la pcr genome dna set  (TaKaRa)


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    Name:
    LA PCR Genome DNA Set
    Description:
    The LA PCR Genome DNA Set is designed for use as controls in long range PCR and enables the optimization of PCR conditions for a wide variety of templates The set includes templates that are highly purified high molecular weight genomic DNA from human and E coli and corresponding primers
    Catalog Number:
    9060
    Price:
    None
    Category:
    Long PCR controls Long range PCR PCR
    Size:
    20 Rxns
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    Structured Review

    TaKaRa la pcr genome dna set
    Tissue-related variation in heteroplasmy levels for maternal (H1a1) and paternal (R0a1) haplogroups in Patient II-4 of Family A. ( A ) Pedigree of Family A. The black-filled symbols indicate the four family members (II-1, II-3, II-4, and III-6) that show biparental mtDNA transmission. The diagonal-filled symbols indicate the three family members (IV-1, IV-2, and IV-3) who carry a high number and level of mtDNA heteroplasmies which underwent normal maternal transmission. ( B ) Variable heteroplasmy levels detected by <t>PCR-NGS</t> in blood, saliva, hair follicle (HF), urine, sperm, and fibroblast (FB) samples collected from Patient II-4. ( C ) Heteroplasmy drift over time increases the proportion of the paternal haplogroup in primary fibroblast cells derived from Patient II-4. ( D, E ) Single cell-derived <t>DNA</t> samples from Patient II-4 were sequenced to measure cell-to-cell variability in heteroplasmy levels. Sanger sequencing for single-sperm samples ( D ) and PCR-NGS for colonies derived from individual primary fibroblast cells ( E ) show marked differences in heteroplasmy levels, even for cells derived from the same tissue.
    The LA PCR Genome DNA Set is designed for use as controls in long range PCR and enables the optimization of PCR conditions for a wide variety of templates The set includes templates that are highly purified high molecular weight genomic DNA from human and E coli and corresponding primers
    https://www.bioz.com/result/la pcr genome dna set/product/TaKaRa
    Average 94 stars, based on 1 article reviews
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    la pcr genome dna set - by Bioz Stars, 2021-03
    94/100 stars

    Images

    1) Product Images from "Heteroplasmy variability in individuals with biparentally inherited mitochondrial DNA"

    Article Title: Heteroplasmy variability in individuals with biparentally inherited mitochondrial DNA

    Journal: bioRxiv

    doi: 10.1101/2020.02.26.939405

    Tissue-related variation in heteroplasmy levels for maternal (H1a1) and paternal (R0a1) haplogroups in Patient II-4 of Family A. ( A ) Pedigree of Family A. The black-filled symbols indicate the four family members (II-1, II-3, II-4, and III-6) that show biparental mtDNA transmission. The diagonal-filled symbols indicate the three family members (IV-1, IV-2, and IV-3) who carry a high number and level of mtDNA heteroplasmies which underwent normal maternal transmission. ( B ) Variable heteroplasmy levels detected by PCR-NGS in blood, saliva, hair follicle (HF), urine, sperm, and fibroblast (FB) samples collected from Patient II-4. ( C ) Heteroplasmy drift over time increases the proportion of the paternal haplogroup in primary fibroblast cells derived from Patient II-4. ( D, E ) Single cell-derived DNA samples from Patient II-4 were sequenced to measure cell-to-cell variability in heteroplasmy levels. Sanger sequencing for single-sperm samples ( D ) and PCR-NGS for colonies derived from individual primary fibroblast cells ( E ) show marked differences in heteroplasmy levels, even for cells derived from the same tissue.
    Figure Legend Snippet: Tissue-related variation in heteroplasmy levels for maternal (H1a1) and paternal (R0a1) haplogroups in Patient II-4 of Family A. ( A ) Pedigree of Family A. The black-filled symbols indicate the four family members (II-1, II-3, II-4, and III-6) that show biparental mtDNA transmission. The diagonal-filled symbols indicate the three family members (IV-1, IV-2, and IV-3) who carry a high number and level of mtDNA heteroplasmies which underwent normal maternal transmission. ( B ) Variable heteroplasmy levels detected by PCR-NGS in blood, saliva, hair follicle (HF), urine, sperm, and fibroblast (FB) samples collected from Patient II-4. ( C ) Heteroplasmy drift over time increases the proportion of the paternal haplogroup in primary fibroblast cells derived from Patient II-4. ( D, E ) Single cell-derived DNA samples from Patient II-4 were sequenced to measure cell-to-cell variability in heteroplasmy levels. Sanger sequencing for single-sperm samples ( D ) and PCR-NGS for colonies derived from individual primary fibroblast cells ( E ) show marked differences in heteroplasmy levels, even for cells derived from the same tissue.

    Techniques Used: Transmission Assay, Polymerase Chain Reaction, Next-Generation Sequencing, Derivative Assay, Sequencing

    2) Product Images from "Spinal muscular atrophy caused by a novel Alu‐mediated deletion of exons 2a‐5 in SMN1 undetectable with routine genetic testing, et al. Spinal muscular atrophy caused by a novel Alu‐mediated deletion of exons 2a‐5 in SMN1 undetectable with routine genetic testing"

    Article Title: Spinal muscular atrophy caused by a novel Alu‐mediated deletion of exons 2a‐5 in SMN1 undetectable with routine genetic testing, et al. Spinal muscular atrophy caused by a novel Alu‐mediated deletion of exons 2a‐5 in SMN1 undetectable with routine genetic testing

    Journal: Molecular Genetics & Genomic Medicine

    doi: 10.1002/mgg3.1238

    Identification of SMN1 variants. (a) qPCR analysis of four retrotransposon‐free SMN genomic regions in the intron 1 (I1), in the exon 3–intron 3 junction (E3I3), in the intron 5–exon 6 junction (I5E6), and ~1 kb downstream from exon 8 (+1 kb). Compared with four copies of SMN genes that are present in controls, we found that the patient has three copies at the I1, I5E6, and +1 kb loci and two copies at the E3I3 loci. The mother has three copies through the I1 to I5E6 loci and two copies at the +1 kb loci. The father has three copies only at the I5E6 loci. (b) Schematic representation of SMN1/2 exon (E)/intron (I) structure. Positions of sequence differences between SMN1 and SMN2 are represented by black vertical bars. The black triangles denote sequence‐specific variants in exons 7, 8 targeted by MLPA probes in routine testing. Locations of Alus in the breakpoint candidate regions in the intron 1 and 5, including the causal AluSp in the intron 1 and AluSq in the intron 5 indicated by vertical text, and primers binding sites for Alu PCR indicated by black arrowheads are shown below the scheme of the SMN structure. Position of the PCR4 spanning exons 5–8 that showed absence of SMN1 sequence‐specific variants indicating disruption of both SMN1 alleles in the patient is represented by yellow box. Range of the paternal deletion of exons 2a‐5 is represented by red box. (c) DNA sequence trace of the Alu PCR, Alu_259_4A, showing a double sequence caused by presence of AluSq wt in intron 5 together with a sequence originating from the intron 1 AluSp . Red arrows indicate the addition of AluSp ‐specific sequence in an Alu PCR product. (d) PCR genotyping of the SMN1Δ(2a‐5) variant showed presence of the deletion‐spanning amplification product in the patient (P) and father (F), but not in mother (M) and control (C). (e) DNA sequence trace of the breakpoint junction‐specific PCR and detail of the Δ2a‐5 breakpoint junction show the new Alu ‐ Alu chimeric element originating from the recombination between the AluSp in the intron 1 and AluSq in the intron 5. A breakpoint microhomology of the AluSp and AluSq is marked with a black box. (f) Schematic representation of SMN1 and SMN2 in the family members. Pink‐marked boxes represent maternal alleles (M) and blue boxes paternal alleles (F). The red crosses denote identified deletions and the dashed vertical lines denote loci of the qPCR (I1, E3I3, I5E6, and +1 kb) and MLPA (exon 7‐E7, exon 8‐E8) probes used for deletion mapping. The black junctions on the box terminals indicate a cis configuration of SMN1 and SMN2 alleles. The model shows (a) a whole deletion of one SMN1 allele in the patient (P) inherited from her mother and detected by the combination of the qPCR and MLPA; (b) a deletion of the second SMN1 allele in the patient inherited from her father and detected by the E3I3 qPCR and transcript analysis (Figure 1a ); and (c) deletion of one copy of one SMN2 allele in the mother detected by the MLPA and the + 1kb qPCR
    Figure Legend Snippet: Identification of SMN1 variants. (a) qPCR analysis of four retrotransposon‐free SMN genomic regions in the intron 1 (I1), in the exon 3–intron 3 junction (E3I3), in the intron 5–exon 6 junction (I5E6), and ~1 kb downstream from exon 8 (+1 kb). Compared with four copies of SMN genes that are present in controls, we found that the patient has three copies at the I1, I5E6, and +1 kb loci and two copies at the E3I3 loci. The mother has three copies through the I1 to I5E6 loci and two copies at the +1 kb loci. The father has three copies only at the I5E6 loci. (b) Schematic representation of SMN1/2 exon (E)/intron (I) structure. Positions of sequence differences between SMN1 and SMN2 are represented by black vertical bars. The black triangles denote sequence‐specific variants in exons 7, 8 targeted by MLPA probes in routine testing. Locations of Alus in the breakpoint candidate regions in the intron 1 and 5, including the causal AluSp in the intron 1 and AluSq in the intron 5 indicated by vertical text, and primers binding sites for Alu PCR indicated by black arrowheads are shown below the scheme of the SMN structure. Position of the PCR4 spanning exons 5–8 that showed absence of SMN1 sequence‐specific variants indicating disruption of both SMN1 alleles in the patient is represented by yellow box. Range of the paternal deletion of exons 2a‐5 is represented by red box. (c) DNA sequence trace of the Alu PCR, Alu_259_4A, showing a double sequence caused by presence of AluSq wt in intron 5 together with a sequence originating from the intron 1 AluSp . Red arrows indicate the addition of AluSp ‐specific sequence in an Alu PCR product. (d) PCR genotyping of the SMN1Δ(2a‐5) variant showed presence of the deletion‐spanning amplification product in the patient (P) and father (F), but not in mother (M) and control (C). (e) DNA sequence trace of the breakpoint junction‐specific PCR and detail of the Δ2a‐5 breakpoint junction show the new Alu ‐ Alu chimeric element originating from the recombination between the AluSp in the intron 1 and AluSq in the intron 5. A breakpoint microhomology of the AluSp and AluSq is marked with a black box. (f) Schematic representation of SMN1 and SMN2 in the family members. Pink‐marked boxes represent maternal alleles (M) and blue boxes paternal alleles (F). The red crosses denote identified deletions and the dashed vertical lines denote loci of the qPCR (I1, E3I3, I5E6, and +1 kb) and MLPA (exon 7‐E7, exon 8‐E8) probes used for deletion mapping. The black junctions on the box terminals indicate a cis configuration of SMN1 and SMN2 alleles. The model shows (a) a whole deletion of one SMN1 allele in the patient (P) inherited from her mother and detected by the combination of the qPCR and MLPA; (b) a deletion of the second SMN1 allele in the patient inherited from her father and detected by the E3I3 qPCR and transcript analysis (Figure 1a ); and (c) deletion of one copy of one SMN2 allele in the mother detected by the MLPA and the + 1kb qPCR

    Techniques Used: Real-time Polymerase Chain Reaction, Sequencing, Multiplex Ligation-dependent Probe Amplification, Binding Assay, Polymerase Chain Reaction, Variant Assay, Amplification

    Related Articles

    Sequencing:

    Article Title: Sp1 Suppresses miR-3178 to Promote the Metastasis Invasion Cascade via Upregulation of TRIOBP
    Article Snippet: Total RNA (1.0 μg) was extracted using TRIzol reagent (Thermo Fisher, Waltham, MA, USA) and used to synthesize cDNA with EasyScript First-Strand cDNA Synthesis SuperMix (Transgen, Beijing, China). qPCR was performed using SYBR Premix Ex Taq II (Takara, Otsu, Shiga, Japan). .. DNA fragment of miR-3178 stem-loop sequence was amplified by PCR from human genome DNA, cloned into pcDNA3.1 or pdsRed2-C1 vector at sites between EcoRI and XhoI (Takara, Otsu, Shiga, Japan). .. The fragment of TRIOBP 3′ UTR was amplified and cloned into pMIR-REPORT vector between EcoRI and BamHI (Takara, Otsu, Shiga, Japan).

    Article Title: Sp1 Suppresses miR-3178 to Promote the Metastasis Invasion Cascade via Upregulation of TRIOBP
    Article Snippet: Real-Time qPCR Total RNA (1.0 μg) was extracted using TRIzol reagent (Thermo Fisher, Waltham, MA, USA) and used to synthesize cDNA with EasyScript First-Strand cDNA Synthesis SuperMix (Transgen, Beijing, China). qPCR was performed using SYBR Premix Ex Taq II (Takara, Otsu, Shiga, Japan). .. Plasmid Construction and Cell Transfection DNA fragment of miR-3178 stem-loop sequence was amplified by PCR from human genome DNA, cloned into pcDNA3.1 or pdsRed2-C1 vector at sites between EcoRI and XhoI (Takara, Otsu, Shiga, Japan). .. The fragment of TRIOBP 3′ UTR was amplified and cloned into pMIR-REPORT vector between EcoRI and BamHI (Takara, Otsu, Shiga, Japan).

    Amplification:

    Article Title: Sp1 Suppresses miR-3178 to Promote the Metastasis Invasion Cascade via Upregulation of TRIOBP
    Article Snippet: Total RNA (1.0 μg) was extracted using TRIzol reagent (Thermo Fisher, Waltham, MA, USA) and used to synthesize cDNA with EasyScript First-Strand cDNA Synthesis SuperMix (Transgen, Beijing, China). qPCR was performed using SYBR Premix Ex Taq II (Takara, Otsu, Shiga, Japan). .. DNA fragment of miR-3178 stem-loop sequence was amplified by PCR from human genome DNA, cloned into pcDNA3.1 or pdsRed2-C1 vector at sites between EcoRI and XhoI (Takara, Otsu, Shiga, Japan). .. The fragment of TRIOBP 3′ UTR was amplified and cloned into pMIR-REPORT vector between EcoRI and BamHI (Takara, Otsu, Shiga, Japan).

    Article Title: Sp1 Suppresses miR-3178 to Promote the Metastasis Invasion Cascade via Upregulation of TRIOBP
    Article Snippet: Real-Time qPCR Total RNA (1.0 μg) was extracted using TRIzol reagent (Thermo Fisher, Waltham, MA, USA) and used to synthesize cDNA with EasyScript First-Strand cDNA Synthesis SuperMix (Transgen, Beijing, China). qPCR was performed using SYBR Premix Ex Taq II (Takara, Otsu, Shiga, Japan). .. Plasmid Construction and Cell Transfection DNA fragment of miR-3178 stem-loop sequence was amplified by PCR from human genome DNA, cloned into pcDNA3.1 or pdsRed2-C1 vector at sites between EcoRI and XhoI (Takara, Otsu, Shiga, Japan). .. The fragment of TRIOBP 3′ UTR was amplified and cloned into pMIR-REPORT vector between EcoRI and BamHI (Takara, Otsu, Shiga, Japan).

    Article Title: PICH promotes sister chromatid disjunction and co-operates with topoisomerase II in mitosis
    Article Snippet: Genomic DNA from DT40 cells was isolated using extraction buffer (100 mM Tris-HCl, pH 7.5, 20 mM EDTA, 150 mM NaCl, 1% SDS and 200 μg ml−1 proteinase K) followed by Phenol–Chloroform extraction. .. The 5′ arm of the targeting vectors was amplified with SH-chPICH-10 and SH-chPICH-33 from DT-40 genomic DNA using a LA-PCR set (Takara). .. The 3′ arm was amplified with SH-chPICH-7 and SH-chPICH-8 using PrimeStarTM (Takara).

    Polymerase Chain Reaction:

    Article Title: Sp1 Suppresses miR-3178 to Promote the Metastasis Invasion Cascade via Upregulation of TRIOBP
    Article Snippet: Total RNA (1.0 μg) was extracted using TRIzol reagent (Thermo Fisher, Waltham, MA, USA) and used to synthesize cDNA with EasyScript First-Strand cDNA Synthesis SuperMix (Transgen, Beijing, China). qPCR was performed using SYBR Premix Ex Taq II (Takara, Otsu, Shiga, Japan). .. DNA fragment of miR-3178 stem-loop sequence was amplified by PCR from human genome DNA, cloned into pcDNA3.1 or pdsRed2-C1 vector at sites between EcoRI and XhoI (Takara, Otsu, Shiga, Japan). .. The fragment of TRIOBP 3′ UTR was amplified and cloned into pMIR-REPORT vector between EcoRI and BamHI (Takara, Otsu, Shiga, Japan).

    Article Title: HuR Maintains a Replicative Life Span by Repressing the ARF Tumor Suppressor
    Article Snippet: Infection was confirmed either by GFP expression or by selection for drug resistance. .. RNAs were prepared from cells or immune complexes using TriPure isolation reagent (Roche, Indianapolis, IN), reverse transcribed using a PrimeScript reverse transcriptase (RT) reagent kit with the genomic DNA (gDNA) Eraser (TaKaRa, Shiga, Japan), and subjected to PCR using the following primers: for PAI-1 , 5′-TCAGAGCAACAAGTTCAACTACACTGAG-3′ (sense) and 5′-CCCACTGTCAAGGCTCCATCACTTGCCCCA-3′ (antisense); for HuR , 5′-TTGGGCTACGGTTTTGTGAAC-3′ (sense) and 5′-CCCACTGATGTATAAGTTGGCAT-3′ (antisense); for ARF , 5′-GCCGCACCGGAATCCT-3′ (sense) and 5′-TTGAGCAGAAGAGCTGCTACGT-3′ (antisense); for Ink4a , 5′-CCCAACGCCCCGAACT-3′ (sense) and 5′-GCAGAAGAGCTGCTACGTGAA-3′ (antisense); for c- myc , 5′-TCTATTTGGGGACAGTGTTC-3′ (sense) and 5′-GGTCATAGTTCCTGTTGGTG-3′ (antisense); for p53 , 5′-TGGAGAGTATTTCACCCTCAAGA-3′ (sense) and 5′-CTCCTCTGTAGCATGGGCATC-3′ (antisense); for β-actin , 5′-CTAAGGCCAACCGTGAAAAG-3′ (sense) and 5′-ACCAGAGGCATACAGGGACA-3′ (antisense); for 18S rRNA 5′-AGTCCCTGCCCTTTGTACACA-3′ (sense) and 5′-GATCCGAGGGCCTCACTAAAC-3′ (antisense); for AUF-1 , 5′-TTTCTCCAGACACACCTGAAGA-3′ (sense) and 5′-CTGTTCCTTTGACATGGCTACTT-3′ (antisense); and for GFP , 5′-TCTGCACCACCGGCAAGCTG-3′ (sense) and 5′-TGCGCTCCTGGACGTAGCCT-3′ (antisense). .. Real-time PCR analysis was carried out on a Chromo4 real-time PCR system (Bio-Rad, Hercules, CA).

    Article Title: Sp1 Suppresses miR-3178 to Promote the Metastasis Invasion Cascade via Upregulation of TRIOBP
    Article Snippet: Real-Time qPCR Total RNA (1.0 μg) was extracted using TRIzol reagent (Thermo Fisher, Waltham, MA, USA) and used to synthesize cDNA with EasyScript First-Strand cDNA Synthesis SuperMix (Transgen, Beijing, China). qPCR was performed using SYBR Premix Ex Taq II (Takara, Otsu, Shiga, Japan). .. Plasmid Construction and Cell Transfection DNA fragment of miR-3178 stem-loop sequence was amplified by PCR from human genome DNA, cloned into pcDNA3.1 or pdsRed2-C1 vector at sites between EcoRI and XhoI (Takara, Otsu, Shiga, Japan). .. The fragment of TRIOBP 3′ UTR was amplified and cloned into pMIR-REPORT vector between EcoRI and BamHI (Takara, Otsu, Shiga, Japan).

    Article Title: Non-invasive in vivo imaging of UCP1 expression in live mice via near-infrared fluorescent protein iRFP720
    Article Snippet: To detect the iRFP720 gene, 5 ng each of the genome DNA was analyzed by PCR in a 5 μl reactions containing 2.5 μl of AmpliTaq Gold 360 Master Mix (ThermoFisher Scientific Inc.) and 1.5 pmoles each of the primers. .. To confirm the correct insertion of the iRFP720 gene at the Ucp1 locus, 10 ng each of the genome DNA was analyzed by PCR using 0.25 unit of PrimeStar GXL DNA Polymerase (TaKaRa Bio Inc.) and 3 pmoles each of the primers in a 10 μl reaction. .. In addition, 20 ng of genome DNA was analyzed by PCR containing 0.1 unit of KOD FX Neo (TOYOBO CO., LTD.) and 1.5 pmoles each of the primers in a 5 μl reaction.

    Clone Assay:

    Article Title: Sp1 Suppresses miR-3178 to Promote the Metastasis Invasion Cascade via Upregulation of TRIOBP
    Article Snippet: Total RNA (1.0 μg) was extracted using TRIzol reagent (Thermo Fisher, Waltham, MA, USA) and used to synthesize cDNA with EasyScript First-Strand cDNA Synthesis SuperMix (Transgen, Beijing, China). qPCR was performed using SYBR Premix Ex Taq II (Takara, Otsu, Shiga, Japan). .. DNA fragment of miR-3178 stem-loop sequence was amplified by PCR from human genome DNA, cloned into pcDNA3.1 or pdsRed2-C1 vector at sites between EcoRI and XhoI (Takara, Otsu, Shiga, Japan). .. The fragment of TRIOBP 3′ UTR was amplified and cloned into pMIR-REPORT vector between EcoRI and BamHI (Takara, Otsu, Shiga, Japan).

    Article Title: Sp1 Suppresses miR-3178 to Promote the Metastasis Invasion Cascade via Upregulation of TRIOBP
    Article Snippet: Real-Time qPCR Total RNA (1.0 μg) was extracted using TRIzol reagent (Thermo Fisher, Waltham, MA, USA) and used to synthesize cDNA with EasyScript First-Strand cDNA Synthesis SuperMix (Transgen, Beijing, China). qPCR was performed using SYBR Premix Ex Taq II (Takara, Otsu, Shiga, Japan). .. Plasmid Construction and Cell Transfection DNA fragment of miR-3178 stem-loop sequence was amplified by PCR from human genome DNA, cloned into pcDNA3.1 or pdsRed2-C1 vector at sites between EcoRI and XhoI (Takara, Otsu, Shiga, Japan). .. The fragment of TRIOBP 3′ UTR was amplified and cloned into pMIR-REPORT vector between EcoRI and BamHI (Takara, Otsu, Shiga, Japan).

    Plasmid Preparation:

    Article Title: Sp1 Suppresses miR-3178 to Promote the Metastasis Invasion Cascade via Upregulation of TRIOBP
    Article Snippet: Total RNA (1.0 μg) was extracted using TRIzol reagent (Thermo Fisher, Waltham, MA, USA) and used to synthesize cDNA with EasyScript First-Strand cDNA Synthesis SuperMix (Transgen, Beijing, China). qPCR was performed using SYBR Premix Ex Taq II (Takara, Otsu, Shiga, Japan). .. DNA fragment of miR-3178 stem-loop sequence was amplified by PCR from human genome DNA, cloned into pcDNA3.1 or pdsRed2-C1 vector at sites between EcoRI and XhoI (Takara, Otsu, Shiga, Japan). .. The fragment of TRIOBP 3′ UTR was amplified and cloned into pMIR-REPORT vector between EcoRI and BamHI (Takara, Otsu, Shiga, Japan).

    Article Title: Sp1 Suppresses miR-3178 to Promote the Metastasis Invasion Cascade via Upregulation of TRIOBP
    Article Snippet: Real-Time qPCR Total RNA (1.0 μg) was extracted using TRIzol reagent (Thermo Fisher, Waltham, MA, USA) and used to synthesize cDNA with EasyScript First-Strand cDNA Synthesis SuperMix (Transgen, Beijing, China). qPCR was performed using SYBR Premix Ex Taq II (Takara, Otsu, Shiga, Japan). .. Plasmid Construction and Cell Transfection DNA fragment of miR-3178 stem-loop sequence was amplified by PCR from human genome DNA, cloned into pcDNA3.1 or pdsRed2-C1 vector at sites between EcoRI and XhoI (Takara, Otsu, Shiga, Japan). .. The fragment of TRIOBP 3′ UTR was amplified and cloned into pMIR-REPORT vector between EcoRI and BamHI (Takara, Otsu, Shiga, Japan).

    Isolation:

    Article Title: HuR Maintains a Replicative Life Span by Repressing the ARF Tumor Suppressor
    Article Snippet: Infection was confirmed either by GFP expression or by selection for drug resistance. .. RNAs were prepared from cells or immune complexes using TriPure isolation reagent (Roche, Indianapolis, IN), reverse transcribed using a PrimeScript reverse transcriptase (RT) reagent kit with the genomic DNA (gDNA) Eraser (TaKaRa, Shiga, Japan), and subjected to PCR using the following primers: for PAI-1 , 5′-TCAGAGCAACAAGTTCAACTACACTGAG-3′ (sense) and 5′-CCCACTGTCAAGGCTCCATCACTTGCCCCA-3′ (antisense); for HuR , 5′-TTGGGCTACGGTTTTGTGAAC-3′ (sense) and 5′-CCCACTGATGTATAAGTTGGCAT-3′ (antisense); for ARF , 5′-GCCGCACCGGAATCCT-3′ (sense) and 5′-TTGAGCAGAAGAGCTGCTACGT-3′ (antisense); for Ink4a , 5′-CCCAACGCCCCGAACT-3′ (sense) and 5′-GCAGAAGAGCTGCTACGTGAA-3′ (antisense); for c- myc , 5′-TCTATTTGGGGACAGTGTTC-3′ (sense) and 5′-GGTCATAGTTCCTGTTGGTG-3′ (antisense); for p53 , 5′-TGGAGAGTATTTCACCCTCAAGA-3′ (sense) and 5′-CTCCTCTGTAGCATGGGCATC-3′ (antisense); for β-actin , 5′-CTAAGGCCAACCGTGAAAAG-3′ (sense) and 5′-ACCAGAGGCATACAGGGACA-3′ (antisense); for 18S rRNA 5′-AGTCCCTGCCCTTTGTACACA-3′ (sense) and 5′-GATCCGAGGGCCTCACTAAAC-3′ (antisense); for AUF-1 , 5′-TTTCTCCAGACACACCTGAAGA-3′ (sense) and 5′-CTGTTCCTTTGACATGGCTACTT-3′ (antisense); and for GFP , 5′-TCTGCACCACCGGCAAGCTG-3′ (sense) and 5′-TGCGCTCCTGGACGTAGCCT-3′ (antisense). .. Real-time PCR analysis was carried out on a Chromo4 real-time PCR system (Bio-Rad, Hercules, CA).

    Transfection:

    Article Title: Sp1 Suppresses miR-3178 to Promote the Metastasis Invasion Cascade via Upregulation of TRIOBP
    Article Snippet: Real-Time qPCR Total RNA (1.0 μg) was extracted using TRIzol reagent (Thermo Fisher, Waltham, MA, USA) and used to synthesize cDNA with EasyScript First-Strand cDNA Synthesis SuperMix (Transgen, Beijing, China). qPCR was performed using SYBR Premix Ex Taq II (Takara, Otsu, Shiga, Japan). .. Plasmid Construction and Cell Transfection DNA fragment of miR-3178 stem-loop sequence was amplified by PCR from human genome DNA, cloned into pcDNA3.1 or pdsRed2-C1 vector at sites between EcoRI and XhoI (Takara, Otsu, Shiga, Japan). .. The fragment of TRIOBP 3′ UTR was amplified and cloned into pMIR-REPORT vector between EcoRI and BamHI (Takara, Otsu, Shiga, Japan).

    Real-time Polymerase Chain Reaction:

    Article Title: Ectopic Expression of a Pak-choi YABBY Gene, BcYAB3, Causes Leaf Curvature and Flowering Stage Delay in Arabidopsis thaliana
    Article Snippet: .. Real-Time PCR Total RNA of Pak-choi and Arabidopsis plants was extracted using an RNAeasy Mini Kit (Tiangen, Beijing, China), and the cDNA for real time PCR was synthesized using a PrimeScript™RT reagent Kit with gDNA Eraser (Takara, Dalian, China). qRT-PCR was carried out with SYBR® Premix Ex TaqTM II (Tli RNaseH Plus) (Takara, Dalian, China) using the ABI StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). .. The PCR procedure was carried out with the following parameters: 95 °C for 30 s, 40 cycles of 95 °C for 5 s, and 60 °C for 30 s. Furthermore, a melting curve was employed to verify the specificity of all reactions.

    Synthesized:

    Article Title: Ectopic Expression of a Pak-choi YABBY Gene, BcYAB3, Causes Leaf Curvature and Flowering Stage Delay in Arabidopsis thaliana
    Article Snippet: .. Real-Time PCR Total RNA of Pak-choi and Arabidopsis plants was extracted using an RNAeasy Mini Kit (Tiangen, Beijing, China), and the cDNA for real time PCR was synthesized using a PrimeScript™RT reagent Kit with gDNA Eraser (Takara, Dalian, China). qRT-PCR was carried out with SYBR® Premix Ex TaqTM II (Tli RNaseH Plus) (Takara, Dalian, China) using the ABI StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). .. The PCR procedure was carried out with the following parameters: 95 °C for 30 s, 40 cycles of 95 °C for 5 s, and 60 °C for 30 s. Furthermore, a melting curve was employed to verify the specificity of all reactions.

    Quantitative RT-PCR:

    Article Title: Ectopic Expression of a Pak-choi YABBY Gene, BcYAB3, Causes Leaf Curvature and Flowering Stage Delay in Arabidopsis thaliana
    Article Snippet: .. Real-Time PCR Total RNA of Pak-choi and Arabidopsis plants was extracted using an RNAeasy Mini Kit (Tiangen, Beijing, China), and the cDNA for real time PCR was synthesized using a PrimeScript™RT reagent Kit with gDNA Eraser (Takara, Dalian, China). qRT-PCR was carried out with SYBR® Premix Ex TaqTM II (Tli RNaseH Plus) (Takara, Dalian, China) using the ABI StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). .. The PCR procedure was carried out with the following parameters: 95 °C for 30 s, 40 cycles of 95 °C for 5 s, and 60 °C for 30 s. Furthermore, a melting curve was employed to verify the specificity of all reactions.

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    Adipose-specific HuR deletion accelerates the senescence of adipocytes. (A) Genotyping of adipose- and tail-derived genomic <t>DNA.</t> (B) HuR mRNA levels in adipose tissue from mice with the indicated genotypes were analyzed by real-time PCR. Values were normalized
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    Adipose-specific HuR deletion accelerates the senescence of adipocytes. (A) Genotyping of adipose- and tail-derived genomic DNA. (B) HuR mRNA levels in adipose tissue from mice with the indicated genotypes were analyzed by real-time PCR. Values were normalized

    Journal: Molecular and Cellular Biology

    Article Title: HuR Maintains a Replicative Life Span by Repressing the ARF Tumor Suppressor

    doi: 10.1128/MCB.01277-12

    Figure Lengend Snippet: Adipose-specific HuR deletion accelerates the senescence of adipocytes. (A) Genotyping of adipose- and tail-derived genomic DNA. (B) HuR mRNA levels in adipose tissue from mice with the indicated genotypes were analyzed by real-time PCR. Values were normalized

    Article Snippet: RNAs were prepared from cells or immune complexes using TriPure isolation reagent (Roche, Indianapolis, IN), reverse transcribed using a PrimeScript reverse transcriptase (RT) reagent kit with the genomic DNA (gDNA) Eraser (TaKaRa, Shiga, Japan), and subjected to PCR using the following primers: for PAI-1 , 5′-TCAGAGCAACAAGTTCAACTACACTGAG-3′ (sense) and 5′-CCCACTGTCAAGGCTCCATCACTTGCCCCA-3′ (antisense); for HuR , 5′-TTGGGCTACGGTTTTGTGAAC-3′ (sense) and 5′-CCCACTGATGTATAAGTTGGCAT-3′ (antisense); for ARF , 5′-GCCGCACCGGAATCCT-3′ (sense) and 5′-TTGAGCAGAAGAGCTGCTACGT-3′ (antisense); for Ink4a , 5′-CCCAACGCCCCGAACT-3′ (sense) and 5′-GCAGAAGAGCTGCTACGTGAA-3′ (antisense); for c- myc , 5′-TCTATTTGGGGACAGTGTTC-3′ (sense) and 5′-GGTCATAGTTCCTGTTGGTG-3′ (antisense); for p53 , 5′-TGGAGAGTATTTCACCCTCAAGA-3′ (sense) and 5′-CTCCTCTGTAGCATGGGCATC-3′ (antisense); for β-actin , 5′-CTAAGGCCAACCGTGAAAAG-3′ (sense) and 5′-ACCAGAGGCATACAGGGACA-3′ (antisense); for 18S rRNA 5′-AGTCCCTGCCCTTTGTACACA-3′ (sense) and 5′-GATCCGAGGGCCTCACTAAAC-3′ (antisense); for AUF-1 , 5′-TTTCTCCAGACACACCTGAAGA-3′ (sense) and 5′-CTGTTCCTTTGACATGGCTACTT-3′ (antisense); and for GFP , 5′-TCTGCACCACCGGCAAGCTG-3′ (sense) and 5′-TGCGCTCCTGGACGTAGCCT-3′ (antisense).

    Techniques: Derivative Assay, Mouse Assay, Real-time Polymerase Chain Reaction

    Identification of SMN1 variants. (a) qPCR analysis of four retrotransposon‐free SMN genomic regions in the intron 1 (I1), in the exon 3–intron 3 junction (E3I3), in the intron 5–exon 6 junction (I5E6), and ~1 kb downstream from exon 8 (+1 kb). Compared with four copies of SMN genes that are present in controls, we found that the patient has three copies at the I1, I5E6, and +1 kb loci and two copies at the E3I3 loci. The mother has three copies through the I1 to I5E6 loci and two copies at the +1 kb loci. The father has three copies only at the I5E6 loci. (b) Schematic representation of SMN1/2 exon (E)/intron (I) structure. Positions of sequence differences between SMN1 and SMN2 are represented by black vertical bars. The black triangles denote sequence‐specific variants in exons 7, 8 targeted by MLPA probes in routine testing. Locations of Alus in the breakpoint candidate regions in the intron 1 and 5, including the causal AluSp in the intron 1 and AluSq in the intron 5 indicated by vertical text, and primers binding sites for Alu PCR indicated by black arrowheads are shown below the scheme of the SMN structure. Position of the PCR4 spanning exons 5–8 that showed absence of SMN1 sequence‐specific variants indicating disruption of both SMN1 alleles in the patient is represented by yellow box. Range of the paternal deletion of exons 2a‐5 is represented by red box. (c) DNA sequence trace of the Alu PCR, Alu_259_4A, showing a double sequence caused by presence of AluSq wt in intron 5 together with a sequence originating from the intron 1 AluSp . Red arrows indicate the addition of AluSp ‐specific sequence in an Alu PCR product. (d) PCR genotyping of the SMN1Δ(2a‐5) variant showed presence of the deletion‐spanning amplification product in the patient (P) and father (F), but not in mother (M) and control (C). (e) DNA sequence trace of the breakpoint junction‐specific PCR and detail of the Δ2a‐5 breakpoint junction show the new Alu ‐ Alu chimeric element originating from the recombination between the AluSp in the intron 1 and AluSq in the intron 5. A breakpoint microhomology of the AluSp and AluSq is marked with a black box. (f) Schematic representation of SMN1 and SMN2 in the family members. Pink‐marked boxes represent maternal alleles (M) and blue boxes paternal alleles (F). The red crosses denote identified deletions and the dashed vertical lines denote loci of the qPCR (I1, E3I3, I5E6, and +1 kb) and MLPA (exon 7‐E7, exon 8‐E8) probes used for deletion mapping. The black junctions on the box terminals indicate a cis configuration of SMN1 and SMN2 alleles. The model shows (a) a whole deletion of one SMN1 allele in the patient (P) inherited from her mother and detected by the combination of the qPCR and MLPA; (b) a deletion of the second SMN1 allele in the patient inherited from her father and detected by the E3I3 qPCR and transcript analysis (Figure 1a ); and (c) deletion of one copy of one SMN2 allele in the mother detected by the MLPA and the + 1kb qPCR

    Journal: Molecular Genetics & Genomic Medicine

    Article Title: Spinal muscular atrophy caused by a novel Alu‐mediated deletion of exons 2a‐5 in SMN1 undetectable with routine genetic testing, et al. Spinal muscular atrophy caused by a novel Alu‐mediated deletion of exons 2a‐5 in SMN1 undetectable with routine genetic testing

    doi: 10.1002/mgg3.1238

    Figure Lengend Snippet: Identification of SMN1 variants. (a) qPCR analysis of four retrotransposon‐free SMN genomic regions in the intron 1 (I1), in the exon 3–intron 3 junction (E3I3), in the intron 5–exon 6 junction (I5E6), and ~1 kb downstream from exon 8 (+1 kb). Compared with four copies of SMN genes that are present in controls, we found that the patient has three copies at the I1, I5E6, and +1 kb loci and two copies at the E3I3 loci. The mother has three copies through the I1 to I5E6 loci and two copies at the +1 kb loci. The father has three copies only at the I5E6 loci. (b) Schematic representation of SMN1/2 exon (E)/intron (I) structure. Positions of sequence differences between SMN1 and SMN2 are represented by black vertical bars. The black triangles denote sequence‐specific variants in exons 7, 8 targeted by MLPA probes in routine testing. Locations of Alus in the breakpoint candidate regions in the intron 1 and 5, including the causal AluSp in the intron 1 and AluSq in the intron 5 indicated by vertical text, and primers binding sites for Alu PCR indicated by black arrowheads are shown below the scheme of the SMN structure. Position of the PCR4 spanning exons 5–8 that showed absence of SMN1 sequence‐specific variants indicating disruption of both SMN1 alleles in the patient is represented by yellow box. Range of the paternal deletion of exons 2a‐5 is represented by red box. (c) DNA sequence trace of the Alu PCR, Alu_259_4A, showing a double sequence caused by presence of AluSq wt in intron 5 together with a sequence originating from the intron 1 AluSp . Red arrows indicate the addition of AluSp ‐specific sequence in an Alu PCR product. (d) PCR genotyping of the SMN1Δ(2a‐5) variant showed presence of the deletion‐spanning amplification product in the patient (P) and father (F), but not in mother (M) and control (C). (e) DNA sequence trace of the breakpoint junction‐specific PCR and detail of the Δ2a‐5 breakpoint junction show the new Alu ‐ Alu chimeric element originating from the recombination between the AluSp in the intron 1 and AluSq in the intron 5. A breakpoint microhomology of the AluSp and AluSq is marked with a black box. (f) Schematic representation of SMN1 and SMN2 in the family members. Pink‐marked boxes represent maternal alleles (M) and blue boxes paternal alleles (F). The red crosses denote identified deletions and the dashed vertical lines denote loci of the qPCR (I1, E3I3, I5E6, and +1 kb) and MLPA (exon 7‐E7, exon 8‐E8) probes used for deletion mapping. The black junctions on the box terminals indicate a cis configuration of SMN1 and SMN2 alleles. The model shows (a) a whole deletion of one SMN1 allele in the patient (P) inherited from her mother and detected by the combination of the qPCR and MLPA; (b) a deletion of the second SMN1 allele in the patient inherited from her father and detected by the E3I3 qPCR and transcript analysis (Figure 1a ); and (c) deletion of one copy of one SMN2 allele in the mother detected by the MLPA and the + 1kb qPCR

    Article Snippet: 2.6 Long‐range PCR Long‐range PCR was performed using four primer pairs amplifying both SMN genes (NG_008691.1, NG_008728.1) in four overlapping PCR products (PCR1‐PCR4, Table ).

    Techniques: Real-time Polymerase Chain Reaction, Sequencing, Multiplex Ligation-dependent Probe Amplification, Binding Assay, Polymerase Chain Reaction, Variant Assay, Amplification

    Generation of Ucp1-iRFP720 KI mice. (A) Targeting of the iRFP720 gene into the Ucp1 locus. The donor vector contains an artificial intron (orange box), iRFP720 gene (red box) and the bovine growth factor gene-derived polyadenylation signal (BGHpA) (green box). Structure of the targeted allele is indicated together with the primers used for genomic PCR (red arrows). (B) Genomic PCR analysis of F0 mice carrying the Ucp1-iRFP720 KI allele. M, marker; wt, wild-type; ctrl, positive control (a DNA fragment that covers the amplicon sequence between the primers c and f in Fig 2A). (C) PCR analysis of the donor vector and the gRNA/Cas9-expression vector randomly integrated into the F0 mouse genome. The table shows the summary of random integration in the F0 mouse genome carrying the Ucp1-iRFP720 allele. (D) Mendelian inheritance of the Ucp1-iRFP720 KI allele. One of the Ucp1-iRFP720 KI mice (#71) was crossed with a WT mouse, and eight F1 mice were born. Their genotypes were analyzed by PCR using the primer sets shown in Fig 2A. Small white and red rectangles show WT Ucp1 and Ucp1-iRFP720 KI alleles, respectively. Rounded rectangles and ellipses indicate male and female mice, respectively.

    Journal: PLoS ONE

    Article Title: Non-invasive in vivo imaging of UCP1 expression in live mice via near-infrared fluorescent protein iRFP720

    doi: 10.1371/journal.pone.0225213

    Figure Lengend Snippet: Generation of Ucp1-iRFP720 KI mice. (A) Targeting of the iRFP720 gene into the Ucp1 locus. The donor vector contains an artificial intron (orange box), iRFP720 gene (red box) and the bovine growth factor gene-derived polyadenylation signal (BGHpA) (green box). Structure of the targeted allele is indicated together with the primers used for genomic PCR (red arrows). (B) Genomic PCR analysis of F0 mice carrying the Ucp1-iRFP720 KI allele. M, marker; wt, wild-type; ctrl, positive control (a DNA fragment that covers the amplicon sequence between the primers c and f in Fig 2A). (C) PCR analysis of the donor vector and the gRNA/Cas9-expression vector randomly integrated into the F0 mouse genome. The table shows the summary of random integration in the F0 mouse genome carrying the Ucp1-iRFP720 allele. (D) Mendelian inheritance of the Ucp1-iRFP720 KI allele. One of the Ucp1-iRFP720 KI mice (#71) was crossed with a WT mouse, and eight F1 mice were born. Their genotypes were analyzed by PCR using the primer sets shown in Fig 2A. Small white and red rectangles show WT Ucp1 and Ucp1-iRFP720 KI alleles, respectively. Rounded rectangles and ellipses indicate male and female mice, respectively.

    Article Snippet: To confirm the correct insertion of the iRFP720 gene at the Ucp1 locus, 10 ng each of the genome DNA was analyzed by PCR using 0.25 unit of PrimeStar GXL DNA Polymerase (TaKaRa Bio Inc.) and 3 pmoles each of the primers in a 10 μl reaction.

    Techniques: Mouse Assay, Plasmid Preparation, Derivative Assay, Polymerase Chain Reaction, Marker, Positive Control, Amplification, Sequencing, Expressing

    Selection of the optimum gRNA sequence to generate Ucp1-iRFP720 KI mice. (A) Structure of the mouse Ucp1 gene. The boxes and the numbers above each box indicate the exons of the mouse Ucp1 gene. Light and dark gray regions within the exons indicate untranslated and translated regions, respectively. The position of the transcription start site is indicated by +1. The DNA sequence around the translation start site of the mouse Ucp1 gene is shown. The red arrows indicate the target sequences of the gRNAs (dashed arrows indicate the complementary sequences). The red boxes show the protospacer adjacent motif (PAM) sequences. (B) The assay system for estimating the functional genome editing efficiency of gRNAs. The Ucp1 gene (+1 ~ +500) containing the gRNA target sites (shown in orange) was inserted into pCAG-EGxxFP vector. When recruited by the gRNA (shown in dark blue) to the target site, the Cas9 nuclease (shown in light blue) introduces a double strand break in the target DNA. Because of homologous recombination between the duplicated sequences (shown in dark green), the EGFP-coding gene is recovered. (C) Fluorescence image of HEK293T cells two days after transfection. The Cas9 nuclease was expressed together with one of the four gRNA candidates (gRNA1~gRNA4) or without gRNA (Cas9 only). Scale bar, 200 μm.

    Journal: PLoS ONE

    Article Title: Non-invasive in vivo imaging of UCP1 expression in live mice via near-infrared fluorescent protein iRFP720

    doi: 10.1371/journal.pone.0225213

    Figure Lengend Snippet: Selection of the optimum gRNA sequence to generate Ucp1-iRFP720 KI mice. (A) Structure of the mouse Ucp1 gene. The boxes and the numbers above each box indicate the exons of the mouse Ucp1 gene. Light and dark gray regions within the exons indicate untranslated and translated regions, respectively. The position of the transcription start site is indicated by +1. The DNA sequence around the translation start site of the mouse Ucp1 gene is shown. The red arrows indicate the target sequences of the gRNAs (dashed arrows indicate the complementary sequences). The red boxes show the protospacer adjacent motif (PAM) sequences. (B) The assay system for estimating the functional genome editing efficiency of gRNAs. The Ucp1 gene (+1 ~ +500) containing the gRNA target sites (shown in orange) was inserted into pCAG-EGxxFP vector. When recruited by the gRNA (shown in dark blue) to the target site, the Cas9 nuclease (shown in light blue) introduces a double strand break in the target DNA. Because of homologous recombination between the duplicated sequences (shown in dark green), the EGFP-coding gene is recovered. (C) Fluorescence image of HEK293T cells two days after transfection. The Cas9 nuclease was expressed together with one of the four gRNA candidates (gRNA1~gRNA4) or without gRNA (Cas9 only). Scale bar, 200 μm.

    Article Snippet: To confirm the correct insertion of the iRFP720 gene at the Ucp1 locus, 10 ng each of the genome DNA was analyzed by PCR using 0.25 unit of PrimeStar GXL DNA Polymerase (TaKaRa Bio Inc.) and 3 pmoles each of the primers in a 10 μl reaction.

    Techniques: Selection, Sequencing, Mouse Assay, Functional Assay, Plasmid Preparation, Homologous Recombination, Fluorescence, Transfection

    Sp1 Negatively Regulates miR-3178 by Binding to Its Promoter Region (A and B) miRNA profiling analysis. LNCaP cells were treated with 100 nM bortezomib (BTZ) (A) or 2.5 μM celastrol (CEL) (B) for 12 hr. miRNAs with over 2-fold changes against control were shown. (C) Expressions of SP1 and miR-3178 post-treatments as (A) and (B) in LNCaP cells are shown. (D) Consensus Sp1 sites and predicted binding sites (BSs) of Sp1 in miR-3178 promoter are shown. Mutations of each BS were indicated by italic red cases. (E) Luciferase reporter assay is shown. Luciferase reporter constructs were generated as schematic depiction and transfected into 1E8 cells in the presence of SP1 or control (Ctrl) plasmid. Wild-type (WT) and mutant Sp1 sites (Mut1–3) were indicated by blank and italic dash, respectively. NS, non-significant difference. (F) ChIP assay is shown. DNA was immunoprecipitated with anti-IgG or anti-Sp1 antibody and amplified by PCR using primer specific for BS3. Input chromatin before immunoprecipitation was used as control. Experiments were repeated three times, and results were shown as mean ± SD in the lower panel. (G) Expressions of miR-3178 in prostate, lung, and breast cancer cell lines are shown. (H) Expression of miR-3178 in lowly metastatic cancer cells after ectopic expression of SP1 is shown. Values were shown as mean ± SD of three independent experiments. *p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Sp1 Suppresses miR-3178 to Promote the Metastasis Invasion Cascade via Upregulation of TRIOBP

    doi: 10.1016/j.omtn.2018.04.008

    Figure Lengend Snippet: Sp1 Negatively Regulates miR-3178 by Binding to Its Promoter Region (A and B) miRNA profiling analysis. LNCaP cells were treated with 100 nM bortezomib (BTZ) (A) or 2.5 μM celastrol (CEL) (B) for 12 hr. miRNAs with over 2-fold changes against control were shown. (C) Expressions of SP1 and miR-3178 post-treatments as (A) and (B) in LNCaP cells are shown. (D) Consensus Sp1 sites and predicted binding sites (BSs) of Sp1 in miR-3178 promoter are shown. Mutations of each BS were indicated by italic red cases. (E) Luciferase reporter assay is shown. Luciferase reporter constructs were generated as schematic depiction and transfected into 1E8 cells in the presence of SP1 or control (Ctrl) plasmid. Wild-type (WT) and mutant Sp1 sites (Mut1–3) were indicated by blank and italic dash, respectively. NS, non-significant difference. (F) ChIP assay is shown. DNA was immunoprecipitated with anti-IgG or anti-Sp1 antibody and amplified by PCR using primer specific for BS3. Input chromatin before immunoprecipitation was used as control. Experiments were repeated three times, and results were shown as mean ± SD in the lower panel. (G) Expressions of miR-3178 in prostate, lung, and breast cancer cell lines are shown. (H) Expression of miR-3178 in lowly metastatic cancer cells after ectopic expression of SP1 is shown. Values were shown as mean ± SD of three independent experiments. *p

    Article Snippet: Plasmid Construction and Cell Transfection DNA fragment of miR-3178 stem-loop sequence was amplified by PCR from human genome DNA, cloned into pcDNA3.1 or pdsRed2-C1 vector at sites between EcoRI and XhoI (Takara, Otsu, Shiga, Japan).

    Techniques: Binding Assay, Luciferase, Reporter Assay, Construct, Generated, Transfection, Plasmid Preparation, Mutagenesis, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, Polymerase Chain Reaction, Expressing