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  • 99
    Thermo Fisher poly a tail length assay
    Poly A Tail Length Assay, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore mrna
    <t>CHOP</t> functions a transcription repressor in myoblasts. (A) A retrovirus encoding a chimera Engrailed-CHOP protein or a retrovirus containing the parental vector was used to infect C2C12 myoblasts. Infected cells were grown in GM and in DM for the indicated time periods and proteins were analyzed by Western blot (left panel). Infected myoblasts were grown in DM for 48 hours and were immunostained with an anti-MyHC antibody (MF20) (right panel). MyHC staining is in red and DAPI is in blue. Percentage of nuclei in myotubes was calculated from three independent experiments. Mean values and standard errors are presented. Bar, 50 µm. (B) Infected cells described in A were grown in DM for 8 hours and were analyzed by immunostaining with antibodies directed against CHOP and MyoD. Control infected cells (left panel) and Engrailed-CHOP infected cells (right panel). Percentage of MyoD-positive nuclei relative to the total number of nuclei was calculated in three independent experiments. Mean values and standard errors are presented. Bar, 50 µm. (C) C2C12 infected cells as in A were grown in DM for 8 hours and total RNA was then extracted. MyoD <t>mRNA</t> levels were analyzed by semi-quantitative RT-PCR and quantified by the posphoimager.
    Mrna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5178 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mrna analysis
    <t>CHOP</t> functions a transcription repressor in myoblasts. (A) A retrovirus encoding a chimera Engrailed-CHOP protein or a retrovirus containing the parental vector was used to infect C2C12 myoblasts. Infected cells were grown in GM and in DM for the indicated time periods and proteins were analyzed by Western blot (left panel). Infected myoblasts were grown in DM for 48 hours and were immunostained with an anti-MyHC antibody (MF20) (right panel). MyHC staining is in red and DAPI is in blue. Percentage of nuclei in myotubes was calculated from three independent experiments. Mean values and standard errors are presented. Bar, 50 µm. (B) Infected cells described in A were grown in DM for 8 hours and were analyzed by immunostaining with antibodies directed against CHOP and MyoD. Control infected cells (left panel) and Engrailed-CHOP infected cells (right panel). Percentage of MyoD-positive nuclei relative to the total number of nuclei was calculated in three independent experiments. Mean values and standard errors are presented. Bar, 50 µm. (C) C2C12 infected cells as in A were grown in DM for 8 hours and total RNA was then extracted. MyoD <t>mRNA</t> levels were analyzed by semi-quantitative RT-PCR and quantified by the posphoimager.
    Mrna Analysis, supplied by Millipore, 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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    94
    Illumina Inc messenger rna mrna
    <t>mRNA</t> levels in bas1 and ino4 mutants. (A and B) Comparisons of <t>RNA-seq</t> read counts (in fragments per kilobase per million mapped reads, FPKM, log2-transformed) between wild type and TF mutants for all annotated genes. (C and D) Proportion of genes that
    Messenger Rna Mrna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 183 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Addgene inc sv40 poly a tail
    <t>mRNA</t> levels in bas1 and ino4 mutants. (A and B) Comparisons of <t>RNA-seq</t> read counts (in fragments per kilobase per million mapped reads, FPKM, log2-transformed) between wild type and TF mutants for all annotated genes. (C and D) Proportion of genes that
    Sv40 Poly A Tail, supplied by Addgene inc, used in various techniques. Bioz Stars score: 86/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher messenger rna mrna
    Transfection of non-phagocytic cells, using <t>mRNA:LPNs</t> and mRNA:CS-PLGA at different ratios performed in epithelial A549 cells for 24 h and 48 h post-transfection using flow cytometer. mRNA complexed LPNs reveal a significant higher transgene expression over CS-PLGA NPs. Both NPs show higher transfection rates with higher mRNA:NP ratios and increasing transfection until 48 h post-transfection N = 4, mean ± SD (*p
    Messenger Rna Mrna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 903 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    TriLink zfn messenger rna mrna
    Hematopoietic recovery following infusion of <t>ZFN</t> <t>mRNA-electroporated</t> HSPCs.
    Zfn Messenger Rna Mrna, supplied by TriLink, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Bioneer Corporation poly a tail rna
    Hematopoietic recovery following infusion of <t>ZFN</t> <t>mRNA-electroporated</t> HSPCs.
    Poly A Tail Rna, supplied by Bioneer Corporation, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore poly a tail addition
    Hematopoietic recovery following infusion of <t>ZFN</t> <t>mRNA-electroporated</t> HSPCs.
    Poly A Tail Addition, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Illumina Inc truseq messenger rna mrna
    Gene expression changes in CD28- vs CD86-silenced cells are consistent with regulation of both distinct and common pathways, including expression of IRF4. (A) Gene set enrichment analysis showing upregulated (top) and downregulated (bottom) gene sets in shCD28- (blue) and shCD86-treated (red) myeloma cells. For each gene set, the enrichment score is shown above the ranked change in gene expression, where genes that overlap the gene set are denoted by blue and red ticks for shCD28 and shCD86, respectively. (B) qRT-PCR showing IRF4 <t>mRNA</t> levels when CD28 or CD86 was silenced in MM.1s or 8226 cells. (C) Representative western blots showing IRF4 levels with silencing of CD28 or CD86 in cell lines indicated at different time points. (D) Integrin levels were measured via qRT-PCR ( ITGB7 and ITGB1 ; left). Representative histograms showing ITGβ1 and ITGβ7 on day 4 after shRNA treatment (right). For qRT-PCR, all data are normalized to β-actin as an endogenous control and then compared relative to mRNA levels in pLKO.1 empty vector control. <t>RNA</t> was extracted at day 3 postinfection. Data shown are mean ± SEM of at least 3 independent experiments. Histograms showing surface levels of indicated molecules. Gray histograms at left represent unstained or isotype controls. Flow cytometry data shown are from day 4 postinfection with lentiviral vectors. Flow cytometry and western blot data shown are representative of at least 3 independent experiments. (E) Cell adhesion 3 days postinfection with lentivirus containing the indicated shRNA. Myeloma cells stained with calcein AM were cocultured with HS-5 cells for 2 hours. Fluorescence was measured (485/528 emission/excitation) with BioTek Synergy H1 multiwell plate reader, and data are presented as fluorescence relative to pLKO.1 controls (mean ± SEM). Prewash readings were taken to ensure similar numbers of live cells were added to each well. All samples were plated in triplicate. Data are mean of 3 independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GX, gene expression changes; RFU, relative fluorescence unit.
    Truseq Messenger Rna Mrna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Roche messenger rna mrna isolation kit
    Quantitative reverse transcription-polymerase chain reaction gene expression profile. Histogram showing the gene expression analysis for untreated HFLS, untreated HFLS-RA, and HFLS-RA treated with MTX IC 50 concentrations (mean ± SD [n = 3]). HFLS indicates human fibroblast-like synoviocytes; <t>mRNA,</t> messenger <t>RNA;</t> RA, rheumatoid arthritis.
    Messenger Rna Mrna Isolation Kit, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    mrna  (TaKaRa)
    99
    TaKaRa mrna
    RAGE PTK profiling of different human tissues. Poly(A) <t>mRNA</t> obtained from Clontech was amplified by <t>RT–PCR</t> as described in Materials and Methods. The 150–170 bp amplicon was purified and used for Mwo I digestion. The completed digested products were separated by a denaturing sequencing gel. Specific PTKs were identified by the digested fragment sizes as indicated.
    Mrna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 9505 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega mrna
    Northern blot analysis of ER mRNAs in tissues of mature female ( n = 3 for α and γ; n = 2 for β) and mature male ( n = 2, testes only) Atlantic croaker. L, liver; O, ovary; B, brain; M, muscle; T, testes. Amounts of <t>mRNA</t> loaded per tissue type: liver, ovary, testes = 3 μg per lane; brain = 2 μg per lane; muscle = 1.3 μg per lane. Lanes shown are one of three loads of mRNA from the same fish. ( A ) Atlantic croaker ERα (acERα). ( B ) Atlantic croaker ERβ (acERβ). ( C ) Atlantic croaker ERγ (acERγ). The size (kb) of each transcript is indicated. <t>RNA</t> size markers (mk) are shown (Millennium Markers, Ambion).
    Mrna, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 6449 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    GE Healthcare mrna
    Stimulation of <t>HIEC/IEC-6</t> cells with LPS induced TNFα, as demonstrated by RT-PCR (A). Already after a stimulation period of three hours TNFα <t>mRNA</t> transcripts were observed in both IEC models. The expression of G3PDH was used as house keeping gene. The translation into the protein product and subsequent secretion of TNFα into the culture medium was analysed in HIEC using a high sensitivity ELISA (B). TNFα was produced in a dose dependent manner after stimulation with LPS. CHX treatment completely suppressed the secretion of TNFα. In keeping, functional experiments with CHX showed in HIEC that the proliferation rate after LPS stimulation remained unchanged once TNFα production was suppressed (C). On the other hand, addition of TNFα in the presence of CHX was still able to suppress HIEC proliferation. Concentrations (x axis) for CHX or LPS in μg/ml, for TNFα in ng/ml. The concentrations of CHX used together with LPS or TNFα were 1 μg/ml.
    Mrna, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 96/100, based on 2646 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Roche poly a rna
    Bone morphogenetic protein (BMP) 6 suppressed secretory leukocyte peptidase inhibitor (SLPI) and whey acid protein (WAP) 14 <t>mRNA</t> expression in cultured human GC. Cultured human granulosa-lutein cells (GCs) were stimulated with BMP-6 (100 ng/mL) for 24 hours. Total <t>RNA</t> was extracted from the cells and subjected to real-time PCR to determine the expression of SLPI and WAP14. Data were normalized by GAPDH mRNA levels to show the relative abundance. Data from 4 different experiments were combined and shown as the mean ± SEM relative to an adjusted value of 1.0 for the mean value of the each control. * P
    Poly A Rna, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 151 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore human alox15 mrna
    Bone morphogenetic protein (BMP) 6 suppressed secretory leukocyte peptidase inhibitor (SLPI) and whey acid protein (WAP) 14 <t>mRNA</t> expression in cultured human GC. Cultured human granulosa-lutein cells (GCs) were stimulated with BMP-6 (100 ng/mL) for 24 hours. Total <t>RNA</t> was extracted from the cells and subjected to real-time PCR to determine the expression of SLPI and WAP14. Data were normalized by GAPDH mRNA levels to show the relative abundance. Data from 4 different experiments were combined and shown as the mean ± SEM relative to an adjusted value of 1.0 for the mean value of the each control. * P
    Human Alox15 Mrna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Illumina Inc high quality poly a tail mrna
    Bone morphogenetic protein (BMP) 6 suppressed secretory leukocyte peptidase inhibitor (SLPI) and whey acid protein (WAP) 14 <t>mRNA</t> expression in cultured human GC. Cultured human granulosa-lutein cells (GCs) were stimulated with BMP-6 (100 ng/mL) for 24 hours. Total <t>RNA</t> was extracted from the cells and subjected to real-time PCR to determine the expression of SLPI and WAP14. Data were normalized by GAPDH mRNA levels to show the relative abundance. Data from 4 different experiments were combined and shown as the mean ± SEM relative to an adjusted value of 1.0 for the mean value of the each control. * P
    High Quality Poly A Tail Mrna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Shanghai Genechem poly a tail
    Bone morphogenetic protein (BMP) 6 suppressed secretory leukocyte peptidase inhibitor (SLPI) and whey acid protein (WAP) 14 <t>mRNA</t> expression in cultured human GC. Cultured human granulosa-lutein cells (GCs) were stimulated with BMP-6 (100 ng/mL) for 24 hours. Total <t>RNA</t> was extracted from the cells and subjected to real-time PCR to determine the expression of SLPI and WAP14. Data were normalized by GAPDH mRNA levels to show the relative abundance. Data from 4 different experiments were combined and shown as the mean ± SEM relative to an adjusted value of 1.0 for the mean value of the each control. * P
    Poly A Tail, supplied by Shanghai Genechem, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Biotechnology Information mrna messenger rna
    Bone morphogenetic protein (BMP) 6 suppressed secretory leukocyte peptidase inhibitor (SLPI) and whey acid protein (WAP) 14 <t>mRNA</t> expression in cultured human GC. Cultured human granulosa-lutein cells (GCs) were stimulated with BMP-6 (100 ng/mL) for 24 hours. Total <t>RNA</t> was extracted from the cells and subjected to real-time PCR to determine the expression of SLPI and WAP14. Data were normalized by GAPDH mRNA levels to show the relative abundance. Data from 4 different experiments were combined and shown as the mean ± SEM relative to an adjusted value of 1.0 for the mean value of the each control. * P
    Mrna Messenger Rna, supplied by Biotechnology Information, used in various techniques. Bioz Stars score: 98/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    SLIT2 LTD messenger rna mrna
    Bone morphogenetic protein (BMP) 6 suppressed secretory leukocyte peptidase inhibitor (SLPI) and whey acid protein (WAP) 14 <t>mRNA</t> expression in cultured human GC. Cultured human granulosa-lutein cells (GCs) were stimulated with BMP-6 (100 ng/mL) for 24 hours. Total <t>RNA</t> was extracted from the cells and subjected to real-time PCR to determine the expression of SLPI and WAP14. Data were normalized by GAPDH mRNA levels to show the relative abundance. Data from 4 different experiments were combined and shown as the mean ± SEM relative to an adjusted value of 1.0 for the mean value of the each control. * P
    Messenger Rna Mrna, supplied by SLIT2 LTD, used in various techniques. Bioz Stars score: 86/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Nanotherapeutics messenger rna mrna
    Bone morphogenetic protein (BMP) 6 suppressed secretory leukocyte peptidase inhibitor (SLPI) and whey acid protein (WAP) 14 <t>mRNA</t> expression in cultured human GC. Cultured human granulosa-lutein cells (GCs) were stimulated with BMP-6 (100 ng/mL) for 24 hours. Total <t>RNA</t> was extracted from the cells and subjected to real-time PCR to determine the expression of SLPI and WAP14. Data were normalized by GAPDH mRNA levels to show the relative abundance. Data from 4 different experiments were combined and shown as the mean ± SEM relative to an adjusted value of 1.0 for the mean value of the each control. * P
    Messenger Rna Mrna, supplied by Nanotherapeutics, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    fluidigm messenger rna mrna
    Bone morphogenetic protein (BMP) 6 suppressed secretory leukocyte peptidase inhibitor (SLPI) and whey acid protein (WAP) 14 <t>mRNA</t> expression in cultured human GC. Cultured human granulosa-lutein cells (GCs) were stimulated with BMP-6 (100 ng/mL) for 24 hours. Total <t>RNA</t> was extracted from the cells and subjected to real-time PCR to determine the expression of SLPI and WAP14. Data were normalized by GAPDH mRNA levels to show the relative abundance. Data from 4 different experiments were combined and shown as the mean ± SEM relative to an adjusted value of 1.0 for the mean value of the each control. * P
    Messenger Rna Mrna, supplied by fluidigm, used in various techniques. Bioz Stars score: 87/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Becton Dickinson saccharomyces cerevisiae messenger rna mrna
    Effects of unincorporated dye precursors on fluorescent nucleic acid analyses. A, B: Comparison of DNA sequencing analyses performed without (A) or with (B) purification of unincorporated dye terminators. Sequencing reactions were prepared from 250 ng pGEMZf(+) plasmid and 4 pmol M13 −47 primer, using BigDye version 3.0 at a 2:10 (μL BigDye:μL total volume) reaction mixture with Better Buffer diluent. Purification B was performed with the RapXtract II Kit. DNA sequences were analyzed on an ABI PRISM 3100 Genetic Analyzer using POP6 polymer and injection parameters recommended for RapXtract Kits (2 kV, 45 s). C, D: Comparison of cDNA-microarray hybridization analyses performed without (C) or with (D) purification of unincorporated dye-labeled nucleotides from the labeling reaction. Labeled cDNA was prepared from 1 μg Saccharomyces <t>cerevisiae</t> <t>mRNA</t> using the CyScribe First Strand Labeling Kit and Cy3-labeled dUTP. Purification D was performed with the RapXtract Fluorescent Nucleic Acid Purification Kit. The cDNA was then hybridized to a yeast collage array at 67°C for 12 h. The microarray was visualized on a ScanArray Lite scanner at a laser output of 80%, PMT gain of 80%, and spatial resolution of 50 μm.
    Saccharomyces Cerevisiae Messenger Rna Mrna, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Miltenyi Biotec stemmacs nuclear egfp messenger rna mrna
    Effects of unincorporated dye precursors on fluorescent nucleic acid analyses. A, B: Comparison of DNA sequencing analyses performed without (A) or with (B) purification of unincorporated dye terminators. Sequencing reactions were prepared from 250 ng pGEMZf(+) plasmid and 4 pmol M13 −47 primer, using BigDye version 3.0 at a 2:10 (μL BigDye:μL total volume) reaction mixture with Better Buffer diluent. Purification B was performed with the RapXtract II Kit. DNA sequences were analyzed on an ABI PRISM 3100 Genetic Analyzer using POP6 polymer and injection parameters recommended for RapXtract Kits (2 kV, 45 s). C, D: Comparison of cDNA-microarray hybridization analyses performed without (C) or with (D) purification of unincorporated dye-labeled nucleotides from the labeling reaction. Labeled cDNA was prepared from 1 μg Saccharomyces <t>cerevisiae</t> <t>mRNA</t> using the CyScribe First Strand Labeling Kit and Cy3-labeled dUTP. Purification D was performed with the RapXtract Fluorescent Nucleic Acid Purification Kit. The cDNA was then hybridized to a yeast collage array at 67°C for 12 h. The microarray was visualized on a ScanArray Lite scanner at a laser output of 80%, PMT gain of 80%, and spatial resolution of 50 μm.
    Stemmacs Nuclear Egfp Messenger Rna Mrna, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 95/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    TaKaRa λ poly a rna
    Effects of unincorporated dye precursors on fluorescent nucleic acid analyses. A, B: Comparison of DNA sequencing analyses performed without (A) or with (B) purification of unincorporated dye terminators. Sequencing reactions were prepared from 250 ng pGEMZf(+) plasmid and 4 pmol M13 −47 primer, using BigDye version 3.0 at a 2:10 (μL BigDye:μL total volume) reaction mixture with Better Buffer diluent. Purification B was performed with the RapXtract II Kit. DNA sequences were analyzed on an ABI PRISM 3100 Genetic Analyzer using POP6 polymer and injection parameters recommended for RapXtract Kits (2 kV, 45 s). C, D: Comparison of cDNA-microarray hybridization analyses performed without (C) or with (D) purification of unincorporated dye-labeled nucleotides from the labeling reaction. Labeled cDNA was prepared from 1 μg Saccharomyces <t>cerevisiae</t> <t>mRNA</t> using the CyScribe First Strand Labeling Kit and Cy3-labeled dUTP. Purification D was performed with the RapXtract Fluorescent Nucleic Acid Purification Kit. The cDNA was then hybridized to a yeast collage array at 67°C for 12 h. The microarray was visualized on a ScanArray Lite scanner at a laser output of 80%, PMT gain of 80%, and spatial resolution of 50 μm.
    λ Poly A Rna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher messenger rna mrna expression data
    Effects of unincorporated dye precursors on fluorescent nucleic acid analyses. A, B: Comparison of DNA sequencing analyses performed without (A) or with (B) purification of unincorporated dye terminators. Sequencing reactions were prepared from 250 ng pGEMZf(+) plasmid and 4 pmol M13 −47 primer, using BigDye version 3.0 at a 2:10 (μL BigDye:μL total volume) reaction mixture with Better Buffer diluent. Purification B was performed with the RapXtract II Kit. DNA sequences were analyzed on an ABI PRISM 3100 Genetic Analyzer using POP6 polymer and injection parameters recommended for RapXtract Kits (2 kV, 45 s). C, D: Comparison of cDNA-microarray hybridization analyses performed without (C) or with (D) purification of unincorporated dye-labeled nucleotides from the labeling reaction. Labeled cDNA was prepared from 1 μg Saccharomyces <t>cerevisiae</t> <t>mRNA</t> using the CyScribe First Strand Labeling Kit and Cy3-labeled dUTP. Purification D was performed with the RapXtract Fluorescent Nucleic Acid Purification Kit. The cDNA was then hybridized to a yeast collage array at 67°C for 12 h. The microarray was visualized on a ScanArray Lite scanner at a laser output of 80%, PMT gain of 80%, and spatial resolution of 50 μm.
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    Effects of unincorporated dye precursors on fluorescent nucleic acid analyses. A, B: Comparison of DNA sequencing analyses performed without (A) or with (B) purification of unincorporated dye terminators. Sequencing reactions were prepared from 250 ng pGEMZf(+) plasmid and 4 pmol M13 −47 primer, using BigDye version 3.0 at a 2:10 (μL BigDye:μL total volume) reaction mixture with Better Buffer diluent. Purification B was performed with the RapXtract II Kit. DNA sequences were analyzed on an ABI PRISM 3100 Genetic Analyzer using POP6 polymer and injection parameters recommended for RapXtract Kits (2 kV, 45 s). C, D: Comparison of cDNA-microarray hybridization analyses performed without (C) or with (D) purification of unincorporated dye-labeled nucleotides from the labeling reaction. Labeled cDNA was prepared from 1 μg Saccharomyces <t>cerevisiae</t> <t>mRNA</t> using the CyScribe First Strand Labeling Kit and Cy3-labeled dUTP. Purification D was performed with the RapXtract Fluorescent Nucleic Acid Purification Kit. The cDNA was then hybridized to a yeast collage array at 67°C for 12 h. The microarray was visualized on a ScanArray Lite scanner at a laser output of 80%, PMT gain of 80%, and spatial resolution of 50 μm.
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    Effects of unincorporated dye precursors on fluorescent nucleic acid analyses. A, B: Comparison of DNA sequencing analyses performed without (A) or with (B) purification of unincorporated dye terminators. Sequencing reactions were prepared from 250 ng pGEMZf(+) plasmid and 4 pmol M13 −47 primer, using BigDye version 3.0 at a 2:10 (μL BigDye:μL total volume) reaction mixture with Better Buffer diluent. Purification B was performed with the RapXtract II Kit. DNA sequences were analyzed on an ABI PRISM 3100 Genetic Analyzer using POP6 polymer and injection parameters recommended for RapXtract Kits (2 kV, 45 s). C, D: Comparison of cDNA-microarray hybridization analyses performed without (C) or with (D) purification of unincorporated dye-labeled nucleotides from the labeling reaction. Labeled cDNA was prepared from 1 μg Saccharomyces <t>cerevisiae</t> <t>mRNA</t> using the CyScribe First Strand Labeling Kit and Cy3-labeled dUTP. Purification D was performed with the RapXtract Fluorescent Nucleic Acid Purification Kit. The cDNA was then hybridized to a yeast collage array at 67°C for 12 h. The microarray was visualized on a ScanArray Lite scanner at a laser output of 80%, PMT gain of 80%, and spatial resolution of 50 μm.
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    Effects of unincorporated dye precursors on fluorescent nucleic acid analyses. A, B: Comparison of DNA sequencing analyses performed without (A) or with (B) purification of unincorporated dye terminators. Sequencing reactions were prepared from 250 ng pGEMZf(+) plasmid and 4 pmol M13 −47 primer, using BigDye version 3.0 at a 2:10 (μL BigDye:μL total volume) reaction mixture with Better Buffer diluent. Purification B was performed with the RapXtract II Kit. DNA sequences were analyzed on an ABI PRISM 3100 Genetic Analyzer using POP6 polymer and injection parameters recommended for RapXtract Kits (2 kV, 45 s). C, D: Comparison of cDNA-microarray hybridization analyses performed without (C) or with (D) purification of unincorporated dye-labeled nucleotides from the labeling reaction. Labeled cDNA was prepared from 1 μg Saccharomyces <t>cerevisiae</t> <t>mRNA</t> using the CyScribe First Strand Labeling Kit and Cy3-labeled dUTP. Purification D was performed with the RapXtract Fluorescent Nucleic Acid Purification Kit. The cDNA was then hybridized to a yeast collage array at 67°C for 12 h. The microarray was visualized on a ScanArray Lite scanner at a laser output of 80%, PMT gain of 80%, and spatial resolution of 50 μm.
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    Mouse multiple tissue blot analysis of the expression of mTR2R1 <t>mRNA.</t> (a) Probe with the entire 3.5 kb mTR2R1 <t>cDNA</t> in clone pBS35. (b) Probe with the 3′ noncoding region of mTR2R1 in clone pBS35.
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    Image Search Results


    CHOP functions a transcription repressor in myoblasts. (A) A retrovirus encoding a chimera Engrailed-CHOP protein or a retrovirus containing the parental vector was used to infect C2C12 myoblasts. Infected cells were grown in GM and in DM for the indicated time periods and proteins were analyzed by Western blot (left panel). Infected myoblasts were grown in DM for 48 hours and were immunostained with an anti-MyHC antibody (MF20) (right panel). MyHC staining is in red and DAPI is in blue. Percentage of nuclei in myotubes was calculated from three independent experiments. Mean values and standard errors are presented. Bar, 50 µm. (B) Infected cells described in A were grown in DM for 8 hours and were analyzed by immunostaining with antibodies directed against CHOP and MyoD. Control infected cells (left panel) and Engrailed-CHOP infected cells (right panel). Percentage of MyoD-positive nuclei relative to the total number of nuclei was calculated in three independent experiments. Mean values and standard errors are presented. Bar, 50 µm. (C) C2C12 infected cells as in A were grown in DM for 8 hours and total RNA was then extracted. MyoD mRNA levels were analyzed by semi-quantitative RT-PCR and quantified by the posphoimager.

    Journal: PLoS ONE

    Article Title: Stress-Induced C/EBP Homology Protein (CHOP) Represses MyoD Transcription to Delay Myoblast Differentiation

    doi: 10.1371/journal.pone.0029498

    Figure Lengend Snippet: CHOP functions a transcription repressor in myoblasts. (A) A retrovirus encoding a chimera Engrailed-CHOP protein or a retrovirus containing the parental vector was used to infect C2C12 myoblasts. Infected cells were grown in GM and in DM for the indicated time periods and proteins were analyzed by Western blot (left panel). Infected myoblasts were grown in DM for 48 hours and were immunostained with an anti-MyHC antibody (MF20) (right panel). MyHC staining is in red and DAPI is in blue. Percentage of nuclei in myotubes was calculated from three independent experiments. Mean values and standard errors are presented. Bar, 50 µm. (B) Infected cells described in A were grown in DM for 8 hours and were analyzed by immunostaining with antibodies directed against CHOP and MyoD. Control infected cells (left panel) and Engrailed-CHOP infected cells (right panel). Percentage of MyoD-positive nuclei relative to the total number of nuclei was calculated in three independent experiments. Mean values and standard errors are presented. Bar, 50 µm. (C) C2C12 infected cells as in A were grown in DM for 8 hours and total RNA was then extracted. MyoD mRNA levels were analyzed by semi-quantitative RT-PCR and quantified by the posphoimager.

    Article Snippet: ShRNA-mediated knockdown of proteins Knockdown of the CHOP protein in myoblasts was achieved by lentiviral infections of 5 viral vectors that express different shRNA directed to CHOP mRNA and were purchased from Sigma-Aldrich (ShRNA MISSION).

    Techniques: Plasmid Preparation, Infection, Western Blot, Staining, Immunostaining, Quantitative RT-PCR

    CHOP represses MyoD transcription. (A) A C2C12 derived cell line expressing a chimera CHOP:ER protein was constructed as is described under “ Materials and Methods ”. (A) Myoblasts were grown in the presence of ethanol or β estradiol (0.1 µM) for 8 hours. Cells were immunostained using anti-MyoD and anti-CHOP antibodies. DAPI in blue, MyoD in green and CHOP in red. Percentage of MyoD-positive nuclei relative to the total number of nuclei was calculated. Bar, 50 µm. (left panel). In another experiment, cells were grown in the presence of ethanol or β estradiol (0.1 µM) for 48 hours and proteins were analyzed by Western blot (right panel). (B) The same cells as above were grown in DM in the presence of ethanol or β estradiol (0.1 µM) for the indicated time periods and mRNA levels of myod and myogenin were determined by semi-quantitative RT-PCR analysis. (C) The same cells as above were grown in DM and in the presence of ethanol or β estradiol for 6 hours in the absence or presence of cycloheximide added to cells one hour before the addition of ethanol or β estradiol. (D) A clone of the above cells (i.e., expressing CHOP:ER) with integrated MyoD reporter gene (6.0 MyoD -nl β Gal) was isolated. These cells were grown in the presence of ethanol or β estradiol for 20 hours. Nuclear expression of β Gal was identified by an enzymatic colorimetric assay, and the expression of CHOP by immunostaining. Arrows point at β Gal-positive nuclei that are CHOP negative. Percentage of β Gal-positive nuclei out of the total number of nuclei was calculated in two independent experiments. Mean values and standard errors are presented. Bar, 50 µm.

    Journal: PLoS ONE

    Article Title: Stress-Induced C/EBP Homology Protein (CHOP) Represses MyoD Transcription to Delay Myoblast Differentiation

    doi: 10.1371/journal.pone.0029498

    Figure Lengend Snippet: CHOP represses MyoD transcription. (A) A C2C12 derived cell line expressing a chimera CHOP:ER protein was constructed as is described under “ Materials and Methods ”. (A) Myoblasts were grown in the presence of ethanol or β estradiol (0.1 µM) for 8 hours. Cells were immunostained using anti-MyoD and anti-CHOP antibodies. DAPI in blue, MyoD in green and CHOP in red. Percentage of MyoD-positive nuclei relative to the total number of nuclei was calculated. Bar, 50 µm. (left panel). In another experiment, cells were grown in the presence of ethanol or β estradiol (0.1 µM) for 48 hours and proteins were analyzed by Western blot (right panel). (B) The same cells as above were grown in DM in the presence of ethanol or β estradiol (0.1 µM) for the indicated time periods and mRNA levels of myod and myogenin were determined by semi-quantitative RT-PCR analysis. (C) The same cells as above were grown in DM and in the presence of ethanol or β estradiol for 6 hours in the absence or presence of cycloheximide added to cells one hour before the addition of ethanol or β estradiol. (D) A clone of the above cells (i.e., expressing CHOP:ER) with integrated MyoD reporter gene (6.0 MyoD -nl β Gal) was isolated. These cells were grown in the presence of ethanol or β estradiol for 20 hours. Nuclear expression of β Gal was identified by an enzymatic colorimetric assay, and the expression of CHOP by immunostaining. Arrows point at β Gal-positive nuclei that are CHOP negative. Percentage of β Gal-positive nuclei out of the total number of nuclei was calculated in two independent experiments. Mean values and standard errors are presented. Bar, 50 µm.

    Article Snippet: ShRNA-mediated knockdown of proteins Knockdown of the CHOP protein in myoblasts was achieved by lentiviral infections of 5 viral vectors that express different shRNA directed to CHOP mRNA and were purchased from Sigma-Aldrich (ShRNA MISSION).

    Techniques: Derivative Assay, Expressing, Construct, Western Blot, Quantitative RT-PCR, Isolation, Enzymatic Colorimetric Assay, Immunostaining

    BCAS2 does not regulate the transcription initiation of Delta but is involved in Delta pre-mRNA splicing. (A) Control of Dl-lacZ (red) in the en > GFP wing disc stained with anti-β-gal antibody. (B) en > GFP , dBCAS2 dsRNA . In the dBCAS2 -depleted posterior compartment, marked by GFP, the expression of Dl-lacZ (red) gives a signal of similar strength as the normal anterior compartment. The expression of GFP and β-galactosidase were merged and displayed in the right panel. Images were taken by confocal microscopy, scale bar, 50 μm. (C). RNA expression of β-galactosidase. RNAs were extracted from wing discs of third instar larvae and subjected to RT-PCR to confirm the RNA expression of β-galactosidase driven by Dl promoter in Act > dBCAS2 dsRNA (lane 2) compared with the control (lane 1). The internal control, rp49 . (D) Schematic diagram of primer design for detecting the intron-containing precursor mRNA (upper) and mRNA of Delta (lower). Primers, exons and introns are denoted with arrowheads, boxes and lines, respectively. (E) Coexpression of dBCAS2 and dBCAS2 dsRNA in larvae can rescue the phenotypes of mRNA decrease and pre-mRNA accumulation caused by dBCAS2 dsRNA . The pre-mRNA and mRNA of Delta were analyzed by quantitative RT-PCR and described in the Materials and Methods. Each genotype was under the control of Act5c-GAL4 driver. White bar: Act5c > +; black bar: Act5c > dBCAS2 dsRNA ; gray bar: Act5c > dBCAS2 dsRNA , dBCAS2 . Data are shown as means and SD relative to the controls from three independent experiments. The P-values was measured by the Student’s t-test. * p

    Journal: PLoS ONE

    Article Title: BCAS2 Regulates Delta-Notch Signaling Activity through Delta Pre-mRNA Splicing in Drosophila Wing Development

    doi: 10.1371/journal.pone.0130706

    Figure Lengend Snippet: BCAS2 does not regulate the transcription initiation of Delta but is involved in Delta pre-mRNA splicing. (A) Control of Dl-lacZ (red) in the en > GFP wing disc stained with anti-β-gal antibody. (B) en > GFP , dBCAS2 dsRNA . In the dBCAS2 -depleted posterior compartment, marked by GFP, the expression of Dl-lacZ (red) gives a signal of similar strength as the normal anterior compartment. The expression of GFP and β-galactosidase were merged and displayed in the right panel. Images were taken by confocal microscopy, scale bar, 50 μm. (C). RNA expression of β-galactosidase. RNAs were extracted from wing discs of third instar larvae and subjected to RT-PCR to confirm the RNA expression of β-galactosidase driven by Dl promoter in Act > dBCAS2 dsRNA (lane 2) compared with the control (lane 1). The internal control, rp49 . (D) Schematic diagram of primer design for detecting the intron-containing precursor mRNA (upper) and mRNA of Delta (lower). Primers, exons and introns are denoted with arrowheads, boxes and lines, respectively. (E) Coexpression of dBCAS2 and dBCAS2 dsRNA in larvae can rescue the phenotypes of mRNA decrease and pre-mRNA accumulation caused by dBCAS2 dsRNA . The pre-mRNA and mRNA of Delta were analyzed by quantitative RT-PCR and described in the Materials and Methods. Each genotype was under the control of Act5c-GAL4 driver. White bar: Act5c > +; black bar: Act5c > dBCAS2 dsRNA ; gray bar: Act5c > dBCAS2 dsRNA , dBCAS2 . Data are shown as means and SD relative to the controls from three independent experiments. The P-values was measured by the Student’s t-test. * p

    Article Snippet: In vivo splicing assays For the detection of mRNA and pre-mRNA in Drosophila , total RNA from imaginal wing discs in 20 third instar larvae was isolated by using TRI reagent (Sigma-Aldrich).

    Techniques: Staining, Expressing, Confocal Microscopy, RNA Expression, Reverse Transcription Polymerase Chain Reaction, Activated Clotting Time Assay, Quantitative RT-PCR

    RT–PCR analysis of HIV-1 mRNA expression. RT–PCR analysis of uncleaved HIV-1 mRNA was carried out using HIV-1 env -specific primers with concomitant amplification of G3PDH. Total RNA was extracted from COS cells transfected with E2-ss- or dsRNAs first, followed by pNL4-3. The relative amounts of HIV-1 transcripts were determined by RT–PCR, as described in Materials and Methods. PCR primers were used to amplify a 529 bp (7072–7600) fragment in HIV-1 transcripts. Lane 1, negative control (no template added); lane 2, relative amount of transcripts in cells transfected with E2-dsRNA; lane 3, relative amount of transcripts in cells transfected with E2-ssRNA; lane 4, relative amount of transcripts in positive control cells transfected with pNL4-3 proviral DNA. The G3PDH RT–PCR was run in parallel to normalize the levels of mRNA in the samples.

    Journal: Nucleic Acids Research

    Article Title: Prevention of HIV-1 infection in human peripheral blood mononuclear cells by specific RNA interference

    doi:

    Figure Lengend Snippet: RT–PCR analysis of HIV-1 mRNA expression. RT–PCR analysis of uncleaved HIV-1 mRNA was carried out using HIV-1 env -specific primers with concomitant amplification of G3PDH. Total RNA was extracted from COS cells transfected with E2-ss- or dsRNAs first, followed by pNL4-3. The relative amounts of HIV-1 transcripts were determined by RT–PCR, as described in Materials and Methods. PCR primers were used to amplify a 529 bp (7072–7600) fragment in HIV-1 transcripts. Lane 1, negative control (no template added); lane 2, relative amount of transcripts in cells transfected with E2-dsRNA; lane 3, relative amount of transcripts in cells transfected with E2-ssRNA; lane 4, relative amount of transcripts in positive control cells transfected with pNL4-3 proviral DNA. The G3PDH RT–PCR was run in parallel to normalize the levels of mRNA in the samples.

    Article Snippet: The reverse transcription and the PCR analysis were performed with total RNA from COS cells (prepared with the GenElute Mammalian Total RNA kit; Sigma) as the template (1 µg/reaction).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Transfection, Polymerase Chain Reaction, Negative Control, Positive Control

    HIF-1α regulates NLRP3 expression and thrombogenesis. Animals were treated with in vivo grade HIF-1α and HIF-2α siRNA. After RNA isolation from indicated groups, real-time PCR was performed for ( A ) HIF-1α, ( B ) NLRP3, ( C ) IL-1β. Thrombus weight ( D ) and clotting time ( E ) were also recorded. ( F ) Chromatin immunoprecipitation with two different HIF-1α antibody (indicated) and primer pairs spanning putative sites (indicated). The enrichment of NLRP3 promoter region in chromatin immunoprecipitation experiments was quantitated and plotted to obtain the bar graph (mean ± SEM) shown in the figure. * P

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

    Article Title: Activation of NLRP3 inflammasome complex potentiates venous thrombosis in response to hypoxia

    doi: 10.1073/pnas.1620458114

    Figure Lengend Snippet: HIF-1α regulates NLRP3 expression and thrombogenesis. Animals were treated with in vivo grade HIF-1α and HIF-2α siRNA. After RNA isolation from indicated groups, real-time PCR was performed for ( A ) HIF-1α, ( B ) NLRP3, ( C ) IL-1β. Thrombus weight ( D ) and clotting time ( E ) were also recorded. ( F ) Chromatin immunoprecipitation with two different HIF-1α antibody (indicated) and primer pairs spanning putative sites (indicated). The enrichment of NLRP3 promoter region in chromatin immunoprecipitation experiments was quantitated and plotted to obtain the bar graph (mean ± SEM) shown in the figure. * P

    Article Snippet: The reverse transcription PCR (RT-PCR) kit for RNA analysis and all other reagents were from Sigma-Aldrich.

    Techniques: Expressing, In Vivo, Isolation, Real-time Polymerase Chain Reaction, Coagulation, Chromatin Immunoprecipitation

    Evidence for involvement of NLRP3 inflammasome components in patients with altitude-induced venous thrombosis. ( A–D ) RNA was isolated from PBMNs from the blood samples of patients ( n = 18), and real-time PCR for indicated genes was performed. β-actin was used as an internal control. The relative expression of NLRP3 ( A ), caspase-1 ( B ), 1L-1β ( C ), and IL-18 ( D ) transcripts. ( E ) Caspase-1/ICE activity in plasma samples from patients and controls ( n = 12). Estimation of NLRP3 ( F ) and IL-1β ( G ) levels in plasma samples from patients and controls ( n ≥ 8) Median values for individual groups are also shown. * P

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

    Article Title: Activation of NLRP3 inflammasome complex potentiates venous thrombosis in response to hypoxia

    doi: 10.1073/pnas.1620458114

    Figure Lengend Snippet: Evidence for involvement of NLRP3 inflammasome components in patients with altitude-induced venous thrombosis. ( A–D ) RNA was isolated from PBMNs from the blood samples of patients ( n = 18), and real-time PCR for indicated genes was performed. β-actin was used as an internal control. The relative expression of NLRP3 ( A ), caspase-1 ( B ), 1L-1β ( C ), and IL-18 ( D ) transcripts. ( E ) Caspase-1/ICE activity in plasma samples from patients and controls ( n = 12). Estimation of NLRP3 ( F ) and IL-1β ( G ) levels in plasma samples from patients and controls ( n ≥ 8) Median values for individual groups are also shown. * P

    Article Snippet: The reverse transcription PCR (RT-PCR) kit for RNA analysis and all other reagents were from Sigma-Aldrich.

    Techniques: Isolation, Real-time Polymerase Chain Reaction, Expressing, Activity Assay

    mRNA levels in bas1 and ino4 mutants. (A and B) Comparisons of RNA-seq read counts (in fragments per kilobase per million mapped reads, FPKM, log2-transformed) between wild type and TF mutants for all annotated genes. (C and D) Proportion of genes that

    Journal: Genetics

    Article Title: High-Resolution Global Analysis of the Influences of Bas1 and Ino4 Transcription Factors on Meiotic DNA Break Distributions in Saccharomyces cerevisiae

    doi: 10.1534/genetics.115.178293

    Figure Lengend Snippet: mRNA levels in bas1 and ino4 mutants. (A and B) Comparisons of RNA-seq read counts (in fragments per kilobase per million mapped reads, FPKM, log2-transformed) between wild type and TF mutants for all annotated genes. (C and D) Proportion of genes that

    Article Snippet: Messenger RNA (mRNA) was enriched by poly-A selection and sequenced using Illumina HiSeq in the Genomics Resources Core Facility of Weill Cornell Medical College.

    Techniques: RNA Sequencing Assay, Transformation Assay

    Diagram illustrating the experimental design and workflow for RNA-seq data analysis. Briefly, tissues from 2 to 3-month-old C57BL/6 conventional (CV) and germ-free (GF) mice were harvested as described previously [ 29 ]. Total RNAs were extracted from each organ ( n = 3 per enterotype per organ). The cDNA libraries were prepared using the poly-A tail selection method and were sequenced using an Illumina HiSeq2000 sequencer (2 × 50 bp paired-end). Fastq files were quality-checked with FastQC and mapped to the mouse reference genome (mm10) using HISAT. The sequence alignment mapping (SAM) files were converted to binary alignment mapping (BAM) files and sorted using SAMtools. The sorted BAM files were subjected to Cufflinks to determine the transcript abundance. Specifically, the transcript abundance of lncRNAs and PCGs was estimated using the mouse NONCODE 2016 lncRNA and UCSC mm10 PCG reference gene transfer format (GTF) files, respectively. The differentially expressed lncRNAs and PCGs were determined by Cuffdiff between CV and GF mice for each organ (FDR-BH

    Journal: BMC Genomics

    Article Title: Coordinate regulation of long non-coding RNAs and protein-coding genes in germ-free mice

    doi: 10.1186/s12864-018-5235-3

    Figure Lengend Snippet: Diagram illustrating the experimental design and workflow for RNA-seq data analysis. Briefly, tissues from 2 to 3-month-old C57BL/6 conventional (CV) and germ-free (GF) mice were harvested as described previously [ 29 ]. Total RNAs were extracted from each organ ( n = 3 per enterotype per organ). The cDNA libraries were prepared using the poly-A tail selection method and were sequenced using an Illumina HiSeq2000 sequencer (2 × 50 bp paired-end). Fastq files were quality-checked with FastQC and mapped to the mouse reference genome (mm10) using HISAT. The sequence alignment mapping (SAM) files were converted to binary alignment mapping (BAM) files and sorted using SAMtools. The sorted BAM files were subjected to Cufflinks to determine the transcript abundance. Specifically, the transcript abundance of lncRNAs and PCGs was estimated using the mouse NONCODE 2016 lncRNA and UCSC mm10 PCG reference gene transfer format (GTF) files, respectively. The differentially expressed lncRNAs and PCGs were determined by Cuffdiff between CV and GF mice for each organ (FDR-BH

    Article Snippet: The complementary DNA (cDNA) libraries were constructed from total RNA samples using a TruSeq RNA Sample Prep Kit with poly-A tail selection (Illumina, San Diego, CA).

    Techniques: RNA Sequencing Assay, Mouse Assay, Selection, Sequencing

    Transfection of non-phagocytic cells, using mRNA:LPNs and mRNA:CS-PLGA at different ratios performed in epithelial A549 cells for 24 h and 48 h post-transfection using flow cytometer. mRNA complexed LPNs reveal a significant higher transgene expression over CS-PLGA NPs. Both NPs show higher transfection rates with higher mRNA:NP ratios and increasing transfection until 48 h post-transfection N = 4, mean ± SD (*p

    Journal: Journal of Nanobiotechnology

    Article Title: Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles

    doi: 10.1186/s12951-018-0401-y

    Figure Lengend Snippet: Transfection of non-phagocytic cells, using mRNA:LPNs and mRNA:CS-PLGA at different ratios performed in epithelial A549 cells for 24 h and 48 h post-transfection using flow cytometer. mRNA complexed LPNs reveal a significant higher transgene expression over CS-PLGA NPs. Both NPs show higher transfection rates with higher mRNA:NP ratios and increasing transfection until 48 h post-transfection N = 4, mean ± SD (*p

    Article Snippet: The encapsulation efficiency (%EE) of bound mRNA:LPNs and mRNA:CS-PLGA NPs was evaluated indirectly by pelleting all samples down at 24,400g for 30 min and determining the concentration of unbound mRNA in the supernatant by measuring absorbance at 260/280 nm with a NanoDrop Spectrophotometer.

    Techniques: Transfection, Flow Cytometry, Cytometry, Expressing

    a1 Representative images depicted from live cell video after nine different time-points. The images are part of a video provided in Additional file 2 : Movie S1. DC2.4 cells were incubated with mRNA:FA-LPNs for a complete time duration of 4 h and with an interval of 3 min/image. Green dots on the images represent the fluorescence signal of labeled LPNs, while the cells signaling in red are the ones with successful mCherry expression. Scale bar = 100 µm. a2 Time-dependent change of the red fluorescence signal resulting from transfected cells and non-transfected, which are correlated with a3 green fluorescence signal of labeled nanoparticles. The tendency of each single cell being transfected is independent of the NPs uptake, as no significant difference between the uptake-behavior of transfected and non-transfected cells was observable. λ em = peak emission wavelength, MFI mean fluorescence intensity (mean ± SD, data from n = 32 fluorescent cells, n = 8 non-fluorescent cells, obtained from 4 independent videos)

    Journal: Journal of Nanobiotechnology

    Article Title: Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles

    doi: 10.1186/s12951-018-0401-y

    Figure Lengend Snippet: a1 Representative images depicted from live cell video after nine different time-points. The images are part of a video provided in Additional file 2 : Movie S1. DC2.4 cells were incubated with mRNA:FA-LPNs for a complete time duration of 4 h and with an interval of 3 min/image. Green dots on the images represent the fluorescence signal of labeled LPNs, while the cells signaling in red are the ones with successful mCherry expression. Scale bar = 100 µm. a2 Time-dependent change of the red fluorescence signal resulting from transfected cells and non-transfected, which are correlated with a3 green fluorescence signal of labeled nanoparticles. The tendency of each single cell being transfected is independent of the NPs uptake, as no significant difference between the uptake-behavior of transfected and non-transfected cells was observable. λ em = peak emission wavelength, MFI mean fluorescence intensity (mean ± SD, data from n = 32 fluorescent cells, n = 8 non-fluorescent cells, obtained from 4 independent videos)

    Article Snippet: The encapsulation efficiency (%EE) of bound mRNA:LPNs and mRNA:CS-PLGA NPs was evaluated indirectly by pelleting all samples down at 24,400g for 30 min and determining the concentration of unbound mRNA in the supernatant by measuring absorbance at 260/280 nm with a NanoDrop Spectrophotometer.

    Techniques: Incubation, Fluorescence, Labeling, Expressing, Transfection

    Representative confocal images of DC2.4 cells transfected with both mRNA complexed NPs and by using jetPRIME ® as a positive control, naked mRNA as negative control. Transfection was analyzed with CLSM a 24 h and b 48 h post-transfection. Red fluorescence reveals cells successfully transfected with the nanoparticles while their morphology remains consistent with non-transfected cells (staining: green: cell membrane; blue: cell nucleus; scale bar: 50 μm)

    Journal: Journal of Nanobiotechnology

    Article Title: Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles

    doi: 10.1186/s12951-018-0401-y

    Figure Lengend Snippet: Representative confocal images of DC2.4 cells transfected with both mRNA complexed NPs and by using jetPRIME ® as a positive control, naked mRNA as negative control. Transfection was analyzed with CLSM a 24 h and b 48 h post-transfection. Red fluorescence reveals cells successfully transfected with the nanoparticles while their morphology remains consistent with non-transfected cells (staining: green: cell membrane; blue: cell nucleus; scale bar: 50 μm)

    Article Snippet: The encapsulation efficiency (%EE) of bound mRNA:LPNs and mRNA:CS-PLGA NPs was evaluated indirectly by pelleting all samples down at 24,400g for 30 min and determining the concentration of unbound mRNA in the supernatant by measuring absorbance at 260/280 nm with a NanoDrop Spectrophotometer.

    Techniques: Transfection, Positive Control, Negative Control, Confocal Laser Scanning Microscopy, Fluorescence, Staining

    Quantification of cellular NP association for fluoresceinamine (FA) labeled blank ( a1 , a2 ) and mRNA complexed nanoparticles ( b1 , b2 ) tested in DC2.4 cells. NPs were incubated for 2 h and 4 h at different concentrations (20, 40 and 60 µg/mL corresponding to the weight ratios 1:10, 1:20 and 1:30 used for transfection). a1 , b1 Green NP fluorescent signal quantified by flow cytometry. Blank LPNs tend to show more uptake then blank CS-PLGA NPs, whereas for mRNA-loaded nanoparticles the opposite was observed. None of the samples showed a significant difference between 2 and 4 h incubation. a2 , b2 Representative graphs obtained for NP samples after 4 h incubation. N = 4, mean ± SD

    Journal: Journal of Nanobiotechnology

    Article Title: Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles

    doi: 10.1186/s12951-018-0401-y

    Figure Lengend Snippet: Quantification of cellular NP association for fluoresceinamine (FA) labeled blank ( a1 , a2 ) and mRNA complexed nanoparticles ( b1 , b2 ) tested in DC2.4 cells. NPs were incubated for 2 h and 4 h at different concentrations (20, 40 and 60 µg/mL corresponding to the weight ratios 1:10, 1:20 and 1:30 used for transfection). a1 , b1 Green NP fluorescent signal quantified by flow cytometry. Blank LPNs tend to show more uptake then blank CS-PLGA NPs, whereas for mRNA-loaded nanoparticles the opposite was observed. None of the samples showed a significant difference between 2 and 4 h incubation. a2 , b2 Representative graphs obtained for NP samples after 4 h incubation. N = 4, mean ± SD

    Article Snippet: The encapsulation efficiency (%EE) of bound mRNA:LPNs and mRNA:CS-PLGA NPs was evaluated indirectly by pelleting all samples down at 24,400g for 30 min and determining the concentration of unbound mRNA in the supernatant by measuring absorbance at 260/280 nm with a NanoDrop Spectrophotometer.

    Techniques: Labeling, Incubation, Transfection, Flow Cytometry, Cytometry

    The kinetics of transfection for both mRNA-loaded NPs was quantified using flow cytometry, which indicated a significant difference between a1 mRNA:LPNs over a2 mRNA:CS-PLGA NPs, while mRNA:LPNs with a ratio of 1:20 and 1:30 elucidated a significant higher transfection rate over 1:10. a3 Control samples for transfection studies were JetPRIME ® as positive control and naked mRNA as negative control. (Note the enlarged y-axis, N = 4, mean ± SD.) a4 Representative graphs for evaluated time-points demonstrate a strong fluorescence shift for mRNA:LPNs with an increment in time and hence a higher transgene expression

    Journal: Journal of Nanobiotechnology

    Article Title: Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles

    doi: 10.1186/s12951-018-0401-y

    Figure Lengend Snippet: The kinetics of transfection for both mRNA-loaded NPs was quantified using flow cytometry, which indicated a significant difference between a1 mRNA:LPNs over a2 mRNA:CS-PLGA NPs, while mRNA:LPNs with a ratio of 1:20 and 1:30 elucidated a significant higher transfection rate over 1:10. a3 Control samples for transfection studies were JetPRIME ® as positive control and naked mRNA as negative control. (Note the enlarged y-axis, N = 4, mean ± SD.) a4 Representative graphs for evaluated time-points demonstrate a strong fluorescence shift for mRNA:LPNs with an increment in time and hence a higher transgene expression

    Article Snippet: The encapsulation efficiency (%EE) of bound mRNA:LPNs and mRNA:CS-PLGA NPs was evaluated indirectly by pelleting all samples down at 24,400g for 30 min and determining the concentration of unbound mRNA in the supernatant by measuring absorbance at 260/280 nm with a NanoDrop Spectrophotometer.

    Techniques: Transfection, Flow Cytometry, Cytometry, Positive Control, Negative Control, Fluorescence, Expressing

    Schematic illustration of all nanoparticles used in this study. Both, LPNs and CS-PLGA NPs either complexed with mRNA or/and labeled with fluoresceinamine are used to quantify their uptake behavior and transfection efficiency in a dendritic cell line (DC2.4)

    Journal: Journal of Nanobiotechnology

    Article Title: Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles

    doi: 10.1186/s12951-018-0401-y

    Figure Lengend Snippet: Schematic illustration of all nanoparticles used in this study. Both, LPNs and CS-PLGA NPs either complexed with mRNA or/and labeled with fluoresceinamine are used to quantify their uptake behavior and transfection efficiency in a dendritic cell line (DC2.4)

    Article Snippet: The encapsulation efficiency (%EE) of bound mRNA:LPNs and mRNA:CS-PLGA NPs was evaluated indirectly by pelleting all samples down at 24,400g for 30 min and determining the concentration of unbound mRNA in the supernatant by measuring absorbance at 260/280 nm with a NanoDrop Spectrophotometer.

    Techniques: Labeling, Transfection

    Gel retardation assay of mRNA complexed with different nanoparticles (NPs) at various ratios: a LPNs and b CS-PLGA NPs. Both images indicate mRNA binding to NPs and their appropriate release using heparin

    Journal: Journal of Nanobiotechnology

    Article Title: Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles

    doi: 10.1186/s12951-018-0401-y

    Figure Lengend Snippet: Gel retardation assay of mRNA complexed with different nanoparticles (NPs) at various ratios: a LPNs and b CS-PLGA NPs. Both images indicate mRNA binding to NPs and their appropriate release using heparin

    Article Snippet: The encapsulation efficiency (%EE) of bound mRNA:LPNs and mRNA:CS-PLGA NPs was evaluated indirectly by pelleting all samples down at 24,400g for 30 min and determining the concentration of unbound mRNA in the supernatant by measuring absorbance at 260/280 nm with a NanoDrop Spectrophotometer.

    Techniques: Electrophoretic Mobility Shift Assay, Binding Assay

    Morphology of mRNA-loaded LPNs ( a1 , a2 ) and CS-PLGA NPs ( b1 , b2 ) visualized using SEM and TEM. a1 , b1 SEM images show a smooth, spherical morphology of the mRNA-loaded nanoparticles and a2 , b2 TEM images show the core–shell structure of the particles after staining with 0.5% (w/V) PTA

    Journal: Journal of Nanobiotechnology

    Article Title: Kinetics of mRNA delivery and protein translation in dendritic cells using lipid-coated PLGA nanoparticles

    doi: 10.1186/s12951-018-0401-y

    Figure Lengend Snippet: Morphology of mRNA-loaded LPNs ( a1 , a2 ) and CS-PLGA NPs ( b1 , b2 ) visualized using SEM and TEM. a1 , b1 SEM images show a smooth, spherical morphology of the mRNA-loaded nanoparticles and a2 , b2 TEM images show the core–shell structure of the particles after staining with 0.5% (w/V) PTA

    Article Snippet: The encapsulation efficiency (%EE) of bound mRNA:LPNs and mRNA:CS-PLGA NPs was evaluated indirectly by pelleting all samples down at 24,400g for 30 min and determining the concentration of unbound mRNA in the supernatant by measuring absorbance at 260/280 nm with a NanoDrop Spectrophotometer.

    Techniques: Transmission Electron Microscopy, Staining

    Hematopoietic recovery following infusion of ZFN mRNA-electroporated HSPCs.

    Journal: Blood

    Article Title: Long-term multilineage engraftment of autologous genome-edited hematopoietic stem cells in nonhuman primates

    doi: 10.1182/blood-2015-09-672337

    Figure Lengend Snippet: Hematopoietic recovery following infusion of ZFN mRNA-electroporated HSPCs.

    Article Snippet: ZFN messenger RNA (mRNA) (TriLink BioTechnologies) was added to cells resuspended to 1 × 107 cells/mL in Cytoporation Media T (Harvard Apparatus, Holliston, MA) at a final concentration of 125 μg/mL for each ZFN mRNA.

    Techniques:

    Gene expression changes in CD28- vs CD86-silenced cells are consistent with regulation of both distinct and common pathways, including expression of IRF4. (A) Gene set enrichment analysis showing upregulated (top) and downregulated (bottom) gene sets in shCD28- (blue) and shCD86-treated (red) myeloma cells. For each gene set, the enrichment score is shown above the ranked change in gene expression, where genes that overlap the gene set are denoted by blue and red ticks for shCD28 and shCD86, respectively. (B) qRT-PCR showing IRF4 mRNA levels when CD28 or CD86 was silenced in MM.1s or 8226 cells. (C) Representative western blots showing IRF4 levels with silencing of CD28 or CD86 in cell lines indicated at different time points. (D) Integrin levels were measured via qRT-PCR ( ITGB7 and ITGB1 ; left). Representative histograms showing ITGβ1 and ITGβ7 on day 4 after shRNA treatment (right). For qRT-PCR, all data are normalized to β-actin as an endogenous control and then compared relative to mRNA levels in pLKO.1 empty vector control. RNA was extracted at day 3 postinfection. Data shown are mean ± SEM of at least 3 independent experiments. Histograms showing surface levels of indicated molecules. Gray histograms at left represent unstained or isotype controls. Flow cytometry data shown are from day 4 postinfection with lentiviral vectors. Flow cytometry and western blot data shown are representative of at least 3 independent experiments. (E) Cell adhesion 3 days postinfection with lentivirus containing the indicated shRNA. Myeloma cells stained with calcein AM were cocultured with HS-5 cells for 2 hours. Fluorescence was measured (485/528 emission/excitation) with BioTek Synergy H1 multiwell plate reader, and data are presented as fluorescence relative to pLKO.1 controls (mean ± SEM). Prewash readings were taken to ensure similar numbers of live cells were added to each well. All samples were plated in triplicate. Data are mean of 3 independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GX, gene expression changes; RFU, relative fluorescence unit.

    Journal: Blood Advances

    Article Title: CD86 regulates myeloma cell survival

    doi: 10.1182/bloodadvances.2017011601

    Figure Lengend Snippet: Gene expression changes in CD28- vs CD86-silenced cells are consistent with regulation of both distinct and common pathways, including expression of IRF4. (A) Gene set enrichment analysis showing upregulated (top) and downregulated (bottom) gene sets in shCD28- (blue) and shCD86-treated (red) myeloma cells. For each gene set, the enrichment score is shown above the ranked change in gene expression, where genes that overlap the gene set are denoted by blue and red ticks for shCD28 and shCD86, respectively. (B) qRT-PCR showing IRF4 mRNA levels when CD28 or CD86 was silenced in MM.1s or 8226 cells. (C) Representative western blots showing IRF4 levels with silencing of CD28 or CD86 in cell lines indicated at different time points. (D) Integrin levels were measured via qRT-PCR ( ITGB7 and ITGB1 ; left). Representative histograms showing ITGβ1 and ITGβ7 on day 4 after shRNA treatment (right). For qRT-PCR, all data are normalized to β-actin as an endogenous control and then compared relative to mRNA levels in pLKO.1 empty vector control. RNA was extracted at day 3 postinfection. Data shown are mean ± SEM of at least 3 independent experiments. Histograms showing surface levels of indicated molecules. Gray histograms at left represent unstained or isotype controls. Flow cytometry data shown are from day 4 postinfection with lentiviral vectors. Flow cytometry and western blot data shown are representative of at least 3 independent experiments. (E) Cell adhesion 3 days postinfection with lentivirus containing the indicated shRNA. Myeloma cells stained with calcein AM were cocultured with HS-5 cells for 2 hours. Fluorescence was measured (485/528 emission/excitation) with BioTek Synergy H1 multiwell plate reader, and data are presented as fluorescence relative to pLKO.1 controls (mean ± SEM). Prewash readings were taken to ensure similar numbers of live cells were added to each well. All samples were plated in triplicate. Data are mean of 3 independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GX, gene expression changes; RFU, relative fluorescence unit.

    Article Snippet: RNA was isolated using the Qiagen RNEasy Kit as described and quality control tested at the Emory Integrated Genomics Core, and 1 µg was sent to Hudson α for RNA sequencing (RNA-seq) library construction using the Illumina TruSeq messenger RNA (mRNA) protocol.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, shRNA, Plasmid Preparation, Flow Cytometry, Cytometry, Staining, Fluorescence

    Quantitative reverse transcription-polymerase chain reaction gene expression profile. Histogram showing the gene expression analysis for untreated HFLS, untreated HFLS-RA, and HFLS-RA treated with MTX IC 50 concentrations (mean ± SD [n = 3]). HFLS indicates human fibroblast-like synoviocytes; mRNA, messenger RNA; RA, rheumatoid arthritis.

    Journal: Biomarker Insights

    Article Title: Identifying Reliable Diagnostic/Predictive Biomarkers for Rheumatoid Arthritis

    doi: 10.1177/1177271918801005

    Figure Lengend Snippet: Quantitative reverse transcription-polymerase chain reaction gene expression profile. Histogram showing the gene expression analysis for untreated HFLS, untreated HFLS-RA, and HFLS-RA treated with MTX IC 50 concentrations (mean ± SD [n = 3]). HFLS indicates human fibroblast-like synoviocytes; mRNA, messenger RNA; RA, rheumatoid arthritis.

    Article Snippet: Messenger RNA isolation, reverse transcription and quantitative reverse transcription-polymerase chain reaction The messenger RNA (mRNA) isolation kit (Roche, West Sussex, UK) was used to extract approximately 1 pg/cell mRNA.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

    RAGE PTK profiling of different human tissues. Poly(A) mRNA obtained from Clontech was amplified by RT–PCR as described in Materials and Methods. The 150–170 bp amplicon was purified and used for Mwo I digestion. The completed digested products were separated by a denaturing sequencing gel. Specific PTKs were identified by the digested fragment sizes as indicated.

    Journal: British Journal of Cancer

    Article Title: Tyrosine-kinase expression profiles in human gastric cancer cell lines and their modulations with retinoic acids

    doi: 10.1038/sj.bjc.6600821

    Figure Lengend Snippet: RAGE PTK profiling of different human tissues. Poly(A) mRNA obtained from Clontech was amplified by RT–PCR as described in Materials and Methods. The 150–170 bp amplicon was purified and used for Mwo I digestion. The completed digested products were separated by a denaturing sequencing gel. Specific PTKs were identified by the digested fragment sizes as indicated.

    Article Snippet: RT–PCR amplification and RAGE profiling of human tissues Poly(A) selected mRNA were purchased from Clontech, including eight adult tissues (brain, kidney, liver, lung, pancreas, placenta, small intestine and stomach) and four foetal tissues (foetal brain, foetal kidney, foetal liver and foetal lung).

    Techniques: Amplification, Reverse Transcription Polymerase Chain Reaction, Purification, Sequencing

    Northern blot analysis of ER mRNAs in tissues of mature female ( n = 3 for α and γ; n = 2 for β) and mature male ( n = 2, testes only) Atlantic croaker. L, liver; O, ovary; B, brain; M, muscle; T, testes. Amounts of mRNA loaded per tissue type: liver, ovary, testes = 3 μg per lane; brain = 2 μg per lane; muscle = 1.3 μg per lane. Lanes shown are one of three loads of mRNA from the same fish. ( A ) Atlantic croaker ERα (acERα). ( B ) Atlantic croaker ERβ (acERβ). ( C ) Atlantic croaker ERγ (acERγ). The size (kb) of each transcript is indicated. RNA size markers (mk) are shown (Millennium Markers, Ambion).

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

    Article Title: Identification of a third distinct estrogen receptor and reclassification of estrogen receptors in teleosts

    doi:

    Figure Lengend Snippet: Northern blot analysis of ER mRNAs in tissues of mature female ( n = 3 for α and γ; n = 2 for β) and mature male ( n = 2, testes only) Atlantic croaker. L, liver; O, ovary; B, brain; M, muscle; T, testes. Amounts of mRNA loaded per tissue type: liver, ovary, testes = 3 μg per lane; brain = 2 μg per lane; muscle = 1.3 μg per lane. Lanes shown are one of three loads of mRNA from the same fish. ( A ) Atlantic croaker ERα (acERα). ( B ) Atlantic croaker ERβ (acERβ). ( C ) Atlantic croaker ERγ (acERγ). The size (kb) of each transcript is indicated. RNA size markers (mk) are shown (Millennium Markers, Ambion).

    Article Snippet: Total liver RNA was extracted ( ) to isolate mRNA (Promega PolyATract System).

    Techniques: Northern Blot, Fluorescence In Situ Hybridization

    Stimulation of HIEC/IEC-6 cells with LPS induced TNFα, as demonstrated by RT-PCR (A). Already after a stimulation period of three hours TNFα mRNA transcripts were observed in both IEC models. The expression of G3PDH was used as house keeping gene. The translation into the protein product and subsequent secretion of TNFα into the culture medium was analysed in HIEC using a high sensitivity ELISA (B). TNFα was produced in a dose dependent manner after stimulation with LPS. CHX treatment completely suppressed the secretion of TNFα. In keeping, functional experiments with CHX showed in HIEC that the proliferation rate after LPS stimulation remained unchanged once TNFα production was suppressed (C). On the other hand, addition of TNFα in the presence of CHX was still able to suppress HIEC proliferation. Concentrations (x axis) for CHX or LPS in μg/ml, for TNFα in ng/ml. The concentrations of CHX used together with LPS or TNFα were 1 μg/ml.

    Journal: Gut

    Article Title: Lipopolysaccharide modulation of normal enterocyte turnover by toll-like receptors is mediated by endogenously produced tumour necrosis factor ?

    doi:

    Figure Lengend Snippet: Stimulation of HIEC/IEC-6 cells with LPS induced TNFα, as demonstrated by RT-PCR (A). Already after a stimulation period of three hours TNFα mRNA transcripts were observed in both IEC models. The expression of G3PDH was used as house keeping gene. The translation into the protein product and subsequent secretion of TNFα into the culture medium was analysed in HIEC using a high sensitivity ELISA (B). TNFα was produced in a dose dependent manner after stimulation with LPS. CHX treatment completely suppressed the secretion of TNFα. In keeping, functional experiments with CHX showed in HIEC that the proliferation rate after LPS stimulation remained unchanged once TNFα production was suppressed (C). On the other hand, addition of TNFα in the presence of CHX was still able to suppress HIEC proliferation. Concentrations (x axis) for CHX or LPS in μg/ml, for TNFα in ng/ml. The concentrations of CHX used together with LPS or TNFα were 1 μg/ml.

    Article Snippet: mRNA was isolated from HIEC and IEC-6 cells at different time points (one to nine hours) after LPS stimulation using a Quickprep mRNA micro purification kit (Amersham Pharmacia Biotech, Freiburg, Germany).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Enzyme-linked Immunosorbent Assay, Produced, Functional Assay

    TLR expression in HIEC: RT-PCR analysis clearly showed TLR2 and TLR4 mRNA transcripts in unstimulated HIEC as well as Caco-2 and HT29 cells (A). Subsequent isolation and sequencing confirmed the identity of TLR2 and TLR4. In addition, all cell lines expressed MD2, a costimulatory molecule associated with TLR4. Western blot analysis (B) confirmed that HIEC express TLR2 (86 kDa) as well as TLR4 (88 kDa). Caco-2 cells served as positive control. In addition, immunofluorescence analysis (C) revealed a clear signal of membrane expressed TLR2 and TLR4 on native HIEC. The regulation of the expression of TLR2 and TLR4 by LPS was analysed by flow cytometry (D). TLR4 expression was higher in unstimulated HIEC compared with TLR2. LPS (10 μg/ml, 48 hours) significantly reduced TLR4 expression without changing TLR2 expression in HIEC. One of three representative experiments is shown.

    Journal: Gut

    Article Title: Lipopolysaccharide modulation of normal enterocyte turnover by toll-like receptors is mediated by endogenously produced tumour necrosis factor ?

    doi:

    Figure Lengend Snippet: TLR expression in HIEC: RT-PCR analysis clearly showed TLR2 and TLR4 mRNA transcripts in unstimulated HIEC as well as Caco-2 and HT29 cells (A). Subsequent isolation and sequencing confirmed the identity of TLR2 and TLR4. In addition, all cell lines expressed MD2, a costimulatory molecule associated with TLR4. Western blot analysis (B) confirmed that HIEC express TLR2 (86 kDa) as well as TLR4 (88 kDa). Caco-2 cells served as positive control. In addition, immunofluorescence analysis (C) revealed a clear signal of membrane expressed TLR2 and TLR4 on native HIEC. The regulation of the expression of TLR2 and TLR4 by LPS was analysed by flow cytometry (D). TLR4 expression was higher in unstimulated HIEC compared with TLR2. LPS (10 μg/ml, 48 hours) significantly reduced TLR4 expression without changing TLR2 expression in HIEC. One of three representative experiments is shown.

    Article Snippet: mRNA was isolated from HIEC and IEC-6 cells at different time points (one to nine hours) after LPS stimulation using a Quickprep mRNA micro purification kit (Amersham Pharmacia Biotech, Freiburg, Germany).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Sequencing, Western Blot, Positive Control, Immunofluorescence, Flow Cytometry, Cytometry

    Bone morphogenetic protein (BMP) 6 suppressed secretory leukocyte peptidase inhibitor (SLPI) and whey acid protein (WAP) 14 mRNA expression in cultured human GC. Cultured human granulosa-lutein cells (GCs) were stimulated with BMP-6 (100 ng/mL) for 24 hours. Total RNA was extracted from the cells and subjected to real-time PCR to determine the expression of SLPI and WAP14. Data were normalized by GAPDH mRNA levels to show the relative abundance. Data from 4 different experiments were combined and shown as the mean ± SEM relative to an adjusted value of 1.0 for the mean value of the each control. * P

    Journal: Reproductive Sciences

    Article Title: The Role of Bone Morphogenetic Protein 6 in Accumulation and Regulation of Neutrophils in the Human Ovary

    doi: 10.1177/1933719113518988

    Figure Lengend Snippet: Bone morphogenetic protein (BMP) 6 suppressed secretory leukocyte peptidase inhibitor (SLPI) and whey acid protein (WAP) 14 mRNA expression in cultured human GC. Cultured human granulosa-lutein cells (GCs) were stimulated with BMP-6 (100 ng/mL) for 24 hours. Total RNA was extracted from the cells and subjected to real-time PCR to determine the expression of SLPI and WAP14. Data were normalized by GAPDH mRNA levels to show the relative abundance. Data from 4 different experiments were combined and shown as the mean ± SEM relative to an adjusted value of 1.0 for the mean value of the each control. * P

    Article Snippet: For the quantification of various messenger RNA (mRNA) levels, real-time polymerase chain reaction (PCR) was performed using a LightCycler (Roche Diagnostic GmbH, Mannheim, Germany), according to the manufacturer’s instructions.

    Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction

    Bone morphogenetic protein (BMP)-6-induced growth-regulated oncogene (GRO) α expression in human granulosa-lutein cells (GCs). Cultured human GCs were stimulated with BMP-6 (0-300 ng/mL) for 24 hours (A) or with BMP-6 (100 ng/mL) for 3 to 48 hours (B). Total RNA was extracted from the cells and subjected to real-time PCR to determine the GRO-α mRNA levels. Data were normalized by GAPDH mRNA levels to show the relative abundance to the control level. Data from at least 4 different experiments were combined and shown as the mean ± SEM relative to an adjusted value of 1.0 for the mean value of the control. * P

    Journal: Reproductive Sciences

    Article Title: The Role of Bone Morphogenetic Protein 6 in Accumulation and Regulation of Neutrophils in the Human Ovary

    doi: 10.1177/1933719113518988

    Figure Lengend Snippet: Bone morphogenetic protein (BMP)-6-induced growth-regulated oncogene (GRO) α expression in human granulosa-lutein cells (GCs). Cultured human GCs were stimulated with BMP-6 (0-300 ng/mL) for 24 hours (A) or with BMP-6 (100 ng/mL) for 3 to 48 hours (B). Total RNA was extracted from the cells and subjected to real-time PCR to determine the GRO-α mRNA levels. Data were normalized by GAPDH mRNA levels to show the relative abundance to the control level. Data from at least 4 different experiments were combined and shown as the mean ± SEM relative to an adjusted value of 1.0 for the mean value of the control. * P

    Article Snippet: For the quantification of various messenger RNA (mRNA) levels, real-time polymerase chain reaction (PCR) was performed using a LightCycler (Roche Diagnostic GmbH, Mannheim, Germany), according to the manufacturer’s instructions.

    Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction

    Effects of unincorporated dye precursors on fluorescent nucleic acid analyses. A, B: Comparison of DNA sequencing analyses performed without (A) or with (B) purification of unincorporated dye terminators. Sequencing reactions were prepared from 250 ng pGEMZf(+) plasmid and 4 pmol M13 −47 primer, using BigDye version 3.0 at a 2:10 (μL BigDye:μL total volume) reaction mixture with Better Buffer diluent. Purification B was performed with the RapXtract II Kit. DNA sequences were analyzed on an ABI PRISM 3100 Genetic Analyzer using POP6 polymer and injection parameters recommended for RapXtract Kits (2 kV, 45 s). C, D: Comparison of cDNA-microarray hybridization analyses performed without (C) or with (D) purification of unincorporated dye-labeled nucleotides from the labeling reaction. Labeled cDNA was prepared from 1 μg Saccharomyces cerevisiae mRNA using the CyScribe First Strand Labeling Kit and Cy3-labeled dUTP. Purification D was performed with the RapXtract Fluorescent Nucleic Acid Purification Kit. The cDNA was then hybridized to a yeast collage array at 67°C for 12 h. The microarray was visualized on a ScanArray Lite scanner at a laser output of 80%, PMT gain of 80%, and spatial resolution of 50 μm.

    Journal: Journal of Biomolecular Techniques : JBT

    Article Title: A Rapid Method for Manual or Automated Purification of Fluorescently Labeled Nucleic Acids for Sequencing, Genotyping, and Microarrays

    doi:

    Figure Lengend Snippet: Effects of unincorporated dye precursors on fluorescent nucleic acid analyses. A, B: Comparison of DNA sequencing analyses performed without (A) or with (B) purification of unincorporated dye terminators. Sequencing reactions were prepared from 250 ng pGEMZf(+) plasmid and 4 pmol M13 −47 primer, using BigDye version 3.0 at a 2:10 (μL BigDye:μL total volume) reaction mixture with Better Buffer diluent. Purification B was performed with the RapXtract II Kit. DNA sequences were analyzed on an ABI PRISM 3100 Genetic Analyzer using POP6 polymer and injection parameters recommended for RapXtract Kits (2 kV, 45 s). C, D: Comparison of cDNA-microarray hybridization analyses performed without (C) or with (D) purification of unincorporated dye-labeled nucleotides from the labeling reaction. Labeled cDNA was prepared from 1 μg Saccharomyces cerevisiae mRNA using the CyScribe First Strand Labeling Kit and Cy3-labeled dUTP. Purification D was performed with the RapXtract Fluorescent Nucleic Acid Purification Kit. The cDNA was then hybridized to a yeast collage array at 67°C for 12 h. The microarray was visualized on a ScanArray Lite scanner at a laser output of 80%, PMT gain of 80%, and spatial resolution of 50 μm.

    Article Snippet: Reactions were prepared using a CyScribe First Strand Labeling Kit (Amersham Biosciences, Piscataway, NJ) and 1 μg Saccharomyces cerevisiae messenger RNA (mRNA) (BD Biosciences Clontech, Palo Alto, CA) or 2 μg mouse brain RNA (Ambion, Austin, TX).

    Techniques: DNA Sequencing, Purification, Sequencing, Plasmid Preparation, Injection, Microarray, Hybridization, Labeling, Nucleic Acid Purification

    Mouse multiple tissue blot analysis of the expression of mTR2R1 mRNA. (a) Probe with the entire 3.5 kb mTR2R1 cDNA in clone pBS35. (b) Probe with the 3′ noncoding region of mTR2R1 in clone pBS35.

    Journal: Gene Expression

    Article Title: Molecular Cloning of a Novel Member of the Nuclear Receptor Superfamily Related to the Orphan Receptor, TR2

    doi:

    Figure Lengend Snippet: Mouse multiple tissue blot analysis of the expression of mTR2R1 mRNA. (a) Probe with the entire 3.5 kb mTR2R1 cDNA in clone pBS35. (b) Probe with the 3′ noncoding region of mTR2R1 in clone pBS35.

    Article Snippet: A λ DR2 cDNA library derived from human prostate mRNA was purchased from Clontech Laboratory, Inc. (Palo Alto, CA).

    Techniques: Expressing