (D, upper) Classification of upregulated genes into Gene Ontology categories; the most significantly over-represented categories are shown. (D, lower) Classification of upregulated genes into MetaCore custom-defined categories. (E, upper and lower) as for (D), but showing downregulated genes. Further details of Gene Ontology and MetaCore assignments are provided in Tables S2 and S3 .
    Pure Link Total Rna Blood Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher leukolock total rna isolation system
    Treatment of human islets with L-WRN+ regulates large numbers of proliferative genes. (A) mRNAs upregulated in response to L-WRN+ by 1.5-fold vs treatment with 8 mM glucose alone, in islets sourced from two different donors. Relative <t>mRNA</t> levels in 5 mM glucose (5), 8 mM glucose (8), 8 mM glucose+LiCl (8+L) and L-WRN+-treated islet aliquots are shown; blue indicates lower abundance, red indicates higher abundance. (B) as for (A), but showing those mRNAs downregulated by 1.5-fold in response to L-WRN+ treatment. (C) qRT-PCR validation of six regulated and four nonregulated mRNAs ( Mafa , Mnx1 , Neurod1 , Pax6 ); fold-changes of L-WRN+ treatment vs 8 mM glucose are shown. Individual gene expression values were normalized to the geometric mean of B2m , Gapdh , Hmbs and Ywhaz [70] . White and black bars, fold-changes observed with <t>RNA-sequencing;</t> red and blue bars, fold-changes observed with qRT-PCR. (D, upper) Classification of upregulated genes into Gene Ontology categories; the most significantly over-represented categories are shown. (D, lower) Classification of upregulated genes into MetaCore custom-defined categories. (E, upper and lower) as for (D), but showing downregulated genes. Further details of Gene Ontology and MetaCore assignments are provided in Tables S2 and S3 .
    Leukolock Total Rna Isolation System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 117 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher total rna solution
    Effect of growth substrate on the expression of XynR, as determined by a nuclease protection assay. Exponentially growing cultures of P. bryantii B 1 4 were pulsed with either 0.05% WS-X (XW-X), 0.05% xylose, or 0.05% glucose for 30 min, and then total <t>RNA</t> was extracted and 10 μg was transferred onto a Hybond-N + positively charged nylon membrane. Two micrograms of S. cerevisiae total RNA (Yeast RNA) was used as a negative control. A 609-bp PCR product (obtained with primers Reg-UP and Reg-DOWN) corresponding to 398 bp of the xynR upstream sequence and 211 bp of xynR (residues 1681 to 2290) was labeled with <t>DIG</t> and used as a probe in the nuclease protection assay.
    Total Rna Solution, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rnaqueous kits
    Effect of growth substrate on the expression of XynR, as determined by a nuclease protection assay. Exponentially growing cultures of P. bryantii B 1 4 were pulsed with either 0.05% WS-X (XW-X), 0.05% xylose, or 0.05% glucose for 30 min, and then total <t>RNA</t> was extracted and 10 μg was transferred onto a Hybond-N + positively charged nylon membrane. Two micrograms of S. cerevisiae total RNA (Yeast RNA) was used as a negative control. A 609-bp PCR product (obtained with primers Reg-UP and Reg-DOWN) corresponding to 398 bp of the xynR upstream sequence and 211 bp of xynR (residues 1681 to 2290) was labeled with <t>DIG</t> and used as a probe in the nuclease protection assay.
    Rnaqueous Kits, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 149 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher mouse liver rna
    MicroRNA (miRNA) and RLuc mRNA analysis of <t>scAAV2-HCV-miR-Cluster</t> 5-injected mice. ( a–e ) Northern blot analyses of miRNA guide strands. Twenty-five µg of total <t>RNA</t> from mice injected with increasing doses of scAAV8-HCV-miR-Cluster 5 (2.5
    Mouse Liver Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 64 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher solid total rna seq kit
    Identification of repressive chromatin-associated lncRNAs using <t>ChRIP-seq.</t> ( a ) The ChRIP-seq analysis pipeline used to identify lncRNAs enriched in repressive chromatin. The pie chart shows 276 lncRNAs enriched in both EZH2 and H3K27me3 ChRIP-seq samples compared with the nuclear <t>RNA</t> (input). The P value was obtained by performing a hypergeometric test using all the lncRNAs in our analysis. ( b ) Bar diagram showing the distribution of T-to-C transitions (indicative of putative RNA–protein contact sites) in input (8,361), EZH2 (18,905) and H3K27me3 (2,651) ChRIP-seq data. Black in the EZH2 bar indicates the number of T-to-C transitions (1,253) that are either present in input or H3K27me3 samples, and blue indicates EZH2-specific T-to-C transitions (17,652). The EZH2-specific T-to-C transitions (17,652) were used to associate with lncRNAs. ( c ) All the possible conversions present in the EZH2 ChRIP-seq sample. T-to-C conversion and the reverse-strand A-to-G conversions were predominant among all the possible conversion events. ( d ) LncRNAs (1,046; annotated and non-annotated) harbour EZH2-specific (17,652) T-to-C conversion site. Seventy repressive chromatin-enriched lncRNAs (out of 276) carry T-to-C transitions, including known PRC2-interacting lncRNAs such as MEG3 , KCNQ1OT1 and BDNF-AS1. The P value was obtained by performing a hypergeometric test using all the lncRNAs considered in our analysis. ( e,f ) The distribution of the sequencing reads on MEG3 and KCNQ1OT1 transcripts from H3K27me3, EZH2-enriched chromatin fractions and input RNA samples. The tags represent the read distribution and the signal represents the intensity of reads over MEG3 and KCNQ1OT1 transcripts. Locations of T-to-C transitions over the exons are depicted below the physical maps. The left panel depicts the RPKM (Reads per kilobase per million) for MEG3 and KCNQ1OT1 in H3K27me3, EZH2 ChRIP RNA and input RNA samples. The fold enrichment (FC) in H3K27me3 and EZH2 ChRIP RNA compared with input is indicated. ( g ) ChRIP validation: RT–qPCR data showing the enrichment of the selected annotated and non-annotated lncRNAs in the EZH2 and H3K27me3 ChRIP pull-downs compared with input. We did not observe any enrichment of these lncRNAs in the H3K4me2 (active chromatin marks) and immunoglobulin G (IgG; nonspecific antibody) ChRIP pull-downs. Actin was used as a negative control. Data represent the mean±s.d. of three independent biological experiments.
    Solid Total Rna Seq Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1121 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher iprep purelink total rna kit
    Identification of repressive chromatin-associated lncRNAs using <t>ChRIP-seq.</t> ( a ) The ChRIP-seq analysis pipeline used to identify lncRNAs enriched in repressive chromatin. The pie chart shows 276 lncRNAs enriched in both EZH2 and H3K27me3 ChRIP-seq samples compared with the nuclear <t>RNA</t> (input). The P value was obtained by performing a hypergeometric test using all the lncRNAs in our analysis. ( b ) Bar diagram showing the distribution of T-to-C transitions (indicative of putative RNA–protein contact sites) in input (8,361), EZH2 (18,905) and H3K27me3 (2,651) ChRIP-seq data. Black in the EZH2 bar indicates the number of T-to-C transitions (1,253) that are either present in input or H3K27me3 samples, and blue indicates EZH2-specific T-to-C transitions (17,652). The EZH2-specific T-to-C transitions (17,652) were used to associate with lncRNAs. ( c ) All the possible conversions present in the EZH2 ChRIP-seq sample. T-to-C conversion and the reverse-strand A-to-G conversions were predominant among all the possible conversion events. ( d ) LncRNAs (1,046; annotated and non-annotated) harbour EZH2-specific (17,652) T-to-C conversion site. Seventy repressive chromatin-enriched lncRNAs (out of 276) carry T-to-C transitions, including known PRC2-interacting lncRNAs such as MEG3 , KCNQ1OT1 and BDNF-AS1. The P value was obtained by performing a hypergeometric test using all the lncRNAs considered in our analysis. ( e,f ) The distribution of the sequencing reads on MEG3 and KCNQ1OT1 transcripts from H3K27me3, EZH2-enriched chromatin fractions and input RNA samples. The tags represent the read distribution and the signal represents the intensity of reads over MEG3 and KCNQ1OT1 transcripts. Locations of T-to-C transitions over the exons are depicted below the physical maps. The left panel depicts the RPKM (Reads per kilobase per million) for MEG3 and KCNQ1OT1 in H3K27me3, EZH2 ChRIP RNA and input RNA samples. The fold enrichment (FC) in H3K27me3 and EZH2 ChRIP RNA compared with input is indicated. ( g ) ChRIP validation: RT–qPCR data showing the enrichment of the selected annotated and non-annotated lncRNAs in the EZH2 and H3K27me3 ChRIP pull-downs compared with input. We did not observe any enrichment of these lncRNAs in the H3K4me2 (active chromatin marks) and immunoglobulin G (IgG; nonspecific antibody) ChRIP pull-downs. Actin was used as a negative control. Data represent the mean±s.d. of three independent biological experiments.
    Iprep Purelink Total Rna Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    Thermo Fisher total rna isolation protocol
    Identification of repressive chromatin-associated lncRNAs using <t>ChRIP-seq.</t> ( a ) The ChRIP-seq analysis pipeline used to identify lncRNAs enriched in repressive chromatin. The pie chart shows 276 lncRNAs enriched in both EZH2 and H3K27me3 ChRIP-seq samples compared with the nuclear <t>RNA</t> (input). The P value was obtained by performing a hypergeometric test using all the lncRNAs in our analysis. ( b ) Bar diagram showing the distribution of T-to-C transitions (indicative of putative RNA–protein contact sites) in input (8,361), EZH2 (18,905) and H3K27me3 (2,651) ChRIP-seq data. Black in the EZH2 bar indicates the number of T-to-C transitions (1,253) that are either present in input or H3K27me3 samples, and blue indicates EZH2-specific T-to-C transitions (17,652). The EZH2-specific T-to-C transitions (17,652) were used to associate with lncRNAs. ( c ) All the possible conversions present in the EZH2 ChRIP-seq sample. T-to-C conversion and the reverse-strand A-to-G conversions were predominant among all the possible conversion events. ( d ) LncRNAs (1,046; annotated and non-annotated) harbour EZH2-specific (17,652) T-to-C conversion site. Seventy repressive chromatin-enriched lncRNAs (out of 276) carry T-to-C transitions, including known PRC2-interacting lncRNAs such as MEG3 , KCNQ1OT1 and BDNF-AS1. The P value was obtained by performing a hypergeometric test using all the lncRNAs considered in our analysis. ( e,f ) The distribution of the sequencing reads on MEG3 and KCNQ1OT1 transcripts from H3K27me3, EZH2-enriched chromatin fractions and input RNA samples. The tags represent the read distribution and the signal represents the intensity of reads over MEG3 and KCNQ1OT1 transcripts. Locations of T-to-C transitions over the exons are depicted below the physical maps. The left panel depicts the RPKM (Reads per kilobase per million) for MEG3 and KCNQ1OT1 in H3K27me3, EZH2 ChRIP RNA and input RNA samples. The fold enrichment (FC) in H3K27me3 and EZH2 ChRIP RNA compared with input is indicated. ( g ) ChRIP validation: RT–qPCR data showing the enrichment of the selected annotated and non-annotated lncRNAs in the EZH2 and H3K27me3 ChRIP pull-downs compared with input. We did not observe any enrichment of these lncRNAs in the H3K4me2 (active chromatin marks) and immunoglobulin G (IgG; nonspecific antibody) ChRIP pull-downs. Actin was used as a negative control. Data represent the mean±s.d. of three independent biological experiments.
    Total Rna Isolation Protocol, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 83/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher human brain total rna
    Strategy for cloning GABAB1k. The top figure represents exon-intron composition of the proposed GABAB1k. Numbered rectangular boxes represent known exons, and lines between boxes are introns. Two arrows on the bottom of the boxes represent ORF. Black bar is intron 4, and the two probe sequence data, NCBI accession numbers W07715 and N80593, are indicated as #1 and #2 above small rectangular boxes, respectively. They are 5′ and 3′ sequences of the microarray probe, clone 300899. A arrow above the black bar is a possible transcription initiation site. Horizontal lines indicate sequences detected after PCR. Based on microarray probe, clone 300899, the following clones were detected in human cultured cells, mouse midbrain, and rat hippocampus: #2, #3, #4, #11, and #18. From the previous microarray probe sequence analysis, the microarray probe aligned to intron 4 and was either 3′ or 5′ UTR of novel isoforms. Because there was no known human GABAB1 isoform that had intron 4 as an exon, the two possible isoform models, GABAB1j and GABAB1k, were proposed. The predicted GABAB1j and GAGAG1k contain intron 4 as 3′ or 5′ UTR, respectively. Because GABAB1j and GABAB1k are only two possible isoform models that contain clone 300899, #2, #3, #4, #11, and #18 can be partial GABAB1j or GABAB1k and show their existences. From the same <t>RNA</t> sources six different N-terminal partial clones, #5-1, #5-2, #6-1, #6-2, #12, and 17 19, indicated the existence of GABAB1k. They share common 5′ UTRs and partial N-terminal <t>ORFs</t> of GABAB1k and suggest that GABAB1k exists in human, mouse, and rat. To clone the full ORF of GABAB1k, additional primers were designed at its possible 3′ UTR region based on other known isoform sequence analyses. Full ORF containing clones were cloned from human brain and mouse midbrain. Bottom two boxes show clones which contain the full ORF (a b, a 7, 3 7 in human and c d in mouse).
    Human Brain Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 183 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Thermo Fisher total rna oligonucleotides
    Strategy for cloning GABAB1k. The top figure represents exon-intron composition of the proposed GABAB1k. Numbered rectangular boxes represent known exons, and lines between boxes are introns. Two arrows on the bottom of the boxes represent ORF. Black bar is intron 4, and the two probe sequence data, NCBI accession numbers W07715 and N80593, are indicated as #1 and #2 above small rectangular boxes, respectively. They are 5′ and 3′ sequences of the microarray probe, clone 300899. A arrow above the black bar is a possible transcription initiation site. Horizontal lines indicate sequences detected after PCR. Based on microarray probe, clone 300899, the following clones were detected in human cultured cells, mouse midbrain, and rat hippocampus: #2, #3, #4, #11, and #18. From the previous microarray probe sequence analysis, the microarray probe aligned to intron 4 and was either 3′ or 5′ UTR of novel isoforms. Because there was no known human GABAB1 isoform that had intron 4 as an exon, the two possible isoform models, GABAB1j and GABAB1k, were proposed. The predicted GABAB1j and GAGAG1k contain intron 4 as 3′ or 5′ UTR, respectively. Because GABAB1j and GABAB1k are only two possible isoform models that contain clone 300899, #2, #3, #4, #11, and #18 can be partial GABAB1j or GABAB1k and show their existences. From the same <t>RNA</t> sources six different N-terminal partial clones, #5-1, #5-2, #6-1, #6-2, #12, and 17 19, indicated the existence of GABAB1k. They share common 5′ UTRs and partial N-terminal <t>ORFs</t> of GABAB1k and suggest that GABAB1k exists in human, mouse, and rat. To clone the full ORF of GABAB1k, additional primers were designed at its possible 3′ UTR region based on other known isoform sequence analyses. Full ORF containing clones were cloned from human brain and mouse midbrain. Bottom two boxes show clones which contain the full ORF (a b, a 7, 3 7 in human and c d in mouse).
    Total Rna Oligonucleotides, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    Thermo Fisher microarray data total rna
    Strategy for cloning GABAB1k. The top figure represents exon-intron composition of the proposed GABAB1k. Numbered rectangular boxes represent known exons, and lines between boxes are introns. Two arrows on the bottom of the boxes represent ORF. Black bar is intron 4, and the two probe sequence data, NCBI accession numbers W07715 and N80593, are indicated as #1 and #2 above small rectangular boxes, respectively. They are 5′ and 3′ sequences of the microarray probe, clone 300899. A arrow above the black bar is a possible transcription initiation site. Horizontal lines indicate sequences detected after PCR. Based on microarray probe, clone 300899, the following clones were detected in human cultured cells, mouse midbrain, and rat hippocampus: #2, #3, #4, #11, and #18. From the previous microarray probe sequence analysis, the microarray probe aligned to intron 4 and was either 3′ or 5′ UTR of novel isoforms. Because there was no known human GABAB1 isoform that had intron 4 as an exon, the two possible isoform models, GABAB1j and GABAB1k, were proposed. The predicted GABAB1j and GAGAG1k contain intron 4 as 3′ or 5′ UTR, respectively. Because GABAB1j and GABAB1k are only two possible isoform models that contain clone 300899, #2, #3, #4, #11, and #18 can be partial GABAB1j or GABAB1k and show their existences. From the same <t>RNA</t> sources six different N-terminal partial clones, #5-1, #5-2, #6-1, #6-2, #12, and 17 19, indicated the existence of GABAB1k. They share common 5′ UTRs and partial N-terminal <t>ORFs</t> of GABAB1k and suggest that GABAB1k exists in human, mouse, and rat. To clone the full ORF of GABAB1k, additional primers were designed at its possible 3′ UTR region based on other known isoform sequence analyses. Full ORF containing clones were cloned from human brain and mouse midbrain. Bottom two boxes show clones which contain the full ORF (a b, a 7, 3 7 in human and c d in mouse).
    Microarray Data Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 83/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Thermo Fisher purelink total rna kit
    Strategy for cloning GABAB1k. The top figure represents exon-intron composition of the proposed GABAB1k. Numbered rectangular boxes represent known exons, and lines between boxes are introns. Two arrows on the bottom of the boxes represent ORF. Black bar is intron 4, and the two probe sequence data, NCBI accession numbers W07715 and N80593, are indicated as #1 and #2 above small rectangular boxes, respectively. They are 5′ and 3′ sequences of the microarray probe, clone 300899. A arrow above the black bar is a possible transcription initiation site. Horizontal lines indicate sequences detected after PCR. Based on microarray probe, clone 300899, the following clones were detected in human cultured cells, mouse midbrain, and rat hippocampus: #2, #3, #4, #11, and #18. From the previous microarray probe sequence analysis, the microarray probe aligned to intron 4 and was either 3′ or 5′ UTR of novel isoforms. Because there was no known human GABAB1 isoform that had intron 4 as an exon, the two possible isoform models, GABAB1j and GABAB1k, were proposed. The predicted GABAB1j and GAGAG1k contain intron 4 as 3′ or 5′ UTR, respectively. Because GABAB1j and GABAB1k are only two possible isoform models that contain clone 300899, #2, #3, #4, #11, and #18 can be partial GABAB1j or GABAB1k and show their existences. From the same <t>RNA</t> sources six different N-terminal partial clones, #5-1, #5-2, #6-1, #6-2, #12, and 17 19, indicated the existence of GABAB1k. They share common 5′ UTRs and partial N-terminal <t>ORFs</t> of GABAB1k and suggest that GABAB1k exists in human, mouse, and rat. To clone the full ORF of GABAB1k, additional primers were designed at its possible 3′ UTR region based on other known isoform sequence analyses. Full ORF containing clones were cloned from human brain and mouse midbrain. Bottom two boxes show clones which contain the full ORF (a b, a 7, 3 7 in human and c d in mouse).
    Purelink Total Rna Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Thermo Fisher total rna isolation reaction
    Strategy for cloning GABAB1k. The top figure represents exon-intron composition of the proposed GABAB1k. Numbered rectangular boxes represent known exons, and lines between boxes are introns. Two arrows on the bottom of the boxes represent ORF. Black bar is intron 4, and the two probe sequence data, NCBI accession numbers W07715 and N80593, are indicated as #1 and #2 above small rectangular boxes, respectively. They are 5′ and 3′ sequences of the microarray probe, clone 300899. A arrow above the black bar is a possible transcription initiation site. Horizontal lines indicate sequences detected after PCR. Based on microarray probe, clone 300899, the following clones were detected in human cultured cells, mouse midbrain, and rat hippocampus: #2, #3, #4, #11, and #18. From the previous microarray probe sequence analysis, the microarray probe aligned to intron 4 and was either 3′ or 5′ UTR of novel isoforms. Because there was no known human GABAB1 isoform that had intron 4 as an exon, the two possible isoform models, GABAB1j and GABAB1k, were proposed. The predicted GABAB1j and GAGAG1k contain intron 4 as 3′ or 5′ UTR, respectively. Because GABAB1j and GABAB1k are only two possible isoform models that contain clone 300899, #2, #3, #4, #11, and #18 can be partial GABAB1j or GABAB1k and show their existences. From the same <t>RNA</t> sources six different N-terminal partial clones, #5-1, #5-2, #6-1, #6-2, #12, and 17 19, indicated the existence of GABAB1k. They share common 5′ UTRs and partial N-terminal <t>ORFs</t> of GABAB1k and suggest that GABAB1k exists in human, mouse, and rat. To clone the full ORF of GABAB1k, additional primers were designed at its possible 3′ UTR region based on other known isoform sequence analyses. Full ORF containing clones were cloned from human brain and mouse midbrain. Bottom two boxes show clones which contain the full ORF (a b, a 7, 3 7 in human and c d in mouse).
    Total Rna Isolation Reaction, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher total rna isolation system
    Strategy for cloning GABAB1k. The top figure represents exon-intron composition of the proposed GABAB1k. Numbered rectangular boxes represent known exons, and lines between boxes are introns. Two arrows on the bottom of the boxes represent ORF. Black bar is intron 4, and the two probe sequence data, NCBI accession numbers W07715 and N80593, are indicated as #1 and #2 above small rectangular boxes, respectively. They are 5′ and 3′ sequences of the microarray probe, clone 300899. A arrow above the black bar is a possible transcription initiation site. Horizontal lines indicate sequences detected after PCR. Based on microarray probe, clone 300899, the following clones were detected in human cultured cells, mouse midbrain, and rat hippocampus: #2, #3, #4, #11, and #18. From the previous microarray probe sequence analysis, the microarray probe aligned to intron 4 and was either 3′ or 5′ UTR of novel isoforms. Because there was no known human GABAB1 isoform that had intron 4 as an exon, the two possible isoform models, GABAB1j and GABAB1k, were proposed. The predicted GABAB1j and GAGAG1k contain intron 4 as 3′ or 5′ UTR, respectively. Because GABAB1j and GABAB1k are only two possible isoform models that contain clone 300899, #2, #3, #4, #11, and #18 can be partial GABAB1j or GABAB1k and show their existences. From the same <t>RNA</t> sources six different N-terminal partial clones, #5-1, #5-2, #6-1, #6-2, #12, and 17 19, indicated the existence of GABAB1k. They share common 5′ UTRs and partial N-terminal <t>ORFs</t> of GABAB1k and suggest that GABAB1k exists in human, mouse, and rat. To clone the full ORF of GABAB1k, additional primers were designed at its possible 3′ UTR region based on other known isoform sequence analyses. Full ORF containing clones were cloned from human brain and mouse midbrain. Bottom two boxes show clones which contain the full ORF (a b, a 7, 3 7 in human and c d in mouse).
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    Thermo Fisher first choice total rna
    Strategy for cloning GABAB1k. The top figure represents exon-intron composition of the proposed GABAB1k. Numbered rectangular boxes represent known exons, and lines between boxes are introns. Two arrows on the bottom of the boxes represent ORF. Black bar is intron 4, and the two probe sequence data, NCBI accession numbers W07715 and N80593, are indicated as #1 and #2 above small rectangular boxes, respectively. They are 5′ and 3′ sequences of the microarray probe, clone 300899. A arrow above the black bar is a possible transcription initiation site. Horizontal lines indicate sequences detected after PCR. Based on microarray probe, clone 300899, the following clones were detected in human cultured cells, mouse midbrain, and rat hippocampus: #2, #3, #4, #11, and #18. From the previous microarray probe sequence analysis, the microarray probe aligned to intron 4 and was either 3′ or 5′ UTR of novel isoforms. Because there was no known human GABAB1 isoform that had intron 4 as an exon, the two possible isoform models, GABAB1j and GABAB1k, were proposed. The predicted GABAB1j and GAGAG1k contain intron 4 as 3′ or 5′ UTR, respectively. Because GABAB1j and GABAB1k are only two possible isoform models that contain clone 300899, #2, #3, #4, #11, and #18 can be partial GABAB1j or GABAB1k and show their existences. From the same <t>RNA</t> sources six different N-terminal partial clones, #5-1, #5-2, #6-1, #6-2, #12, and 17 19, indicated the existence of GABAB1k. They share common 5′ UTRs and partial N-terminal <t>ORFs</t> of GABAB1k and suggest that GABAB1k exists in human, mouse, and rat. To clone the full ORF of GABAB1k, additional primers were designed at its possible 3′ UTR region based on other known isoform sequence analyses. Full ORF containing clones were cloned from human brain and mouse midbrain. Bottom two boxes show clones which contain the full ORF (a b, a 7, 3 7 in human and c d in mouse).
    First Choice Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 83/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher murine standard total rna
    Strategy for cloning GABAB1k. The top figure represents exon-intron composition of the proposed GABAB1k. Numbered rectangular boxes represent known exons, and lines between boxes are introns. Two arrows on the bottom of the boxes represent ORF. Black bar is intron 4, and the two probe sequence data, NCBI accession numbers W07715 and N80593, are indicated as #1 and #2 above small rectangular boxes, respectively. They are 5′ and 3′ sequences of the microarray probe, clone 300899. A arrow above the black bar is a possible transcription initiation site. Horizontal lines indicate sequences detected after PCR. Based on microarray probe, clone 300899, the following clones were detected in human cultured cells, mouse midbrain, and rat hippocampus: #2, #3, #4, #11, and #18. From the previous microarray probe sequence analysis, the microarray probe aligned to intron 4 and was either 3′ or 5′ UTR of novel isoforms. Because there was no known human GABAB1 isoform that had intron 4 as an exon, the two possible isoform models, GABAB1j and GABAB1k, were proposed. The predicted GABAB1j and GAGAG1k contain intron 4 as 3′ or 5′ UTR, respectively. Because GABAB1j and GABAB1k are only two possible isoform models that contain clone 300899, #2, #3, #4, #11, and #18 can be partial GABAB1j or GABAB1k and show their existences. From the same <t>RNA</t> sources six different N-terminal partial clones, #5-1, #5-2, #6-1, #6-2, #12, and 17 19, indicated the existence of GABAB1k. They share common 5′ UTRs and partial N-terminal <t>ORFs</t> of GABAB1k and suggest that GABAB1k exists in human, mouse, and rat. To clone the full ORF of GABAB1k, additional primers were designed at its possible 3′ UTR region based on other known isoform sequence analyses. Full ORF containing clones were cloned from human brain and mouse midbrain. Bottom two boxes show clones which contain the full ORF (a b, a 7, 3 7 in human and c d in mouse).
    Murine Standard Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    The expression of PAQR3 partially rescued miR-137-enhanced cell proliferation and invasion. (A) Western blot analysis showed that transfection of small interfering RNA against PAQR3 into T24 cells led to dramatically decreased PAQR3 protein expression. GAPDH was also detected as a loading control. (B) Silencing of PAQR3 significantly enhanced the proliferation of bladder cancer cells. The growth index as assessed at 0, 24, 48 and 72 h. (C) Western blot analysis of PAQR3 in T24 cells co-transfected with either miR-137 mimic or scramble and 2.0 µg pCDNA- PAQR3 or pCDNA empty vector. GAPDH was also detected as a loading control. (D) Cell growth curves in T24 cells transfected with different combinations at 0, 24, 48 and 72 h. (E) Transwell analysis of T24 cells treated with different combinations. The relative ratio of invasive cells per field is shown right. *p

    Journal: PLoS ONE

    Article Title: MicroRNA-137 Upregulation Increases Bladder Cancer Cell Proliferation and Invasion by Targeting PAQR3

    doi: 10.1371/journal.pone.0109734

    Figure Lengend Snippet: The expression of PAQR3 partially rescued miR-137-enhanced cell proliferation and invasion. (A) Western blot analysis showed that transfection of small interfering RNA against PAQR3 into T24 cells led to dramatically decreased PAQR3 protein expression. GAPDH was also detected as a loading control. (B) Silencing of PAQR3 significantly enhanced the proliferation of bladder cancer cells. The growth index as assessed at 0, 24, 48 and 72 h. (C) Western blot analysis of PAQR3 in T24 cells co-transfected with either miR-137 mimic or scramble and 2.0 µg pCDNA- PAQR3 or pCDNA empty vector. GAPDH was also detected as a loading control. (D) Cell growth curves in T24 cells transfected with different combinations at 0, 24, 48 and 72 h. (E) Transwell analysis of T24 cells treated with different combinations. The relative ratio of invasive cells per field is shown right. *p

    Article Snippet: For the reporter gene assay, the cells were cotransfected with 0.5 µg of pGL3-PAQR3-3′UTR or pGL3- PAQR3-3′UTR Mut plasmid, 0.05 ng of the phRL-SV40 control vector (Promega, USA), and 100 nM miR-137 or control RNA using Lipofectamine 2000 (Invitrogen, USA).

    Techniques: Expressing, Western Blot, Transfection, Small Interfering RNA, Plasmid Preparation

    Changes in the RNA and DNA content (fg cell −1 ) of each strain across temperatures, with different symbols for each level of supply C:P. Error bars represent one standard error of the mean.

    Journal: Frontiers in Microbiology

    Article Title: The Effects of Nutrient Imbalances and Temperature on the Biomass Stoichiometry of Freshwater Bacteria

    doi: 10.3389/fmicb.2017.01692

    Figure Lengend Snippet: Changes in the RNA and DNA content (fg cell −1 ) of each strain across temperatures, with different symbols for each level of supply C:P. Error bars represent one standard error of the mean.

    Article Snippet: Bacterial samples on the filters, negative control samples (containing all reagents but no bacteria), and standards of RNA (E. coli , Invitrogen) and DNA (calf thymus, Sigma) were prepared for analysis as described in Makino et al. ( ).

    Techniques:

    Overview of the miRNA microarray procedure. RNA species smaller than ~40 nt are purified by a rapid electrophoretic gel fractionation method. miRNAs are 3′-end labeled with poly(A) polymerase, amine-modified nucleotides, and amine-reactive dyes.

    Journal: RNA

    Article Title: An optimized isolation and labeling platform for accurate microRNA expression profiling

    doi: 10.1261/rna.2610405

    Figure Lengend Snippet: Overview of the miRNA microarray procedure. RNA species smaller than ~40 nt are purified by a rapid electrophoretic gel fractionation method. miRNAs are 3′-end labeled with poly(A) polymerase, amine-modified nucleotides, and amine-reactive dyes.

    Article Snippet: For miRNA expression profiling in normal human tissues, miRNA certified First- Choice Total RNA (Ambion) were used.

    Techniques: Microarray, Purification, Fractionation, Labeling, Modification

    Comparison between miRNA microarray data and other detection methods. Total RNA was isolated from human bladder, lung, or uterus. miRNA populations in 10 μg of each sample were independently fractionated, labeled, and compared by pairs on two

    Journal: RNA

    Article Title: An optimized isolation and labeling platform for accurate microRNA expression profiling

    doi: 10.1261/rna.2610405

    Figure Lengend Snippet: Comparison between miRNA microarray data and other detection methods. Total RNA was isolated from human bladder, lung, or uterus. miRNA populations in 10 μg of each sample were independently fractionated, labeled, and compared by pairs on two

    Article Snippet: For miRNA expression profiling in normal human tissues, miRNA certified First- Choice Total RNA (Ambion) were used.

    Techniques: Microarray, Isolation, Labeling

    Comparison between enriched and gel purified miRNA fractions. One, 5, or 10 μg of total RNA from human bladder or lung were used to gel purify miRNAs (GP1, GP5, GP10) or to prepare fractions enriched in small RNA (E1, E5, E10). RNA species in

    Journal: RNA

    Article Title: An optimized isolation and labeling platform for accurate microRNA expression profiling

    doi: 10.1261/rna.2610405

    Figure Lengend Snippet: Comparison between enriched and gel purified miRNA fractions. One, 5, or 10 μg of total RNA from human bladder or lung were used to gel purify miRNAs (GP1, GP5, GP10) or to prepare fractions enriched in small RNA (E1, E5, E10). RNA species in

    Article Snippet: For miRNA expression profiling in normal human tissues, miRNA certified First- Choice Total RNA (Ambion) were used.

    Techniques: Purification

    miRNA microarray reproducibility. Total RNA was isolated from a single human prostate sample and a single human colon sample. Each total RNA sample was split into six independent samples (10 μg each). miRNAs in each sample were independently fractionated

    Journal: RNA

    Article Title: An optimized isolation and labeling platform for accurate microRNA expression profiling

    doi: 10.1261/rna.2610405

    Figure Lengend Snippet: miRNA microarray reproducibility. Total RNA was isolated from a single human prostate sample and a single human colon sample. Each total RNA sample was split into six independent samples (10 μg each). miRNAs in each sample were independently fractionated

    Article Snippet: For miRNA expression profiling in normal human tissues, miRNA certified First- Choice Total RNA (Ambion) were used.

    Techniques: Microarray, Isolation

    miRNA microarray accuracy and precision. ( A ) Self versus self analysis. A human thymus total RNA sample (20 μg) was split in half and miRNAs were independently gel purified, labeled either with Cy3 or Cy5 dyes, and hybridized to the same array

    Journal: RNA

    Article Title: An optimized isolation and labeling platform for accurate microRNA expression profiling

    doi: 10.1261/rna.2610405

    Figure Lengend Snippet: miRNA microarray accuracy and precision. ( A ) Self versus self analysis. A human thymus total RNA sample (20 μg) was split in half and miRNAs were independently gel purified, labeled either with Cy3 or Cy5 dyes, and hybridized to the same array

    Article Snippet: For miRNA expression profiling in normal human tissues, miRNA certified First- Choice Total RNA (Ambion) were used.

    Techniques: Microarray, Purification, Labeling

    Validation of miRNA expression data. Total RNA was isolated from the 10 indicated tissues, independent from the ones used in the microarray profiling study (Fig. 6). Expression levels of let-7C and miR- 200B were analyzed by Northern blot (2

    Journal: RNA

    Article Title: An optimized isolation and labeling platform for accurate microRNA expression profiling

    doi: 10.1261/rna.2610405

    Figure Lengend Snippet: Validation of miRNA expression data. Total RNA was isolated from the 10 indicated tissues, independent from the ones used in the microarray profiling study (Fig. 6). Expression levels of let-7C and miR- 200B were analyzed by Northern blot (2

    Article Snippet: For miRNA expression profiling in normal human tissues, miRNA certified First- Choice Total RNA (Ambion) were used.

    Techniques: Expressing, Isolation, Microarray, Northern Blot

    miRNA expression profiles across 26 human normal tissues. miRNAs from 10 μg of the indicated total RNA samples were gel purified, labeled with Cy5, and compared on 26 independent microarrays against Cy3-labeled miRNAs isolated from 10 μg

    Journal: RNA

    Article Title: An optimized isolation and labeling platform for accurate microRNA expression profiling

    doi: 10.1261/rna.2610405

    Figure Lengend Snippet: miRNA expression profiles across 26 human normal tissues. miRNAs from 10 μg of the indicated total RNA samples were gel purified, labeled with Cy5, and compared on 26 independent microarrays against Cy3-labeled miRNAs isolated from 10 μg

    Article Snippet: For miRNA expression profiling in normal human tissues, miRNA certified First- Choice Total RNA (Ambion) were used.

    Techniques: Expressing, Purification, Labeling, Isolation

    Limit of blank study. ( a ) Summary of the MFI probe signals obtained from repeat testing with the MMA of a no RNA control sample, a total RNA sample purified from the translocation-negative HL60 cell line, and eight total RNA samples purified from asymptomatic control donors' white blood cells (WBC control RNA). Minimum (MIN), maximum (MAX), median, mean and s.d. (STDEV) values for the 11 fusion-transcript-specific probes combined are shown for each sample type and overall. ( b ) Results by probe type for all sample types combined. The graph shows the mean, maximum (MAX) and twice the limit of blank (2 × LOB) values for each of the 11 fusion-transcript-specific probes relative to the qualitative 350 MFI cutoff value (dash line). The error bars represent the s.d. of each probe-specific distribution. The complete data set is presented in Supplementary Table 2 .

    Journal: Blood Cancer Journal

    Article Title: Risk-based classification of leukemia by cytogenetic and multiplex molecular methods: results from a multicenter validation study

    doi: 10.1038/bcj.2012.24

    Figure Lengend Snippet: Limit of blank study. ( a ) Summary of the MFI probe signals obtained from repeat testing with the MMA of a no RNA control sample, a total RNA sample purified from the translocation-negative HL60 cell line, and eight total RNA samples purified from asymptomatic control donors' white blood cells (WBC control RNA). Minimum (MIN), maximum (MAX), median, mean and s.d. (STDEV) values for the 11 fusion-transcript-specific probes combined are shown for each sample type and overall. ( b ) Results by probe type for all sample types combined. The graph shows the mean, maximum (MAX) and twice the limit of blank (2 × LOB) values for each of the 11 fusion-transcript-specific probes relative to the qualitative 350 MFI cutoff value (dash line). The error bars represent the s.d. of each probe-specific distribution. The complete data set is presented in Supplementary Table 2 .

    Article Snippet: When indicated, total RNA was diluted in purified HL60 total RNA (Applied Biosystems) keeping the concentration of total RNA constant.

    Techniques: Purification, Translocation Assay

    Panel expansion. Representative example of results (MFI) with two prototype assays detecting 23 different targets prepared by in vitro transcription and spiked in a background of translocation- and mutation-negative HL60 RNA (400 ng input). ( a ) Specific detection of 13 fusion transcripts commonly found in CML and ALL. Results for the two samples false negative for MLL–AFF1 with the MMA at site 3 (study ID no. 146 and no. 184) are also shown. ( b ) Specific detection of three NPM1 mutant transcripts and seven fusion transcripts commonly found in AML. For this assay, the NPM1 wild-type sequence (NPM1 WT) is used as an endogenous control. Target-specific positive signals above the qualitative cutoff (350 MFI) are highlighted.

    Journal: Blood Cancer Journal

    Article Title: Risk-based classification of leukemia by cytogenetic and multiplex molecular methods: results from a multicenter validation study

    doi: 10.1038/bcj.2012.24

    Figure Lengend Snippet: Panel expansion. Representative example of results (MFI) with two prototype assays detecting 23 different targets prepared by in vitro transcription and spiked in a background of translocation- and mutation-negative HL60 RNA (400 ng input). ( a ) Specific detection of 13 fusion transcripts commonly found in CML and ALL. Results for the two samples false negative for MLL–AFF1 with the MMA at site 3 (study ID no. 146 and no. 184) are also shown. ( b ) Specific detection of three NPM1 mutant transcripts and seven fusion transcripts commonly found in AML. For this assay, the NPM1 wild-type sequence (NPM1 WT) is used as an endogenous control. Target-specific positive signals above the qualitative cutoff (350 MFI) are highlighted.

    Article Snippet: When indicated, total RNA was diluted in purified HL60 total RNA (Applied Biosystems) keeping the concentration of total RNA constant.

    Techniques: In Vitro, Translocation Assay, Mutagenesis, Sequencing

    Representative MMA output. Each row represents the results from a single multiplex PCR amplification hybridized onto 12 target-specific probes in a single reaction. The resulting MFI signals generated by each probe-bound PCR product are shown for three control samples, a total RNA sample isolated from a translocation-negative cell line (HL60), 12 different synthetic fusion transcripts prepared by in vitro transcription and spiked in a background of HL60 RNA (400 ng input) and eight total RNA samples purified from translocation-positive cell lines (400 ng input). Target-specific positive signals above the qualitative cutoff (350 MFI) are highlighted. The three controls are designed to assess the validity of the multiplex amplification, hybridization and detection steps in every batch/run.

    Journal: Blood Cancer Journal

    Article Title: Risk-based classification of leukemia by cytogenetic and multiplex molecular methods: results from a multicenter validation study

    doi: 10.1038/bcj.2012.24

    Figure Lengend Snippet: Representative MMA output. Each row represents the results from a single multiplex PCR amplification hybridized onto 12 target-specific probes in a single reaction. The resulting MFI signals generated by each probe-bound PCR product are shown for three control samples, a total RNA sample isolated from a translocation-negative cell line (HL60), 12 different synthetic fusion transcripts prepared by in vitro transcription and spiked in a background of HL60 RNA (400 ng input) and eight total RNA samples purified from translocation-positive cell lines (400 ng input). Target-specific positive signals above the qualitative cutoff (350 MFI) are highlighted. The three controls are designed to assess the validity of the multiplex amplification, hybridization and detection steps in every batch/run.

    Article Snippet: When indicated, total RNA was diluted in purified HL60 total RNA (Applied Biosystems) keeping the concentration of total RNA constant.

    Techniques: Multiplex Assay, Polymerase Chain Reaction, Amplification, Generated, Isolation, Translocation Assay, In Vitro, Purification, Hybridization

    Evaluation of analytical sensitivity. ( a ) Total RNA purified from the indicated translocation-positive cell lines was tested with the MMA at 1000, 100, 10 or 1 ng per RT reaction. ( b ) The same total RNA samples were tested either undiluted (100%) or diluted at 10, 1% or 0.1% in a background of total RNA isolated from the translocation-negative cell line HL60 at a final input of 400 ng per RT reaction. ( c ) Same experiment as in ( b ) at 100, 10 or 1% dilution and 100 ng input. ( d ) BCR–ABL1 sensitivity controls tested in duplicate with the MMA at 600 ng input. The graphs show the average MFI signals generated by the target-specific probes (black bars) and by the GAPDH endogenous control probe (white bars) relative to the 350 MFI cutoff value (dash lines). The complete data set is presented in Supplementary Table 2 .

    Journal: Blood Cancer Journal

    Article Title: Risk-based classification of leukemia by cytogenetic and multiplex molecular methods: results from a multicenter validation study

    doi: 10.1038/bcj.2012.24

    Figure Lengend Snippet: Evaluation of analytical sensitivity. ( a ) Total RNA purified from the indicated translocation-positive cell lines was tested with the MMA at 1000, 100, 10 or 1 ng per RT reaction. ( b ) The same total RNA samples were tested either undiluted (100%) or diluted at 10, 1% or 0.1% in a background of total RNA isolated from the translocation-negative cell line HL60 at a final input of 400 ng per RT reaction. ( c ) Same experiment as in ( b ) at 100, 10 or 1% dilution and 100 ng input. ( d ) BCR–ABL1 sensitivity controls tested in duplicate with the MMA at 600 ng input. The graphs show the average MFI signals generated by the target-specific probes (black bars) and by the GAPDH endogenous control probe (white bars) relative to the 350 MFI cutoff value (dash lines). The complete data set is presented in Supplementary Table 2 .

    Article Snippet: When indicated, total RNA was diluted in purified HL60 total RNA (Applied Biosystems) keeping the concentration of total RNA constant.

    Techniques: Purification, Translocation Assay, Isolation, Generated

    HprK Xcc has a broad regulatory role in Xcc . Functional categories of differential expressed genes (DEGs) in hprK Xcc mutant ΔhprK Xcc . Genome‐scale transcriptome profiling of Xcc strains cultured in nutrition rich medium NYG were investigated by RNA‐sequencing, and 256 genes were found differentially expressed by two‐fold or more in hprK Xcc mutant (Table S2 ). These genes were broadly categorized according to their biological function (He et al ., 2007 ).

    Journal: Environmental Microbiology

    Article Title: HprKXcc is a serine kinase that regulates virulence in the Gram‐negative phytopathogen Xanthomonas campestris

    doi: 10.1111/1462-2920.14740

    Figure Lengend Snippet: HprK Xcc has a broad regulatory role in Xcc . Functional categories of differential expressed genes (DEGs) in hprK Xcc mutant ΔhprK Xcc . Genome‐scale transcriptome profiling of Xcc strains cultured in nutrition rich medium NYG were investigated by RNA‐sequencing, and 256 genes were found differentially expressed by two‐fold or more in hprK Xcc mutant (Table S2 ). These genes were broadly categorized according to their biological function (He et al ., 2007 ).

    Article Snippet: The total RNAs were extracted from the cultures of the Xcc strains with a total‐RNA extraction kit (Invitrogen, Waltham, MA, USA) and cDNA generated using a cDNA synthesis kit (Invitrogen).

    Techniques: Functional Assay, Mutagenesis, Cell Culture, RNA Sequencing Assay

    Overexpression of PtsH protein reduces the EPS production, activity of extracellular enzymes and cell motility in Xcc . A. Plate assays were used to test the EPS production (a), the activity of extracellular enzymes (b, c) and motility (d). Here an overnight culture (2 μl, OD 600 = 1.0) of each Xcc strain was spotted onto a tested plate containing 0.2% (wt/vol) L‐arabinose. For EPS production, bacteria on NY plates containing 2.0% (wt/vol) glucose were incubated at 28°C for 5 days. The strain 8004/pBptsH displayed small colonies to the control strain 8004/pBBad22K, indicating the EPS yield of 8004/pBptsH strain was less compared to that of the control strain. For estimation of the activity of extracellular enzymes, strains on NYG plates 0.5% (wt/vol) skim milk (for protease) or 0.25% (wt/vol) carboxymethylcellulose (for endoglucanase) were incubated at 28°C for 24 h (endoglucanase) or 48 h (protease). Zones of clearance around the spot, which due to the degradation of the substrate, from strain 8004/pBptsH were small compared to the control strain, indicating the activity of extracellular enzymes of strain 8004/pBptsH was less to that of the wild type. Similar results were obtained in two other independent experiments. To detect swimming motility, an overnight culture (OD 600 of 1.0) of each Xcc strain was stabbed into 0.28% agar plates composed of 0.03% Bacto peptone and 0.03% yeast extract followed by incubation at 28°C for 4 days. B. ptsH driven by arabinose‐inducible ( ara ) promoter in Xcc produces high concentration of PtsH protein. (i) Reverse‐transcription PCR (RT‐PCR) assay to examine the transcription level of ptsH gene in Xcc strains. RT‐PCR was performed using the synthesized cDNAs from the extracted total RNAs of the Xcc strains grown in NY medium for 20 h as templates to amplify the internal sequence of ptsH gene with primer set 1305NF/R. PCR fragment of ptsH from strain 8004/pBptsH was diluted in 10 times before electrophoresis analysis. The 16S rRNA gene in Xcc strains was used as a control. (ii) Western blot assay to examine the translation level of PtsH protein in Xcc strains. The recombinant plasmids pHisptsH lac and pHisptsH ara , which contains the PtsH coding sequence fused with 6 × His tag in its C‐terminus, were introduced into Xcc strain 8004. The resulting recombinant strains were cultured in NYG medium with (for strain 8004/pHisptsH ara ) or without (for strain 8004/pHisptsH lac ) L‐arabinose for 12 h, and the total proteins in Xcc cells were prepared as previously described (Zang et al ., 2007 ). Thirty micrograms of cell protein was electrophoresed in SDS‐PAGE gel and transferred to a PVDF membrane. The presence of PtsH protein subject to lac or ara promoter was detected by anti‐6 × His monoclonal antibody (a). As a loading reference, the blot was also probed with an anti‐RNA polymerase β‐antibody (b). [Color figure can be viewed at http://wileyonlinelibrary.com ]

    Journal: Environmental Microbiology

    Article Title: HprKXcc is a serine kinase that regulates virulence in the Gram‐negative phytopathogen Xanthomonas campestris

    doi: 10.1111/1462-2920.14740

    Figure Lengend Snippet: Overexpression of PtsH protein reduces the EPS production, activity of extracellular enzymes and cell motility in Xcc . A. Plate assays were used to test the EPS production (a), the activity of extracellular enzymes (b, c) and motility (d). Here an overnight culture (2 μl, OD 600 = 1.0) of each Xcc strain was spotted onto a tested plate containing 0.2% (wt/vol) L‐arabinose. For EPS production, bacteria on NY plates containing 2.0% (wt/vol) glucose were incubated at 28°C for 5 days. The strain 8004/pBptsH displayed small colonies to the control strain 8004/pBBad22K, indicating the EPS yield of 8004/pBptsH strain was less compared to that of the control strain. For estimation of the activity of extracellular enzymes, strains on NYG plates 0.5% (wt/vol) skim milk (for protease) or 0.25% (wt/vol) carboxymethylcellulose (for endoglucanase) were incubated at 28°C for 24 h (endoglucanase) or 48 h (protease). Zones of clearance around the spot, which due to the degradation of the substrate, from strain 8004/pBptsH were small compared to the control strain, indicating the activity of extracellular enzymes of strain 8004/pBptsH was less to that of the wild type. Similar results were obtained in two other independent experiments. To detect swimming motility, an overnight culture (OD 600 of 1.0) of each Xcc strain was stabbed into 0.28% agar plates composed of 0.03% Bacto peptone and 0.03% yeast extract followed by incubation at 28°C for 4 days. B. ptsH driven by arabinose‐inducible ( ara ) promoter in Xcc produces high concentration of PtsH protein. (i) Reverse‐transcription PCR (RT‐PCR) assay to examine the transcription level of ptsH gene in Xcc strains. RT‐PCR was performed using the synthesized cDNAs from the extracted total RNAs of the Xcc strains grown in NY medium for 20 h as templates to amplify the internal sequence of ptsH gene with primer set 1305NF/R. PCR fragment of ptsH from strain 8004/pBptsH was diluted in 10 times before electrophoresis analysis. The 16S rRNA gene in Xcc strains was used as a control. (ii) Western blot assay to examine the translation level of PtsH protein in Xcc strains. The recombinant plasmids pHisptsH lac and pHisptsH ara , which contains the PtsH coding sequence fused with 6 × His tag in its C‐terminus, were introduced into Xcc strain 8004. The resulting recombinant strains were cultured in NYG medium with (for strain 8004/pHisptsH ara ) or without (for strain 8004/pHisptsH lac ) L‐arabinose for 12 h, and the total proteins in Xcc cells were prepared as previously described (Zang et al ., 2007 ). Thirty micrograms of cell protein was electrophoresed in SDS‐PAGE gel and transferred to a PVDF membrane. The presence of PtsH protein subject to lac or ara promoter was detected by anti‐6 × His monoclonal antibody (a). As a loading reference, the blot was also probed with an anti‐RNA polymerase β‐antibody (b). [Color figure can be viewed at http://wileyonlinelibrary.com ]

    Article Snippet: The total RNAs were extracted from the cultures of the Xcc strains with a total‐RNA extraction kit (Invitrogen, Waltham, MA, USA) and cDNA generated using a cDNA synthesis kit (Invitrogen).

    Techniques: Over Expression, Activity Assay, Incubation, Acetylene Reduction Assay, Concentration Assay, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Synthesized, Sequencing, Electrophoresis, Western Blot, Recombinant, Cell Culture, SDS Page

    RNA analysis following immunostaining. (A) An agarose gel of 5 µg HeLa total RNA (lane 1) and 5 µg HeLa total RNA incubated with primary antibody for anti–cytokeratin AE1/AE3 for 20 min (lane 2). (B) A BioAnalyzer image of total

    Journal: Journal of Histochemistry and Cytochemistry

    Article Title: Effect of Immunohistochemistry on Molecular Analysis of Tissue Samples

    doi: 10.1369/0022155411404704

    Figure Lengend Snippet: RNA analysis following immunostaining. (A) An agarose gel of 5 µg HeLa total RNA (lane 1) and 5 µg HeLa total RNA incubated with primary antibody for anti–cytokeratin AE1/AE3 for 20 min (lane 2). (B) A BioAnalyzer image of total

    Article Snippet: Commercially available HeLa cell total RNA (Invitrogen) and total human heart RNA (Ambion/Applied Biosystems, Austin, TX) were used for the RNA quality studies.

    Techniques: Immunostaining, Agarose Gel Electrophoresis, Incubation

    Engrafted human umbilical cord-derived mesenchymal stem cells (HUMSCs) differentiate into osteoblasts and inhibit the number of osteoclasts at the distal femur. TRAP staining was performed to detect osteoclasts (arrows) in the metaphysis of the distal femur in the normal + phosphate-buffered saline (PBS; A), normal + HUMSCs (B), ovariectomy (OVX) + PBS (C), and OVX + HUMSCs (D) groups. The number of osteoclasts per trabecular bone area (no./mm 2 ) was quantified. The results indicated no difference in the osteoclast number among the normal + PBS, normal + HUMSCs, and OVX + HUMSCs groups. However, the osteoclast number was significantly higher in the OVX + PBS group than in the other 3 groups (E). Histological sections were immunologically stained with anti-osteocalcin antibody to identify osteocalcin expression near the trabeculae in the metaphysis in each group. The results revealed considerably osteocalcin expression around trabeculae in the normal + HUMSCs group (G). Some osteocalcin expression was detected in the normal + PBS and OVX + HUMSCs groups (F and I). In the OVX + PBS group, only slight expression was detected (H, arrows). Histological sections obtained from the left distal femur were stained with antihuman nuclei antibody to label HUMSCs. Human nuclei were found within the bone marrow cavity (J2) and trabeculae (J1). Double immunofluorescence revealed that numerous HUMSCs (rhodamine, red in K) and abundant osterix (fluorescein, green in L) existed in the rat femur. The merged image showed that many HUMSCs did differentiate into osterix-expressing osteoblasts (M). The results of RT-PCR further demonstrated that the grafted HUMSCs expressed human osteocalcin at the messenger RNA level (N). * Versus normal + PBS group, P

    Journal: Cell Transplantation

    Article Title: Xenograft of Human Umbilical Mesenchymal Stem Cells from Wharton’s Jelly Differentiating into Osteocytes and Reducing Osteoclast Activity Reverses Osteoporosis in Ovariectomized Rats

    doi: 10.1177/0963689717750666

    Figure Lengend Snippet: Engrafted human umbilical cord-derived mesenchymal stem cells (HUMSCs) differentiate into osteoblasts and inhibit the number of osteoclasts at the distal femur. TRAP staining was performed to detect osteoclasts (arrows) in the metaphysis of the distal femur in the normal + phosphate-buffered saline (PBS; A), normal + HUMSCs (B), ovariectomy (OVX) + PBS (C), and OVX + HUMSCs (D) groups. The number of osteoclasts per trabecular bone area (no./mm 2 ) was quantified. The results indicated no difference in the osteoclast number among the normal + PBS, normal + HUMSCs, and OVX + HUMSCs groups. However, the osteoclast number was significantly higher in the OVX + PBS group than in the other 3 groups (E). Histological sections were immunologically stained with anti-osteocalcin antibody to identify osteocalcin expression near the trabeculae in the metaphysis in each group. The results revealed considerably osteocalcin expression around trabeculae in the normal + HUMSCs group (G). Some osteocalcin expression was detected in the normal + PBS and OVX + HUMSCs groups (F and I). In the OVX + PBS group, only slight expression was detected (H, arrows). Histological sections obtained from the left distal femur were stained with antihuman nuclei antibody to label HUMSCs. Human nuclei were found within the bone marrow cavity (J2) and trabeculae (J1). Double immunofluorescence revealed that numerous HUMSCs (rhodamine, red in K) and abundant osterix (fluorescein, green in L) existed in the rat femur. The merged image showed that many HUMSCs did differentiate into osterix-expressing osteoblasts (M). The results of RT-PCR further demonstrated that the grafted HUMSCs expressed human osteocalcin at the messenger RNA level (N). * Versus normal + PBS group, P

    Article Snippet: Reverse Transcription Polymerase Chain Reaction for Detecting Human Osteocalcin in the Femur of the Rats Total RNA was freshly isolated from the rat femur using the TRIzol Reagent (Invitrogen, USA) and reverse transcribed into complementary DNA with SuperScript III (18,080, Invitrogen).

    Techniques: Derivative Assay, Staining, Expressing, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction

    Treatment of human islets with L-WRN+ regulates large numbers of proliferative genes. (A) mRNAs upregulated in response to L-WRN+ by 1.5-fold vs treatment with 8 mM glucose alone, in islets sourced from two different donors. Relative mRNA levels in 5 mM glucose (5), 8 mM glucose (8), 8 mM glucose+LiCl (8+L) and L-WRN+-treated islet aliquots are shown; blue indicates lower abundance, red indicates higher abundance. (B) as for (A), but showing those mRNAs downregulated by 1.5-fold in response to L-WRN+ treatment. (C) qRT-PCR validation of six regulated and four nonregulated mRNAs ( Mafa , Mnx1 , Neurod1 , Pax6 ); fold-changes of L-WRN+ treatment vs 8 mM glucose are shown. Individual gene expression values were normalized to the geometric mean of B2m , Gapdh , Hmbs and Ywhaz [70] . White and black bars, fold-changes observed with RNA-sequencing; red and blue bars, fold-changes observed with qRT-PCR. (D, upper) Classification of upregulated genes into Gene Ontology categories; the most significantly over-represented categories are shown. (D, lower) Classification of upregulated genes into MetaCore custom-defined categories. (E, upper and lower) as for (D), but showing downregulated genes. Further details of Gene Ontology and MetaCore assignments are provided in Tables S2 and S3 .

    Journal: PLoS ONE

    Article Title: A Novel Strategy to Increase the Proliferative Potential of Adult Human ?-Cells While Maintaining Their Differentiated Phenotype

    doi: 10.1371/journal.pone.0066131

    Figure Lengend Snippet: Treatment of human islets with L-WRN+ regulates large numbers of proliferative genes. (A) mRNAs upregulated in response to L-WRN+ by 1.5-fold vs treatment with 8 mM glucose alone, in islets sourced from two different donors. Relative mRNA levels in 5 mM glucose (5), 8 mM glucose (8), 8 mM glucose+LiCl (8+L) and L-WRN+-treated islet aliquots are shown; blue indicates lower abundance, red indicates higher abundance. (B) as for (A), but showing those mRNAs downregulated by 1.5-fold in response to L-WRN+ treatment. (C) qRT-PCR validation of six regulated and four nonregulated mRNAs ( Mafa , Mnx1 , Neurod1 , Pax6 ); fold-changes of L-WRN+ treatment vs 8 mM glucose are shown. Individual gene expression values were normalized to the geometric mean of B2m , Gapdh , Hmbs and Ywhaz [70] . White and black bars, fold-changes observed with RNA-sequencing; red and blue bars, fold-changes observed with qRT-PCR. (D, upper) Classification of upregulated genes into Gene Ontology categories; the most significantly over-represented categories are shown. (D, lower) Classification of upregulated genes into MetaCore custom-defined categories. (E, upper and lower) as for (D), but showing downregulated genes. Further details of Gene Ontology and MetaCore assignments are provided in Tables S2 and S3 .

    Article Snippet: Polyadenylated mRNA was obtained from 1 µg of human islet total RNA using oligo(dT)-coupled Dynabeads (Invitrogen), and mRNA-sequencing library preparation was performed essentially as described , , using Illumina HiSeq 2000 sequencers and 3′ library indexing rather than 5′ bar-coding.

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

    Inclusion of L-WRN+-regulated mRNAs in Wnt-related, proliferative and TGF-β signaling networks. (A) Partial network of Wnt signaling; red squares designate mRNAs upregulated by at least 1.5-fold in response to L-WRN+. (B) Proliferative signaling network focused on Ki-67 and Sp1. (C) Partial network of TGF-β signaling. Up and downregulated mRNAs in panels A–C were defined using RNA-sequencing; red squares designate mRNAs upregulated by at least 1.5-fold in respone to L-WRN+; blue squares designate downregulated mRNAs. Green connecting lines indicate activation, red connecting lines indicate inhibition, and gray lines indicate mixed or uncertain effects, according to previously published studies contained in the curated MetaCore database. (D E) Wnt pathway targets Axin2 and c-Myc compared amongst treated islets using qRT-PCR. Islets (75) were cultured for indicated times in CMRL-1066 medium containing 10% FBS and 5 mM glucose ± 5 mM LiCl or in 50% Wnt3a, R-spondin-3 and Noggin conditioned medium (L-WRN) ± Y-27632 and SB-431542 inhibitors as indicated. Samples were processed for qRT-PCR and levels of mRNA for Axin2 and c-Myc were determined as described in Materials and Methods . Transcripts were normalized to β-actin at the corresponding time point. Data are the means ± SE of n = 3 biological replicates. * P

    Journal: PLoS ONE

    Article Title: A Novel Strategy to Increase the Proliferative Potential of Adult Human ?-Cells While Maintaining Their Differentiated Phenotype

    doi: 10.1371/journal.pone.0066131

    Figure Lengend Snippet: Inclusion of L-WRN+-regulated mRNAs in Wnt-related, proliferative and TGF-β signaling networks. (A) Partial network of Wnt signaling; red squares designate mRNAs upregulated by at least 1.5-fold in response to L-WRN+. (B) Proliferative signaling network focused on Ki-67 and Sp1. (C) Partial network of TGF-β signaling. Up and downregulated mRNAs in panels A–C were defined using RNA-sequencing; red squares designate mRNAs upregulated by at least 1.5-fold in respone to L-WRN+; blue squares designate downregulated mRNAs. Green connecting lines indicate activation, red connecting lines indicate inhibition, and gray lines indicate mixed or uncertain effects, according to previously published studies contained in the curated MetaCore database. (D E) Wnt pathway targets Axin2 and c-Myc compared amongst treated islets using qRT-PCR. Islets (75) were cultured for indicated times in CMRL-1066 medium containing 10% FBS and 5 mM glucose ± 5 mM LiCl or in 50% Wnt3a, R-spondin-3 and Noggin conditioned medium (L-WRN) ± Y-27632 and SB-431542 inhibitors as indicated. Samples were processed for qRT-PCR and levels of mRNA for Axin2 and c-Myc were determined as described in Materials and Methods . Transcripts were normalized to β-actin at the corresponding time point. Data are the means ± SE of n = 3 biological replicates. * P

    Article Snippet: Polyadenylated mRNA was obtained from 1 µg of human islet total RNA using oligo(dT)-coupled Dynabeads (Invitrogen), and mRNA-sequencing library preparation was performed essentially as described , , using Illumina HiSeq 2000 sequencers and 3′ library indexing rather than 5′ bar-coding.

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

    Effect of growth substrate on the expression of XynR, as determined by a nuclease protection assay. Exponentially growing cultures of P. bryantii B 1 4 were pulsed with either 0.05% WS-X (XW-X), 0.05% xylose, or 0.05% glucose for 30 min, and then total RNA was extracted and 10 μg was transferred onto a Hybond-N + positively charged nylon membrane. Two micrograms of S. cerevisiae total RNA (Yeast RNA) was used as a negative control. A 609-bp PCR product (obtained with primers Reg-UP and Reg-DOWN) corresponding to 398 bp of the xynR upstream sequence and 211 bp of xynR (residues 1681 to 2290) was labeled with DIG and used as a probe in the nuclease protection assay.

    Journal: Journal of Bacteriology

    Article Title: Involvement of the Multidomain Regulatory Protein XynR in Positive Control of Xylanase Gene Expression in the Ruminal Anaerobe Prevotella bryantii B14

    doi: 10.1128/JB.185.7.2219-2226.2003

    Figure Lengend Snippet: Effect of growth substrate on the expression of XynR, as determined by a nuclease protection assay. Exponentially growing cultures of P. bryantii B 1 4 were pulsed with either 0.05% WS-X (XW-X), 0.05% xylose, or 0.05% glucose for 30 min, and then total RNA was extracted and 10 μg was transferred onto a Hybond-N + positively charged nylon membrane. Two micrograms of S. cerevisiae total RNA (Yeast RNA) was used as a negative control. A 609-bp PCR product (obtained with primers Reg-UP and Reg-DOWN) corresponding to 398 bp of the xynR upstream sequence and 211 bp of xynR (residues 1681 to 2290) was labeled with DIG and used as a probe in the nuclease protection assay.

    Article Snippet: A total-RNA solution was mixed with 600 pg of DIG-labeled probe and 2 μg of yeast RNA (Ambion Inc.), and this was followed by ethanol precipitation.

    Techniques: Expressing, Negative Control, Polymerase Chain Reaction, Sequencing, Labeling

    MicroRNA (miRNA) and RLuc mRNA analysis of scAAV2-HCV-miR-Cluster 5-injected mice. ( a–e ) Northern blot analyses of miRNA guide strands. Twenty-five µg of total RNA from mice injected with increasing doses of scAAV8-HCV-miR-Cluster 5 (2.5

    Journal: Molecular Therapy

    Article Title: Preclinical Evaluation of An Anti-HCV miRNA Cluster for Treatment of HCV Infection

    doi: 10.1038/mt.2012.247

    Figure Lengend Snippet: MicroRNA (miRNA) and RLuc mRNA analysis of scAAV2-HCV-miR-Cluster 5-injected mice. ( a–e ) Northern blot analyses of miRNA guide strands. Twenty-five µg of total RNA from mice injected with increasing doses of scAAV8-HCV-miR-Cluster 5 (2.5

    Article Snippet: Quantitation of the anti-HCV miRNAs in Huh-7.5 cell extracts and mouse liver RNA was performed using custom TaqMan small RNA QRT-PCR assays (Applied Biosystems) according to the manufacturer's instructions, using synthetic oligonucleotides to generate standard curves.

    Techniques: Injection, Mouse Assay, Northern Blot

    Identification of repressive chromatin-associated lncRNAs using ChRIP-seq. ( a ) The ChRIP-seq analysis pipeline used to identify lncRNAs enriched in repressive chromatin. The pie chart shows 276 lncRNAs enriched in both EZH2 and H3K27me3 ChRIP-seq samples compared with the nuclear RNA (input). The P value was obtained by performing a hypergeometric test using all the lncRNAs in our analysis. ( b ) Bar diagram showing the distribution of T-to-C transitions (indicative of putative RNA–protein contact sites) in input (8,361), EZH2 (18,905) and H3K27me3 (2,651) ChRIP-seq data. Black in the EZH2 bar indicates the number of T-to-C transitions (1,253) that are either present in input or H3K27me3 samples, and blue indicates EZH2-specific T-to-C transitions (17,652). The EZH2-specific T-to-C transitions (17,652) were used to associate with lncRNAs. ( c ) All the possible conversions present in the EZH2 ChRIP-seq sample. T-to-C conversion and the reverse-strand A-to-G conversions were predominant among all the possible conversion events. ( d ) LncRNAs (1,046; annotated and non-annotated) harbour EZH2-specific (17,652) T-to-C conversion site. Seventy repressive chromatin-enriched lncRNAs (out of 276) carry T-to-C transitions, including known PRC2-interacting lncRNAs such as MEG3 , KCNQ1OT1 and BDNF-AS1. The P value was obtained by performing a hypergeometric test using all the lncRNAs considered in our analysis. ( e,f ) The distribution of the sequencing reads on MEG3 and KCNQ1OT1 transcripts from H3K27me3, EZH2-enriched chromatin fractions and input RNA samples. The tags represent the read distribution and the signal represents the intensity of reads over MEG3 and KCNQ1OT1 transcripts. Locations of T-to-C transitions over the exons are depicted below the physical maps. The left panel depicts the RPKM (Reads per kilobase per million) for MEG3 and KCNQ1OT1 in H3K27me3, EZH2 ChRIP RNA and input RNA samples. The fold enrichment (FC) in H3K27me3 and EZH2 ChRIP RNA compared with input is indicated. ( g ) ChRIP validation: RT–qPCR data showing the enrichment of the selected annotated and non-annotated lncRNAs in the EZH2 and H3K27me3 ChRIP pull-downs compared with input. We did not observe any enrichment of these lncRNAs in the H3K4me2 (active chromatin marks) and immunoglobulin G (IgG; nonspecific antibody) ChRIP pull-downs. Actin was used as a negative control. Data represent the mean±s.d. of three independent biological experiments.

    Journal: Nature Communications

    Article Title: MEG3 long noncoding RNA regulates the TGF-β pathway genes through formation of RNA–DNA triplex structures

    doi: 10.1038/ncomms8743

    Figure Lengend Snippet: Identification of repressive chromatin-associated lncRNAs using ChRIP-seq. ( a ) The ChRIP-seq analysis pipeline used to identify lncRNAs enriched in repressive chromatin. The pie chart shows 276 lncRNAs enriched in both EZH2 and H3K27me3 ChRIP-seq samples compared with the nuclear RNA (input). The P value was obtained by performing a hypergeometric test using all the lncRNAs in our analysis. ( b ) Bar diagram showing the distribution of T-to-C transitions (indicative of putative RNA–protein contact sites) in input (8,361), EZH2 (18,905) and H3K27me3 (2,651) ChRIP-seq data. Black in the EZH2 bar indicates the number of T-to-C transitions (1,253) that are either present in input or H3K27me3 samples, and blue indicates EZH2-specific T-to-C transitions (17,652). The EZH2-specific T-to-C transitions (17,652) were used to associate with lncRNAs. ( c ) All the possible conversions present in the EZH2 ChRIP-seq sample. T-to-C conversion and the reverse-strand A-to-G conversions were predominant among all the possible conversion events. ( d ) LncRNAs (1,046; annotated and non-annotated) harbour EZH2-specific (17,652) T-to-C conversion site. Seventy repressive chromatin-enriched lncRNAs (out of 276) carry T-to-C transitions, including known PRC2-interacting lncRNAs such as MEG3 , KCNQ1OT1 and BDNF-AS1. The P value was obtained by performing a hypergeometric test using all the lncRNAs considered in our analysis. ( e,f ) The distribution of the sequencing reads on MEG3 and KCNQ1OT1 transcripts from H3K27me3, EZH2-enriched chromatin fractions and input RNA samples. The tags represent the read distribution and the signal represents the intensity of reads over MEG3 and KCNQ1OT1 transcripts. Locations of T-to-C transitions over the exons are depicted below the physical maps. The left panel depicts the RPKM (Reads per kilobase per million) for MEG3 and KCNQ1OT1 in H3K27me3, EZH2 ChRIP RNA and input RNA samples. The fold enrichment (FC) in H3K27me3 and EZH2 ChRIP RNA compared with input is indicated. ( g ) ChRIP validation: RT–qPCR data showing the enrichment of the selected annotated and non-annotated lncRNAs in the EZH2 and H3K27me3 ChRIP pull-downs compared with input. We did not observe any enrichment of these lncRNAs in the H3K4me2 (active chromatin marks) and immunoglobulin G (IgG; nonspecific antibody) ChRIP pull-downs. Actin was used as a negative control. Data represent the mean±s.d. of three independent biological experiments.

    Article Snippet: Since the chromatin-bound RNA yield from one single experiment is suboptimal for high-throughput sequencing, we pooled RNA from 6 to 8 ChRIP pull-downs, and the sequencing library was made using SOLiD Total RNA-Seq Kit and sequenced the library using SOLiD platform (Applied Biosystem).

    Techniques: Sequencing, Quantitative RT-PCR, Negative Control

    Strategy for cloning GABAB1k. The top figure represents exon-intron composition of the proposed GABAB1k. Numbered rectangular boxes represent known exons, and lines between boxes are introns. Two arrows on the bottom of the boxes represent ORF. Black bar is intron 4, and the two probe sequence data, NCBI accession numbers W07715 and N80593, are indicated as #1 and #2 above small rectangular boxes, respectively. They are 5′ and 3′ sequences of the microarray probe, clone 300899. A arrow above the black bar is a possible transcription initiation site. Horizontal lines indicate sequences detected after PCR. Based on microarray probe, clone 300899, the following clones were detected in human cultured cells, mouse midbrain, and rat hippocampus: #2, #3, #4, #11, and #18. From the previous microarray probe sequence analysis, the microarray probe aligned to intron 4 and was either 3′ or 5′ UTR of novel isoforms. Because there was no known human GABAB1 isoform that had intron 4 as an exon, the two possible isoform models, GABAB1j and GABAB1k, were proposed. The predicted GABAB1j and GAGAG1k contain intron 4 as 3′ or 5′ UTR, respectively. Because GABAB1j and GABAB1k are only two possible isoform models that contain clone 300899, #2, #3, #4, #11, and #18 can be partial GABAB1j or GABAB1k and show their existences. From the same RNA sources six different N-terminal partial clones, #5-1, #5-2, #6-1, #6-2, #12, and 17 19, indicated the existence of GABAB1k. They share common 5′ UTRs and partial N-terminal ORFs of GABAB1k and suggest that GABAB1k exists in human, mouse, and rat. To clone the full ORF of GABAB1k, additional primers were designed at its possible 3′ UTR region based on other known isoform sequence analyses. Full ORF containing clones were cloned from human brain and mouse midbrain. Bottom two boxes show clones which contain the full ORF (a b, a 7, 3 7 in human and c d in mouse).

    Journal: PLoS ONE

    Article Title: Intron 4 Containing Novel GABAB1 Isoforms Impair GABAB Receptor Function

    doi: 10.1371/journal.pone.0014044

    Figure Lengend Snippet: Strategy for cloning GABAB1k. The top figure represents exon-intron composition of the proposed GABAB1k. Numbered rectangular boxes represent known exons, and lines between boxes are introns. Two arrows on the bottom of the boxes represent ORF. Black bar is intron 4, and the two probe sequence data, NCBI accession numbers W07715 and N80593, are indicated as #1 and #2 above small rectangular boxes, respectively. They are 5′ and 3′ sequences of the microarray probe, clone 300899. A arrow above the black bar is a possible transcription initiation site. Horizontal lines indicate sequences detected after PCR. Based on microarray probe, clone 300899, the following clones were detected in human cultured cells, mouse midbrain, and rat hippocampus: #2, #3, #4, #11, and #18. From the previous microarray probe sequence analysis, the microarray probe aligned to intron 4 and was either 3′ or 5′ UTR of novel isoforms. Because there was no known human GABAB1 isoform that had intron 4 as an exon, the two possible isoform models, GABAB1j and GABAB1k, were proposed. The predicted GABAB1j and GAGAG1k contain intron 4 as 3′ or 5′ UTR, respectively. Because GABAB1j and GABAB1k are only two possible isoform models that contain clone 300899, #2, #3, #4, #11, and #18 can be partial GABAB1j or GABAB1k and show their existences. From the same RNA sources six different N-terminal partial clones, #5-1, #5-2, #6-1, #6-2, #12, and 17 19, indicated the existence of GABAB1k. They share common 5′ UTRs and partial N-terminal ORFs of GABAB1k and suggest that GABAB1k exists in human, mouse, and rat. To clone the full ORF of GABAB1k, additional primers were designed at its possible 3′ UTR region based on other known isoform sequence analyses. Full ORF containing clones were cloned from human brain and mouse midbrain. Bottom two boxes show clones which contain the full ORF (a b, a 7, 3 7 in human and c d in mouse).

    Article Snippet: Cloning novel isoforms, GABAB1k, GABAB1l, GABAB1m, and GABAB1n To clone full ORFs of the novel isoforms, cDNAs were generated with oligo(dT) primer from human brain total RNA (Ambion, Austin, TX, USA) and mouse midbrain total RNA.

    Techniques: Clone Assay, Sequencing, Microarray, Polymerase Chain Reaction, Cell Culture