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  • 99
    Thermo Fisher complementary dna cdna synthesis kit
    Overexpression of AMF1 affects cellular responses under etoposide treatment. (A) Overexpression of AMF1 in U2OS holds more cells in G 1 phase and inhibits etoposide-induced S-G 2 arrest and apoptosis. U2OS/AMF1 or U2OS/β-Gal cells were treated with 10 μM etoposide for 0, 24, and 48 h. Cells were harvested, processed, and subjected to flow cytometric analysis. Cell cycle stages are represented by the cellular DNA content, which was analyzed by PI staining and fluorescence-activated cell sorting. Boundaries for G 1 and S-G 2 phases are labeled on the top pair of graphs. (B) Western blotting analysis of AMF1, p53, and p21 WAF1/CIP1 in U2OS cells at 0, 4, 8, and 24 h after addition of 10 μM etoposide into culture medium. Protein concentrations in cell extracts were determined, and equal amounts were loaded in each lane as judged by the level of α-tubulin. Intensity of bands at 0 h shows the baseline level of each protein. (C) <t>RT-PCR</t> analysis of p21 WAF1/CIP1 transcripts in U2OS cells at 0, 4, 8, and 24 h after addition of 10 μM etoposide into culture medium. The 370-bp product was amplified using p21 oligonucleotides. As a control, the <t>cDNA</t> encoding GAPDH was amplified with specific oligonucleotides.
    Complementary Dna Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 692 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore complementary dna cdna synthesis
    Overexpression of AMF1 affects cellular responses under etoposide treatment. (A) Overexpression of AMF1 in U2OS holds more cells in G 1 phase and inhibits etoposide-induced S-G 2 arrest and apoptosis. U2OS/AMF1 or U2OS/β-Gal cells were treated with 10 μM etoposide for 0, 24, and 48 h. Cells were harvested, processed, and subjected to flow cytometric analysis. Cell cycle stages are represented by the cellular DNA content, which was analyzed by PI staining and fluorescence-activated cell sorting. Boundaries for G 1 and S-G 2 phases are labeled on the top pair of graphs. (B) Western blotting analysis of AMF1, p53, and p21 WAF1/CIP1 in U2OS cells at 0, 4, 8, and 24 h after addition of 10 μM etoposide into culture medium. Protein concentrations in cell extracts were determined, and equal amounts were loaded in each lane as judged by the level of α-tubulin. Intensity of bands at 0 h shows the baseline level of each protein. (C) <t>RT-PCR</t> analysis of p21 WAF1/CIP1 transcripts in U2OS cells at 0, 4, 8, and 24 h after addition of 10 μM etoposide into culture medium. The 370-bp product was amplified using p21 oligonucleotides. As a control, the <t>cDNA</t> encoding GAPDH was amplified with specific oligonucleotides.
    Complementary Dna Cdna Synthesis, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Illumina Inc complementary dna cdna synthesis
    ( A ) Comparison of average mapping statistics of transcriptomic content among three datasets using 50 bp reads from Genome Analyzer IIx (Illumina, Inc.): <t>cDNA</t> libraries made from single RNA samples of mouse testis tissue in which mRNA was enriched from total RNA either by (i) TruSeq™ poly-A selection or by (ii) RiboMinus™ rRNA depletion and (iii) cDNA library in which cDNA was synthesized directly from total RNA (DNase treated and left untreated) using the Ovation® RNA-Seq system. ( B ) Complexity of libraries (percent of unique start sites, unique pairs out of total mapped reads): cDNA libraries of TruSeq™ poly-A selection, RiboMinus™ rRNA depletion and Ovation® RNA-seq amplification system (different input amounts of total RNA). The sample prepared using the Ovation® RNA-Seq system provided slightly higher percentage of unique start sites.
    Complementary Dna Cdna Synthesis, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 96/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher complementary dna cdna synthesis
    Expression levels of type I collagen, Smad2/3, and TGF-β1 genes after ES-P treatment in aged mice. (A) The left ears of fourteen-week old mice were treated daily with 30 μg T . spiralis ES-P for 14 days (red color). The right ears of were treated daily with PBS for 14 days as control (blue color). After 14 days, ears were collected. Total <t>RNA</t> was extracted from each ear tissue and <t>cDNA</t> was constructed. (B) The gene expression levels of collagen I , TGF-β1 , and Smad2/3 were analyzed by real-time PCR. The gene expression value of each group was normalized by control value, 14 weeks group. (**; P
    Complementary Dna Cdna Synthesis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10167 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Viogene complementary dna cdna synthesis
    Expression levels of type I collagen, Smad2/3, and TGF-β1 genes after ES-P treatment in aged mice. (A) The left ears of fourteen-week old mice were treated daily with 30 μg T . spiralis ES-P for 14 days (red color). The right ears of were treated daily with PBS for 14 days as control (blue color). After 14 days, ears were collected. Total <t>RNA</t> was extracted from each ear tissue and <t>cDNA</t> was constructed. (B) The gene expression levels of collagen I , TGF-β1 , and Smad2/3 were analyzed by real-time PCR. The gene expression value of each group was normalized by control value, 14 weeks group. (**; P
    Complementary Dna Cdna Synthesis, supplied by Viogene, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa cdna synthesis
    LIRKO mice show quantitative and qualitative defects in bile acid synthesis on a chow diet (a) Real-time <t>PCR</t> analysis of <t>cDNA</t> prepared from six-week old mice sacrificed during the light cycle following a 24 hour fast; the expression of each gene was normalized to its value in Lox mice (n=5-8, *p=0.05 versus Lox). (b) Since the synthesis of bile acids is equivalent to fecal excretion in the steady state, we assessed bile acid synthesis by collecting feces from 3 month old Lox and LIRKO mice for 72 hours and measuring total bile acid outputs (n=4, p=0.02). Hepatic bile from 5-6 month old Lox and LIRKO mice was subjected to HPLC analysis as described in Methods (n=4-5). (c) Hydrophobicity indices were calculated as described in Methods (*p=0.002). (d) The ratio of muricholates to cholate was calculated using the molar percentages of tauroα- and tauroβ- muricholate and taurocholate (*p=0.001). (e) Three-month old mice were gavaged with an FXR agonist (GW4064) at 100 mg/kg/d or vehicle for 14 days. Mice were fasted overnight, and sacrificed two hours after the last dose of agonist. Bile was expressed from the gallbladder and ratio of muricholates to cholate was determined as above. *p
    Cdna Synthesis, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 11844 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Promega complementary dna cdna synthesis kit
    A, Ethidium bromide-stained agarose gel of dsRNA. Lanes a and b contain dsRNA extracts from two representative strains of Lentinula edodes FMRI0339 <t>mycovirus</t> (LeV-FMRI0339) and Pleurotus ostreatus ASI2792 mycovirus (ASI2792-PoV) with the characteristic 12-kb viral genome of LeV and approximately 8.0-kb viral genome of ASI2792-PoV, respectively. Lanes 1 to 14 contain dsRNA extracts from 14 isolates obtained by the mycelia fragmentation method described in “Materials and Methods”; B, Northern blot analysis of the corresponding gel with the dsRNA using a partial <t>cDNA</t> clone (792 bp) encoding the RNA-dependent RNA polymerase (RdRp) protein in the PoV-ASI2792 as a probe. Arrowheads indicate the mycovirus genome segment from PoV-ASI2792.
    Complementary Dna Cdna Synthesis Kit, supplied by Promega, used in various techniques. Bioz Stars score: 86/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    TaKaRa complementary dna cdna synthesis kit
    Bioactivity and diagram of AGSs isolated from mouse hypertrophic heart. In A and B , data are presented in three panels to illustrate the viability of the transformed yeast and the galactose-dependent growth under the selective pressure of exclusion of histidine from the medium. Galactose promotes the expression of each <t>cDNA</t> in the pYES2-containing GAL1 promoter. About 2000 cells were suspended in H 2 O and spotted on medium with glucose plus histidine ( left ; no selection), glucose minus histidine ( center ; selection without induction), or galactose plus histidine ( right ; selection plus induction). A , epistasis analysis of isolated clones. Transformants in a yeast strain expressing human Gα 16 (Gpa1(1–41)) and yeast lacking Gα, Gβ, or downstream signaling molecules (Δ G α, yeast lacking Gα; Δ G β, yeast lacking Gβ; Δ Ste20 , yeast lacking p21-activated kinase; Δ Ste5 , yeast lacking the kinase scaffold protein). B , effect of isolated <t>cDNAs</t> in yeast expressing various types of Gα. C , schematic diagram of the sequences of TFE3, TFEB, and MITF in mouse. The line above the sequence refers to cDNA isolated by the yeast-based functional screen. HLH , helix-loop-helix. D , bioactivity of full-length TFE3, TFEB, and MITF. The full-length clones were transformed into yeast expressing Gα 16 . The magnitude of activation of G-protein signaling pathway was monitored by β-galactosidase activity. Data are presented as the mean S.E. of five experiments with duplicate determinations. *, p
    Complementary Dna Cdna Synthesis Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Bioneer Corporation complementary dna cdna synthesis kit
    Bioactivity and diagram of AGSs isolated from mouse hypertrophic heart. In A and B , data are presented in three panels to illustrate the viability of the transformed yeast and the galactose-dependent growth under the selective pressure of exclusion of histidine from the medium. Galactose promotes the expression of each <t>cDNA</t> in the pYES2-containing GAL1 promoter. About 2000 cells were suspended in H 2 O and spotted on medium with glucose plus histidine ( left ; no selection), glucose minus histidine ( center ; selection without induction), or galactose plus histidine ( right ; selection plus induction). A , epistasis analysis of isolated clones. Transformants in a yeast strain expressing human Gα 16 (Gpa1(1–41)) and yeast lacking Gα, Gβ, or downstream signaling molecules (Δ G α, yeast lacking Gα; Δ G β, yeast lacking Gβ; Δ Ste20 , yeast lacking p21-activated kinase; Δ Ste5 , yeast lacking the kinase scaffold protein). B , effect of isolated <t>cDNAs</t> in yeast expressing various types of Gα. C , schematic diagram of the sequences of TFE3, TFEB, and MITF in mouse. The line above the sequence refers to cDNA isolated by the yeast-based functional screen. HLH , helix-loop-helix. D , bioactivity of full-length TFE3, TFEB, and MITF. The full-length clones were transformed into yeast expressing Gα 16 . The magnitude of activation of G-protein signaling pathway was monitored by β-galactosidase activity. Data are presented as the mean S.E. of five experiments with duplicate determinations. *, p
    Complementary Dna Cdna Synthesis Kit, supplied by Bioneer Corporation, used in various techniques. Bioz Stars score: 89/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad complementary dna cdna synthesis kit
    Breast tumor kinase expression in breast cancer cell lines. (a) Total RNA was isolated from the breast cancer cell lines using the RNeasy kit (Qiagen), and the <t>cDNA</t> was generated using <t>iScript</t> cDNA synthesis (Bio-Rad). The cDNA was amplified using primers specifically for breast tumor kinase (Brk) or β-actin. The relative level of Brk mRNA was determined using densitometric analysis and was normalized to the amount of β-actin. The level of Brk mRNA in the MDA-MB-468 cell line was set to 1; numbers indicate the fold increase compared with the 468 cells. (b) Whole-cell lysates were prepared from the breast cancer cell lines. Lysates were immunoblotted with anti-Brk antibody (top) and with anti-Ran antibody (bottom). The relative expression of Brk protein was determined using densitometric analysis and was normalized to the amount of Ran. The expression of Brk protein in the MDA-MB-468 cell line was set to 1; numbers indicate the fold increase compared with the 468 cells.
    Complementary Dna Cdna Synthesis Kit, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 102 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Roche cdna synthesis kit
    Messenger <t>RNA</t> expression of TNC receptors of MCF-7 cells after TGF-β and TNC treatments. ( a ) mRNA was extracted from cells co-stimulated with TNC (10 μg/ml) and TGF-β1 (5 ng/ml) for synthesis of <t>cDNA</t> and performance of real-time PCR determining the cycle threshold ( C t ) values for each subunit. Differences in C t values of TNC receptor mRNAs from that of GAPDH mRNA in TGF-β1/TNC-treated cells are shown using qPCR. C t values of α7/8/9 and β3 integrin subunits were very high, indicating only limited expression. Calibration curves were obtained using each target gene in the samples. ( b ) Changes of receptor mRNA expression for αv/2 and β1/6 after treatment with TGF-β1 and/or TNC. The results were normalized to GAPDH expression. Integrin αv showed significant increase after TGF-β1/TNC treatment and β6 was dramatically increased after TGF-β1 addition, with or without TNC (mean±s.d.; * P
    Cdna Synthesis Kit, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 977 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    TransGen biotech co cdna synthesis kit
    Messenger <t>RNA</t> expression of TNC receptors of MCF-7 cells after TGF-β and TNC treatments. ( a ) mRNA was extracted from cells co-stimulated with TNC (10 μg/ml) and TGF-β1 (5 ng/ml) for synthesis of <t>cDNA</t> and performance of real-time PCR determining the cycle threshold ( C t ) values for each subunit. Differences in C t values of TNC receptor mRNAs from that of GAPDH mRNA in TGF-β1/TNC-treated cells are shown using qPCR. C t values of α7/8/9 and β3 integrin subunits were very high, indicating only limited expression. Calibration curves were obtained using each target gene in the samples. ( b ) Changes of receptor mRNA expression for αv/2 and β1/6 after treatment with TGF-β1 and/or TNC. The results were normalized to GAPDH expression. Integrin αv showed significant increase after TGF-β1/TNC treatment and β6 was dramatically increased after TGF-β1 addition, with or without TNC (mean±s.d.; * P
    Cdna Synthesis Kit, supplied by TransGen biotech co, used in various techniques. Bioz Stars score: 96/100, based on 132 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher complementary dna cdna synthesis supermix kit
    Messenger <t>RNA</t> expression of TNC receptors of MCF-7 cells after TGF-β and TNC treatments. ( a ) mRNA was extracted from cells co-stimulated with TNC (10 μg/ml) and TGF-β1 (5 ng/ml) for synthesis of <t>cDNA</t> and performance of real-time PCR determining the cycle threshold ( C t ) values for each subunit. Differences in C t values of TNC receptor mRNAs from that of GAPDH mRNA in TGF-β1/TNC-treated cells are shown using qPCR. C t values of α7/8/9 and β3 integrin subunits were very high, indicating only limited expression. Calibration curves were obtained using each target gene in the samples. ( b ) Changes of receptor mRNA expression for αv/2 and β1/6 after treatment with TGF-β1 and/or TNC. The results were normalized to GAPDH expression. Integrin αv showed significant increase after TGF-β1/TNC treatment and β6 was dramatically increased after TGF-β1 addition, with or without TNC (mean±s.d.; * P
    Complementary Dna Cdna Synthesis Supermix Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher cdna synthesis system
    Expression pattern of HERF1 . Northern blots of adult tissues (A), murine embryos (B), or MEL cells following treatment with the differentiation-inducing agent DMSO (D) were hybridized with a full-length HERF1 <t>cDNA.</t> Filters were stripped and rehybridized with a probe for glycerol-3-phosphate dehydrogenase (GPDH) to assess the integrity and amount of <t>RNA</t> and with a probe for β-globin (D; numbers above the lanes represent days in DMSO). (C) In situ hybridization was performed with a 271-bp restriction fragment from the unique 3′ region of HERF1 labeled with [ 33 P]UTP. The left side shows the bright-field view of a 12-μm sagittal section from an E15 embryo stained with hematoxylin and eosin; the right side is a dark-field view of the same section hybridized with the HERF1 probe. Sense control shows no specific hybridization (data not shown).
    Cdna Synthesis System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 219 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Stratagene complementary dna cdna synthesis complementary dna
    Analysis of SMU.2080 transcription factor activity A) Illustrated here are the sequences of the <t>DNA</t> fragments used for electrophoretic mobility shift assays (EMSA) of SMU.2080 and the promoter regions of SMU.150, SMU.423, and SMU.1906. Sequences labeled DR+ contain the wild-type direct repeat sequence, whereas those labeled DR- contain a rearrangement of the direct repeat nucleotides. All other bases in the DNA fragments are identical. EMSA analysis was performed using purified recombinant SMU.2080 and both wild-type (DR+) and mutant (DR-) direct repeat DNA for B) SMU.150, C) SMU.423, and D) SMU.1906. As described in Experimental Procedures, increasing concentrations of SMU.2080 were incubated with 10 ng of the indicated DNA before running the reactions on a nondenaturing polyacrylamide gel. For each series of four EMSA reactions, the first lane contained no protein. E) Rapid amplification of <t>cDNA</t> ends (RACE) PCR was used to determine the transcription start sites of SMU.150, SMU.423, and SMU.1906. The LytTR family direct repeats are shown in bold, while the -35 and extended -10 sequences are underlined. The transcription start sites are indicated with an arrow over the +1 site.
    Complementary Dna Cdna Synthesis Complementary Dna, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Meridian Life Science cdna synthesis kit
    Analysis of SMU.2080 transcription factor activity A) Illustrated here are the sequences of the <t>DNA</t> fragments used for electrophoretic mobility shift assays (EMSA) of SMU.2080 and the promoter regions of SMU.150, SMU.423, and SMU.1906. Sequences labeled DR+ contain the wild-type direct repeat sequence, whereas those labeled DR- contain a rearrangement of the direct repeat nucleotides. All other bases in the DNA fragments are identical. EMSA analysis was performed using purified recombinant SMU.2080 and both wild-type (DR+) and mutant (DR-) direct repeat DNA for B) SMU.150, C) SMU.423, and D) SMU.1906. As described in Experimental Procedures, increasing concentrations of SMU.2080 were incubated with 10 ng of the indicated DNA before running the reactions on a nondenaturing polyacrylamide gel. For each series of four EMSA reactions, the first lane contained no protein. E) Rapid amplification of <t>cDNA</t> ends (RACE) PCR was used to determine the transcription start sites of SMU.150, SMU.423, and SMU.1906. The LytTR family direct repeats are shown in bold, while the -35 and extended -10 sequences are underlined. The transcription start sites are indicated with an arrow over the +1 site.
    Cdna Synthesis Kit, supplied by Meridian Life Science, used in various techniques. Bioz Stars score: 93/100, based on 448 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher cdna synthesis kit
    Expression analysis of mecA in JE2 and mutant strains. <t>RNA</t> was isolated from the exponentially grown (0.5–1.2 OD 600nm ) cultures treated with or without oxicillin (Ox) 4 μg/ml for 60 min in MHB. <t>cDNA</t> was prepared from RNA samples and used as template with appropriate primer sets, and relative expression levels were calculated as mentioned in the Materials and Methods section. Each bar shows the mean and standard deviation of values obtained from three replicates. The effect of mutation and oxacillin on expression of pbp2 and mecA was analyzed by performing multiple pairwise comparisons for pbp2 (uppercase letters) and mecA (lowercase letters) using ANOVA followed by Tukey's post-hoc test, and p -values
    Cdna Synthesis Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8801 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Quanta Biosciences cdna synthesis kit
    Expression analysis of mecA in JE2 and mutant strains. <t>RNA</t> was isolated from the exponentially grown (0.5–1.2 OD 600nm ) cultures treated with or without oxicillin (Ox) 4 μg/ml for 60 min in MHB. <t>cDNA</t> was prepared from RNA samples and used as template with appropriate primer sets, and relative expression levels were calculated as mentioned in the Materials and Methods section. Each bar shows the mean and standard deviation of values obtained from three replicates. The effect of mutation and oxacillin on expression of pbp2 and mecA was analyzed by performing multiple pairwise comparisons for pbp2 (uppercase letters) and mecA (lowercase letters) using ANOVA followed by Tukey's post-hoc test, and p -values
    Cdna Synthesis Kit, supplied by Quanta Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 196 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Promega goscript complementary dna cdna synthesis kit
    Expression analysis of mecA in JE2 and mutant strains. <t>RNA</t> was isolated from the exponentially grown (0.5–1.2 OD 600nm ) cultures treated with or without oxicillin (Ox) 4 μg/ml for 60 min in MHB. <t>cDNA</t> was prepared from RNA samples and used as template with appropriate primer sets, and relative expression levels were calculated as mentioned in the Materials and Methods section. Each bar shows the mean and standard deviation of values obtained from three replicates. The effect of mutation and oxacillin on expression of pbp2 and mecA was analyzed by performing multiple pairwise comparisons for pbp2 (uppercase letters) and mecA (lowercase letters) using ANOVA followed by Tukey's post-hoc test, and p -values
    Goscript Complementary Dna Cdna Synthesis Kit, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Quanta Biosciences superscript complementary dna cdna synthesis kit
    Expression analysis of mecA in JE2 and mutant strains. <t>RNA</t> was isolated from the exponentially grown (0.5–1.2 OD 600nm ) cultures treated with or without oxicillin (Ox) 4 μg/ml for 60 min in MHB. <t>cDNA</t> was prepared from RNA samples and used as template with appropriate primer sets, and relative expression levels were calculated as mentioned in the Materials and Methods section. Each bar shows the mean and standard deviation of values obtained from three replicates. The effect of mutation and oxacillin on expression of pbp2 and mecA was analyzed by performing multiple pairwise comparisons for pbp2 (uppercase letters) and mecA (lowercase letters) using ANOVA followed by Tukey's post-hoc test, and p -values
    Superscript Complementary Dna Cdna Synthesis Kit, supplied by Quanta Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies cdna synthesis kit
    Isolation of IgM-binding subclones and identification of <t>cDNA</t> inserts. (A) Cells transduced by the retroviral expression construct containing CLL-derived (top) or PMA-activated 697 pre–B cell–derived (bottom) cDNA libraries were enriched for IgM binding by FACS and subcloned for limiting dilution. Three representative subclones from each library are shown for their IgM-binding activity or lack of binding, as determined by flow cytometry. (B) Agarose gel electrophoresis analysis of RT-PCR products. <t>RNA</t> isolated from nontransduced control BW5147 T cells (lane 1) and from IgM-binding (lanes 3–5 and 7–9) or IgM-nonbinding (lanes 2 and 6) subclones from CLL-derived (lanes 2–5) and PMA-activated 697 pre–B cell–derived (lanes 6–9) cDNA libraries were subjected to RT-PCR as described in Materials and methods. Amplified products were electrophoresed in 0.7% agarose and stained with ethidium bromide. Lane 10 is a PCR control without a first-strand cDNA template. Hin dIII-digested λ DNA was used as a size marker. The experiments were performed once for A and twice for B.
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    Stratagene cdna synthesis kit
    Primary structure of CAP and mRNA expression. (A) <t>3T3-L1</t> adipocyte <t>cDNA</t> clones isolated from the yeast two-hybrid screen with c-Cbl as bait are shown (black lines) aligned below a schematic of the domain organization of the CAP protein. Numbers in parentheses refer to the number of times each clone was isolated from the yeast two-hybrid screen. The letters A, B, and C indicate the presence of alternative spliced inserts. (B) Total RNA (20 μg) isolated from 3T3-L1 adipocytes or fibroblasts was hybridized to a probe prepared from the DNA insert of clone 2.1 labeled with [α- 32 P]dCTP by random-primer extension (upper). The filter was then stripped and rehybridized with a β-actin probe to estimate the relative amount of RNA in each lane (lower). The migration positions of RNA size markers (in kilobases) are on the left.
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    Thermo Fisher complementary dna cdna synthesis trizol reagent
    Primary structure of CAP and mRNA expression. (A) <t>3T3-L1</t> adipocyte <t>cDNA</t> clones isolated from the yeast two-hybrid screen with c-Cbl as bait are shown (black lines) aligned below a schematic of the domain organization of the CAP protein. Numbers in parentheses refer to the number of times each clone was isolated from the yeast two-hybrid screen. The letters A, B, and C indicate the presence of alternative spliced inserts. (B) Total RNA (20 μg) isolated from 3T3-L1 adipocytes or fibroblasts was hybridized to a probe prepared from the DNA insert of clone 2.1 labeled with [α- 32 P]dCTP by random-primer extension (upper). The filter was then stripped and rehybridized with a β-actin probe to estimate the relative amount of RNA in each lane (lower). The migration positions of RNA size markers (in kilobases) are on the left.
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    UTX mutations in human T-ALL. (A) <t>DNA</t> sequencing chromatogram showing a UTX mutation in the gDNA of a male primary T-ALL patient sample. The mutation is absent in remission material of the same patient. (B) Graphical representation of the localization of genetic lesions in the UTX protein structure. In-frame deletion/insertion mutations are depicted in orange circles and frameshift mutations in blue circles. (C) Genotyping and allelic expression analysis of SNP rs181547731 in gDNA and <t>cDNA</t> derived from female T-ALL lymphoblasts. (D) Graphical representation of the different mutations (dark orange rectangles) and deletions (light orange rectangles) present in a set of T-ALL oncogenes and tumor suppressor genes in 35 primary T-ALL patient samples. The different T-ALL subgroups include TAL-LMO, TLX3, TLX1, HOXA, and patients for whom the subgroup is unknown. The age subgroups include children (age ≤15 years; dark red rectangles) and adults (age > 15 years; light red rectangles). Male and female T-ALL patient samples are presented in dark blue and light blue rectangles, respectively. JmjC, Jumonji C; TPR, tetratricopeptide repeat.
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    Bioneer Corporation complementary dna cdna synthesis
    UTX mutations in human T-ALL. (A) <t>DNA</t> sequencing chromatogram showing a UTX mutation in the gDNA of a male primary T-ALL patient sample. The mutation is absent in remission material of the same patient. (B) Graphical representation of the localization of genetic lesions in the UTX protein structure. In-frame deletion/insertion mutations are depicted in orange circles and frameshift mutations in blue circles. (C) Genotyping and allelic expression analysis of SNP rs181547731 in gDNA and <t>cDNA</t> derived from female T-ALL lymphoblasts. (D) Graphical representation of the different mutations (dark orange rectangles) and deletions (light orange rectangles) present in a set of T-ALL oncogenes and tumor suppressor genes in 35 primary T-ALL patient samples. The different T-ALL subgroups include TAL-LMO, TLX3, TLX1, HOXA, and patients for whom the subgroup is unknown. The age subgroups include children (age ≤15 years; dark red rectangles) and adults (age > 15 years; light red rectangles). Male and female T-ALL patient samples are presented in dark blue and light blue rectangles, respectively. JmjC, Jumonji C; TPR, tetratricopeptide repeat.
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    Evrogen complementary dna cdna synthesis
    UTX mutations in human T-ALL. (A) <t>DNA</t> sequencing chromatogram showing a UTX mutation in the gDNA of a male primary T-ALL patient sample. The mutation is absent in remission material of the same patient. (B) Graphical representation of the localization of genetic lesions in the UTX protein structure. In-frame deletion/insertion mutations are depicted in orange circles and frameshift mutations in blue circles. (C) Genotyping and allelic expression analysis of SNP rs181547731 in gDNA and <t>cDNA</t> derived from female T-ALL lymphoblasts. (D) Graphical representation of the different mutations (dark orange rectangles) and deletions (light orange rectangles) present in a set of T-ALL oncogenes and tumor suppressor genes in 35 primary T-ALL patient samples. The different T-ALL subgroups include TAL-LMO, TLX3, TLX1, HOXA, and patients for whom the subgroup is unknown. The age subgroups include children (age ≤15 years; dark red rectangles) and adults (age > 15 years; light red rectangles). Male and female T-ALL patient samples are presented in dark blue and light blue rectangles, respectively. JmjC, Jumonji C; TPR, tetratricopeptide repeat.
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    Pyrosequencing assay for detection of correctly spliced transcript in fibroblasts of patients with the c.610 + 364G > A mutation . The N158S-forward primer binds in exon 3, the N158S-biot-reverse primer at the junction of exon 4b and exon 5. This primer setting exclusively amplifies <t>cDNA</t> of correctly spliced OPA1 transcripts (top bar, wildtype allele) but no transcripts with retained cryptic exon c between exons 4b/5 (middle bar, mutant allele, and upper pyrosequencing scheme). The sequencing primer (N158S-seq) binds two bases upstream of the heterozygous SNP rs7624750 in exon 4. The A allele of rs7624750 cosegregates with the mutation and cannot be detected in cDNA from mutant untreated cells using this assay. Upon AON treatment, cryptic exon c is skipped and the mutant transcript can be amplified (lower bar) and hence its allelic contribution can be measured via pyrosequencing (lower left scheme). A relative ratio of 35.3% versus 64.7% (A-allele versus G-allele) corresponds to a rescue of 55% of all mutant alleles in the cDNA of treated fibroblasts (example), which corresponds to a shift of total correctly spliced OPA1 mRNA of 77.5% (lower graph) For further graphical illustration of the principle compare Supplementary Figure S2 . AON, antisense oligonucleotides; cDNA, complementary <t>DNA.</t>
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    Pyrosequencing assay for detection of correctly spliced transcript in fibroblasts of patients with the c.610 + 364G > A mutation . The N158S-forward primer binds in exon 3, the N158S-biot-reverse primer at the junction of exon 4b and exon 5. This primer setting exclusively amplifies <t>cDNA</t> of correctly spliced OPA1 transcripts (top bar, wildtype allele) but no transcripts with retained cryptic exon c between exons 4b/5 (middle bar, mutant allele, and upper pyrosequencing scheme). The sequencing primer (N158S-seq) binds two bases upstream of the heterozygous SNP rs7624750 in exon 4. The A allele of rs7624750 cosegregates with the mutation and cannot be detected in cDNA from mutant untreated cells using this assay. Upon AON treatment, cryptic exon c is skipped and the mutant transcript can be amplified (lower bar) and hence its allelic contribution can be measured via pyrosequencing (lower left scheme). A relative ratio of 35.3% versus 64.7% (A-allele versus G-allele) corresponds to a rescue of 55% of all mutant alleles in the cDNA of treated fibroblasts (example), which corresponds to a shift of total correctly spliced OPA1 mRNA of 77.5% (lower graph) For further graphical illustration of the principle compare Supplementary Figure S2 . AON, antisense oligonucleotides; cDNA, complementary <t>DNA.</t>
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    Pyrosequencing assay for detection of correctly spliced transcript in fibroblasts of patients with the c.610 + 364G > A mutation . The N158S-forward primer binds in exon 3, the N158S-biot-reverse primer at the junction of exon 4b and exon 5. This primer setting exclusively amplifies <t>cDNA</t> of correctly spliced OPA1 transcripts (top bar, wildtype allele) but no transcripts with retained cryptic exon c between exons 4b/5 (middle bar, mutant allele, and upper pyrosequencing scheme). The sequencing primer (N158S-seq) binds two bases upstream of the heterozygous SNP rs7624750 in exon 4. The A allele of rs7624750 cosegregates with the mutation and cannot be detected in cDNA from mutant untreated cells using this assay. Upon AON treatment, cryptic exon c is skipped and the mutant transcript can be amplified (lower bar) and hence its allelic contribution can be measured via pyrosequencing (lower left scheme). A relative ratio of 35.3% versus 64.7% (A-allele versus G-allele) corresponds to a rescue of 55% of all mutant alleles in the cDNA of treated fibroblasts (example), which corresponds to a shift of total correctly spliced OPA1 mRNA of 77.5% (lower graph) For further graphical illustration of the principle compare Supplementary Figure S2 . AON, antisense oligonucleotides; cDNA, complementary <t>DNA.</t>
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    MACHEREY NAGEL complementary dna cdna synthesis
    Clostridial flagellin transcript and gene copies during colitis and recovery. <t>RNA</t> and DNA were recovered from the same nucleic acids preparation, RNA was reverse transcribed into single-stranded <t>cDNA.</t> Gene and transcript copies were determined with qPCR. The ratio of flagellin to gene copies was calculated for individual mice and log-transformed. Means were compared by One-Way ANOVA, different letters indicate significance ( P
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    Image Search Results


    Overexpression of AMF1 affects cellular responses under etoposide treatment. (A) Overexpression of AMF1 in U2OS holds more cells in G 1 phase and inhibits etoposide-induced S-G 2 arrest and apoptosis. U2OS/AMF1 or U2OS/β-Gal cells were treated with 10 μM etoposide for 0, 24, and 48 h. Cells were harvested, processed, and subjected to flow cytometric analysis. Cell cycle stages are represented by the cellular DNA content, which was analyzed by PI staining and fluorescence-activated cell sorting. Boundaries for G 1 and S-G 2 phases are labeled on the top pair of graphs. (B) Western blotting analysis of AMF1, p53, and p21 WAF1/CIP1 in U2OS cells at 0, 4, 8, and 24 h after addition of 10 μM etoposide into culture medium. Protein concentrations in cell extracts were determined, and equal amounts were loaded in each lane as judged by the level of α-tubulin. Intensity of bands at 0 h shows the baseline level of each protein. (C) RT-PCR analysis of p21 WAF1/CIP1 transcripts in U2OS cells at 0, 4, 8, and 24 h after addition of 10 μM etoposide into culture medium. The 370-bp product was amplified using p21 oligonucleotides. As a control, the cDNA encoding GAPDH was amplified with specific oligonucleotides.

    Journal: Molecular and Cellular Biology

    Article Title: AMF1 (GPS2) Modulates p53 Transactivation

    doi: 10.1128/MCB.21.17.5913-5924.2001

    Figure Lengend Snippet: Overexpression of AMF1 affects cellular responses under etoposide treatment. (A) Overexpression of AMF1 in U2OS holds more cells in G 1 phase and inhibits etoposide-induced S-G 2 arrest and apoptosis. U2OS/AMF1 or U2OS/β-Gal cells were treated with 10 μM etoposide for 0, 24, and 48 h. Cells were harvested, processed, and subjected to flow cytometric analysis. Cell cycle stages are represented by the cellular DNA content, which was analyzed by PI staining and fluorescence-activated cell sorting. Boundaries for G 1 and S-G 2 phases are labeled on the top pair of graphs. (B) Western blotting analysis of AMF1, p53, and p21 WAF1/CIP1 in U2OS cells at 0, 4, 8, and 24 h after addition of 10 μM etoposide into culture medium. Protein concentrations in cell extracts were determined, and equal amounts were loaded in each lane as judged by the level of α-tubulin. Intensity of bands at 0 h shows the baseline level of each protein. (C) RT-PCR analysis of p21 WAF1/CIP1 transcripts in U2OS cells at 0, 4, 8, and 24 h after addition of 10 μM etoposide into culture medium. The 370-bp product was amplified using p21 oligonucleotides. As a control, the cDNA encoding GAPDH was amplified with specific oligonucleotides.

    Article Snippet: U2OS/AMF1 and U2OS/β-Gal cells were cultured in 60-mm-diameter dishes and treated with etoposide for 0, 4, 8, or 24 h. Total RNA was isolated using TRIZOL reagent (GIBCO/BRL), and 5 μg of total RNA was reverse transcribed with oligo(dT) by using the Thermoscript RT-PCR system for first-strand cDNA synthesis (GIBCO/BRL).

    Techniques: Over Expression, Flow Cytometry, Staining, Fluorescence, FACS, Labeling, Western Blot, Reverse Transcription Polymerase Chain Reaction, Amplification

    ( A ) Comparison of average mapping statistics of transcriptomic content among three datasets using 50 bp reads from Genome Analyzer IIx (Illumina, Inc.): cDNA libraries made from single RNA samples of mouse testis tissue in which mRNA was enriched from total RNA either by (i) TruSeq™ poly-A selection or by (ii) RiboMinus™ rRNA depletion and (iii) cDNA library in which cDNA was synthesized directly from total RNA (DNase treated and left untreated) using the Ovation® RNA-Seq system. ( B ) Complexity of libraries (percent of unique start sites, unique pairs out of total mapped reads): cDNA libraries of TruSeq™ poly-A selection, RiboMinus™ rRNA depletion and Ovation® RNA-seq amplification system (different input amounts of total RNA). The sample prepared using the Ovation® RNA-Seq system provided slightly higher percentage of unique start sites.

    Journal: Nucleic Acids Research

    Article Title: Whole-transcriptome RNAseq analysis from minute amount of total RNA

    doi: 10.1093/nar/gkr547

    Figure Lengend Snippet: ( A ) Comparison of average mapping statistics of transcriptomic content among three datasets using 50 bp reads from Genome Analyzer IIx (Illumina, Inc.): cDNA libraries made from single RNA samples of mouse testis tissue in which mRNA was enriched from total RNA either by (i) TruSeq™ poly-A selection or by (ii) RiboMinus™ rRNA depletion and (iii) cDNA library in which cDNA was synthesized directly from total RNA (DNase treated and left untreated) using the Ovation® RNA-Seq system. ( B ) Complexity of libraries (percent of unique start sites, unique pairs out of total mapped reads): cDNA libraries of TruSeq™ poly-A selection, RiboMinus™ rRNA depletion and Ovation® RNA-seq amplification system (different input amounts of total RNA). The sample prepared using the Ovation® RNA-Seq system provided slightly higher percentage of unique start sites.

    Article Snippet: The seven aliquots of total RNA [500, 100 (with technical replicates), 50, 10 ng, 500 pg, 50 pg] was treated by DNase and subjected to cDNA synthesis.

    Techniques: Selection, cDNA Library Assay, Synthesized, RNA Sequencing Assay, Amplification

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

    Journal: PLoS Neglected Tropical Diseases

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

    doi: 10.1371/journal.pntd.0006516

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

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

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

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

    Journal: Nature medicine

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

    doi: 10.1038/nm1785

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

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

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

    Cell-cycle-dependent co-localization of TorR-GFP focus with the nucleoid affecting expression of several genes. Synchronization, induction of TorR-GFP expression, and preparation of W3110Δ torRdnaC2 cells carrying pTorR-GFP were as described in the legend of Figure 2 . Total RNA isolation, complementary DNA (cDNA) synthesis and performance of reverse transcriptional quantitative PCR (RT-qPCR) were as described in Materials and Methods. Each gene expression was normalized using the Ct value corresponding to the E. coli rplO gene as an internal reference and the relative expression at every time point to the 0 min was calculated. The expression values are the mean of three individual experiments and the standard errors are given as the error bars. The X-axis indicates the time intervals after shift-down of the culture to the permissive temperature (30 °C) and the Y-axis represents the relative expression of each gene. The grey bars are for expressions of genes in the cells with TorR-GFP and the black bars are for that in the cells without TorR-GFP. The values are the average of three experiments. The error bars are as indicated. All the genes analyzed are as indicated ( a – h ).

    Journal: Genes

    Article Title: A Spatial Control for Correct Timing of Gene Expression during the Escherichia coli Cell Cycle

    doi: 10.3390/genes8010001

    Figure Lengend Snippet: Cell-cycle-dependent co-localization of TorR-GFP focus with the nucleoid affecting expression of several genes. Synchronization, induction of TorR-GFP expression, and preparation of W3110Δ torRdnaC2 cells carrying pTorR-GFP were as described in the legend of Figure 2 . Total RNA isolation, complementary DNA (cDNA) synthesis and performance of reverse transcriptional quantitative PCR (RT-qPCR) were as described in Materials and Methods. Each gene expression was normalized using the Ct value corresponding to the E. coli rplO gene as an internal reference and the relative expression at every time point to the 0 min was calculated. The expression values are the mean of three individual experiments and the standard errors are given as the error bars. The X-axis indicates the time intervals after shift-down of the culture to the permissive temperature (30 °C) and the Y-axis represents the relative expression of each gene. The grey bars are for expressions of genes in the cells with TorR-GFP and the black bars are for that in the cells without TorR-GFP. The values are the average of three experiments. The error bars are as indicated. All the genes analyzed are as indicated ( a – h ).

    Article Snippet: Complementary DNA (cDNA) synthesis was performed with 5 μg of total RNA as template in a 25 μL reaction mixture including 4 μmol/L of each primer using the PrimeScript™ 1st Strand cDNA Synthesis Kit (TaKaRa, Otsu, Japan) as described in the manufacturer’s protocol.

    Techniques: Expressing, Isolation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    A, Ethidium bromide-stained agarose gel of dsRNA. Lanes a and b contain dsRNA extracts from two representative strains of Lentinula edodes FMRI0339 mycovirus (LeV-FMRI0339) and Pleurotus ostreatus ASI2792 mycovirus (ASI2792-PoV) with the characteristic 12-kb viral genome of LeV and approximately 8.0-kb viral genome of ASI2792-PoV, respectively. Lanes 1 to 14 contain dsRNA extracts from 14 isolates obtained by the mycelia fragmentation method described in “Materials and Methods”; B, Northern blot analysis of the corresponding gel with the dsRNA using a partial cDNA clone (792 bp) encoding the RNA-dependent RNA polymerase (RdRp) protein in the PoV-ASI2792 as a probe. Arrowheads indicate the mycovirus genome segment from PoV-ASI2792.

    Journal: Mycobiology

    Article Title: Viral Effects of a dsRNA Mycovirus (PoV-ASI2792) on the Vegetative Growth of the Edible Mushroom Pleurotus ostreatus

    doi: 10.5941/MYCO.2016.44.4.283

    Figure Lengend Snippet: A, Ethidium bromide-stained agarose gel of dsRNA. Lanes a and b contain dsRNA extracts from two representative strains of Lentinula edodes FMRI0339 mycovirus (LeV-FMRI0339) and Pleurotus ostreatus ASI2792 mycovirus (ASI2792-PoV) with the characteristic 12-kb viral genome of LeV and approximately 8.0-kb viral genome of ASI2792-PoV, respectively. Lanes 1 to 14 contain dsRNA extracts from 14 isolates obtained by the mycelia fragmentation method described in “Materials and Methods”; B, Northern blot analysis of the corresponding gel with the dsRNA using a partial cDNA clone (792 bp) encoding the RNA-dependent RNA polymerase (RdRp) protein in the PoV-ASI2792 as a probe. Arrowheads indicate the mycovirus genome segment from PoV-ASI2792.

    Article Snippet: Cloning and sequence analysis of the partial RdRp gene To obtain a cDNA clone that corresponded to the mycovirus RdRp, cDNA synthesis was conducted as described procedure using a cDNA synthesis kit (Promega, Fitchburg, WI, USA) and reverse transcription-PCR (RT-PCR) was performed as described previously [ ].

    Techniques: Staining, Agarose Gel Electrophoresis, Northern Blot

    Bioactivity and diagram of AGSs isolated from mouse hypertrophic heart. In A and B , data are presented in three panels to illustrate the viability of the transformed yeast and the galactose-dependent growth under the selective pressure of exclusion of histidine from the medium. Galactose promotes the expression of each cDNA in the pYES2-containing GAL1 promoter. About 2000 cells were suspended in H 2 O and spotted on medium with glucose plus histidine ( left ; no selection), glucose minus histidine ( center ; selection without induction), or galactose plus histidine ( right ; selection plus induction). A , epistasis analysis of isolated clones. Transformants in a yeast strain expressing human Gα 16 (Gpa1(1–41)) and yeast lacking Gα, Gβ, or downstream signaling molecules (Δ G α, yeast lacking Gα; Δ G β, yeast lacking Gβ; Δ Ste20 , yeast lacking p21-activated kinase; Δ Ste5 , yeast lacking the kinase scaffold protein). B , effect of isolated cDNAs in yeast expressing various types of Gα. C , schematic diagram of the sequences of TFE3, TFEB, and MITF in mouse. The line above the sequence refers to cDNA isolated by the yeast-based functional screen. HLH , helix-loop-helix. D , bioactivity of full-length TFE3, TFEB, and MITF. The full-length clones were transformed into yeast expressing Gα 16 . The magnitude of activation of G-protein signaling pathway was monitored by β-galactosidase activity. Data are presented as the mean S.E. of five experiments with duplicate determinations. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of Transcription Factor E3 (TFE3) as a Receptor-independent Activator of G?16

    doi: 10.1074/jbc.M111.219816

    Figure Lengend Snippet: Bioactivity and diagram of AGSs isolated from mouse hypertrophic heart. In A and B , data are presented in three panels to illustrate the viability of the transformed yeast and the galactose-dependent growth under the selective pressure of exclusion of histidine from the medium. Galactose promotes the expression of each cDNA in the pYES2-containing GAL1 promoter. About 2000 cells were suspended in H 2 O and spotted on medium with glucose plus histidine ( left ; no selection), glucose minus histidine ( center ; selection without induction), or galactose plus histidine ( right ; selection plus induction). A , epistasis analysis of isolated clones. Transformants in a yeast strain expressing human Gα 16 (Gpa1(1–41)) and yeast lacking Gα, Gβ, or downstream signaling molecules (Δ G α, yeast lacking Gα; Δ G β, yeast lacking Gβ; Δ Ste20 , yeast lacking p21-activated kinase; Δ Ste5 , yeast lacking the kinase scaffold protein). B , effect of isolated cDNAs in yeast expressing various types of Gα. C , schematic diagram of the sequences of TFE3, TFEB, and MITF in mouse. The line above the sequence refers to cDNA isolated by the yeast-based functional screen. HLH , helix-loop-helix. D , bioactivity of full-length TFE3, TFEB, and MITF. The full-length clones were transformed into yeast expressing Gα 16 . The magnitude of activation of G-protein signaling pathway was monitored by β-galactosidase activity. Data are presented as the mean S.E. of five experiments with duplicate determinations. *, p

    Article Snippet: mRNA isolated from the left ventricle in the TAC or tachycardia models was used to synthesize cDNAs using a cDNA Synthesis kit (Takara); cDNAs were cloned into the pYES2 yeast expression vector.

    Techniques: Isolation, Transformation Assay, Expressing, Selection, Clone Assay, Sequencing, Functional Assay, Activation Assay, Activity Assay

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

    Journal: Breast Cancer Research : BCR

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

    doi: 10.1186/bcr1794

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

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

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

    Immunohistologic analysis of TELi. (A–B): H E staining of native liver and TELi. An asterisk is placed near a blood vessel containing erythrocytes in TELi. White arrows indicate incompletely degraded scaffold material. (C): Immunofluorescence staining for albumin (red) and CK8 (cholangiocyte and periductular hepatocyte marker, green) in native liver and TELi. Arrowhead shows double positive cells. (D): Immunofluorescence staining for HNF4α (hepatocyte specific transcription factor, red) and CK8 (green). Nuclei stained with 4′,6‐diamidino‐2‐phenylindole (blue). (E): Reverse transcription‐polymerase chain reaction for albumin expression. cDNA prepared from whole liver RNA. Scale bars = 50 μm (A) , 25 μm (B–D) . Data represent more than three independent experiments. Abbreviations: H E, hematoxylin and eosin; L, ladder; NC, negative control (water); PC, positive control; TELi, tissue‐engineered liver.

    Journal: Stem Cells Translational Medicine

    Article Title: Functional Human and Murine Tissue‐Engineered Liver Is Generated from Adult Stem/Progenitor Cells

    doi: 10.5966/sctm.2016-0205

    Figure Lengend Snippet: Immunohistologic analysis of TELi. (A–B): H E staining of native liver and TELi. An asterisk is placed near a blood vessel containing erythrocytes in TELi. White arrows indicate incompletely degraded scaffold material. (C): Immunofluorescence staining for albumin (red) and CK8 (cholangiocyte and periductular hepatocyte marker, green) in native liver and TELi. Arrowhead shows double positive cells. (D): Immunofluorescence staining for HNF4α (hepatocyte specific transcription factor, red) and CK8 (green). Nuclei stained with 4′,6‐diamidino‐2‐phenylindole (blue). (E): Reverse transcription‐polymerase chain reaction for albumin expression. cDNA prepared from whole liver RNA. Scale bars = 50 μm (A) , 25 μm (B–D) . Data represent more than three independent experiments. Abbreviations: H E, hematoxylin and eosin; L, ladder; NC, negative control (water); PC, positive control; TELi, tissue‐engineered liver.

    Article Snippet: Reverse Transcription‐Polymerase Chain Reaction RNA was isolated from frozen TELi with the Qiagen RNA purification kit. cDNA was prepared from 1000 ng of RNA using Bio‐Rad cDNA synthesis kit (Bio‐Rad, Hercules, CA, http://www.bio-rad.com ).

    Techniques: Staining, Immunofluorescence, Marker, Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control, Positive Control

    RNA immunoprecipitation of (TMG)-capped primary miRNAs (Pri-miRNAs) in quiescent human foreskin fibroblasts (HFFs) RT-PCR data shows the RNA immunoprecipitation of Pri-miR-34a and Pri-miR-3188 in quiescent HFFs (as well as the positive control snRNA U7), but not Pri-miR-423 using an antibody against (TMG)-capped RNAs. Total RNA was extracted using TRIzol Reagent, and 10 μg of RNA was diluted in NET-2 buffer, precleared and incubated with Protein G Sepharose 4 Fast Flow beads loaded with 15 μl of control antibody (Normal Rabbit Serum, EMD-Millipore) or with antibody recognizing the (TMG)-cap (Anti-m3G-cap, rabbit polyclonal, Synaptic Systems). Beads were rinsed five times with NET-2 buffer and were resuspended in G-50 buffer. RNA was extracted from the beads by phenol-chloroform-isoamyl alcohol extraction and resuspended in 20 μl of nuclease-free water. Immunoprecipitated tri-methylated capped RNA was converted to cDNA using iScript cDNA synthesis kit (Bio-Rad), followed by RT-PCR, and visualized after gel electrophoresis.

    Journal: Bio-protocol

    Article Title: Immunoprecipitation of Tri-methylated Capped RNA

    doi: 10.21769/BioProtoc.2717

    Figure Lengend Snippet: RNA immunoprecipitation of (TMG)-capped primary miRNAs (Pri-miRNAs) in quiescent human foreskin fibroblasts (HFFs) RT-PCR data shows the RNA immunoprecipitation of Pri-miR-34a and Pri-miR-3188 in quiescent HFFs (as well as the positive control snRNA U7), but not Pri-miR-423 using an antibody against (TMG)-capped RNAs. Total RNA was extracted using TRIzol Reagent, and 10 μg of RNA was diluted in NET-2 buffer, precleared and incubated with Protein G Sepharose 4 Fast Flow beads loaded with 15 μl of control antibody (Normal Rabbit Serum, EMD-Millipore) or with antibody recognizing the (TMG)-cap (Anti-m3G-cap, rabbit polyclonal, Synaptic Systems). Beads were rinsed five times with NET-2 buffer and were resuspended in G-50 buffer. RNA was extracted from the beads by phenol-chloroform-isoamyl alcohol extraction and resuspended in 20 μl of nuclease-free water. Immunoprecipitated tri-methylated capped RNA was converted to cDNA using iScript cDNA synthesis kit (Bio-Rad), followed by RT-PCR, and visualized after gel electrophoresis.

    Article Snippet: Protein G Sepharose® 4 Fast Flow Beads (GE Healthcare, catalog number: 17061801) Normal rabbit serum (control, EMD Millipore, catalog number: NS01L-1ML) Sodium chloride (NaCl) (Fisher Scientific, catalog number: S671-3) NP-40 (Thermo Fisher Scientific, catalog number: 28324) Tris base (Fisher Scientific, catalog number: BP152-5) Hydrochloric acid (HCl) (VWR, catalog number: BDH7204-1) RNasin™ Plus RNase inhibitor (Promega, catalog number: N2611) NaOAc (AMRESCO, catalog number: 0602) Ethylenediaminetetraacetate acid (EDTA), pH 8 (Thermo Fisher Scientific, catalog number: AM9260G) Ethylenediaminetetraacetate acid (EDTA) (AMRESCO, catalog number: 0105) Sodium dodecyl sulfate (SDS) (Thermo Fisher Scientific, catalog number: AM9822) Phenol/Chloroform/Isoamyl Alcohol; 125:24:1 mixture, pH 4.5 (Thermo Fisher Scientific, catalog number: AM9720) Agarose LE (Denville Scientific, catalog number: CA3510-8) iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, catalog number: 1708891) Sso Advanced™ Universal SYBR® Green Supermix (Bio-Rad Laboratories, catalog number: 1725274) PARIS™ Kit (Thermo Fisher Scientific, catalog number: AM1921) Boric acid (Fisher Scientific, catalog number: A73-1) NET-2 Buffer (see Recipes) G-50 Buffer (see Recipes) 1× TBE (see Recipes)

    Techniques: Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Positive Control, Incubation, Flow Cytometry, Methylation, Nucleic Acid Electrophoresis

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

    Journal: Oncogenesis

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

    doi: 10.1038/oncsis.2013.27

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

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

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

    Expression pattern of HERF1 . Northern blots of adult tissues (A), murine embryos (B), or MEL cells following treatment with the differentiation-inducing agent DMSO (D) were hybridized with a full-length HERF1 cDNA. Filters were stripped and rehybridized with a probe for glycerol-3-phosphate dehydrogenase (GPDH) to assess the integrity and amount of RNA and with a probe for β-globin (D; numbers above the lanes represent days in DMSO). (C) In situ hybridization was performed with a 271-bp restriction fragment from the unique 3′ region of HERF1 labeled with [ 33 P]UTP. The left side shows the bright-field view of a 12-μm sagittal section from an E15 embryo stained with hematoxylin and eosin; the right side is a dark-field view of the same section hybridized with the HERF1 probe. Sense control shows no specific hybridization (data not shown).

    Journal: Molecular and Cellular Biology

    Article Title: HERF1, a Novel Hematopoiesis-Specific RING Finger Protein, Is Required for Terminal Differentiation of Erythroid Cells

    doi:

    Figure Lengend Snippet: Expression pattern of HERF1 . Northern blots of adult tissues (A), murine embryos (B), or MEL cells following treatment with the differentiation-inducing agent DMSO (D) were hybridized with a full-length HERF1 cDNA. Filters were stripped and rehybridized with a probe for glycerol-3-phosphate dehydrogenase (GPDH) to assess the integrity and amount of RNA and with a probe for β-globin (D; numbers above the lanes represent days in DMSO). (C) In situ hybridization was performed with a 271-bp restriction fragment from the unique 3′ region of HERF1 labeled with [ 33 P]UTP. The left side shows the bright-field view of a 12-μm sagittal section from an E15 embryo stained with hematoxylin and eosin; the right side is a dark-field view of the same section hybridized with the HERF1 probe. Sense control shows no specific hybridization (data not shown).

    Article Snippet: Oligo(dT)-primed double-stranded cDNA was synthesized from 5 μg of poly(A)+ RNA by using a cDNA synthesis system (GIBCO BRL) according to the manufacturer’s instructions.

    Techniques: Expressing, Northern Blot, In Situ Hybridization, Labeling, Staining, Hybridization

    Analysis of SMU.2080 transcription factor activity A) Illustrated here are the sequences of the DNA fragments used for electrophoretic mobility shift assays (EMSA) of SMU.2080 and the promoter regions of SMU.150, SMU.423, and SMU.1906. Sequences labeled DR+ contain the wild-type direct repeat sequence, whereas those labeled DR- contain a rearrangement of the direct repeat nucleotides. All other bases in the DNA fragments are identical. EMSA analysis was performed using purified recombinant SMU.2080 and both wild-type (DR+) and mutant (DR-) direct repeat DNA for B) SMU.150, C) SMU.423, and D) SMU.1906. As described in Experimental Procedures, increasing concentrations of SMU.2080 were incubated with 10 ng of the indicated DNA before running the reactions on a nondenaturing polyacrylamide gel. For each series of four EMSA reactions, the first lane contained no protein. E) Rapid amplification of cDNA ends (RACE) PCR was used to determine the transcription start sites of SMU.150, SMU.423, and SMU.1906. The LytTR family direct repeats are shown in bold, while the -35 and extended -10 sequences are underlined. The transcription start sites are indicated with an arrow over the +1 site.

    Journal: Molecular microbiology

    Article Title: Identification of a Novel Bacteriocin Regulatory System in Streptococcus mutans

    doi: 10.1111/j.1365-2958.2010.07417.x

    Figure Lengend Snippet: Analysis of SMU.2080 transcription factor activity A) Illustrated here are the sequences of the DNA fragments used for electrophoretic mobility shift assays (EMSA) of SMU.2080 and the promoter regions of SMU.150, SMU.423, and SMU.1906. Sequences labeled DR+ contain the wild-type direct repeat sequence, whereas those labeled DR- contain a rearrangement of the direct repeat nucleotides. All other bases in the DNA fragments are identical. EMSA analysis was performed using purified recombinant SMU.2080 and both wild-type (DR+) and mutant (DR-) direct repeat DNA for B) SMU.150, C) SMU.423, and D) SMU.1906. As described in Experimental Procedures, increasing concentrations of SMU.2080 were incubated with 10 ng of the indicated DNA before running the reactions on a nondenaturing polyacrylamide gel. For each series of four EMSA reactions, the first lane contained no protein. E) Rapid amplification of cDNA ends (RACE) PCR was used to determine the transcription start sites of SMU.150, SMU.423, and SMU.1906. The LytTR family direct repeats are shown in bold, while the -35 and extended -10 sequences are underlined. The transcription start sites are indicated with an arrow over the +1 site.

    Article Snippet: Total RNA (300 ng) was used for complementary DNA (cDNA) synthesis using Stratascript reverse transcriptase (Stratagene) according to the manufacturer’s protocol.

    Techniques: Activity Assay, Electrophoretic Mobility Shift Assay, Labeling, Sequencing, Purification, Recombinant, Mutagenesis, Incubation, Rapid Amplification of cDNA Ends, Polymerase Chain Reaction

    Expression of Dgrca mRNA in various organs of D. glomerata (A), in nodules with or without light treatment (B, lanes N e , N w , and N w ) and in oligo(dT)-cellulose fractions of nodule RNA (B, lanes A− and A+). Sizes in nucleotide of rRNA species are shown on the right of each panel. A, Total RNA from leaves (lane L), flowers (lane Fl), immature fruits (lane Fr), roots (lane R), and nodules harvested 4 to 11 weeks after Frankia inoculation (N 4 –N 11 ) was hybridized to radiolabeled Dgrca cDNA insert. B, Total RNA from nodules harvested from excised roots (lane N e ) or from roots of whole plants (lanes N w ) after treatment with white light (Lt) or darkness (Dk) were hybridized to the full-length Dgrca cDNA probe. Total N 11 RNA (lane N 11 ), root RNA (lane R), and leaf RNA (lane L) are included for a comparison. The unbound fraction of total N 11 RNA (lane A−) and the bound N 11 RNA fraction (lane A+) after oligo(dT)-cellulose treatment were also hybridized to the full-length Dgrca cDNA probe. C, Total RNA from roots (lane R), N 4 nodules, or N 7 nodules was hybridized to a radiolabeled intron-A probe. The asterisk indicates the predicted position of the 1700-nucleotide mRNA species. For A and C and the first seven lanes of B, approximately 5 μg of total RNA was loaded into each lane; for lane A+ in B, 2.5 μg of poly(A + ) RNA was used. Autoradiography was carried out with preflashed Kodak XAR 5 film at −80°C for 16.5 h (A), 6 d (B), and 5 d (C).

    Journal: Plant Physiology

    Article Title: Symbiotic Root Nodules of the Actinorhizal Plant Datisca glomerata Express Rubisco Activase mRNA 1

    doi:

    Figure Lengend Snippet: Expression of Dgrca mRNA in various organs of D. glomerata (A), in nodules with or without light treatment (B, lanes N e , N w , and N w ) and in oligo(dT)-cellulose fractions of nodule RNA (B, lanes A− and A+). Sizes in nucleotide of rRNA species are shown on the right of each panel. A, Total RNA from leaves (lane L), flowers (lane Fl), immature fruits (lane Fr), roots (lane R), and nodules harvested 4 to 11 weeks after Frankia inoculation (N 4 –N 11 ) was hybridized to radiolabeled Dgrca cDNA insert. B, Total RNA from nodules harvested from excised roots (lane N e ) or from roots of whole plants (lanes N w ) after treatment with white light (Lt) or darkness (Dk) were hybridized to the full-length Dgrca cDNA probe. Total N 11 RNA (lane N 11 ), root RNA (lane R), and leaf RNA (lane L) are included for a comparison. The unbound fraction of total N 11 RNA (lane A−) and the bound N 11 RNA fraction (lane A+) after oligo(dT)-cellulose treatment were also hybridized to the full-length Dgrca cDNA probe. C, Total RNA from roots (lane R), N 4 nodules, or N 7 nodules was hybridized to a radiolabeled intron-A probe. The asterisk indicates the predicted position of the 1700-nucleotide mRNA species. For A and C and the first seven lanes of B, approximately 5 μg of total RNA was loaded into each lane; for lane A+ in B, 2.5 μg of poly(A + ) RNA was used. Autoradiography was carried out with preflashed Kodak XAR 5 film at −80°C for 16.5 h (A), 6 d (B), and 5 d (C).

    Article Snippet: Two Lambda Zap cDNA libraries were made representing mRNAs from D. glomerata nodules harvested 4, 5, and 7 weeks after inoculation with Frankia . cDNA synthesis and ligation was carried out according to the protocol for Lambda Zap Express cDNA synthesis (Stratagene).

    Techniques: Expressing, Autoradiography

    Expression analysis of mecA in JE2 and mutant strains. RNA was isolated from the exponentially grown (0.5–1.2 OD 600nm ) cultures treated with or without oxicillin (Ox) 4 μg/ml for 60 min in MHB. cDNA was prepared from RNA samples and used as template with appropriate primer sets, and relative expression levels were calculated as mentioned in the Materials and Methods section. Each bar shows the mean and standard deviation of values obtained from three replicates. The effect of mutation and oxacillin on expression of pbp2 and mecA was analyzed by performing multiple pairwise comparisons for pbp2 (uppercase letters) and mecA (lowercase letters) using ANOVA followed by Tukey's post-hoc test, and p -values

    Journal: Frontiers in Microbiology

    Article Title: RelQ Mediates the Expression of β-Lactam Resistance in Methicillin-Resistant Staphylococcus aureus

    doi: 10.3389/fmicb.2019.00339

    Figure Lengend Snippet: Expression analysis of mecA in JE2 and mutant strains. RNA was isolated from the exponentially grown (0.5–1.2 OD 600nm ) cultures treated with or without oxicillin (Ox) 4 μg/ml for 60 min in MHB. cDNA was prepared from RNA samples and used as template with appropriate primer sets, and relative expression levels were calculated as mentioned in the Materials and Methods section. Each bar shows the mean and standard deviation of values obtained from three replicates. The effect of mutation and oxacillin on expression of pbp2 and mecA was analyzed by performing multiple pairwise comparisons for pbp2 (uppercase letters) and mecA (lowercase letters) using ANOVA followed by Tukey's post-hoc test, and p -values

    Article Snippet: For relative quantification, 2 μg total RNA was reverse transcribed using a cDNA Synthesis Kit (Fermentas, USA), and 1 μl of 10x diluted cDNA was used as template in 20 μl reaction volume.

    Techniques: Expressing, Mutagenesis, Isolation, Standard Deviation

    Effect of relQ expression on mecA transcription in parent and mutant strains. Transcript level was monitored by RT-PCR. cDNA was prepared by reverse-transcription of the RNA samples isolated from exponentially grown cultures treated with or without oxicillin (Ox) 4 μg/ml for 60 min in MHB. The relative expression levels were calculated using the 2 - Δ Δ C T method. Each bar shows the mean and standard deviation of values obtained from three replicates. The effect of relQ expression on mecA transcription level (lowercase letters) in different strains was analyzed by performing multiple pairwise comparisons using ANOVA followed by Tukey's post-hoc test, and p -values

    Journal: Frontiers in Microbiology

    Article Title: RelQ Mediates the Expression of β-Lactam Resistance in Methicillin-Resistant Staphylococcus aureus

    doi: 10.3389/fmicb.2019.00339

    Figure Lengend Snippet: Effect of relQ expression on mecA transcription in parent and mutant strains. Transcript level was monitored by RT-PCR. cDNA was prepared by reverse-transcription of the RNA samples isolated from exponentially grown cultures treated with or without oxicillin (Ox) 4 μg/ml for 60 min in MHB. The relative expression levels were calculated using the 2 - Δ Δ C T method. Each bar shows the mean and standard deviation of values obtained from three replicates. The effect of relQ expression on mecA transcription level (lowercase letters) in different strains was analyzed by performing multiple pairwise comparisons using ANOVA followed by Tukey's post-hoc test, and p -values

    Article Snippet: For relative quantification, 2 μg total RNA was reverse transcribed using a cDNA Synthesis Kit (Fermentas, USA), and 1 μl of 10x diluted cDNA was used as template in 20 μl reaction volume.

    Techniques: Expressing, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Isolation, Standard Deviation

    Differential expressions of genes modulating the mechanism of resistance to A. alternata in naturally infected mango fruits. (A) Effect of HWB on alternaria black spot (ABS) symptom development on mango cvs. Palmer, Kent, Tommy Atkins, Keitt, Lily and Shelly. (B) ABS symptom development on naturally infected fruits cv. Keitt following HWB treatment. (C) qRT-PCR differential expression profiling of genes Syn121 , glutardoxin , IT1K2 and AOS of cv. Shelly. Fruit peel tissues were sampled at four different time points after HWB treatment. RNA was extracted and served as a template for cDNA followed by qRT-PCR analysis of the genes of interest. Proportional increases in relative expression values were normalized against the samples of untreated mango fruits at 0 h. Expression data are means of two replicates. ABS-covered area was evaluated after 4 weeks of storage at 12°C. Average values followed by different letters differ significantly at P

    Journal: BMC Genomics

    Article Title: De-novo assembly of mango fruit peel transcriptome reveals mechanisms of mango response to hot water treatment

    doi: 10.1186/1471-2164-15-957

    Figure Lengend Snippet: Differential expressions of genes modulating the mechanism of resistance to A. alternata in naturally infected mango fruits. (A) Effect of HWB on alternaria black spot (ABS) symptom development on mango cvs. Palmer, Kent, Tommy Atkins, Keitt, Lily and Shelly. (B) ABS symptom development on naturally infected fruits cv. Keitt following HWB treatment. (C) qRT-PCR differential expression profiling of genes Syn121 , glutardoxin , IT1K2 and AOS of cv. Shelly. Fruit peel tissues were sampled at four different time points after HWB treatment. RNA was extracted and served as a template for cDNA followed by qRT-PCR analysis of the genes of interest. Proportional increases in relative expression values were normalized against the samples of untreated mango fruits at 0 h. Expression data are means of two replicates. ABS-covered area was evaluated after 4 weeks of storage at 12°C. Average values followed by different letters differ significantly at P

    Article Snippet: Analysis by qRT-PCR Single-stranded cDNA was synthesized from 1 μg of total RNA by means of the Verso cDNA synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA).

    Techniques: Infection, Quantitative RT-PCR, Expressing

    Isolation of IgM-binding subclones and identification of cDNA inserts. (A) Cells transduced by the retroviral expression construct containing CLL-derived (top) or PMA-activated 697 pre–B cell–derived (bottom) cDNA libraries were enriched for IgM binding by FACS and subcloned for limiting dilution. Three representative subclones from each library are shown for their IgM-binding activity or lack of binding, as determined by flow cytometry. (B) Agarose gel electrophoresis analysis of RT-PCR products. RNA isolated from nontransduced control BW5147 T cells (lane 1) and from IgM-binding (lanes 3–5 and 7–9) or IgM-nonbinding (lanes 2 and 6) subclones from CLL-derived (lanes 2–5) and PMA-activated 697 pre–B cell–derived (lanes 6–9) cDNA libraries were subjected to RT-PCR as described in Materials and methods. Amplified products were electrophoresed in 0.7% agarose and stained with ethidium bromide. Lane 10 is a PCR control without a first-strand cDNA template. Hin dIII-digested λ DNA was used as a size marker. The experiments were performed once for A and twice for B.

    Journal: The Journal of Experimental Medicine

    Article Title: Identity of the elusive IgM Fc receptor (Fc?R) in humans

    doi: 10.1084/jem.20091107

    Figure Lengend Snippet: Isolation of IgM-binding subclones and identification of cDNA inserts. (A) Cells transduced by the retroviral expression construct containing CLL-derived (top) or PMA-activated 697 pre–B cell–derived (bottom) cDNA libraries were enriched for IgM binding by FACS and subcloned for limiting dilution. Three representative subclones from each library are shown for their IgM-binding activity or lack of binding, as determined by flow cytometry. (B) Agarose gel electrophoresis analysis of RT-PCR products. RNA isolated from nontransduced control BW5147 T cells (lane 1) and from IgM-binding (lanes 3–5 and 7–9) or IgM-nonbinding (lanes 2 and 6) subclones from CLL-derived (lanes 2–5) and PMA-activated 697 pre–B cell–derived (lanes 6–9) cDNA libraries were subjected to RT-PCR as described in Materials and methods. Amplified products were electrophoresed in 0.7% agarose and stained with ethidium bromide. Lane 10 is a PCR control without a first-strand cDNA template. Hin dIII-digested λ DNA was used as a size marker. The experiments were performed once for A and twice for B.

    Article Snippet: The cDNA libraries were constructed by a cDNA synthesis kit (Agilent Technologies) using poly(A)+ RNA isolated by an Oligotex mRNA purification kit (QIAGEN) from (a) blood MNCs from a patient with CLL and (b) the 697 pre–B cell line preactivated with 10 nM PMA for 8 h, as previously described ( ).

    Techniques: Isolation, Binding Assay, Expressing, Construct, Derivative Assay, FACS, Activity Assay, Flow Cytometry, Cytometry, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Amplification, Staining, Polymerase Chain Reaction, Marker

    Primary structure of CAP and mRNA expression. (A) 3T3-L1 adipocyte cDNA clones isolated from the yeast two-hybrid screen with c-Cbl as bait are shown (black lines) aligned below a schematic of the domain organization of the CAP protein. Numbers in parentheses refer to the number of times each clone was isolated from the yeast two-hybrid screen. The letters A, B, and C indicate the presence of alternative spliced inserts. (B) Total RNA (20 μg) isolated from 3T3-L1 adipocytes or fibroblasts was hybridized to a probe prepared from the DNA insert of clone 2.1 labeled with [α- 32 P]dCTP by random-primer extension (upper). The filter was then stripped and rehybridized with a β-actin probe to estimate the relative amount of RNA in each lane (lower). The migration positions of RNA size markers (in kilobases) are on the left.

    Journal: Molecular and Cellular Biology

    Article Title: A Novel, Multifunctional c-Cbl Binding Protein in Insulin Receptor Signaling in 3T3-L1 Adipocytes

    doi:

    Figure Lengend Snippet: Primary structure of CAP and mRNA expression. (A) 3T3-L1 adipocyte cDNA clones isolated from the yeast two-hybrid screen with c-Cbl as bait are shown (black lines) aligned below a schematic of the domain organization of the CAP protein. Numbers in parentheses refer to the number of times each clone was isolated from the yeast two-hybrid screen. The letters A, B, and C indicate the presence of alternative spliced inserts. (B) Total RNA (20 μg) isolated from 3T3-L1 adipocytes or fibroblasts was hybridized to a probe prepared from the DNA insert of clone 2.1 labeled with [α- 32 P]dCTP by random-primer extension (upper). The filter was then stripped and rehybridized with a β-actin probe to estimate the relative amount of RNA in each lane (lower). The migration positions of RNA size markers (in kilobases) are on the left.

    Article Snippet: A 3T3-L1 adipocyte cDNA library was synthesized with a cDNA synthesis kit (Stratagene) and constructed in the pGAD-GH GAL4 vector (Clontech).

    Techniques: Expressing, Clone Assay, Isolation, Two Hybrid Screening, Labeling, Migration

    UTX mutations in human T-ALL. (A) DNA sequencing chromatogram showing a UTX mutation in the gDNA of a male primary T-ALL patient sample. The mutation is absent in remission material of the same patient. (B) Graphical representation of the localization of genetic lesions in the UTX protein structure. In-frame deletion/insertion mutations are depicted in orange circles and frameshift mutations in blue circles. (C) Genotyping and allelic expression analysis of SNP rs181547731 in gDNA and cDNA derived from female T-ALL lymphoblasts. (D) Graphical representation of the different mutations (dark orange rectangles) and deletions (light orange rectangles) present in a set of T-ALL oncogenes and tumor suppressor genes in 35 primary T-ALL patient samples. The different T-ALL subgroups include TAL-LMO, TLX3, TLX1, HOXA, and patients for whom the subgroup is unknown. The age subgroups include children (age ≤15 years; dark red rectangles) and adults (age > 15 years; light red rectangles). Male and female T-ALL patient samples are presented in dark blue and light blue rectangles, respectively. JmjC, Jumonji C; TPR, tetratricopeptide repeat.

    Journal: Blood

    Article Title: The H3K27me3 demethylase UTX is a gender-specific tumor suppressor in T-cell acute lymphoblastic leukemia

    doi: 10.1182/blood-2014-05-577270

    Figure Lengend Snippet: UTX mutations in human T-ALL. (A) DNA sequencing chromatogram showing a UTX mutation in the gDNA of a male primary T-ALL patient sample. The mutation is absent in remission material of the same patient. (B) Graphical representation of the localization of genetic lesions in the UTX protein structure. In-frame deletion/insertion mutations are depicted in orange circles and frameshift mutations in blue circles. (C) Genotyping and allelic expression analysis of SNP rs181547731 in gDNA and cDNA derived from female T-ALL lymphoblasts. (D) Graphical representation of the different mutations (dark orange rectangles) and deletions (light orange rectangles) present in a set of T-ALL oncogenes and tumor suppressor genes in 35 primary T-ALL patient samples. The different T-ALL subgroups include TAL-LMO, TLX3, TLX1, HOXA, and patients for whom the subgroup is unknown. The age subgroups include children (age ≤15 years; dark red rectangles) and adults (age > 15 years; light red rectangles). Male and female T-ALL patient samples are presented in dark blue and light blue rectangles, respectively. JmjC, Jumonji C; TPR, tetratricopeptide repeat.

    Article Snippet: After RNA quality assessment, complementary DNA (cDNA) synthesis was performed using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories).

    Techniques: DNA Sequencing, Mutagenesis, Expressing, Derivative Assay

    The TatNrf2mer peptide induces antioxidant genes and protects cells against oxidative stress. ( A ) Map of the plasmid containing the TatNrf2mer sequence. A DNA sequence coding for the HIV-1 Tat peptide fused to the Nrf2 peptide was designed and codon optimized for expression in humans and mice. This sequence was cloned in a lentiviral vector plasmid fused to the puromycin resistance gene by a T2A self-cleaving peptide sequence. ( B ) Detection of TatNrf2mer mRNA in stably transfected ARPE-19 cells expressing TatNrf2mer. ARPE-19 cells transduced with lentiviral vectors delivering either TatNrf2mer-PuroR or PuroR were selected in puromycin. Total RNA was isolated from both stable cells, and a cDNA library was generated to detect the presence of TatNrf2mer by RT-PCR. Lentiviral plasmid containing the TatNrf2mer sequence was used as a positive control. ( C ) Constitutive expression of TatNrf2mer induces the expression of ARE genes. Quantitative RT-PCR detecting the GSTM1 and NqO1 mRNA was performed using the cDNA library described in ( B ), using β-actin as a control. ARPE-19 stably expressing TatNrf2mer had greater expression of ARE genes. Assays were performed in triplicate. ( D ) TatNrf2mer expression protects ARPE-19 cells from paraquat and H 2 O 2 induced oxidative stress. Stably transfected ARPE-19 cells were incubated with 55, 166, or 500 μM paraquat for 48 hours or with 800 μM H 2 O 2 for 6 hours to induce oxidative damage. Afterwards, cell viability was assessed with the Cell Titration or MTT assay, respectively. Assays were performed in triplicate. Values are reported as average ± SD.

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Targeting the Nrf2 Signaling Pathway in the Retina With a Gene-Delivered Secretable and Cell-Penetrating Peptide

    doi: 10.1167/iovs.15-17703

    Figure Lengend Snippet: The TatNrf2mer peptide induces antioxidant genes and protects cells against oxidative stress. ( A ) Map of the plasmid containing the TatNrf2mer sequence. A DNA sequence coding for the HIV-1 Tat peptide fused to the Nrf2 peptide was designed and codon optimized for expression in humans and mice. This sequence was cloned in a lentiviral vector plasmid fused to the puromycin resistance gene by a T2A self-cleaving peptide sequence. ( B ) Detection of TatNrf2mer mRNA in stably transfected ARPE-19 cells expressing TatNrf2mer. ARPE-19 cells transduced with lentiviral vectors delivering either TatNrf2mer-PuroR or PuroR were selected in puromycin. Total RNA was isolated from both stable cells, and a cDNA library was generated to detect the presence of TatNrf2mer by RT-PCR. Lentiviral plasmid containing the TatNrf2mer sequence was used as a positive control. ( C ) Constitutive expression of TatNrf2mer induces the expression of ARE genes. Quantitative RT-PCR detecting the GSTM1 and NqO1 mRNA was performed using the cDNA library described in ( B ), using β-actin as a control. ARPE-19 stably expressing TatNrf2mer had greater expression of ARE genes. Assays were performed in triplicate. ( D ) TatNrf2mer expression protects ARPE-19 cells from paraquat and H 2 O 2 induced oxidative stress. Stably transfected ARPE-19 cells were incubated with 55, 166, or 500 μM paraquat for 48 hours or with 800 μM H 2 O 2 for 6 hours to induce oxidative damage. Afterwards, cell viability was assessed with the Cell Titration or MTT assay, respectively. Assays were performed in triplicate. Values are reported as average ± SD.

    Article Snippet: cDNA Synthesis Complementary DNA was synthesized with the iScript cDNA synthesis kit (Bio-Rad, Hercules CA, USA).

    Techniques: Plasmid Preparation, Sequencing, Expressing, Mouse Assay, Clone Assay, Stable Transfection, Transfection, Transduction, Isolation, cDNA Library Assay, Generated, Reverse Transcription Polymerase Chain Reaction, Positive Control, Quantitative RT-PCR, Incubation, Titration, MTT Assay

    Adora2b -deficient mice have increased inflammation with impaired induction of Tregs after LPS-induced lung injury. LPS-induced inflammation was initiated in 9–12 wk-old male Adora2b +/+ (C57BL/6J) or age and weight matched Adora2b −/− mice by intratracheal instillation of LPS or saline. Bronchoalveolar lavage (BAL) fluids were collected 1 d post treatment. A) Leukocyte cell counts of BAL fluid were determined utilizing a cellometer (mean ± SEM, n = 2–4). B) Protein content of BAL fluid was determined with BCA assay. Fold increase of protein concentration in LPS treated BAL samples over respective saline BAL samples is displayed (mean ± SEM, n = 3–10). C) Analysis of cell infiltration into the lung airspace (BAL) of mice exposed to inhaled LPS. Mice ( Adora2b +/+ or Adora2b −/−) were exposed to nebulized saline or lipopolysaccharide (LPS); 24 hours post-exposure, mice were euthanized, and BAL was harvested. Cells were then subjected to flow cytometric analysis to identify neutrophils (highly granular, Gr1+ cells). D) Analysis of FoxP3 gene expression in lungs of mice exposed to inhaled LPS. Mice ( Adora2b +/+ or −/−) were exposed to nebulized saline or lipopolysaccharide (LPS); 24 hours post-exposure, mice were euthanized, and perfused lungs were harvested. Total RNA was harvested from lungs, cDNA prepared and analyzed by real-time PCR. Data depict mean (± SEM) fold changes in indicated genes. Fold change was calculated based on primer efficiencies, standardized to changes in actin, with Adora2b +/+ Saline mean value defined as 1. E) Fold change in cell number of either regulatory T cells (lymphocyte size, CD3+ CD4+ FoxP3+) in the lung airspace (BAL) of mice exposed to inhaled saline or LPS as above. Data are from 2–6 mice; 2 independent experiments. F) Relative abundance of CD4 effector T cells (Teffs, defined as lymphocyte size, CD3+ CD4+ CD44high cells) to Tregs based on cell counts from either Adora2b +/+ or Adora2b −/− mice, with pie charts depicting mean cell count for the indicated cell populations from 2–6 mice; 2 independent expts. Analysis of total cellular infiltration into airways and protein leakage (panels A–B) was done in animals treated with intratracheal LPS. Analysis of neutrophil infiltration and Tregs (panels C–F) was done in animals treated with inhaled LPS. Statistically significant differences are indicated and were calculated either by one-way ANOVA (A) or by unpaired t test (B–E) with a focus on whether Adora2b −/− LPS treated animals were statistically different from either saline treated or Adora2b +/+ LPS treated mice.

    Journal: PLoS ONE

    Article Title: Adora2b Adenosine Receptor Engagement Enhances Regulatory T Cell Abundance during Endotoxin-Induced Pulmonary Inflammation

    doi: 10.1371/journal.pone.0032416

    Figure Lengend Snippet: Adora2b -deficient mice have increased inflammation with impaired induction of Tregs after LPS-induced lung injury. LPS-induced inflammation was initiated in 9–12 wk-old male Adora2b +/+ (C57BL/6J) or age and weight matched Adora2b −/− mice by intratracheal instillation of LPS or saline. Bronchoalveolar lavage (BAL) fluids were collected 1 d post treatment. A) Leukocyte cell counts of BAL fluid were determined utilizing a cellometer (mean ± SEM, n = 2–4). B) Protein content of BAL fluid was determined with BCA assay. Fold increase of protein concentration in LPS treated BAL samples over respective saline BAL samples is displayed (mean ± SEM, n = 3–10). C) Analysis of cell infiltration into the lung airspace (BAL) of mice exposed to inhaled LPS. Mice ( Adora2b +/+ or Adora2b −/−) were exposed to nebulized saline or lipopolysaccharide (LPS); 24 hours post-exposure, mice were euthanized, and BAL was harvested. Cells were then subjected to flow cytometric analysis to identify neutrophils (highly granular, Gr1+ cells). D) Analysis of FoxP3 gene expression in lungs of mice exposed to inhaled LPS. Mice ( Adora2b +/+ or −/−) were exposed to nebulized saline or lipopolysaccharide (LPS); 24 hours post-exposure, mice were euthanized, and perfused lungs were harvested. Total RNA was harvested from lungs, cDNA prepared and analyzed by real-time PCR. Data depict mean (± SEM) fold changes in indicated genes. Fold change was calculated based on primer efficiencies, standardized to changes in actin, with Adora2b +/+ Saline mean value defined as 1. E) Fold change in cell number of either regulatory T cells (lymphocyte size, CD3+ CD4+ FoxP3+) in the lung airspace (BAL) of mice exposed to inhaled saline or LPS as above. Data are from 2–6 mice; 2 independent experiments. F) Relative abundance of CD4 effector T cells (Teffs, defined as lymphocyte size, CD3+ CD4+ CD44high cells) to Tregs based on cell counts from either Adora2b +/+ or Adora2b −/− mice, with pie charts depicting mean cell count for the indicated cell populations from 2–6 mice; 2 independent expts. Analysis of total cellular infiltration into airways and protein leakage (panels A–B) was done in animals treated with intratracheal LPS. Analysis of neutrophil infiltration and Tregs (panels C–F) was done in animals treated with inhaled LPS. Statistically significant differences are indicated and were calculated either by one-way ANOVA (A) or by unpaired t test (B–E) with a focus on whether Adora2b −/− LPS treated animals were statistically different from either saline treated or Adora2b +/+ LPS treated mice.

    Article Snippet: RNA isolation and Real-time PCR Total RNA was extracted from cells or tissue by Trizol, followed by cDNA synthesis using iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Inc, Munich, Germany) according to the manufacturer's instructions.

    Techniques: Mouse Assay, BIA-KA, Protein Concentration, Flow Cytometry, Expressing, Real-time Polymerase Chain Reaction, Cell Counting

    Pyrosequencing assay for detection of correctly spliced transcript in fibroblasts of patients with the c.610 + 364G > A mutation . The N158S-forward primer binds in exon 3, the N158S-biot-reverse primer at the junction of exon 4b and exon 5. This primer setting exclusively amplifies cDNA of correctly spliced OPA1 transcripts (top bar, wildtype allele) but no transcripts with retained cryptic exon c between exons 4b/5 (middle bar, mutant allele, and upper pyrosequencing scheme). The sequencing primer (N158S-seq) binds two bases upstream of the heterozygous SNP rs7624750 in exon 4. The A allele of rs7624750 cosegregates with the mutation and cannot be detected in cDNA from mutant untreated cells using this assay. Upon AON treatment, cryptic exon c is skipped and the mutant transcript can be amplified (lower bar) and hence its allelic contribution can be measured via pyrosequencing (lower left scheme). A relative ratio of 35.3% versus 64.7% (A-allele versus G-allele) corresponds to a rescue of 55% of all mutant alleles in the cDNA of treated fibroblasts (example), which corresponds to a shift of total correctly spliced OPA1 mRNA of 77.5% (lower graph) For further graphical illustration of the principle compare Supplementary Figure S2 . AON, antisense oligonucleotides; cDNA, complementary DNA.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Antisense Oligonucleotide Mediated Splice Correction of a Deep Intronic Mutation in OPA1

    doi: 10.1038/mtna.2016.93

    Figure Lengend Snippet: Pyrosequencing assay for detection of correctly spliced transcript in fibroblasts of patients with the c.610 + 364G > A mutation . The N158S-forward primer binds in exon 3, the N158S-biot-reverse primer at the junction of exon 4b and exon 5. This primer setting exclusively amplifies cDNA of correctly spliced OPA1 transcripts (top bar, wildtype allele) but no transcripts with retained cryptic exon c between exons 4b/5 (middle bar, mutant allele, and upper pyrosequencing scheme). The sequencing primer (N158S-seq) binds two bases upstream of the heterozygous SNP rs7624750 in exon 4. The A allele of rs7624750 cosegregates with the mutation and cannot be detected in cDNA from mutant untreated cells using this assay. Upon AON treatment, cryptic exon c is skipped and the mutant transcript can be amplified (lower bar) and hence its allelic contribution can be measured via pyrosequencing (lower left scheme). A relative ratio of 35.3% versus 64.7% (A-allele versus G-allele) corresponds to a rescue of 55% of all mutant alleles in the cDNA of treated fibroblasts (example), which corresponds to a shift of total correctly spliced OPA1 mRNA of 77.5% (lower graph) For further graphical illustration of the principle compare Supplementary Figure S2 . AON, antisense oligonucleotides; cDNA, complementary DNA.

    Article Snippet: Cells were harvested at different time points, using the Peq Gold total RNA kit (Peqlab, Erlangen, Germany), followed by complementary DNA (cDNA) synthesis with the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Mannheim, Germany).

    Techniques: Pyrosequencing Assay, Mutagenesis, Sequencing, Amplification

    DNA methylation in the CLDN3 and CLDN4 promoters in ovarian cell lines and NOSE cells. ( A ) CLDN4 and ( B ) CLDN3 DNA methylation status by MSP analysis in ovarian cell lines and NOSE cells. The location of CpG islands and regions for MSP and bisulfite sequencing PCR (BSP) are indicated in the genomic sequence of CLDN3 and CLDN4 (top). The transcript and protein levels for CLDN3 and CLDN4 were measured by quantitative real-time reverse transcription–PCR (qRT–PCR) and western blot, respectively (middle). The qRT–PCR values are represented as mean ± SD. DNA methylation status was analyzed by MSP and quantitative MSP (qMSP) (bottom). Unmethylated (UM) and methylated (M) bands are shown and the quantitative methylation levels by qMSP are represented as percentage of methylated reference (PMR, %). Human leukocytes (h.L), methylated in vitro by Sss I treatment (h.L.M), were used as a positive control. ( C ) CLDN4 and ( D ) CLDN3 DNA methylation status by bisulfite sequencing in ovarian cell lines. CpG sites analyzed by BSP in the CLDN4 (21 CpG sites) and CLDN3 promoter region (47 CpG sites) are indicated. At least 10 clones were sequenced and each row represents the methylation pattern of an individual cloned PCR product (filled square, methylated CpG sites; open square, unmethylated CpG sites).

    Journal: Carcinogenesis

    Article Title: Derepression of CLDN3 and CLDN4 during ovarian tumorigenesis is associated with loss of repressive histone modifications

    doi: 10.1093/carcin/bgp336

    Figure Lengend Snippet: DNA methylation in the CLDN3 and CLDN4 promoters in ovarian cell lines and NOSE cells. ( A ) CLDN4 and ( B ) CLDN3 DNA methylation status by MSP analysis in ovarian cell lines and NOSE cells. The location of CpG islands and regions for MSP and bisulfite sequencing PCR (BSP) are indicated in the genomic sequence of CLDN3 and CLDN4 (top). The transcript and protein levels for CLDN3 and CLDN4 were measured by quantitative real-time reverse transcription–PCR (qRT–PCR) and western blot, respectively (middle). The qRT–PCR values are represented as mean ± SD. DNA methylation status was analyzed by MSP and quantitative MSP (qMSP) (bottom). Unmethylated (UM) and methylated (M) bands are shown and the quantitative methylation levels by qMSP are represented as percentage of methylated reference (PMR, %). Human leukocytes (h.L), methylated in vitro by Sss I treatment (h.L.M), were used as a positive control. ( C ) CLDN4 and ( D ) CLDN3 DNA methylation status by bisulfite sequencing in ovarian cell lines. CpG sites analyzed by BSP in the CLDN4 (21 CpG sites) and CLDN3 promoter region (47 CpG sites) are indicated. At least 10 clones were sequenced and each row represents the methylation pattern of an individual cloned PCR product (filled square, methylated CpG sites; open square, unmethylated CpG sites).

    Article Snippet: Following complementary DNA synthesis, quantitative polymerase chain reaction (PCR) was carried out as described previously ( ) in a dual system LightCycler (Roche Applied Science, Mannheim, Germany) using the primers, Universal Probe Library (Roche Applied Science) probe sequences are listed in supplementary Table S1 (available at Carcinogenesis Online), with the HPRT1 Taqman probe (TIB MOLBIOL, Berlin, Germany) used as a ‘reference gene’ to normalize gene expression.

    Techniques: DNA Methylation Assay, Methylation Sequencing, Polymerase Chain Reaction, Sequencing, Quantitative RT-PCR, Western Blot, Methylation, In Vitro, Positive Control, Clone Assay

    Clostridial flagellin transcript and gene copies during colitis and recovery. RNA and DNA were recovered from the same nucleic acids preparation, RNA was reverse transcribed into single-stranded cDNA. Gene and transcript copies were determined with qPCR. The ratio of flagellin to gene copies was calculated for individual mice and log-transformed. Means were compared by One-Way ANOVA, different letters indicate significance ( P

    Journal: The ISME Journal

    Article Title: Longitudinal study of murine microbiota activity and interactions with the host during acute inflammation and recovery

    doi: 10.1038/ismej.2013.223

    Figure Lengend Snippet: Clostridial flagellin transcript and gene copies during colitis and recovery. RNA and DNA were recovered from the same nucleic acids preparation, RNA was reverse transcribed into single-stranded cDNA. Gene and transcript copies were determined with qPCR. The ratio of flagellin to gene copies was calculated for individual mice and log-transformed. Means were compared by One-Way ANOVA, different letters indicate significance ( P

    Article Snippet: RNA isolation and cDNA synthesis from colon tissue For RNA preparation from colon tissue, pieces were homogenized in 700 μl RA1 buffer of the NucleoSpin II RNA isolation kit (Macherey–Nagel, Graz, Austria) and processed according to manufacturer's instructions. cDNA was prepared as described previously ( ).

    Techniques: Real-time Polymerase Chain Reaction, Mouse Assay, Transformation Assay