pcr based site directed mutagenesis kit  (Agilent technologies)

 
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    Absolutely RNA Purification Magnetic Bead
    Description:
    Absolutely mRNA Purification Kit produces high yields of pure mRNA using a fast 20 minute protocol RNA magnetic bead based purification includes customized oligo dT magnetic particles that exclusively bind mRNA to maximize yield and quality and minimize rRNA contamination
    Catalog Number:
    ABSOLUTELY-RNA-PURIFICATION-MAGNETIC-BEAD
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    Products Real Time Pcr Qpcr Real Time Pcr Nucleic Acid Isolation And Purification Kits Rna Kit For Qpcr Absolutely Rna Purification Magnetic Bead
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    Structured Review

    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.
    Absolutely mRNA Purification Kit produces high yields of pure mRNA using a fast 20 minute protocol RNA magnetic bead based purification includes customized oligo dT magnetic particles that exclusively bind mRNA to maximize yield and quality and minimize rRNA contamination
    https://www.bioz.com/result/pcr based site directed mutagenesis kit/product/Agilent technologies
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    pcr based site directed mutagenesis kit - by Bioz Stars, 2021-06
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    Images

    1) Product Images from "Epigenetic silencing of miR-520c leads to induced S100A4 expression and its mediated colorectal cancer progression"

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

    Journal: Oncotarget

    doi: 10.18632/oncotarget.15499

    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.
    Figure Legend 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.

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

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

    2) Product Images from "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"

    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

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.16453

    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
    Figure Legend 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

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

    3) Product Images from "Role of the CXCR4-LASP1 Axis in the Stabilization of Snail1 in Triple-Negative Breast Cancer"

    Article Title: Role of the CXCR4-LASP1 Axis in the Stabilization of Snail1 in Triple-Negative Breast Cancer

    Journal: Cancers

    doi: 10.3390/cancers12092372

    ( A ) Nuclear LASP1 co-immunoprecipitated with Snail1 and proteins that regulate the stability of Snail1 endogenously in response to CXCL12; ( A ) Co-immunoprecipitation of LASP1 with Snail1 and its regulators—Top panel: Serum-starved MDA-Bone-Un cells were stimulated with 20 nM CXCL12 for 20 min. In some conditions, Bone-Un cells were pre-incubated with the CXCR4 antagonist AMD-3465 for 30 min prior to the addition of the ligand CXCL12. LASP1 was immunoprecipitated with mouse anti-LASP1 (8C6 clone) antibodies from 250 µg of the nuclear extracts from MDA-Bone-Un cells and analyzed by 10% SDS-PAGE followed by immunoblotting for associated Snail1, A20, GSK-3β, and LSD1. ‘M’ represents the mock co-immunoprecipitation performed with isotype control IgG1 antibodies. Bottom panel: 15 µg of nuclear lysates were resolved by 10% SDS-PAGE, and immunoblotted for Snail1, A20, GSK-3β, LSD1 and LASP1 blot. ( B ) Differential association of phosphorylated forms of LASP1 to endogenous Snail1 and its regulators—1.5 nmol of each of the GST, LASP1-WT and its phosphomimetic (S146D and Y171D) and phosphonull (S146A and Y171A) mutants fused to GST were incubated with 250 µg of nuclear lysate derived from CXCL12-stimulated MDA-Bone-Un cells. The differential association of endogenous Snail1, GSK-3β, A20 and LSD1 with full length LASP1-WT and its mutants were analyzed by 10% SDS-PAGE, followed by immunoblotting; n = 3. ( C ) Occupancy of LASP1 at the E-cadherin promoter region—Serum-starved MDA-Bone-Un cells were stimulated with 20 nM CXCL12 for 20 min. In some conditions, Bone-Un cells were pre-incubated with the CXCR4 antagonist AMD-3465 for 30 min prior to the addition of the ligand CXCL12. The chromatin fragments were prepared and subjected to chromatin immunoprecipitation (ChIP) analysis. The data from quantitative, real-time PCR were statistically analyzed and shown as the Mean ± SD, n = 3 independent biological repeats; * p
    Figure Legend Snippet: ( A ) Nuclear LASP1 co-immunoprecipitated with Snail1 and proteins that regulate the stability of Snail1 endogenously in response to CXCL12; ( A ) Co-immunoprecipitation of LASP1 with Snail1 and its regulators—Top panel: Serum-starved MDA-Bone-Un cells were stimulated with 20 nM CXCL12 for 20 min. In some conditions, Bone-Un cells were pre-incubated with the CXCR4 antagonist AMD-3465 for 30 min prior to the addition of the ligand CXCL12. LASP1 was immunoprecipitated with mouse anti-LASP1 (8C6 clone) antibodies from 250 µg of the nuclear extracts from MDA-Bone-Un cells and analyzed by 10% SDS-PAGE followed by immunoblotting for associated Snail1, A20, GSK-3β, and LSD1. ‘M’ represents the mock co-immunoprecipitation performed with isotype control IgG1 antibodies. Bottom panel: 15 µg of nuclear lysates were resolved by 10% SDS-PAGE, and immunoblotted for Snail1, A20, GSK-3β, LSD1 and LASP1 blot. ( B ) Differential association of phosphorylated forms of LASP1 to endogenous Snail1 and its regulators—1.5 nmol of each of the GST, LASP1-WT and its phosphomimetic (S146D and Y171D) and phosphonull (S146A and Y171A) mutants fused to GST were incubated with 250 µg of nuclear lysate derived from CXCL12-stimulated MDA-Bone-Un cells. The differential association of endogenous Snail1, GSK-3β, A20 and LSD1 with full length LASP1-WT and its mutants were analyzed by 10% SDS-PAGE, followed by immunoblotting; n = 3. ( C ) Occupancy of LASP1 at the E-cadherin promoter region—Serum-starved MDA-Bone-Un cells were stimulated with 20 nM CXCL12 for 20 min. In some conditions, Bone-Un cells were pre-incubated with the CXCR4 antagonist AMD-3465 for 30 min prior to the addition of the ligand CXCL12. The chromatin fragments were prepared and subjected to chromatin immunoprecipitation (ChIP) analysis. The data from quantitative, real-time PCR were statistically analyzed and shown as the Mean ± SD, n = 3 independent biological repeats; * p

    Techniques Used: Immunoprecipitation, Multiple Displacement Amplification, Incubation, SDS Page, Derivative Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

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    Isolation:

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    SYBR Green Assay:

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    Amplification:

    Article Title: Subepithelial telocytes are an important source of Wnts that supports intestinal crypts
    Article Snippet: A single-cell suspension was obtained by filtrating the supernatant through a 40μm strainer and washing with 5mM EDTA in HBSS, before cell sorting using a BD influx instrument (BD Biosciences, San Jose, CA). .. For RNA isolation, GFP+ and Tomato+ cells for Foxl1+ and Foxl1− mesenchymal cells, respectively, were lysed and total RNA was isolated by column purification (Absolutely RNA Nanoprep Kit; Agilent Technologies). cDNA was synthesized and amplified using Ribo-SPIA technology (Ovation RNA-Seq System V2 #7102 NuGEN). cDNA was sonicated and an mRNA sequencing library was prepared using the NEBNext RNA library prep kit (New England BioLabs, Inc). .. For RNA isolation, GFP+ and Tomato+ cells for Foxl1+ and Foxl1− mesenchymal cells, respectively, were lysed and total RNA was isolated by column purification (Absolutely RNA Nanoprep Kit; Agilent Technologies). cDNA was synthesized and amplified using Ribo-SPIA technology (Ovation RNA-Seq System V2 #7102 NuGEN). cDNA was sonicated and an mRNA sequencing library was prepared using the NEBNext RNA library prep kit (New England BioLabs, Inc).

    RNA Sequencing Assay:

    Article Title: Subepithelial telocytes are an important source of Wnts that supports intestinal crypts
    Article Snippet: A single-cell suspension was obtained by filtrating the supernatant through a 40μm strainer and washing with 5mM EDTA in HBSS, before cell sorting using a BD influx instrument (BD Biosciences, San Jose, CA). .. For RNA isolation, GFP+ and Tomato+ cells for Foxl1+ and Foxl1− mesenchymal cells, respectively, were lysed and total RNA was isolated by column purification (Absolutely RNA Nanoprep Kit; Agilent Technologies). cDNA was synthesized and amplified using Ribo-SPIA technology (Ovation RNA-Seq System V2 #7102 NuGEN). cDNA was sonicated and an mRNA sequencing library was prepared using the NEBNext RNA library prep kit (New England BioLabs, Inc). .. For RNA isolation, GFP+ and Tomato+ cells for Foxl1+ and Foxl1− mesenchymal cells, respectively, were lysed and total RNA was isolated by column purification (Absolutely RNA Nanoprep Kit; Agilent Technologies). cDNA was synthesized and amplified using Ribo-SPIA technology (Ovation RNA-Seq System V2 #7102 NuGEN). cDNA was sonicated and an mRNA sequencing library was prepared using the NEBNext RNA library prep kit (New England BioLabs, Inc).

    Sonication:

    Article Title: Subepithelial telocytes are an important source of Wnts that supports intestinal crypts
    Article Snippet: A single-cell suspension was obtained by filtrating the supernatant through a 40μm strainer and washing with 5mM EDTA in HBSS, before cell sorting using a BD influx instrument (BD Biosciences, San Jose, CA). .. For RNA isolation, GFP+ and Tomato+ cells for Foxl1+ and Foxl1− mesenchymal cells, respectively, were lysed and total RNA was isolated by column purification (Absolutely RNA Nanoprep Kit; Agilent Technologies). cDNA was synthesized and amplified using Ribo-SPIA technology (Ovation RNA-Seq System V2 #7102 NuGEN). cDNA was sonicated and an mRNA sequencing library was prepared using the NEBNext RNA library prep kit (New England BioLabs, Inc). .. For RNA isolation, GFP+ and Tomato+ cells for Foxl1+ and Foxl1− mesenchymal cells, respectively, were lysed and total RNA was isolated by column purification (Absolutely RNA Nanoprep Kit; Agilent Technologies). cDNA was synthesized and amplified using Ribo-SPIA technology (Ovation RNA-Seq System V2 #7102 NuGEN). cDNA was sonicated and an mRNA sequencing library was prepared using the NEBNext RNA library prep kit (New England BioLabs, Inc).

    Sequencing:

    Article Title: Subepithelial telocytes are an important source of Wnts that supports intestinal crypts
    Article Snippet: A single-cell suspension was obtained by filtrating the supernatant through a 40μm strainer and washing with 5mM EDTA in HBSS, before cell sorting using a BD influx instrument (BD Biosciences, San Jose, CA). .. For RNA isolation, GFP+ and Tomato+ cells for Foxl1+ and Foxl1− mesenchymal cells, respectively, were lysed and total RNA was isolated by column purification (Absolutely RNA Nanoprep Kit; Agilent Technologies). cDNA was synthesized and amplified using Ribo-SPIA technology (Ovation RNA-Seq System V2 #7102 NuGEN). cDNA was sonicated and an mRNA sequencing library was prepared using the NEBNext RNA library prep kit (New England BioLabs, Inc). .. For RNA isolation, GFP+ and Tomato+ cells for Foxl1+ and Foxl1− mesenchymal cells, respectively, were lysed and total RNA was isolated by column purification (Absolutely RNA Nanoprep Kit; Agilent Technologies). cDNA was synthesized and amplified using Ribo-SPIA technology (Ovation RNA-Seq System V2 #7102 NuGEN). cDNA was sonicated and an mRNA sequencing library was prepared using the NEBNext RNA library prep kit (New England BioLabs, Inc).

    Mouse Assay:

    Article Title: AhR Ligands Modulate the Differentiation of Innate Lymphoid Cells and T Helper Cell Subsets That Control the Severity of a Pulmonary Fungal Infection
    Article Snippet: .. Real-Time PCR (qPCR) RNA isolation from lung macerates of AhR ligand-treated and untreated mice was performed as previously described ( ). .. A NanoDrop ND-1000 spectrophotometer was used to determine RNA purity and concentration.

    cDNA Library Assay:

    Article Title: High Quality Aspergillus aculeatus Genomes and Transcriptomes: A Platform for Cellulase Activity Optimization Toward Industrial Applications
    Article Snippet: In short, ribosomal RNA was degraded from 10 μg of total RNA using the Ribominus™ Eukaryotic kit (Life Technologies, USA). .. The RNA was enriched using the Absolutely mRNA Purification kit (Agilent Technologies, USA) and further quantified by BioAnalyzer 2100 (Agilent Technologies, USA) before cDNA library construction. .. The cDNA libraries were constructed using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA).

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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
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    Agilent technologies quikchange lightning multi 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.
    Quikchange Lightning Multi Site Directed Mutagenesis Kit, 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/quikchange lightning multi site directed mutagenesis kit/product/Agilent technologies
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    quikchange lightning multi site directed mutagenesis kit - by Bioz Stars, 2021-06
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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

    ( A ) Nuclear LASP1 co-immunoprecipitated with Snail1 and proteins that regulate the stability of Snail1 endogenously in response to CXCL12; ( A ) Co-immunoprecipitation of LASP1 with Snail1 and its regulators—Top panel: Serum-starved MDA-Bone-Un cells were stimulated with 20 nM CXCL12 for 20 min. In some conditions, Bone-Un cells were pre-incubated with the CXCR4 antagonist AMD-3465 for 30 min prior to the addition of the ligand CXCL12. LASP1 was immunoprecipitated with mouse anti-LASP1 (8C6 clone) antibodies from 250 µg of the nuclear extracts from MDA-Bone-Un cells and analyzed by 10% SDS-PAGE followed by immunoblotting for associated Snail1, A20, GSK-3β, and LSD1. ‘M’ represents the mock co-immunoprecipitation performed with isotype control IgG1 antibodies. Bottom panel: 15 µg of nuclear lysates were resolved by 10% SDS-PAGE, and immunoblotted for Snail1, A20, GSK-3β, LSD1 and LASP1 blot. ( B ) Differential association of phosphorylated forms of LASP1 to endogenous Snail1 and its regulators—1.5 nmol of each of the GST, LASP1-WT and its phosphomimetic (S146D and Y171D) and phosphonull (S146A and Y171A) mutants fused to GST were incubated with 250 µg of nuclear lysate derived from CXCL12-stimulated MDA-Bone-Un cells. The differential association of endogenous Snail1, GSK-3β, A20 and LSD1 with full length LASP1-WT and its mutants were analyzed by 10% SDS-PAGE, followed by immunoblotting; n = 3. ( C ) Occupancy of LASP1 at the E-cadherin promoter region—Serum-starved MDA-Bone-Un cells were stimulated with 20 nM CXCL12 for 20 min. In some conditions, Bone-Un cells were pre-incubated with the CXCR4 antagonist AMD-3465 for 30 min prior to the addition of the ligand CXCL12. The chromatin fragments were prepared and subjected to chromatin immunoprecipitation (ChIP) analysis. The data from quantitative, real-time PCR were statistically analyzed and shown as the Mean ± SD, n = 3 independent biological repeats; * p

    Journal: Cancers

    Article Title: Role of the CXCR4-LASP1 Axis in the Stabilization of Snail1 in Triple-Negative Breast Cancer

    doi: 10.3390/cancers12092372

    Figure Lengend Snippet: ( A ) Nuclear LASP1 co-immunoprecipitated with Snail1 and proteins that regulate the stability of Snail1 endogenously in response to CXCL12; ( A ) Co-immunoprecipitation of LASP1 with Snail1 and its regulators—Top panel: Serum-starved MDA-Bone-Un cells were stimulated with 20 nM CXCL12 for 20 min. In some conditions, Bone-Un cells were pre-incubated with the CXCR4 antagonist AMD-3465 for 30 min prior to the addition of the ligand CXCL12. LASP1 was immunoprecipitated with mouse anti-LASP1 (8C6 clone) antibodies from 250 µg of the nuclear extracts from MDA-Bone-Un cells and analyzed by 10% SDS-PAGE followed by immunoblotting for associated Snail1, A20, GSK-3β, and LSD1. ‘M’ represents the mock co-immunoprecipitation performed with isotype control IgG1 antibodies. Bottom panel: 15 µg of nuclear lysates were resolved by 10% SDS-PAGE, and immunoblotted for Snail1, A20, GSK-3β, LSD1 and LASP1 blot. ( B ) Differential association of phosphorylated forms of LASP1 to endogenous Snail1 and its regulators—1.5 nmol of each of the GST, LASP1-WT and its phosphomimetic (S146D and Y171D) and phosphonull (S146A and Y171A) mutants fused to GST were incubated with 250 µg of nuclear lysate derived from CXCL12-stimulated MDA-Bone-Un cells. The differential association of endogenous Snail1, GSK-3β, A20 and LSD1 with full length LASP1-WT and its mutants were analyzed by 10% SDS-PAGE, followed by immunoblotting; n = 3. ( C ) Occupancy of LASP1 at the E-cadherin promoter region—Serum-starved MDA-Bone-Un cells were stimulated with 20 nM CXCL12 for 20 min. In some conditions, Bone-Un cells were pre-incubated with the CXCR4 antagonist AMD-3465 for 30 min prior to the addition of the ligand CXCL12. The chromatin fragments were prepared and subjected to chromatin immunoprecipitation (ChIP) analysis. The data from quantitative, real-time PCR were statistically analyzed and shown as the Mean ± SD, n = 3 independent biological repeats; * p

    Article Snippet: These were differentially mutated into either phosphomimetic (S146D and Y171D) or phosphonull (S146A and Y171F) substitutions, by employing PCR based site-directed mutagenesis kit (QuikChange II, Agilent Tech., Santa Clara, CA, USA).

    Techniques: Immunoprecipitation, Multiple Displacement Amplification, Incubation, SDS Page, Derivative Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction