lysis buffer  (Qiagen)


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    Structured Review

    Qiagen lysis buffer
    Lysis Buffer, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1734 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lysis buffer/product/Qiagen
    Average 99 stars, based on 1734 article reviews
    Price from $9.99 to $1999.99
    lysis buffer - by Bioz Stars, 2020-04
    99/100 stars

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    Related Articles

    Centrifugation:

    Article Title: Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1 [C]Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1 [C] [W]
    Article Snippet: After 12 h of growth, cells were collected by centrifugation, crushed with liquid nitrogen using a pestle and mortar, and resuspended in lysis buffer (50 m m Na2 PO4 , 300 m m sodium chloride, 20 m m imidazole, pH 8.0 with 0.1 m m phenylmethylsulfonyl fluoride and protease inhibitor cocktail; Sigma-Aldrich). .. After dialysis for 12 h in lysis buffer, poly(His)-tagged proteins were purified using nickel-nitrilotriacetic acid agarose beads according to the manufacturer’s protocol (Qiagen).

    Amplification:

    Article Title: Reprogramming of CaCo2 colorectal cancer cells after using the complex of poly-(N-vinylpyrrolidone) with small non-coding RNAs
    Article Snippet: .. One portion of the cells were removed at 11, 21 and 30 days, treated with a lysis buffer from an RNAeasy mini kit (Qiagen, USA), and frozen at -20 °C for further total RNA isolation, reverse transcription reaction, and specific cDNA transcript amplification. .. Another portion of the cells was used for staining using the Leishman-Romanowsky method.

    Synthesized:

    Article Title: The CREB coactivator CRTC2 promotes oncogenesis in LKB1-mutant non–small cell lung cancer
    Article Snippet: .. Real-time qRT-PCR and RNA-seq Cultured cells were disrupted in lysis buffer from the RNeasy Mini Kit (Qiagen), and mRNA was purified following the manufacturer’s instructions. cDNA was synthesized using the Transcriptor First Strand cDNA Synthesis Kit (Roche). .. Quantitative polymerase chain reaction real-time qRT-PCR was carried out with diluted cDNA, appropriate primers, and LightCycler 480 SYBR Green I Master (Roche) on a Lightcycler 480 instrument (Roche).

    Quantitative RT-PCR:

    Article Title: The CREB coactivator CRTC2 promotes oncogenesis in LKB1-mutant non–small cell lung cancer
    Article Snippet: .. Real-time qRT-PCR and RNA-seq Cultured cells were disrupted in lysis buffer from the RNeasy Mini Kit (Qiagen), and mRNA was purified following the manufacturer’s instructions. cDNA was synthesized using the Transcriptor First Strand cDNA Synthesis Kit (Roche). .. Quantitative polymerase chain reaction real-time qRT-PCR was carried out with diluted cDNA, appropriate primers, and LightCycler 480 SYBR Green I Master (Roche) on a Lightcycler 480 instrument (Roche).

    Article Title: Multi‐omics identify xanthine as a pro‐survival metabolite for nematodes with mitochondrial dysfunction
    Article Snippet: Paragraph title: qRT–PCR ... Pellets were then pestle‐homogenized in the lysis buffer of the RNA Extraction Kit (RNeasy, Qiagen).

    Real-time Polymerase Chain Reaction:

    Article Title: Differential Responses of Bovine Monocyte-Derived Macrophages to Infection by Neospora caninum Isolates of High and Low Virulence
    Article Snippet: Proliferation Assays Proliferation kinetics of Nc-Spain7 and Nc-Spain1H isolates in MØs were determined by quantifying the number of tachyzoites at specific times (8, 24, 36, 48, 60, and 72 hpi) using quantitative real-time PCR (qPCR). .. Samples were collected by adding 200 μl of PBS, 180 μl of lysis buffer, and 20 μl of proteinase K (Qiagen, Germany) to each well and were stored at −80°C until DNA extraction.

    Article Title: Rapid antibiotic susceptibility testing from blood culture bottles with species agnostic real-time polymerase chain reaction
    Article Snippet: Paragraph title: Assessment of antibiotic susceptibility using qPCR ... Pellets were resuspended in 200 μL lysis buffer (10 mM Tris-HCl, 1 mM disodium EDTA, pH 8.0, 20 mg/mL lysozyme, 300 U/mL mutanolysin, 1.2% Triton X-100) and incubated at 37°C with shaking at 1,200 rpm in a ThermoMixer for 20 min. Glass beads (0.1 mm PowerBeads; Qiagen, Hilden, Germany) were then added to the samples to an approximate volume of 20 μL and the samples were vortexed on the fastest setting for 5 min. Buffers and spin columns from a QIaAMP DNA Mini Kit (Qiagen) were then used to complete the DNA extraction.

    Article Title: The CREB coactivator CRTC2 promotes oncogenesis in LKB1-mutant non–small cell lung cancer
    Article Snippet: Real-time qRT-PCR and RNA-seq Cultured cells were disrupted in lysis buffer from the RNeasy Mini Kit (Qiagen), and mRNA was purified following the manufacturer’s instructions. cDNA was synthesized using the Transcriptor First Strand cDNA Synthesis Kit (Roche). .. Quantitative polymerase chain reaction real-time qRT-PCR was carried out with diluted cDNA, appropriate primers, and LightCycler 480 SYBR Green I Master (Roche) on a Lightcycler 480 instrument (Roche).

    Article Title: Multi‐omics identify xanthine as a pro‐survival metabolite for nematodes with mitochondrial dysfunction
    Article Snippet: Pellets were then pestle‐homogenized in the lysis buffer of the RNA Extraction Kit (RNeasy, Qiagen). .. Each set of primers was verified through a two‐step protocol that included (i) testing of nine combinations of primer concentrations against 10 ng of wt cDNA, choosing the combination which gave the lowest C T value and only the expected product when the qPCR was run on a 2% agarose gel, and (ii) the linearity of C T values given by the primer combination identified in step (i) with a serial dilution of cDNA ranging from 10 to 0.31 ng.

    Microarray:

    Article Title: 1,25-Dihydroxyvitamin D3 affects gastric cancer progression by repressing BMP3 promoter methylation
    Article Snippet: Paragraph title: DNA extraction, DNA methylation microarray, and analysis ... Then, gastric tissues were dissolved in 200 µL of lysis buffer from the DNA Micro Kit (catalog no. 56304; Qiagen NV, Venlo, the Netherlands) and incubated with proteinase K overnight at 56°C for two nights.

    Incubation:

    Article Title: Nanocomplexes of Graphene Oxide and Platinum Nanoparticles against Colorectal Cancer Colo205, HT-29, HTC-116, SW480, Liver Cancer HepG2, Human Breast Cancer MCF-7, and Adenocarcinoma LNCaP and Human Cervical Hela B Cell Lines
    Article Snippet: Isolation of Total RNA For the isolation of total RNA, MCF-7, Colo205, and HepG2 cells (1 × 105 cells per well) were incubated for 24 h. Subsequently, the medium was removed, and GO (100 µg/mL), NP-Pt (25 µg/mL), and GO-NP-Pt (GO100 :Pt25 µg/mL) in the culture medium were added to the cells and incubated for an additional 24 h. Total RNA was isolated using a PureLink® RNA Mini Kit (Ambion™ Life Technologies, Foster City, CA, USA). .. The resulting cell pellet was resuspended in lysis buffer containing 1% 2-mercaptoethanol, and subsequently, the frozen metal balls were added to the probe and homogenized in a TissueLyser ball mill (Qiagen, Germantown, MD, USA) for 5 min at 50 Hz.

    Article Title: Rapid antibiotic susceptibility testing from blood culture bottles with species agnostic real-time polymerase chain reaction
    Article Snippet: .. Pellets were resuspended in 200 μL lysis buffer (10 mM Tris-HCl, 1 mM disodium EDTA, pH 8.0, 20 mg/mL lysozyme, 300 U/mL mutanolysin, 1.2% Triton X-100) and incubated at 37°C with shaking at 1,200 rpm in a ThermoMixer for 20 min. Glass beads (0.1 mm PowerBeads; Qiagen, Hilden, Germany) were then added to the samples to an approximate volume of 20 μL and the samples were vortexed on the fastest setting for 5 min. Buffers and spin columns from a QIaAMP DNA Mini Kit (Qiagen) were then used to complete the DNA extraction. ..

    Article Title: 1,25-Dihydroxyvitamin D3 affects gastric cancer progression by repressing BMP3 promoter methylation
    Article Snippet: .. Then, gastric tissues were dissolved in 200 µL of lysis buffer from the DNA Micro Kit (catalog no. 56304; Qiagen NV, Venlo, the Netherlands) and incubated with proteinase K overnight at 56°C for two nights. .. DNA was extracted according to the manufacturer’s protocol (QIAamp DNA Micro Kit; Qiagen NV) and stored at −80°C to prevent degradation.

    Article Title: Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1 [C]Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1 [C] [W]
    Article Snippet: Cells were then washed twice with MilliQ water, resuspended in synthetic complete medium lacking uracil, containing 2% (w/v) Gal and 1% (w/v) raffinose, and incubated at 30°C for protein induction. .. After dialysis for 12 h in lysis buffer, poly(His)-tagged proteins were purified using nickel-nitrilotriacetic acid agarose beads according to the manufacturer’s protocol (Qiagen).

    Formalin-fixed Paraffin-Embedded:

    Article Title: 1,25-Dihydroxyvitamin D3 affects gastric cancer progression by repressing BMP3 promoter methylation
    Article Snippet: Before DNA extraction, gastric specimens were formalin-fixed, paraffin-embedded and microscopically examined using H & E staining. .. Then, gastric tissues were dissolved in 200 µL of lysis buffer from the DNA Micro Kit (catalog no. 56304; Qiagen NV, Venlo, the Netherlands) and incubated with proteinase K overnight at 56°C for two nights.

    Expressing:

    Article Title: Tsg101, an Inactive Homologue of Ubiquitin Ligase E2, Interacts Specifically with Human Immunodeficiency Virus Type 2 Gag Polyprotein and Results in Increased Levels of Ubiquitinated Gag
    Article Snippet: pH6 M-Ub was introduced into 293T cells by cotransfection with plasmids expressing HIV-2 Gag and Tsg101. .. The remaining cells were resuspended in lysis buffer (6 M guanidine hydrochloride, 100 mM NaH2 PO4 , 20 mM imidazole, 10 mM Tris-Cl [pH 8]), sonicated for 1 min on ice, and then rotated at room temperature for 1 h. The His-tagged proteins were purified on Ni-nitrotriacetate (NTA) spin columns (Qiagen), washed four times with 600 μl of wash buffer (8 M urea, 100 mM NaH2 PO4 , 20 mM imidazole, 10 mM Tris-Cl [pH 6.2]), and eluted once with 200 μl of elution buffer (8 M urea, 100 mM NaH2 PO4 , 20 mM imidazole, 10 mM Tris-Cl [pH 4.0]) and once with 200 μl of elution buffer containing 250 mM imidazole.

    Article Title: Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1 [C]Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1 [C] [W]
    Article Snippet: Paragraph title: Yeast Expression, Protein Purification, and Analysis of Yeast Extracts ... After dialysis for 12 h in lysis buffer, poly(His)-tagged proteins were purified using nickel-nitrilotriacetic acid agarose beads according to the manufacturer’s protocol (Qiagen).

    Modification:

    Article Title: 1,25-Dihydroxyvitamin D3 affects gastric cancer progression by repressing BMP3 promoter methylation
    Article Snippet: Then, gastric tissues were dissolved in 200 µL of lysis buffer from the DNA Micro Kit (catalog no. 56304; Qiagen NV, Venlo, the Netherlands) and incubated with proteinase K overnight at 56°C for two nights. .. Bisulfite modification of 500 ng of DNA from each sample was performed using the EZ DNA Methylation Kit (Zymo Research, Orange, CA, USA).

    Western Blot:

    Article Title: Tsg101, an Inactive Homologue of Ubiquitin Ligase E2, Interacts Specifically with Human Immunodeficiency Virus Type 2 Gag Polyprotein and Results in Increased Levels of Ubiquitinated Gag
    Article Snippet: At 24 h later, the transfected cultures were treated with MG132 (2 μM) for an additional 16 h. The cells were harvested, and 10% of the cells were resuspended in SDS-PAGE loading buffer and analyzed by Western blotting, using anti-26 monoclonal antibody, to confirm that equivalent levels of Gag protein were expressed in each sample. .. The remaining cells were resuspended in lysis buffer (6 M guanidine hydrochloride, 100 mM NaH2 PO4 , 20 mM imidazole, 10 mM Tris-Cl [pH 8]), sonicated for 1 min on ice, and then rotated at room temperature for 1 h. The His-tagged proteins were purified on Ni-nitrotriacetate (NTA) spin columns (Qiagen), washed four times with 600 μl of wash buffer (8 M urea, 100 mM NaH2 PO4 , 20 mM imidazole, 10 mM Tris-Cl [pH 6.2]), and eluted once with 200 μl of elution buffer (8 M urea, 100 mM NaH2 PO4 , 20 mM imidazole, 10 mM Tris-Cl [pH 4.0]) and once with 200 μl of elution buffer containing 250 mM imidazole.

    Transformation Assay:

    Article Title: Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1 [C]Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1 [C] [W]
    Article Snippet: After verifying the integrity of the gene through sequencing, expression plasmids were transformed into S. cerevisiae (strain BY4741) following the standard lithium acetate method. .. After dialysis for 12 h in lysis buffer, poly(His)-tagged proteins were purified using nickel-nitrilotriacetic acid agarose beads according to the manufacturer’s protocol (Qiagen).

    Transfection:

    Article Title: Tsg101, an Inactive Homologue of Ubiquitin Ligase E2, Interacts Specifically with Human Immunodeficiency Virus Type 2 Gag Polyprotein and Results in Increased Levels of Ubiquitinated Gag
    Article Snippet: At 24 h later, the transfected cultures were treated with MG132 (2 μM) for an additional 16 h. The cells were harvested, and 10% of the cells were resuspended in SDS-PAGE loading buffer and analyzed by Western blotting, using anti-26 monoclonal antibody, to confirm that equivalent levels of Gag protein were expressed in each sample. .. The remaining cells were resuspended in lysis buffer (6 M guanidine hydrochloride, 100 mM NaH2 PO4 , 20 mM imidazole, 10 mM Tris-Cl [pH 8]), sonicated for 1 min on ice, and then rotated at room temperature for 1 h. The His-tagged proteins were purified on Ni-nitrotriacetate (NTA) spin columns (Qiagen), washed four times with 600 μl of wash buffer (8 M urea, 100 mM NaH2 PO4 , 20 mM imidazole, 10 mM Tris-Cl [pH 6.2]), and eluted once with 200 μl of elution buffer (8 M urea, 100 mM NaH2 PO4 , 20 mM imidazole, 10 mM Tris-Cl [pH 4.0]) and once with 200 μl of elution buffer containing 250 mM imidazole.

    Sequencing:

    Article Title: Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1 [C]Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1 [C] [W]
    Article Snippet: After verifying the integrity of the gene through sequencing, expression plasmids were transformed into S. cerevisiae (strain BY4741) following the standard lithium acetate method. .. After dialysis for 12 h in lysis buffer, poly(His)-tagged proteins were purified using nickel-nitrilotriacetic acid agarose beads according to the manufacturer’s protocol (Qiagen).

    Serial Dilution:

    Article Title: Multi‐omics identify xanthine as a pro‐survival metabolite for nematodes with mitochondrial dysfunction
    Article Snippet: Pellets were then pestle‐homogenized in the lysis buffer of the RNA Extraction Kit (RNeasy, Qiagen). .. Each set of primers was verified through a two‐step protocol that included (i) testing of nine combinations of primer concentrations against 10 ng of wt cDNA, choosing the combination which gave the lowest C T value and only the expected product when the qPCR was run on a 2% agarose gel, and (ii) the linearity of C T values given by the primer combination identified in step (i) with a serial dilution of cDNA ranging from 10 to 0.31 ng.

    Cell Culture:

    Article Title: The CREB coactivator CRTC2 promotes oncogenesis in LKB1-mutant non–small cell lung cancer
    Article Snippet: .. Real-time qRT-PCR and RNA-seq Cultured cells were disrupted in lysis buffer from the RNeasy Mini Kit (Qiagen), and mRNA was purified following the manufacturer’s instructions. cDNA was synthesized using the Transcriptor First Strand cDNA Synthesis Kit (Roche). .. Quantitative polymerase chain reaction real-time qRT-PCR was carried out with diluted cDNA, appropriate primers, and LightCycler 480 SYBR Green I Master (Roche) on a Lightcycler 480 instrument (Roche).

    Polymerase Chain Reaction:

    Article Title: Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1 [C]Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1 [C] [W]
    Article Snippet: Transformants were selected on synthetic dextrose medium without uracil after 3 d of incubation at 30°C and were confirmed by colony PCR. .. After dialysis for 12 h in lysis buffer, poly(His)-tagged proteins were purified using nickel-nitrilotriacetic acid agarose beads according to the manufacturer’s protocol (Qiagen).

    Sonication:

    Article Title: Tsg101, an Inactive Homologue of Ubiquitin Ligase E2, Interacts Specifically with Human Immunodeficiency Virus Type 2 Gag Polyprotein and Results in Increased Levels of Ubiquitinated Gag
    Article Snippet: .. The remaining cells were resuspended in lysis buffer (6 M guanidine hydrochloride, 100 mM NaH2 PO4 , 20 mM imidazole, 10 mM Tris-Cl [pH 8]), sonicated for 1 min on ice, and then rotated at room temperature for 1 h. The His-tagged proteins were purified on Ni-nitrotriacetate (NTA) spin columns (Qiagen), washed four times with 600 μl of wash buffer (8 M urea, 100 mM NaH2 PO4 , 20 mM imidazole, 10 mM Tris-Cl [pH 6.2]), and eluted once with 200 μl of elution buffer (8 M urea, 100 mM NaH2 PO4 , 20 mM imidazole, 10 mM Tris-Cl [pH 4.0]) and once with 200 μl of elution buffer containing 250 mM imidazole. .. The eluted samples were pooled, precipitated with 10% trichloroacetic acid, and analyzed by Western blotting using anti-p26 monoclonal antibody.

    Injection:

    Article Title: Rapid antibiotic susceptibility testing from blood culture bottles with species agnostic real-time polymerase chain reaction
    Article Snippet: The overnight cultures were used to spike 10 mL of human whole blood (BioIVT, Maryland, USA), and the entire aliquots of blood were injected into BD BACTEC standard/10 aerobic/F bottles (Becton Dickinson, New Jersey, USA). .. Pellets were resuspended in 200 μL lysis buffer (10 mM Tris-HCl, 1 mM disodium EDTA, pH 8.0, 20 mg/mL lysozyme, 300 U/mL mutanolysin, 1.2% Triton X-100) and incubated at 37°C with shaking at 1,200 rpm in a ThermoMixer for 20 min. Glass beads (0.1 mm PowerBeads; Qiagen, Hilden, Germany) were then added to the samples to an approximate volume of 20 μL and the samples were vortexed on the fastest setting for 5 min. Buffers and spin columns from a QIaAMP DNA Mini Kit (Qiagen) were then used to complete the DNA extraction.

    DNA Extraction:

    Article Title: Differential Responses of Bovine Monocyte-Derived Macrophages to Infection by Neospora caninum Isolates of High and Low Virulence
    Article Snippet: .. Samples were collected by adding 200 μl of PBS, 180 μl of lysis buffer, and 20 μl of proteinase K (Qiagen, Germany) to each well and were stored at −80°C until DNA extraction. ..

    Article Title: Rapid antibiotic susceptibility testing from blood culture bottles with species agnostic real-time polymerase chain reaction
    Article Snippet: .. Pellets were resuspended in 200 μL lysis buffer (10 mM Tris-HCl, 1 mM disodium EDTA, pH 8.0, 20 mg/mL lysozyme, 300 U/mL mutanolysin, 1.2% Triton X-100) and incubated at 37°C with shaking at 1,200 rpm in a ThermoMixer for 20 min. Glass beads (0.1 mm PowerBeads; Qiagen, Hilden, Germany) were then added to the samples to an approximate volume of 20 μL and the samples were vortexed on the fastest setting for 5 min. Buffers and spin columns from a QIaAMP DNA Mini Kit (Qiagen) were then used to complete the DNA extraction. ..

    Article Title: 1,25-Dihydroxyvitamin D3 affects gastric cancer progression by repressing BMP3 promoter methylation
    Article Snippet: Paragraph title: DNA extraction, DNA methylation microarray, and analysis ... Then, gastric tissues were dissolved in 200 µL of lysis buffer from the DNA Micro Kit (catalog no. 56304; Qiagen NV, Venlo, the Netherlands) and incubated with proteinase K overnight at 56°C for two nights.

    In Vivo:

    Article Title: Tsg101, an Inactive Homologue of Ubiquitin Ligase E2, Interacts Specifically with Human Immunodeficiency Virus Type 2 Gag Polyprotein and Results in Increased Levels of Ubiquitinated Gag
    Article Snippet: Paragraph title: In vivo ubiquitination of HIV-2 Gag. ... The remaining cells were resuspended in lysis buffer (6 M guanidine hydrochloride, 100 mM NaH2 PO4 , 20 mM imidazole, 10 mM Tris-Cl [pH 8]), sonicated for 1 min on ice, and then rotated at room temperature for 1 h. The His-tagged proteins were purified on Ni-nitrotriacetate (NTA) spin columns (Qiagen), washed four times with 600 μl of wash buffer (8 M urea, 100 mM NaH2 PO4 , 20 mM imidazole, 10 mM Tris-Cl [pH 6.2]), and eluted once with 200 μl of elution buffer (8 M urea, 100 mM NaH2 PO4 , 20 mM imidazole, 10 mM Tris-Cl [pH 4.0]) and once with 200 μl of elution buffer containing 250 mM imidazole.

    RNA Sequencing Assay:

    Article Title: The CREB coactivator CRTC2 promotes oncogenesis in LKB1-mutant non–small cell lung cancer
    Article Snippet: .. Real-time qRT-PCR and RNA-seq Cultured cells were disrupted in lysis buffer from the RNeasy Mini Kit (Qiagen), and mRNA was purified following the manufacturer’s instructions. cDNA was synthesized using the Transcriptor First Strand cDNA Synthesis Kit (Roche). .. Quantitative polymerase chain reaction real-time qRT-PCR was carried out with diluted cDNA, appropriate primers, and LightCycler 480 SYBR Green I Master (Roche) on a Lightcycler 480 instrument (Roche).

    Methylation:

    Article Title: 1,25-Dihydroxyvitamin D3 affects gastric cancer progression by repressing BMP3 promoter methylation
    Article Snippet: DNA extraction, DNA methylation microarray, and analysis The method for DNA extraction and methylation analysis is described previously. .. Then, gastric tissues were dissolved in 200 µL of lysis buffer from the DNA Micro Kit (catalog no. 56304; Qiagen NV, Venlo, the Netherlands) and incubated with proteinase K overnight at 56°C for two nights.

    Isolation:

    Article Title: Nanocomplexes of Graphene Oxide and Platinum Nanoparticles against Colorectal Cancer Colo205, HT-29, HTC-116, SW480, Liver Cancer HepG2, Human Breast Cancer MCF-7, and Adenocarcinoma LNCaP and Human Cervical Hela B Cell Lines
    Article Snippet: Paragraph title: 2.7.1. Isolation of Total RNA ... The resulting cell pellet was resuspended in lysis buffer containing 1% 2-mercaptoethanol, and subsequently, the frozen metal balls were added to the probe and homogenized in a TissueLyser ball mill (Qiagen, Germantown, MD, USA) for 5 min at 50 Hz.

    Article Title: Reprogramming of CaCo2 colorectal cancer cells after using the complex of poly-(N-vinylpyrrolidone) with small non-coding RNAs
    Article Snippet: .. One portion of the cells were removed at 11, 21 and 30 days, treated with a lysis buffer from an RNAeasy mini kit (Qiagen, USA), and frozen at -20 °C for further total RNA isolation, reverse transcription reaction, and specific cDNA transcript amplification. .. Another portion of the cells was used for staining using the Leishman-Romanowsky method.

    Article Title: The CREB coactivator CRTC2 promotes oncogenesis in LKB1-mutant non–small cell lung cancer
    Article Snippet: Real-time qRT-PCR and RNA-seq Cultured cells were disrupted in lysis buffer from the RNeasy Mini Kit (Qiagen), and mRNA was purified following the manufacturer’s instructions. cDNA was synthesized using the Transcriptor First Strand cDNA Synthesis Kit (Roche). .. RNA-seq libraries were prepared using the mRNA isolation protocol and NEBNext Ultra kits from New England BioLabs following the manufacturer’s protocols.

    Article Title: Multi‐omics identify xanthine as a pro‐survival metabolite for nematodes with mitochondrial dysfunction
    Article Snippet: Pellets were then pestle‐homogenized in the lysis buffer of the RNA Extraction Kit (RNeasy, Qiagen). .. RNA was isolated as per the manufacturer's instructions.

    Article Title: AntagomiR-103 and -107 Treatment Affects Cardiac Function and Metabolism
    Article Snippet: RNA and RT-PCR Cardiac tissue was lysed with stainless steel beads (QIAGEN, Venlo, the Netherlands) in lysis buffer with the TissueLyser LT (QIAGEN, Venlo, the Netherlands). .. RNA was isolated from the lysate with mirVANA kit (Thermo Fisher Scientific, Waltham, MA), according to the manufacturer’s instructions.

    Purification:

    Article Title: The CREB coactivator CRTC2 promotes oncogenesis in LKB1-mutant non–small cell lung cancer
    Article Snippet: .. Real-time qRT-PCR and RNA-seq Cultured cells were disrupted in lysis buffer from the RNeasy Mini Kit (Qiagen), and mRNA was purified following the manufacturer’s instructions. cDNA was synthesized using the Transcriptor First Strand cDNA Synthesis Kit (Roche). .. Quantitative polymerase chain reaction real-time qRT-PCR was carried out with diluted cDNA, appropriate primers, and LightCycler 480 SYBR Green I Master (Roche) on a Lightcycler 480 instrument (Roche).

    Article Title: Tsg101, an Inactive Homologue of Ubiquitin Ligase E2, Interacts Specifically with Human Immunodeficiency Virus Type 2 Gag Polyprotein and Results in Increased Levels of Ubiquitinated Gag
    Article Snippet: .. The remaining cells were resuspended in lysis buffer (6 M guanidine hydrochloride, 100 mM NaH2 PO4 , 20 mM imidazole, 10 mM Tris-Cl [pH 8]), sonicated for 1 min on ice, and then rotated at room temperature for 1 h. The His-tagged proteins were purified on Ni-nitrotriacetate (NTA) spin columns (Qiagen), washed four times with 600 μl of wash buffer (8 M urea, 100 mM NaH2 PO4 , 20 mM imidazole, 10 mM Tris-Cl [pH 6.2]), and eluted once with 200 μl of elution buffer (8 M urea, 100 mM NaH2 PO4 , 20 mM imidazole, 10 mM Tris-Cl [pH 4.0]) and once with 200 μl of elution buffer containing 250 mM imidazole. .. The eluted samples were pooled, precipitated with 10% trichloroacetic acid, and analyzed by Western blotting using anti-p26 monoclonal antibody.

    Article Title: Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1 [C]Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1 [C] [W]
    Article Snippet: .. After dialysis for 12 h in lysis buffer, poly(His)-tagged proteins were purified using nickel-nitrilotriacetic acid agarose beads according to the manufacturer’s protocol (Qiagen). ..

    Protein Purification:

    Article Title: Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1 [C]Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1 [C] [W]
    Article Snippet: Paragraph title: Yeast Expression, Protein Purification, and Analysis of Yeast Extracts ... After dialysis for 12 h in lysis buffer, poly(His)-tagged proteins were purified using nickel-nitrilotriacetic acid agarose beads according to the manufacturer’s protocol (Qiagen).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: AntagomiR-103 and -107 Treatment Affects Cardiac Function and Metabolism
    Article Snippet: .. RNA and RT-PCR Cardiac tissue was lysed with stainless steel beads (QIAGEN, Venlo, the Netherlands) in lysis buffer with the TissueLyser LT (QIAGEN, Venlo, the Netherlands). .. RNA was isolated from the lysate with mirVANA kit (Thermo Fisher Scientific, Waltham, MA), according to the manufacturer’s instructions.

    Protein Extraction:

    Article Title: Bcl-2 associated athanogene 5 (Bag5) is overexpressed in prostate cancer and inhibits ER-stress induced apoptosis
    Article Snippet: .. For protein extraction from patient material, 8 μm-thick frozen tissue sections were homogenized in lysis buffer with the TissueLyser (Qiagen, Hilden, Germany) and frozen at -80°C. .. Endoplamsic reticulum fractionation Endoplasmic Reticulum fractionation was performed with the ER enrichment kit from Imgenex (Hamburg, Germany).

    Cotransfection:

    Article Title: Tsg101, an Inactive Homologue of Ubiquitin Ligase E2, Interacts Specifically with Human Immunodeficiency Virus Type 2 Gag Polyprotein and Results in Increased Levels of Ubiquitinated Gag
    Article Snippet: pH6 M-Ub was introduced into 293T cells by cotransfection with plasmids expressing HIV-2 Gag and Tsg101. .. The remaining cells were resuspended in lysis buffer (6 M guanidine hydrochloride, 100 mM NaH2 PO4 , 20 mM imidazole, 10 mM Tris-Cl [pH 8]), sonicated for 1 min on ice, and then rotated at room temperature for 1 h. The His-tagged proteins were purified on Ni-nitrotriacetate (NTA) spin columns (Qiagen), washed four times with 600 μl of wash buffer (8 M urea, 100 mM NaH2 PO4 , 20 mM imidazole, 10 mM Tris-Cl [pH 6.2]), and eluted once with 200 μl of elution buffer (8 M urea, 100 mM NaH2 PO4 , 20 mM imidazole, 10 mM Tris-Cl [pH 4.0]) and once with 200 μl of elution buffer containing 250 mM imidazole.

    Staining:

    Article Title: Reprogramming of CaCo2 colorectal cancer cells after using the complex of poly-(N-vinylpyrrolidone) with small non-coding RNAs
    Article Snippet: One portion of the cells were removed at 11, 21 and 30 days, treated with a lysis buffer from an RNAeasy mini kit (Qiagen, USA), and frozen at -20 °C for further total RNA isolation, reverse transcription reaction, and specific cDNA transcript amplification. .. Another portion of the cells was used for staining using the Leishman-Romanowsky method.

    Article Title: 1,25-Dihydroxyvitamin D3 affects gastric cancer progression by repressing BMP3 promoter methylation
    Article Snippet: Before DNA extraction, gastric specimens were formalin-fixed, paraffin-embedded and microscopically examined using H & E staining. .. Then, gastric tissues were dissolved in 200 µL of lysis buffer from the DNA Micro Kit (catalog no. 56304; Qiagen NV, Venlo, the Netherlands) and incubated with proteinase K overnight at 56°C for two nights.

    SDS Page:

    Article Title: Tsg101, an Inactive Homologue of Ubiquitin Ligase E2, Interacts Specifically with Human Immunodeficiency Virus Type 2 Gag Polyprotein and Results in Increased Levels of Ubiquitinated Gag
    Article Snippet: At 24 h later, the transfected cultures were treated with MG132 (2 μM) for an additional 16 h. The cells were harvested, and 10% of the cells were resuspended in SDS-PAGE loading buffer and analyzed by Western blotting, using anti-26 monoclonal antibody, to confirm that equivalent levels of Gag protein were expressed in each sample. .. The remaining cells were resuspended in lysis buffer (6 M guanidine hydrochloride, 100 mM NaH2 PO4 , 20 mM imidazole, 10 mM Tris-Cl [pH 8]), sonicated for 1 min on ice, and then rotated at room temperature for 1 h. The His-tagged proteins were purified on Ni-nitrotriacetate (NTA) spin columns (Qiagen), washed four times with 600 μl of wash buffer (8 M urea, 100 mM NaH2 PO4 , 20 mM imidazole, 10 mM Tris-Cl [pH 6.2]), and eluted once with 200 μl of elution buffer (8 M urea, 100 mM NaH2 PO4 , 20 mM imidazole, 10 mM Tris-Cl [pH 4.0]) and once with 200 μl of elution buffer containing 250 mM imidazole.

    Software:

    Article Title: Rapid antibiotic susceptibility testing from blood culture bottles with species agnostic real-time polymerase chain reaction
    Article Snippet: Blood culture (BC) bottles were incubated in a BD BACTEC FX40 instrument until flagged as positive by the instrument software. .. Pellets were resuspended in 200 μL lysis buffer (10 mM Tris-HCl, 1 mM disodium EDTA, pH 8.0, 20 mg/mL lysozyme, 300 U/mL mutanolysin, 1.2% Triton X-100) and incubated at 37°C with shaking at 1,200 rpm in a ThermoMixer for 20 min. Glass beads (0.1 mm PowerBeads; Qiagen, Hilden, Germany) were then added to the samples to an approximate volume of 20 μL and the samples were vortexed on the fastest setting for 5 min. Buffers and spin columns from a QIaAMP DNA Mini Kit (Qiagen) were then used to complete the DNA extraction.

    SYBR Green Assay:

    Article Title: The CREB coactivator CRTC2 promotes oncogenesis in LKB1-mutant non–small cell lung cancer
    Article Snippet: Real-time qRT-PCR and RNA-seq Cultured cells were disrupted in lysis buffer from the RNeasy Mini Kit (Qiagen), and mRNA was purified following the manufacturer’s instructions. cDNA was synthesized using the Transcriptor First Strand cDNA Synthesis Kit (Roche). .. Quantitative polymerase chain reaction real-time qRT-PCR was carried out with diluted cDNA, appropriate primers, and LightCycler 480 SYBR Green I Master (Roche) on a Lightcycler 480 instrument (Roche).

    Article Title: Multi‐omics identify xanthine as a pro‐survival metabolite for nematodes with mitochondrial dysfunction
    Article Snippet: Pellets were then pestle‐homogenized in the lysis buffer of the RNA Extraction Kit (RNeasy, Qiagen). .. The RNA concentration was quantified with a NanoDrop 2000C (Thermo Fisher Scientific), and 100 ng was used for cDNA synthesis with the qScript Kit (Quantabio) as per the manufacturer's instructions. qRT–PCRs were performed on 1 μl of cDNA with a Fast SYBR Green Master Mix (Applied Biosystems) on an Applied Biosystems qRT–PCR cycler.

    RNA Extraction:

    Article Title: Multi‐omics identify xanthine as a pro‐survival metabolite for nematodes with mitochondrial dysfunction
    Article Snippet: .. Pellets were then pestle‐homogenized in the lysis buffer of the RNA Extraction Kit (RNeasy, Qiagen). .. RNA was isolated as per the manufacturer's instructions.

    Article Title: Identification of biomarkers that distinguish chemical contaminants based on gene expression profiles
    Article Snippet: Paragraph title: Total RNA extraction ... Cells were homogenized in the lysis buffer with FAST Prep-24 from MP at speed 6.0/s twice, each for 30s before using RNeasy kits (Qiagen).

    Agarose Gel Electrophoresis:

    Article Title: Multi‐omics identify xanthine as a pro‐survival metabolite for nematodes with mitochondrial dysfunction
    Article Snippet: Pellets were then pestle‐homogenized in the lysis buffer of the RNA Extraction Kit (RNeasy, Qiagen). .. Each set of primers was verified through a two‐step protocol that included (i) testing of nine combinations of primer concentrations against 10 ng of wt cDNA, choosing the combination which gave the lowest C T value and only the expected product when the qPCR was run on a 2% agarose gel, and (ii) the linearity of C T values given by the primer combination identified in step (i) with a serial dilution of cDNA ranging from 10 to 0.31 ng.

    Spectrophotometry:

    Article Title: Nanocomplexes of Graphene Oxide and Platinum Nanoparticles against Colorectal Cancer Colo205, HT-29, HTC-116, SW480, Liver Cancer HepG2, Human Breast Cancer MCF-7, and Adenocarcinoma LNCaP and Human Cervical Hela B Cell Lines
    Article Snippet: The resulting cell pellet was resuspended in lysis buffer containing 1% 2-mercaptoethanol, and subsequently, the frozen metal balls were added to the probe and homogenized in a TissueLyser ball mill (Qiagen, Germantown, MD, USA) for 5 min at 50 Hz. .. The isolated RNA was measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA).

    Article Title: 1,25-Dihydroxyvitamin D3 affects gastric cancer progression by repressing BMP3 promoter methylation
    Article Snippet: Then, gastric tissues were dissolved in 200 µL of lysis buffer from the DNA Micro Kit (catalog no. 56304; Qiagen NV, Venlo, the Netherlands) and incubated with proteinase K overnight at 56°C for two nights. .. DNA concentration was determined at 260 nm using a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies Inc., Wilmington, NC, USA).

    Article Title: Identification of biomarkers that distinguish chemical contaminants based on gene expression profiles
    Article Snippet: Cells were homogenized in the lysis buffer with FAST Prep-24 from MP at speed 6.0/s twice, each for 30s before using RNeasy kits (Qiagen). .. Total RNA concentrations were measured using NanoDrop® ND-1000 Spectrophotometer (NanoDrop technologies, Wilmington, DE, USA).

    DNA Methylation Assay:

    Article Title: 1,25-Dihydroxyvitamin D3 affects gastric cancer progression by repressing BMP3 promoter methylation
    Article Snippet: Paragraph title: DNA extraction, DNA methylation microarray, and analysis ... Then, gastric tissues were dissolved in 200 µL of lysis buffer from the DNA Micro Kit (catalog no. 56304; Qiagen NV, Venlo, the Netherlands) and incubated with proteinase K overnight at 56°C for two nights.

    Concentration Assay:

    Article Title: 1,25-Dihydroxyvitamin D3 affects gastric cancer progression by repressing BMP3 promoter methylation
    Article Snippet: Then, gastric tissues were dissolved in 200 µL of lysis buffer from the DNA Micro Kit (catalog no. 56304; Qiagen NV, Venlo, the Netherlands) and incubated with proteinase K overnight at 56°C for two nights. .. DNA concentration was determined at 260 nm using a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies Inc., Wilmington, NC, USA).

    Article Title: Multi‐omics identify xanthine as a pro‐survival metabolite for nematodes with mitochondrial dysfunction
    Article Snippet: Pellets were then pestle‐homogenized in the lysis buffer of the RNA Extraction Kit (RNeasy, Qiagen). .. The RNA concentration was quantified with a NanoDrop 2000C (Thermo Fisher Scientific), and 100 ng was used for cDNA synthesis with the qScript Kit (Quantabio) as per the manufacturer's instructions. qRT–PCRs were performed on 1 μl of cDNA with a Fast SYBR Green Master Mix (Applied Biosystems) on an Applied Biosystems qRT–PCR cycler.

    Protease Inhibitor:

    Article Title: Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1 [C]Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1 [C] [W]
    Article Snippet: After 12 h of growth, cells were collected by centrifugation, crushed with liquid nitrogen using a pestle and mortar, and resuspended in lysis buffer (50 m m Na2 PO4 , 300 m m sodium chloride, 20 m m imidazole, pH 8.0 with 0.1 m m phenylmethylsulfonyl fluoride and protease inhibitor cocktail; Sigma-Aldrich). .. After dialysis for 12 h in lysis buffer, poly(His)-tagged proteins were purified using nickel-nitrilotriacetic acid agarose beads according to the manufacturer’s protocol (Qiagen).

    Lysis:

    Article Title: Differential Responses of Bovine Monocyte-Derived Macrophages to Infection by Neospora caninum Isolates of High and Low Virulence
    Article Snippet: .. Samples were collected by adding 200 μl of PBS, 180 μl of lysis buffer, and 20 μl of proteinase K (Qiagen, Germany) to each well and were stored at −80°C until DNA extraction. ..

    Article Title: Nanocomplexes of Graphene Oxide and Platinum Nanoparticles against Colorectal Cancer Colo205, HT-29, HTC-116, SW480, Liver Cancer HepG2, Human Breast Cancer MCF-7, and Adenocarcinoma LNCaP and Human Cervical Hela B Cell Lines
    Article Snippet: .. The resulting cell pellet was resuspended in lysis buffer containing 1% 2-mercaptoethanol, and subsequently, the frozen metal balls were added to the probe and homogenized in a TissueLyser ball mill (Qiagen, Germantown, MD, USA) for 5 min at 50 Hz. ..

    Article Title: Bcl-2 associated athanogene 5 (Bag5) is overexpressed in prostate cancer and inhibits ER-stress induced apoptosis
    Article Snippet: .. For protein extraction from patient material, 8 μm-thick frozen tissue sections were homogenized in lysis buffer with the TissueLyser (Qiagen, Hilden, Germany) and frozen at -80°C. .. Endoplamsic reticulum fractionation Endoplasmic Reticulum fractionation was performed with the ER enrichment kit from Imgenex (Hamburg, Germany).

    Article Title: Rapid antibiotic susceptibility testing from blood culture bottles with species agnostic real-time polymerase chain reaction
    Article Snippet: .. Pellets were resuspended in 200 μL lysis buffer (10 mM Tris-HCl, 1 mM disodium EDTA, pH 8.0, 20 mg/mL lysozyme, 300 U/mL mutanolysin, 1.2% Triton X-100) and incubated at 37°C with shaking at 1,200 rpm in a ThermoMixer for 20 min. Glass beads (0.1 mm PowerBeads; Qiagen, Hilden, Germany) were then added to the samples to an approximate volume of 20 μL and the samples were vortexed on the fastest setting for 5 min. Buffers and spin columns from a QIaAMP DNA Mini Kit (Qiagen) were then used to complete the DNA extraction. ..

    Article Title: Reprogramming of CaCo2 colorectal cancer cells after using the complex of poly-(N-vinylpyrrolidone) with small non-coding RNAs
    Article Snippet: .. One portion of the cells were removed at 11, 21 and 30 days, treated with a lysis buffer from an RNAeasy mini kit (Qiagen, USA), and frozen at -20 °C for further total RNA isolation, reverse transcription reaction, and specific cDNA transcript amplification. .. Another portion of the cells was used for staining using the Leishman-Romanowsky method.

    Article Title: 1,25-Dihydroxyvitamin D3 affects gastric cancer progression by repressing BMP3 promoter methylation
    Article Snippet: .. Then, gastric tissues were dissolved in 200 µL of lysis buffer from the DNA Micro Kit (catalog no. 56304; Qiagen NV, Venlo, the Netherlands) and incubated with proteinase K overnight at 56°C for two nights. .. DNA was extracted according to the manufacturer’s protocol (QIAamp DNA Micro Kit; Qiagen NV) and stored at −80°C to prevent degradation.

    Article Title: The CREB coactivator CRTC2 promotes oncogenesis in LKB1-mutant non–small cell lung cancer
    Article Snippet: .. Real-time qRT-PCR and RNA-seq Cultured cells were disrupted in lysis buffer from the RNeasy Mini Kit (Qiagen), and mRNA was purified following the manufacturer’s instructions. cDNA was synthesized using the Transcriptor First Strand cDNA Synthesis Kit (Roche). .. Quantitative polymerase chain reaction real-time qRT-PCR was carried out with diluted cDNA, appropriate primers, and LightCycler 480 SYBR Green I Master (Roche) on a Lightcycler 480 instrument (Roche).

    Article Title: Multi‐omics identify xanthine as a pro‐survival metabolite for nematodes with mitochondrial dysfunction
    Article Snippet: .. Pellets were then pestle‐homogenized in the lysis buffer of the RNA Extraction Kit (RNeasy, Qiagen). .. RNA was isolated as per the manufacturer's instructions.

    Article Title: Identification of biomarkers that distinguish chemical contaminants based on gene expression profiles
    Article Snippet: .. Cells were homogenized in the lysis buffer with FAST Prep-24 from MP at speed 6.0/s twice, each for 30s before using RNeasy kits (Qiagen). .. Total RNA concentrations were measured using NanoDrop® ND-1000 Spectrophotometer (NanoDrop technologies, Wilmington, DE, USA).

    Article Title: Tsg101, an Inactive Homologue of Ubiquitin Ligase E2, Interacts Specifically with Human Immunodeficiency Virus Type 2 Gag Polyprotein and Results in Increased Levels of Ubiquitinated Gag
    Article Snippet: .. The remaining cells were resuspended in lysis buffer (6 M guanidine hydrochloride, 100 mM NaH2 PO4 , 20 mM imidazole, 10 mM Tris-Cl [pH 8]), sonicated for 1 min on ice, and then rotated at room temperature for 1 h. The His-tagged proteins were purified on Ni-nitrotriacetate (NTA) spin columns (Qiagen), washed four times with 600 μl of wash buffer (8 M urea, 100 mM NaH2 PO4 , 20 mM imidazole, 10 mM Tris-Cl [pH 6.2]), and eluted once with 200 μl of elution buffer (8 M urea, 100 mM NaH2 PO4 , 20 mM imidazole, 10 mM Tris-Cl [pH 4.0]) and once with 200 μl of elution buffer containing 250 mM imidazole. .. The eluted samples were pooled, precipitated with 10% trichloroacetic acid, and analyzed by Western blotting using anti-p26 monoclonal antibody.

    Article Title: Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1 [C]Methyl Jasmonate-Elicited Transcriptional Responses and Pentacyclic Triterpene Biosynthesis in Sweet Basil 1 [C] [W]
    Article Snippet: .. After dialysis for 12 h in lysis buffer, poly(His)-tagged proteins were purified using nickel-nitrilotriacetic acid agarose beads according to the manufacturer’s protocol (Qiagen). ..

    Article Title: AntagomiR-103 and -107 Treatment Affects Cardiac Function and Metabolism
    Article Snippet: .. RNA and RT-PCR Cardiac tissue was lysed with stainless steel beads (QIAGEN, Venlo, the Netherlands) in lysis buffer with the TissueLyser LT (QIAGEN, Venlo, the Netherlands). .. RNA was isolated from the lysate with mirVANA kit (Thermo Fisher Scientific, Waltham, MA), according to the manufacturer’s instructions.

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    Qiagen rlt lysis buffer
    Asymmetric competition is dependent on direct interactions between resident and challenger pneumococci. a, Day 4–5 old pups were inoculated with a heat-killed serotype 23F resident strain (10 5 CFU) for 6h before introduction of the live isogenic challenger strain with increasing CFU (10 1 -10 3 ). Nasal lavages were cultured 3 days later to determine challenger colonization densities, n=3–4. Median values are shown. b, Pups were inoculated with live (10 3 CFU) or heat-killed (10 5 CFU) resident strain for 6h and nasal lavages with <t>RLT</t> lysis buffer were obtained to isolate host <t>RNA.</t> Early inflammatory cytokine expression levels were assessed by SYBR green real-time quantitative RT-PCR. Cytokine expression induced by heat-killed S. pneumoniae were shown as fold change relative to that elicited by live bacteria, n=5–6. Data are shown as mean ± SEM. c-d, Pups were inoculated with either a streptomycin sensitive ( c ) or resistant ( d ) resident strain for 3 days before intranasal administration of streptomycin. An isogenic streptomycin resistant challenger was inoculated 6h later and its colonization density determined after 3 days. Median values are shown. Colonization levels by 10 1 CFU challenger alone, and in resident-colonized pups treated with either phosphate buffered saline (PBS) or streptomycin, were compared by Kruskal–Wallis one-way analysis of variance in ( d ), n=4–8, * p
    Rlt Lysis Buffer, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 92 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen rna lysis buffer
    Conventional GFP derivatives are not compatible with laser-directed microdissection. The cellular signal of a nuclear-targeted EYFP (EYFPnuc) is preserved in freeze-dried cryosections obtained from transgenic mice. <t>RNA</t> isolated from cryosections is intact. A , The cellular resolution of EYFP expressed in transgenic mice is lost after cryosectioning. Sagittal sections of mouse brains from mice expressing cytoplasmic localized EYFP under the control of the Thy-1 promoter were either fixed with 4% buffered PFA or immediately frozen on dry ice. Vibratome sections (PFA, left) obtained from PFA fixed tissue (30 μm) and cryosections (15 μm; Cryo, middle) from frozen tissue, were analyzed for EYFP fluorescence. After cryosectioning, the cellular resolution of the EYFP fluorescent signal is lost. The EYFP signal is retained but appears to be diffuse and faded. Staining of the cryosection with thionin (Cryo, right) reveals that the cellular integrity of the tissue specimen is intact. B , Nuclear targeted fluorescent proteins appear to be promising candidates for a combined in vivo labeling and microdissection approach. COS7 or HeLa cells were transiently transfected with expression constructs coding for GFP derivatives localized to different cellular compartments: EGFP (cytoplasmic localization), EGFP carrying a farnesylation signal (EGFP-F, attached to membranes), EGFP fused to the N terminus of a synthetic transmembrane protein (TM-EGFP), and EYFP fused to three nuclear localization signals (EYFPnuc). Forty-eight hours after transfection, COS7 cells were either fixed with a PBS-buffered 4% PFA or fixed with 70% ethanol (EtOH) and subsequently dry mounted. HeLa cells transfected with EYFPnuc were either dry mounted (DRY) or fixed with 70% ethanol (EtOH) and subsequently frozen at −80°C before analysis. C , Transgenic mice were generated that express a nuclear-targeted EYFP (EYFPnuc) under control of the Thy-1 Promoter (TYNC mice). The Thy-1 minigene was modified by replacing the Thy-1 ORF with sequences coding for EYFPnuc. D , E , Vibratome sections (30 μm) from brains of PFA-fixed TYNC mice were analyzed for YFP fluorescence and for cellular marker gene expression with immunohistochemistry. D , Within the hippocampus, the YFP expression is most prominent in pyramidal neurons of the CA1 field (CA1) and in the granular cell layer of the dentate gyrus (DG). In the CA3 field (CA3), fewer cells are YFP positive. The YFP fluorescence appears to be strictly overlapping with the indirect GFP immune fluorescence analysis (α-GFP), although it is less sensitive. The pan-neuronal marker NeuN (α-NeuN) colocalizes with the nuclear YFP signal. Staining with GFAP (α-GFAP) and parvalbumin (α-Parv) does not reveal YFP overlapping signals. E , Higher-magnification photographs obtained from vibratome sections at the level of the primary motor and sensory cortex. YFP-positive nuclei (YFP) do not colocalize with the interneuronal marker parvalbumin (α-Parv) and GAD67 (α-GAD67) and also not with the astrocyte marker GFAP (α-GFAP). Arrows mark parvalbumin, GAD67, or GFAP-positive cells stained in red in the left panel. No overlap with YFP fluorescence can be detected in the right panel with arrows pointing to identical positions as in the left panel. Scale bar, 50 μm. F–H , Cryosections (8 μm; Cryo) were mounted on PEN foils (Leica) and analyzed for YFP fluorescence. F , In the hippocampus, YFP-positive nuclei can be detected in the granular cell layer (DG) and in the CA1 field of the pyramidal cell layer (CA1). In the CA3 field (CA3), fewer cells are YFP positive. The inset shows the morphology of the hippocampus in bright field. G , Higher magnification of YFP-positive nuclei in the somatosensory cortex. Scale bar, 50 μm. H , In the cortex, cells located in deeper layers show a nuclear YFP signal. The inset shows the morphology of the section at the level of the somatosensory cortex in bright field. I , Total RNA analyzed with a Bioanalyzer (Agilent); each lane represents half of the RNA isolated from one coronal. brain section. Adjacent 8-μ-thick sections were cryomounted on PEN foil slides, dried, and treated as follows: (1) stored for 2 h at room temperature, (2) stored for 8 h at room temperature, (3) stored for 24 h at room temperature, (4) stored for 48 h at room temperature, (5) frozen at −80°C and then kept at room temperature for 2 h, and (6) <t>microdissected</t> regions pooled from eight sections (total area size isolated comparable with one section). The ratio of the 28S versus the 18S rRNA bands was determined with the Bioanalyzer software and for all samples was 1.3± 0.1. The amount of RNA isolated from one coronal 8 μ brain cryosection was ∼50 ng.
    Rna Lysis Buffer, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Targeted hydroxymethylation of four different aberrantly methylated genes by dCas9-TET3CD fusion protein in human kidney cells. a Schematic representing hypermethylated RASAL1 promoter region (upper panel) and reactivated RASAL1 expression through induction of RASAL1 promoter hydroxymethylation by dCas9-TET3CD fusion protein in complex with a sgRNA binding to its target region (lower panel). b Schematic of domain structure of the dCas9-TET3CD (upper panel) and dCas9-TETCDi (lower panel) fusion protein. c – e Locations for RASAL1/EYA1/LRFN2- sgRNAs are indicated by thick lines with corresponding PAM in magenta within the human RASAL1/EYA1/LRFN2 gene locus, respectively. Human fibrotic TK188 fibroblasts were transduced with lentivirus expressing demethylation constructs guided by RASAL1 -sgRNAs 1–10, EYA1-sgRNA 1–6 , LRFN2-sgRNA 1–8 , or by LacZ control sgRNA. Results were normalized to reference gene GAPDH. f , g <t>MeDIP</t> and hMeDIP analysis of TK188 cells were transduced with dCas9-TET3CD- RASAL1- sgRNA3. The results were calculated relative to the input. h Bisulfite sequencing summary of promoter methylation status of the RASAL1 gene in TK188 cells transduced with demethylation constructs guided by RASAL1 -sgRNA3, by LacZ control sgRNA or <t>DNA</t> treated with M.SssI serving as positive control. Each data point represents the mean of three independent transduction experiments with error bars indicating the standard error of the mean for six or more bisulfite sequencing results. i Locations for KL- sgRNAs are indicated by thick lines with corresponding PAM in magenta within the human KL gene locus. Three days TGFβ1-treated HK2 cells were transduced with lentivirus expressing demethylation constructs guided by KL -sgRNAs 1–8 or by LacZ control sgRNA. j , k MeDIP and hMeDIP analysis of HK2 cells were transduction with dCas9-TET3CD- KL- sgRNA2. The results were calculated relative to the input. All data are presented as mean value; error bars represent S.D.; n = 3 independent biological replicates, n.s. not significant; * p
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    Asymmetric competition is dependent on direct interactions between resident and challenger pneumococci. a, Day 4–5 old pups were inoculated with a heat-killed serotype 23F resident strain (10 5 CFU) for 6h before introduction of the live isogenic challenger strain with increasing CFU (10 1 -10 3 ). Nasal lavages were cultured 3 days later to determine challenger colonization densities, n=3–4. Median values are shown. b, Pups were inoculated with live (10 3 CFU) or heat-killed (10 5 CFU) resident strain for 6h and nasal lavages with RLT lysis buffer were obtained to isolate host RNA. Early inflammatory cytokine expression levels were assessed by SYBR green real-time quantitative RT-PCR. Cytokine expression induced by heat-killed S. pneumoniae were shown as fold change relative to that elicited by live bacteria, n=5–6. Data are shown as mean ± SEM. c-d, Pups were inoculated with either a streptomycin sensitive ( c ) or resistant ( d ) resident strain for 3 days before intranasal administration of streptomycin. An isogenic streptomycin resistant challenger was inoculated 6h later and its colonization density determined after 3 days. Median values are shown. Colonization levels by 10 1 CFU challenger alone, and in resident-colonized pups treated with either phosphate buffered saline (PBS) or streptomycin, were compared by Kruskal–Wallis one-way analysis of variance in ( d ), n=4–8, * p

    Journal: Nature microbiology

    Article Title: Pneumococcal quorum sensing drives an asymmetric owner-intruder competitive strategy during carriage via the competence regulon

    doi: 10.1038/s41564-018-0314-4

    Figure Lengend Snippet: Asymmetric competition is dependent on direct interactions between resident and challenger pneumococci. a, Day 4–5 old pups were inoculated with a heat-killed serotype 23F resident strain (10 5 CFU) for 6h before introduction of the live isogenic challenger strain with increasing CFU (10 1 -10 3 ). Nasal lavages were cultured 3 days later to determine challenger colonization densities, n=3–4. Median values are shown. b, Pups were inoculated with live (10 3 CFU) or heat-killed (10 5 CFU) resident strain for 6h and nasal lavages with RLT lysis buffer were obtained to isolate host RNA. Early inflammatory cytokine expression levels were assessed by SYBR green real-time quantitative RT-PCR. Cytokine expression induced by heat-killed S. pneumoniae were shown as fold change relative to that elicited by live bacteria, n=5–6. Data are shown as mean ± SEM. c-d, Pups were inoculated with either a streptomycin sensitive ( c ) or resistant ( d ) resident strain for 3 days before intranasal administration of streptomycin. An isogenic streptomycin resistant challenger was inoculated 6h later and its colonization density determined after 3 days. Median values are shown. Colonization levels by 10 1 CFU challenger alone, and in resident-colonized pups treated with either phosphate buffered saline (PBS) or streptomycin, were compared by Kruskal–Wallis one-way analysis of variance in ( d ), n=4–8, * p

    Article Snippet: To obtain host RNA, nasal lavages with RLT lysis buffer (Qiagen) were performed and RNA isolated with the RNeasy minikit (Qiagen) as previously described . cDNA was reverse transcribed using a high-capacity cDNA kit (Applied Biosystems) following the manufacturer’s instructions.

    Techniques: Cell Culture, Lysis, Expressing, SYBR Green Assay, Quantitative RT-PCR

    Conventional GFP derivatives are not compatible with laser-directed microdissection. The cellular signal of a nuclear-targeted EYFP (EYFPnuc) is preserved in freeze-dried cryosections obtained from transgenic mice. RNA isolated from cryosections is intact. A , The cellular resolution of EYFP expressed in transgenic mice is lost after cryosectioning. Sagittal sections of mouse brains from mice expressing cytoplasmic localized EYFP under the control of the Thy-1 promoter were either fixed with 4% buffered PFA or immediately frozen on dry ice. Vibratome sections (PFA, left) obtained from PFA fixed tissue (30 μm) and cryosections (15 μm; Cryo, middle) from frozen tissue, were analyzed for EYFP fluorescence. After cryosectioning, the cellular resolution of the EYFP fluorescent signal is lost. The EYFP signal is retained but appears to be diffuse and faded. Staining of the cryosection with thionin (Cryo, right) reveals that the cellular integrity of the tissue specimen is intact. B , Nuclear targeted fluorescent proteins appear to be promising candidates for a combined in vivo labeling and microdissection approach. COS7 or HeLa cells were transiently transfected with expression constructs coding for GFP derivatives localized to different cellular compartments: EGFP (cytoplasmic localization), EGFP carrying a farnesylation signal (EGFP-F, attached to membranes), EGFP fused to the N terminus of a synthetic transmembrane protein (TM-EGFP), and EYFP fused to three nuclear localization signals (EYFPnuc). Forty-eight hours after transfection, COS7 cells were either fixed with a PBS-buffered 4% PFA or fixed with 70% ethanol (EtOH) and subsequently dry mounted. HeLa cells transfected with EYFPnuc were either dry mounted (DRY) or fixed with 70% ethanol (EtOH) and subsequently frozen at −80°C before analysis. C , Transgenic mice were generated that express a nuclear-targeted EYFP (EYFPnuc) under control of the Thy-1 Promoter (TYNC mice). The Thy-1 minigene was modified by replacing the Thy-1 ORF with sequences coding for EYFPnuc. D , E , Vibratome sections (30 μm) from brains of PFA-fixed TYNC mice were analyzed for YFP fluorescence and for cellular marker gene expression with immunohistochemistry. D , Within the hippocampus, the YFP expression is most prominent in pyramidal neurons of the CA1 field (CA1) and in the granular cell layer of the dentate gyrus (DG). In the CA3 field (CA3), fewer cells are YFP positive. The YFP fluorescence appears to be strictly overlapping with the indirect GFP immune fluorescence analysis (α-GFP), although it is less sensitive. The pan-neuronal marker NeuN (α-NeuN) colocalizes with the nuclear YFP signal. Staining with GFAP (α-GFAP) and parvalbumin (α-Parv) does not reveal YFP overlapping signals. E , Higher-magnification photographs obtained from vibratome sections at the level of the primary motor and sensory cortex. YFP-positive nuclei (YFP) do not colocalize with the interneuronal marker parvalbumin (α-Parv) and GAD67 (α-GAD67) and also not with the astrocyte marker GFAP (α-GFAP). Arrows mark parvalbumin, GAD67, or GFAP-positive cells stained in red in the left panel. No overlap with YFP fluorescence can be detected in the right panel with arrows pointing to identical positions as in the left panel. Scale bar, 50 μm. F–H , Cryosections (8 μm; Cryo) were mounted on PEN foils (Leica) and analyzed for YFP fluorescence. F , In the hippocampus, YFP-positive nuclei can be detected in the granular cell layer (DG) and in the CA1 field of the pyramidal cell layer (CA1). In the CA3 field (CA3), fewer cells are YFP positive. The inset shows the morphology of the hippocampus in bright field. G , Higher magnification of YFP-positive nuclei in the somatosensory cortex. Scale bar, 50 μm. H , In the cortex, cells located in deeper layers show a nuclear YFP signal. The inset shows the morphology of the section at the level of the somatosensory cortex in bright field. I , Total RNA analyzed with a Bioanalyzer (Agilent); each lane represents half of the RNA isolated from one coronal. brain section. Adjacent 8-μ-thick sections were cryomounted on PEN foil slides, dried, and treated as follows: (1) stored for 2 h at room temperature, (2) stored for 8 h at room temperature, (3) stored for 24 h at room temperature, (4) stored for 48 h at room temperature, (5) frozen at −80°C and then kept at room temperature for 2 h, and (6) microdissected regions pooled from eight sections (total area size isolated comparable with one section). The ratio of the 28S versus the 18S rRNA bands was determined with the Bioanalyzer software and for all samples was 1.3± 0.1. The amount of RNA isolated from one coronal 8 μ brain cryosection was ∼50 ng.

    Journal: The Journal of Neuroscience

    Article Title: Global Transcriptome Analysis of Genetically Identified Neurons in the Adult Cortex

    doi: 10.1523/JNEUROSCI.0468-06.2006

    Figure Lengend Snippet: Conventional GFP derivatives are not compatible with laser-directed microdissection. The cellular signal of a nuclear-targeted EYFP (EYFPnuc) is preserved in freeze-dried cryosections obtained from transgenic mice. RNA isolated from cryosections is intact. A , The cellular resolution of EYFP expressed in transgenic mice is lost after cryosectioning. Sagittal sections of mouse brains from mice expressing cytoplasmic localized EYFP under the control of the Thy-1 promoter were either fixed with 4% buffered PFA or immediately frozen on dry ice. Vibratome sections (PFA, left) obtained from PFA fixed tissue (30 μm) and cryosections (15 μm; Cryo, middle) from frozen tissue, were analyzed for EYFP fluorescence. After cryosectioning, the cellular resolution of the EYFP fluorescent signal is lost. The EYFP signal is retained but appears to be diffuse and faded. Staining of the cryosection with thionin (Cryo, right) reveals that the cellular integrity of the tissue specimen is intact. B , Nuclear targeted fluorescent proteins appear to be promising candidates for a combined in vivo labeling and microdissection approach. COS7 or HeLa cells were transiently transfected with expression constructs coding for GFP derivatives localized to different cellular compartments: EGFP (cytoplasmic localization), EGFP carrying a farnesylation signal (EGFP-F, attached to membranes), EGFP fused to the N terminus of a synthetic transmembrane protein (TM-EGFP), and EYFP fused to three nuclear localization signals (EYFPnuc). Forty-eight hours after transfection, COS7 cells were either fixed with a PBS-buffered 4% PFA or fixed with 70% ethanol (EtOH) and subsequently dry mounted. HeLa cells transfected with EYFPnuc were either dry mounted (DRY) or fixed with 70% ethanol (EtOH) and subsequently frozen at −80°C before analysis. C , Transgenic mice were generated that express a nuclear-targeted EYFP (EYFPnuc) under control of the Thy-1 Promoter (TYNC mice). The Thy-1 minigene was modified by replacing the Thy-1 ORF with sequences coding for EYFPnuc. D , E , Vibratome sections (30 μm) from brains of PFA-fixed TYNC mice were analyzed for YFP fluorescence and for cellular marker gene expression with immunohistochemistry. D , Within the hippocampus, the YFP expression is most prominent in pyramidal neurons of the CA1 field (CA1) and in the granular cell layer of the dentate gyrus (DG). In the CA3 field (CA3), fewer cells are YFP positive. The YFP fluorescence appears to be strictly overlapping with the indirect GFP immune fluorescence analysis (α-GFP), although it is less sensitive. The pan-neuronal marker NeuN (α-NeuN) colocalizes with the nuclear YFP signal. Staining with GFAP (α-GFAP) and parvalbumin (α-Parv) does not reveal YFP overlapping signals. E , Higher-magnification photographs obtained from vibratome sections at the level of the primary motor and sensory cortex. YFP-positive nuclei (YFP) do not colocalize with the interneuronal marker parvalbumin (α-Parv) and GAD67 (α-GAD67) and also not with the astrocyte marker GFAP (α-GFAP). Arrows mark parvalbumin, GAD67, or GFAP-positive cells stained in red in the left panel. No overlap with YFP fluorescence can be detected in the right panel with arrows pointing to identical positions as in the left panel. Scale bar, 50 μm. F–H , Cryosections (8 μm; Cryo) were mounted on PEN foils (Leica) and analyzed for YFP fluorescence. F , In the hippocampus, YFP-positive nuclei can be detected in the granular cell layer (DG) and in the CA1 field of the pyramidal cell layer (CA1). In the CA3 field (CA3), fewer cells are YFP positive. The inset shows the morphology of the hippocampus in bright field. G , Higher magnification of YFP-positive nuclei in the somatosensory cortex. Scale bar, 50 μm. H , In the cortex, cells located in deeper layers show a nuclear YFP signal. The inset shows the morphology of the section at the level of the somatosensory cortex in bright field. I , Total RNA analyzed with a Bioanalyzer (Agilent); each lane represents half of the RNA isolated from one coronal. brain section. Adjacent 8-μ-thick sections were cryomounted on PEN foil slides, dried, and treated as follows: (1) stored for 2 h at room temperature, (2) stored for 8 h at room temperature, (3) stored for 24 h at room temperature, (4) stored for 48 h at room temperature, (5) frozen at −80°C and then kept at room temperature for 2 h, and (6) microdissected regions pooled from eight sections (total area size isolated comparable with one section). The ratio of the 28S versus the 18S rRNA bands was determined with the Bioanalyzer software and for all samples was 1.3± 0.1. The amount of RNA isolated from one coronal 8 μ brain cryosection was ∼50 ng.

    Article Snippet: Microdissected samples were collected in 100 μl of RNA lysis buffer (Qiagen, Hilden, Germany) and stored at room temperature until further processing.

    Techniques: Laser Capture Microdissection, Transgenic Assay, Mouse Assay, Isolation, Expressing, Fluorescence, Staining, In Vivo, Labeling, Transfection, Construct, Generated, Modification, Marker, Immunohistochemistry, Software

    Targeted hydroxymethylation of four different aberrantly methylated genes by dCas9-TET3CD fusion protein in human kidney cells. a Schematic representing hypermethylated RASAL1 promoter region (upper panel) and reactivated RASAL1 expression through induction of RASAL1 promoter hydroxymethylation by dCas9-TET3CD fusion protein in complex with a sgRNA binding to its target region (lower panel). b Schematic of domain structure of the dCas9-TET3CD (upper panel) and dCas9-TETCDi (lower panel) fusion protein. c – e Locations for RASAL1/EYA1/LRFN2- sgRNAs are indicated by thick lines with corresponding PAM in magenta within the human RASAL1/EYA1/LRFN2 gene locus, respectively. Human fibrotic TK188 fibroblasts were transduced with lentivirus expressing demethylation constructs guided by RASAL1 -sgRNAs 1–10, EYA1-sgRNA 1–6 , LRFN2-sgRNA 1–8 , or by LacZ control sgRNA. Results were normalized to reference gene GAPDH. f , g MeDIP and hMeDIP analysis of TK188 cells were transduced with dCas9-TET3CD- RASAL1- sgRNA3. The results were calculated relative to the input. h Bisulfite sequencing summary of promoter methylation status of the RASAL1 gene in TK188 cells transduced with demethylation constructs guided by RASAL1 -sgRNA3, by LacZ control sgRNA or DNA treated with M.SssI serving as positive control. Each data point represents the mean of three independent transduction experiments with error bars indicating the standard error of the mean for six or more bisulfite sequencing results. i Locations for KL- sgRNAs are indicated by thick lines with corresponding PAM in magenta within the human KL gene locus. Three days TGFβ1-treated HK2 cells were transduced with lentivirus expressing demethylation constructs guided by KL -sgRNAs 1–8 or by LacZ control sgRNA. j , k MeDIP and hMeDIP analysis of HK2 cells were transduction with dCas9-TET3CD- KL- sgRNA2. The results were calculated relative to the input. All data are presented as mean value; error bars represent S.D.; n = 3 independent biological replicates, n.s. not significant; * p

    Journal: Nature Communications

    Article Title: High-fidelity CRISPR/Cas9- based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis

    doi: 10.1038/s41467-018-05766-5

    Figure Lengend Snippet: Targeted hydroxymethylation of four different aberrantly methylated genes by dCas9-TET3CD fusion protein in human kidney cells. a Schematic representing hypermethylated RASAL1 promoter region (upper panel) and reactivated RASAL1 expression through induction of RASAL1 promoter hydroxymethylation by dCas9-TET3CD fusion protein in complex with a sgRNA binding to its target region (lower panel). b Schematic of domain structure of the dCas9-TET3CD (upper panel) and dCas9-TETCDi (lower panel) fusion protein. c – e Locations for RASAL1/EYA1/LRFN2- sgRNAs are indicated by thick lines with corresponding PAM in magenta within the human RASAL1/EYA1/LRFN2 gene locus, respectively. Human fibrotic TK188 fibroblasts were transduced with lentivirus expressing demethylation constructs guided by RASAL1 -sgRNAs 1–10, EYA1-sgRNA 1–6 , LRFN2-sgRNA 1–8 , or by LacZ control sgRNA. Results were normalized to reference gene GAPDH. f , g MeDIP and hMeDIP analysis of TK188 cells were transduced with dCas9-TET3CD- RASAL1- sgRNA3. The results were calculated relative to the input. h Bisulfite sequencing summary of promoter methylation status of the RASAL1 gene in TK188 cells transduced with demethylation constructs guided by RASAL1 -sgRNA3, by LacZ control sgRNA or DNA treated with M.SssI serving as positive control. Each data point represents the mean of three independent transduction experiments with error bars indicating the standard error of the mean for six or more bisulfite sequencing results. i Locations for KL- sgRNAs are indicated by thick lines with corresponding PAM in magenta within the human KL gene locus. Three days TGFβ1-treated HK2 cells were transduced with lentivirus expressing demethylation constructs guided by KL -sgRNAs 1–8 or by LacZ control sgRNA. j , k MeDIP and hMeDIP analysis of HK2 cells were transduction with dCas9-TET3CD- KL- sgRNA2. The results were calculated relative to the input. All data are presented as mean value; error bars represent S.D.; n = 3 independent biological replicates, n.s. not significant; * p

    Article Snippet: DNA isolation, MeDIP, and hMeDIP assay Animal tissues or cell pellets were lysed by DNA lysis buffer (Qiagen, Hilden, Germany) and precipitated and purified using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol.

    Techniques: Methylation, Expressing, Binding Assay, Transduction, Construct, Methylated DNA Immunoprecipitation, Methylation Sequencing, Positive Control