superscript iii first strand synthesis system  (Thermo Fisher)


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    Thermo Fisher superscript iii first strand synthesis system
    mRNA expression levels of ATRAP and SIRT1 in ciRPTEC in response to angiotensin II (Ang II) treatment or serum withdrawal. The ciRPTEC were treated with 10 −6 M of Ang II (+) for 24 hours ( a,b ) or serum withdrawal (−) for 24 hours ( c,d ). The relative mRNA levels of ATRAP and SIRT1 in the ciRPTEC were determined by RT-qPCR, normalized to 18 S ribosomal <t>RNA.</t> mRNA levels in the absence of Ang II treatment (−; for a,b ) or presence of serum (+; for c,d ) were set to 1. All data were obtained with <t>three</t> biologically independent experiments. Values represent the means ± standard error. ( a ) **p
    Superscript Iii First Strand Synthesis System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2970 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Angiotensin II type 1 receptor-associated protein deficiency attenuates sirtuin1 expression in an immortalised human renal proximal tubule cell line"

    Article Title: Angiotensin II type 1 receptor-associated protein deficiency attenuates sirtuin1 expression in an immortalised human renal proximal tubule cell line

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-52566-y

    mRNA expression levels of ATRAP and SIRT1 in ciRPTEC in response to angiotensin II (Ang II) treatment or serum withdrawal. The ciRPTEC were treated with 10 −6 M of Ang II (+) for 24 hours ( a,b ) or serum withdrawal (−) for 24 hours ( c,d ). The relative mRNA levels of ATRAP and SIRT1 in the ciRPTEC were determined by RT-qPCR, normalized to 18 S ribosomal RNA. mRNA levels in the absence of Ang II treatment (−; for a,b ) or presence of serum (+; for c,d ) were set to 1. All data were obtained with three biologically independent experiments. Values represent the means ± standard error. ( a ) **p
    Figure Legend Snippet: mRNA expression levels of ATRAP and SIRT1 in ciRPTEC in response to angiotensin II (Ang II) treatment or serum withdrawal. The ciRPTEC were treated with 10 −6 M of Ang II (+) for 24 hours ( a,b ) or serum withdrawal (−) for 24 hours ( c,d ). The relative mRNA levels of ATRAP and SIRT1 in the ciRPTEC were determined by RT-qPCR, normalized to 18 S ribosomal RNA. mRNA levels in the absence of Ang II treatment (−; for a,b ) or presence of serum (+; for c,d ) were set to 1. All data were obtained with three biologically independent experiments. Values represent the means ± standard error. ( a ) **p

    Techniques Used: Expressing, Quantitative RT-PCR

    Effect of ATRAP knockdown on SIRT1 mRNA and protein expression with or without serum-withdrawal. The ciRPTEC were treated with negative control siRNA (ATRAP-control, a–f ), ATRAP siRNA #1 (ATRAP-KD1, a–f ) for 48 hours, followed by serum withdrawal for 24 hours ( a–e ). ( a,c ) The relative mRNA levels of ATRAP ( a ) or SIRT1 ( c ) on ciRPTEC were determined by RT-qPCR, normalized to 18 S ribosomal RNA. mRNA levels in the presence of serum (+) and the control siRNA were set to 1. ( b,d ) The relative protein expression of ATRAP and SIRT1 in the ciRPTEC was determined by western blot analysis, normalized to β-actin expression. Protein levels in the presence of serum (+) and control siRNA were set to 100. SIRT1 proteins were detected with an antibody towards the N-terminal 1–131 amino acids (SIRT1_N lot 2465249). ( e ) SIRT1 protein detected with an antibody recognising the C-terminal region (SIRT1_C). ( f ) Half-life analysis of the SIRT1 protein was performed 48 hours after siRNA transfection of ciRPTEC cells and treatment with emetine to repress de-novo protein synthesis. Cell lysates were collected at 0, 2, 4 and 8 hours after emetine treatment. SIRT1 proteins were detected with the SIRT1_C antibody. Since the SIRT1 expression level at time 0 was decreased in ATRAP-KD1, the signal intensities of the SIRT1 proteins at 0 hours were set to similar levels visually between the ATRAP-control and ATRAP-KD1 by showing the short exposure (ATRAP-control) and the long exposure (ATRAP-KD1) images. Original gel images are presented in Supplementary Fig. S12 . All data were obtained with three biologically independent experiments (except for ( h ) where two replicates were used) and were analysed by two-way ANOVA. Values represent the means ± standard error. ( a,c ) **p
    Figure Legend Snippet: Effect of ATRAP knockdown on SIRT1 mRNA and protein expression with or without serum-withdrawal. The ciRPTEC were treated with negative control siRNA (ATRAP-control, a–f ), ATRAP siRNA #1 (ATRAP-KD1, a–f ) for 48 hours, followed by serum withdrawal for 24 hours ( a–e ). ( a,c ) The relative mRNA levels of ATRAP ( a ) or SIRT1 ( c ) on ciRPTEC were determined by RT-qPCR, normalized to 18 S ribosomal RNA. mRNA levels in the presence of serum (+) and the control siRNA were set to 1. ( b,d ) The relative protein expression of ATRAP and SIRT1 in the ciRPTEC was determined by western blot analysis, normalized to β-actin expression. Protein levels in the presence of serum (+) and control siRNA were set to 100. SIRT1 proteins were detected with an antibody towards the N-terminal 1–131 amino acids (SIRT1_N lot 2465249). ( e ) SIRT1 protein detected with an antibody recognising the C-terminal region (SIRT1_C). ( f ) Half-life analysis of the SIRT1 protein was performed 48 hours after siRNA transfection of ciRPTEC cells and treatment with emetine to repress de-novo protein synthesis. Cell lysates were collected at 0, 2, 4 and 8 hours after emetine treatment. SIRT1 proteins were detected with the SIRT1_C antibody. Since the SIRT1 expression level at time 0 was decreased in ATRAP-KD1, the signal intensities of the SIRT1 proteins at 0 hours were set to similar levels visually between the ATRAP-control and ATRAP-KD1 by showing the short exposure (ATRAP-control) and the long exposure (ATRAP-KD1) images. Original gel images are presented in Supplementary Fig. S12 . All data were obtained with three biologically independent experiments (except for ( h ) where two replicates were used) and were analysed by two-way ANOVA. Values represent the means ± standard error. ( a,c ) **p

    Techniques Used: Expressing, Negative Control, Quantitative RT-PCR, Western Blot, Transfection

    mRNA expression of the proximal tubule markers, AT1R and ATRAP , in clonal immortalised cells. ( a–d ) The relative mRNA levels of SGLT2 , DPP4 , ATRAP and AT1R in 12 clonal immortalized cell (ciRPTEC) clones were determined by RT-qPCR, normalized to 18S ribosomal RNA. The mRNA levels of the original RPTEC (RPTEC-Ori) were set to 1. Data were obtained with three biologically independent experiments. Values represent the means ± standard error.
    Figure Legend Snippet: mRNA expression of the proximal tubule markers, AT1R and ATRAP , in clonal immortalised cells. ( a–d ) The relative mRNA levels of SGLT2 , DPP4 , ATRAP and AT1R in 12 clonal immortalized cell (ciRPTEC) clones were determined by RT-qPCR, normalized to 18S ribosomal RNA. The mRNA levels of the original RPTEC (RPTEC-Ori) were set to 1. Data were obtained with three biologically independent experiments. Values represent the means ± standard error.

    Techniques Used: Expressing, Quantitative RT-PCR

    Effect of ATRAP knockout generated using CRISPR-CAS9 on SIRT1 mRNA and protein expression levels under serum-starvation. ATRAP-KO (ciRPTEC expressing CAS9 and gRNA targeted towards ATRAP) or ATRAP-control (ciRPTEC expressing only CAS9) cells were cultured with or without serum for 24 hours. ( a,c ) The relative protein expression of ATRAP and SIRT1 in the ciRPTEC was determined by western blot analysis, normalized to β-actin expression. Protein levels of ATRAP-control with serum (+) were set to 100. ( b ) The relative mRNA levels of SIRT1 in the ciRPTEC was determined by RT-qPCR, normalized to 18 S ribosomal RNA. The mRNA level of ATRAP-control with serum (+) was set to 1. All data were obtained with three biologically independent experiments. Values represent the means ± standard error. All data were analysed by two-way ANOVA. ( a ) # p
    Figure Legend Snippet: Effect of ATRAP knockout generated using CRISPR-CAS9 on SIRT1 mRNA and protein expression levels under serum-starvation. ATRAP-KO (ciRPTEC expressing CAS9 and gRNA targeted towards ATRAP) or ATRAP-control (ciRPTEC expressing only CAS9) cells were cultured with or without serum for 24 hours. ( a,c ) The relative protein expression of ATRAP and SIRT1 in the ciRPTEC was determined by western blot analysis, normalized to β-actin expression. Protein levels of ATRAP-control with serum (+) were set to 100. ( b ) The relative mRNA levels of SIRT1 in the ciRPTEC was determined by RT-qPCR, normalized to 18 S ribosomal RNA. The mRNA level of ATRAP-control with serum (+) was set to 1. All data were obtained with three biologically independent experiments. Values represent the means ± standard error. All data were analysed by two-way ANOVA. ( a ) # p

    Techniques Used: Knock-Out, Generated, CRISPR, Expressing, Cell Culture, Western Blot, Quantitative RT-PCR

    Comparison of mRNA expression levels of distal and proximal tubule markers in the ciRPTEC 2B-1 cell line, and reactivity of NHE3 in this cell line to angiotensin II (Ang II) treatment. ( a,b ) The relative mRNA levels of CALB1 and AQP2 in the original RPTEC (RPTEC-Ori) cell line and the clonal immortalized cell line 2B1 (ciRPTEC 2B1) were determined by RT-qPCR, normalized to 18S ribosomal RNA. mRNA levels of SGLT2 were set to 1. ( c ) The relative mRNA levels of NHE3 in ciRPTEC 2B1 after 24 hours of treatment with a range of Ang II concentrations were determined by RT-qPCR, normalized to 18S ribosomal RNA. mRNA levels obtained without Ang II (concentration 0 M) were set to 1. Data were obtained with three biologically independent experiments. Values represent the means ± standard error. *p
    Figure Legend Snippet: Comparison of mRNA expression levels of distal and proximal tubule markers in the ciRPTEC 2B-1 cell line, and reactivity of NHE3 in this cell line to angiotensin II (Ang II) treatment. ( a,b ) The relative mRNA levels of CALB1 and AQP2 in the original RPTEC (RPTEC-Ori) cell line and the clonal immortalized cell line 2B1 (ciRPTEC 2B1) were determined by RT-qPCR, normalized to 18S ribosomal RNA. mRNA levels of SGLT2 were set to 1. ( c ) The relative mRNA levels of NHE3 in ciRPTEC 2B1 after 24 hours of treatment with a range of Ang II concentrations were determined by RT-qPCR, normalized to 18S ribosomal RNA. mRNA levels obtained without Ang II (concentration 0 M) were set to 1. Data were obtained with three biologically independent experiments. Values represent the means ± standard error. *p

    Techniques Used: Expressing, Quantitative RT-PCR, Concentration Assay

    2) Product Images from "Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction"

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction

    Journal: Cell stem cell

    doi: 10.1016/j.stem.2019.09.010

    The Dynamic Changes of ATAC-seq, ChIP-seq, and RNA-seq Peaks Are Sequentially Correlated (A) Transcripts per million (TPM) (log2, averaging over three biological replicates) per time point of marker genes (neural, mesoderm, endoderm, neural crest, pluripotent, nodal/BMP targets, and immediate early genes). (B–D) Heatmap of scaled read counts (log2, averaged over three biological replicates and standardized per row) of temporal genes and genomic regions, showing data from RNA-seq (B), H3K27ac ChIP-seq (C), and ATAC-seq (D). The loci in each assay were clustered into six groups based on their temporal patterns. (E) Overlap between the temporal clusters in the three data modalities (Bonferroni-corrected p values of a hypergeometric test). Circle sizes represent the proportion of overlap between every two clusters. The overlap is computed either at the region level (ATAC-seq versus ChIP-seq) or at the gene level (ATAC/ChIP-seq versus RNA-seq; regions in the former assays are represented by their nearest gene).
    Figure Legend Snippet: The Dynamic Changes of ATAC-seq, ChIP-seq, and RNA-seq Peaks Are Sequentially Correlated (A) Transcripts per million (TPM) (log2, averaging over three biological replicates) per time point of marker genes (neural, mesoderm, endoderm, neural crest, pluripotent, nodal/BMP targets, and immediate early genes). (B–D) Heatmap of scaled read counts (log2, averaged over three biological replicates and standardized per row) of temporal genes and genomic regions, showing data from RNA-seq (B), H3K27ac ChIP-seq (C), and ATAC-seq (D). The loci in each assay were clustered into six groups based on their temporal patterns. (E) Overlap between the temporal clusters in the three data modalities (Bonferroni-corrected p values of a hypergeometric test). Circle sizes represent the proportion of overlap between every two clusters. The overlap is computed either at the region level (ATAC-seq versus ChIP-seq) or at the gene level (ATAC/ChIP-seq versus RNA-seq; regions in the former assays are represented by their nearest gene).

    Techniques Used: Chromatin Immunoprecipitation, RNA Sequencing Assay, Marker

    Validation of Enhancers by Luciferase Assays and CRISPRa (A) Relative luciferase activity for each enhancer compared to Renilla luciferase activity at 0, 12, and 72 h post-neural induction. Five ESC enhancers, four immediate response (IR) enhancers, four NPC enhancers, and empty pLS-mP-Luc vector (negative control) were tested. (B) MPRA signal (RNA/DNA ratio) at 0, 12, and 72 h post-neural induction. (C–F) Functional validation of enhancers by CRISPRa. sgRNAs that target enhancers nearby SOX1 (C), OTX2 (D), IRX3 (E), and PAX6 alternative promoters (F) or negative control sgRNA (NC_sgRNA) were infected into hESCs that stably express dCas9-VP64. Upregulation of respective genes relative to HPRT were examined by qPCR and shown as bar charts on the right. Data are presented as means ± SD of three independent experiments.
    Figure Legend Snippet: Validation of Enhancers by Luciferase Assays and CRISPRa (A) Relative luciferase activity for each enhancer compared to Renilla luciferase activity at 0, 12, and 72 h post-neural induction. Five ESC enhancers, four immediate response (IR) enhancers, four NPC enhancers, and empty pLS-mP-Luc vector (negative control) were tested. (B) MPRA signal (RNA/DNA ratio) at 0, 12, and 72 h post-neural induction. (C–F) Functional validation of enhancers by CRISPRa. sgRNAs that target enhancers nearby SOX1 (C), OTX2 (D), IRX3 (E), and PAX6 alternative promoters (F) or negative control sgRNA (NC_sgRNA) were infected into hESCs that stably express dCas9-VP64. Upregulation of respective genes relative to HPRT were examined by qPCR and shown as bar charts on the right. Data are presented as means ± SD of three independent experiments.

    Techniques Used: Luciferase, Activity Assay, Plasmid Preparation, Negative Control, Functional Assay, Infection, Stable Transfection, Real-time Polymerase Chain Reaction

    Activity of Temporal CRS: Comparing lentiMPRA to the Endogenous Signals (A–D) Temporal MPRA signal (RNA/DNA ratio; A), normalized read count of the closest gene detected by RNA-seq (B), H3K27ac ChIP-seq (C), and ATAC-seq (D), clustered into four temporal groups separately. Rows are standardized. (E) Overlap between the lentiMPRA clusters and the three genomic data modalities. Shown are Bonferroni-corrected p values of a hypergeometric test. Circle sizes represent the proportion of overlap between every two clusters. The overlap is computed either at the region level (lentiMPRA versus ATAC-seq or ChIP-seq) or at the gene level (lentiMPRA versus RNA-seq; using the nearest gene to represent each lentiMPRA region).
    Figure Legend Snippet: Activity of Temporal CRS: Comparing lentiMPRA to the Endogenous Signals (A–D) Temporal MPRA signal (RNA/DNA ratio; A), normalized read count of the closest gene detected by RNA-seq (B), H3K27ac ChIP-seq (C), and ATAC-seq (D), clustered into four temporal groups separately. Rows are standardized. (E) Overlap between the lentiMPRA clusters and the three genomic data modalities. Shown are Bonferroni-corrected p values of a hypergeometric test. Circle sizes represent the proportion of overlap between every two clusters. The overlap is computed either at the region level (lentiMPRA versus ATAC-seq or ChIP-seq) or at the gene level (lentiMPRA versus RNA-seq; using the nearest gene to represent each lentiMPRA region).

    Techniques Used: Activity Assay, RNA Sequencing Assay, Chromatin Immunoprecipitation

    3) Product Images from "Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction"

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction

    Journal: Cell stem cell

    doi: 10.1016/j.stem.2019.09.010

    The Dynamic Changes of ATAC-seq, ChIP-seq, and RNA-seq Peaks Are Sequentially Correlated (A) Transcripts per million (TPM) (log2, averaging over three biological replicates) per time point of marker genes (neural, mesoderm, endoderm, neural crest, pluripotent, nodal/BMP targets, and immediate early genes). (B–D) Heatmap of scaled read counts (log2, averaged over three biological replicates and standardized per row) of temporal genes and genomic regions, showing data from RNA-seq (B), H3K27ac ChIP-seq (C), and ATAC-seq (D). The loci in each assay were clustered into six groups based on their temporal patterns. (E) Overlap between the temporal clusters in the three data modalities (Bonferroni-corrected p values of a hypergeometric test). Circle sizes represent the proportion of overlap between every two clusters. The overlap is computed either at the region level (ATAC-seq versus ChIP-seq) or at the gene level (ATAC/ChIP-seq versus RNA-seq; regions in the former assays are represented by their nearest gene).
    Figure Legend Snippet: The Dynamic Changes of ATAC-seq, ChIP-seq, and RNA-seq Peaks Are Sequentially Correlated (A) Transcripts per million (TPM) (log2, averaging over three biological replicates) per time point of marker genes (neural, mesoderm, endoderm, neural crest, pluripotent, nodal/BMP targets, and immediate early genes). (B–D) Heatmap of scaled read counts (log2, averaged over three biological replicates and standardized per row) of temporal genes and genomic regions, showing data from RNA-seq (B), H3K27ac ChIP-seq (C), and ATAC-seq (D). The loci in each assay were clustered into six groups based on their temporal patterns. (E) Overlap between the temporal clusters in the three data modalities (Bonferroni-corrected p values of a hypergeometric test). Circle sizes represent the proportion of overlap between every two clusters. The overlap is computed either at the region level (ATAC-seq versus ChIP-seq) or at the gene level (ATAC/ChIP-seq versus RNA-seq; regions in the former assays are represented by their nearest gene).

    Techniques Used: Chromatin Immunoprecipitation, RNA Sequencing Assay, Marker

    Validation of Enhancers by Luciferase Assays and CRISPRa (A) Relative luciferase activity for each enhancer compared to Renilla luciferase activity at 0, 12, and 72 h post-neural induction. Five ESC enhancers, four immediate response (IR) enhancers, four NPC enhancers, and empty pLS-mP-Luc vector (negative control) were tested. (B) MPRA signal (RNA/DNA ratio) at 0, 12, and 72 h post-neural induction. (C–F) Functional validation of enhancers by CRISPRa. sgRNAs that target enhancers nearby SOX1 (C), OTX2 (D), IRX3 (E), and PAX6 alternative promoters (F) or negative control sgRNA (NC_sgRNA) were infected into hESCs that stably express dCas9-VP64. Upregulation of respective genes relative to HPRT were examined by qPCR and shown as bar charts on the right. Data are presented as means ± SD of three independent experiments.
    Figure Legend Snippet: Validation of Enhancers by Luciferase Assays and CRISPRa (A) Relative luciferase activity for each enhancer compared to Renilla luciferase activity at 0, 12, and 72 h post-neural induction. Five ESC enhancers, four immediate response (IR) enhancers, four NPC enhancers, and empty pLS-mP-Luc vector (negative control) were tested. (B) MPRA signal (RNA/DNA ratio) at 0, 12, and 72 h post-neural induction. (C–F) Functional validation of enhancers by CRISPRa. sgRNAs that target enhancers nearby SOX1 (C), OTX2 (D), IRX3 (E), and PAX6 alternative promoters (F) or negative control sgRNA (NC_sgRNA) were infected into hESCs that stably express dCas9-VP64. Upregulation of respective genes relative to HPRT were examined by qPCR and shown as bar charts on the right. Data are presented as means ± SD of three independent experiments.

    Techniques Used: Luciferase, Activity Assay, Plasmid Preparation, Negative Control, Functional Assay, Infection, Stable Transfection, Real-time Polymerase Chain Reaction

    Activity of Temporal CRS: Comparing lentiMPRA to the Endogenous Signals (A–D) Temporal MPRA signal (RNA/DNA ratio; A), normalized read count of the closest gene detected by RNA-seq (B), H3K27ac ChIP-seq (C), and ATAC-seq (D), clustered into four temporal groups separately. Rows are standardized. (E) Overlap between the lentiMPRA clusters and the three genomic data modalities. Shown are Bonferroni-corrected p values of a hypergeometric test. Circle sizes represent the proportion of overlap between every two clusters. The overlap is computed either at the region level (lentiMPRA versus ATAC-seq or ChIP-seq) or at the gene level (lentiMPRA versus RNA-seq; using the nearest gene to represent each lentiMPRA region).
    Figure Legend Snippet: Activity of Temporal CRS: Comparing lentiMPRA to the Endogenous Signals (A–D) Temporal MPRA signal (RNA/DNA ratio; A), normalized read count of the closest gene detected by RNA-seq (B), H3K27ac ChIP-seq (C), and ATAC-seq (D), clustered into four temporal groups separately. Rows are standardized. (E) Overlap between the lentiMPRA clusters and the three genomic data modalities. Shown are Bonferroni-corrected p values of a hypergeometric test. Circle sizes represent the proportion of overlap between every two clusters. The overlap is computed either at the region level (lentiMPRA versus ATAC-seq or ChIP-seq) or at the gene level (lentiMPRA versus RNA-seq; using the nearest gene to represent each lentiMPRA region).

    Techniques Used: Activity Assay, RNA Sequencing Assay, Chromatin Immunoprecipitation

    4) Product Images from "Semaphorin 3E/PlexinD1 signaling is required for cardiac ventricular compaction"

    Article Title: Semaphorin 3E/PlexinD1 signaling is required for cardiac ventricular compaction

    Journal: JCI Insight

    doi: 10.1172/jci.insight.125908

    Endothelium-specific Plxnd1 overexpression does not affect cardiac chamber development but can rescue the hypertrabeculation and noncompaction defects when crossed to a Plxnd1 –/– background. Scheme of the endothelial-specific Plxnd1 -transgenic construct ( A ). Transgenic founder (F0) mice were identified through PCR screening. Three 325-bp transgene-positive founders (white dotted box) were used for generating F1 ( B ). Western blot analysis of PlexinD1 for F2 control (transgene negative) and Tie2-Plxnd1-Tg embryo lysate. Anti-vinculin antibody is used as a loading control ( C ). Real-time qPCR for Plxnd1 using RNA isolated from endothelial cells of E10.5 control and Tie2-Plxnd1-Tg embryo ( D ). A transgenic line showing elevated levels of Plexind1 proteins was used for subsequent experiments. H E staining of paraffin sections of E12.5 ( n = 5 for each genotype) and E14.5 ( n = 6 for each genotype) control and Tie2-Plxnd1-Tg hearts ( E ). H E staining of paraffin sections of E15.5 Plxnd1 –/– and Plxnd1 –/– Tie2-Plxnd1-Tg hearts ( F ). ( n = 4 for each genotype). LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle. Scale bars: 100 μm.
    Figure Legend Snippet: Endothelium-specific Plxnd1 overexpression does not affect cardiac chamber development but can rescue the hypertrabeculation and noncompaction defects when crossed to a Plxnd1 –/– background. Scheme of the endothelial-specific Plxnd1 -transgenic construct ( A ). Transgenic founder (F0) mice were identified through PCR screening. Three 325-bp transgene-positive founders (white dotted box) were used for generating F1 ( B ). Western blot analysis of PlexinD1 for F2 control (transgene negative) and Tie2-Plxnd1-Tg embryo lysate. Anti-vinculin antibody is used as a loading control ( C ). Real-time qPCR for Plxnd1 using RNA isolated from endothelial cells of E10.5 control and Tie2-Plxnd1-Tg embryo ( D ). A transgenic line showing elevated levels of Plexind1 proteins was used for subsequent experiments. H E staining of paraffin sections of E12.5 ( n = 5 for each genotype) and E14.5 ( n = 6 for each genotype) control and Tie2-Plxnd1-Tg hearts ( E ). H E staining of paraffin sections of E15.5 Plxnd1 –/– and Plxnd1 –/– Tie2-Plxnd1-Tg hearts ( F ). ( n = 4 for each genotype). LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle. Scale bars: 100 μm.

    Techniques Used: Over Expression, Transgenic Assay, Construct, Mouse Assay, Polymerase Chain Reaction, Western Blot, Real-time Polymerase Chain Reaction, Isolation, Staining

    5) Product Images from "Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction"

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction

    Journal: Cell stem cell

    doi: 10.1016/j.stem.2019.09.010

    Validation of Enhancers by Luciferase Assays and CRISPRa (A) Relative luciferase activity for each enhancer compared to Renilla luciferase activity at 0, 12, and 72 h post-neural induction. Five ESC enhancers, four immediate response (IR) enhancers, four NPC enhancers, and empty pLS-mP-Luc vector (negative control) were tested. (B) MPRA signal (RNA/DNA ratio) at 0, 12, and 72 h post-neural induction. (C–F) Functional validation of enhancers by CRISPRa. sgRNAs that target enhancers nearby SOX1 (C), OTX2 (D), IRX3 (E), and PAX6 alternative promoters (F) or negative control sgRNA (NC_sgRNA) were infected into hESCs that stably express dCas9-VP64. Upregulation of respective genes relative to HPRT were examined by qPCR and shown as bar charts on the right. Data are presented as means ± SD of three independent experiments.
    Figure Legend Snippet: Validation of Enhancers by Luciferase Assays and CRISPRa (A) Relative luciferase activity for each enhancer compared to Renilla luciferase activity at 0, 12, and 72 h post-neural induction. Five ESC enhancers, four immediate response (IR) enhancers, four NPC enhancers, and empty pLS-mP-Luc vector (negative control) were tested. (B) MPRA signal (RNA/DNA ratio) at 0, 12, and 72 h post-neural induction. (C–F) Functional validation of enhancers by CRISPRa. sgRNAs that target enhancers nearby SOX1 (C), OTX2 (D), IRX3 (E), and PAX6 alternative promoters (F) or negative control sgRNA (NC_sgRNA) were infected into hESCs that stably express dCas9-VP64. Upregulation of respective genes relative to HPRT were examined by qPCR and shown as bar charts on the right. Data are presented as means ± SD of three independent experiments.

    Techniques Used: Luciferase, Activity Assay, Plasmid Preparation, Negative Control, Functional Assay, Infection, Stable Transfection, Real-time Polymerase Chain Reaction

    6) Product Images from "Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction"

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction

    Journal: Cell stem cell

    doi: 10.1016/j.stem.2019.09.010

    Validation of Enhancers by Luciferase Assays and CRISPRa (A) Relative luciferase activity for each enhancer compared to Renilla luciferase activity at 0, 12, and 72 h post-neural induction. Five ESC enhancers, four immediate response (IR) enhancers, four NPC enhancers, and empty pLS-mP-Luc vector (negative control) were tested. (B) MPRA signal (RNA/DNA ratio) at 0, 12, and 72 h post-neural induction. (C–F) Functional validation of enhancers by CRISPRa. sgRNAs that target enhancers nearby SOX1 (C), OTX2 (D), IRX3 (E), and PAX6 alternative promoters (F) or negative control sgRNA (NC_sgRNA) were infected into hESCs that stably express dCas9-VP64. Upregulation of respective genes relative to HPRT were examined by qPCR and shown as bar charts on the right. Data are presented as means ± SD of three independent experiments.
    Figure Legend Snippet: Validation of Enhancers by Luciferase Assays and CRISPRa (A) Relative luciferase activity for each enhancer compared to Renilla luciferase activity at 0, 12, and 72 h post-neural induction. Five ESC enhancers, four immediate response (IR) enhancers, four NPC enhancers, and empty pLS-mP-Luc vector (negative control) were tested. (B) MPRA signal (RNA/DNA ratio) at 0, 12, and 72 h post-neural induction. (C–F) Functional validation of enhancers by CRISPRa. sgRNAs that target enhancers nearby SOX1 (C), OTX2 (D), IRX3 (E), and PAX6 alternative promoters (F) or negative control sgRNA (NC_sgRNA) were infected into hESCs that stably express dCas9-VP64. Upregulation of respective genes relative to HPRT were examined by qPCR and shown as bar charts on the right. Data are presented as means ± SD of three independent experiments.

    Techniques Used: Luciferase, Activity Assay, Plasmid Preparation, Negative Control, Functional Assay, Infection, Stable Transfection, Real-time Polymerase Chain Reaction

    7) Product Images from "Visualization of Positive and Negative Sense Viral RNA for Probing the Mechanism of Direct-Acting Antivirals against Hepatitis C Virus"

    Article Title: Visualization of Positive and Negative Sense Viral RNA for Probing the Mechanism of Direct-Acting Antivirals against Hepatitis C Virus

    Journal: Viruses

    doi: 10.3390/v11111039

    Early impact of different classes of HCV inhibitors on HCV RNA and NS5A. ( A ) Huh-7.5.1 cells were infected with HCV Jc1/Gluc2A at an MOI of 0.5. After 48 h, the cells were treated with various inhibitors (Danoprevir, 0.32 μM; Ledipasvir, 3 μM; Daclatasvir, 3.2 nM; Sofosbuvir, 20 μM), then after a further 8 h cells were fixed and probed sequentially for (−)RNA, (+)RNA and NS5A. Finally, nuclei were stained with DAPI. Cells were imaged on a Leica SP8 confocal microscope using a 63× oil-immersion objective. ( B ) Representative images from each inhibitor showing (−)RNA in green, (+)RNA in red, NS5A in gray and nuclei in blue. Boxed regions shown enlarged. Scale bars represent 10 µm. Early impact of different classes of HCV inhibitors on HCV RNA and NS5A. (C and D) Fields of view were captured as described in Figure 3 , then analyzed using Gen5 software (BioTek). Abundance of (+) and (−)RNA strands was quantified by: ( C ) number of RNA foci per cell; ( D ) RNA fluorescence intensity per cell in the field of view; and ( E ) RT-qPCR to determine copy numbers of the of (+) and (−)RNA using specific primers. All values are expressed as a percentage of the DMSO-treated sample. Error bars indicate standard error of the mean for three independent experiments. Significance of differences between DMSO and drug treatments was calculated using one-way ANOVA and Tukey’s post-test for multiple comparisons. Differences were not significant unless otherwise indicated. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. P values shown on columns indicate comparison to DMSO. Lines indicate comparisons between test samples. Lines group samples, and are color coordinated for (+)RNA and (−)RNA. Flat lines group all samples beneath them, inverted Vs indicate comparison of the samples under the ends of the lines. DNV, Danoprevir; LDV, Ledipasvir; DCV, Daclatasvir; SOF, Sofosbuvir. ( F ) Comparison of intracellular HCV transcript number under control (DMSO) conditions as determined by strand-specific RT-qPCR and bDNA FISH. Error bars indicate standard error of the mean; no additional statistical analyses were performed.
    Figure Legend Snippet: Early impact of different classes of HCV inhibitors on HCV RNA and NS5A. ( A ) Huh-7.5.1 cells were infected with HCV Jc1/Gluc2A at an MOI of 0.5. After 48 h, the cells were treated with various inhibitors (Danoprevir, 0.32 μM; Ledipasvir, 3 μM; Daclatasvir, 3.2 nM; Sofosbuvir, 20 μM), then after a further 8 h cells were fixed and probed sequentially for (−)RNA, (+)RNA and NS5A. Finally, nuclei were stained with DAPI. Cells were imaged on a Leica SP8 confocal microscope using a 63× oil-immersion objective. ( B ) Representative images from each inhibitor showing (−)RNA in green, (+)RNA in red, NS5A in gray and nuclei in blue. Boxed regions shown enlarged. Scale bars represent 10 µm. Early impact of different classes of HCV inhibitors on HCV RNA and NS5A. (C and D) Fields of view were captured as described in Figure 3 , then analyzed using Gen5 software (BioTek). Abundance of (+) and (−)RNA strands was quantified by: ( C ) number of RNA foci per cell; ( D ) RNA fluorescence intensity per cell in the field of view; and ( E ) RT-qPCR to determine copy numbers of the of (+) and (−)RNA using specific primers. All values are expressed as a percentage of the DMSO-treated sample. Error bars indicate standard error of the mean for three independent experiments. Significance of differences between DMSO and drug treatments was calculated using one-way ANOVA and Tukey’s post-test for multiple comparisons. Differences were not significant unless otherwise indicated. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. P values shown on columns indicate comparison to DMSO. Lines indicate comparisons between test samples. Lines group samples, and are color coordinated for (+)RNA and (−)RNA. Flat lines group all samples beneath them, inverted Vs indicate comparison of the samples under the ends of the lines. DNV, Danoprevir; LDV, Ledipasvir; DCV, Daclatasvir; SOF, Sofosbuvir. ( F ) Comparison of intracellular HCV transcript number under control (DMSO) conditions as determined by strand-specific RT-qPCR and bDNA FISH. Error bars indicate standard error of the mean; no additional statistical analyses were performed.

    Techniques Used: Infection, Staining, Microscopy, Software, Fluorescence, Quantitative RT-PCR, Fluorescence In Situ Hybridization

    8) Product Images from "The HER2 S310F Mutant Can Form an Active Heterodimer with the EGFR, Which Can Be Inhibited by Cetuximab but Not by Trastuzumab as well as Pertuzumab"

    Article Title: The HER2 S310F Mutant Can Form an Active Heterodimer with the EGFR, Which Can Be Inhibited by Cetuximab but Not by Trastuzumab as well as Pertuzumab

    Journal: Biomolecules

    doi: 10.3390/biom9100629

    Inhibition of 5637 cell proliferation and receptor activation by anti-HER2 and anti-EGFR agents. Cells were treated with HER2 or EGFR inhibitors for 96 h. ( a ) Cell viability was measured by WST-1 kit as described in materials and methods. Relative cell viability as a percent was determined as ((absorbance in each treatment set—absorbance in untreated set)/absorbance in non-treated set X 100). Results represent the mean ± SD obtained from three independent experiments. * p
    Figure Legend Snippet: Inhibition of 5637 cell proliferation and receptor activation by anti-HER2 and anti-EGFR agents. Cells were treated with HER2 or EGFR inhibitors for 96 h. ( a ) Cell viability was measured by WST-1 kit as described in materials and methods. Relative cell viability as a percent was determined as ((absorbance in each treatment set—absorbance in untreated set)/absorbance in non-treated set X 100). Results represent the mean ± SD obtained from three independent experiments. * p

    Techniques Used: Inhibition, Activation Assay

    9) Product Images from "The Dietary Flavone Luteolin Epigenetically Activates the Nrf2 Pathway and Blocks Cell Transformation in Human Colorectal Cancer HCT116 Cells"

    Article Title: The Dietary Flavone Luteolin Epigenetically Activates the Nrf2 Pathway and Blocks Cell Transformation in Human Colorectal Cancer HCT116 Cells

    Journal: Journal of cellular biochemistry

    doi: 10.1002/jcb.27275

    LUT activated Nrf2 and its downstream genes. ( A-C ) mRNA expression of Nrf2, HO-1, and NQO1 in HCT116 cells treated with LUT. HCT116 cells were treated with various concentrations of LUT for 3 days, and total RNA was extracted and converted to cDNA. Relative gene expression was assessed by qPCR. ( D-E ) Protein expression of Nrf2, HO-1, and NQO-1 in HCT116 cells treated with LUT. HCT116 cells were treated with various concentrations of LUT for 3 days, and then protein levels were measured by western blotting. Representative images of blots are shown in D. The relative densities of the blots were measured by ImageJ software. The data are presented as the mean ± SD from three independent experiments, as shown in E. * P
    Figure Legend Snippet: LUT activated Nrf2 and its downstream genes. ( A-C ) mRNA expression of Nrf2, HO-1, and NQO1 in HCT116 cells treated with LUT. HCT116 cells were treated with various concentrations of LUT for 3 days, and total RNA was extracted and converted to cDNA. Relative gene expression was assessed by qPCR. ( D-E ) Protein expression of Nrf2, HO-1, and NQO-1 in HCT116 cells treated with LUT. HCT116 cells were treated with various concentrations of LUT for 3 days, and then protein levels were measured by western blotting. Representative images of blots are shown in D. The relative densities of the blots were measured by ImageJ software. The data are presented as the mean ± SD from three independent experiments, as shown in E. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Software

    Related Articles

    Clone Assay:

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: The total RNA was reverse-transcribed using SuperScript III first-strand synthesis system (Invitrogen) according to manufacturer’s protocol. cDNA of ELF3 , FOXB1 , HOMEZ , ID4 , IRX3 , LHX5 , OTX2 , PAX6a , SMAD1 , SMAD4 and SOX1 were amplified. .. BARHL1 (catalog#, MHS6278-213245170), MAF (catalog#, MHS6278-202806268), NR2F2 (catalog#, MHS6278-202800802), NR3C1 (catalog#, MHS6278-202832263), POU3F2 (catalog#, OHS6271-213587035) and SOX2 (catalog#, MHS6278-202826163) cDNA clones were obtained from Dharmacon.

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: The sequences of cloned cDNA were confirmed by Sanger sequencing. .. Reverse-transcribed using Superscript III first-strand synthesis system (Invitrogen). qPCR was performed using SsoFast EvaGreen supermix (Bio Rad) according to manufacturer’s instruction.

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
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    Article Title: Humanization of Murine Neutralizing Antibodies against Human Herpesvirus 6B
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    Article Title: Combinatorial genetic replenishments in myocardial and outflow tract tissues restore heart function in tnnt2 mutant zebrafish
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    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: Individual colonies were isolated to obtain clonal cell populations and expanded for 2 weeks in blasticidin media. sgRNA sequences for SOX1 , IRX3 , OTX2 enhancers, PAX6 promoter and non-targeting negative control sequence were amplified as a part of PCR primers ( , sheet 3) using the pLG1 plasmid (gift from Prof. Jonathan Weissman) as a template and cloned into XhoI and BstXI site of the pLG1. .. Reverse-transcription was carried out using SuperScript III first-strand synthesis system (Invitrogen). qPCR was performed using SsoFast EvaGreen supermix (Bio Rad) according to the manufacturer’s protocol.

    Amplification:

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: .. The total RNA was reverse-transcribed using SuperScript III first-strand synthesis system (Invitrogen) according to manufacturer’s protocol. cDNA of ELF3 , FOXB1 , HOMEZ , ID4 , IRX3 , LHX5 , OTX2 , PAX6a , SMAD1 , SMAD4 and SOX1 were amplified. .. BARHL1 (catalog#, MHS6278-213245170), MAF (catalog#, MHS6278-202806268), NR2F2 (catalog#, MHS6278-202800802), NR3C1 (catalog#, MHS6278-202832263), POU3F2 (catalog#, OHS6271-213587035) and SOX2 (catalog#, MHS6278-202826163) cDNA clones were obtained from Dharmacon.

    Article Title: Visualization of Positive and Negative Sense Viral RNA for Probing the Mechanism of Direct-Acting Antivirals against Hepatitis C Virus
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    Article Title: Generation of a Nebulizable CDR-Modified MERS-CoV Neutralizing Human Antibody
    Article Snippet: The RNA was used to synthesize cDNA using Superscript III First-Strand Synthesis system (Invitrogen) with oligo(dT) primers according to the manufacturer’s instructions. .. Using the cDNA as a template, the genes encoding the variable regions of heavy and light chains (VH and VΚ /Vλ ) were amplified and used for the construction of a human scFv phage-display libraries as described previously [ , ].

    Article Title: The HER2 S310F Mutant Can Form an Active Heterodimer with the EGFR, Which Can Be Inhibited by Cetuximab but Not by Trastuzumab as well as Pertuzumab
    Article Snippet: .. After cDNA was synthesized using a SuperScript III First-Strand Synthesis system (Invitrogen), the HER2 gene fragment encoding from N302 residue to R340 residue was amplified using specific primer sets (HER2 forward: 5′-GCCTCCACTTCAACCACAGTGGC-3′ and HER2 reverse: 5′-CTGTGATCTCTTCCAGAGTCTCAAAC-3′). .. The PCR conditions were as follows: Preliminary denaturation at 95 °C for 7 min, followed by 25 cycles of 30 s at 95 °C, 30 s at 54 °C, and 1 min at 72 °C.

    Article Title: Humanization of Murine Neutralizing Antibodies against Human Herpesvirus 6B
    Article Snippet: The cDNA of each MAb was constructed by reverse transcription-PCR (RT-PCR) using an oligo(dT)20 primer and the SuperScript III first-strand synthesis system (Invitrogen). .. The cDNAs of the heavy-chain variable domains were amplified by PCR with high-degeneracy primers for the mouse heavy-chain constant region and mouse heavy-chain framework 1 (FR1) region ( ).

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: Individual colonies were isolated to obtain clonal cell populations and expanded for 2 weeks in blasticidin media. sgRNA sequences for SOX1 , IRX3 , OTX2 enhancers, PAX6 promoter and non-targeting negative control sequence were amplified as a part of PCR primers ( , sheet 3) using the pLG1 plasmid (gift from Prof. Jonathan Weissman) as a template and cloned into XhoI and BstXI site of the pLG1. .. Reverse-transcription was carried out using SuperScript III first-strand synthesis system (Invitrogen). qPCR was performed using SsoFast EvaGreen supermix (Bio Rad) according to the manufacturer’s protocol.

    Stable Transfection:

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: To generate a H1 hESC line that stably expresses dCas9-VP64, the lenti dCAS-VP64_Blast vector (Addgene, #61425, RRID:Addgene_61425) was transduced into H1 hESCs via lentivirus at a MOI of 0.2 along with 8 μg/mL polybrene (Sigma) and incubated for 2 days to allow genomic integration. .. Reverse-transcription was carried out using SuperScript III first-strand synthesis system (Invitrogen). qPCR was performed using SsoFast EvaGreen supermix (Bio Rad) according to the manufacturer’s protocol.

    Synthesized:

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: The total RNA was reverse-transcribed using SuperScript III first-strand synthesis system (Invitrogen) according to manufacturer’s protocol. cDNA of ELF3 , FOXB1 , HOMEZ , ID4 , IRX3 , LHX5 , OTX2 , PAX6a , SMAD1 , SMAD4 and SOX1 were amplified. .. BACH2 , DMBX1 , FOSL2 , NFE2 , OTX1 , SOX5 and TCF7L2 cDNA sequences were synthesized by Twist Bioscience.

    Article Title: Generation of a Nebulizable CDR-Modified MERS-CoV Neutralizing Human Antibody
    Article Snippet: The RNA was used to synthesize cDNA using Superscript III First-Strand Synthesis system (Invitrogen) with oligo(dT) primers according to the manufacturer’s instructions. .. For the construction of the first randomization library, a set of degenerate Ultramer DNA oligonucleotides (Integrated DNA Technologies, Coralville, IA, USA) encoding residues from H1 to H65 of clone C-8 (VHN1 ) was chemically synthesized to contain either a codon encoding the wild-type amino acid or a GAK degenerate codon at the H29, H32, H51, H52, H53, and H54 residues ( ).

    Article Title: Sertoli cell-only phenotype and scRNA-seq define PRAMEF12 as a factor essential for spermatogenesis in mice
    Article Snippet: .. RNA isolation and quantitative real-time RT-PCR Total RNA was isolated from mouse tissues using RNeasy Mini Kit (Qiagen) and cDNA was synthesized with SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific). .. Quantitative RT-PCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad) and QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific).

    Article Title: The HER2 S310F Mutant Can Form an Active Heterodimer with the EGFR, Which Can Be Inhibited by Cetuximab but Not by Trastuzumab as well as Pertuzumab
    Article Snippet: .. After cDNA was synthesized using a SuperScript III First-Strand Synthesis system (Invitrogen), the HER2 gene fragment encoding from N302 residue to R340 residue was amplified using specific primer sets (HER2 forward: 5′-GCCTCCACTTCAACCACAGTGGC-3′ and HER2 reverse: 5′-CTGTGATCTCTTCCAGAGTCTCAAAC-3′). .. The PCR conditions were as follows: Preliminary denaturation at 95 °C for 7 min, followed by 25 cycles of 30 s at 95 °C, 30 s at 54 °C, and 1 min at 72 °C.

    TA Cloning:

    Article Title: Humanization of Murine Neutralizing Antibodies against Human Herpesvirus 6B
    Article Snippet: The cDNA of each MAb was constructed by reverse transcription-PCR (RT-PCR) using an oligo(dT)20 primer and the SuperScript III first-strand synthesis system (Invitrogen). .. The PCR products were cloned into a pCR4 TOPO vector using a TOPO TA cloning kit (Invitrogen), and the vectors were used to transform Escherichia coli strain DH5α.

    Quantitative RT-PCR:

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: Paragraph title: Overexpression and RT-qPCR ... The total RNA was reverse-transcribed using SuperScript III first-strand synthesis system (Invitrogen) according to manufacturer’s protocol. cDNA of ELF3 , FOXB1 , HOMEZ , ID4 , IRX3 , LHX5 , OTX2 , PAX6a , SMAD1 , SMAD4 and SOX1 were amplified.

    Article Title: Visualization of Positive and Negative Sense Viral RNA for Probing the Mechanism of Direct-Acting Antivirals against Hepatitis C Virus
    Article Snippet: Paragraph title: 2.3. Strand-Specific Quantitative RT-PCR ... RC21 and RC1 were replaced by Tag-ZK21 primer (5′-ggccgtcatggtggcgaataaCCTGACAACACTAAaATTGGTGC-3′) and Tag-ZK1 primer (5′-ggccgtcatggtggcgaataaAGGATCATAGGTGATGAAGAAAAGT-3′). cDNA synthesis was performed using SuperScript III First-Strand Synthesis System (Invitrogen), following the manufacturers’ instructions. qPCR amplification was conducted using PowerUp SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA), in a PikoReal 96 Real-Time PCR system (ThermoFisher Scientific).

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: .. At day 9 and 12 (72 hours and 6 days post neural induction), total RNA was collected using RNeasy mini kit (QIAGEN) and reverse-transcribed using SuperScript III first-strand synthesis system (Invitrogen). qPCR was performed using SsoFast EvaGreen supermix (Bio Rad) according to the manufacturer’s protocol. sgRNA sequences and primers used for the plasmid construction and RT-qPCR are shown in , sheet 5. .. For RNA-seq, reads were aligned to the hg19 human genome assembly with Tophat2 (Version 2.1.1) , and low quality reads were trimmed or removed with Trimmomatic (Version 0.3.2) ( ).

    Article Title: Sertoli cell-only phenotype and scRNA-seq define PRAMEF12 as a factor essential for spermatogenesis in mice
    Article Snippet: .. RNA isolation and quantitative real-time RT-PCR Total RNA was isolated from mouse tissues using RNeasy Mini Kit (Qiagen) and cDNA was synthesized with SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific). .. Quantitative RT-PCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad) and QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific).

    Article Title: A Stress-Associated Protein, PtSAP13, From Populus trichocarpa Provides Tolerance to Salt Stress
    Article Snippet: Paragraph title: 4.3. RNA Isolation and Quantitative Real-Time PCR (qRT-PCR) Analysis ... The SuperScript III first-strand synthesis system (Life technologies, Carlsbad, CA, USA) was used to synthesize the first-strand cDNA with 3 μg mRNA.

    Article Title: Semaphorin 3E/PlexinD1 signaling is required for cardiac ventricular compaction
    Article Snippet: Paragraph title: RNA extraction and quantitative RT-PCR. ... Total RNA was isolated from embryonic hearts using TRIzol (Life Technologies, catalog 15596-018). cDNA synthesis was performed with 1-μg RNA using random hexamers and the SuperScript-III First-Strand Synthesis system (Life Technologies, catalog 18080-051).

    Article Title: Angiotensin II type 1 receptor-associated protein deficiency attenuates sirtuin1 expression in an immortalised human renal proximal tubule cell line
    Article Snippet: .. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis Total RNA was extracted from the RPTEC with the NucleoSpin RNA Plus kit (Macherey Nagel), and cDNA was produced with the SuperScript III First-Strand Synthesis System (Invitrogen). .. RT-qPCR was performed with a Bio-Rad CFX96 Touch Real-Time PCR Detection System by incubating the reverse transcript product with TaqMan PCR Master Mix and a designed TaqMan probe (Applied Biosystems), essentially as described previously , , .

    SYBR Green Assay:

    Article Title: Visualization of Positive and Negative Sense Viral RNA for Probing the Mechanism of Direct-Acting Antivirals against Hepatitis C Virus
    Article Snippet: .. RC21 and RC1 were replaced by Tag-ZK21 primer (5′-ggccgtcatggtggcgaataaCCTGACAACACTAAaATTGGTGC-3′) and Tag-ZK1 primer (5′-ggccgtcatggtggcgaataaAGGATCATAGGTGATGAAGAAAAGT-3′). cDNA synthesis was performed using SuperScript III First-Strand Synthesis System (Invitrogen), following the manufacturers’ instructions. qPCR amplification was conducted using PowerUp SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA), in a PikoReal 96 Real-Time PCR system (ThermoFisher Scientific). .. The cycle conditions were uracil-DNA glycosylase (UDG) activation at 50 °C for 2 min, dual-lock DNA polymerase at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 15 sec, annealing at 55 °C for 15 sec, and extension at 72 °C for 1 min. An MR766 infectious clone [ ] was used to generate a standard curve, and was subject to the same strand specific RT-qPCR protocol.

    Article Title: Sertoli cell-only phenotype and scRNA-seq define PRAMEF12 as a factor essential for spermatogenesis in mice
    Article Snippet: RNA isolation and quantitative real-time RT-PCR Total RNA was isolated from mouse tissues using RNeasy Mini Kit (Qiagen) and cDNA was synthesized with SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific). .. Quantitative RT-PCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad) and QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific).

    Article Title: Protein lysine 43 methylation by EZH1 promotes AML1-ETO transcriptional repression in leukemia
    Article Snippet: The first strand cDNA synthesis was conducted using the SuperScript® III First-Strand Synthesis System (Invitrogen). .. The expression of EZH1 and other genes was measured using Power SYBR® Green PCR Master Mix (Applied Biosystems).

    Incubation:

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: H1-ESCs cultured in a 24-well plate for 24 hours were infected with the lentivirus with a MOI of 5 along with 8 μg/mL polybrene (Sigma) and incubated for 2 days to allow genomic integration. .. Reverse-transcribed using Superscript III first-strand synthesis system (Invitrogen). qPCR was performed using SsoFast EvaGreen supermix (Bio Rad) according to manufacturer’s instruction.

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: Cells were incubated for 2 days to allow genomic integration and further cultured for 2 days in mTeSR media supplemented with 2 μg/mL puromycin for selection. .. At day 9 and 12 (72 hours and 6 days post neural induction), total RNA was collected using RNeasy mini kit (QIAGEN) and reverse-transcribed using SuperScript III first-strand synthesis system (Invitrogen). qPCR was performed using SsoFast EvaGreen supermix (Bio Rad) according to the manufacturer’s protocol. sgRNA sequences and primers used for the plasmid construction and RT-qPCR are shown in , sheet 5.

    Article Title: The Dietary Flavone Luteolin Epigenetically Activates the Nrf2 Pathway and Blocks Cell Transformation in Human Colorectal Cancer HCT116 Cells
    Article Snippet: After incubation for 24 h, the cells were treated with 0.1% DMSO (control), 2.5 μM 5-Aza and 100 nM TSA, or LUT at 15 and 30 μM for 3 days. .. Total RNA was extracted from the treated HCT116 cells using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA), and 1 μg of total RNA was reverse-transcribed to cDNA using the SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA).

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: The sgRNA plasmids were transduced into dCas9-VP64 ESCs via lentivirus at a MOI of 5 along with 8 μg/mL polybrene (Sigma) and incubated for 2 days to allow genomic integration. .. Reverse-transcription was carried out using SuperScript III first-strand synthesis system (Invitrogen). qPCR was performed using SsoFast EvaGreen supermix (Bio Rad) according to the manufacturer’s protocol.

    Cell Culture:

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: H1-ESCs cultured in a 24-well plate for 24 hours were infected with the lentivirus with a MOI of 5 along with 8 μg/mL polybrene (Sigma) and incubated for 2 days to allow genomic integration. .. Reverse-transcribed using Superscript III first-strand synthesis system (Invitrogen). qPCR was performed using SsoFast EvaGreen supermix (Bio Rad) according to manufacturer’s instruction.

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: At day 4 after infection, the cells were replated and cultured in mTeSR supplemented with puromycin. .. At day 9 and 12 (72 hours and 6 days post neural induction), total RNA was collected using RNeasy mini kit (QIAGEN) and reverse-transcribed using SuperScript III first-strand synthesis system (Invitrogen). qPCR was performed using SsoFast EvaGreen supermix (Bio Rad) according to the manufacturer’s protocol. sgRNA sequences and primers used for the plasmid construction and RT-qPCR are shown in , sheet 5.

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: The cells were further cultured for 5 days in a media supplemented with 2 μg/mL blasticidin for selection. .. Reverse-transcription was carried out using SuperScript III first-strand synthesis system (Invitrogen). qPCR was performed using SsoFast EvaGreen supermix (Bio Rad) according to the manufacturer’s protocol.

    Expressing:

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: The overexpression vectors were individually packaged into lentivirus using Lenti-Pac HIV expression packaging kit (GeneCopoeia) and the lentivirus was concentrated using Lenti-X concentrator (Takara) according to the manufacturer’s protocol. .. Reverse-transcribed using Superscript III first-strand synthesis system (Invitrogen). qPCR was performed using SsoFast EvaGreen supermix (Bio Rad) according to manufacturer’s instruction.

    Article Title: Sertoli cell-only phenotype and scRNA-seq define PRAMEF12 as a factor essential for spermatogenesis in mice
    Article Snippet: RNA isolation and quantitative real-time RT-PCR Total RNA was isolated from mouse tissues using RNeasy Mini Kit (Qiagen) and cDNA was synthesized with SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific). .. The relative abundance of each transcript was calculated by the 2−ΔΔCt normalized to endogenous β-actin expression .

    Article Title: Protein lysine 43 methylation by EZH1 promotes AML1-ETO transcriptional repression in leukemia
    Article Snippet: The first strand cDNA synthesis was conducted using the SuperScript® III First-Strand Synthesis System (Invitrogen). .. AML1-ETO expression was detected using the TaqMan® Gene Expression Assay (Applied Biosystems, Foster City, CA).

    Article Title: A Stress-Associated Protein, PtSAP13, From Populus trichocarpa Provides Tolerance to Salt Stress
    Article Snippet: The SuperScript III first-strand synthesis system (Life technologies, Carlsbad, CA, USA) was used to synthesize the first-strand cDNA with 3 μg mRNA. .. The 2−ΔΔCT method was used to calculate the relative expression levels of each gene [ ].

    Article Title: Semaphorin 3E/PlexinD1 signaling is required for cardiac ventricular compaction
    Article Snippet: Total RNA was isolated from embryonic hearts using TRIzol (Life Technologies, catalog 15596-018). cDNA synthesis was performed with 1-μg RNA using random hexamers and the SuperScript-III First-Strand Synthesis system (Life Technologies, catalog 18080-051). .. Gene expression was measured by quantitative RT-PCR using HOT FIREPol EvaGreen qPCR supermix.

    Article Title: The Dietary Flavone Luteolin Epigenetically Activates the Nrf2 Pathway and Blocks Cell Transformation in Human Colorectal Cancer HCT116 Cells
    Article Snippet: Total RNA was extracted from the treated HCT116 cells using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA), and 1 μg of total RNA was reverse-transcribed to cDNA using the SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). .. The relative mRNA expression levels of Nrf2, HO-1, and NQO1 were determined by qPCR on an ABI7900HT system.

    Knock-In:

    Article Title: Combinatorial genetic replenishments in myocardial and outflow tract tissues restore heart function in tnnt2 mutant zebrafish
    Article Snippet: Generation of Tg(myl7:TetOn;tre:tnnt2a-p2a-mKate2 ) zebrafish via Tol2 transposons RNA was extracted from zebrafish at 3 dpf (N =50) and cDNA was obtained using SuperScript® III First-Strand Synthesis System (Invitrogen). .. Knockin plasmid was constructed by attL/attR recombination (LR) reaction.

    Over Expression:

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: Paragraph title: Overexpression and RT-qPCR ... The total RNA was reverse-transcribed using SuperScript III first-strand synthesis system (Invitrogen) according to manufacturer’s protocol. cDNA of ELF3 , FOXB1 , HOMEZ , ID4 , IRX3 , LHX5 , OTX2 , PAX6a , SMAD1 , SMAD4 and SOX1 were amplified.

    Sequencing:

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: The sequences of cloned cDNA were confirmed by Sanger sequencing. .. Reverse-transcribed using Superscript III first-strand synthesis system (Invitrogen). qPCR was performed using SsoFast EvaGreen supermix (Bio Rad) according to manufacturer’s instruction.

    Article Title: The HER2 S310F Mutant Can Form an Active Heterodimer with the EGFR, Which Can Be Inhibited by Cetuximab but Not by Trastuzumab as well as Pertuzumab
    Article Snippet: Paragraph title: 2.4. Sequencing of Amplified HER2 Gene Fragments of 5637 Cell ... After cDNA was synthesized using a SuperScript III First-Strand Synthesis system (Invitrogen), the HER2 gene fragment encoding from N302 residue to R340 residue was amplified using specific primer sets (HER2 forward: 5′-GCCTCCACTTCAACCACAGTGGC-3′ and HER2 reverse: 5′-CTGTGATCTCTTCCAGAGTCTCAAAC-3′).

    Article Title: Humanization of Murine Neutralizing Antibodies against Human Herpesvirus 6B
    Article Snippet: Paragraph title: Sequence determination. ... The cDNA of each MAb was constructed by reverse transcription-PCR (RT-PCR) using an oligo(dT)20 primer and the SuperScript III first-strand synthesis system (Invitrogen).

    Article Title: Semaphorin 3E/PlexinD1 signaling is required for cardiac ventricular compaction
    Article Snippet: Total RNA was isolated from embryonic hearts using TRIzol (Life Technologies, catalog 15596-018). cDNA synthesis was performed with 1-μg RNA using random hexamers and the SuperScript-III First-Strand Synthesis system (Life Technologies, catalog 18080-051). .. All qPCRs were conducted in 384 plates using the 7900HT Sequence Detection System and the results were analyzed using SDS2.0 software (Applied Biosystem).

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: Individual colonies were isolated to obtain clonal cell populations and expanded for 2 weeks in blasticidin media. sgRNA sequences for SOX1 , IRX3 , OTX2 enhancers, PAX6 promoter and non-targeting negative control sequence were amplified as a part of PCR primers ( , sheet 3) using the pLG1 plasmid (gift from Prof. Jonathan Weissman) as a template and cloned into XhoI and BstXI site of the pLG1. .. Reverse-transcription was carried out using SuperScript III first-strand synthesis system (Invitrogen). qPCR was performed using SsoFast EvaGreen supermix (Bio Rad) according to the manufacturer’s protocol.

    Infection:

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: H1-ESCs cultured in a 24-well plate for 24 hours were infected with the lentivirus with a MOI of 5 along with 8 μg/mL polybrene (Sigma) and incubated for 2 days to allow genomic integration. .. Reverse-transcribed using Superscript III first-strand synthesis system (Invitrogen). qPCR was performed using SsoFast EvaGreen supermix (Bio Rad) according to manufacturer’s instruction.

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: At day 4 after infection, the cells were replated and cultured in mTeSR supplemented with puromycin. .. At day 9 and 12 (72 hours and 6 days post neural induction), total RNA was collected using RNeasy mini kit (QIAGEN) and reverse-transcribed using SuperScript III first-strand synthesis system (Invitrogen). qPCR was performed using SsoFast EvaGreen supermix (Bio Rad) according to the manufacturer’s protocol. sgRNA sequences and primers used for the plasmid construction and RT-qPCR are shown in , sheet 5.

    Inhibition:

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: Total RNA was collected from neural progenitor cells differentiated from hESCs by dual-Smad inhibition as described above. .. The total RNA was reverse-transcribed using SuperScript III first-strand synthesis system (Invitrogen) according to manufacturer’s protocol. cDNA of ELF3 , FOXB1 , HOMEZ , ID4 , IRX3 , LHX5 , OTX2 , PAX6a , SMAD1 , SMAD4 and SOX1 were amplified.

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: At day six, cells were induced into a neural lineage by dual-Smad inhibition. .. At day 9 and 12 (72 hours and 6 days post neural induction), total RNA was collected using RNeasy mini kit (QIAGEN) and reverse-transcribed using SuperScript III first-strand synthesis system (Invitrogen). qPCR was performed using SsoFast EvaGreen supermix (Bio Rad) according to the manufacturer’s protocol. sgRNA sequences and primers used for the plasmid construction and RT-qPCR are shown in , sheet 5.

    Polymerase Chain Reaction:

    Article Title: Generation of a Nebulizable CDR-Modified MERS-CoV Neutralizing Human Antibody
    Article Snippet: The RNA was used to synthesize cDNA using Superscript III First-Strand Synthesis system (Invitrogen) with oligo(dT) primers according to the manufacturer’s instructions. .. Then, the gene fragment (VHC ) encoding residues from H58 to H113 of clone C-8 was amplified by PCR using primer set 1 ( ) in a T100 Thermal Cycler (Bio-Rad, Carlsbad, CA, USA).

    Article Title: Protein lysine 43 methylation by EZH1 promotes AML1-ETO transcriptional repression in leukemia
    Article Snippet: The first strand cDNA synthesis was conducted using the SuperScript® III First-Strand Synthesis System (Invitrogen). .. The expression of EZH1 and other genes was measured using Power SYBR® Green PCR Master Mix (Applied Biosystems).

    Article Title: The HER2 S310F Mutant Can Form an Active Heterodimer with the EGFR, Which Can Be Inhibited by Cetuximab but Not by Trastuzumab as well as Pertuzumab
    Article Snippet: After cDNA was synthesized using a SuperScript III First-Strand Synthesis system (Invitrogen), the HER2 gene fragment encoding from N302 residue to R340 residue was amplified using specific primer sets (HER2 forward: 5′-GCCTCCACTTCAACCACAGTGGC-3′ and HER2 reverse: 5′-CTGTGATCTCTTCCAGAGTCTCAAAC-3′). .. The PCR conditions were as follows: Preliminary denaturation at 95 °C for 7 min, followed by 25 cycles of 30 s at 95 °C, 30 s at 54 °C, and 1 min at 72 °C.

    Article Title: Humanization of Murine Neutralizing Antibodies against Human Herpesvirus 6B
    Article Snippet: The cDNA of each MAb was constructed by reverse transcription-PCR (RT-PCR) using an oligo(dT)20 primer and the SuperScript III first-strand synthesis system (Invitrogen). .. The cDNAs of the heavy-chain variable domains were amplified by PCR with high-degeneracy primers for the mouse heavy-chain constant region and mouse heavy-chain framework 1 (FR1) region ( ).

    Article Title: Combinatorial genetic replenishments in myocardial and outflow tract tissues restore heart function in tnnt2 mutant zebrafish
    Article Snippet: Generation of Tg(myl7:TetOn;tre:tnnt2a-p2a-mKate2 ) zebrafish via Tol2 transposons RNA was extracted from zebrafish at 3 dpf (N =50) and cDNA was obtained using SuperScript® III First-Strand Synthesis System (Invitrogen). .. The tnnt2a cDNA was successfully cloned into the PCR® II vector to generate pPCRII_ tnnt2a cDNA.

    Article Title: Angiotensin II type 1 receptor-associated protein deficiency attenuates sirtuin1 expression in an immortalised human renal proximal tubule cell line
    Article Snippet: Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis Total RNA was extracted from the RPTEC with the NucleoSpin RNA Plus kit (Macherey Nagel), and cDNA was produced with the SuperScript III First-Strand Synthesis System (Invitrogen). .. RT-qPCR was performed with a Bio-Rad CFX96 Touch Real-Time PCR Detection System by incubating the reverse transcript product with TaqMan PCR Master Mix and a designed TaqMan probe (Applied Biosystems), essentially as described previously , , .

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: Individual colonies were isolated to obtain clonal cell populations and expanded for 2 weeks in blasticidin media. sgRNA sequences for SOX1 , IRX3 , OTX2 enhancers, PAX6 promoter and non-targeting negative control sequence were amplified as a part of PCR primers ( , sheet 3) using the pLG1 plasmid (gift from Prof. Jonathan Weissman) as a template and cloned into XhoI and BstXI site of the pLG1. .. Reverse-transcription was carried out using SuperScript III first-strand synthesis system (Invitrogen). qPCR was performed using SsoFast EvaGreen supermix (Bio Rad) according to the manufacturer’s protocol.

    Isolation:

    Article Title: Generation of a Nebulizable CDR-Modified MERS-CoV Neutralizing Human Antibody
    Article Snippet: The PBMCs were subjected to total RNA isolation using the TRI Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. .. The RNA was used to synthesize cDNA using Superscript III First-Strand Synthesis system (Invitrogen) with oligo(dT) primers according to the manufacturer’s instructions.

    Article Title: Sertoli cell-only phenotype and scRNA-seq define PRAMEF12 as a factor essential for spermatogenesis in mice
    Article Snippet: .. RNA isolation and quantitative real-time RT-PCR Total RNA was isolated from mouse tissues using RNeasy Mini Kit (Qiagen) and cDNA was synthesized with SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific). .. Quantitative RT-PCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad) and QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific).

    Article Title: Protein lysine 43 methylation by EZH1 promotes AML1-ETO transcriptional repression in leukemia
    Article Snippet: Paragraph title: RNA isolation and quantitative PCR (qPCR) ... The first strand cDNA synthesis was conducted using the SuperScript® III First-Strand Synthesis System (Invitrogen).

    Article Title: A Stress-Associated Protein, PtSAP13, From Populus trichocarpa Provides Tolerance to Salt Stress
    Article Snippet: Paragraph title: 4.3. RNA Isolation and Quantitative Real-Time PCR (qRT-PCR) Analysis ... The SuperScript III first-strand synthesis system (Life technologies, Carlsbad, CA, USA) was used to synthesize the first-strand cDNA with 3 μg mRNA.

    Article Title: The HER2 S310F Mutant Can Form an Active Heterodimer with the EGFR, Which Can Be Inhibited by Cetuximab but Not by Trastuzumab as well as Pertuzumab
    Article Snippet: Sequencing of Amplified HER2 Gene Fragments of 5637 Cell Total RNA was isolated from the 5637 cells using TRIzol reagent (Invitrogen) as previously described [ ]. .. After cDNA was synthesized using a SuperScript III First-Strand Synthesis system (Invitrogen), the HER2 gene fragment encoding from N302 residue to R340 residue was amplified using specific primer sets (HER2 forward: 5′-GCCTCCACTTCAACCACAGTGGC-3′ and HER2 reverse: 5′-CTGTGATCTCTTCCAGAGTCTCAAAC-3′).

    Article Title: Semaphorin 3E/PlexinD1 signaling is required for cardiac ventricular compaction
    Article Snippet: .. Total RNA was isolated from embryonic hearts using TRIzol (Life Technologies, catalog 15596-018). cDNA synthesis was performed with 1-μg RNA using random hexamers and the SuperScript-III First-Strand Synthesis system (Life Technologies, catalog 18080-051). .. Samples were treated with DNaseI before reverse transcription to remove any DNA contamination (Thermo Fisher Scientific, catalog 18068015).

    Article Title: The Dietary Flavone Luteolin Epigenetically Activates the Nrf2 Pathway and Blocks Cell Transformation in Human Colorectal Cancer HCT116 Cells
    Article Snippet: Paragraph title: 2.5. RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction (qPCR) ... Total RNA was extracted from the treated HCT116 cells using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA), and 1 μg of total RNA was reverse-transcribed to cDNA using the SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA).

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: Individual colonies were isolated to obtain clonal cell populations and expanded for 2 weeks in blasticidin media. sgRNA sequences for SOX1 , IRX3 , OTX2 enhancers, PAX6 promoter and non-targeting negative control sequence were amplified as a part of PCR primers ( , sheet 3) using the pLG1 plasmid (gift from Prof. Jonathan Weissman) as a template and cloned into XhoI and BstXI site of the pLG1. .. Reverse-transcription was carried out using SuperScript III first-strand synthesis system (Invitrogen). qPCR was performed using SsoFast EvaGreen supermix (Bio Rad) according to the manufacturer’s protocol.

    Size-exclusion Chromatography:

    Article Title: Visualization of Positive and Negative Sense Viral RNA for Probing the Mechanism of Direct-Acting Antivirals against Hepatitis C Virus
    Article Snippet: RC21 and RC1 were replaced by Tag-ZK21 primer (5′-ggccgtcatggtggcgaataaCCTGACAACACTAAaATTGGTGC-3′) and Tag-ZK1 primer (5′-ggccgtcatggtggcgaataaAGGATCATAGGTGATGAAGAAAAGT-3′). cDNA synthesis was performed using SuperScript III First-Strand Synthesis System (Invitrogen), following the manufacturers’ instructions. qPCR amplification was conducted using PowerUp SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA), in a PikoReal 96 Real-Time PCR system (ThermoFisher Scientific). .. The cycle conditions were uracil-DNA glycosylase (UDG) activation at 50 °C for 2 min, dual-lock DNA polymerase at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 15 sec, annealing at 55 °C for 15 sec, and extension at 72 °C for 1 min. An MR766 infectious clone [ ] was used to generate a standard curve, and was subject to the same strand specific RT-qPCR protocol.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Humanization of Murine Neutralizing Antibodies against Human Herpesvirus 6B
    Article Snippet: .. The cDNA of each MAb was constructed by reverse transcription-PCR (RT-PCR) using an oligo(dT)20 primer and the SuperScript III first-strand synthesis system (Invitrogen). .. The cDNAs of the heavy-chain variable domains were amplified by PCR with high-degeneracy primers for the mouse heavy-chain constant region and mouse heavy-chain framework 1 (FR1) region ( ).

    Construct:

    Article Title: Humanization of Murine Neutralizing Antibodies against Human Herpesvirus 6B
    Article Snippet: .. The cDNA of each MAb was constructed by reverse transcription-PCR (RT-PCR) using an oligo(dT)20 primer and the SuperScript III first-strand synthesis system (Invitrogen). .. The cDNAs of the heavy-chain variable domains were amplified by PCR with high-degeneracy primers for the mouse heavy-chain constant region and mouse heavy-chain framework 1 (FR1) region ( ).

    Article Title: Combinatorial genetic replenishments in myocardial and outflow tract tissues restore heart function in tnnt2 mutant zebrafish
    Article Snippet: Generation of Tg(myl7:TetOn;tre:tnnt2a-p2a-mKate2 ) zebrafish via Tol2 transposons RNA was extracted from zebrafish at 3 dpf (N =50) and cDNA was obtained using SuperScript® III First-Strand Synthesis System (Invitrogen). .. Knockin plasmid was constructed by attL/attR recombination (LR) reaction.

    IA:

    Article Title: Generation of a Nebulizable CDR-Modified MERS-CoV Neutralizing Human Antibody
    Article Snippet: The RNA was used to synthesize cDNA using Superscript III First-Strand Synthesis system (Invitrogen) with oligo(dT) primers according to the manufacturer’s instructions. .. For the construction of the first randomization library, a set of degenerate Ultramer DNA oligonucleotides (Integrated DNA Technologies, Coralville, IA, USA) encoding residues from H1 to H65 of clone C-8 (VHN1 ) was chemically synthesized to contain either a codon encoding the wild-type amino acid or a GAK degenerate codon at the H29, H32, H51, H52, H53, and H54 residues ( ).

    RNA Extraction:

    Article Title: Semaphorin 3E/PlexinD1 signaling is required for cardiac ventricular compaction
    Article Snippet: Paragraph title: RNA extraction and quantitative RT-PCR. ... Total RNA was isolated from embryonic hearts using TRIzol (Life Technologies, catalog 15596-018). cDNA synthesis was performed with 1-μg RNA using random hexamers and the SuperScript-III First-Strand Synthesis system (Life Technologies, catalog 18080-051).

    Plasmid Preparation:

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: .. At day 9 and 12 (72 hours and 6 days post neural induction), total RNA was collected using RNeasy mini kit (QIAGEN) and reverse-transcribed using SuperScript III first-strand synthesis system (Invitrogen). qPCR was performed using SsoFast EvaGreen supermix (Bio Rad) according to the manufacturer’s protocol. sgRNA sequences and primers used for the plasmid construction and RT-qPCR are shown in , sheet 5. .. For RNA-seq, reads were aligned to the hg19 human genome assembly with Tophat2 (Version 2.1.1) , and low quality reads were trimmed or removed with Trimmomatic (Version 0.3.2) ( ).

    Article Title: Humanization of Murine Neutralizing Antibodies against Human Herpesvirus 6B
    Article Snippet: The cDNA of each MAb was constructed by reverse transcription-PCR (RT-PCR) using an oligo(dT)20 primer and the SuperScript III first-strand synthesis system (Invitrogen). .. The PCR products were cloned into a pCR4 TOPO vector using a TOPO TA cloning kit (Invitrogen), and the vectors were used to transform Escherichia coli strain DH5α.

    Article Title: Combinatorial genetic replenishments in myocardial and outflow tract tissues restore heart function in tnnt2 mutant zebrafish
    Article Snippet: Generation of Tg(myl7:TetOn;tre:tnnt2a-p2a-mKate2 ) zebrafish via Tol2 transposons RNA was extracted from zebrafish at 3 dpf (N =50) and cDNA was obtained using SuperScript® III First-Strand Synthesis System (Invitrogen). .. The tnnt2a cDNA was successfully cloned into the PCR® II vector to generate pPCRII_ tnnt2a cDNA.

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: Individual colonies were isolated to obtain clonal cell populations and expanded for 2 weeks in blasticidin media. sgRNA sequences for SOX1 , IRX3 , OTX2 enhancers, PAX6 promoter and non-targeting negative control sequence were amplified as a part of PCR primers ( , sheet 3) using the pLG1 plasmid (gift from Prof. Jonathan Weissman) as a template and cloned into XhoI and BstXI site of the pLG1. .. Reverse-transcription was carried out using SuperScript III first-strand synthesis system (Invitrogen). qPCR was performed using SsoFast EvaGreen supermix (Bio Rad) according to the manufacturer’s protocol.

    Software:

    Article Title: Semaphorin 3E/PlexinD1 signaling is required for cardiac ventricular compaction
    Article Snippet: Total RNA was isolated from embryonic hearts using TRIzol (Life Technologies, catalog 15596-018). cDNA synthesis was performed with 1-μg RNA using random hexamers and the SuperScript-III First-Strand Synthesis system (Life Technologies, catalog 18080-051). .. All qPCRs were conducted in 384 plates using the 7900HT Sequence Detection System and the results were analyzed using SDS2.0 software (Applied Biosystem).

    Real-time Polymerase Chain Reaction:

    Article Title: Visualization of Positive and Negative Sense Viral RNA for Probing the Mechanism of Direct-Acting Antivirals against Hepatitis C Virus
    Article Snippet: .. RC21 and RC1 were replaced by Tag-ZK21 primer (5′-ggccgtcatggtggcgaataaCCTGACAACACTAAaATTGGTGC-3′) and Tag-ZK1 primer (5′-ggccgtcatggtggcgaataaAGGATCATAGGTGATGAAGAAAAGT-3′). cDNA synthesis was performed using SuperScript III First-Strand Synthesis System (Invitrogen), following the manufacturers’ instructions. qPCR amplification was conducted using PowerUp SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA), in a PikoReal 96 Real-Time PCR system (ThermoFisher Scientific). .. The cycle conditions were uracil-DNA glycosylase (UDG) activation at 50 °C for 2 min, dual-lock DNA polymerase at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 15 sec, annealing at 55 °C for 15 sec, and extension at 72 °C for 1 min. An MR766 infectious clone [ ] was used to generate a standard curve, and was subject to the same strand specific RT-qPCR protocol.

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: .. Reverse-transcribed using Superscript III first-strand synthesis system (Invitrogen). qPCR was performed using SsoFast EvaGreen supermix (Bio Rad) according to manufacturer’s instruction. .. Primer sequences used for qPCR are shown in , sheet 5.

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: .. At day 9 and 12 (72 hours and 6 days post neural induction), total RNA was collected using RNeasy mini kit (QIAGEN) and reverse-transcribed using SuperScript III first-strand synthesis system (Invitrogen). qPCR was performed using SsoFast EvaGreen supermix (Bio Rad) according to the manufacturer’s protocol. sgRNA sequences and primers used for the plasmid construction and RT-qPCR are shown in , sheet 5. .. For RNA-seq, reads were aligned to the hg19 human genome assembly with Tophat2 (Version 2.1.1) , and low quality reads were trimmed or removed with Trimmomatic (Version 0.3.2) ( ).

    Article Title: Sertoli cell-only phenotype and scRNA-seq define PRAMEF12 as a factor essential for spermatogenesis in mice
    Article Snippet: RNA isolation and quantitative real-time RT-PCR Total RNA was isolated from mouse tissues using RNeasy Mini Kit (Qiagen) and cDNA was synthesized with SuperScript III First-Strand Synthesis System (Thermo Fisher Scientific). .. Quantitative RT-PCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad) and QuantStudio 6 Flex Real-Time PCR System (Thermo Fisher Scientific).

    Article Title: Protein lysine 43 methylation by EZH1 promotes AML1-ETO transcriptional repression in leukemia
    Article Snippet: Paragraph title: RNA isolation and quantitative PCR (qPCR) ... The first strand cDNA synthesis was conducted using the SuperScript® III First-Strand Synthesis System (Invitrogen).

    Article Title: A Stress-Associated Protein, PtSAP13, From Populus trichocarpa Provides Tolerance to Salt Stress
    Article Snippet: Paragraph title: 4.3. RNA Isolation and Quantitative Real-Time PCR (qRT-PCR) Analysis ... The SuperScript III first-strand synthesis system (Life technologies, Carlsbad, CA, USA) was used to synthesize the first-strand cDNA with 3 μg mRNA.

    Article Title: Semaphorin 3E/PlexinD1 signaling is required for cardiac ventricular compaction
    Article Snippet: Total RNA was isolated from embryonic hearts using TRIzol (Life Technologies, catalog 15596-018). cDNA synthesis was performed with 1-μg RNA using random hexamers and the SuperScript-III First-Strand Synthesis system (Life Technologies, catalog 18080-051). .. Gene expression was measured by quantitative RT-PCR using HOT FIREPol EvaGreen qPCR supermix.

    Article Title: Angiotensin II type 1 receptor-associated protein deficiency attenuates sirtuin1 expression in an immortalised human renal proximal tubule cell line
    Article Snippet: .. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis Total RNA was extracted from the RPTEC with the NucleoSpin RNA Plus kit (Macherey Nagel), and cDNA was produced with the SuperScript III First-Strand Synthesis System (Invitrogen). .. RT-qPCR was performed with a Bio-Rad CFX96 Touch Real-Time PCR Detection System by incubating the reverse transcript product with TaqMan PCR Master Mix and a designed TaqMan probe (Applied Biosystems), essentially as described previously , , .

    Article Title: The Dietary Flavone Luteolin Epigenetically Activates the Nrf2 Pathway and Blocks Cell Transformation in Human Colorectal Cancer HCT116 Cells
    Article Snippet: Paragraph title: 2.5. RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction (qPCR) ... Total RNA was extracted from the treated HCT116 cells using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA), and 1 μg of total RNA was reverse-transcribed to cDNA using the SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA).

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: .. Reverse-transcription was carried out using SuperScript III first-strand synthesis system (Invitrogen). qPCR was performed using SsoFast EvaGreen supermix (Bio Rad) according to the manufacturer’s protocol. .. Primer sequences used for qPCR are shown in , sheet 5.

    Negative Control:

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: Individual colonies were isolated to obtain clonal cell populations and expanded for 2 weeks in blasticidin media. sgRNA sequences for SOX1 , IRX3 , OTX2 enhancers, PAX6 promoter and non-targeting negative control sequence were amplified as a part of PCR primers ( , sheet 3) using the pLG1 plasmid (gift from Prof. Jonathan Weissman) as a template and cloned into XhoI and BstXI site of the pLG1. .. Reverse-transcription was carried out using SuperScript III first-strand synthesis system (Invitrogen). qPCR was performed using SsoFast EvaGreen supermix (Bio Rad) according to the manufacturer’s protocol.

    Selection:

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: Cells were incubated for 2 days to allow genomic integration and further cultured for 2 days in mTeSR media supplemented with 2 μg/mL puromycin for selection. .. At day 9 and 12 (72 hours and 6 days post neural induction), total RNA was collected using RNeasy mini kit (QIAGEN) and reverse-transcribed using SuperScript III first-strand synthesis system (Invitrogen). qPCR was performed using SsoFast EvaGreen supermix (Bio Rad) according to the manufacturer’s protocol. sgRNA sequences and primers used for the plasmid construction and RT-qPCR are shown in , sheet 5.

    Article Title: Identification and Massively Parallel Characterization of Regulatory Elements Driving Neural Induction
    Article Snippet: The cells were further cultured for 5 days in a media supplemented with 2 μg/mL blasticidin for selection. .. Reverse-transcription was carried out using SuperScript III first-strand synthesis system (Invitrogen). qPCR was performed using SsoFast EvaGreen supermix (Bio Rad) according to the manufacturer’s protocol.

    Agarose Gel Electrophoresis:

    Article Title: The HER2 S310F Mutant Can Form an Active Heterodimer with the EGFR, Which Can Be Inhibited by Cetuximab but Not by Trastuzumab as well as Pertuzumab
    Article Snippet: After cDNA was synthesized using a SuperScript III First-Strand Synthesis system (Invitrogen), the HER2 gene fragment encoding from N302 residue to R340 residue was amplified using specific primer sets (HER2 forward: 5′-GCCTCCACTTCAACCACAGTGGC-3′ and HER2 reverse: 5′-CTGTGATCTCTTCCAGAGTCTCAAAC-3′). .. After agarose gel electrophoresis, the amplified DNA was extracted using the Qiagen Gel Extraction Kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany) and subjected to Sanger sequencing (Macrogen, Seoul, Korea).

    In Vitro:

    Article Title: Visualization of Positive and Negative Sense Viral RNA for Probing the Mechanism of Direct-Acting Antivirals against Hepatitis C Virus
    Article Snippet: In vitro transcribed RNA from the HCV infectious clone was used to generate a standard curve. .. RC21 and RC1 were replaced by Tag-ZK21 primer (5′-ggccgtcatggtggcgaataaCCTGACAACACTAAaATTGGTGC-3′) and Tag-ZK1 primer (5′-ggccgtcatggtggcgaataaAGGATCATAGGTGATGAAGAAAAGT-3′). cDNA synthesis was performed using SuperScript III First-Strand Synthesis System (Invitrogen), following the manufacturers’ instructions. qPCR amplification was conducted using PowerUp SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA), in a PikoReal 96 Real-Time PCR system (ThermoFisher Scientific).

    Produced:

    Article Title: Angiotensin II type 1 receptor-associated protein deficiency attenuates sirtuin1 expression in an immortalised human renal proximal tubule cell line
    Article Snippet: .. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis Total RNA was extracted from the RPTEC with the NucleoSpin RNA Plus kit (Macherey Nagel), and cDNA was produced with the SuperScript III First-Strand Synthesis System (Invitrogen). .. RT-qPCR was performed with a Bio-Rad CFX96 Touch Real-Time PCR Detection System by incubating the reverse transcript product with TaqMan PCR Master Mix and a designed TaqMan probe (Applied Biosystems), essentially as described previously , , .

    Activation Assay:

    Article Title: Visualization of Positive and Negative Sense Viral RNA for Probing the Mechanism of Direct-Acting Antivirals against Hepatitis C Virus
    Article Snippet: RC21 and RC1 were replaced by Tag-ZK21 primer (5′-ggccgtcatggtggcgaataaCCTGACAACACTAAaATTGGTGC-3′) and Tag-ZK1 primer (5′-ggccgtcatggtggcgaataaAGGATCATAGGTGATGAAGAAAAGT-3′). cDNA synthesis was performed using SuperScript III First-Strand Synthesis System (Invitrogen), following the manufacturers’ instructions. qPCR amplification was conducted using PowerUp SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA), in a PikoReal 96 Real-Time PCR system (ThermoFisher Scientific). .. The cycle conditions were uracil-DNA glycosylase (UDG) activation at 50 °C for 2 min, dual-lock DNA polymerase at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 15 sec, annealing at 55 °C for 15 sec, and extension at 72 °C for 1 min. An MR766 infectious clone [ ] was used to generate a standard curve, and was subject to the same strand specific RT-qPCR protocol.

    Gel Extraction:

    Article Title: The HER2 S310F Mutant Can Form an Active Heterodimer with the EGFR, Which Can Be Inhibited by Cetuximab but Not by Trastuzumab as well as Pertuzumab
    Article Snippet: After cDNA was synthesized using a SuperScript III First-Strand Synthesis system (Invitrogen), the HER2 gene fragment encoding from N302 residue to R340 residue was amplified using specific primer sets (HER2 forward: 5′-GCCTCCACTTCAACCACAGTGGC-3′ and HER2 reverse: 5′-CTGTGATCTCTTCCAGAGTCTCAAAC-3′). .. After agarose gel electrophoresis, the amplified DNA was extracted using the Qiagen Gel Extraction Kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany) and subjected to Sanger sequencing (Macrogen, Seoul, Korea).

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