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  • 99
    Thermo Fisher power sybr green pcr master mix
    Effect of CIB1 knockdown on de novo KSHV infection. (A) Untransduced, control or CIB1-shRNA transduced HMVEC-d cells were infected with KSHV (20 DNA copies/cell). At 24 h p.i., cells were harvested, total RNA was isolated, and viral gene expression was determined by real-time <t>RT-PCR</t> with KSHV ORF73 gene specific primers. Results are presented as percentage of inhibition of KSHV gene expression by sh-CIB1 or control compared with the infected untransduced cells. ***P = 0.0001. (B) (i) Control or CIB1-shRNA transduced HMVEC-d cells were mock or KSHV infected (20 DNA copies/cell) for 2 h at 37°C, washed to remove unbound viruses, and continued to culture for another 46 h. At 48 h p.i., cells were processed for immunofluorescence analysis using mouse anti-LANA-1 antibodies and co-stained with DAPI. Representative images are shown. (B) (ii) The percentage of cells observed positive for characteristic punctate LANA-1 staining in IFA is presented graphically. A minimum of three independent fields, each with at least 10 cells were chosen. Error bars show ± SD. (C) Control or CIB1-shRNA transduced HMVEC-d cells were mock or HSV-1 infected (3 pfu/cell) for 2 h at 37°C, washed to remove unbound viruses, and incubated for another 6 h. At 8 h p.i., cells were harvested, total RNA was isolated, and HSV-1 gene expression was quantified by <t>SYBR</t> green q-PCR method with ICP0 and ICP4 gene specific primers. Results are presented as fold HSV-1 gene expression normalized to internal tubulin control. Error bars show ± SD. (D) 293 cells were either mock-transfected or transfected with CIB1 overexpressing vector pcDNA-CIB1-Myc using lipofectamine. At 48 h post-transfection, CIB1 overexpression was examined by Western blotting with rabbit anti-CIB1 and mouse anti-Myc antibodies. Actin was used as loading control. (E) At 48 h post-transfection, transfected 293 cells were infected with KSHV (20 DNA copies/cell) for 2 h at 37°C for entry experiments. Post-washing, total DNA was isolated and KSHV entry was determined by real-time DNA-PCR for the ORF73 gene. Each reaction was done in triplicate and each bar represents the average ± SD of three independent experiments. Results are presented as percentage increase in KSHV DNA internalization in pcDNA-CIB1-Myc expressing cells compared with the control vector transfected cells, which is considered as 100%.
    Power Sybr Green Pcr Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 72942 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher power sybr green rna to ct 1 step kit
    Metanephric mesenchymal (MM) cell expression of <t>RNA</t> encoding mesenchymal and endothelial markers by RT-PCR. Total RNA was extracted from MM and ureteric bud (UB) cell lysates and reverse transcribed to synthesize cDNA with the use of the <t>SYBR</t> Green method. Ratio of cDNA transcript of marker: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown for each cell line. The results show high transcript levels of mesenchymal markers forkhead box D1 (Foxd1), glioma-associated oncogene homolog 1 (Gli-1), and ANG-1 ( A–C ), as well as endothelial markers aquaporin 1 (AQP1), Flt-1 (vascular endothelial growth factor receptor 1; VEGFR1), Tie-2 (angiopoietin receptor), and platelet endothelial cell adhesion molecule (PECAM) ( D–G ) in MM cells relative to UB cells. Data are expressed as means ± SEM. n = 3 independent experiments. ∗ P
    Power Sybr Green Rna To Ct 1 Step Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2838 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher power sybr green pcr master mix kit
    Metanephric mesenchymal (MM) cell expression of <t>RNA</t> encoding mesenchymal and endothelial markers by RT-PCR. Total RNA was extracted from MM and ureteric bud (UB) cell lysates and reverse transcribed to synthesize cDNA with the use of the <t>SYBR</t> Green method. Ratio of cDNA transcript of marker: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown for each cell line. The results show high transcript levels of mesenchymal markers forkhead box D1 (Foxd1), glioma-associated oncogene homolog 1 (Gli-1), and ANG-1 ( A–C ), as well as endothelial markers aquaporin 1 (AQP1), Flt-1 (vascular endothelial growth factor receptor 1; VEGFR1), Tie-2 (angiopoietin receptor), and platelet endothelial cell adhesion molecule (PECAM) ( D–G ) in MM cells relative to UB cells. Data are expressed as means ± SEM. n = 3 independent experiments. ∗ P
    Power Sybr Green Pcr Master Mix Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1797 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Intratracheal instillation of IL-36α increased the mRNA expression of proinflammatory mediators in the lungs of IL-1αβ −/− mice. A–F) Transcript expression of early response cytokines (TNFα and IL-36γ), the classical IL-1 receptor IL-1R1, the novel IL-1 cytokine cluster receptor IL-36R and the neutrophil specific chemokines CXCL1 and CXCL2 in the lungs of IL-1αβ −/− mice 24 h following a single i.t instillation of PBS or 10 µg IL-36α. Transcript expression was evaluated by <t>SYBR-Green</t> based quantitative real-time <t>PCR.</t> G–H) Protein expression of TNFα and CXCL1 in the BAL fluid recovered from IL-1αβ −/− mice 24 h following a single i.t instillation of PBS or 10 µg IL-36α. Protein expression was quantified by multiplexed cytometric bead arrays. *Indicates significant differences ( P
    Power Sybr Green Cells To Ct Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1229 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PEMF stimulated expression of miR21-5p in differentiated human osteoblasts. Total RNAs from control or PEMF-treated hBMSCs of females (24 × 2, 27, 29, and 30 years old, n = 5) at day 23 of differentiation or (31, 36, 58, and 68 years old) at day 23 or 33 of differentiation were isolated and subjected to RT-qPCR using the miScript II kit with miScript HiSpec Buffer and miScript <t>SYBR</t> Green <t>PCR</t> Kit. snoR10-1 was used to normalize miR21-5p expression and the expression is shown as a percentage of the relevant control samples. ∗ indicates significant increase compared to control using one-way ANOVA.
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    PEMF stimulated expression of miR21-5p in differentiated human osteoblasts. Total RNAs from control or PEMF-treated hBMSCs of females (24 × 2, 27, 29, and 30 years old, n = 5) at day 23 of differentiation or (31, 36, 58, and 68 years old) at day 23 or 33 of differentiation were isolated and subjected to RT-qPCR using the miScript II kit with miScript HiSpec Buffer and miScript <t>SYBR</t> Green <t>PCR</t> Kit. snoR10-1 was used to normalize miR21-5p expression and the expression is shown as a percentage of the relevant control samples. ∗ indicates significant increase compared to control using one-way ANOVA.
    Power Sybr Green Pcr Master Mix, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 355 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PEMF stimulated expression of miR21-5p in differentiated human osteoblasts. Total RNAs from control or PEMF-treated hBMSCs of females (24 × 2, 27, 29, and 30 years old, n = 5) at day 23 of differentiation or (31, 36, 58, and 68 years old) at day 23 or 33 of differentiation were isolated and subjected to RT-qPCR using the miScript II kit with miScript HiSpec Buffer and miScript <t>SYBR</t> Green <t>PCR</t> Kit. snoR10-1 was used to normalize miR21-5p expression and the expression is shown as a percentage of the relevant control samples. ∗ indicates significant increase compared to control using one-way ANOVA.
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    Image Search Results


    Effect of CIB1 knockdown on de novo KSHV infection. (A) Untransduced, control or CIB1-shRNA transduced HMVEC-d cells were infected with KSHV (20 DNA copies/cell). At 24 h p.i., cells were harvested, total RNA was isolated, and viral gene expression was determined by real-time RT-PCR with KSHV ORF73 gene specific primers. Results are presented as percentage of inhibition of KSHV gene expression by sh-CIB1 or control compared with the infected untransduced cells. ***P = 0.0001. (B) (i) Control or CIB1-shRNA transduced HMVEC-d cells were mock or KSHV infected (20 DNA copies/cell) for 2 h at 37°C, washed to remove unbound viruses, and continued to culture for another 46 h. At 48 h p.i., cells were processed for immunofluorescence analysis using mouse anti-LANA-1 antibodies and co-stained with DAPI. Representative images are shown. (B) (ii) The percentage of cells observed positive for characteristic punctate LANA-1 staining in IFA is presented graphically. A minimum of three independent fields, each with at least 10 cells were chosen. Error bars show ± SD. (C) Control or CIB1-shRNA transduced HMVEC-d cells were mock or HSV-1 infected (3 pfu/cell) for 2 h at 37°C, washed to remove unbound viruses, and incubated for another 6 h. At 8 h p.i., cells were harvested, total RNA was isolated, and HSV-1 gene expression was quantified by SYBR green q-PCR method with ICP0 and ICP4 gene specific primers. Results are presented as fold HSV-1 gene expression normalized to internal tubulin control. Error bars show ± SD. (D) 293 cells were either mock-transfected or transfected with CIB1 overexpressing vector pcDNA-CIB1-Myc using lipofectamine. At 48 h post-transfection, CIB1 overexpression was examined by Western blotting with rabbit anti-CIB1 and mouse anti-Myc antibodies. Actin was used as loading control. (E) At 48 h post-transfection, transfected 293 cells were infected with KSHV (20 DNA copies/cell) for 2 h at 37°C for entry experiments. Post-washing, total DNA was isolated and KSHV entry was determined by real-time DNA-PCR for the ORF73 gene. Each reaction was done in triplicate and each bar represents the average ± SD of three independent experiments. Results are presented as percentage increase in KSHV DNA internalization in pcDNA-CIB1-Myc expressing cells compared with the control vector transfected cells, which is considered as 100%.

    Journal: PLoS Pathogens

    Article Title: CIB1 Synergizes with EphrinA2 to Regulate Kaposi's Sarcoma-Associated Herpesvirus Macropinocytic Entry in Human Microvascular Dermal Endothelial Cells

    doi: 10.1371/journal.ppat.1003941

    Figure Lengend Snippet: Effect of CIB1 knockdown on de novo KSHV infection. (A) Untransduced, control or CIB1-shRNA transduced HMVEC-d cells were infected with KSHV (20 DNA copies/cell). At 24 h p.i., cells were harvested, total RNA was isolated, and viral gene expression was determined by real-time RT-PCR with KSHV ORF73 gene specific primers. Results are presented as percentage of inhibition of KSHV gene expression by sh-CIB1 or control compared with the infected untransduced cells. ***P = 0.0001. (B) (i) Control or CIB1-shRNA transduced HMVEC-d cells were mock or KSHV infected (20 DNA copies/cell) for 2 h at 37°C, washed to remove unbound viruses, and continued to culture for another 46 h. At 48 h p.i., cells were processed for immunofluorescence analysis using mouse anti-LANA-1 antibodies and co-stained with DAPI. Representative images are shown. (B) (ii) The percentage of cells observed positive for characteristic punctate LANA-1 staining in IFA is presented graphically. A minimum of three independent fields, each with at least 10 cells were chosen. Error bars show ± SD. (C) Control or CIB1-shRNA transduced HMVEC-d cells were mock or HSV-1 infected (3 pfu/cell) for 2 h at 37°C, washed to remove unbound viruses, and incubated for another 6 h. At 8 h p.i., cells were harvested, total RNA was isolated, and HSV-1 gene expression was quantified by SYBR green q-PCR method with ICP0 and ICP4 gene specific primers. Results are presented as fold HSV-1 gene expression normalized to internal tubulin control. Error bars show ± SD. (D) 293 cells were either mock-transfected or transfected with CIB1 overexpressing vector pcDNA-CIB1-Myc using lipofectamine. At 48 h post-transfection, CIB1 overexpression was examined by Western blotting with rabbit anti-CIB1 and mouse anti-Myc antibodies. Actin was used as loading control. (E) At 48 h post-transfection, transfected 293 cells were infected with KSHV (20 DNA copies/cell) for 2 h at 37°C for entry experiments. Post-washing, total DNA was isolated and KSHV entry was determined by real-time DNA-PCR for the ORF73 gene. Each reaction was done in triplicate and each bar represents the average ± SD of three independent experiments. Results are presented as percentage increase in KSHV DNA internalization in pcDNA-CIB1-Myc expressing cells compared with the control vector transfected cells, which is considered as 100%.

    Article Snippet: Equal volumes of prepared cDNA were used for quantification of HSV-1 gene ICP0 and ICP4 transcripts with the Power SYBR Green PCR master mix (Applied biosystems) according to the manufacturer's protocol.

    Techniques: Infection, shRNA, Isolation, Expressing, Quantitative RT-PCR, Inhibition, Immunofluorescence, Staining, Incubation, SYBR Green Assay, Polymerase Chain Reaction, Transfection, Plasmid Preparation, Over Expression, Western Blot

    Promoter methylation is involved in β4 repression during TGFβ-induced EMT. (A,B) Methylation status of the entire CpG island in the β4 promoter in the absence (A) or presence (B) of TGFβ for 11 days. Each circle represents a CpG dinucleotide from the first (1) to the last (66) CpG of randomly selected clones that were sequenced. White circles, unmethylated CpG; black circles, methylated CpG. (C) Immunoblot analysis of β4 expression in the presence of TGFβ alone or TGFβ plus 5-aza for the times indicated. (D) Analysis of β4 mRNA expression by SYBR Green qRT-PCR in the absence of TGFβ (UNT), with TGFβ alone (TGFβ) or with TGFβ and 5-aza (5aza) for 48 hours. Results are reported as the relative expression normalized to that of GAPDH. (E) Bisulfite conversion and methylation analysis of integrin α6 promoter after TGFβ treatment for 11 days as described in A.

    Journal: Journal of Cell Science

    Article Title: Regulation of ?4-integrin expression by epigenetic modifications in the mammary gland and during the epithelial-to-mesenchymal transition

    doi: 10.1242/jcs.049148

    Figure Lengend Snippet: Promoter methylation is involved in β4 repression during TGFβ-induced EMT. (A,B) Methylation status of the entire CpG island in the β4 promoter in the absence (A) or presence (B) of TGFβ for 11 days. Each circle represents a CpG dinucleotide from the first (1) to the last (66) CpG of randomly selected clones that were sequenced. White circles, unmethylated CpG; black circles, methylated CpG. (C) Immunoblot analysis of β4 expression in the presence of TGFβ alone or TGFβ plus 5-aza for the times indicated. (D) Analysis of β4 mRNA expression by SYBR Green qRT-PCR in the absence of TGFβ (UNT), with TGFβ alone (TGFβ) or with TGFβ and 5-aza (5aza) for 48 hours. Results are reported as the relative expression normalized to that of GAPDH. (E) Bisulfite conversion and methylation analysis of integrin α6 promoter after TGFβ treatment for 11 days as described in A.

    Article Snippet: For quantitative RT-PCR (qRT-PCR), reverse-transcribed cDNA was used for real-time PCR using Power SYBR Green Master Mix (Applied Biosystems) with the HT7900 Fast Real Time PCR machine (Applied Biosystems).

    Techniques: Methylation, Clone Assay, Expressing, SYBR Green Assay, Quantitative RT-PCR

    TGFβ withdrawal reverses EMT and restores both E-cadherin and β4 integrin expression. (A) Phase-contrast photomicrographs of NMuMG cells either untreated (UNT), treated with TGFβ for 11 days (EMT) or treated with TGFβ for 11 days and subsequently in the absence of TGFβ for 13 days (MET). (B) Immunoblot analysis of E-cadherin (E-cad) and β4 integrin (β4) expression under conditions described in A. Tubulin or actin served as a loading control. (C) Analysis of β4 mRNA expression normalized to that of GAPDH in the cells described in A by SYBR Green qRT-PCR. (D) Extracts from the cells described in A were immunoprecipitated with either a nonspecific IgG or a monoclonal antibody against α6 (GOH3), and the immunoprecipitates were blotted with a polyclonal antibody against β4.

    Journal: Journal of Cell Science

    Article Title: Regulation of ?4-integrin expression by epigenetic modifications in the mammary gland and during the epithelial-to-mesenchymal transition

    doi: 10.1242/jcs.049148

    Figure Lengend Snippet: TGFβ withdrawal reverses EMT and restores both E-cadherin and β4 integrin expression. (A) Phase-contrast photomicrographs of NMuMG cells either untreated (UNT), treated with TGFβ for 11 days (EMT) or treated with TGFβ for 11 days and subsequently in the absence of TGFβ for 13 days (MET). (B) Immunoblot analysis of E-cadherin (E-cad) and β4 integrin (β4) expression under conditions described in A. Tubulin or actin served as a loading control. (C) Analysis of β4 mRNA expression normalized to that of GAPDH in the cells described in A by SYBR Green qRT-PCR. (D) Extracts from the cells described in A were immunoprecipitated with either a nonspecific IgG or a monoclonal antibody against α6 (GOH3), and the immunoprecipitates were blotted with a polyclonal antibody against β4.

    Article Snippet: For quantitative RT-PCR (qRT-PCR), reverse-transcribed cDNA was used for real-time PCR using Power SYBR Green Master Mix (Applied Biosystems) with the HT7900 Fast Real Time PCR machine (Applied Biosystems).

    Techniques: Expressing, SYBR Green Assay, Quantitative RT-PCR, Immunoprecipitation

    Inhibition of deacetylase activity fails to rescue the replication of pUL29/28 or pUL38-deficient viruses. (A) Trichostatin A does not rescue pUL29/28 or pUL38-deficient viruses. Fibroblasts were infected at a multiplicity of 0.1 pfu/cell using wild-type (BAD wt ) or inoculum containing equivalent numbers of viral genomes for BAD sub UL29 ( sub UL29) or BAD sub UL38 ( sub UL38) viruses. Cultures were treated with 300 nM TSA for the duration of the experiment and viral titers from culture supernatants were determined at 10 dpi. Data is from duplicate experiments. (B) BAD sub UL29 ( sub UL29) virus is deficient in immediate-early gene expression irrespective of treatment with TSA. Cells pretreated with 500 nM of TSA were infected using 0.1 pfu/cell of wild-type (BAD w t) or BAD sub UL29 ( sub UL29) virus as described above, and total RNA was harvested at the indicated times. Expression of IE1 RNA was determined by qRT-PCR using cDNA produced from DNase-treated RNA and random hexamers. Quantitation was completed with SYBR green and primers specific to exon 4 of IE1. Samples were normalized to cellular GAPDH RNA levels.

    Journal: PLoS Pathogens

    Article Title: Human Cytomegalovirus UL29/28 Protein Interacts with Components of the NuRD Complex Which Promote Accumulation of Immediate-Early RNA

    doi: 10.1371/journal.ppat.1000965

    Figure Lengend Snippet: Inhibition of deacetylase activity fails to rescue the replication of pUL29/28 or pUL38-deficient viruses. (A) Trichostatin A does not rescue pUL29/28 or pUL38-deficient viruses. Fibroblasts were infected at a multiplicity of 0.1 pfu/cell using wild-type (BAD wt ) or inoculum containing equivalent numbers of viral genomes for BAD sub UL29 ( sub UL29) or BAD sub UL38 ( sub UL38) viruses. Cultures were treated with 300 nM TSA for the duration of the experiment and viral titers from culture supernatants were determined at 10 dpi. Data is from duplicate experiments. (B) BAD sub UL29 ( sub UL29) virus is deficient in immediate-early gene expression irrespective of treatment with TSA. Cells pretreated with 500 nM of TSA were infected using 0.1 pfu/cell of wild-type (BAD w t) or BAD sub UL29 ( sub UL29) virus as described above, and total RNA was harvested at the indicated times. Expression of IE1 RNA was determined by qRT-PCR using cDNA produced from DNase-treated RNA and random hexamers. Quantitation was completed with SYBR green and primers specific to exon 4 of IE1. Samples were normalized to cellular GAPDH RNA levels.

    Article Snippet: The immunocomplexes were captured with protein G-agarose, eluted from the beads at room temperature with buffer containing 1% SDS and 100 mM NaHCO3 , crosslinks were reversed by incubation at 65°C for 5 h, proteins were digested with proteinase K (40 µg/ml) for 2 h at 55°C, and DNA recovered by using QiaQuick purification columns (Qiagen) and assayed by quantitative PCR (qPCR) with a Power SYBR green PCR kit (Applied Biosystems) and a primer pair specific for the HCMV MIEP ( 5′-AACAGCGTGGATGGCGTCTCC-3′ and 5′-GGCACCAAAATCAACGGGACTTT-3′ ).

    Techniques: Inhibition, Histone Deacetylase Assay, Activity Assay, Infection, Expressing, Quantitative RT-PCR, Produced, Quantitation Assay, SYBR Green Assay

    Reduced expression of components of the NuRD complex inhibits HCMV replication. (A) Disruption of NuRD complex in primary fibroblasts. Short hairpin RNA (shRNA) sequences to a scrambled control, CHD4, RBBP4 or both CHD4 and RBBP4 were delivered to fibroblasts using lentivirus vectors and expressing cells were isolated by puromycin resistance. Expression of CHD4 and RBBP4 was quantified by qRT-PCR using two separate sets of gene-specific primers and total cellular RNA. The data was normalized to GAPDH RNA levels and includes the average percent reduction for both primer sets to each gene. (B) shRNA-expressing cells were infected at a multiplicity of 0.25 pfu/cell with a BAD wt derivative expressing GFP. Images of infected cells were captured at 96 hpi. (C) Expression of immediate-early and early RNAs at 10 hpi was determined by qRT-PCR using cDNA produced from DNase-treated RNA and random hexamers. Quantitation was completed with SYBR green and sequence-specific primers as indicated. Expression was normalized to cellular GAPDH RNA levels. (D) HCMV genome replication was quantified by qPCR at 96 hpi. qPCR data was normalized using primers to β-actin. Data is derived from replicate experiments.

    Journal: PLoS Pathogens

    Article Title: Human Cytomegalovirus UL29/28 Protein Interacts with Components of the NuRD Complex Which Promote Accumulation of Immediate-Early RNA

    doi: 10.1371/journal.ppat.1000965

    Figure Lengend Snippet: Reduced expression of components of the NuRD complex inhibits HCMV replication. (A) Disruption of NuRD complex in primary fibroblasts. Short hairpin RNA (shRNA) sequences to a scrambled control, CHD4, RBBP4 or both CHD4 and RBBP4 were delivered to fibroblasts using lentivirus vectors and expressing cells were isolated by puromycin resistance. Expression of CHD4 and RBBP4 was quantified by qRT-PCR using two separate sets of gene-specific primers and total cellular RNA. The data was normalized to GAPDH RNA levels and includes the average percent reduction for both primer sets to each gene. (B) shRNA-expressing cells were infected at a multiplicity of 0.25 pfu/cell with a BAD wt derivative expressing GFP. Images of infected cells were captured at 96 hpi. (C) Expression of immediate-early and early RNAs at 10 hpi was determined by qRT-PCR using cDNA produced from DNase-treated RNA and random hexamers. Quantitation was completed with SYBR green and sequence-specific primers as indicated. Expression was normalized to cellular GAPDH RNA levels. (D) HCMV genome replication was quantified by qPCR at 96 hpi. qPCR data was normalized using primers to β-actin. Data is derived from replicate experiments.

    Article Snippet: The immunocomplexes were captured with protein G-agarose, eluted from the beads at room temperature with buffer containing 1% SDS and 100 mM NaHCO3 , crosslinks were reversed by incubation at 65°C for 5 h, proteins were digested with proteinase K (40 µg/ml) for 2 h at 55°C, and DNA recovered by using QiaQuick purification columns (Qiagen) and assayed by quantitative PCR (qPCR) with a Power SYBR green PCR kit (Applied Biosystems) and a primer pair specific for the HCMV MIEP ( 5′-AACAGCGTGGATGGCGTCTCC-3′ and 5′-GGCACCAAAATCAACGGGACTTT-3′ ).

    Techniques: Expressing, shRNA, Isolation, Quantitative RT-PCR, Infection, Produced, Quantitation Assay, SYBR Green Assay, Sequencing, Real-time Polymerase Chain Reaction, Derivative Assay

    The expression profile of all three Kindlin paralogs was quantified by real-time RT-PCR using SYBR Green. Data are presented as the relative expression of Kindlins (fold change) normalized by a housekeeping gene, β-actin. Note: The error bars indicate the standard deviation.

    Journal: Evolutionary Bioinformatics Online

    Article Title: Phylogenetic Analysis of Kindlins Suggests Subfunctionalization of an Ancestral Unduplicated Kindlin into Three Paralogs in Vertebrates

    doi: 10.4137/EBO.S6179

    Figure Lengend Snippet: The expression profile of all three Kindlin paralogs was quantified by real-time RT-PCR using SYBR Green. Data are presented as the relative expression of Kindlins (fold change) normalized by a housekeeping gene, β-actin. Note: The error bars indicate the standard deviation.

    Article Snippet: Triplicate samples of each PCR mixture, each containing 4.7 μl of POWER SYBR Green PCR master mixture (Applied Biosystems), 0.3 μl of a 10 pmol/μl of primer mixture, 0.3 μl of cDNA, and water to a total volume of 10 μl were transferred into a 96-well plate on an ABI 7500 Fast Real Time PCR System (Applied Biosystems).

    Techniques: Expressing, Quantitative RT-PCR, SYBR Green Assay, Standard Deviation

    Metanephric mesenchymal (MM) cell expression of RNA encoding mesenchymal and endothelial markers by RT-PCR. Total RNA was extracted from MM and ureteric bud (UB) cell lysates and reverse transcribed to synthesize cDNA with the use of the SYBR Green method. Ratio of cDNA transcript of marker: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown for each cell line. The results show high transcript levels of mesenchymal markers forkhead box D1 (Foxd1), glioma-associated oncogene homolog 1 (Gli-1), and ANG-1 ( A–C ), as well as endothelial markers aquaporin 1 (AQP1), Flt-1 (vascular endothelial growth factor receptor 1; VEGFR1), Tie-2 (angiopoietin receptor), and platelet endothelial cell adhesion molecule (PECAM) ( D–G ) in MM cells relative to UB cells. Data are expressed as means ± SEM. n = 3 independent experiments. ∗ P

    Journal: The American Journal of Pathology

    Article Title: Mouse Metanephric Mesenchymal Cell–Derived Angioblasts Undergo Vasculogenesis in Three-Dimensional Culture

    doi: 10.1016/j.ajpath.2017.10.022

    Figure Lengend Snippet: Metanephric mesenchymal (MM) cell expression of RNA encoding mesenchymal and endothelial markers by RT-PCR. Total RNA was extracted from MM and ureteric bud (UB) cell lysates and reverse transcribed to synthesize cDNA with the use of the SYBR Green method. Ratio of cDNA transcript of marker: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown for each cell line. The results show high transcript levels of mesenchymal markers forkhead box D1 (Foxd1), glioma-associated oncogene homolog 1 (Gli-1), and ANG-1 ( A–C ), as well as endothelial markers aquaporin 1 (AQP1), Flt-1 (vascular endothelial growth factor receptor 1; VEGFR1), Tie-2 (angiopoietin receptor), and platelet endothelial cell adhesion molecule (PECAM) ( D–G ) in MM cells relative to UB cells. Data are expressed as means ± SEM. n = 3 independent experiments. ∗ P

    Article Snippet: Twenty nanograms of total RNA from each sample was amplified with specific primers with the use of the Power SYBR Green RNA-to-CT 1-Step Kit and 7900HT fast Real-Time PCR System (Applied Biosystems, Foster City, CA).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, SYBR Green Assay, Marker

    Intratracheal instillation of IL-36α increased the mRNA expression of proinflammatory mediators in the lungs of IL-1αβ −/− mice. A–F) Transcript expression of early response cytokines (TNFα and IL-36γ), the classical IL-1 receptor IL-1R1, the novel IL-1 cytokine cluster receptor IL-36R and the neutrophil specific chemokines CXCL1 and CXCL2 in the lungs of IL-1αβ −/− mice 24 h following a single i.t instillation of PBS or 10 µg IL-36α. Transcript expression was evaluated by SYBR-Green based quantitative real-time PCR. G–H) Protein expression of TNFα and CXCL1 in the BAL fluid recovered from IL-1αβ −/− mice 24 h following a single i.t instillation of PBS or 10 µg IL-36α. Protein expression was quantified by multiplexed cytometric bead arrays. *Indicates significant differences ( P

    Journal: PLoS ONE

    Article Title: IL-36? Exerts Pro-Inflammatory Effects in the Lungs of Mice

    doi: 10.1371/journal.pone.0045784

    Figure Lengend Snippet: Intratracheal instillation of IL-36α increased the mRNA expression of proinflammatory mediators in the lungs of IL-1αβ −/− mice. A–F) Transcript expression of early response cytokines (TNFα and IL-36γ), the classical IL-1 receptor IL-1R1, the novel IL-1 cytokine cluster receptor IL-36R and the neutrophil specific chemokines CXCL1 and CXCL2 in the lungs of IL-1αβ −/− mice 24 h following a single i.t instillation of PBS or 10 µg IL-36α. Transcript expression was evaluated by SYBR-Green based quantitative real-time PCR. G–H) Protein expression of TNFα and CXCL1 in the BAL fluid recovered from IL-1αβ −/− mice 24 h following a single i.t instillation of PBS or 10 µg IL-36α. Protein expression was quantified by multiplexed cytometric bead arrays. *Indicates significant differences ( P

    Article Snippet: Equal concentrations of DNaseI treated RNA was reverse transcribed into cDNA using Taqman Reverse Transcription reagents (Applied Biosystems) and used as input for quantitative real-time PCR using Power SYBR Green kit (Applied Biosystems).

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

    Intratracheal instillation of IL-36α increased the mRNA expression of proinflammatory mediators in the lungs of wild-type C57BL/6 mice. A–H) Transcript expression of early response cytokines (TNFα, IL-1α, IL-1β, IL-36γ), the classical IL-1 receptor IL-1R1, the novel IL-1 cytokine cluster receptor IL-36R and the neutrophil specific chemokines CXCL1 and CXCL2 in the lungs of mice 24 h following a single i.t instillation of PBS or 10 µg IL-36α. Transcript expression was evaluated by SYBR-Green based quantitative real-time PCR. I–K) Protein expression of TNFα, IL-1α, IL-1β and CXCL1 in the BAL fluid recovered from mice 24 h following a single i.t instillation of PBS or 10 µg IL-36α. Protein expression was quantified by multiplexed cytometric bead arrays. *Indicates significant differences ( P

    Journal: PLoS ONE

    Article Title: IL-36? Exerts Pro-Inflammatory Effects in the Lungs of Mice

    doi: 10.1371/journal.pone.0045784

    Figure Lengend Snippet: Intratracheal instillation of IL-36α increased the mRNA expression of proinflammatory mediators in the lungs of wild-type C57BL/6 mice. A–H) Transcript expression of early response cytokines (TNFα, IL-1α, IL-1β, IL-36γ), the classical IL-1 receptor IL-1R1, the novel IL-1 cytokine cluster receptor IL-36R and the neutrophil specific chemokines CXCL1 and CXCL2 in the lungs of mice 24 h following a single i.t instillation of PBS or 10 µg IL-36α. Transcript expression was evaluated by SYBR-Green based quantitative real-time PCR. I–K) Protein expression of TNFα, IL-1α, IL-1β and CXCL1 in the BAL fluid recovered from mice 24 h following a single i.t instillation of PBS or 10 µg IL-36α. Protein expression was quantified by multiplexed cytometric bead arrays. *Indicates significant differences ( P

    Article Snippet: Equal concentrations of DNaseI treated RNA was reverse transcribed into cDNA using Taqman Reverse Transcription reagents (Applied Biosystems) and used as input for quantitative real-time PCR using Power SYBR Green kit (Applied Biosystems).

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

    IL-36α induced the expression of proinflammatory cytokines and chemokines in splenic CD11c + cells. A–H) Transcript expression of early response cytokines (TNFα, IL-1α, IL-1β, IL-36γ), the classical IL-1 receptor IL-1R1, the novel IL-1 cytokine cluster receptor IL-36R as well as the neutrophil specific chemokines CXCL1 and CXCL2 in splenic CD11c + cells 2 h following incubation with increasing concentrations of IL-36α. Transcript expression was evaluated by SYBR-Green based quantitative real-time PCR. *Indicates significant differences ( P

    Journal: PLoS ONE

    Article Title: IL-36? Exerts Pro-Inflammatory Effects in the Lungs of Mice

    doi: 10.1371/journal.pone.0045784

    Figure Lengend Snippet: IL-36α induced the expression of proinflammatory cytokines and chemokines in splenic CD11c + cells. A–H) Transcript expression of early response cytokines (TNFα, IL-1α, IL-1β, IL-36γ), the classical IL-1 receptor IL-1R1, the novel IL-1 cytokine cluster receptor IL-36R as well as the neutrophil specific chemokines CXCL1 and CXCL2 in splenic CD11c + cells 2 h following incubation with increasing concentrations of IL-36α. Transcript expression was evaluated by SYBR-Green based quantitative real-time PCR. *Indicates significant differences ( P

    Article Snippet: Equal concentrations of DNaseI treated RNA was reverse transcribed into cDNA using Taqman Reverse Transcription reagents (Applied Biosystems) and used as input for quantitative real-time PCR using Power SYBR Green kit (Applied Biosystems).

    Techniques: Expressing, Incubation, SYBR Green Assay, Real-time Polymerase Chain Reaction

    IL-36α induced the expression of T cell costimulatory molecules in splenic CD11c + cells. A–C) Transcript expression of the co-stimulatory molecules CD80, CD86 and CD40 in splenic CD11c + cells 2 h following incubation with increasing concentrations of IL-36α. Transcript expression was evaluated by SYBR-Green based quantitative real-time PCR. *Indicates significant differences ( P

    Journal: PLoS ONE

    Article Title: IL-36? Exerts Pro-Inflammatory Effects in the Lungs of Mice

    doi: 10.1371/journal.pone.0045784

    Figure Lengend Snippet: IL-36α induced the expression of T cell costimulatory molecules in splenic CD11c + cells. A–C) Transcript expression of the co-stimulatory molecules CD80, CD86 and CD40 in splenic CD11c + cells 2 h following incubation with increasing concentrations of IL-36α. Transcript expression was evaluated by SYBR-Green based quantitative real-time PCR. *Indicates significant differences ( P

    Article Snippet: Equal concentrations of DNaseI treated RNA was reverse transcribed into cDNA using Taqman Reverse Transcription reagents (Applied Biosystems) and used as input for quantitative real-time PCR using Power SYBR Green kit (Applied Biosystems).

    Techniques: Expressing, Incubation, SYBR Green Assay, Real-time Polymerase Chain Reaction

    PEMF stimulated expression of miR21-5p in differentiated human osteoblasts. Total RNAs from control or PEMF-treated hBMSCs of females (24 × 2, 27, 29, and 30 years old, n = 5) at day 23 of differentiation or (31, 36, 58, and 68 years old) at day 23 or 33 of differentiation were isolated and subjected to RT-qPCR using the miScript II kit with miScript HiSpec Buffer and miScript SYBR Green PCR Kit. snoR10-1 was used to normalize miR21-5p expression and the expression is shown as a percentage of the relevant control samples. ∗ indicates significant increase compared to control using one-way ANOVA.

    Journal: Stem Cells International

    Article Title: Pulsed Electromagnetic Field Regulates MicroRNA 21 Expression to Activate TGF-β Signaling in Human Bone Marrow Stromal Cells to Enhance Osteoblast Differentiation

    doi: 10.1155/2017/2450327

    Figure Lengend Snippet: PEMF stimulated expression of miR21-5p in differentiated human osteoblasts. Total RNAs from control or PEMF-treated hBMSCs of females (24 × 2, 27, 29, and 30 years old, n = 5) at day 23 of differentiation or (31, 36, 58, and 68 years old) at day 23 or 33 of differentiation were isolated and subjected to RT-qPCR using the miScript II kit with miScript HiSpec Buffer and miScript SYBR Green PCR Kit. snoR10-1 was used to normalize miR21-5p expression and the expression is shown as a percentage of the relevant control samples. ∗ indicates significant increase compared to control using one-way ANOVA.

    Article Snippet: The Power SYBR green master mix kit for PCR reactions was purchased from Invitrogen.

    Techniques: Expressing, Isolation, Quantitative RT-PCR, SYBR Green Assay, Polymerase Chain Reaction