transcription polymerase chain reaction pcr  (Thermo Fisher)


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    Structured Review

    Thermo Fisher transcription polymerase chain reaction pcr
    EGF-induced COX-2 enhances expression of MMP-1, MMP-2, MMP-3 and MMP-9 HONE1, shLacZ and shCOX-2 cells were treated with 10 μM PGE 2 or 50 ng/ml EGF in serum-free medium for the indicated period of time. (A, B) Total <t>RNA</t> was extracted for reverse-transcription <t>PCR</t> with MMP-1, MMP-3, COX-2 and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) primers. (C–E) Cells were transfected with MMP-1 , MMP-3 and MMP-9 promoters using lipofection. Cells were treated with 50 ng/ml EGF and 10 or 20 μM PGE 2 in serum-free medium for 24 h. Luciferase activity and protein concentrations were then determined and normalized. Values represent means ± S.E.M. of three determinations. (F) Cells were treated with 50 ng/ml EGF or 10 μM PGE 2 in serum-free medium for 9 h. The mRNA level of MMP-2 was measured and normalized to GAPDH by real-time PCR. Values represent means ± S.E.M. of three determinations.
    Transcription Polymerase Chain Reaction Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    transcription polymerase chain reaction pcr - by Bioz Stars, 2020-04
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    Images

    1) Product Images from "Epidermal growth factor-induced cyclooxygenase-2 enhances head and neck squamous cell carcinoma metastasis through fibronectin up-regulation"

    Article Title: Epidermal growth factor-induced cyclooxygenase-2 enhances head and neck squamous cell carcinoma metastasis through fibronectin up-regulation

    Journal: Oncotarget

    doi:

    EGF-induced COX-2 enhances expression of MMP-1, MMP-2, MMP-3 and MMP-9 HONE1, shLacZ and shCOX-2 cells were treated with 10 μM PGE 2 or 50 ng/ml EGF in serum-free medium for the indicated period of time. (A, B) Total RNA was extracted for reverse-transcription PCR with MMP-1, MMP-3, COX-2 and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) primers. (C–E) Cells were transfected with MMP-1 , MMP-3 and MMP-9 promoters using lipofection. Cells were treated with 50 ng/ml EGF and 10 or 20 μM PGE 2 in serum-free medium for 24 h. Luciferase activity and protein concentrations were then determined and normalized. Values represent means ± S.E.M. of three determinations. (F) Cells were treated with 50 ng/ml EGF or 10 μM PGE 2 in serum-free medium for 9 h. The mRNA level of MMP-2 was measured and normalized to GAPDH by real-time PCR. Values represent means ± S.E.M. of three determinations.
    Figure Legend Snippet: EGF-induced COX-2 enhances expression of MMP-1, MMP-2, MMP-3 and MMP-9 HONE1, shLacZ and shCOX-2 cells were treated with 10 μM PGE 2 or 50 ng/ml EGF in serum-free medium for the indicated period of time. (A, B) Total RNA was extracted for reverse-transcription PCR with MMP-1, MMP-3, COX-2 and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) primers. (C–E) Cells were transfected with MMP-1 , MMP-3 and MMP-9 promoters using lipofection. Cells were treated with 50 ng/ml EGF and 10 or 20 μM PGE 2 in serum-free medium for 24 h. Luciferase activity and protein concentrations were then determined and normalized. Values represent means ± S.E.M. of three determinations. (F) Cells were treated with 50 ng/ml EGF or 10 μM PGE 2 in serum-free medium for 9 h. The mRNA level of MMP-2 was measured and normalized to GAPDH by real-time PCR. Values represent means ± S.E.M. of three determinations.

    Techniques Used: Expressing, Polymerase Chain Reaction, Transfection, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction

    2) Product Images from "FOXA1 potentiates lineage-specific enhancer activation through modulating TET1 expression and function"

    Article Title: FOXA1 potentiates lineage-specific enhancer activation through modulating TET1 expression and function

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw498

    TET1 is a direct transcriptional target of FOXA1. ( A ) ChIP-seq showing FOXA1 binding events at TET1 promoter and enhancer. FOXA1 ChIP-seq was conducted in LNCaP cells and FOXA1 binding events were identified by HOMER and visualized in UCSC Genome Browser. FKHD motifs (indicated by red box) near FXBS were determined by JASPAR. DNA fragments containing FXBS at the TET1 promoter (pTET1) and enhancer (eTET1) were each cloned into pGL4 luciferase reporter construct with wild-type (WT) or mutated (mut) FKHD motif (mutated nt shown in red at the bottom panel). ( B ) ChIP-PCR validation of FOXA1 binding to TET1 enhancer and promoter in LNCaP cells. ChIP was performed using anti-FOXA1 and anti-IgG antibodies in LNCaP cells. ChIP-qPCR was performed using primers flanking the FOXA1 binding peaks at the TET1 enhancer (eTET1) and promoter (pTET). PSA is used as a positive control while KIAA0066 a negative control. Data shown are mean ± SEM of technical replicates from one representative experiment out of three. ( C ) FOXA1 occupancy at TET1 promoter and enhancer was decreased by FOXA1 knockdown. ChIP-qPCR using anti-FOXA1 antibody was carried out in control and FOXA1-depleted LNCaP cells. Data shown are mean ± SEM of technical replicates from one representative experiment out of three. * P
    Figure Legend Snippet: TET1 is a direct transcriptional target of FOXA1. ( A ) ChIP-seq showing FOXA1 binding events at TET1 promoter and enhancer. FOXA1 ChIP-seq was conducted in LNCaP cells and FOXA1 binding events were identified by HOMER and visualized in UCSC Genome Browser. FKHD motifs (indicated by red box) near FXBS were determined by JASPAR. DNA fragments containing FXBS at the TET1 promoter (pTET1) and enhancer (eTET1) were each cloned into pGL4 luciferase reporter construct with wild-type (WT) or mutated (mut) FKHD motif (mutated nt shown in red at the bottom panel). ( B ) ChIP-PCR validation of FOXA1 binding to TET1 enhancer and promoter in LNCaP cells. ChIP was performed using anti-FOXA1 and anti-IgG antibodies in LNCaP cells. ChIP-qPCR was performed using primers flanking the FOXA1 binding peaks at the TET1 enhancer (eTET1) and promoter (pTET). PSA is used as a positive control while KIAA0066 a negative control. Data shown are mean ± SEM of technical replicates from one representative experiment out of three. ( C ) FOXA1 occupancy at TET1 promoter and enhancer was decreased by FOXA1 knockdown. ChIP-qPCR using anti-FOXA1 antibody was carried out in control and FOXA1-depleted LNCaP cells. Data shown are mean ± SEM of technical replicates from one representative experiment out of three. * P

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Clone Assay, Luciferase, Construct, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Positive Control, Negative Control

    FOXA1 induces TET1 gene expression. ( A and B ) Correlated FOXA1 and TET1 gene expression in prostate cells. RNA was extracted from a panel of 12 prostate cell lines and analyzed by qRT-PCR for FOXA1 (A) and TET1 (B) gene expression. Data shown are mean ± SEM of technical replicates from one representative experiment out of three. ( C and D ) TET1 transcript (C) and protein (D) are downregulated following FOXA1 knockdown in LNCaP cells. LNCaP cells were infected with shCtrl or shFOXA1 lentivirus and subsequently subjected to qRT-PCR and western blot analysis. Data shown are one representative out of triplicate experiments. ( E ) TET1 is downregulated by FOXA1 knockdown in C4-2B cells. C4-2B cells were infected with shCtrl or shFOXA1 lentivirus for 8 h followed by puromycin selection for 4 days, and subsequently subjected to qRT-PCR and western blot analysis. Data shown are one representative out of triplicate experiments. ( F and G ) TET1 is upregulated following FOXA1 overexpression. The 22Rv1 (F) and DU145 (G) cells were infected with LacZ or FOXA1 adenovirus for 48 h and immunoblot was performed to assess FOXA1 and TET1 protein levels. ( H ) Positive TET1 staining in FOXA1-expressing cells. DU145 cells were infected with LacZ control (i–iii) or Flag-tagged FOXA1 (iv–vi) adenovirus for 48 h and then subjected to Immunofluorescence co-staining of FOXA1 and TET1. Bottom panel shows zoomed-in region containing both FOXA1-uninfected and -infected cells.
    Figure Legend Snippet: FOXA1 induces TET1 gene expression. ( A and B ) Correlated FOXA1 and TET1 gene expression in prostate cells. RNA was extracted from a panel of 12 prostate cell lines and analyzed by qRT-PCR for FOXA1 (A) and TET1 (B) gene expression. Data shown are mean ± SEM of technical replicates from one representative experiment out of three. ( C and D ) TET1 transcript (C) and protein (D) are downregulated following FOXA1 knockdown in LNCaP cells. LNCaP cells were infected with shCtrl or shFOXA1 lentivirus and subsequently subjected to qRT-PCR and western blot analysis. Data shown are one representative out of triplicate experiments. ( E ) TET1 is downregulated by FOXA1 knockdown in C4-2B cells. C4-2B cells were infected with shCtrl or shFOXA1 lentivirus for 8 h followed by puromycin selection for 4 days, and subsequently subjected to qRT-PCR and western blot analysis. Data shown are one representative out of triplicate experiments. ( F and G ) TET1 is upregulated following FOXA1 overexpression. The 22Rv1 (F) and DU145 (G) cells were infected with LacZ or FOXA1 adenovirus for 48 h and immunoblot was performed to assess FOXA1 and TET1 protein levels. ( H ) Positive TET1 staining in FOXA1-expressing cells. DU145 cells were infected with LacZ control (i–iii) or Flag-tagged FOXA1 (iv–vi) adenovirus for 48 h and then subjected to Immunofluorescence co-staining of FOXA1 and TET1. Bottom panel shows zoomed-in region containing both FOXA1-uninfected and -infected cells.

    Techniques Used: Expressing, Quantitative RT-PCR, Infection, Western Blot, Selection, Over Expression, Staining, Immunofluorescence

    Related Articles

    Clone Assay:

    Article Title: FOXA1 potentiates lineage-specific enhancer activation through modulating TET1 expression and function
    Article Snippet: .. For FOXA1 and TET1 FL and domain constructs, human FOXA1 and TET1 cDNA were amplified by reverse transcription polymerase chain reaction (PCR) from LNCaP cells and pENTR223 TET1 (Harvard Plasmid), respectively, and cloned into the entry vector pCR8/GW/TOPO (Invitrogen). .. Adenoviral construct expressing FOXA1 was generated by recombining pCR8-FOXA1 with pAD/CMV/V5 using LR Clonase II (Invitrogen).

    Article Title: miR-375 inhibits the proliferation of gastric cancer cells by repressing ERBB2 expression
    Article Snippet: .. ERBB2 expression vector construction The full length coding region of human ERBB2 was amplified by reverse transcription polymerase chain reaction (PCR) and cloned into the pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA), which was then designated as pcDNA3.1-ERBB2. .. This vector and the control vector, pcDNA3.1, were transfected into cells using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions.

    Article Title: An Algal Nucleus-encoded Subunit of Mitochondrial ATP Synthase Rescues a Defect in the Analogous Human Mitochondrial-encoded Subunit
    Article Snippet: First-strand cDNA was obtained using the SuperScript First Strand Synthesis System for reverse transcription-polymerase chain reaction (PCR) (Invitrogen, Carlsbad, CA). .. Isolated clones were screened by automated sequencing using the ABI Prism Big Dye kit (PerkinElmer Life Sciences, Boston, MA).

    Article Title: The Toll-like receptor 2 (TLR2) ligand FSL-1 is internalized via the clathrin-dependent endocytic pathway triggered by CD14 and CD36 but not by TLR2
    Article Snippet: .. The complementary DNAs (cDNAs) of human CD14, CD36 and TLR2 were obtained as described previously., , Briefly, they were obtained by reverse transcription–polymerase chain reaction (PCR) of total RNA isolated from a human monocyte/macrophage cell line, THP-1 cells, and then they were cloned into a pEF6/V5-His TOPO vector (Invitrogen, Carlsbad, CA) or pcDNA3.1-His-TOPO vector (Invitrogen). .. Their transfection into wild-type HEK293 cells (HEK293WT) was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

    Amplification:

    Article Title: FOXA1 potentiates lineage-specific enhancer activation through modulating TET1 expression and function
    Article Snippet: .. For FOXA1 and TET1 FL and domain constructs, human FOXA1 and TET1 cDNA were amplified by reverse transcription polymerase chain reaction (PCR) from LNCaP cells and pENTR223 TET1 (Harvard Plasmid), respectively, and cloned into the entry vector pCR8/GW/TOPO (Invitrogen). .. Adenoviral construct expressing FOXA1 was generated by recombining pCR8-FOXA1 with pAD/CMV/V5 using LR Clonase II (Invitrogen).

    Article Title: miR-375 inhibits the proliferation of gastric cancer cells by repressing ERBB2 expression
    Article Snippet: .. ERBB2 expression vector construction The full length coding region of human ERBB2 was amplified by reverse transcription polymerase chain reaction (PCR) and cloned into the pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA), which was then designated as pcDNA3.1-ERBB2. .. This vector and the control vector, pcDNA3.1, were transfected into cells using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions.

    Article Title: Heterochromatin Protein 1? Epigenetically Regulates Cell Differentiation and Exhibits Potential as a Therapeutic Target for Various Types of Cancers
    Article Snippet: .. The cDNAs encoding full-length and deletion mutant forms (deleted at 1 to 213 nucleotides, ΔCD; and at 318 to 519 nucleotides, ΔCSD in the open reading frame) of the HP1γ gene were amplified by reverse transcription-polymerase chain reaction (PCR) using total RNA from HCT116 cells, and was subcloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA), forming the plasmid pCRHP1γ, pCRHPΔCSD, and pCRHPΔCD, respectively. .. The plasmids were then digested with Eco RI, and the Eco RI fragment containing the full-length and deletion mutants of HP1γ cDNA were subcloned into the Eco RI site of pGene-V5 (Invitrogen), forming the plasmid pGHP1γ, pGHPΔCSD, or pGHPΔCD, respectively.

    Article Title: An Algal Nucleus-encoded Subunit of Mitochondrial ATP Synthase Rescues a Defect in the Analogous Human Mitochondrial-encoded Subunit
    Article Snippet: First-strand cDNA was obtained using the SuperScript First Strand Synthesis System for reverse transcription-polymerase chain reaction (PCR) (Invitrogen, Carlsbad, CA). .. The SMART RACE cDNA Amplification kit ( CLONTECH , Palo Alto, CA) was used for determination of the 5′- and 3′-untranslated regions (UTRs).

    Article Title: PURIFICATION, CHARACTERIZATION AND CLONING OF A RICIN B-LIKE LECTIN FROM MUSHROOM Clitocybe nebularis WITH ANTIPROLIFERATIVE ACTIVITY AGAINST HUMAN LEUKEMIC T CELLS
    Article Snippet: First strand complementary DNA (cDNA) was synthesized from the total RNA by reverse transcription-polymerase chain reaction (PCR) using a GeneAmp RNA PCR Core Kit (Applied Biosystems, Foster city, CA) with anchored oligo(dT)-adapter primer dT(17)3’RACE ( , Supplementary data). .. For PCR amplification of the partial cnl gene and cDNA sequences, genomic DNA and reverse transcript were used as templates.

    Article Title: Inhibitory Effects of Short-Chain Fatty Acids and ω-3 Polyunsaturated Fatty Acids on Profibrotic Factors in Dermal Fibroblasts
    Article Snippet: First-strand cDNAs were synthesized from the isolated RNAs with the First-Strand cDNA Synthesis System for reverse transcription-polymerase chain reaction (PCR) (Invitrogen). cDNAs were used for subsequent quantitative real-time PCR analysis using the SYBR Premix Ex Taq II (Takara Bio, Otsu, Japan) with appropriate primers ( ). .. Post-PCR melting curves were confirmed by the specificity of single-target amplification.

    Synthesized:

    Article Title: Aristolochic acid I determine the phenotype and activation of macrophages in acute and chronic kidney disease
    Article Snippet: .. RNA extraction, reverse transcription and qRT-PCR Pure Link RNA Mini Kit (Ambion, Germany) was used to extract total RNA from renal tissue stored in RNA later, according to the manufacturer’s instructions. cDNA was synthesized from 1 µg of total RNA by reverse transcription polymerase chain reaction (PCR) using Superscript II reverse transcriptase (Thermo Fisher, Germany) according to manufacturer instructions. .. Quantitative real-time PCR (qRT-PCR) from cDNA was performed a Light Cycler 480 (Roche, Germany).

    Article Title: PURIFICATION, CHARACTERIZATION AND CLONING OF A RICIN B-LIKE LECTIN FROM MUSHROOM Clitocybe nebularis WITH ANTIPROLIFERATIVE ACTIVITY AGAINST HUMAN LEUKEMIC T CELLS
    Article Snippet: .. First strand complementary DNA (cDNA) was synthesized from the total RNA by reverse transcription-polymerase chain reaction (PCR) using a GeneAmp RNA PCR Core Kit (Applied Biosystems, Foster city, CA) with anchored oligo(dT)-adapter primer dT(17)3’RACE ( , Supplementary data). .. For PCR amplification of the partial cnl gene and cDNA sequences, genomic DNA and reverse transcript were used as templates.

    Article Title: Pancreatic fibroblast growth factor 21 protects against type 2 diabetes in mice by promoting insulin expression and secretion in a PI3K/Akt signaling‐dependent manner, et al. Pancreatic fibroblast growth factor 21 protects against type 2 diabetes in mice by promoting insulin expression and secretion in a PI3K/Akt signaling‐dependent manner
    Article Snippet: Total RNA was extracted from tissue or isolated islets using TRIzol reagent (Invitrogen). cDNA was synthesized from 0.5 μg of total RNA using a supermix consisting of oligo (dT), random hexamer primers and reverse transcriptase (Invitrogen). .. Relative gene expression levels were determined by reverse transcription–polymerase chain reaction (PCR) (Applied Biosystems, Foster City, CA, USA) using SYBR Green with normalization to GAPDH.

    Article Title: Inhibitory Effects of Short-Chain Fatty Acids and ω-3 Polyunsaturated Fatty Acids on Profibrotic Factors in Dermal Fibroblasts
    Article Snippet: .. First-strand cDNAs were synthesized from the isolated RNAs with the First-Strand cDNA Synthesis System for reverse transcription-polymerase chain reaction (PCR) (Invitrogen). cDNAs were used for subsequent quantitative real-time PCR analysis using the SYBR Premix Ex Taq II (Takara Bio, Otsu, Japan) with appropriate primers ( ). .. The PCR reactions were run on an iCycler IQ (Bio-Rad, Hercules, Calif) for 40 cycles at 95°C for 30 seconds, at an annealing temperature ( ) for 30 seconds, and at 72°C for 30 seconds.

    Construct:

    Article Title: FOXA1 potentiates lineage-specific enhancer activation through modulating TET1 expression and function
    Article Snippet: .. For FOXA1 and TET1 FL and domain constructs, human FOXA1 and TET1 cDNA were amplified by reverse transcription polymerase chain reaction (PCR) from LNCaP cells and pENTR223 TET1 (Harvard Plasmid), respectively, and cloned into the entry vector pCR8/GW/TOPO (Invitrogen). .. Adenoviral construct expressing FOXA1 was generated by recombining pCR8-FOXA1 with pAD/CMV/V5 using LR Clonase II (Invitrogen).

    Real-time Polymerase Chain Reaction:

    Article Title: Paracrine Action of Mesenchymal Stem Cells Revealed by Single Cell Gene Profiling in Infarcted Murine Hearts
    Article Snippet: .. Total RNA were extracted from these tissues, and 0.5 μg RNA was used in reverse transcription–polymerase chain reaction(PCR), followed by real-time PCR (Applied biosystems, CA, USA) to examine the expression level of paracrine factors. .. In addition, different zones including remote zone, border zone and infarcted zone were digested into single cells by collagenase type I(1 mg/ml, Sigma), then eGFP+ cells were collected to determine the distribution of MSCs in infarcted hearts.

    Article Title: Aristolochic acid I determine the phenotype and activation of macrophages in acute and chronic kidney disease
    Article Snippet: RNA extraction, reverse transcription and qRT-PCR Pure Link RNA Mini Kit (Ambion, Germany) was used to extract total RNA from renal tissue stored in RNA later, according to the manufacturer’s instructions. cDNA was synthesized from 1 µg of total RNA by reverse transcription polymerase chain reaction (PCR) using Superscript II reverse transcriptase (Thermo Fisher, Germany) according to manufacturer instructions. .. Quantitative real-time PCR (qRT-PCR) from cDNA was performed a Light Cycler 480 (Roche, Germany).

    Article Title: Pancreatic fibroblast growth factor 21 protects against type 2 diabetes in mice by promoting insulin expression and secretion in a PI3K/Akt signaling‐dependent manner, et al. Pancreatic fibroblast growth factor 21 protects against type 2 diabetes in mice by promoting insulin expression and secretion in a PI3K/Akt signaling‐dependent manner
    Article Snippet: Paragraph title: 2.7. Real‐time PCR analysis ... Relative gene expression levels were determined by reverse transcription–polymerase chain reaction (PCR) (Applied Biosystems, Foster City, CA, USA) using SYBR Green with normalization to GAPDH.

    Article Title: Inhibitory Effects of Short-Chain Fatty Acids and ω-3 Polyunsaturated Fatty Acids on Profibrotic Factors in Dermal Fibroblasts
    Article Snippet: .. First-strand cDNAs were synthesized from the isolated RNAs with the First-Strand cDNA Synthesis System for reverse transcription-polymerase chain reaction (PCR) (Invitrogen). cDNAs were used for subsequent quantitative real-time PCR analysis using the SYBR Premix Ex Taq II (Takara Bio, Otsu, Japan) with appropriate primers ( ). .. The PCR reactions were run on an iCycler IQ (Bio-Rad, Hercules, Calif) for 40 cycles at 95°C for 30 seconds, at an annealing temperature ( ) for 30 seconds, and at 72°C for 30 seconds.

    Transplantation Assay:

    Article Title: Paracrine Action of Mesenchymal Stem Cells Revealed by Single Cell Gene Profiling in Infarcted Murine Hearts
    Article Snippet: Laser capture micro-dissection and qRT-PCR analysis Mice were euthanized and hearts were removed at day 5 after transplantation, embedded in optimal cutting temperature, and then frozen in liquid nitrogen. .. Total RNA were extracted from these tissues, and 0.5 μg RNA was used in reverse transcription–polymerase chain reaction(PCR), followed by real-time PCR (Applied biosystems, CA, USA) to examine the expression level of paracrine factors.

    Random Hexamer Labeling:

    Article Title: Pancreatic fibroblast growth factor 21 protects against type 2 diabetes in mice by promoting insulin expression and secretion in a PI3K/Akt signaling‐dependent manner, et al. Pancreatic fibroblast growth factor 21 protects against type 2 diabetes in mice by promoting insulin expression and secretion in a PI3K/Akt signaling‐dependent manner
    Article Snippet: Total RNA was extracted from tissue or isolated islets using TRIzol reagent (Invitrogen). cDNA was synthesized from 0.5 μg of total RNA using a supermix consisting of oligo (dT), random hexamer primers and reverse transcriptase (Invitrogen). .. Relative gene expression levels were determined by reverse transcription–polymerase chain reaction (PCR) (Applied Biosystems, Foster City, CA, USA) using SYBR Green with normalization to GAPDH.

    Expressing:

    Article Title: Paracrine Action of Mesenchymal Stem Cells Revealed by Single Cell Gene Profiling in Infarcted Murine Hearts
    Article Snippet: .. Total RNA were extracted from these tissues, and 0.5 μg RNA was used in reverse transcription–polymerase chain reaction(PCR), followed by real-time PCR (Applied biosystems, CA, USA) to examine the expression level of paracrine factors. .. In addition, different zones including remote zone, border zone and infarcted zone were digested into single cells by collagenase type I(1 mg/ml, Sigma), then eGFP+ cells were collected to determine the distribution of MSCs in infarcted hearts.

    Article Title: FOXA1 potentiates lineage-specific enhancer activation through modulating TET1 expression and function
    Article Snippet: For FOXA1 and TET1 FL and domain constructs, human FOXA1 and TET1 cDNA were amplified by reverse transcription polymerase chain reaction (PCR) from LNCaP cells and pENTR223 TET1 (Harvard Plasmid), respectively, and cloned into the entry vector pCR8/GW/TOPO (Invitrogen). .. Adenoviral construct expressing FOXA1 was generated by recombining pCR8-FOXA1 with pAD/CMV/V5 using LR Clonase II (Invitrogen).

    Article Title: miR-375 inhibits the proliferation of gastric cancer cells by repressing ERBB2 expression
    Article Snippet: .. ERBB2 expression vector construction The full length coding region of human ERBB2 was amplified by reverse transcription polymerase chain reaction (PCR) and cloned into the pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA), which was then designated as pcDNA3.1-ERBB2. .. This vector and the control vector, pcDNA3.1, were transfected into cells using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions.

    Article Title: Heterochromatin Protein 1? Epigenetically Regulates Cell Differentiation and Exhibits Potential as a Therapeutic Target for Various Types of Cancers
    Article Snippet: The cDNAs encoding full-length and deletion mutant forms (deleted at 1 to 213 nucleotides, ΔCD; and at 318 to 519 nucleotides, ΔCSD in the open reading frame) of the HP1γ gene were amplified by reverse transcription-polymerase chain reaction (PCR) using total RNA from HCT116 cells, and was subcloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA), forming the plasmid pCRHP1γ, pCRHPΔCSD, and pCRHPΔCD, respectively. .. To produce a cell line with ectopic expression of HP1γ and the HP1γ deletion mutants under control of a mifepristone-inducible promoter, 3T3L1 cells in 100-mm cell culture dishes were transfected with 3 μg of a regulatory plasmid, the pSwitch vector, and 7 μg of pGHP1γ, pGHPΔCSD (termed as ΔCSD), pGHPΔCD (termed as ΔCD), or an empty vector pGene-V5 as a control, by using DoFect GT1 transfection reagent (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s recommendations.

    Article Title: Pancreatic fibroblast growth factor 21 protects against type 2 diabetes in mice by promoting insulin expression and secretion in a PI3K/Akt signaling‐dependent manner, et al. Pancreatic fibroblast growth factor 21 protects against type 2 diabetes in mice by promoting insulin expression and secretion in a PI3K/Akt signaling‐dependent manner
    Article Snippet: .. Relative gene expression levels were determined by reverse transcription–polymerase chain reaction (PCR) (Applied Biosystems, Foster City, CA, USA) using SYBR Green with normalization to GAPDH. .. A TUNEL assay was performed to detect apoptosis using a TMR green in situ cell death detection kit (Roche Applied Science), according to the manufacturer's instructions.

    Modification:

    Article Title: FOXA1 potentiates lineage-specific enhancer activation through modulating TET1 expression and function
    Article Snippet: Cell lines, plasmids and antibodies Prostate cancer cell lines LNCaP, VCaP, 22Rv1, BPH1, RWPE-1, DU145 and human embryonic kidney cell line HEK293T cells were obtained from American Type Culture Collection and cultured in either RPMI1640 or Dulbecco's modified Eagle's medium with 10% fetal bovine serum (FBS). .. For FOXA1 and TET1 FL and domain constructs, human FOXA1 and TET1 cDNA were amplified by reverse transcription polymerase chain reaction (PCR) from LNCaP cells and pENTR223 TET1 (Harvard Plasmid), respectively, and cloned into the entry vector pCR8/GW/TOPO (Invitrogen).

    Over Expression:

    Article Title: FOXA1 potentiates lineage-specific enhancer activation through modulating TET1 expression and function
    Article Snippet: For FOXA1 and TET1 FL and domain constructs, human FOXA1 and TET1 cDNA were amplified by reverse transcription polymerase chain reaction (PCR) from LNCaP cells and pENTR223 TET1 (Harvard Plasmid), respectively, and cloned into the entry vector pCR8/GW/TOPO (Invitrogen). .. Overexpression constructs for TET1 were generated by recombination of pCR8-TET1 with NTSFB destination vector or pLenti CMV/TO Puro DEST (Addgene plasmid 17 293).

    Transfection:

    Article Title: miR-375 inhibits the proliferation of gastric cancer cells by repressing ERBB2 expression
    Article Snippet: ERBB2 expression vector construction The full length coding region of human ERBB2 was amplified by reverse transcription polymerase chain reaction (PCR) and cloned into the pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA), which was then designated as pcDNA3.1-ERBB2. .. This vector and the control vector, pcDNA3.1, were transfected into cells using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions.

    Article Title: Heterochromatin Protein 1? Epigenetically Regulates Cell Differentiation and Exhibits Potential as a Therapeutic Target for Various Types of Cancers
    Article Snippet: Paragraph title: Plasmid Construction and Transfection ... The cDNAs encoding full-length and deletion mutant forms (deleted at 1 to 213 nucleotides, ΔCD; and at 318 to 519 nucleotides, ΔCSD in the open reading frame) of the HP1γ gene were amplified by reverse transcription-polymerase chain reaction (PCR) using total RNA from HCT116 cells, and was subcloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA), forming the plasmid pCRHP1γ, pCRHPΔCSD, and pCRHPΔCD, respectively.

    Article Title: The Toll-like receptor 2 (TLR2) ligand FSL-1 is internalized via the clathrin-dependent endocytic pathway triggered by CD14 and CD36 but not by TLR2
    Article Snippet: The complementary DNAs (cDNAs) of human CD14, CD36 and TLR2 were obtained as described previously., , Briefly, they were obtained by reverse transcription–polymerase chain reaction (PCR) of total RNA isolated from a human monocyte/macrophage cell line, THP-1 cells, and then they were cloned into a pEF6/V5-His TOPO vector (Invitrogen, Carlsbad, CA) or pcDNA3.1-His-TOPO vector (Invitrogen). .. Their transfection into wild-type HEK293 cells (HEK293WT) was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

    Cell Culture:

    Article Title: FOXA1 potentiates lineage-specific enhancer activation through modulating TET1 expression and function
    Article Snippet: Cell lines, plasmids and antibodies Prostate cancer cell lines LNCaP, VCaP, 22Rv1, BPH1, RWPE-1, DU145 and human embryonic kidney cell line HEK293T cells were obtained from American Type Culture Collection and cultured in either RPMI1640 or Dulbecco's modified Eagle's medium with 10% fetal bovine serum (FBS). .. For FOXA1 and TET1 FL and domain constructs, human FOXA1 and TET1 cDNA were amplified by reverse transcription polymerase chain reaction (PCR) from LNCaP cells and pENTR223 TET1 (Harvard Plasmid), respectively, and cloned into the entry vector pCR8/GW/TOPO (Invitrogen).

    Article Title: Heterochromatin Protein 1? Epigenetically Regulates Cell Differentiation and Exhibits Potential as a Therapeutic Target for Various Types of Cancers
    Article Snippet: The cDNAs encoding full-length and deletion mutant forms (deleted at 1 to 213 nucleotides, ΔCD; and at 318 to 519 nucleotides, ΔCSD in the open reading frame) of the HP1γ gene were amplified by reverse transcription-polymerase chain reaction (PCR) using total RNA from HCT116 cells, and was subcloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA), forming the plasmid pCRHP1γ, pCRHPΔCSD, and pCRHPΔCD, respectively. .. To produce a cell line with ectopic expression of HP1γ and the HP1γ deletion mutants under control of a mifepristone-inducible promoter, 3T3L1 cells in 100-mm cell culture dishes were transfected with 3 μg of a regulatory plasmid, the pSwitch vector, and 7 μg of pGHP1γ, pGHPΔCSD (termed as ΔCSD), pGHPΔCD (termed as ΔCD), or an empty vector pGene-V5 as a control, by using DoFect GT1 transfection reagent (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s recommendations.

    Article Title: An Algal Nucleus-encoded Subunit of Mitochondrial ATP Synthase Rescues a Defect in the Analogous Human Mitochondrial-encoded Subunit
    Article Snippet: C. reinhardtii strain 21 gr (mt+) (CC-1690) was obtained from the Chlamydomonas Genetic Center (Duke University, Durham, NC) and was cultured under continuous light at room temperature ( ). .. First-strand cDNA was obtained using the SuperScript First Strand Synthesis System for reverse transcription-polymerase chain reaction (PCR) (Invitrogen, Carlsbad, CA).

    Generated:

    Article Title: FOXA1 potentiates lineage-specific enhancer activation through modulating TET1 expression and function
    Article Snippet: For FOXA1 and TET1 FL and domain constructs, human FOXA1 and TET1 cDNA were amplified by reverse transcription polymerase chain reaction (PCR) from LNCaP cells and pENTR223 TET1 (Harvard Plasmid), respectively, and cloned into the entry vector pCR8/GW/TOPO (Invitrogen). .. Adenoviral construct expressing FOXA1 was generated by recombining pCR8-FOXA1 with pAD/CMV/V5 using LR Clonase II (Invitrogen).

    Polymerase Chain Reaction:

    Article Title: Aristolochic acid I determine the phenotype and activation of macrophages in acute and chronic kidney disease
    Article Snippet: .. RNA extraction, reverse transcription and qRT-PCR Pure Link RNA Mini Kit (Ambion, Germany) was used to extract total RNA from renal tissue stored in RNA later, according to the manufacturer’s instructions. cDNA was synthesized from 1 µg of total RNA by reverse transcription polymerase chain reaction (PCR) using Superscript II reverse transcriptase (Thermo Fisher, Germany) according to manufacturer instructions. .. Quantitative real-time PCR (qRT-PCR) from cDNA was performed a Light Cycler 480 (Roche, Germany).

    Article Title: FOXA1 potentiates lineage-specific enhancer activation through modulating TET1 expression and function
    Article Snippet: .. For FOXA1 and TET1 FL and domain constructs, human FOXA1 and TET1 cDNA were amplified by reverse transcription polymerase chain reaction (PCR) from LNCaP cells and pENTR223 TET1 (Harvard Plasmid), respectively, and cloned into the entry vector pCR8/GW/TOPO (Invitrogen). .. Adenoviral construct expressing FOXA1 was generated by recombining pCR8-FOXA1 with pAD/CMV/V5 using LR Clonase II (Invitrogen).

    Article Title: miR-375 inhibits the proliferation of gastric cancer cells by repressing ERBB2 expression
    Article Snippet: .. ERBB2 expression vector construction The full length coding region of human ERBB2 was amplified by reverse transcription polymerase chain reaction (PCR) and cloned into the pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA), which was then designated as pcDNA3.1-ERBB2. .. This vector and the control vector, pcDNA3.1, were transfected into cells using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions.

    Article Title: Heterochromatin Protein 1? Epigenetically Regulates Cell Differentiation and Exhibits Potential as a Therapeutic Target for Various Types of Cancers
    Article Snippet: .. The cDNAs encoding full-length and deletion mutant forms (deleted at 1 to 213 nucleotides, ΔCD; and at 318 to 519 nucleotides, ΔCSD in the open reading frame) of the HP1γ gene were amplified by reverse transcription-polymerase chain reaction (PCR) using total RNA from HCT116 cells, and was subcloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA), forming the plasmid pCRHP1γ, pCRHPΔCSD, and pCRHPΔCD, respectively. .. The plasmids were then digested with Eco RI, and the Eco RI fragment containing the full-length and deletion mutants of HP1γ cDNA were subcloned into the Eco RI site of pGene-V5 (Invitrogen), forming the plasmid pGHP1γ, pGHPΔCSD, or pGHPΔCD, respectively.

    Article Title: Epidermal growth factor-induced cyclooxygenase-2 enhances head and neck squamous cell carcinoma metastasis through fibronectin up-regulation
    Article Snippet: .. Reverse transcription–polymerase chain reaction Total RNA was isolated using the TRIzol RNA extraction kit (Invitrogen), and 2 μg of RNA was subjected to reverse transcription–polymerase chain reaction (PCR) with SuperScriptTM II (Invitrogen). ..

    Article Title: An Algal Nucleus-encoded Subunit of Mitochondrial ATP Synthase Rescues a Defect in the Analogous Human Mitochondrial-encoded Subunit
    Article Snippet: .. First-strand cDNA was obtained using the SuperScript First Strand Synthesis System for reverse transcription-polymerase chain reaction (PCR) (Invitrogen, Carlsbad, CA). .. The SMART RACE cDNA Amplification kit ( CLONTECH , Palo Alto, CA) was used for determination of the 5′- and 3′-untranslated regions (UTRs).

    Article Title: PURIFICATION, CHARACTERIZATION AND CLONING OF A RICIN B-LIKE LECTIN FROM MUSHROOM Clitocybe nebularis WITH ANTIPROLIFERATIVE ACTIVITY AGAINST HUMAN LEUKEMIC T CELLS
    Article Snippet: .. First strand complementary DNA (cDNA) was synthesized from the total RNA by reverse transcription-polymerase chain reaction (PCR) using a GeneAmp RNA PCR Core Kit (Applied Biosystems, Foster city, CA) with anchored oligo(dT)-adapter primer dT(17)3’RACE ( , Supplementary data). .. For PCR amplification of the partial cnl gene and cDNA sequences, genomic DNA and reverse transcript were used as templates.

    Article Title: Pancreatic fibroblast growth factor 21 protects against type 2 diabetes in mice by promoting insulin expression and secretion in a PI3K/Akt signaling‐dependent manner, et al. Pancreatic fibroblast growth factor 21 protects against type 2 diabetes in mice by promoting insulin expression and secretion in a PI3K/Akt signaling‐dependent manner
    Article Snippet: .. Relative gene expression levels were determined by reverse transcription–polymerase chain reaction (PCR) (Applied Biosystems, Foster City, CA, USA) using SYBR Green with normalization to GAPDH. .. A TUNEL assay was performed to detect apoptosis using a TMR green in situ cell death detection kit (Roche Applied Science), according to the manufacturer's instructions.

    Article Title: The Toll-like receptor 2 (TLR2) ligand FSL-1 is internalized via the clathrin-dependent endocytic pathway triggered by CD14 and CD36 but not by TLR2
    Article Snippet: .. The complementary DNAs (cDNAs) of human CD14, CD36 and TLR2 were obtained as described previously., , Briefly, they were obtained by reverse transcription–polymerase chain reaction (PCR) of total RNA isolated from a human monocyte/macrophage cell line, THP-1 cells, and then they were cloned into a pEF6/V5-His TOPO vector (Invitrogen, Carlsbad, CA) or pcDNA3.1-His-TOPO vector (Invitrogen). .. Their transfection into wild-type HEK293 cells (HEK293WT) was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

    Article Title: Inhibitory Effects of Short-Chain Fatty Acids and ω-3 Polyunsaturated Fatty Acids on Profibrotic Factors in Dermal Fibroblasts
    Article Snippet: .. First-strand cDNAs were synthesized from the isolated RNAs with the First-Strand cDNA Synthesis System for reverse transcription-polymerase chain reaction (PCR) (Invitrogen). cDNAs were used for subsequent quantitative real-time PCR analysis using the SYBR Premix Ex Taq II (Takara Bio, Otsu, Japan) with appropriate primers ( ). .. The PCR reactions were run on an iCycler IQ (Bio-Rad, Hercules, Calif) for 40 cycles at 95°C for 30 seconds, at an annealing temperature ( ) for 30 seconds, and at 72°C for 30 seconds.

    Sequencing:

    Article Title: An Algal Nucleus-encoded Subunit of Mitochondrial ATP Synthase Rescues a Defect in the Analogous Human Mitochondrial-encoded Subunit
    Article Snippet: Using the Chlamydomonas ) ( , ), we identified a number of overlapping expressed sequence tags (ESTs) encoding the putative ATP6 mRNA. .. First-strand cDNA was obtained using the SuperScript First Strand Synthesis System for reverse transcription-polymerase chain reaction (PCR) (Invitrogen, Carlsbad, CA).

    Article Title: PURIFICATION, CHARACTERIZATION AND CLONING OF A RICIN B-LIKE LECTIN FROM MUSHROOM Clitocybe nebularis WITH ANTIPROLIFERATIVE ACTIVITY AGAINST HUMAN LEUKEMIC T CELLS
    Article Snippet: First strand complementary DNA (cDNA) was synthesized from the total RNA by reverse transcription-polymerase chain reaction (PCR) using a GeneAmp RNA PCR Core Kit (Applied Biosystems, Foster city, CA) with anchored oligo(dT)-adapter primer dT(17)3’RACE ( , Supplementary data). .. Forward (CNL-D-1f) and reverse (CNL-D-1r) degenerate primers ( ) were designed based on the partial amino acid sequence in regions with the lowest degeneracy.

    Injection:

    Article Title: Paracrine Action of Mesenchymal Stem Cells Revealed by Single Cell Gene Profiling in Infarcted Murine Hearts
    Article Snippet: Green fluorescence visualized under laser microscopy was used as an indicator for micro-dissection. eGFP+ MSCs, para-eGFP+ cells, remote cells and cells from PBS injected hearts were independently captured by a LMD6000 system (Leica, Germany). .. Total RNA were extracted from these tissues, and 0.5 μg RNA was used in reverse transcription–polymerase chain reaction(PCR), followed by real-time PCR (Applied biosystems, CA, USA) to examine the expression level of paracrine factors.

    Molecular Weight:

    Article Title: PURIFICATION, CHARACTERIZATION AND CLONING OF A RICIN B-LIKE LECTIN FROM MUSHROOM Clitocybe nebularis WITH ANTIPROLIFERATIVE ACTIVITY AGAINST HUMAN LEUKEMIC T CELLS
    Article Snippet: High molecular weight genomic DNA was isolated from frozen powdered C. nebularis fruiting bodies as described [ ]. .. First strand complementary DNA (cDNA) was synthesized from the total RNA by reverse transcription-polymerase chain reaction (PCR) using a GeneAmp RNA PCR Core Kit (Applied Biosystems, Foster city, CA) with anchored oligo(dT)-adapter primer dT(17)3’RACE ( , Supplementary data).

    Molecular Cloning:

    Article Title: PURIFICATION, CHARACTERIZATION AND CLONING OF A RICIN B-LIKE LECTIN FROM MUSHROOM Clitocybe nebularis WITH ANTIPROLIFERATIVE ACTIVITY AGAINST HUMAN LEUKEMIC T CELLS
    Article Snippet: Paragraph title: 2.7. Molecular cloning of the gene and cDNA encoding CNL ... First strand complementary DNA (cDNA) was synthesized from the total RNA by reverse transcription-polymerase chain reaction (PCR) using a GeneAmp RNA PCR Core Kit (Applied Biosystems, Foster city, CA) with anchored oligo(dT)-adapter primer dT(17)3’RACE ( , Supplementary data).

    Fluorescence:

    Article Title: Paracrine Action of Mesenchymal Stem Cells Revealed by Single Cell Gene Profiling in Infarcted Murine Hearts
    Article Snippet: Green fluorescence visualized under laser microscopy was used as an indicator for micro-dissection. eGFP+ MSCs, para-eGFP+ cells, remote cells and cells from PBS injected hearts were independently captured by a LMD6000 system (Leica, Germany). .. Total RNA were extracted from these tissues, and 0.5 μg RNA was used in reverse transcription–polymerase chain reaction(PCR), followed by real-time PCR (Applied biosystems, CA, USA) to examine the expression level of paracrine factors.

    Mutagenesis:

    Article Title: Heterochromatin Protein 1? Epigenetically Regulates Cell Differentiation and Exhibits Potential as a Therapeutic Target for Various Types of Cancers
    Article Snippet: .. The cDNAs encoding full-length and deletion mutant forms (deleted at 1 to 213 nucleotides, ΔCD; and at 318 to 519 nucleotides, ΔCSD in the open reading frame) of the HP1γ gene were amplified by reverse transcription-polymerase chain reaction (PCR) using total RNA from HCT116 cells, and was subcloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA), forming the plasmid pCRHP1γ, pCRHPΔCSD, and pCRHPΔCD, respectively. .. The plasmids were then digested with Eco RI, and the Eco RI fragment containing the full-length and deletion mutants of HP1γ cDNA were subcloned into the Eco RI site of pGene-V5 (Invitrogen), forming the plasmid pGHP1γ, pGHPΔCSD, or pGHPΔCD, respectively.

    Isolation:

    Article Title: Epidermal growth factor-induced cyclooxygenase-2 enhances head and neck squamous cell carcinoma metastasis through fibronectin up-regulation
    Article Snippet: .. Reverse transcription–polymerase chain reaction Total RNA was isolated using the TRIzol RNA extraction kit (Invitrogen), and 2 μg of RNA was subjected to reverse transcription–polymerase chain reaction (PCR) with SuperScriptTM II (Invitrogen). ..

    Article Title: An Algal Nucleus-encoded Subunit of Mitochondrial ATP Synthase Rescues a Defect in the Analogous Human Mitochondrial-encoded Subunit
    Article Snippet: Paragraph title: Isolation of C. reinhardtii ATP6 ... First-strand cDNA was obtained using the SuperScript First Strand Synthesis System for reverse transcription-polymerase chain reaction (PCR) (Invitrogen, Carlsbad, CA).

    Article Title: PURIFICATION, CHARACTERIZATION AND CLONING OF A RICIN B-LIKE LECTIN FROM MUSHROOM Clitocybe nebularis WITH ANTIPROLIFERATIVE ACTIVITY AGAINST HUMAN LEUKEMIC T CELLS
    Article Snippet: Total RNA was extracted using RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol for isolation from plant tissues and filamentous fungi. .. First strand complementary DNA (cDNA) was synthesized from the total RNA by reverse transcription-polymerase chain reaction (PCR) using a GeneAmp RNA PCR Core Kit (Applied Biosystems, Foster city, CA) with anchored oligo(dT)-adapter primer dT(17)3’RACE ( , Supplementary data).

    Article Title: Pancreatic fibroblast growth factor 21 protects against type 2 diabetes in mice by promoting insulin expression and secretion in a PI3K/Akt signaling‐dependent manner, et al. Pancreatic fibroblast growth factor 21 protects against type 2 diabetes in mice by promoting insulin expression and secretion in a PI3K/Akt signaling‐dependent manner
    Article Snippet: Total RNA was extracted from tissue or isolated islets using TRIzol reagent (Invitrogen). cDNA was synthesized from 0.5 μg of total RNA using a supermix consisting of oligo (dT), random hexamer primers and reverse transcriptase (Invitrogen). .. Relative gene expression levels were determined by reverse transcription–polymerase chain reaction (PCR) (Applied Biosystems, Foster City, CA, USA) using SYBR Green with normalization to GAPDH.

    Article Title: The Toll-like receptor 2 (TLR2) ligand FSL-1 is internalized via the clathrin-dependent endocytic pathway triggered by CD14 and CD36 but not by TLR2
    Article Snippet: .. The complementary DNAs (cDNAs) of human CD14, CD36 and TLR2 were obtained as described previously., , Briefly, they were obtained by reverse transcription–polymerase chain reaction (PCR) of total RNA isolated from a human monocyte/macrophage cell line, THP-1 cells, and then they were cloned into a pEF6/V5-His TOPO vector (Invitrogen, Carlsbad, CA) or pcDNA3.1-His-TOPO vector (Invitrogen). .. Their transfection into wild-type HEK293 cells (HEK293WT) was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

    Article Title: Inhibitory Effects of Short-Chain Fatty Acids and ω-3 Polyunsaturated Fatty Acids on Profibrotic Factors in Dermal Fibroblasts
    Article Snippet: .. First-strand cDNAs were synthesized from the isolated RNAs with the First-Strand cDNA Synthesis System for reverse transcription-polymerase chain reaction (PCR) (Invitrogen). cDNAs were used for subsequent quantitative real-time PCR analysis using the SYBR Premix Ex Taq II (Takara Bio, Otsu, Japan) with appropriate primers ( ). .. The PCR reactions were run on an iCycler IQ (Bio-Rad, Hercules, Calif) for 40 cycles at 95°C for 30 seconds, at an annealing temperature ( ) for 30 seconds, and at 72°C for 30 seconds.

    Microscopy:

    Article Title: Paracrine Action of Mesenchymal Stem Cells Revealed by Single Cell Gene Profiling in Infarcted Murine Hearts
    Article Snippet: Green fluorescence visualized under laser microscopy was used as an indicator for micro-dissection. eGFP+ MSCs, para-eGFP+ cells, remote cells and cells from PBS injected hearts were independently captured by a LMD6000 system (Leica, Germany). .. Total RNA were extracted from these tissues, and 0.5 μg RNA was used in reverse transcription–polymerase chain reaction(PCR), followed by real-time PCR (Applied biosystems, CA, USA) to examine the expression level of paracrine factors.

    Mouse Assay:

    Article Title: Paracrine Action of Mesenchymal Stem Cells Revealed by Single Cell Gene Profiling in Infarcted Murine Hearts
    Article Snippet: Laser capture micro-dissection and qRT-PCR analysis Mice were euthanized and hearts were removed at day 5 after transplantation, embedded in optimal cutting temperature, and then frozen in liquid nitrogen. .. Total RNA were extracted from these tissues, and 0.5 μg RNA was used in reverse transcription–polymerase chain reaction(PCR), followed by real-time PCR (Applied biosystems, CA, USA) to examine the expression level of paracrine factors.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Aristolochic acid I determine the phenotype and activation of macrophages in acute and chronic kidney disease
    Article Snippet: .. RNA extraction, reverse transcription and qRT-PCR Pure Link RNA Mini Kit (Ambion, Germany) was used to extract total RNA from renal tissue stored in RNA later, according to the manufacturer’s instructions. cDNA was synthesized from 1 µg of total RNA by reverse transcription polymerase chain reaction (PCR) using Superscript II reverse transcriptase (Thermo Fisher, Germany) according to manufacturer instructions. .. Quantitative real-time PCR (qRT-PCR) from cDNA was performed a Light Cycler 480 (Roche, Germany).

    Article Title: FOXA1 potentiates lineage-specific enhancer activation through modulating TET1 expression and function
    Article Snippet: .. For FOXA1 and TET1 FL and domain constructs, human FOXA1 and TET1 cDNA were amplified by reverse transcription polymerase chain reaction (PCR) from LNCaP cells and pENTR223 TET1 (Harvard Plasmid), respectively, and cloned into the entry vector pCR8/GW/TOPO (Invitrogen). .. Adenoviral construct expressing FOXA1 was generated by recombining pCR8-FOXA1 with pAD/CMV/V5 using LR Clonase II (Invitrogen).

    Article Title: miR-375 inhibits the proliferation of gastric cancer cells by repressing ERBB2 expression
    Article Snippet: .. ERBB2 expression vector construction The full length coding region of human ERBB2 was amplified by reverse transcription polymerase chain reaction (PCR) and cloned into the pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA), which was then designated as pcDNA3.1-ERBB2. .. This vector and the control vector, pcDNA3.1, were transfected into cells using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions.

    Article Title: Heterochromatin Protein 1? Epigenetically Regulates Cell Differentiation and Exhibits Potential as a Therapeutic Target for Various Types of Cancers
    Article Snippet: .. The cDNAs encoding full-length and deletion mutant forms (deleted at 1 to 213 nucleotides, ΔCD; and at 318 to 519 nucleotides, ΔCSD in the open reading frame) of the HP1γ gene were amplified by reverse transcription-polymerase chain reaction (PCR) using total RNA from HCT116 cells, and was subcloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA), forming the plasmid pCRHP1γ, pCRHPΔCSD, and pCRHPΔCD, respectively. .. The plasmids were then digested with Eco RI, and the Eco RI fragment containing the full-length and deletion mutants of HP1γ cDNA were subcloned into the Eco RI site of pGene-V5 (Invitrogen), forming the plasmid pGHP1γ, pGHPΔCSD, or pGHPΔCD, respectively.

    Article Title: Epidermal growth factor-induced cyclooxygenase-2 enhances head and neck squamous cell carcinoma metastasis through fibronectin up-regulation
    Article Snippet: .. Reverse transcription–polymerase chain reaction Total RNA was isolated using the TRIzol RNA extraction kit (Invitrogen), and 2 μg of RNA was subjected to reverse transcription–polymerase chain reaction (PCR) with SuperScriptTM II (Invitrogen). ..

    Article Title: An Algal Nucleus-encoded Subunit of Mitochondrial ATP Synthase Rescues a Defect in the Analogous Human Mitochondrial-encoded Subunit
    Article Snippet: .. First-strand cDNA was obtained using the SuperScript First Strand Synthesis System for reverse transcription-polymerase chain reaction (PCR) (Invitrogen, Carlsbad, CA). .. The SMART RACE cDNA Amplification kit ( CLONTECH , Palo Alto, CA) was used for determination of the 5′- and 3′-untranslated regions (UTRs).

    Article Title: PURIFICATION, CHARACTERIZATION AND CLONING OF A RICIN B-LIKE LECTIN FROM MUSHROOM Clitocybe nebularis WITH ANTIPROLIFERATIVE ACTIVITY AGAINST HUMAN LEUKEMIC T CELLS
    Article Snippet: .. First strand complementary DNA (cDNA) was synthesized from the total RNA by reverse transcription-polymerase chain reaction (PCR) using a GeneAmp RNA PCR Core Kit (Applied Biosystems, Foster city, CA) with anchored oligo(dT)-adapter primer dT(17)3’RACE ( , Supplementary data). .. For PCR amplification of the partial cnl gene and cDNA sequences, genomic DNA and reverse transcript were used as templates.

    Article Title: Pancreatic fibroblast growth factor 21 protects against type 2 diabetes in mice by promoting insulin expression and secretion in a PI3K/Akt signaling‐dependent manner, et al. Pancreatic fibroblast growth factor 21 protects against type 2 diabetes in mice by promoting insulin expression and secretion in a PI3K/Akt signaling‐dependent manner
    Article Snippet: .. Relative gene expression levels were determined by reverse transcription–polymerase chain reaction (PCR) (Applied Biosystems, Foster City, CA, USA) using SYBR Green with normalization to GAPDH. .. A TUNEL assay was performed to detect apoptosis using a TMR green in situ cell death detection kit (Roche Applied Science), according to the manufacturer's instructions.

    Article Title: The Toll-like receptor 2 (TLR2) ligand FSL-1 is internalized via the clathrin-dependent endocytic pathway triggered by CD14 and CD36 but not by TLR2
    Article Snippet: .. The complementary DNAs (cDNAs) of human CD14, CD36 and TLR2 were obtained as described previously., , Briefly, they were obtained by reverse transcription–polymerase chain reaction (PCR) of total RNA isolated from a human monocyte/macrophage cell line, THP-1 cells, and then they were cloned into a pEF6/V5-His TOPO vector (Invitrogen, Carlsbad, CA) or pcDNA3.1-His-TOPO vector (Invitrogen). .. Their transfection into wild-type HEK293 cells (HEK293WT) was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

    Article Title: Inhibitory Effects of Short-Chain Fatty Acids and ω-3 Polyunsaturated Fatty Acids on Profibrotic Factors in Dermal Fibroblasts
    Article Snippet: .. First-strand cDNAs were synthesized from the isolated RNAs with the First-Strand cDNA Synthesis System for reverse transcription-polymerase chain reaction (PCR) (Invitrogen). cDNAs were used for subsequent quantitative real-time PCR analysis using the SYBR Premix Ex Taq II (Takara Bio, Otsu, Japan) with appropriate primers ( ). .. The PCR reactions were run on an iCycler IQ (Bio-Rad, Hercules, Calif) for 40 cycles at 95°C for 30 seconds, at an annealing temperature ( ) for 30 seconds, and at 72°C for 30 seconds.

    Quantitative RT-PCR:

    Article Title: Paracrine Action of Mesenchymal Stem Cells Revealed by Single Cell Gene Profiling in Infarcted Murine Hearts
    Article Snippet: Paragraph title: Laser capture micro-dissection and qRT-PCR analysis ... Total RNA were extracted from these tissues, and 0.5 μg RNA was used in reverse transcription–polymerase chain reaction(PCR), followed by real-time PCR (Applied biosystems, CA, USA) to examine the expression level of paracrine factors.

    Article Title: Aristolochic acid I determine the phenotype and activation of macrophages in acute and chronic kidney disease
    Article Snippet: .. RNA extraction, reverse transcription and qRT-PCR Pure Link RNA Mini Kit (Ambion, Germany) was used to extract total RNA from renal tissue stored in RNA later, according to the manufacturer’s instructions. cDNA was synthesized from 1 µg of total RNA by reverse transcription polymerase chain reaction (PCR) using Superscript II reverse transcriptase (Thermo Fisher, Germany) according to manufacturer instructions. .. Quantitative real-time PCR (qRT-PCR) from cDNA was performed a Light Cycler 480 (Roche, Germany).

    Lysis:

    Article Title: Paracrine Action of Mesenchymal Stem Cells Revealed by Single Cell Gene Profiling in Infarcted Murine Hearts
    Article Snippet: The dissected tissues were placed on the caps of micro-centrifuge tubes with 50μl lysis enhanced buffer. .. Total RNA were extracted from these tissues, and 0.5 μg RNA was used in reverse transcription–polymerase chain reaction(PCR), followed by real-time PCR (Applied biosystems, CA, USA) to examine the expression level of paracrine factors.

    Plasmid Preparation:

    Article Title: FOXA1 potentiates lineage-specific enhancer activation through modulating TET1 expression and function
    Article Snippet: .. For FOXA1 and TET1 FL and domain constructs, human FOXA1 and TET1 cDNA were amplified by reverse transcription polymerase chain reaction (PCR) from LNCaP cells and pENTR223 TET1 (Harvard Plasmid), respectively, and cloned into the entry vector pCR8/GW/TOPO (Invitrogen). .. Adenoviral construct expressing FOXA1 was generated by recombining pCR8-FOXA1 with pAD/CMV/V5 using LR Clonase II (Invitrogen).

    Article Title: miR-375 inhibits the proliferation of gastric cancer cells by repressing ERBB2 expression
    Article Snippet: .. ERBB2 expression vector construction The full length coding region of human ERBB2 was amplified by reverse transcription polymerase chain reaction (PCR) and cloned into the pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA), which was then designated as pcDNA3.1-ERBB2. .. This vector and the control vector, pcDNA3.1, were transfected into cells using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions.

    Article Title: Heterochromatin Protein 1? Epigenetically Regulates Cell Differentiation and Exhibits Potential as a Therapeutic Target for Various Types of Cancers
    Article Snippet: .. The cDNAs encoding full-length and deletion mutant forms (deleted at 1 to 213 nucleotides, ΔCD; and at 318 to 519 nucleotides, ΔCSD in the open reading frame) of the HP1γ gene were amplified by reverse transcription-polymerase chain reaction (PCR) using total RNA from HCT116 cells, and was subcloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA), forming the plasmid pCRHP1γ, pCRHPΔCSD, and pCRHPΔCD, respectively. .. The plasmids were then digested with Eco RI, and the Eco RI fragment containing the full-length and deletion mutants of HP1γ cDNA were subcloned into the Eco RI site of pGene-V5 (Invitrogen), forming the plasmid pGHP1γ, pGHPΔCSD, or pGHPΔCD, respectively.

    Article Title: An Algal Nucleus-encoded Subunit of Mitochondrial ATP Synthase Rescues a Defect in the Analogous Human Mitochondrial-encoded Subunit
    Article Snippet: First-strand cDNA was obtained using the SuperScript First Strand Synthesis System for reverse transcription-polymerase chain reaction (PCR) (Invitrogen, Carlsbad, CA). .. Amplified PCR products were inserted into pCRII-TOPO Vector (Invitrogen) for sequencing.

    Article Title: The Toll-like receptor 2 (TLR2) ligand FSL-1 is internalized via the clathrin-dependent endocytic pathway triggered by CD14 and CD36 but not by TLR2
    Article Snippet: .. The complementary DNAs (cDNAs) of human CD14, CD36 and TLR2 were obtained as described previously., , Briefly, they were obtained by reverse transcription–polymerase chain reaction (PCR) of total RNA isolated from a human monocyte/macrophage cell line, THP-1 cells, and then they were cloned into a pEF6/V5-His TOPO vector (Invitrogen, Carlsbad, CA) or pcDNA3.1-His-TOPO vector (Invitrogen). .. Their transfection into wild-type HEK293 cells (HEK293WT) was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions.

    SYBR Green Assay:

    Article Title: Pancreatic fibroblast growth factor 21 protects against type 2 diabetes in mice by promoting insulin expression and secretion in a PI3K/Akt signaling‐dependent manner, et al. Pancreatic fibroblast growth factor 21 protects against type 2 diabetes in mice by promoting insulin expression and secretion in a PI3K/Akt signaling‐dependent manner
    Article Snippet: .. Relative gene expression levels were determined by reverse transcription–polymerase chain reaction (PCR) (Applied Biosystems, Foster City, CA, USA) using SYBR Green with normalization to GAPDH. .. A TUNEL assay was performed to detect apoptosis using a TMR green in situ cell death detection kit (Roche Applied Science), according to the manufacturer's instructions.

    RNA Extraction:

    Article Title: Aristolochic acid I determine the phenotype and activation of macrophages in acute and chronic kidney disease
    Article Snippet: .. RNA extraction, reverse transcription and qRT-PCR Pure Link RNA Mini Kit (Ambion, Germany) was used to extract total RNA from renal tissue stored in RNA later, according to the manufacturer’s instructions. cDNA was synthesized from 1 µg of total RNA by reverse transcription polymerase chain reaction (PCR) using Superscript II reverse transcriptase (Thermo Fisher, Germany) according to manufacturer instructions. .. Quantitative real-time PCR (qRT-PCR) from cDNA was performed a Light Cycler 480 (Roche, Germany).

    Article Title: Epidermal growth factor-induced cyclooxygenase-2 enhances head and neck squamous cell carcinoma metastasis through fibronectin up-regulation
    Article Snippet: .. Reverse transcription–polymerase chain reaction Total RNA was isolated using the TRIzol RNA extraction kit (Invitrogen), and 2 μg of RNA was subjected to reverse transcription–polymerase chain reaction (PCR) with SuperScriptTM II (Invitrogen). ..

    Agarose Gel Electrophoresis:

    Article Title: Epidermal growth factor-induced cyclooxygenase-2 enhances head and neck squamous cell carcinoma metastasis through fibronectin up-regulation
    Article Snippet: Reverse transcription–polymerase chain reaction Total RNA was isolated using the TRIzol RNA extraction kit (Invitrogen), and 2 μg of RNA was subjected to reverse transcription–polymerase chain reaction (PCR) with SuperScriptTM II (Invitrogen). .. The PCR products were separated by 1% agarose gel electrophoresis and visualized with ethidium bromide staining.

    Staining:

    Article Title: Epidermal growth factor-induced cyclooxygenase-2 enhances head and neck squamous cell carcinoma metastasis through fibronectin up-regulation
    Article Snippet: Reverse transcription–polymerase chain reaction Total RNA was isolated using the TRIzol RNA extraction kit (Invitrogen), and 2 μg of RNA was subjected to reverse transcription–polymerase chain reaction (PCR) with SuperScriptTM II (Invitrogen). .. The PCR products were separated by 1% agarose gel electrophoresis and visualized with ethidium bromide staining.

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  • 99
    Thermo Fisher dna digestion mix
    Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary <t>DNA</t> (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease <t>(DNase</t> I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)
    Dna Digestion Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher transcriptome
    Maternal genotype and prenatal stress affect the <t>transcriptome</t> of developing embryos. ( A ) Venn diagram showing overlap between differentially expressed genes (DEGs) in wild-type (WT) and maternal Slc6a4 +/− (SERT) embryos in response to stress. ( B ) Validation of subset of DEGs identified by RNA-seq using qPCR correlated with fold change from RNA-seq data (Pearson R = 0.995, p
    Transcriptome, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 195 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher single embryo transcriptome sequencing
    Reduced Mat2a mRNA Levels and Embryonic Lethality around Implantation Stage in the Mettl16 Mutant Mice (A) Generation of a Mettl16 . C–S4F. (C) Genotyping of E2.5 embryos from Mettl16 +/− x Mettl16 +/− crosses confirmed the expected Mendelian ratios among the genotypes. Scale bar in μm is indicated. (D) <t>Transcriptome</t> of individual isolated E2.5 embryos of Mettl16 −/− (KO), Mettl16 +/− (HET), and Mettl16 +/+ . (E) Heatmap shows the expression of genes with significant differential expression between any two genotypes (adjusted p ≤ 0.1). Genes differentially expressed in Mettl16 −/− (KO) when compared to both Mettl16 +/− (HET) and Mettl16 +/+ (WT) are marked by red arrowhead. (F) The boxplots show the expected downregulation of the targeted gene ( Mettl16 ) in KO samples, as well as the downregulation of Mat2a C. (G) Normalized read coverage along the Mat2a locus demonstrates the overall depletion in the KO. Note that the gene is on the Crick strand, so it goes from right to left. (H) Lack of METTL16 results in aberrant splicing of the last intron. The reads spanning the splice junction (SJ) of last Mat2a (ENSMUST00000059472.9) intron are significantly depleted in the KO even when normalized to overall Mat2a D.
    Single Embryo Transcriptome Sequencing, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)

    Journal: Nature Communications

    Article Title: Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs

    doi: 10.1038/s41467-018-02866-0

    Figure Lengend Snippet: Overview of RT-RamDA and single-cell RamDA-seq. a Schematic diagram of RT-RamDA. 1. RT primers (oligo-dT and not-so-random primers) anneal to a RNA template. 2. Complementary DNA (cDNA) is synthesized by the RNA-dependent DNA polymerase activity of RNase H minus reverse transcriptase (RTase). 3. Endonuclease (DNase I) selectively nicks the cDNA of the RNA:cDNA hybrid strand. 4. The 3′ cDNA strand is displaced by the strand displacement activity of RTase mediated by the T4 gene 32 protein (gp32), starting from the nick randomly introduced by DNase I. cDNA is amplified as a displaced strand and protected by gp32 from DNase I. b Relative yield of cDNA molecules using RT-qPCR ( n = 4). Mouse ESC total RNA (10 pg) was used as a template, and 1/10 the amount of cDNA was used for qPCR. The relative yield was calculated by averaging the amplification efficiency of four mESC ( Nanog , Pou5f1 , Zfp42 , and Sox2 ) and three housekeeping ( Gnb2l1 , Atp5a1 , and Tubb5 ) genes using a conventional method (−) as a standard. c Schematic diagram of RamDA-seq and C1-RamDA-seq. For details, please refer to the Methods section. d Number of detected transcripts with twofold or lower expression changes against rdRNA-seq (count ≥ 10). For the boxplots in b and d , the center line, and lower and upper bounds of each box represent the median, and first and third quartiles, respectively. The lower (upper) whisker extends to smallest (largest) values no further than 1.5 × interquartile range (IQR) from the first (third) quartile. e Squared coefficient of variation of the read count. All conditions were adjusted, and 10 million reads were used in d and e . Transcripts were annotated by GENCODE gene annotation (vM9)

    Article Snippet: To eliminate genomic DNA contamination, 1 μL of genomic DNA digestion mix (0.5× PrimeScript Buffer, 0.2 U of DNase I Amplification Grade, 1: 5 000 000 ERCC RNA Spike-In Mix I (Thermo Fisher) in RNase-free water) was added to 1 μL of the denatured sample.

    Techniques: Synthesized, Activity Assay, Amplification, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Expressing, Whisker Assay

    Maternal genotype and prenatal stress affect the transcriptome of developing embryos. ( A ) Venn diagram showing overlap between differentially expressed genes (DEGs) in wild-type (WT) and maternal Slc6a4 +/− (SERT) embryos in response to stress. ( B ) Validation of subset of DEGs identified by RNA-seq using qPCR correlated with fold change from RNA-seq data (Pearson R = 0.995, p

    Journal: Scientific Reports

    Article Title: Interplay between maternal Slc6a4 mutation and prenatal stress: a possible mechanism for autistic behavior development

    doi: 10.1038/s41598-017-07405-3

    Figure Lengend Snippet: Maternal genotype and prenatal stress affect the transcriptome of developing embryos. ( A ) Venn diagram showing overlap between differentially expressed genes (DEGs) in wild-type (WT) and maternal Slc6a4 +/− (SERT) embryos in response to stress. ( B ) Validation of subset of DEGs identified by RNA-seq using qPCR correlated with fold change from RNA-seq data (Pearson R = 0.995, p

    Article Snippet: Library preparation and sequencing Nucleic acids were collected from placenta and embryo brains and used as the template for methylome, transcriptome and miRNA library construction, followed by sequencing on the Ion Proton System (Thermofisher Scientific, Carlsbad, CA).

    Techniques: RNA Sequencing Assay, Real-time Polymerase Chain Reaction

    DMF and MMF induced DNMT-mediated methylation-silencing of GSDMD gene. ( A ) Whole transcriptome analysis data showed increase in DNMT3A and DNMT3B in DMF- or MMF-treated NK92 cells, compared to control cells. ( B ) Western blotting data also showed an increase in the expression of DNMT3A and DNMT3B in DMF or MMF treated cells but not in LPS treated cells. ( C ) DMF and MMF increased the expression of DNMT3A and DNMT3B in cells incubated with LPS. ( D ) Schematic representation of the presence of CpG Islands in the promoter region of GSDMD gene and repression of expression is observed upon methylation of those CGIs. ( E ) Data from MSP-PCR showed the methylation levels of GSDMD in cells either untreated (Control), or treated with LPS, or LPS plus DMF or MMF in the presence of IL-2 for 24 h. *P

    Journal: ImmunoTargets and Therapy

    Article Title: Gasdermin D Hypermethylation Inhibits Pyroptosis And LPS-Induced IL-1β Release From NK92 Cells

    doi: 10.2147/ITT.S219867

    Figure Lengend Snippet: DMF and MMF induced DNMT-mediated methylation-silencing of GSDMD gene. ( A ) Whole transcriptome analysis data showed increase in DNMT3A and DNMT3B in DMF- or MMF-treated NK92 cells, compared to control cells. ( B ) Western blotting data also showed an increase in the expression of DNMT3A and DNMT3B in DMF or MMF treated cells but not in LPS treated cells. ( C ) DMF and MMF increased the expression of DNMT3A and DNMT3B in cells incubated with LPS. ( D ) Schematic representation of the presence of CpG Islands in the promoter region of GSDMD gene and repression of expression is observed upon methylation of those CGIs. ( E ) Data from MSP-PCR showed the methylation levels of GSDMD in cells either untreated (Control), or treated with LPS, or LPS plus DMF or MMF in the presence of IL-2 for 24 h. *P

    Article Snippet: Whole Transcriptome Analysis RNA extracted from NK92 cells incubated with either 100 μM DMF, 100 μM MMF, DMSO, or 10 μg/mL LPS were analyzed for whole transcriptome profiling using targeted whole RNA-seq with AmpliSeq whole transcriptome on S5 system (Thermo Fisher Scientific).

    Techniques: Methylation, Western Blot, Expressing, Incubation, Polymerase Chain Reaction

    Reduced Mat2a mRNA Levels and Embryonic Lethality around Implantation Stage in the Mettl16 Mutant Mice (A) Generation of a Mettl16 . C–S4F. (C) Genotyping of E2.5 embryos from Mettl16 +/− x Mettl16 +/− crosses confirmed the expected Mendelian ratios among the genotypes. Scale bar in μm is indicated. (D) Transcriptome of individual isolated E2.5 embryos of Mettl16 −/− (KO), Mettl16 +/− (HET), and Mettl16 +/+ . (E) Heatmap shows the expression of genes with significant differential expression between any two genotypes (adjusted p ≤ 0.1). Genes differentially expressed in Mettl16 −/− (KO) when compared to both Mettl16 +/− (HET) and Mettl16 +/+ (WT) are marked by red arrowhead. (F) The boxplots show the expected downregulation of the targeted gene ( Mettl16 ) in KO samples, as well as the downregulation of Mat2a C. (G) Normalized read coverage along the Mat2a locus demonstrates the overall depletion in the KO. Note that the gene is on the Crick strand, so it goes from right to left. (H) Lack of METTL16 results in aberrant splicing of the last intron. The reads spanning the splice junction (SJ) of last Mat2a (ENSMUST00000059472.9) intron are significantly depleted in the KO even when normalized to overall Mat2a D.

    Journal: Molecular Cell

    Article Title: Methylation of Structured RNA by the m6A Writer METTL16 Is Essential for Mouse Embryonic Development

    doi: 10.1016/j.molcel.2018.08.004

    Figure Lengend Snippet: Reduced Mat2a mRNA Levels and Embryonic Lethality around Implantation Stage in the Mettl16 Mutant Mice (A) Generation of a Mettl16 . C–S4F. (C) Genotyping of E2.5 embryos from Mettl16 +/− x Mettl16 +/− crosses confirmed the expected Mendelian ratios among the genotypes. Scale bar in μm is indicated. (D) Transcriptome of individual isolated E2.5 embryos of Mettl16 −/− (KO), Mettl16 +/− (HET), and Mettl16 +/+ . (E) Heatmap shows the expression of genes with significant differential expression between any two genotypes (adjusted p ≤ 0.1). Genes differentially expressed in Mettl16 −/− (KO) when compared to both Mettl16 +/− (HET) and Mettl16 +/+ (WT) are marked by red arrowhead. (F) The boxplots show the expected downregulation of the targeted gene ( Mettl16 ) in KO samples, as well as the downregulation of Mat2a C. (G) Normalized read coverage along the Mat2a locus demonstrates the overall depletion in the KO. Note that the gene is on the Crick strand, so it goes from right to left. (H) Lack of METTL16 results in aberrant splicing of the last intron. The reads spanning the splice junction (SJ) of last Mat2a (ENSMUST00000059472.9) intron are significantly depleted in the KO even when normalized to overall Mat2a D.

    Article Snippet: For single-embryo transcriptome sequencing, the isolated E2.5 and E3.5 embryos were visually examined for viability and cell number, and transferred separately into single tubes of 0.2 mL thin-walled 8-tube PCR strips (Thermo, AB-0451).

    Techniques: Mutagenesis, Mouse Assay, Isolation, Expressing

    E3.5 Mettl16 −/− Blastocysts Display Normal Morphology but Vast Transcriptome Dysregulation (A) E3.5 Mettl16 −/− KO embryos display normal morphology and their counts from Mettl16 +/− x Mettl16 +/− crosses correspond to expected Mendelian ratios among the genotypes. Scale bar in μm is indicated. (B) The boxplots show the expected downregulation of the targeted gene ( Mettl16 ) in KO samples, as well as the downregulation of Mat2a A. (C) MA plots comparing the expression between the genotypes reveal that the vast number of genes are dysregulated in the Mettl16 −/− KO embryos. The genes with significantly different expression are shown as red dots (adjusted p ≤ 0.1). B–S6D. (E) Venn diagrams compare the lists of dysregulated genes when Mettl16 −/− expression is compared to Mettl16 +/− or to Mettl16 +/+ . (F) Comparison of proportion of reads encompassing splice junctions does not reveal a difference in splicing between individual genotypes. (G) Global transcription from exons, introns, and repeats is not affected in Mettl16 −/− . Error bars refer to SD.

    Journal: Molecular Cell

    Article Title: Methylation of Structured RNA by the m6A Writer METTL16 Is Essential for Mouse Embryonic Development

    doi: 10.1016/j.molcel.2018.08.004

    Figure Lengend Snippet: E3.5 Mettl16 −/− Blastocysts Display Normal Morphology but Vast Transcriptome Dysregulation (A) E3.5 Mettl16 −/− KO embryos display normal morphology and their counts from Mettl16 +/− x Mettl16 +/− crosses correspond to expected Mendelian ratios among the genotypes. Scale bar in μm is indicated. (B) The boxplots show the expected downregulation of the targeted gene ( Mettl16 ) in KO samples, as well as the downregulation of Mat2a A. (C) MA plots comparing the expression between the genotypes reveal that the vast number of genes are dysregulated in the Mettl16 −/− KO embryos. The genes with significantly different expression are shown as red dots (adjusted p ≤ 0.1). B–S6D. (E) Venn diagrams compare the lists of dysregulated genes when Mettl16 −/− expression is compared to Mettl16 +/− or to Mettl16 +/+ . (F) Comparison of proportion of reads encompassing splice junctions does not reveal a difference in splicing between individual genotypes. (G) Global transcription from exons, introns, and repeats is not affected in Mettl16 −/− . Error bars refer to SD.

    Article Snippet: For single-embryo transcriptome sequencing, the isolated E2.5 and E3.5 embryos were visually examined for viability and cell number, and transferred separately into single tubes of 0.2 mL thin-walled 8-tube PCR strips (Thermo, AB-0451).

    Techniques: Expressing