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    SNCA mRNA and α-Synuclein Protein Levels in the MD NPC Lines Carrying the gRNA-dCas9-DNMT3A Transgenes (A) Levels of SNCA mRNA were assessed using <t>qRT-PCR.</t> The SNCA mRNA levels in the different lines were measured by <t>TaqMan-based</t> gene expression assays and calculated relative to the geometric mean of GAPDH mRNA and PPIA mRNA reference controls using the 2 −ΔΔCt method. Each bar represents the mean ± SEM of four biological and two technical replicates for a particular MD NPC line. The calibrator was a particular RNA sample of the control MD NPCs that was included in all runs. (B and C) The levels of α-synuclein protein were semi-quantified by western blot using the α-synuclein rabbit monoclonal antibody normalized to β-actin mAb. (B) The two lanes represent the analysis performed using the samples of the following MD NPC lines: gRNA4-dCas9-DNMT3A and dCas9-DNMT3A no-gRNA. (C) Quantification of the α-synuclein protein signals for each MD NPC line using ImageJ. Bars represents the intensity of the bands ± SEM of two biological and technical repeats. (D–G) Representative immunocytochemistry images for the α-synuclein signal (D and F) and the α-synuclein and Nestin double-staining signals (E and G) of the MD NPC lines. Scale bars, 10 μm. (H) Quantification of the α-synuclein protein signal in the MD NPC line carrying the gRNA4-dCas9-DNMT3A vector and the control line with the no-gRNA vector. 50 cells were imaged in two independent experiments. Bars represent the means ± SEM of the intensity of α-synuclein staining in a total of 100 cells. *p
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    SNCA mRNA and α-Synuclein Protein Levels in the MD NPC Lines Carrying the gRNA-dCas9-DNMT3A Transgenes (A) Levels of SNCA mRNA were assessed using <t>qRT-PCR.</t> The SNCA mRNA levels in the different lines were measured by <t>TaqMan-based</t> gene expression assays and calculated relative to the geometric mean of GAPDH mRNA and PPIA mRNA reference controls using the 2 −ΔΔCt method. Each bar represents the mean ± SEM of four biological and two technical replicates for a particular MD NPC line. The calibrator was a particular RNA sample of the control MD NPCs that was included in all runs. (B and C) The levels of α-synuclein protein were semi-quantified by western blot using the α-synuclein rabbit monoclonal antibody normalized to β-actin mAb. (B) The two lanes represent the analysis performed using the samples of the following MD NPC lines: gRNA4-dCas9-DNMT3A and dCas9-DNMT3A no-gRNA. (C) Quantification of the α-synuclein protein signals for each MD NPC line using ImageJ. Bars represents the intensity of the bands ± SEM of two biological and technical repeats. (D–G) Representative immunocytochemistry images for the α-synuclein signal (D and F) and the α-synuclein and Nestin double-staining signals (E and G) of the MD NPC lines. Scale bars, 10 μm. (H) Quantification of the α-synuclein protein signal in the MD NPC line carrying the gRNA4-dCas9-DNMT3A vector and the control line with the no-gRNA vector. 50 cells were imaged in two independent experiments. Bars represent the means ± SEM of the intensity of α-synuclein staining in a total of 100 cells. *p
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    Quantitative real-time <t>PCR</t> validation of miRNAs. To validate differential expression of the miRNAs, 4 of the miRNAs with the largest fold-changes (2 up- and 2 down-regulated; see Table 1 ), were validated using quantitative real-time PCR with either Taqman or <t>SYBR</t> Green. In addition, we performed qRT-PCR on miR-423-5p, which was the only miRNA differentially expressed at both 1 and 6 hours, and miR-466f-3p, which was chosen for subsequent investigations. If significant (FDR of 10%), miRNA fold-change on the array is given below the corresponding stretch duration. PCR results are qualitatively consistent in direction and magnitude with the array. N = 4-6/group. ANOVA and post-hoc Tukey results: *p
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    <t>qRT-PCR</t> validation of a subset of differentially expressed genes in mESC and iMOP cells. Graphs show linear fold change of differentially expressed genes by <t>Taqman</t> qPCR detection in Atoh1-expressing cells (blue) and Atoh1/miR-183 family-expressing cells (red) compared to control in mESCs (A) and iMOP cells (B). Data were normalized to detection of 18S ribosomal RNA. All values represent statistically significant differences for mESCs except for Ndnf expression in Atoh1/miR-183 family-expressing cells, and statistically significant differences are indicated by asterisks for iMOP cells (n = 3, Student’s t-test p
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    <t>qRT-PCR</t> validation of a subset of differentially expressed genes in mESC and iMOP cells. Graphs show linear fold change of differentially expressed genes by <t>Taqman</t> qPCR detection in Atoh1-expressing cells (blue) and Atoh1/miR-183 family-expressing cells (red) compared to control in mESCs (A) and iMOP cells (B). Data were normalized to detection of 18S ribosomal RNA. All values represent statistically significant differences for mESCs except for Ndnf expression in Atoh1/miR-183 family-expressing cells, and statistically significant differences are indicated by asterisks for iMOP cells (n = 3, Student’s t-test p
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    <t>qRT-PCR</t> validation of a subset of differentially expressed genes in mESC and iMOP cells. Graphs show linear fold change of differentially expressed genes by <t>Taqman</t> qPCR detection in Atoh1-expressing cells (blue) and Atoh1/miR-183 family-expressing cells (red) compared to control in mESCs (A) and iMOP cells (B). Data were normalized to detection of 18S ribosomal RNA. All values represent statistically significant differences for mESCs except for Ndnf expression in Atoh1/miR-183 family-expressing cells, and statistically significant differences are indicated by asterisks for iMOP cells (n = 3, Student’s t-test p
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    Image Search Results


    SNCA mRNA and α-Synuclein Protein Levels in the MD NPC Lines Carrying the gRNA-dCas9-DNMT3A Transgenes (A) Levels of SNCA mRNA were assessed using qRT-PCR. The SNCA mRNA levels in the different lines were measured by TaqMan-based gene expression assays and calculated relative to the geometric mean of GAPDH mRNA and PPIA mRNA reference controls using the 2 −ΔΔCt method. Each bar represents the mean ± SEM of four biological and two technical replicates for a particular MD NPC line. The calibrator was a particular RNA sample of the control MD NPCs that was included in all runs. (B and C) The levels of α-synuclein protein were semi-quantified by western blot using the α-synuclein rabbit monoclonal antibody normalized to β-actin mAb. (B) The two lanes represent the analysis performed using the samples of the following MD NPC lines: gRNA4-dCas9-DNMT3A and dCas9-DNMT3A no-gRNA. (C) Quantification of the α-synuclein protein signals for each MD NPC line using ImageJ. Bars represents the intensity of the bands ± SEM of two biological and technical repeats. (D–G) Representative immunocytochemistry images for the α-synuclein signal (D and F) and the α-synuclein and Nestin double-staining signals (E and G) of the MD NPC lines. Scale bars, 10 μm. (H) Quantification of the α-synuclein protein signal in the MD NPC line carrying the gRNA4-dCas9-DNMT3A vector and the control line with the no-gRNA vector. 50 cells were imaged in two independent experiments. Bars represent the means ± SEM of the intensity of α-synuclein staining in a total of 100 cells. *p

    Journal: Molecular Therapy

    Article Title: Downregulation of SNCA Expression by Targeted Editing of DNA Methylation: A Potential Strategy for Precision Therapy in PD

    doi: 10.1016/j.ymthe.2018.08.019

    Figure Lengend Snippet: SNCA mRNA and α-Synuclein Protein Levels in the MD NPC Lines Carrying the gRNA-dCas9-DNMT3A Transgenes (A) Levels of SNCA mRNA were assessed using qRT-PCR. The SNCA mRNA levels in the different lines were measured by TaqMan-based gene expression assays and calculated relative to the geometric mean of GAPDH mRNA and PPIA mRNA reference controls using the 2 −ΔΔCt method. Each bar represents the mean ± SEM of four biological and two technical replicates for a particular MD NPC line. The calibrator was a particular RNA sample of the control MD NPCs that was included in all runs. (B and C) The levels of α-synuclein protein were semi-quantified by western blot using the α-synuclein rabbit monoclonal antibody normalized to β-actin mAb. (B) The two lanes represent the analysis performed using the samples of the following MD NPC lines: gRNA4-dCas9-DNMT3A and dCas9-DNMT3A no-gRNA. (C) Quantification of the α-synuclein protein signals for each MD NPC line using ImageJ. Bars represents the intensity of the bands ± SEM of two biological and technical repeats. (D–G) Representative immunocytochemistry images for the α-synuclein signal (D and F) and the α-synuclein and Nestin double-staining signals (E and G) of the MD NPC lines. Scale bars, 10 μm. (H) Quantification of the α-synuclein protein signal in the MD NPC line carrying the gRNA4-dCas9-DNMT3A vector and the control line with the no-gRNA vector. 50 cells were imaged in two independent experiments. Bars represent the means ± SEM of the intensity of α-synuclein staining in a total of 100 cells. *p

    Article Snippet: Each cDNA (20 ng) was amplified in duplicate in at least two independent runs for two independent experiments (overall ≥8 repeats), using TaqMan Universal PCR master mix reagent (Applied Biosystems) and the following conditions: 2 min at 50°C, 10 min at 95°C, and 40 cycles of 15 s at 95°C and 1 min at 60°C.

    Techniques: Quantitative RT-PCR, Expressing, Western Blot, Immunocytochemistry, Double Staining, Plasmid Preparation, Staining

    Characterization of the Stably Transduced SNCA-Tri MD NPCs Representative immunocytochemistry images of the SNCA-Tri MD NPCs carrying the gRNA-dCas9-DNMT3A transgene. (A–J) MD NPCs showed the expression of Nestin (A–E) and FoxA2 (F–J) with no gRNA (A and F), gRNA1 (B and G), gRNA2 (C and H), gRNA3 (D and I), or gRNA4 (E and J). Scale bars, 10 μm. (K and L) MD NPC markers were evaluated using real-time qRT-PCR for (K) Nestin and (L) FoxA2. The levels of mRNAs were measured by TaqMan expression assays and calculated relative to the geometric mean of GAPDH mRNA and PPIA mRNA reference controls using the 2 −ΔΔCT method. Each column represents the mean of two biological and technical replicates. The error bars represent the SEM.

    Journal: Molecular Therapy

    Article Title: Downregulation of SNCA Expression by Targeted Editing of DNA Methylation: A Potential Strategy for Precision Therapy in PD

    doi: 10.1016/j.ymthe.2018.08.019

    Figure Lengend Snippet: Characterization of the Stably Transduced SNCA-Tri MD NPCs Representative immunocytochemistry images of the SNCA-Tri MD NPCs carrying the gRNA-dCas9-DNMT3A transgene. (A–J) MD NPCs showed the expression of Nestin (A–E) and FoxA2 (F–J) with no gRNA (A and F), gRNA1 (B and G), gRNA2 (C and H), gRNA3 (D and I), or gRNA4 (E and J). Scale bars, 10 μm. (K and L) MD NPC markers were evaluated using real-time qRT-PCR for (K) Nestin and (L) FoxA2. The levels of mRNAs were measured by TaqMan expression assays and calculated relative to the geometric mean of GAPDH mRNA and PPIA mRNA reference controls using the 2 −ΔΔCT method. Each column represents the mean of two biological and technical replicates. The error bars represent the SEM.

    Article Snippet: Each cDNA (20 ng) was amplified in duplicate in at least two independent runs for two independent experiments (overall ≥8 repeats), using TaqMan Universal PCR master mix reagent (Applied Biosystems) and the following conditions: 2 min at 50°C, 10 min at 95°C, and 40 cycles of 15 s at 95°C and 1 min at 60°C.

    Techniques: Stable Transfection, Immunocytochemistry, Expressing, Quantitative RT-PCR

    Quantitative real-time PCR validation of miRNAs. To validate differential expression of the miRNAs, 4 of the miRNAs with the largest fold-changes (2 up- and 2 down-regulated; see Table 1 ), were validated using quantitative real-time PCR with either Taqman or SYBR Green. In addition, we performed qRT-PCR on miR-423-5p, which was the only miRNA differentially expressed at both 1 and 6 hours, and miR-466f-3p, which was chosen for subsequent investigations. If significant (FDR of 10%), miRNA fold-change on the array is given below the corresponding stretch duration. PCR results are qualitatively consistent in direction and magnitude with the array. N = 4-6/group. ANOVA and post-hoc Tukey results: *p

    Journal: BMC Genomics

    Article Title: MicroRNA modulate alveolar epithelial response to cyclic stretch

    doi: 10.1186/1471-2164-13-154

    Figure Lengend Snippet: Quantitative real-time PCR validation of miRNAs. To validate differential expression of the miRNAs, 4 of the miRNAs with the largest fold-changes (2 up- and 2 down-regulated; see Table 1 ), were validated using quantitative real-time PCR with either Taqman or SYBR Green. In addition, we performed qRT-PCR on miR-423-5p, which was the only miRNA differentially expressed at both 1 and 6 hours, and miR-466f-3p, which was chosen for subsequent investigations. If significant (FDR of 10%), miRNA fold-change on the array is given below the corresponding stretch duration. PCR results are qualitatively consistent in direction and magnitude with the array. N = 4-6/group. ANOVA and post-hoc Tukey results: *p

    Article Snippet: Real-time PCR was performed (Taqman universal master mix or Applied Biosystems power SYBR green PCR master mix).

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

    qRT-PCR validation of a subset of differentially expressed genes in mESC and iMOP cells. Graphs show linear fold change of differentially expressed genes by Taqman qPCR detection in Atoh1-expressing cells (blue) and Atoh1/miR-183 family-expressing cells (red) compared to control in mESCs (A) and iMOP cells (B). Data were normalized to detection of 18S ribosomal RNA. All values represent statistically significant differences for mESCs except for Ndnf expression in Atoh1/miR-183 family-expressing cells, and statistically significant differences are indicated by asterisks for iMOP cells (n = 3, Student’s t-test p

    Journal: PLoS ONE

    Article Title: Transcriptome-wide comparison of the impact of Atoh1 and miR-183 family on pluripotent stem cells and multipotent otic progenitor cells

    doi: 10.1371/journal.pone.0180855

    Figure Lengend Snippet: qRT-PCR validation of a subset of differentially expressed genes in mESC and iMOP cells. Graphs show linear fold change of differentially expressed genes by Taqman qPCR detection in Atoh1-expressing cells (blue) and Atoh1/miR-183 family-expressing cells (red) compared to control in mESCs (A) and iMOP cells (B). Data were normalized to detection of 18S ribosomal RNA. All values represent statistically significant differences for mESCs except for Ndnf expression in Atoh1/miR-183 family-expressing cells, and statistically significant differences are indicated by asterisks for iMOP cells (n = 3, Student’s t-test p

    Article Snippet: Reverse transcription and quantitative PCR analysis (qRT-PCR) For mRNA detection, the TaqMan Reverse Transcription Kit (Life technologies) was used to reverse-transcribe RNA according to the manufacturer protocol. qPCR was performed using Taqman Fast Advanced Universal PCR Master Mix (Life Technologies), and reactions were analyzed using a 7500 Fast Real-Time PCR Instrument (Applied Biosystems).

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