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    Structured Review

    Thermo Fisher superscript iii
    Planar cell polarity in the developing cochlea of <t>Ripor2</t> deficient mice. (A) Representative single plane confocal images showing the kinocilium position and the bundle orientation of outer and inner hair cells in cochleae from wild type and Ripor2 deficient mice at E18.5 and P5. Kinocilia and the stereociliary bundles were stained with an acetylated-α-tubulin antibody (green signals) and phalloidin (red signals), respectively. (B) Bundle orientation plots generated using Oriana 4 circular graphing software (Kovach Computing Services). Each dot represent individual stereociliary bundle orientation angles with 90º indicating the normal angle formed between the longitudinal axis and mediolateral axis. Measurements (n > 100 per each plot) were taken in all <t>three</t> different portions of the length of the cochlea (base, middle and apex) from Ripor2 KO (red) and wild type (green) mice at E18.5 and P5. Statistical significance was analyzed by chi-square.*p
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    Images

    1) Product Images from "Ripor2 is involved in auditory hair cell stereociliary bundle structure and orientation"

    Article Title: Ripor2 is involved in auditory hair cell stereociliary bundle structure and orientation

    Journal: Journal of molecular medicine (Berlin, Germany)

    doi: 10.1007/s00109-018-1694-x

    Planar cell polarity in the developing cochlea of Ripor2 deficient mice. (A) Representative single plane confocal images showing the kinocilium position and the bundle orientation of outer and inner hair cells in cochleae from wild type and Ripor2 deficient mice at E18.5 and P5. Kinocilia and the stereociliary bundles were stained with an acetylated-α-tubulin antibody (green signals) and phalloidin (red signals), respectively. (B) Bundle orientation plots generated using Oriana 4 circular graphing software (Kovach Computing Services). Each dot represent individual stereociliary bundle orientation angles with 90º indicating the normal angle formed between the longitudinal axis and mediolateral axis. Measurements (n > 100 per each plot) were taken in all three different portions of the length of the cochlea (base, middle and apex) from Ripor2 KO (red) and wild type (green) mice at E18.5 and P5. Statistical significance was analyzed by chi-square.*p
    Figure Legend Snippet: Planar cell polarity in the developing cochlea of Ripor2 deficient mice. (A) Representative single plane confocal images showing the kinocilium position and the bundle orientation of outer and inner hair cells in cochleae from wild type and Ripor2 deficient mice at E18.5 and P5. Kinocilia and the stereociliary bundles were stained with an acetylated-α-tubulin antibody (green signals) and phalloidin (red signals), respectively. (B) Bundle orientation plots generated using Oriana 4 circular graphing software (Kovach Computing Services). Each dot represent individual stereociliary bundle orientation angles with 90º indicating the normal angle formed between the longitudinal axis and mediolateral axis. Measurements (n > 100 per each plot) were taken in all three different portions of the length of the cochlea (base, middle and apex) from Ripor2 KO (red) and wild type (green) mice at E18.5 and P5. Statistical significance was analyzed by chi-square.*p

    Techniques Used: Mouse Assay, Staining, Generated, Software

    Abundance and expression of phospho-Myh9 and Acetyl-α-tubulin in cochlea of wild type and Ripor2 deficient mouse. (A) Representative Z-stack merged projections showing that the pattern of expression of phosphorylated-Myh9 is similar to the kinocilium of inner and outer hair cells and the primary cilium of supporting cells. Whole mount cochlea from P4 wild type mice was immunostained using an anti-Myh9-phospho-S1943 rabbit polyclonal antibody (ab2974, EMD Millipore). Scale bar: 8 μm. (B) Representative Z-stack merged projections showing expression of phospho-Myh9, alpha-acetyl tubulin, and stereociliary actin in P4 WT and Ripor2 KO mice. A SEM image is presented to indicate the base to tip orientation of the kinocilium. Scale bars: 2 μm. (C) Amount of phospho-Myh9 in wild type and Ripor2 KO mice. Cell lysates from P4 mouse cochleae were analyzed by western blotting using an anti-Myh9-phospho-S1943 rabbit polyclonal antibody (ab2974, EMD Millipore). The loading control β-tubulin was detected using a rabbit polyclonal antibody (ab15708, EMD Millipore). (D) Relative density of bands from three independent experiments were measured using Image J. Results are presented as average ± SD, * p
    Figure Legend Snippet: Abundance and expression of phospho-Myh9 and Acetyl-α-tubulin in cochlea of wild type and Ripor2 deficient mouse. (A) Representative Z-stack merged projections showing that the pattern of expression of phosphorylated-Myh9 is similar to the kinocilium of inner and outer hair cells and the primary cilium of supporting cells. Whole mount cochlea from P4 wild type mice was immunostained using an anti-Myh9-phospho-S1943 rabbit polyclonal antibody (ab2974, EMD Millipore). Scale bar: 8 μm. (B) Representative Z-stack merged projections showing expression of phospho-Myh9, alpha-acetyl tubulin, and stereociliary actin in P4 WT and Ripor2 KO mice. A SEM image is presented to indicate the base to tip orientation of the kinocilium. Scale bars: 2 μm. (C) Amount of phospho-Myh9 in wild type and Ripor2 KO mice. Cell lysates from P4 mouse cochleae were analyzed by western blotting using an anti-Myh9-phospho-S1943 rabbit polyclonal antibody (ab2974, EMD Millipore). The loading control β-tubulin was detected using a rabbit polyclonal antibody (ab15708, EMD Millipore). (D) Relative density of bands from three independent experiments were measured using Image J. Results are presented as average ± SD, * p

    Techniques Used: Expressing, Mouse Assay, Western Blot

    Localization and abundance of Myh9 in cochlea of wild type and Ripor2 deficient mouse. ( A and B ) Representative single plane confocal images showing expression of Ripor2 and Myh9 to the stereocilia of outer and inner hair cells in wild type (A) and Ripor2 deficient mice (B) . Sample preparation was performed in parallel for both specimens. The conditions for the capture of images and the laser intensities were the same for both samples. Whole mount cochleae from P4 mice were immunostained using an anti-Ripor2 rabbit polyclonal (HPA031245, Sigma) and an anti-Myh9 mouse monoclonal antibody (H00004627-M01, Abnova). Alexa Fluor 647 Phalloidin (Blue) was utilized for F-actin staining to visualize the stereociliary bundle. Scale bar: 5 μm. (C) Amount of Myh9 in wild type and Ripor2 KO mouse cochleae. Cell lysates from P4 mouse cochleae were analyzed by western blotting using a primary mouse monoclonal antibody (H00004627-M01, Abnova) to detect total Myh9. The loading control β-tubulin was detected using a rabbit polyclonal antibody (ab15708, EMD Millipore). (D) Relative density of bands from three western blot independent experiments were measured using Image J. Results are presented as average ± SD, * p
    Figure Legend Snippet: Localization and abundance of Myh9 in cochlea of wild type and Ripor2 deficient mouse. ( A and B ) Representative single plane confocal images showing expression of Ripor2 and Myh9 to the stereocilia of outer and inner hair cells in wild type (A) and Ripor2 deficient mice (B) . Sample preparation was performed in parallel for both specimens. The conditions for the capture of images and the laser intensities were the same for both samples. Whole mount cochleae from P4 mice were immunostained using an anti-Ripor2 rabbit polyclonal (HPA031245, Sigma) and an anti-Myh9 mouse monoclonal antibody (H00004627-M01, Abnova). Alexa Fluor 647 Phalloidin (Blue) was utilized for F-actin staining to visualize the stereociliary bundle. Scale bar: 5 μm. (C) Amount of Myh9 in wild type and Ripor2 KO mouse cochleae. Cell lysates from P4 mouse cochleae were analyzed by western blotting using a primary mouse monoclonal antibody (H00004627-M01, Abnova) to detect total Myh9. The loading control β-tubulin was detected using a rabbit polyclonal antibody (ab15708, EMD Millipore). (D) Relative density of bands from three western blot independent experiments were measured using Image J. Results are presented as average ± SD, * p

    Techniques Used: Expressing, Mouse Assay, Sample Prep, Staining, Western Blot

    2) Product Images from "Activation of RNase L in Egyptian Rousette Bat-Derived RoNi/7 Cells Is Dependent Primarily on OAS3 and Independent of MAVS Signaling"

    Article Title: Activation of RNase L in Egyptian Rousette Bat-Derived RoNi/7 Cells Is Dependent Primarily on OAS3 and Independent of MAVS Signaling

    Journal: mBio

    doi: 10.1128/mBio.02414-19

    Induction of OAS1, OAS2, OAS3, and IFIT1 mRNA expression in RoNi/7 cells, following treatment with IFN-α or viral infection. RoNi/7 cells were treated with 1,000 U of human universal IFN-α (A) or infected with SINV (B) or SeV (C) (MOI = 10), cells were lysed at 24 h posttreatment or 12 (SINV) or 24 (SeV) h postinfection, and RNA was isolated. The mRNA levels were determined by qRT-PCR, calculated relative to β-actin mRNA, and expressed as fold over levels of mock treatment using the formula 2 −Δ(Δ CT ) (Δ C T = C T gene of interest − C T actin ). The data are pooled from three independent experiments.
    Figure Legend Snippet: Induction of OAS1, OAS2, OAS3, and IFIT1 mRNA expression in RoNi/7 cells, following treatment with IFN-α or viral infection. RoNi/7 cells were treated with 1,000 U of human universal IFN-α (A) or infected with SINV (B) or SeV (C) (MOI = 10), cells were lysed at 24 h posttreatment or 12 (SINV) or 24 (SeV) h postinfection, and RNA was isolated. The mRNA levels were determined by qRT-PCR, calculated relative to β-actin mRNA, and expressed as fold over levels of mock treatment using the formula 2 −Δ(Δ CT ) (Δ C T = C T gene of interest − C T actin ). The data are pooled from three independent experiments.

    Techniques Used: Expressing, Infection, Isolation, Quantitative RT-PCR

    Activation of RNase L during SINV infection of RoNi/7 cells requires OAS3 expression. (A) bOas1 , bOas2 , bOas3 , and bRNase L -KO RoNi/7 cells were mock treated or treated with 1,000 U of IFN-α overnight. Cells were lysed, and proteins were analyzed by polyacrylamide gel electrophoresis followed by Western immunoblotting with rabbit polyclonal antibodies against bOAS3. The arrowhead indicates bOAS2. The data are from one representative of two independent experiments. (B) DNA sequences encompassing the mutations from the Oas1 gene of bOAS1- KO cells and the RNase L gene of bRNase L- KO cells were amplified, sequenced, and compared with the reference sequences of the genes. (C) WT and KO RoNi/7 cells were infected with SINV (MOI = 10 PFU/cell); at 12 h postinfection; cells were lysed; and RNA integrity was assessed on a Bioanalyzer. The positions of 18S and 28S rRNA are indicated. The data are from one representative of two independent experiments. (D) Cells were infected with SINV (MOI = 1 PFU/cell); at the indicated time points, the supernatants were harvested; and infectious viruses were titrated by plaque assay on Vero cells. The viral titer data are pooled from two independent experiments with three biological replicates in each experiment and expressed as means ± SDs (***, P
    Figure Legend Snippet: Activation of RNase L during SINV infection of RoNi/7 cells requires OAS3 expression. (A) bOas1 , bOas2 , bOas3 , and bRNase L -KO RoNi/7 cells were mock treated or treated with 1,000 U of IFN-α overnight. Cells were lysed, and proteins were analyzed by polyacrylamide gel electrophoresis followed by Western immunoblotting with rabbit polyclonal antibodies against bOAS3. The arrowhead indicates bOAS2. The data are from one representative of two independent experiments. (B) DNA sequences encompassing the mutations from the Oas1 gene of bOAS1- KO cells and the RNase L gene of bRNase L- KO cells were amplified, sequenced, and compared with the reference sequences of the genes. (C) WT and KO RoNi/7 cells were infected with SINV (MOI = 10 PFU/cell); at 12 h postinfection; cells were lysed; and RNA integrity was assessed on a Bioanalyzer. The positions of 18S and 28S rRNA are indicated. The data are from one representative of two independent experiments. (D) Cells were infected with SINV (MOI = 1 PFU/cell); at the indicated time points, the supernatants were harvested; and infectious viruses were titrated by plaque assay on Vero cells. The viral titer data are pooled from two independent experiments with three biological replicates in each experiment and expressed as means ± SDs (***, P

    Techniques Used: Activation Assay, Infection, Expressing, Polyacrylamide Gel Electrophoresis, Western Blot, Amplification, Plaque Assay

    3) Product Images from "New Type of Sendai Virus Vector Provides Transgene-Free iPS Cells Derived from Chimpanzee Blood"

    Article Title: New Type of Sendai Virus Vector Provides Transgene-Free iPS Cells Derived from Chimpanzee Blood

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0113052

    Generation of a new temperature-sensitive Sendai virus vector, TS12KOS. ( a ) Comparison of schematic structures among the newly constructed Sendai virus (SeV) vector, TS12KOS, and previous vectors. The TS12KOS vector contains three point mutations in the RNA polymerase–related gene (P) and carries the coding sequences of KLF4 (K), OCT3/4 (O), and SOX2 (S) in the KOS direction. In comparison, the HNL/TS15 c-Myc vector carries two additional mutations, L1361C and L1558I, in the large polymerase (L) gene and an exogenous c- MYC cDNA sequence inserted between the hemagglutinin-neuraminidase (HN) and L genes, and the conventional vectors individually carry three reprogramming factors as indicated. ( b ) iPS cell generation from human skin-derived fibroblasts. The efficiency of iPS cell generation was significantly higher using the TS12KOS vector than with the conventional vectors at all multiplicities of infection (MOI) tested. iPSC colonies were identified on day 28 of induction by the appearance of alkaline phosphatase-positive (AP + ) colonies with embryonic stem (ES) cell-like colony morphology. N1, N2, and N3 represent individual healthy volunteers. Experiments were conducted in triplicate (mean ± SD). * P
    Figure Legend Snippet: Generation of a new temperature-sensitive Sendai virus vector, TS12KOS. ( a ) Comparison of schematic structures among the newly constructed Sendai virus (SeV) vector, TS12KOS, and previous vectors. The TS12KOS vector contains three point mutations in the RNA polymerase–related gene (P) and carries the coding sequences of KLF4 (K), OCT3/4 (O), and SOX2 (S) in the KOS direction. In comparison, the HNL/TS15 c-Myc vector carries two additional mutations, L1361C and L1558I, in the large polymerase (L) gene and an exogenous c- MYC cDNA sequence inserted between the hemagglutinin-neuraminidase (HN) and L genes, and the conventional vectors individually carry three reprogramming factors as indicated. ( b ) iPS cell generation from human skin-derived fibroblasts. The efficiency of iPS cell generation was significantly higher using the TS12KOS vector than with the conventional vectors at all multiplicities of infection (MOI) tested. iPSC colonies were identified on day 28 of induction by the appearance of alkaline phosphatase-positive (AP + ) colonies with embryonic stem (ES) cell-like colony morphology. N1, N2, and N3 represent individual healthy volunteers. Experiments were conducted in triplicate (mean ± SD). * P

    Techniques Used: Plasmid Preparation, Construct, Sequencing, Derivative Assay, Infection

    4) Product Images from "AtPR5K2, a PR5-Like Receptor Kinase, Modulates Plant Responses to Drought Stress by Phosphorylating Protein Phosphatase 2Cs"

    Article Title: AtPR5K2, a PR5-Like Receptor Kinase, Modulates Plant Responses to Drought Stress by Phosphorylating Protein Phosphatase 2Cs

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2019.01146

    Expression patterns and subcellular localization of AtPR5K2. (A) The expression of AtPR5K2 in various tissues of Arabidopsis thaliana . Total RNA was extracted from the roots, rosette leaves, cauline leaves, stems, flowers, and siliques of the wild-type plants. The transcript levels of AtPR5K2 were measured using quantitative reverse transcription PCR (qRT-PCR) and calculated relative to the expression of the endogenous control gene, TUBULIN2 . Error bars represent the ± SD from three independent experiments. (B) Subcellular localization of AtPR5K2 in Arabidopsis protoplasts. 35S:AtPR5K2-GFP and Aquaporin-RFP were coexpressed in Arabidopsis protoplasts, which were analyzed using confocal fluorescence microscopy and photographed after 24 h of incubation at 22°C. Aquaporin-RFP is a plasma-membrane marker. Scale bars represent 10 μm. (C) Subcellular localization of AtPR5K2 in the epidermal cells of tobacco ( Nicotiana benthamiana ) leave expressing 35S:AtPR5K2-GFP before and after plasmolysis. The epidermal cells were analyzed using confocal fluorescence microscopy and photographed after 48 h of incubation at 25°C. Scale bars represent 20 μm. Red asterisk indicates that AtPR5K2-GFP signal remains in the Hechtian strands. Red arrowheads point to the retracted plasma membrane. (D) In vitro kinase assays of AtPR5K2. The upper panel indicates the schematic structure of the GST-fused AtPR5K2 kinase domain (wPR5K2KD) and the GST-fused mutagenized AtPR5K2 kinase domain (mPR5K2KD). Each kinase domain was individually expressed in Escherichia coli , and 2 μg purified proteins was incubated in kinase assay buffer. Radioactive-labeled products were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and detected using radioactivity (bottom right). After electrophoresis, the purified products were stained with Coomassie brilliant blue (bottom left).
    Figure Legend Snippet: Expression patterns and subcellular localization of AtPR5K2. (A) The expression of AtPR5K2 in various tissues of Arabidopsis thaliana . Total RNA was extracted from the roots, rosette leaves, cauline leaves, stems, flowers, and siliques of the wild-type plants. The transcript levels of AtPR5K2 were measured using quantitative reverse transcription PCR (qRT-PCR) and calculated relative to the expression of the endogenous control gene, TUBULIN2 . Error bars represent the ± SD from three independent experiments. (B) Subcellular localization of AtPR5K2 in Arabidopsis protoplasts. 35S:AtPR5K2-GFP and Aquaporin-RFP were coexpressed in Arabidopsis protoplasts, which were analyzed using confocal fluorescence microscopy and photographed after 24 h of incubation at 22°C. Aquaporin-RFP is a plasma-membrane marker. Scale bars represent 10 μm. (C) Subcellular localization of AtPR5K2 in the epidermal cells of tobacco ( Nicotiana benthamiana ) leave expressing 35S:AtPR5K2-GFP before and after plasmolysis. The epidermal cells were analyzed using confocal fluorescence microscopy and photographed after 48 h of incubation at 25°C. Scale bars represent 20 μm. Red asterisk indicates that AtPR5K2-GFP signal remains in the Hechtian strands. Red arrowheads point to the retracted plasma membrane. (D) In vitro kinase assays of AtPR5K2. The upper panel indicates the schematic structure of the GST-fused AtPR5K2 kinase domain (wPR5K2KD) and the GST-fused mutagenized AtPR5K2 kinase domain (mPR5K2KD). Each kinase domain was individually expressed in Escherichia coli , and 2 μg purified proteins was incubated in kinase assay buffer. Radioactive-labeled products were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and detected using radioactivity (bottom right). After electrophoresis, the purified products were stained with Coomassie brilliant blue (bottom left).

    Techniques Used: Expressing, Polymerase Chain Reaction, Quantitative RT-PCR, Fluorescence, Microscopy, Incubation, Marker, In Vitro, Purification, Kinase Assay, Labeling, Polyacrylamide Gel Electrophoresis, SDS Page, Radioactivity, Electrophoresis, Staining

    5) Product Images from "Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors"

    Article Title: Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors

    Journal: Nature

    doi: 10.1038/s41586-019-1161-z

    Additional analysis of data showing transcriptome-wide off-target RNA editing in HEK293T cells by BE3 with two different gRNAs. (a) Percentages of different predicted effects and locations of edited cytosines in each RNA-seq replicate from . (b) Percentages (x-axis) and numbers (shown inside the bars) of expressed genes in each RNA-seq replicate from same dataset as described in (a) that show at least one edited cytosine. (c) Jitter plots of cytosines modified by BE3 expression with the RNF2 or the EMX1 . The percentage of all modified cytosines in each category is also shown. (d) Sequence logos derived from edited cytosines identified in each RNA-seq replicate. Analysis done using RNA-seq data generated from cDNA, thus every T depicted should be considered a U in actual RNA. (e) Venn diagram showing numbers of cytosines edited with the RNF2 and EMX1 gRNAs. For each gRNA, the number of cytosines represents the union of those identified in the three replicates. Extended Data Fig. 2c
    Figure Legend Snippet: Additional analysis of data showing transcriptome-wide off-target RNA editing in HEK293T cells by BE3 with two different gRNAs. (a) Percentages of different predicted effects and locations of edited cytosines in each RNA-seq replicate from . (b) Percentages (x-axis) and numbers (shown inside the bars) of expressed genes in each RNA-seq replicate from same dataset as described in (a) that show at least one edited cytosine. (c) Jitter plots of cytosines modified by BE3 expression with the RNF2 or the EMX1 . The percentage of all modified cytosines in each category is also shown. (d) Sequence logos derived from edited cytosines identified in each RNA-seq replicate. Analysis done using RNA-seq data generated from cDNA, thus every T depicted should be considered a U in actual RNA. (e) Venn diagram showing numbers of cytosines edited with the RNF2 and EMX1 gRNAs. For each gRNA, the number of cytosines represents the union of those identified in the three replicates. Extended Data Fig. 2c

    Techniques Used: RNA Sequencing Assay, Modification, Expressing, Sequencing, Derivative Assay, Generated

    6) Product Images from "Generation of targeted homozygosity in the genome of human induced pluripotent stem cells"

    Article Title: Generation of targeted homozygosity in the genome of human induced pluripotent stem cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0225740

    Establishment of a BLM transcript regulation system and detection of crossovers by a reporter cassette in hiPSCs. (A) Schematic representation of a crossover induced by a double-stranded DNA break during the 4N stage of the cell cycle in hiPSCs. X is a dominant mutation. After segregation, case #a cells have the disease phenotype, while case #b cells are normal. (B) Targeting of the Tet-Off cassette to both alleles of the BLM locus with the assistance of TALEN ( BLM tet-puro/tet-puro ). The selection cassettes were removed by Flippase (Flpo), the hiPSC- BLM tet/tet cells were established. (C) Electrophoresis of qRT-PCR products. Total RNA from 2 × 10 5 hiPSC- BLM tet/tet cells was collected at days 0, 5, and 7 after dox treatment, and qRT-PCR was performed. (D) Histogram data represent the mean ± SEM of three independent experiments. Comparison of two groups with normally distributed variables was performed using the Student’s t -test by Kaleida Graph software. (E) Targeting of the double selection cassette ( cNP ) to the AAVS1 locus (hiPSC- BLM tet/tet AAVS cNP/+ ). (F) Schematic representation of mitotic crossover during the 4N stage. DSBs were introduced by CRISPR/Cas9.
    Figure Legend Snippet: Establishment of a BLM transcript regulation system and detection of crossovers by a reporter cassette in hiPSCs. (A) Schematic representation of a crossover induced by a double-stranded DNA break during the 4N stage of the cell cycle in hiPSCs. X is a dominant mutation. After segregation, case #a cells have the disease phenotype, while case #b cells are normal. (B) Targeting of the Tet-Off cassette to both alleles of the BLM locus with the assistance of TALEN ( BLM tet-puro/tet-puro ). The selection cassettes were removed by Flippase (Flpo), the hiPSC- BLM tet/tet cells were established. (C) Electrophoresis of qRT-PCR products. Total RNA from 2 × 10 5 hiPSC- BLM tet/tet cells was collected at days 0, 5, and 7 after dox treatment, and qRT-PCR was performed. (D) Histogram data represent the mean ± SEM of three independent experiments. Comparison of two groups with normally distributed variables was performed using the Student’s t -test by Kaleida Graph software. (E) Targeting of the double selection cassette ( cNP ) to the AAVS1 locus (hiPSC- BLM tet/tet AAVS cNP/+ ). (F) Schematic representation of mitotic crossover during the 4N stage. DSBs were introduced by CRISPR/Cas9.

    Techniques Used: Mutagenesis, Selection, Electrophoresis, Quantitative RT-PCR, Software, CRISPR

    7) Product Images from "CDK12 phosphorylates 4E-BP1 to enable mTORC1-dependent translation and mitotic genome stability"

    Article Title: CDK12 phosphorylates 4E-BP1 to enable mTORC1-dependent translation and mitotic genome stability

    Journal: Genes & Development

    doi: 10.1101/gad.322339.118

    Genome-wide ribosome profiling identifies CDK12-dependent translation target genes, including mRNAs encoding critical centromere and kinetochore complexes. ( A ) Scatter plot representation of the genome-wide fold-changes of RPFs and mRNA abundance in U2OS cells depleted of CDK12. The differential translation efficiency (log 2 fold change ≥ 1 and ≤−1; false discovery rate [FDR] ≤ 0.1) was plotted for mRNAs subject to transcriptional regulation (“transcription-only”; blue points), translational regulation (“translation-only”; red points), and homodirectional changes in both processes (“homodirectional; green points). The CHK1 gene was identified in the down-regulated group of “translation-only” CDK12 targets. ( B ) Venn diagram displays the number of genes down-regulated at the level of transcription (green), translation (blue), or both (overlapping) from A . Note that CHK1 was present in the group of “translation-only” target genes. ( C ) BedGraphs of total mRNA levels (RNA-seq) and RPFs (Ribo-seq) for the RPL5 , CHEK1 , EIF1 , IFT88 , and RPL30 genes in U2OS cells treated with control (CTL) or CDK12-specific siRNAs. The five genes shown fall in the category of “translation-only” targets of CDK12. Scales represent the genomic DNA size and structure of the coding region of the individual genes. ( D ) RIP analysis of selected candidate “translation-only” CDK12 target mRNAs that were identified by Ribo-seq. The top panel shows total steady-state mRNA levels in cells transfected with control (CTL) or CDK12-siRNAs by qRT–PCR, and the bottom panel displays the mRNAs bound to eIF4G in each condition, as determined by RIP. All values represent the mean ± SD from three independent experiments ( n = 3). ( E ) Immunoblot analysis of protein expression for select CDK12 “translation-only” target genes. Several genes from D were tested by immunoblot to monitor protein expression using the indicated antisera.
    Figure Legend Snippet: Genome-wide ribosome profiling identifies CDK12-dependent translation target genes, including mRNAs encoding critical centromere and kinetochore complexes. ( A ) Scatter plot representation of the genome-wide fold-changes of RPFs and mRNA abundance in U2OS cells depleted of CDK12. The differential translation efficiency (log 2 fold change ≥ 1 and ≤−1; false discovery rate [FDR] ≤ 0.1) was plotted for mRNAs subject to transcriptional regulation (“transcription-only”; blue points), translational regulation (“translation-only”; red points), and homodirectional changes in both processes (“homodirectional; green points). The CHK1 gene was identified in the down-regulated group of “translation-only” CDK12 targets. ( B ) Venn diagram displays the number of genes down-regulated at the level of transcription (green), translation (blue), or both (overlapping) from A . Note that CHK1 was present in the group of “translation-only” target genes. ( C ) BedGraphs of total mRNA levels (RNA-seq) and RPFs (Ribo-seq) for the RPL5 , CHEK1 , EIF1 , IFT88 , and RPL30 genes in U2OS cells treated with control (CTL) or CDK12-specific siRNAs. The five genes shown fall in the category of “translation-only” targets of CDK12. Scales represent the genomic DNA size and structure of the coding region of the individual genes. ( D ) RIP analysis of selected candidate “translation-only” CDK12 target mRNAs that were identified by Ribo-seq. The top panel shows total steady-state mRNA levels in cells transfected with control (CTL) or CDK12-siRNAs by qRT–PCR, and the bottom panel displays the mRNAs bound to eIF4G in each condition, as determined by RIP. All values represent the mean ± SD from three independent experiments ( n = 3). ( E ) Immunoblot analysis of protein expression for select CDK12 “translation-only” target genes. Several genes from D were tested by immunoblot to monitor protein expression using the indicated antisera.

    Techniques Used: Genome Wide, RNA Sequencing Assay, CTL Assay, Transfection, Quantitative RT-PCR, Expressing

    8) Product Images from "MOZ is essential for maintenance of hematopoietic stem cells"

    Article Title: MOZ is essential for maintenance of hematopoietic stem cells

    Journal:

    doi: 10.1101/gad.1393106

    Changes in populations of myeloid and erythroid cells in MOZ −/− fetal liver. ( A ) Population of myeloid progenitors. Cells obtained from E14.5 fetal liver cells were stained with FcγR III/II-FITC, CD34-PE, Lin./Sca-1-Biotin-streptavidin-PerCP-Cy5.5,
    Figure Legend Snippet: Changes in populations of myeloid and erythroid cells in MOZ −/− fetal liver. ( A ) Population of myeloid progenitors. Cells obtained from E14.5 fetal liver cells were stained with FcγR III/II-FITC, CD34-PE, Lin./Sca-1-Biotin-streptavidin-PerCP-Cy5.5,

    Techniques Used: Staining

    Related Articles

    Clone Assay:

    Article Title: Ripor2 is involved in auditory hair cell stereociliary bundle structure and orientation
    Article Snippet: Wild type RIPOR2 cDNA was synthesized from control blood RNA using SuperScript III (Invitrogen) and random hexamers (Promega). .. The resulting amplicon was cloned into a pcDNA3.1/CT-GFP-TOPO vector (Invitrogen) [ ].

    Article Title: Activation of RNase L in Egyptian Rousette Bat-Derived RoNi/7 Cells Is Dependent Primarily on OAS3 and Independent of MAVS Signaling
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    Amplification:

    Article Title: Ripor2 is involved in auditory hair cell stereociliary bundle structure and orientation
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    Article Snippet: For unbiased repertoire analysis, 5′-RACE was performed with SMARTer RACE cDNA Amplification Kit (Clontech, Mountain View, CA). .. For ZK2B10 gene-specific lineage analysis, reverse transcription (RT) was performed with SuperScript III (Life Technologies) and oligo (dT).

    Article Title: Identification of novel predictive factors for post surgical corneal haze
    Article Snippet: In addition, cDNA was synthesized using Superscript III (Life Technologies, Carlsbad, CA) cDNA conversion kit. .. The quantitative real-time PCR cycle includes pre-incubation at 95 °C for 3 minutes, followed by 40 amplification cycles at 95 °C for 10 seconds, annealing and extension at 58 °C for 30 sec with dissociation curve analysis at 65 °C to 95 °C increment 0.5 °C for 0.05 minutes using a CFX ConnectTM real-time PCR detection system (Bio-Rad, Philadelphia, PA) using SYBR Green Assay (KAPA SYBR Fast qPCR master mix: KAPA Biosystems, Wilmington, MA).

    Article Title: Activation of RNase L in Egyptian Rousette Bat-Derived RoNi/7 Cells Is Dependent Primarily on OAS3 and Independent of MAVS Signaling
    Article Snippet: Briefly, the cells were treated with 1,000 U of IFN-α overnight, and total cellular RNA was extracted using an RNeasy Plus Mini kit (Qiagen). cDNA was synthesized by reverse transcription using SuperScript III (Invitrogen) and mRNA-specific reverse primers. bOAS1-rev and bOAS2-rev ( ) primers were used for reverse transcription to synthesize bOAS1 and bOAS2 cDNA, respectively. .. The DNA fragments of bOAS1, bOAS2, bOAS3-F2, and bOAS3-F3 were amplified by using AccuPrime DNA polymerase (Invitrogen) with the forward primers and reverse primers (see in the supplemental material), and were cloned into pCR2.1 TOPO vectors (Invitrogen).

    Synthesized:

    Article Title: Ripor2 is involved in auditory hair cell stereociliary bundle structure and orientation
    Article Snippet: .. Wild type RIPOR2 cDNA was synthesized from control blood RNA using SuperScript III (Invitrogen) and random hexamers (Promega). .. Full-length wild type RIPOR2 lacking the stop codon was amplified by high-fidelity PCR using Long Expand Taq Polymerase (Roche) and a primer pair (RIPOR2-TOPO-F, RIPOR2-TOPO-R, ).

    Article Title: Generation of targeted homozygosity in the genome of human induced pluripotent stem cells
    Article Snippet: .. First-strand cDNA was synthesized from 800 ng total RNA with random hexamer primers using SuperScript III (Invitrogen) at 50°C for 60 min. qRT-PCR was performed to measure BLM gene expression. .. Total RNA was reverse transcribed to cDNA using the SuperScript III First-Strand Synthesis System for qRT-PCR (Invitrogen). qRT-PCR was performed with the Applied Biosystems 7900HT Fast Real-Time PCR System using the following PCR primer sets: RTPCR-leader-F and RTPCR-exon3-R, hActb-RTPCR-F, and hActb-RTPCR-R ( ).

    Article Title: Identification of novel predictive factors for post surgical corneal haze
    Article Snippet: .. In addition, cDNA was synthesized using Superscript III (Life Technologies, Carlsbad, CA) cDNA conversion kit. .. The quantitative real-time PCR cycle includes pre-incubation at 95 °C for 3 minutes, followed by 40 amplification cycles at 95 °C for 10 seconds, annealing and extension at 58 °C for 30 sec with dissociation curve analysis at 65 °C to 95 °C increment 0.5 °C for 0.05 minutes using a CFX ConnectTM real-time PCR detection system (Bio-Rad, Philadelphia, PA) using SYBR Green Assay (KAPA SYBR Fast qPCR master mix: KAPA Biosystems, Wilmington, MA).

    Article Title: Activation of RNase L in Egyptian Rousette Bat-Derived RoNi/7 Cells Is Dependent Primarily on OAS3 and Independent of MAVS Signaling
    Article Snippet: .. Briefly, the cells were treated with 1,000 U of IFN-α overnight, and total cellular RNA was extracted using an RNeasy Plus Mini kit (Qiagen). cDNA was synthesized by reverse transcription using SuperScript III (Invitrogen) and mRNA-specific reverse primers. bOAS1-rev and bOAS2-rev ( ) primers were used for reverse transcription to synthesize bOAS1 and bOAS2 cDNA, respectively. .. Three cDNA fragments were cloned to assemble the full length of the bOAS3 open reading frame. bOAS3-F1-rev, bOAS3-F2-rev, and bOAS3-F3-rev ( ) were used for reverse transcription reactions to synthesize three cDNA fragments (bOAS3-F1, bOAS3-F2, and bOAS3-F3, respectively).

    Construct:

    Article Title: Ripor2 is involved in auditory hair cell stereociliary bundle structure and orientation
    Article Snippet: Paragraph title: Generation of expression constructs ... Wild type RIPOR2 cDNA was synthesized from control blood RNA using SuperScript III (Invitrogen) and random hexamers (Promega).

    SYBR Green Assay:

    Article Title: AtPR5K2, a PR5-Like Receptor Kinase, Modulates Plant Responses to Drought Stress by Phosphorylating Protein Phosphatase 2Cs
    Article Snippet: For the RT-PCR and quantitative RT-PCR (qRT-PCR) analyses, 2 µg total RNA was used for cDNA synthesis using SuperScript III (Thermo Fisher Scientific, MA, USA), in accordance with the manufacturer’s protocol. .. The qRT-PCR analysis was performed using a SYBR Green Supermix kit (Bio-Rad Laboratories, Hercules, CA, USA), and the relative gene expression levels were automatically calculated using the CFX384 real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA).

    Article Title: CDK2-mediated site-specific phosphorylation of EZH2 drives and maintains triple-negative breast cancer
    Article Snippet: Quantitative RT–PCR (qRT–PCR) was performed using SYBR Green dye on a Bio-Rad PCR machine . .. In brief, 1 μg of total RNA was reverse transcribed into cDNA using SuperScript III (Invitrogen) in the presence of random hexamers.

    Article Title: eIF4A2 drives repression of translation at initiation by Ccr4-Not through purine-rich motifs in the 5′UTR
    Article Snippet: RT-qPCR For IP validations (Additional file : Figure S4A, S9A), RT-PCR was conducted on 50 ng of the RNA extracted from the IPs and the 10% input RNA using SuperScript III (Invitrogen). .. Primers were designed for RNAs found to be enriched in each of the IPs as well as RNAs enriched/depleted in all IPs (Additional file : Table S2). qPCR was conducted using Fast SYBR Green PCR Master Mix on a 7500 Fast Real Time PCR System (Applied Biosystems) with three technical replicates for two biological replicates.

    Article Title: CDK12 phosphorylates 4E-BP1 to enable mTORC1-dependent translation and mitotic genome stability
    Article Snippet: .. The total RNA from each sample (1 µg) was subjected to cDNA synthesis with CHK1 -specific primers (GSP1; 5′-ACTTCATGAGGCAATTTCTG-3′ and GSP2: 5′-AGTGCATGTTAAAGAAGATC-3′) using SuperScript III (Life Technologies). qPCR measurements of CHK1 mRNA were performed in triplicate using the SYBR Green master mix (Life Technologies), and the average of the technical replicates was normalized to GAPDH levels using the comparative CT method. ..

    Article Title: Identification of novel predictive factors for post surgical corneal haze
    Article Snippet: In addition, cDNA was synthesized using Superscript III (Life Technologies, Carlsbad, CA) cDNA conversion kit. .. The quantitative real-time PCR cycle includes pre-incubation at 95 °C for 3 minutes, followed by 40 amplification cycles at 95 °C for 10 seconds, annealing and extension at 58 °C for 30 sec with dissociation curve analysis at 65 °C to 95 °C increment 0.5 °C for 0.05 minutes using a CFX ConnectTM real-time PCR detection system (Bio-Rad, Philadelphia, PA) using SYBR Green Assay (KAPA SYBR Fast qPCR master mix: KAPA Biosystems, Wilmington, MA).

    Microarray:

    Article Title: MOZ is essential for maintenance of hematopoietic stem cells
    Article Snippet: Paragraph title: Microarray analysis and RT–PCR ... Reverse transcription of total RNA isolated from E12.5 fetal liver and E14.5 Lin− /Sca-1+ cells was performed using SuperScript III (Invitrogen Life Technologies).

    Quantitation Assay:

    Article Title: Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors
    Article Snippet: RNA library preparation was performed with the TruSeq Stranded Total RNA Library Prep Gold kit (Illumina) with initial input of ~500ng of extracted RNA per sample, using SuperScript III (Invitrogen) for first-strand synthesis. .. Ribosomal RNA (rRNA) depletion was confirmed after the initial rRNA removal step by fluorometric quantitation using the Qubit RNA HS Assay Kit (Invitrogen).

    Expressing:

    Article Title: Ripor2 is involved in auditory hair cell stereociliary bundle structure and orientation
    Article Snippet: Paragraph title: Generation of expression constructs ... Wild type RIPOR2 cDNA was synthesized from control blood RNA using SuperScript III (Invitrogen) and random hexamers (Promega).

    Article Title: AtPR5K2, a PR5-Like Receptor Kinase, Modulates Plant Responses to Drought Stress by Phosphorylating Protein Phosphatase 2Cs
    Article Snippet: For the RT-PCR and quantitative RT-PCR (qRT-PCR) analyses, 2 µg total RNA was used for cDNA synthesis using SuperScript III (Thermo Fisher Scientific, MA, USA), in accordance with the manufacturer’s protocol. .. The qRT-PCR analysis was performed using a SYBR Green Supermix kit (Bio-Rad Laboratories, Hercules, CA, USA), and the relative gene expression levels were automatically calculated using the CFX384 real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA).

    Article Title: Generation of targeted homozygosity in the genome of human induced pluripotent stem cells
    Article Snippet: .. First-strand cDNA was synthesized from 800 ng total RNA with random hexamer primers using SuperScript III (Invitrogen) at 50°C for 60 min. qRT-PCR was performed to measure BLM gene expression. .. Total RNA was reverse transcribed to cDNA using the SuperScript III First-Strand Synthesis System for qRT-PCR (Invitrogen). qRT-PCR was performed with the Applied Biosystems 7900HT Fast Real-Time PCR System using the following PCR primer sets: RTPCR-leader-F and RTPCR-exon3-R, hActb-RTPCR-F, and hActb-RTPCR-R ( ).

    Article Title: CDK2-mediated site-specific phosphorylation of EZH2 drives and maintains triple-negative breast cancer
    Article Snippet: In brief, 1 μg of total RNA was reverse transcribed into cDNA using SuperScript III (Invitrogen) in the presence of random hexamers. .. The quantity of the target gene for each sample was divided by the average sample quantity of the GAPDH to obtain the relative level of the gene expression.

    Article Title: MOZ is essential for maintenance of hematopoietic stem cells
    Article Snippet: The expression value (Signal) of each gene was calculated and normalized using GeneChip Operating Software version 1.2 (Affymetrix). .. Reverse transcription of total RNA isolated from E12.5 fetal liver and E14.5 Lin− /Sca-1+ cells was performed using SuperScript III (Invitrogen Life Technologies).

    Article Title: Differential Effects of Heparin and Hyaluronic Acid on Neural Patterning of Human Induced Pluripotent Stem Cells
    Article Snippet: Reverse transcription was carried out using 2 μ g of total RNA, anchored oligo-dT primers (Operon, Huntsville, AL), and Superscript III (Invitrogen, Carlsbad, CA; according to the protocol of the manufacturer). .. The gene β -actin was used as an endogenous control for normalization of expression levels.

    Western Blot:

    Article Title: Zc3h10 Acts as a Transcription Factor and Is Phosphorylated to Activate the Thermogenic Program
    Article Snippet: .. RT-PCR Analysis and western blotting Reverse transcription was performed with 500ng of total RNA using SuperScript III (Invitrogen). .. RT-qPCR was performed in triplicate with an ABI PRISM 7500 sequence detection system (Applied Biosystems) to quantify the relative mRNA levels for various genes.

    Transfection:

    Article Title: CDK12 phosphorylates 4E-BP1 to enable mTORC1-dependent translation and mitotic genome stability
    Article Snippet: , U2OS cells (5 × 105 ) in a six-well dish were transfected with the indicated siRNAs. .. The total RNA from each sample (1 µg) was subjected to cDNA synthesis with CHK1 -specific primers (GSP1; 5′-ACTTCATGAGGCAATTTCTG-3′ and GSP2: 5′-AGTGCATGTTAAAGAAGATC-3′) using SuperScript III (Life Technologies). qPCR measurements of CHK1 mRNA were performed in triplicate using the SYBR Green master mix (Life Technologies), and the average of the technical replicates was normalized to GAPDH levels using the comparative CT method.

    Polymerase Chain Reaction:

    Article Title: Ripor2 is involved in auditory hair cell stereociliary bundle structure and orientation
    Article Snippet: Wild type RIPOR2 cDNA was synthesized from control blood RNA using SuperScript III (Invitrogen) and random hexamers (Promega). .. Full-length wild type RIPOR2 lacking the stop codon was amplified by high-fidelity PCR using Long Expand Taq Polymerase (Roche) and a primer pair (RIPOR2-TOPO-F, RIPOR2-TOPO-R, ).

    Article Title: Development of a Potent and Protective Germline-Like Antibody Lineage Against Zika Virus in a Convalescent Human
    Article Snippet: Paragraph title: Sample Preparation Using 5′-RACE PCR ... For ZK2B10 gene-specific lineage analysis, reverse transcription (RT) was performed with SuperScript III (Life Technologies) and oligo (dT).

    Article Title: New Type of Sendai Virus Vector Provides Transgene-Free iPS Cells Derived from Chimpanzee Blood
    Article Snippet: Paragraph title: DNA and RNA Isolations and PCR ... Total RNA was transcribed to DNA with Superscript III (Invitrogen) and randam primers (Invitrogen).

    Article Title: Generation of targeted homozygosity in the genome of human induced pluripotent stem cells
    Article Snippet: First-strand cDNA was synthesized from 800 ng total RNA with random hexamer primers using SuperScript III (Invitrogen) at 50°C for 60 min. qRT-PCR was performed to measure BLM gene expression. .. Total RNA was reverse transcribed to cDNA using the SuperScript III First-Strand Synthesis System for qRT-PCR (Invitrogen). qRT-PCR was performed with the Applied Biosystems 7900HT Fast Real-Time PCR System using the following PCR primer sets: RTPCR-leader-F and RTPCR-exon3-R, hActb-RTPCR-F, and hActb-RTPCR-R ( ).

    Article Title: CDK2-mediated site-specific phosphorylation of EZH2 drives and maintains triple-negative breast cancer
    Article Snippet: Quantitative RT–PCR (qRT–PCR) was performed using SYBR Green dye on a Bio-Rad PCR machine . .. In brief, 1 μg of total RNA was reverse transcribed into cDNA using SuperScript III (Invitrogen) in the presence of random hexamers.

    Article Title: eIF4A2 drives repression of translation at initiation by Ccr4-Not through purine-rich motifs in the 5′UTR
    Article Snippet: RT-qPCR For IP validations (Additional file : Figure S4A, S9A), RT-PCR was conducted on 50 ng of the RNA extracted from the IPs and the 10% input RNA using SuperScript III (Invitrogen). .. Primers were designed for RNAs found to be enriched in each of the IPs as well as RNAs enriched/depleted in all IPs (Additional file : Table S2). qPCR was conducted using Fast SYBR Green PCR Master Mix on a 7500 Fast Real Time PCR System (Applied Biosystems) with three technical replicates for two biological replicates.

    Article Title: Differential Effects of Heparin and Hyaluronic Acid on Neural Patterning of Human Induced Pluripotent Stem Cells
    Article Snippet: Reverse transcription was carried out using 2 μ g of total RNA, anchored oligo-dT primers (Operon, Huntsville, AL), and Superscript III (Invitrogen, Carlsbad, CA; according to the protocol of the manufacturer). .. Real-time RT-PCR reactions were performed on an ABI7500 instrument (Applied Biosystems, Foster City, CA), using SYBR1 Green PCR Master Mix (Applied Biosystems).

    Article Title: Activation of RNase L in Egyptian Rousette Bat-Derived RoNi/7 Cells Is Dependent Primarily on OAS3 and Independent of MAVS Signaling
    Article Snippet: Briefly, the cells were treated with 1,000 U of IFN-α overnight, and total cellular RNA was extracted using an RNeasy Plus Mini kit (Qiagen). cDNA was synthesized by reverse transcription using SuperScript III (Invitrogen) and mRNA-specific reverse primers. bOAS1-rev and bOAS2-rev ( ) primers were used for reverse transcription to synthesize bOAS1 and bOAS2 cDNA, respectively. .. The DNA fragment of bOAS3-F1 was amplified by using Q5 DNA polymerase (NEB) and was cloned into pCR II Blunt-TOPO vectors (Invitrogen).

    Sequencing:

    Article Title: Development of a Potent and Protective Germline-Like Antibody Lineage Against Zika Virus in a Convalescent Human
    Article Snippet: For ZK2B10 gene-specific lineage analysis, reverse transcription (RT) was performed with SuperScript III (Life Technologies) and oligo (dT). .. To facilitate deep sequencing on the Ion GeneStudio S5 system, the forward primers (both 5′-RACE and gene-specific) contained a P1 adaptor, while the reverse primer contained an A adaptor and an Ion XpressTM barcode (Life Technologies) to differentiate the libraries from various time points.

    Article Title: Zc3h10 Acts as a Transcription Factor and Is Phosphorylated to Activate the Thermogenic Program
    Article Snippet: RT-PCR Analysis and western blotting Reverse transcription was performed with 500ng of total RNA using SuperScript III (Invitrogen). .. RT-qPCR was performed in triplicate with an ABI PRISM 7500 sequence detection system (Applied Biosystems) to quantify the relative mRNA levels for various genes.

    Article Title: Activation of RNase L in Egyptian Rousette Bat-Derived RoNi/7 Cells Is Dependent Primarily on OAS3 and Independent of MAVS Signaling
    Article Snippet: Paragraph title: Cloning and sequencing of bat OAS cDNAs. ... Briefly, the cells were treated with 1,000 U of IFN-α overnight, and total cellular RNA was extracted using an RNeasy Plus Mini kit (Qiagen). cDNA was synthesized by reverse transcription using SuperScript III (Invitrogen) and mRNA-specific reverse primers. bOAS1-rev and bOAS2-rev ( ) primers were used for reverse transcription to synthesize bOAS1 and bOAS2 cDNA, respectively.

    RNA Sequencing Assay:

    Article Title: Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors
    Article Snippet: Paragraph title: RNA-seq experiments. ... RNA library preparation was performed with the TruSeq Stranded Total RNA Library Prep Gold kit (Illumina) with initial input of ~500ng of extracted RNA per sample, using SuperScript III (Invitrogen) for first-strand synthesis.

    RNA HS Assay:

    Article Title: Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors
    Article Snippet: RNA library preparation was performed with the TruSeq Stranded Total RNA Library Prep Gold kit (Illumina) with initial input of ~500ng of extracted RNA per sample, using SuperScript III (Invitrogen) for first-strand synthesis. .. Ribosomal RNA (rRNA) depletion was confirmed after the initial rRNA removal step by fluorometric quantitation using the Qubit RNA HS Assay Kit (Invitrogen).

    Isolation:

    Article Title: Zc3h10 Acts as a Transcription Factor and Is Phosphorylated to Activate the Thermogenic Program
    Article Snippet: RT-PCR Analysis and western blotting Reverse transcription was performed with 500ng of total RNA using SuperScript III (Invitrogen). .. For western blot analysis, total cell lysates were prepared using RIPA buffer, and nuclear extracts were isolated using the NE-PER Nuclear and Cytoplasmic Extraction kit (Thermo).

    Article Title: MOZ is essential for maintenance of hematopoietic stem cells
    Article Snippet: .. Reverse transcription of total RNA isolated from E12.5 fetal liver and E14.5 Lin− /Sca-1+ cells was performed using SuperScript III (Invitrogen Life Technologies). ..

    Article Title: Identification of novel predictive factors for post surgical corneal haze
    Article Snippet: Paragraph title: Isolation of RNA, cDNA synthesis and real-time PCR ... In addition, cDNA was synthesized using Superscript III (Life Technologies, Carlsbad, CA) cDNA conversion kit.

    Article Title: Differential Effects of Heparin and Hyaluronic Acid on Neural Patterning of Human Induced Pluripotent Stem Cells
    Article Snippet: Total RNA was isolated from neural cell samples using the RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol followed by treatment with the DNA-Free RNA Kit (Zymo, Irvine, CA). .. Reverse transcription was carried out using 2 μ g of total RNA, anchored oligo-dT primers (Operon, Huntsville, AL), and Superscript III (Invitrogen, Carlsbad, CA; according to the protocol of the manufacturer).

    Size-exclusion Chromatography:

    Article Title: MOZ is essential for maintenance of hematopoietic stem cells
    Article Snippet: Reverse transcription of total RNA isolated from E12.5 fetal liver and E14.5 Lin− /Sca-1+ cells was performed using SuperScript III (Invitrogen Life Technologies). .. Primers and conditions used in the RT–PCR study were as follows: for HoxA9, 5′-ACAATGCCGAGAATGAGAGC-3′ and 5′-CATTTTCATC CTGCGGTTCT-3′, with 36 cycles at 95°C for 45 sec, 57°C for 45 sec, and 72°C for 45 sec; for c-Mpl, 5′-ACCTTTGGAACCC GGTATGTG-3′ and 5′-CTGTGGAGAGCAGCTGAATG-3′, with 35 cycles at 95°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec; for MOZ, 5′-TGTATCTGCTGCCTGTGGAG-3′ and 5′-CCCGGTTCTGCTCTTTTGTA-3′, with 35 cycles at 95°C for 1 min, 60°C for 1 min, 72°C for 1 min; for c-Kit, 5′-ACAGGAG CAGAGCAAAGGTG-3′ and 5′-CGACCACAAAGCCAATGA GC-3′, with 35 cycles at 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min; and for β-actin, 5′-GAGAGGGAAATCGTGC GTGA-3′ and 5′-ACATCTGCTGGAAGGTGGAC-3′, with 25 cycles at 95°C for 30 sec, 57°C for 30 sec, and 72°C for 1 min.

    Article Title: Identification of novel predictive factors for post surgical corneal haze
    Article Snippet: In addition, cDNA was synthesized using Superscript III (Life Technologies, Carlsbad, CA) cDNA conversion kit. .. The quantitative real-time PCR cycle includes pre-incubation at 95 °C for 3 minutes, followed by 40 amplification cycles at 95 °C for 10 seconds, annealing and extension at 58 °C for 30 sec with dissociation curve analysis at 65 °C to 95 °C increment 0.5 °C for 0.05 minutes using a CFX ConnectTM real-time PCR detection system (Bio-Rad, Philadelphia, PA) using SYBR Green Assay (KAPA SYBR Fast qPCR master mix: KAPA Biosystems, Wilmington, MA).

    Purification:

    Article Title: Development of a Potent and Protective Germline-Like Antibody Lineage Against Zika Virus in a Convalescent Human
    Article Snippet: For ZK2B10 gene-specific lineage analysis, reverse transcription (RT) was performed with SuperScript III (Life Technologies) and oligo (dT). .. In both cases, the cDNA was purified and eluted in 20 μl of elution buffer (NucleoSpin PCR Clean-up Kit, Clontech).

    Article Title: AtPR5K2, a PR5-Like Receptor Kinase, Modulates Plant Responses to Drought Stress by Phosphorylating Protein Phosphatase 2Cs
    Article Snippet: Quantitative RT-PCR Analysis Total RNA was extracted and purified from different Arabidopsis tissues using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions, and treated with DNaseI (Sigma-Aldrich, St. Louis, MO, USA) to remove any genomic DNA contaminants. .. For the RT-PCR and quantitative RT-PCR (qRT-PCR) analyses, 2 µg total RNA was used for cDNA synthesis using SuperScript III (Thermo Fisher Scientific, MA, USA), in accordance with the manufacturer’s protocol.

    Article Title: New Type of Sendai Virus Vector Provides Transgene-Free iPS Cells Derived from Chimpanzee Blood
    Article Snippet: Total RNA was purified with Sepasol Super G reagent (Nacalai Tesque, Japan). .. Total RNA was transcribed to DNA with Superscript III (Invitrogen) and randam primers (Invitrogen).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: AtPR5K2, a PR5-Like Receptor Kinase, Modulates Plant Responses to Drought Stress by Phosphorylating Protein Phosphatase 2Cs
    Article Snippet: .. For the RT-PCR and quantitative RT-PCR (qRT-PCR) analyses, 2 µg total RNA was used for cDNA synthesis using SuperScript III (Thermo Fisher Scientific, MA, USA), in accordance with the manufacturer’s protocol. .. The qRT-PCR analysis was performed using a SYBR Green Supermix kit (Bio-Rad Laboratories, Hercules, CA, USA), and the relative gene expression levels were automatically calculated using the CFX384 real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA).

    Article Title: Zc3h10 Acts as a Transcription Factor and Is Phosphorylated to Activate the Thermogenic Program
    Article Snippet: .. RT-PCR Analysis and western blotting Reverse transcription was performed with 500ng of total RNA using SuperScript III (Invitrogen). .. RT-qPCR was performed in triplicate with an ABI PRISM 7500 sequence detection system (Applied Biosystems) to quantify the relative mRNA levels for various genes.

    Article Title: New Type of Sendai Virus Vector Provides Transgene-Free iPS Cells Derived from Chimpanzee Blood
    Article Snippet: Total RNA was transcribed to DNA with Superscript III (Invitrogen) and randam primers (Invitrogen). .. Genomic PCR and RT-PCR was performed with QuickTaq (TOYOBO, Japan) as described previously , .

    Article Title: Generation of targeted homozygosity in the genome of human induced pluripotent stem cells
    Article Snippet: First-strand cDNA was synthesized from 800 ng total RNA with random hexamer primers using SuperScript III (Invitrogen) at 50°C for 60 min. qRT-PCR was performed to measure BLM gene expression. .. Total RNA was reverse transcribed to cDNA using the SuperScript III First-Strand Synthesis System for qRT-PCR (Invitrogen). qRT-PCR was performed with the Applied Biosystems 7900HT Fast Real-Time PCR System using the following PCR primer sets: RTPCR-leader-F and RTPCR-exon3-R, hActb-RTPCR-F, and hActb-RTPCR-R ( ).

    Article Title: eIF4A2 drives repression of translation at initiation by Ccr4-Not through purine-rich motifs in the 5′UTR
    Article Snippet: .. RT-qPCR For IP validations (Additional file : Figure S4A, S9A), RT-PCR was conducted on 50 ng of the RNA extracted from the IPs and the 10% input RNA using SuperScript III (Invitrogen). .. Primers were designed for RNAs found to be enriched in each of the IPs as well as RNAs enriched/depleted in all IPs (Additional file : Table S2). qPCR was conducted using Fast SYBR Green PCR Master Mix on a 7500 Fast Real Time PCR System (Applied Biosystems) with three technical replicates for two biological replicates.

    Article Title: MOZ is essential for maintenance of hematopoietic stem cells
    Article Snippet: Paragraph title: Microarray analysis and RT–PCR ... Reverse transcription of total RNA isolated from E12.5 fetal liver and E14.5 Lin− /Sca-1+ cells was performed using SuperScript III (Invitrogen Life Technologies).

    Article Title: Differential Effects of Heparin and Hyaluronic Acid on Neural Patterning of Human Induced Pluripotent Stem Cells
    Article Snippet: Paragraph title: Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis. ... Reverse transcription was carried out using 2 μ g of total RNA, anchored oligo-dT primers (Operon, Huntsville, AL), and Superscript III (Invitrogen, Carlsbad, CA; according to the protocol of the manufacturer).

    Quantitative RT-PCR:

    Article Title: AtPR5K2, a PR5-Like Receptor Kinase, Modulates Plant Responses to Drought Stress by Phosphorylating Protein Phosphatase 2Cs
    Article Snippet: .. For the RT-PCR and quantitative RT-PCR (qRT-PCR) analyses, 2 µg total RNA was used for cDNA synthesis using SuperScript III (Thermo Fisher Scientific, MA, USA), in accordance with the manufacturer’s protocol. .. The qRT-PCR analysis was performed using a SYBR Green Supermix kit (Bio-Rad Laboratories, Hercules, CA, USA), and the relative gene expression levels were automatically calculated using the CFX384 real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA).

    Article Title: Zc3h10 Acts as a Transcription Factor and Is Phosphorylated to Activate the Thermogenic Program
    Article Snippet: RT-PCR Analysis and western blotting Reverse transcription was performed with 500ng of total RNA using SuperScript III (Invitrogen). .. RT-qPCR was performed in triplicate with an ABI PRISM 7500 sequence detection system (Applied Biosystems) to quantify the relative mRNA levels for various genes.

    Article Title: Generation of targeted homozygosity in the genome of human induced pluripotent stem cells
    Article Snippet: .. First-strand cDNA was synthesized from 800 ng total RNA with random hexamer primers using SuperScript III (Invitrogen) at 50°C for 60 min. qRT-PCR was performed to measure BLM gene expression. .. Total RNA was reverse transcribed to cDNA using the SuperScript III First-Strand Synthesis System for qRT-PCR (Invitrogen). qRT-PCR was performed with the Applied Biosystems 7900HT Fast Real-Time PCR System using the following PCR primer sets: RTPCR-leader-F and RTPCR-exon3-R, hActb-RTPCR-F, and hActb-RTPCR-R ( ).

    Article Title: CDK2-mediated site-specific phosphorylation of EZH2 drives and maintains triple-negative breast cancer
    Article Snippet: Paragraph title: Quantitative real-time RT–PCR ... In brief, 1 μg of total RNA was reverse transcribed into cDNA using SuperScript III (Invitrogen) in the presence of random hexamers.

    Article Title: eIF4A2 drives repression of translation at initiation by Ccr4-Not through purine-rich motifs in the 5′UTR
    Article Snippet: .. RT-qPCR For IP validations (Additional file : Figure S4A, S9A), RT-PCR was conducted on 50 ng of the RNA extracted from the IPs and the 10% input RNA using SuperScript III (Invitrogen). .. Primers were designed for RNAs found to be enriched in each of the IPs as well as RNAs enriched/depleted in all IPs (Additional file : Table S2). qPCR was conducted using Fast SYBR Green PCR Master Mix on a 7500 Fast Real Time PCR System (Applied Biosystems) with three technical replicates for two biological replicates.

    Article Title: Differential Effects of Heparin and Hyaluronic Acid on Neural Patterning of Human Induced Pluripotent Stem Cells
    Article Snippet: Reverse transcription was carried out using 2 μ g of total RNA, anchored oligo-dT primers (Operon, Huntsville, AL), and Superscript III (Invitrogen, Carlsbad, CA; according to the protocol of the manufacturer). .. Real-time RT-PCR reactions were performed on an ABI7500 instrument (Applied Biosystems, Foster City, CA), using SYBR1 Green PCR Master Mix (Applied Biosystems).

    Article Title: SUR-8 interacts with PP1-87B to stabilize PERIOD and regulate circadian rhythms in Drosophila
    Article Snippet: .. RNA extraction and qRT-PCR analysis Total RNA was extracted from fly heads and then subject to reverse transcription using superscript III (Thermo Fisher). .. Quantitative real-time PCR was carried out using SYBR Master Mix (Thermo Fisher).

    Plasmid Preparation:

    Article Title: Ripor2 is involved in auditory hair cell stereociliary bundle structure and orientation
    Article Snippet: Wild type RIPOR2 cDNA was synthesized from control blood RNA using SuperScript III (Invitrogen) and random hexamers (Promega). .. The resulting amplicon was cloned into a pcDNA3.1/CT-GFP-TOPO vector (Invitrogen) [ ].

    Software:

    Article Title: MOZ is essential for maintenance of hematopoietic stem cells
    Article Snippet: The change value (Signal Log Ratio) and change call (Increase, Marginal Increase, No Change, Marginal Decrease, or Decrease) for each gene was calculated by Comparison Analysis of the software. .. Reverse transcription of total RNA isolated from E12.5 fetal liver and E14.5 Lin− /Sca-1+ cells was performed using SuperScript III (Invitrogen Life Technologies).

    Article Title: Differential Effects of Heparin and Hyaluronic Acid on Neural Patterning of Human Induced Pluripotent Stem Cells
    Article Snippet: Reverse transcription was carried out using 2 μ g of total RNA, anchored oligo-dT primers (Operon, Huntsville, AL), and Superscript III (Invitrogen, Carlsbad, CA; according to the protocol of the manufacturer). .. Primers specific for target genes ( ) were designed using the software Oligo Explorer 1.2 (Genelink, Hawthorne, NY).

    Real-time Polymerase Chain Reaction:

    Article Title: AtPR5K2, a PR5-Like Receptor Kinase, Modulates Plant Responses to Drought Stress by Phosphorylating Protein Phosphatase 2Cs
    Article Snippet: For the RT-PCR and quantitative RT-PCR (qRT-PCR) analyses, 2 µg total RNA was used for cDNA synthesis using SuperScript III (Thermo Fisher Scientific, MA, USA), in accordance with the manufacturer’s protocol. .. The qRT-PCR analysis was performed using a SYBR Green Supermix kit (Bio-Rad Laboratories, Hercules, CA, USA), and the relative gene expression levels were automatically calculated using the CFX384 real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA).

    Article Title: Zc3h10 Acts as a Transcription Factor and Is Phosphorylated to Activate the Thermogenic Program
    Article Snippet: RT-PCR Analysis and western blotting Reverse transcription was performed with 500ng of total RNA using SuperScript III (Invitrogen). .. Statistical analysis of the qPCR was obtained using the ΔΔCt method with 18 s as the control.

    Article Title: Generation of targeted homozygosity in the genome of human induced pluripotent stem cells
    Article Snippet: Paragraph title: RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) ... First-strand cDNA was synthesized from 800 ng total RNA with random hexamer primers using SuperScript III (Invitrogen) at 50°C for 60 min. qRT-PCR was performed to measure BLM gene expression.

    Article Title: Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors
    Article Snippet: RNA library preparation was performed with the TruSeq Stranded Total RNA Library Prep Gold kit (Illumina) with initial input of ~500ng of extracted RNA per sample, using SuperScript III (Invitrogen) for first-strand synthesis. .. RNA-seq libraries were examined on a High-resolution QIAxcel (Qiagen) and pooled based on qPCR quantification with the KAPA Library Quantification Kit Illumina (KAPA Biosystems) or the NEBNext Library Quant Kit for Illumina (NEB).

    Article Title: eIF4A2 drives repression of translation at initiation by Ccr4-Not through purine-rich motifs in the 5′UTR
    Article Snippet: RT-qPCR For IP validations (Additional file : Figure S4A, S9A), RT-PCR was conducted on 50 ng of the RNA extracted from the IPs and the 10% input RNA using SuperScript III (Invitrogen). .. Primers were designed for RNAs found to be enriched in each of the IPs as well as RNAs enriched/depleted in all IPs (Additional file : Table S2). qPCR was conducted using Fast SYBR Green PCR Master Mix on a 7500 Fast Real Time PCR System (Applied Biosystems) with three technical replicates for two biological replicates.

    Article Title: CDK12 phosphorylates 4E-BP1 to enable mTORC1-dependent translation and mitotic genome stability
    Article Snippet: .. The total RNA from each sample (1 µg) was subjected to cDNA synthesis with CHK1 -specific primers (GSP1; 5′-ACTTCATGAGGCAATTTCTG-3′ and GSP2: 5′-AGTGCATGTTAAAGAAGATC-3′) using SuperScript III (Life Technologies). qPCR measurements of CHK1 mRNA were performed in triplicate using the SYBR Green master mix (Life Technologies), and the average of the technical replicates was normalized to GAPDH levels using the comparative CT method. ..

    Article Title: Identification of novel predictive factors for post surgical corneal haze
    Article Snippet: Paragraph title: Isolation of RNA, cDNA synthesis and real-time PCR ... In addition, cDNA was synthesized using Superscript III (Life Technologies, Carlsbad, CA) cDNA conversion kit.

    Article Title: SUR-8 interacts with PP1-87B to stabilize PERIOD and regulate circadian rhythms in Drosophila
    Article Snippet: RNA extraction and qRT-PCR analysis Total RNA was extracted from fly heads and then subject to reverse transcription using superscript III (Thermo Fisher). .. Quantitative real-time PCR was carried out using SYBR Master Mix (Thermo Fisher).

    RNA Extraction:

    Article Title: Generation of targeted homozygosity in the genome of human induced pluripotent stem cells
    Article Snippet: Paragraph title: RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) ... First-strand cDNA was synthesized from 800 ng total RNA with random hexamer primers using SuperScript III (Invitrogen) at 50°C for 60 min. qRT-PCR was performed to measure BLM gene expression.

    Article Title: SUR-8 interacts with PP1-87B to stabilize PERIOD and regulate circadian rhythms in Drosophila
    Article Snippet: .. RNA extraction and qRT-PCR analysis Total RNA was extracted from fly heads and then subject to reverse transcription using superscript III (Thermo Fisher). .. Quantitative real-time PCR was carried out using SYBR Master Mix (Thermo Fisher).

    Sample Prep:

    Article Title: Development of a Potent and Protective Germline-Like Antibody Lineage Against Zika Virus in a Convalescent Human
    Article Snippet: Paragraph title: Sample Preparation Using 5′-RACE PCR ... For ZK2B10 gene-specific lineage analysis, reverse transcription (RT) was performed with SuperScript III (Life Technologies) and oligo (dT).

    Random Hexamer Labeling:

    Article Title: Generation of targeted homozygosity in the genome of human induced pluripotent stem cells
    Article Snippet: .. First-strand cDNA was synthesized from 800 ng total RNA with random hexamer primers using SuperScript III (Invitrogen) at 50°C for 60 min. qRT-PCR was performed to measure BLM gene expression. .. Total RNA was reverse transcribed to cDNA using the SuperScript III First-Strand Synthesis System for qRT-PCR (Invitrogen). qRT-PCR was performed with the Applied Biosystems 7900HT Fast Real-Time PCR System using the following PCR primer sets: RTPCR-leader-F and RTPCR-exon3-R, hActb-RTPCR-F, and hActb-RTPCR-R ( ).

    Knock-Out:

    Article Title: MOZ is essential for maintenance of hematopoietic stem cells
    Article Snippet: To identify genes that were significantly affected by MOZ knockout, we selected genes that showed a change call of Increase and a Signal Log Ratio of more than 1 (more than twofold increase) or genes that showed a change call of Decrease and a Signal Log Ratio of less than −1 (more than twofold decrease) in all four comparisons between MOZ−/− and MOZ+/+ fetal livers. .. Reverse transcription of total RNA isolated from E12.5 fetal liver and E14.5 Lin− /Sca-1+ cells was performed using SuperScript III (Invitrogen Life Technologies).

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