pcr based site directed mutagenesis  (Agilent technologies)

 
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    InfinityLab Quick Change Valves
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    InfinityLab Quick Change Valves enable automation of a wide variety of applications such as column or solvent selection column regeneration or sample cleanup and enrichment The valves design with separate valve heads and drives provides the flexibility to choose combinations that match any laboratory s individual application requirements Quick Change Valves can be installed in the externally mounted Agilent 1290 Infinity Valve Drive or modules containing valves including the Agilent 1260 Infinity II Multicolumn Thermostat the Agilent 1290 Infinity II Multicolumn Thermostat each with one valve or the Agilent 1290 Infinity Flexible Cube with up to two valves
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    INFINITYLAB-QUICK-CHANGE-VALVES
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    Agilent technologies pcr based site directed mutagenesis
    Effect of Aβ on ADAM10 and miR-140-5p (A) Neuronal SHSY5Y cells were transfected with ADAM10 promoter (ADAM10p) and ADAM10 <t>promoter-3’UTR</t> (ADAM10p+3’utr) reporter constructs, 48 h post-transfection is followed by treatment with Aβ (5 µM) for 24 hrs. Results are presented as Firefly/Renilla luciferase activity of the respective constructs with or without Aβ as control. Data represent at least six independent experiments with triplicate measurements. (B) Neuronal SHSY5Y cells with miR-140-5p expression either without or with Aβ (5 µM) treatment and transfected with miRNA-140-5p inhibitor. MiRNA-140-5p expression was measured by <t>qRT-PCR</t> and represented as fold change compared with untreated control. Results are represented by 2^-ΔΔCT. Black line indicates Fisher exact t-test p-value
    InfinityLab Quick Change Valves enable automation of a wide variety of applications such as column or solvent selection column regeneration or sample cleanup and enrichment The valves design with separate valve heads and drives provides the flexibility to choose combinations that match any laboratory s individual application requirements Quick Change Valves can be installed in the externally mounted Agilent 1290 Infinity Valve Drive or modules containing valves including the Agilent 1260 Infinity II Multicolumn Thermostat the Agilent 1290 Infinity II Multicolumn Thermostat each with one valve or the Agilent 1290 Infinity Flexible Cube with up to two valves
    https://www.bioz.com/result/pcr based site directed mutagenesis/product/Agilent technologies
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pcr based site directed mutagenesis - by Bioz Stars, 2021-06
    86/100 stars

    Images

    1) Product Images from "Regulation of ADAM10 by miR-140-5p and potential relevance for Alzheimer’s Disease"

    Article Title: Regulation of ADAM10 by miR-140-5p and potential relevance for Alzheimer’s Disease

    Journal: Neurobiology of aging

    doi: 10.1016/j.neurobiolaging.2017.11.007

    Effect of Aβ on ADAM10 and miR-140-5p (A) Neuronal SHSY5Y cells were transfected with ADAM10 promoter (ADAM10p) and ADAM10 promoter-3’UTR (ADAM10p+3’utr) reporter constructs, 48 h post-transfection is followed by treatment with Aβ (5 µM) for 24 hrs. Results are presented as Firefly/Renilla luciferase activity of the respective constructs with or without Aβ as control. Data represent at least six independent experiments with triplicate measurements. (B) Neuronal SHSY5Y cells with miR-140-5p expression either without or with Aβ (5 µM) treatment and transfected with miRNA-140-5p inhibitor. MiRNA-140-5p expression was measured by qRT-PCR and represented as fold change compared with untreated control. Results are represented by 2^-ΔΔCT. Black line indicates Fisher exact t-test p-value
    Figure Legend Snippet: Effect of Aβ on ADAM10 and miR-140-5p (A) Neuronal SHSY5Y cells were transfected with ADAM10 promoter (ADAM10p) and ADAM10 promoter-3’UTR (ADAM10p+3’utr) reporter constructs, 48 h post-transfection is followed by treatment with Aβ (5 µM) for 24 hrs. Results are presented as Firefly/Renilla luciferase activity of the respective constructs with or without Aβ as control. Data represent at least six independent experiments with triplicate measurements. (B) Neuronal SHSY5Y cells with miR-140-5p expression either without or with Aβ (5 µM) treatment and transfected with miRNA-140-5p inhibitor. MiRNA-140-5p expression was measured by qRT-PCR and represented as fold change compared with untreated control. Results are represented by 2^-ΔΔCT. Black line indicates Fisher exact t-test p-value

    Techniques Used: Transfection, Construct, Luciferase, Activity Assay, Expressing, Quantitative RT-PCR

    MiRNA expression levels in human AD hippocampus compared to controls (A) The expression profile of ADAM10 candidate miRNAs in microarray represented by 2^-ΔΔCT`. (B) Quantitative RT-PCR analysis of ADAM10 candidate miRNA-140, miRNA-182, miRNA-194 and control miRNA-126 in Control vs AD. Data represent three independent experiments with triplicate measurements. All p-values were corrected with the Bonferroni correction for multiple comparisons.
    Figure Legend Snippet: MiRNA expression levels in human AD hippocampus compared to controls (A) The expression profile of ADAM10 candidate miRNAs in microarray represented by 2^-ΔΔCT`. (B) Quantitative RT-PCR analysis of ADAM10 candidate miRNA-140, miRNA-182, miRNA-194 and control miRNA-126 in Control vs AD. Data represent three independent experiments with triplicate measurements. All p-values were corrected with the Bonferroni correction for multiple comparisons.

    Techniques Used: Expressing, Microarray, Quantitative RT-PCR

    2) Product Images from "Frequency of COL4A3/COL4A4 Mutations amongst Families Segregating Glomerular Microscopic Hematuria and Evidence for Activation of the Unfolded Protein Response. Focal and Segmental Glomerulosclerosis Is a Frequent Development during Ageing"

    Article Title: Frequency of COL4A3/COL4A4 Mutations amongst Families Segregating Glomerular Microscopic Hematuria and Evidence for Activation of the Unfolded Protein Response. Focal and Segmental Glomerulosclerosis Is a Frequent Development during Ageing

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0115015

    Overexpression of wild type or mutant COL4A3 chains induces XBP1 splicing in AB8/13 cells. (a) Representative experiment of reverse transcription-PCR using XBP1 mRNA as template, from AB8/13 cells transiently expressing COL4A3 -WT (A3/WT) or the mutant chains G1334E, G871C, G484R ( COL4A3 ). PCR products were run on 3% agarose gel. It is apparent that over-expression of all chains induces XBP1 splicing, as evidenced by the appearance of the spliced band (s) when the PCR product is cut with the restriction enzyme Pst1. (h) hybrid, (u) unspliced (b) RT-PCR is quantified via densitometric analysis of the bands after PstI digestion as follows: ratio of the spliced band and the sum of the two PstI digest bands (s/(u1+u2), with PstI digestion. Hybrid band (h) was considered as equally contributing to unspliced and spliced bands. There is statistically significant XBP1 splicing when either WT or any of the mutant chains is overexpressed in AB8/13 cells. L19 was used as an internal PCR control. Data are means ± SEM of three independent experiments.
    Figure Legend Snippet: Overexpression of wild type or mutant COL4A3 chains induces XBP1 splicing in AB8/13 cells. (a) Representative experiment of reverse transcription-PCR using XBP1 mRNA as template, from AB8/13 cells transiently expressing COL4A3 -WT (A3/WT) or the mutant chains G1334E, G871C, G484R ( COL4A3 ). PCR products were run on 3% agarose gel. It is apparent that over-expression of all chains induces XBP1 splicing, as evidenced by the appearance of the spliced band (s) when the PCR product is cut with the restriction enzyme Pst1. (h) hybrid, (u) unspliced (b) RT-PCR is quantified via densitometric analysis of the bands after PstI digestion as follows: ratio of the spliced band and the sum of the two PstI digest bands (s/(u1+u2), with PstI digestion. Hybrid band (h) was considered as equally contributing to unspliced and spliced bands. There is statistically significant XBP1 splicing when either WT or any of the mutant chains is overexpressed in AB8/13 cells. L19 was used as an internal PCR control. Data are means ± SEM of three independent experiments.

    Techniques Used: Over Expression, Mutagenesis, Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction

    3) Product Images from "Phosphorylation at tyrosine 114 of Proliferating Cell Nuclear Antigen (PCNA) is required for adipogenesis in response to high fat diet"

    Article Title: Phosphorylation at tyrosine 114 of Proliferating Cell Nuclear Antigen (PCNA) is required for adipogenesis in response to high fat diet

    Journal: Biochemical and biophysical research communications

    doi: 10.1016/j.bbrc.2012.11.047

    Y114 phosphorylation of PCNA and the knock-in mouse model A , The Y114 residue of PCNA is evolutionarily conserved. B , HKE293T cells were transfected with FLAG-tagged PCNA of WT or Y114F mutant. The ectopic PCNA was immunoprecipitated with an anti-FLAG antibody and the blot was probed with the anti-phospho-Y114 PCNA antibody. C , Targeting strategy to generate Y114F knock-in mutation. D , PCR analysis to confirm the WT and the Y114F allele in WT, heterozygous (W/F) and homozygous (F/F) mutant mice. E , Protein extracts of WT and F/F MEF cells were examined for total PCNA and phospho-Y114 PCNA (p-PCNA) by Western analysis.
    Figure Legend Snippet: Y114 phosphorylation of PCNA and the knock-in mouse model A , The Y114 residue of PCNA is evolutionarily conserved. B , HKE293T cells were transfected with FLAG-tagged PCNA of WT or Y114F mutant. The ectopic PCNA was immunoprecipitated with an anti-FLAG antibody and the blot was probed with the anti-phospho-Y114 PCNA antibody. C , Targeting strategy to generate Y114F knock-in mutation. D , PCR analysis to confirm the WT and the Y114F allele in WT, heterozygous (W/F) and homozygous (F/F) mutant mice. E , Protein extracts of WT and F/F MEF cells were examined for total PCNA and phospho-Y114 PCNA (p-PCNA) by Western analysis.

    Techniques Used: Knock-In, Transfection, Mutagenesis, Immunoprecipitation, Polymerase Chain Reaction, Mouse Assay, Western Blot

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    Agilent technologies pcr based site directed mutagenesis kit
    Methylation status of the <t>miR-520c</t> regulatory region and expression of miR-520c-3p and its target S100A4 in a cohort of CRC tumor specimens ( A ) Methylation specific <t>PCR</t> products of metachronous metastasis positive and metachronous negative CRC tumor specimens were analyzed using agarose gel electrophoresis. The miR-520c regulatory element was methylated in all specimens. The two cell lines SW620 and Colo206f served as internal controls. ( B ) miR-520c-3p expression in metachronous metastasis positive ( n = 10) and negative ( n = 10) CRC tumor specimens in comparison to representative normal mucosa were quantified using qRT-PCR. ( C , D ) miR-520c-3p and S100A4 expression in CRC tumor specimens in comparison to representative normal mucosa were quantified using qRT-PCR. RPII and RNUB6 served as internal controls.
    Pcr Based Site Directed Mutagenesis Kit, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr based site directed mutagenesis kit/product/Agilent technologies
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pcr based site directed mutagenesis kit - by Bioz Stars, 2021-06
    99/100 stars
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    86
    Agilent technologies pcr based site directed mutagenesis
    Effect of Aβ on ADAM10 and miR-140-5p (A) Neuronal SHSY5Y cells were transfected with ADAM10 promoter (ADAM10p) and ADAM10 <t>promoter-3’UTR</t> (ADAM10p+3’utr) reporter constructs, 48 h post-transfection is followed by treatment with Aβ (5 µM) for 24 hrs. Results are presented as Firefly/Renilla luciferase activity of the respective constructs with or without Aβ as control. Data represent at least six independent experiments with triplicate measurements. (B) Neuronal SHSY5Y cells with miR-140-5p expression either without or with Aβ (5 µM) treatment and transfected with miRNA-140-5p inhibitor. MiRNA-140-5p expression was measured by <t>qRT-PCR</t> and represented as fold change compared with untreated control. Results are represented by 2^-ΔΔCT. Black line indicates Fisher exact t-test p-value
    Pcr Based Site Directed Mutagenesis, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr based site directed mutagenesis/product/Agilent technologies
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pcr based site directed mutagenesis - by Bioz Stars, 2021-06
    86/100 stars
      Buy from Supplier

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    Methylation status of the miR-520c regulatory region and expression of miR-520c-3p and its target S100A4 in a cohort of CRC tumor specimens ( A ) Methylation specific PCR products of metachronous metastasis positive and metachronous negative CRC tumor specimens were analyzed using agarose gel electrophoresis. The miR-520c regulatory element was methylated in all specimens. The two cell lines SW620 and Colo206f served as internal controls. ( B ) miR-520c-3p expression in metachronous metastasis positive ( n = 10) and negative ( n = 10) CRC tumor specimens in comparison to representative normal mucosa were quantified using qRT-PCR. ( C , D ) miR-520c-3p and S100A4 expression in CRC tumor specimens in comparison to representative normal mucosa were quantified using qRT-PCR. RPII and RNUB6 served as internal controls.

    Journal: Oncotarget

    Article Title: Epigenetic silencing of miR-520c leads to induced S100A4 expression and its mediated colorectal cancer progression

    doi: 10.18632/oncotarget.15499

    Figure Lengend Snippet: Methylation status of the miR-520c regulatory region and expression of miR-520c-3p and its target S100A4 in a cohort of CRC tumor specimens ( A ) Methylation specific PCR products of metachronous metastasis positive and metachronous negative CRC tumor specimens were analyzed using agarose gel electrophoresis. The miR-520c regulatory element was methylated in all specimens. The two cell lines SW620 and Colo206f served as internal controls. ( B ) miR-520c-3p expression in metachronous metastasis positive ( n = 10) and negative ( n = 10) CRC tumor specimens in comparison to representative normal mucosa were quantified using qRT-PCR. ( C , D ) miR-520c-3p and S100A4 expression in CRC tumor specimens in comparison to representative normal mucosa were quantified using qRT-PCR. RPII and RNUB6 served as internal controls.

    Article Snippet: Specific miR-505-5p and miR-520c-3p seed sequences were mutated using PCR based site directed mutagenesis kit (#210518, Agilent technologies, USA).

    Techniques: Methylation, Expressing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Quantitative RT-PCR

    miR-505-5p and miR-520c-3p inhibit the S100A4 mediated migration and invasion in vitro ( A ) HCT116 cells were co-transfected with control-miR, miR-505-5p and miR-520c-3p along with either vector-control or -S100A4, respectively. After 48 h cells were plated on top of the Boyden chambers for migration and matrigel-coated Boyden chambers for invasion. After 16 h the migrated or invaded cells were measured as described in the materials and methods. ( B ) SW620 cells were co-transfected with control-miR, miR-505-5p and miR-520c-3p along with either si-RNA-control (si-control) or si-RNA-S100A4 (si-S100A4), respectively. After 48 h the cells were used for migration and invasion assay as stated above. ( C , D ) Transfection efficiency of ectopic overexpression and knock-down of S100A4 on mRNA level was analyzed using qRT-PCR. ( E , F ) S100A4 protein expression after transfection was analyzed by Western blots. RPII was used as internal control for S100A4 mRNA expression and β-actin for Western blotting. ( *p

    Journal: Oncotarget

    Article Title: Epigenetic silencing of miR-520c leads to induced S100A4 expression and its mediated colorectal cancer progression

    doi: 10.18632/oncotarget.15499

    Figure Lengend Snippet: miR-505-5p and miR-520c-3p inhibit the S100A4 mediated migration and invasion in vitro ( A ) HCT116 cells were co-transfected with control-miR, miR-505-5p and miR-520c-3p along with either vector-control or -S100A4, respectively. After 48 h cells were plated on top of the Boyden chambers for migration and matrigel-coated Boyden chambers for invasion. After 16 h the migrated or invaded cells were measured as described in the materials and methods. ( B ) SW620 cells were co-transfected with control-miR, miR-505-5p and miR-520c-3p along with either si-RNA-control (si-control) or si-RNA-S100A4 (si-S100A4), respectively. After 48 h the cells were used for migration and invasion assay as stated above. ( C , D ) Transfection efficiency of ectopic overexpression and knock-down of S100A4 on mRNA level was analyzed using qRT-PCR. ( E , F ) S100A4 protein expression after transfection was analyzed by Western blots. RPII was used as internal control for S100A4 mRNA expression and β-actin for Western blotting. ( *p

    Article Snippet: Specific miR-505-5p and miR-520c-3p seed sequences were mutated using PCR based site directed mutagenesis kit (#210518, Agilent technologies, USA).

    Techniques: Migration, In Vitro, Transfection, Plasmid Preparation, Invasion Assay, Over Expression, Quantitative RT-PCR, Expressing, Western Blot

    miR-520c regulatory region is hypermethylated in CRC cell lines and treatment with 5-Aza induces the expression of miR-520c-3p and downregulates S100A4 expression ( A , B ) SW480, SW620 and HCT116 cells were treated with 5-Aza (2 mM) for 3 days. qRT-PCR and Western blots were performed to analyze the impact of 5-Aza treatment on miR-520c-3p and S100A4 expression. RNUB6, RPII and β-actin served as internal controls. ( C ) HCT116 and SW620 cells transfected with the wild type S100A4-3′-UTR were treated with 5-Aza (2 μM) for 24 h and the luciferase activity was measured. Renilla luciferase activity was used for normalization. Percentage luciferase activity was significantly reduced after 5-Aza. ( *p

    Journal: Oncotarget

    Article Title: Epigenetic silencing of miR-520c leads to induced S100A4 expression and its mediated colorectal cancer progression

    doi: 10.18632/oncotarget.15499

    Figure Lengend Snippet: miR-520c regulatory region is hypermethylated in CRC cell lines and treatment with 5-Aza induces the expression of miR-520c-3p and downregulates S100A4 expression ( A , B ) SW480, SW620 and HCT116 cells were treated with 5-Aza (2 mM) for 3 days. qRT-PCR and Western blots were performed to analyze the impact of 5-Aza treatment on miR-520c-3p and S100A4 expression. RNUB6, RPII and β-actin served as internal controls. ( C ) HCT116 and SW620 cells transfected with the wild type S100A4-3′-UTR were treated with 5-Aza (2 μM) for 24 h and the luciferase activity was measured. Renilla luciferase activity was used for normalization. Percentage luciferase activity was significantly reduced after 5-Aza. ( *p

    Article Snippet: Specific miR-505-5p and miR-520c-3p seed sequences were mutated using PCR based site directed mutagenesis kit (#210518, Agilent technologies, USA).

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Luciferase, Activity Assay

    COBRA and bisulfite sequencing analysis of miR-520c regulatory region ( A , B ) COBRA analysis using the restriction enzyme BstUI was performed to estimate the methylation status of miR-520c-3p in CRC cell lines. Colo206f, Colo320DM and WiDr were also analyzed after 5-Aza (2 μm) treatment for 3 days. ( C ) Colo320DM cells were treated with 2 μM 5-Aza for 3 days and the methylation of miR-520c-3p was analyzed by bisulfite sequencing. From each group 10 representative clones were sequenced. Each circle represents CpG islands in the sequenced region, and the black circle represents the methylated and the empty white circle represents unmethylated CpG island. ( D , E ) qRT-PCR of miR-520c-3p, S100A4 and Western blot analysis of S100A4 were performed after 5-Aza treatment of Colo206f, Colo320DM for 3 days (20 μg of total protein used for Western blot) and WiDr (5 μg of total protein used for Western blot) cell lines (from low to moderate S100A4 expressing cells). RNUB6, RPII and β-actin served as internal controls. ( *p

    Journal: Oncotarget

    Article Title: Epigenetic silencing of miR-520c leads to induced S100A4 expression and its mediated colorectal cancer progression

    doi: 10.18632/oncotarget.15499

    Figure Lengend Snippet: COBRA and bisulfite sequencing analysis of miR-520c regulatory region ( A , B ) COBRA analysis using the restriction enzyme BstUI was performed to estimate the methylation status of miR-520c-3p in CRC cell lines. Colo206f, Colo320DM and WiDr were also analyzed after 5-Aza (2 μm) treatment for 3 days. ( C ) Colo320DM cells were treated with 2 μM 5-Aza for 3 days and the methylation of miR-520c-3p was analyzed by bisulfite sequencing. From each group 10 representative clones were sequenced. Each circle represents CpG islands in the sequenced region, and the black circle represents the methylated and the empty white circle represents unmethylated CpG island. ( D , E ) qRT-PCR of miR-520c-3p, S100A4 and Western blot analysis of S100A4 were performed after 5-Aza treatment of Colo206f, Colo320DM for 3 days (20 μg of total protein used for Western blot) and WiDr (5 μg of total protein used for Western blot) cell lines (from low to moderate S100A4 expressing cells). RNUB6, RPII and β-actin served as internal controls. ( *p

    Article Snippet: Specific miR-505-5p and miR-520c-3p seed sequences were mutated using PCR based site directed mutagenesis kit (#210518, Agilent technologies, USA).

    Techniques: Combined Bisulfite Restriction Analysis Assay, Methylation Sequencing, Methylation, Clone Assay, Quantitative RT-PCR, Western Blot, Expressing

    Overexpression of miR-520c-3p inhibits metastasis formation in vivo ( A ) HCT116 cells stably expressing miR-520c-3p or control-miR were generated. The overexpression of the miR was quantified by qRT-PCR and compared to control-miR. The expression of the target gene S100A4 in the miR overexpressing cells was downregulated as indicated by Western blotting. ( B ) The cells were intrasplenically injected in SCID beige mice and sacrificed after 20 days. The expression of miR-520c-3p in the shock-frozen primary tumor in the spleen was quantified by qRT-PCR. ( C ) Metastasized cells into the liver of the animals were analyzed using specific primers to exclusively detect HMD by qRT-PCR. Less amounts of HMD were detectable in the liver sections of the miR-520c-3p overexpressing group. ( D ) The cells were intrasplenically injected in SCID beige mice and tumor and metastasis formation was monitored by bioluminescence imaging. Representative mice from each group are shown on the day the animals were sacrificed. ( *p

    Journal: Oncotarget

    Article Title: Epigenetic silencing of miR-520c leads to induced S100A4 expression and its mediated colorectal cancer progression

    doi: 10.18632/oncotarget.15499

    Figure Lengend Snippet: Overexpression of miR-520c-3p inhibits metastasis formation in vivo ( A ) HCT116 cells stably expressing miR-520c-3p or control-miR were generated. The overexpression of the miR was quantified by qRT-PCR and compared to control-miR. The expression of the target gene S100A4 in the miR overexpressing cells was downregulated as indicated by Western blotting. ( B ) The cells were intrasplenically injected in SCID beige mice and sacrificed after 20 days. The expression of miR-520c-3p in the shock-frozen primary tumor in the spleen was quantified by qRT-PCR. ( C ) Metastasized cells into the liver of the animals were analyzed using specific primers to exclusively detect HMD by qRT-PCR. Less amounts of HMD were detectable in the liver sections of the miR-520c-3p overexpressing group. ( D ) The cells were intrasplenically injected in SCID beige mice and tumor and metastasis formation was monitored by bioluminescence imaging. Representative mice from each group are shown on the day the animals were sacrificed. ( *p

    Article Snippet: Specific miR-505-5p and miR-520c-3p seed sequences were mutated using PCR based site directed mutagenesis kit (#210518, Agilent technologies, USA).

    Techniques: Over Expression, In Vivo, Stable Transfection, Expressing, Generated, Quantitative RT-PCR, Western Blot, Injection, Mouse Assay, Imaging

    miR-505-5p and miR-520c-3p target the S100A4-3′UTR, and downregulate S100A4 expression ( A ) The seed sequences for miR-505-5p and miR-520c-3p in S100A4-3′-UTR (wild type) were detected by in silico predictions. The wild type and mutated seed sequence were cloned into the dual-luciferase vector and luciferase assays were performed to show direct regulation of the S100A4-3′-UTR by these miRs. ( B ) Luciferase reporter assays of HCT116 and SW620 cells transfected with control-miR (Control), miR-505-5p, anti-miR-505-5p, miR-520c-3p, anti-miR-520c-3p and co-transfected with either S100A4-3′-UTR wild type or the mutated constructs were performed. Renilla luciferase values of the vector were used for normalization. Percent luciferase activity was calculated based on the control values. ( C ) S100A4 mRNA expression was quantified using qRT-PCR and RPII was used as internal control. ( D ) S100A4 Western blotting was performed and bands were densitometrically quantified by normalizing to the internal control β-actin. Mean values of triplicates were represented. ( *p

    Journal: Oncotarget

    Article Title: Epigenetic silencing of miR-520c leads to induced S100A4 expression and its mediated colorectal cancer progression

    doi: 10.18632/oncotarget.15499

    Figure Lengend Snippet: miR-505-5p and miR-520c-3p target the S100A4-3′UTR, and downregulate S100A4 expression ( A ) The seed sequences for miR-505-5p and miR-520c-3p in S100A4-3′-UTR (wild type) were detected by in silico predictions. The wild type and mutated seed sequence were cloned into the dual-luciferase vector and luciferase assays were performed to show direct regulation of the S100A4-3′-UTR by these miRs. ( B ) Luciferase reporter assays of HCT116 and SW620 cells transfected with control-miR (Control), miR-505-5p, anti-miR-505-5p, miR-520c-3p, anti-miR-520c-3p and co-transfected with either S100A4-3′-UTR wild type or the mutated constructs were performed. Renilla luciferase values of the vector were used for normalization. Percent luciferase activity was calculated based on the control values. ( C ) S100A4 mRNA expression was quantified using qRT-PCR and RPII was used as internal control. ( D ) S100A4 Western blotting was performed and bands were densitometrically quantified by normalizing to the internal control β-actin. Mean values of triplicates were represented. ( *p

    Article Snippet: Specific miR-505-5p and miR-520c-3p seed sequences were mutated using PCR based site directed mutagenesis kit (#210518, Agilent technologies, USA).

    Techniques: Expressing, In Silico, Sequencing, Clone Assay, Luciferase, Plasmid Preparation, Transfection, Construct, Activity Assay, Quantitative RT-PCR, Western Blot

    Decreased stability of LDHB mRNA caused by HYOU1 silencing is related to the increase of miR‐375‐3p. A, The expression of miR‐375 was analysed using RT‐PCR in control or HYOU1‐silenced IHH4, TPC1 and K1 cells. B, Luciferase activity was measured in control or HYOU1‐silenced IHH4 cells transfected with luciferase reporter vector bearing wild‐type or mutant 3′UTR of LDHB mRNA. C, D, Western blot analysis of LDHB in control or HYOU1‐silenced IHH4 or K1 cells transfected miR‐375 mimics and antagomir. E, Representative images of immunostaining of HYOU1 and LDHB in PTC tissues. F‐H, HYOU1 mRNA, LDHB mRNA and miR‐375 levels were measured in 115 PTC tissues using RT‐qPCR. The correlation among them was assessed by Spearman's rank correlation

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: HYOU1 facilitates proliferation, invasion and glycolysis of papillary thyroid cancer via stabilizing LDHB mRNA. HYOU1 facilitates proliferation, invasion and glycolysis of papillary thyroid cancer via stabilizing LDHB mRNA

    doi: 10.1111/jcmm.16453

    Figure Lengend Snippet: Decreased stability of LDHB mRNA caused by HYOU1 silencing is related to the increase of miR‐375‐3p. A, The expression of miR‐375 was analysed using RT‐PCR in control or HYOU1‐silenced IHH4, TPC1 and K1 cells. B, Luciferase activity was measured in control or HYOU1‐silenced IHH4 cells transfected with luciferase reporter vector bearing wild‐type or mutant 3′UTR of LDHB mRNA. C, D, Western blot analysis of LDHB in control or HYOU1‐silenced IHH4 or K1 cells transfected miR‐375 mimics and antagomir. E, Representative images of immunostaining of HYOU1 and LDHB in PTC tissues. F‐H, HYOU1 mRNA, LDHB mRNA and miR‐375 levels were measured in 115 PTC tissues using RT‐qPCR. The correlation among them was assessed by Spearman's rank correlation

    Article Snippet: The construct containing mutant for the seed sequence of miR‐375‐3p was obtained through PCR‐based site‐directed mutagenesis kit (Agilent Technologies).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Immunostaining, Quantitative RT-PCR

    Effect of Aβ on ADAM10 and miR-140-5p (A) Neuronal SHSY5Y cells were transfected with ADAM10 promoter (ADAM10p) and ADAM10 promoter-3’UTR (ADAM10p+3’utr) reporter constructs, 48 h post-transfection is followed by treatment with Aβ (5 µM) for 24 hrs. Results are presented as Firefly/Renilla luciferase activity of the respective constructs with or without Aβ as control. Data represent at least six independent experiments with triplicate measurements. (B) Neuronal SHSY5Y cells with miR-140-5p expression either without or with Aβ (5 µM) treatment and transfected with miRNA-140-5p inhibitor. MiRNA-140-5p expression was measured by qRT-PCR and represented as fold change compared with untreated control. Results are represented by 2^-ΔΔCT. Black line indicates Fisher exact t-test p-value

    Journal: Neurobiology of aging

    Article Title: Regulation of ADAM10 by miR-140-5p and potential relevance for Alzheimer’s Disease

    doi: 10.1016/j.neurobiolaging.2017.11.007

    Figure Lengend Snippet: Effect of Aβ on ADAM10 and miR-140-5p (A) Neuronal SHSY5Y cells were transfected with ADAM10 promoter (ADAM10p) and ADAM10 promoter-3’UTR (ADAM10p+3’utr) reporter constructs, 48 h post-transfection is followed by treatment with Aβ (5 µM) for 24 hrs. Results are presented as Firefly/Renilla luciferase activity of the respective constructs with or without Aβ as control. Data represent at least six independent experiments with triplicate measurements. (B) Neuronal SHSY5Y cells with miR-140-5p expression either without or with Aβ (5 µM) treatment and transfected with miRNA-140-5p inhibitor. MiRNA-140-5p expression was measured by qRT-PCR and represented as fold change compared with untreated control. Results are represented by 2^-ΔΔCT. Black line indicates Fisher exact t-test p-value

    Article Snippet: ADAM10 and SOX2 reporter constructs with mutations in promoter or 3’UTR were generated by incorporating mutations into the miR-140 binding site on ADAM10 3’UTR and SOX2 3’UTR as well as in SOX2 binding site on ADAM10 promoter by PCR-based site directed mutagenesis using Pfu Turbo DNA polymerase (Quickchange II Site Directed Mutagenesis Kit; Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s protocol and were verified by sequencing.

    Techniques: Transfection, Construct, Luciferase, Activity Assay, Expressing, Quantitative RT-PCR

    MiRNA expression levels in human AD hippocampus compared to controls (A) The expression profile of ADAM10 candidate miRNAs in microarray represented by 2^-ΔΔCT`. (B) Quantitative RT-PCR analysis of ADAM10 candidate miRNA-140, miRNA-182, miRNA-194 and control miRNA-126 in Control vs AD. Data represent three independent experiments with triplicate measurements. All p-values were corrected with the Bonferroni correction for multiple comparisons.

    Journal: Neurobiology of aging

    Article Title: Regulation of ADAM10 by miR-140-5p and potential relevance for Alzheimer’s Disease

    doi: 10.1016/j.neurobiolaging.2017.11.007

    Figure Lengend Snippet: MiRNA expression levels in human AD hippocampus compared to controls (A) The expression profile of ADAM10 candidate miRNAs in microarray represented by 2^-ΔΔCT`. (B) Quantitative RT-PCR analysis of ADAM10 candidate miRNA-140, miRNA-182, miRNA-194 and control miRNA-126 in Control vs AD. Data represent three independent experiments with triplicate measurements. All p-values were corrected with the Bonferroni correction for multiple comparisons.

    Article Snippet: ADAM10 and SOX2 reporter constructs with mutations in promoter or 3’UTR were generated by incorporating mutations into the miR-140 binding site on ADAM10 3’UTR and SOX2 3’UTR as well as in SOX2 binding site on ADAM10 promoter by PCR-based site directed mutagenesis using Pfu Turbo DNA polymerase (Quickchange II Site Directed Mutagenesis Kit; Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s protocol and were verified by sequencing.

    Techniques: Expressing, Microarray, Quantitative RT-PCR

    Overexpression of wild type or mutant COL4A3 chains induces XBP1 splicing in AB8/13 cells. (a) Representative experiment of reverse transcription-PCR using XBP1 mRNA as template, from AB8/13 cells transiently expressing COL4A3 -WT (A3/WT) or the mutant chains G1334E, G871C, G484R ( COL4A3 ). PCR products were run on 3% agarose gel. It is apparent that over-expression of all chains induces XBP1 splicing, as evidenced by the appearance of the spliced band (s) when the PCR product is cut with the restriction enzyme Pst1. (h) hybrid, (u) unspliced (b) RT-PCR is quantified via densitometric analysis of the bands after PstI digestion as follows: ratio of the spliced band and the sum of the two PstI digest bands (s/(u1+u2), with PstI digestion. Hybrid band (h) was considered as equally contributing to unspliced and spliced bands. There is statistically significant XBP1 splicing when either WT or any of the mutant chains is overexpressed in AB8/13 cells. L19 was used as an internal PCR control. Data are means ± SEM of three independent experiments.

    Journal: PLoS ONE

    Article Title: Frequency of COL4A3/COL4A4 Mutations amongst Families Segregating Glomerular Microscopic Hematuria and Evidence for Activation of the Unfolded Protein Response. Focal and Segmental Glomerulosclerosis Is a Frequent Development during Ageing

    doi: 10.1371/journal.pone.0115015

    Figure Lengend Snippet: Overexpression of wild type or mutant COL4A3 chains induces XBP1 splicing in AB8/13 cells. (a) Representative experiment of reverse transcription-PCR using XBP1 mRNA as template, from AB8/13 cells transiently expressing COL4A3 -WT (A3/WT) or the mutant chains G1334E, G871C, G484R ( COL4A3 ). PCR products were run on 3% agarose gel. It is apparent that over-expression of all chains induces XBP1 splicing, as evidenced by the appearance of the spliced band (s) when the PCR product is cut with the restriction enzyme Pst1. (h) hybrid, (u) unspliced (b) RT-PCR is quantified via densitometric analysis of the bands after PstI digestion as follows: ratio of the spliced band and the sum of the two PstI digest bands (s/(u1+u2), with PstI digestion. Hybrid band (h) was considered as equally contributing to unspliced and spliced bands. There is statistically significant XBP1 splicing when either WT or any of the mutant chains is overexpressed in AB8/13 cells. L19 was used as an internal PCR control. Data are means ± SEM of three independent experiments.

    Article Snippet: The COL4A3 -p.(G484R), COL4A3 -p.(G871C) and COL4A3 -p.(A587G) point mutations were introduced in the WT COL4A3 by PCR-based site-directed mutagenesis (QuickChange Site-Directed Mutagenesis, Stratagene, La Jolla, CA).

    Techniques: Over Expression, Mutagenesis, Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction