rlt buffer  (Qiagen)


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    Name:
    Buffer RLT
    Description:
    For lysis of cells and tissues before RNA isolation. Kit contents: Qiagen Buffer RLT, 220mL, For RNeasy Lysis Buffer for Lysing Cells and Tissues Prior to RNA Isolation and Simultaneous RNA/DNA/Protein Isolation.
    Catalog Number:
    79216
    Price:
    None
    Category:
    Buffer RLT
    Score:
    85
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    Structured Review

    Qiagen rlt buffer
    Buffer RLT
    For lysis of cells and tissues before RNA isolation. Kit contents: Qiagen Buffer RLT, 220mL, For RNeasy Lysis Buffer for Lysing Cells and Tissues Prior to RNA Isolation and Simultaneous RNA/DNA/Protein Isolation.
    https://www.bioz.com/result/rlt buffer/product/Qiagen
    Average 99 stars, based on 677 article reviews
    Price from $9.99 to $1999.99
    rlt buffer - by Bioz Stars, 2020-01
    99/100 stars

    Images

    1) Product Images from "Clearance of Pneumococcal Colonization in Infants Is Delayed through Altered Macrophage Trafficking"

    Article Title: Clearance of Pneumococcal Colonization in Infants Is Delayed through Altered Macrophage Trafficking

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005004

    Depleting the microbiota limits infant Ccl2 expression and accelerates pneumococcal clearance. (A) Breeding pairs were placed on sterile filtered tap water or water containing 5 antibiotics (Abx) for at least 5 days prior to giving birth, and continued until the infant mice were 10d old. Mice were sacrificed, and nasal lavages obtained with PBS. DNA was isolated from lavage fluid, and qPCR used to measure the number of copies of 16S bacterial ribosomal DNA. (B-C) Adult mice were maintained on sterile filtered tap water or water containing 5 antibiotics for at least 2 weeks. Breeding pairs were provided water as above. Mice were sacrificed, and nasal lavages obtained with RLT lysis buffer. RNA was isolated and reverse-transcribed into cDNA, and qRT-PCR used to measure relative expression of Ccl2 (B) and Ccl7 (C) in the nasopharynx. (D) Infant mice were exposed to tap water or antibiotics as in the previously described protocol. Treatments were continued until infant mice were 6d old, then all mice were given tap water to drink. 24 hrs later, mice were inoculated with pneumococci. At 7 dpi, mice were sacrificed and nasal lavages fixed and stained for flow cytometry to measure the number of macrophages present in the airway lumen. (E-F) Infant mice were treated as in (D). At 14d (E) and 7d (F) post-inoculation, mice were sacrificed. Nasal lavages were obtained and plated on media containing neomycin and catalase to measure pneumococcal density in the URT. (G) Ccl2 expression was measured in the nasopharynx of tap- or antibiotic-water exposed conventionally-reared (Conv), or germ-free infant mice. Data are represented as mean +/- SEM. Horizontal lines indicate median values. n.s., not significant. * = p
    Figure Legend Snippet: Depleting the microbiota limits infant Ccl2 expression and accelerates pneumococcal clearance. (A) Breeding pairs were placed on sterile filtered tap water or water containing 5 antibiotics (Abx) for at least 5 days prior to giving birth, and continued until the infant mice were 10d old. Mice were sacrificed, and nasal lavages obtained with PBS. DNA was isolated from lavage fluid, and qPCR used to measure the number of copies of 16S bacterial ribosomal DNA. (B-C) Adult mice were maintained on sterile filtered tap water or water containing 5 antibiotics for at least 2 weeks. Breeding pairs were provided water as above. Mice were sacrificed, and nasal lavages obtained with RLT lysis buffer. RNA was isolated and reverse-transcribed into cDNA, and qRT-PCR used to measure relative expression of Ccl2 (B) and Ccl7 (C) in the nasopharynx. (D) Infant mice were exposed to tap water or antibiotics as in the previously described protocol. Treatments were continued until infant mice were 6d old, then all mice were given tap water to drink. 24 hrs later, mice were inoculated with pneumococci. At 7 dpi, mice were sacrificed and nasal lavages fixed and stained for flow cytometry to measure the number of macrophages present in the airway lumen. (E-F) Infant mice were treated as in (D). At 14d (E) and 7d (F) post-inoculation, mice were sacrificed. Nasal lavages were obtained and plated on media containing neomycin and catalase to measure pneumococcal density in the URT. (G) Ccl2 expression was measured in the nasopharynx of tap- or antibiotic-water exposed conventionally-reared (Conv), or germ-free infant mice. Data are represented as mean +/- SEM. Horizontal lines indicate median values. n.s., not significant. * = p

    Techniques Used: Expressing, Mouse Assay, Isolation, Real-time Polymerase Chain Reaction, Lysis, Quantitative RT-PCR, Staining, Flow Cytometry, Cytometry

    Infant mice do not form a gradient of CCL2 expression during colonization. (A) Adult (42d old) and infant (7d old) mice were inoculated with PBS (mock) or pneumococci. At 3d post-inoculation, nasal lavages were obtained with RLT lysis buffer, and RNA was isolated and reverse transcribed to cDNA. qRT-PCR was performed to measure relative expression of Ccl2 . (B) Serum was obtained from adult and infant mice, and ELISA used to measure CCL2 levels. (C-D) Peritoneal macrophages from adult and infant mice were lysed with RLT buffer and RNA isolated. qRT-PCR was used to measure relative expression of Ccl2 (C) and Ccr2 (D). In (C), cultured macrophages were incubated overnight with PBS (Mock) or heat-killed bacterial lysates (Stim) prior to lysis. (E-H) Uncolonized adult and infant mice were sacrificed, and nasal lavages obtained with RLT lysis buffer. RNA was isolated and reverse-transcribed into cDNA. qRT-PCR was used to measure relative expression of Ccl7 , (E) Il6 , (F) Cxcl1 , (G) and Cxcl2 (H). Data are represented as mean +/- SEM. n.s., not significant. * = p
    Figure Legend Snippet: Infant mice do not form a gradient of CCL2 expression during colonization. (A) Adult (42d old) and infant (7d old) mice were inoculated with PBS (mock) or pneumococci. At 3d post-inoculation, nasal lavages were obtained with RLT lysis buffer, and RNA was isolated and reverse transcribed to cDNA. qRT-PCR was performed to measure relative expression of Ccl2 . (B) Serum was obtained from adult and infant mice, and ELISA used to measure CCL2 levels. (C-D) Peritoneal macrophages from adult and infant mice were lysed with RLT buffer and RNA isolated. qRT-PCR was used to measure relative expression of Ccl2 (C) and Ccr2 (D). In (C), cultured macrophages were incubated overnight with PBS (Mock) or heat-killed bacterial lysates (Stim) prior to lysis. (E-H) Uncolonized adult and infant mice were sacrificed, and nasal lavages obtained with RLT lysis buffer. RNA was isolated and reverse-transcribed into cDNA. qRT-PCR was used to measure relative expression of Ccl7 , (E) Il6 , (F) Cxcl1 , (G) and Cxcl2 (H). Data are represented as mean +/- SEM. n.s., not significant. * = p

    Techniques Used: Mouse Assay, Expressing, Lysis, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Incubation

    CCL2 overexpression increases macrophage recruitment and pneumococcal clearance. (A-C) Mice were inoculated with a control (GFP-expressing) or a CCL2-expressing AAV5 vector at 4d of age, followed by pneumococcal colonization at 7d old. Seven and 21 days later, mice were sacrificed and nasal lavages were obtained with RLT lysis buffer. (A) RNA was isolated and cDNA reverse-transcribed, followed by qRT-PCR to measure the relative expression of Ccl2 in the upper respiratory tract, using primers Ccl2 ORF-F and Ccl2 ORF-R. ELISA was used to measure CCL2 protein levels in nasal lavage (B) and serum (C) at 7 and 21 dpi. (D) Mice were inoculated with a control (GFP-expressing) or a CCL2-expressing AAV5 vector at 4d of age, followed by pneumococcal colonization at 7d of age. Seven days later, mice were sacrificed, nasal lavages obtained and flow cytometry used to measure macrophage recruitment. (E) At 21d post-inoculation, nasal lavages were obtained and plated to measure the pneumococcal load in the nasopharynx. Data are represented as mean +/- SEM. n.s., not significant. * = p
    Figure Legend Snippet: CCL2 overexpression increases macrophage recruitment and pneumococcal clearance. (A-C) Mice were inoculated with a control (GFP-expressing) or a CCL2-expressing AAV5 vector at 4d of age, followed by pneumococcal colonization at 7d old. Seven and 21 days later, mice were sacrificed and nasal lavages were obtained with RLT lysis buffer. (A) RNA was isolated and cDNA reverse-transcribed, followed by qRT-PCR to measure the relative expression of Ccl2 in the upper respiratory tract, using primers Ccl2 ORF-F and Ccl2 ORF-R. ELISA was used to measure CCL2 protein levels in nasal lavage (B) and serum (C) at 7 and 21 dpi. (D) Mice were inoculated with a control (GFP-expressing) or a CCL2-expressing AAV5 vector at 4d of age, followed by pneumococcal colonization at 7d of age. Seven days later, mice were sacrificed, nasal lavages obtained and flow cytometry used to measure macrophage recruitment. (E) At 21d post-inoculation, nasal lavages were obtained and plated to measure the pneumococcal load in the nasopharynx. Data are represented as mean +/- SEM. n.s., not significant. * = p

    Techniques Used: Over Expression, Mouse Assay, Expressing, Plasmid Preparation, Lysis, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry

    2) Product Images from "Influenza promotes pneumococcal growth during co-infection by providing host sialylated substrates as a nutrient source"

    Article Title: Influenza promotes pneumococcal growth during co-infection by providing host sialylated substrates as a nutrient source

    Journal:

    doi: 10.1016/j.chom.2014.06.005

    Influenza promotes pneumococcal colonization, growth and aspiration (A) Mice were infected with influenza for the indicated number of days, nasal lavages with RLT RNA lysis buffer obtained, and qRT-PCR performed to measure the relative expression of x31 viral nucleoprotein (NP). (B) Mice given PBS (mock, open symbols) or influenza (closed symbols) were weighed daily for 7 days, and the percent change in weight from baseline graphed. (C) Mice were infected with influenza or PBS, followed 7 days later by intranasal inoculation with bacteria of the indicated serotype. After 24 hrs post-bacterial inoculation (hpi), nasal lavages were obtained and plated for quantitative culture of colonizing pneumococci. (D) Mice were inoculated with influenza or PBS, followed 7 days later by challenge with strain P1121. Within 1 hr, nasal lavages were obtained and plated for quantitative culture (CFUs). (E) After 7 days of mock (black line) or influenza (grey shaded) infection, mice were inoculated with CFSE-labeled pneumococci for 8 hrs. Nasal lavages were obtained, fixed and stained for pneumococcal capsule, and flow cytometry performed to compare bacterial replication. (F) The median fluorescence intensity (MFI) of CFSE per bacterial cell was calculated for each condition, and displayed as 1/MFI, or number of divisions per cell (division index). (G) Mock or influenza-infected mice were challenged 7 days later with WT pneumococci for 24 hrs. Bronchoalveolar lavages were obtained and plated for quantitative culture. Horizontal lines indicate median values, and data in F are represented as mean +/− SD. n.s. = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001. All experiments were performed at least twice, with 4-11 mice per group. See also .
    Figure Legend Snippet: Influenza promotes pneumococcal colonization, growth and aspiration (A) Mice were infected with influenza for the indicated number of days, nasal lavages with RLT RNA lysis buffer obtained, and qRT-PCR performed to measure the relative expression of x31 viral nucleoprotein (NP). (B) Mice given PBS (mock, open symbols) or influenza (closed symbols) were weighed daily for 7 days, and the percent change in weight from baseline graphed. (C) Mice were infected with influenza or PBS, followed 7 days later by intranasal inoculation with bacteria of the indicated serotype. After 24 hrs post-bacterial inoculation (hpi), nasal lavages were obtained and plated for quantitative culture of colonizing pneumococci. (D) Mice were inoculated with influenza or PBS, followed 7 days later by challenge with strain P1121. Within 1 hr, nasal lavages were obtained and plated for quantitative culture (CFUs). (E) After 7 days of mock (black line) or influenza (grey shaded) infection, mice were inoculated with CFSE-labeled pneumococci for 8 hrs. Nasal lavages were obtained, fixed and stained for pneumococcal capsule, and flow cytometry performed to compare bacterial replication. (F) The median fluorescence intensity (MFI) of CFSE per bacterial cell was calculated for each condition, and displayed as 1/MFI, or number of divisions per cell (division index). (G) Mock or influenza-infected mice were challenged 7 days later with WT pneumococci for 24 hrs. Bronchoalveolar lavages were obtained and plated for quantitative culture. Horizontal lines indicate median values, and data in F are represented as mean +/− SD. n.s. = not significant, * = p < 0.05, ** = p < 0.01, *** = p < 0.001. All experiments were performed at least twice, with 4-11 mice per group. See also .

    Techniques Used: Mouse Assay, Infection, Lysis, Quantitative RT-PCR, Expressing, Labeling, Staining, Flow Cytometry, Cytometry, Fluorescence

    3) Product Images from "Defining a role for sphingosine kinase 1 in p53-dependent tumors"

    Article Title: Defining a role for sphingosine kinase 1 in p53-dependent tumors

    Journal: Oncogene

    doi: 10.1038/onc.2011.302

    Primary mouse cell lines respond to genotoxic stress through p53-dependent, caspase-2 mediated proteolysis of SK1 to alter the balance of pro- and anti-growth sphingolipids. (a) Mouse embryonic fibroblasts (MEFs) expressing WT p53 or with p53 knocked out, were treated with indicated doses of UV radiation (J/m 2 ), cultured for 48 hours, and then whole cell lysate was examined by (a) Western blotting with antibodies to the indicated proteins, or, (b) harvested in RLT buffer, prepared for qRT-PCR, with enzyme expression normalized to β-actin expression for each reaction performed in triplicate, or, (c) WT MEFs were pre-treated with 20 uM z-VDVAD-fmk (caspase-2 inhibitor) dissolved in DMSO or DMSO control in fresh media and incubated for one hour prior to treatment with 0 or 20 J/m 2 UV radiation, incubated for 48 hours before being harvested by scraping on ice using Tris-Triton lysis buffer supplemented with protease and phosphatase inhibitors from Sigma for western blotting with antibodies to the indicated proteins (n=3), or (d) harvested for sphingolipidomic analysis to measure S1P and C 16 -ceramide by LC/MS, expressed as pmol lipid per mg protein ± SEM (n=3, and *p
    Figure Legend Snippet: Primary mouse cell lines respond to genotoxic stress through p53-dependent, caspase-2 mediated proteolysis of SK1 to alter the balance of pro- and anti-growth sphingolipids. (a) Mouse embryonic fibroblasts (MEFs) expressing WT p53 or with p53 knocked out, were treated with indicated doses of UV radiation (J/m 2 ), cultured for 48 hours, and then whole cell lysate was examined by (a) Western blotting with antibodies to the indicated proteins, or, (b) harvested in RLT buffer, prepared for qRT-PCR, with enzyme expression normalized to β-actin expression for each reaction performed in triplicate, or, (c) WT MEFs were pre-treated with 20 uM z-VDVAD-fmk (caspase-2 inhibitor) dissolved in DMSO or DMSO control in fresh media and incubated for one hour prior to treatment with 0 or 20 J/m 2 UV radiation, incubated for 48 hours before being harvested by scraping on ice using Tris-Triton lysis buffer supplemented with protease and phosphatase inhibitors from Sigma for western blotting with antibodies to the indicated proteins (n=3), or (d) harvested for sphingolipidomic analysis to measure S1P and C 16 -ceramide by LC/MS, expressed as pmol lipid per mg protein ± SEM (n=3, and *p

    Techniques Used: Expressing, Cell Culture, Western Blot, Quantitative RT-PCR, Incubation, Lysis, Liquid Chromatography, Mass Spectrometry

    Related Articles

    Methylation Sequencing:

    Article Title: Unique cell-type specific patterns of DNA methylation in the root meristem
    Article Snippet: Sorted cells were collected directly into specific lysis buffers that were compatible with downstream applications. .. Cells used for bisulfite sequencing, mRNA-seq, smRNA-seq were lysed in Buffer AP1 (Qiagen), Buffer RLT (Qiagen), Trizol (Invitrogen). .. All samples were immediately stored at −80 °C until gDNA and RNA was extracted using DNeasy Plant mini kit (Qiagen) and RNeasy Plant mini kit (Qiagen) or Trizol, respectively.

    Cell Isolation:

    Article Title: Unique cell-type specific patterns of DNA methylation in the root meristem
    Article Snippet: Paragraph title: Cell isolation ... Cells used for bisulfite sequencing, mRNA-seq, smRNA-seq were lysed in Buffer AP1 (Qiagen), Buffer RLT (Qiagen), Trizol (Invitrogen).

    Amplification:

    Article Title: Targeted Mutation of NGN3 Gene Disrupts Pancreatic Endocrine Cell Development in Pigs
    Article Snippet: Approximately 30 mg of tissue was lysed using Qiagen buffer RLT with Betamercaptoethanol (BME) and polytron benchtop tissue homogenizer. .. Approximately 30 mg of tissue was lysed using Qiagen buffer RLT with Betamercaptoethanol (BME) and polytron benchtop tissue homogenizer.

    Centrifugation:

    Article Title: In vivo genome-wide profiling reveals a tissue-specific role for 5-formylcytosine
    Article Snippet: Tissues were collected from timed matings with the day of the copulation plug as E0.5. .. Embryonic tissues were lysed in RLT plus buffer (Qiagen), homogenised via centrifugation through a QIAshreddred column (Qiagen) at full speed for 2 min in a microcentrifuge. .. DNA and RNA were isolated using AllPrep DNA/RNA kit (Qiagen) following the manufacturer’s instructions.

    Article Title: Allele-specific transcription factor binding to common and rare variants associated with disease and gene expression
    Article Snippet: Peripheral blood mononuclear cells (PBMCs) were purified from healthy donor buffy coats (Department of Transfusion Medicine, Uppsala University Hospital, Sweden) using Ficoll density-gradient centrifugation. .. The harvested cells were stored in RLT buffer (QIAGEN) at −80 °C.

    Quantitative RT-PCR:

    Article Title: Targeting Endothelial Adhesion Molecule Transcription for Treatment of Inflammatory Disease: A Proof-of-Concept Study
    Article Snippet: At the conclusion of retinal endothelial cell manipulations, culture medium was replaced with Buffer RLT (Qiagen, Hilden, Germany) with 0.55 mM β -mercaptoethanol (Sigma-Aldrich, St. Louis, MO), and cells were frozen at −80°C prior to RNA extraction. .. RNA concentration was determined by spectrophotometry on the NanoDrop 2000 instrument (ThermoFisher Scientific, Wilmington, DE).

    Article Title: Versatility of Biofilm Matrix Molecules in Staphylococcus epidermidis Clinical Isolates and Importance of Polysaccharide Intercellular Adhesin Expression during High Shear Stress
    Article Snippet: Biofilms were harvested by removing TSB from the flow cell chamber and resuspending biofilm cells in 900 µl of RLT buffer (Qiagen)–1% β-mercaptoethanol. .. Biofilms were harvested by removing TSB from the flow cell chamber and resuspending biofilm cells in 900 µl of RLT buffer (Qiagen)–1% β-mercaptoethanol.

    SYBR Green Assay:

    Article Title: Versatility of Biofilm Matrix Molecules in Staphylococcus epidermidis Clinical Isolates and Importance of Polysaccharide Intercellular Adhesin Expression during High Shear Stress
    Article Snippet: Biofilms were harvested by removing TSB from the flow cell chamber and resuspending biofilm cells in 900 µl of RLT buffer (Qiagen)–1% β-mercaptoethanol. .. Biofilms were harvested by removing TSB from the flow cell chamber and resuspending biofilm cells in 900 µl of RLT buffer (Qiagen)–1% β-mercaptoethanol.

    Incubation:

    Article Title: Genetic encoding of DNA nanostructures and their self-assembly in living bacteria
    Article Snippet: The RFP ratio is calculated by dividing the fluorescence with the expression of the RT by that in the absence of the RT (cells containing the same plasmid, including inducible system, but lacking the HIVRT genes). .. Cells were inoculated in 500 μl LB Miller Broth with antibiotics in a 96-well plate covered with a breathable membrane (AeraSeal, Excel Scientific) at 37 °C and 1,000 r.p.m. for 16 h. Overnight cultures were then diluted 1,000-fold by mixing 10 μl of the culture into 10 ml of LB Miller Broth containing the appropriate inducer and incubated at 37 °C and 250 r.p.m. for 18 h. After incubation, the DNA nanostructures were purified using the following protocol: (i) Cells were centrifuged at 5,000g for 7 min at 4 °C; (ii) the supernatant was removed; (iii) cells were resuspended in 200 μl of TE buffer (10 mM Tris-EDTA) containing 3 mg ml−1 of Lysozyme; (iv) 700 μl of RLT buffer (Qiagen, #79216) was added; (v) the resulting solution was centrifuged at 21,130 g for 2 min in order to remove the insoluble materials and the supernatant is transferred to a clean tube; (vi) 500 μl of 100% ethanol was added to the supernatant; (vii) 700 μl of the sample was transferred into a QIAquick Spin Column (Qiagen, #1018215) and centrifuged at 15,000g for 15 s; (viii) step vii was repeated until all the supernatant solution from step vi has passed through the same column tube (the flow through was discarded after each step); (ix) 700 μl of RW1 buffer (Qiagen, # 1053394) was added to the collection tube and centrifuged for 15 s at 15,000g (the flow through was discarded); (x) 500 μl RPE buffer (Qiagen, # 1018013) was pipetted into the column tube and centrifuged for 15 s at 15,000g (the flow through was discarded); (xi) 500 μl Buffer RPE (Mat. .. 1018013 Qiagen) was pipetted into the column tube and centrifuged for 2 min at 15,000g (the flow through was discarded); (xii) the empty column tube was centrifuged for 1 min at 15,000g ; (xiii) the column was placed into a clean tube and 50 μl of water was added; (xiv) the resulting solution was then incubated with RNase A (100 μg ml−1 , Qiagen) in the presence of 150 mM NaCl to recover the DNA–RNA chimera or without salt to recover just the ssDNA.

    Article Title: Allele-specific transcription factor binding to common and rare variants associated with disease and gene expression
    Article Snippet: The cells were stimulated with a phosphorothioate-modified CpG A oligonucleotide ODN2216 (CyberGene) at the concentration of 3 μg/ml and incubated for 5 h at 37 °C with 5 % CO2 . .. The harvested cells were stored in RLT buffer (QIAGEN) at −80 °C.

    Expressing:

    Article Title: Targeted Mutation of NGN3 Gene Disrupts Pancreatic Endocrine Cell Development in Pigs
    Article Snippet: Paragraph title: Gene expression analysis using RT-PCR ... Approximately 30 mg of tissue was lysed using Qiagen buffer RLT with Betamercaptoethanol (BME) and polytron benchtop tissue homogenizer.

    Article Title: S100A8 promotes migration and infiltration of inflammatory cells in acute anterior uveitis
    Article Snippet: Next, the iris-ciliary body tissue was isolated from the stabilized eye, and then homogenized with a rotor- stator homogenizer in buffer RLT (Qiagen). .. Next, the iris-ciliary body tissue was isolated from the stabilized eye, and then homogenized with a rotor- stator homogenizer in buffer RLT (Qiagen).

    Article Title: Does Caspase-6 Have a Role in Perinatal Brain Injury?
    Article Snippet: The entire supernatant was collected (1 ml) at 6, 24 or 48 h; 300 μl was used immediately for a lactate dehydrogenase assay (Roche, USA) and the remaining supernatant stored at −80°C until further analysis. .. Cells were washed with RNAse-free PBS and then lysed with 350-μl buffer RLT (Qiagen; containing β-mercaptoethanol at a concentration of 1:100), harvested and stored at −80°C for gene expression analysis. .. All time points were run in each independent experiment and all group experiments were performed a minimum of 3-6 independent times.

    Article Title: A phylogenomic study quantifies competing mechanisms for pseudogenization in prokaryotes—The Mycobacterium leprae case
    Article Snippet: Paragraph title: Expression analysis ... Briefly, frozen tissue sections of nine lepromatous leprosy skin lesions, taken at the time of diagnosis after written consent was obtained and before any treatment was started, were lysed in Qiagen RLT buffer (Qiagen, Hilden, Germany) and homogenized with silica beads.

    Article Title: Gene expression of INPP5F as an independent prognostic marker in fludarabine-based therapy of chronic lymphocytic leukemia
    Article Snippet: Paragraph title: Gene expression profiling ... CLL samples were positively enriched by magnetic cell sorting using CD19 microbeads and MACS columns (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and resuspended in RLT buffer (Qiagen, Crawley, West Sussex, UK).

    Article Title: Versatility of Biofilm Matrix Molecules in Staphylococcus epidermidis Clinical Isolates and Importance of Polysaccharide Intercellular Adhesin Expression during High Shear Stress
    Article Snippet: Biofilms were harvested by removing TSB from the flow cell chamber and resuspending biofilm cells in 900 µl of RLT buffer (Qiagen)–1% β-mercaptoethanol. .. Biofilms were harvested by removing TSB from the flow cell chamber and resuspending biofilm cells in 900 µl of RLT buffer (Qiagen)–1% β-mercaptoethanol.

    Article Title: Cardiac Non-myocyte Cells Show Enhanced Pharmacological Function Suggestive of Contractile Maturity in Stem Cell Derived Cardiomyocyte Microtissues
    Article Snippet: Paragraph title: Gene expression analysis ... All primary cells were detached with prewarmed Accutase (Sigma, A6964) for 5 min at 37°C, 5% CO2 , centrifuged for 3 min at 1200 ×g before lysis in RLT buffer (Qiagen) and stored at −80 °C until processing.

    Flow Cytometry:

    Article Title: Genetic encoding of DNA nanostructures and their self-assembly in living bacteria
    Article Snippet: The RFP ratio is calculated by dividing the fluorescence with the expression of the RT by that in the absence of the RT (cells containing the same plasmid, including inducible system, but lacking the HIVRT genes). .. Cells were inoculated in 500 μl LB Miller Broth with antibiotics in a 96-well plate covered with a breathable membrane (AeraSeal, Excel Scientific) at 37 °C and 1,000 r.p.m. for 16 h. Overnight cultures were then diluted 1,000-fold by mixing 10 μl of the culture into 10 ml of LB Miller Broth containing the appropriate inducer and incubated at 37 °C and 250 r.p.m. for 18 h. After incubation, the DNA nanostructures were purified using the following protocol: (i) Cells were centrifuged at 5,000g for 7 min at 4 °C; (ii) the supernatant was removed; (iii) cells were resuspended in 200 μl of TE buffer (10 mM Tris-EDTA) containing 3 mg ml−1 of Lysozyme; (iv) 700 μl of RLT buffer (Qiagen, #79216) was added; (v) the resulting solution was centrifuged at 21,130 g for 2 min in order to remove the insoluble materials and the supernatant is transferred to a clean tube; (vi) 500 μl of 100% ethanol was added to the supernatant; (vii) 700 μl of the sample was transferred into a QIAquick Spin Column (Qiagen, #1018215) and centrifuged at 15,000g for 15 s; (viii) step vii was repeated until all the supernatant solution from step vi has passed through the same column tube (the flow through was discarded after each step); (ix) 700 μl of RW1 buffer (Qiagen, # 1053394) was added to the collection tube and centrifuged for 15 s at 15,000g (the flow through was discarded); (x) 500 μl RPE buffer (Qiagen, # 1018013) was pipetted into the column tube and centrifuged for 15 s at 15,000g (the flow through was discarded); (xi) 500 μl Buffer RPE (Mat. .. 1018013 Qiagen) was pipetted into the column tube and centrifuged for 2 min at 15,000g (the flow through was discarded); (xii) the empty column tube was centrifuged for 1 min at 15,000g ; (xiii) the column was placed into a clean tube and 50 μl of water was added; (xiv) the resulting solution was then incubated with RNase A (100 μg ml−1 , Qiagen) in the presence of 150 mM NaCl to recover the DNA–RNA chimera or without salt to recover just the ssDNA.

    Article Title: Versatility of Biofilm Matrix Molecules in Staphylococcus epidermidis Clinical Isolates and Importance of Polysaccharide Intercellular Adhesin Expression during High Shear Stress
    Article Snippet: Biofilms were grown at 37°C in 100% TSB under conditions of constant flow for 24 h at a rate of either 0.25 ml/min (“slow”) or 2 ml/min (“fast”). .. Biofilms were harvested by removing TSB from the flow cell chamber and resuspending biofilm cells in 900 µl of RLT buffer (Qiagen)–1% β-mercaptoethanol. .. Cells were disrupted using a FastPrep FP120 device (Thermo Scientific).

    Cell Culture:

    Article Title: The transcription factors SOX9 and SOX5/SOX6 cooperate genome-wide through super-enhancers to drive chondrogenesis
    Article Snippet: RCS cells were harvested at 95% confluence in Buffer RLT (Qiagen). .. Total RNA was extracted and purified using RNeasy Mini Kit (Qiagen).

    Article Title: Allele-specific transcription factor binding to common and rare variants associated with disease and gene expression
    Article Snippet: The cells were cultured in 1 ml volumes in 24-well plates (Nunc) in macrophage serum-free medium (Life Technologies) at the concentration of 3 × 106 B cells/ml. .. The harvested cells were stored in RLT buffer (QIAGEN) at −80 °C.

    Article Title: Cardiac Non-myocyte Cells Show Enhanced Pharmacological Function Suggestive of Contractile Maturity in Stem Cell Derived Cardiomyocyte Microtissues
    Article Snippet: Adult left ventricle heart total RNA was obtained from one 21-year old normal male donor with no reported concomitant disease. hESC-CM total RNA was obtained from hESC-CMs cultured as described in . .. All primary cells were detached with prewarmed Accutase (Sigma, A6964) for 5 min at 37°C, 5% CO2 , centrifuged for 3 min at 1200 ×g before lysis in RLT buffer (Qiagen) and stored at −80 °C until processing.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Targeted Mutation of NGN3 Gene Disrupts Pancreatic Endocrine Cell Development in Pigs
    Article Snippet: Paragraph title: Gene expression analysis using RT-PCR ... Approximately 30 mg of tissue was lysed using Qiagen buffer RLT with Betamercaptoethanol (BME) and polytron benchtop tissue homogenizer.

    Lactate Dehydrogenase Assay:

    Article Title: Does Caspase-6 Have a Role in Perinatal Brain Injury?
    Article Snippet: Cells were washed with RNAse-free PBS and then lysed with 350-μl buffer RLT (Qiagen; containing β-mercaptoethanol at a concentration of 1:100), harvested and stored at −80°C for gene expression analysis. .. Cells were washed with RNAse-free PBS and then lysed with 350-μl buffer RLT (Qiagen; containing β-mercaptoethanol at a concentration of 1:100), harvested and stored at −80°C for gene expression analysis.

    Generated:

    Article Title: Versatility of Biofilm Matrix Molecules in Staphylococcus epidermidis Clinical Isolates and Importance of Polysaccharide Intercellular Adhesin Expression during High Shear Stress
    Article Snippet: Biofilms were harvested by removing TSB from the flow cell chamber and resuspending biofilm cells in 900 µl of RLT buffer (Qiagen)–1% β-mercaptoethanol. .. Biofilms were harvested by removing TSB from the flow cell chamber and resuspending biofilm cells in 900 µl of RLT buffer (Qiagen)–1% β-mercaptoethanol.

    other:

    Article Title: Adaptation of Laser Microdissection Technique for the Study of a Spontaneous Metastatic Mammary Carcinoma Mouse Model by NanoString Technologies
    Article Snippet: The decrease of lysate volume (below 4 μl) during removal of cutout membranes from the tube can be adjusted with buffer RLT.

    Polymerase Chain Reaction:

    Article Title: Targeted Mutation of NGN3 Gene Disrupts Pancreatic Endocrine Cell Development in Pigs
    Article Snippet: Approximately 30 mg of tissue was lysed using Qiagen buffer RLT with Betamercaptoethanol (BME) and polytron benchtop tissue homogenizer. .. Approximately 30 mg of tissue was lysed using Qiagen buffer RLT with Betamercaptoethanol (BME) and polytron benchtop tissue homogenizer.

    Article Title: Targeting Endothelial Adhesion Molecule Transcription for Treatment of Inflammatory Disease: A Proof-of-Concept Study
    Article Snippet: Paragraph title: 2.3. RNA Isolation, Reverse Transcription, and Polymerase Chain Reaction ... At the conclusion of retinal endothelial cell manipulations, culture medium was replaced with Buffer RLT (Qiagen, Hilden, Germany) with 0.55 mM β -mercaptoethanol (Sigma-Aldrich, St. Louis, MO), and cells were frozen at −80°C prior to RNA extraction.

    Magnetic Cell Separation:

    Article Title: Gene expression of INPP5F as an independent prognostic marker in fludarabine-based therapy of chronic lymphocytic leukemia
    Article Snippet: Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll density gradient centrifugation. .. CLL samples were positively enriched by magnetic cell sorting using CD19 microbeads and MACS columns (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and resuspended in RLT buffer (Qiagen, Crawley, West Sussex, UK). .. In addition, as a validation set, PBMC samples (n =92) were processed into RLT buffer without CD19 enrichment.

    Nucleic Acid Electrophoresis:

    Article Title: Normalization with Corresponding Naïve Tissue Minimizes Bias Caused by Commercial Reverse Transcription Kits on Quantitative Real-Time PCR Results
    Article Snippet: For RNA extraction, brain tissue samples were lysed with RLT-buffer (Qiagen, Hilden, Germany) and homogenized with a MM300 mill mixer (Retsch, Haan, Germany) at 30 Hz for 2 minutes. .. Then, we isolated the total RNA from the lysed and homogenized cells by using a RNeasy Lipid Tissue Mini Kit (Qiagen) according to the manufacturer's instructions and eluted these with 30 μL to 50 μL RNase-free water.

    RNA Sequencing Assay:

    Article Title: The transcription factors SOX9 and SOX5/SOX6 cooperate genome-wide through super-enhancers to drive chondrogenesis
    Article Snippet: Paragraph title: RNA-seq assay and data analysis ... RCS cells were harvested at 95% confluence in Buffer RLT (Qiagen).

    Article Title: A phylogenomic study quantifies competing mechanisms for pseudogenization in prokaryotes—The Mycobacterium leprae case
    Article Snippet: RNA sequencing data of leprosy skin lesions was analyzed from [ ]. .. Briefly, frozen tissue sections of nine lepromatous leprosy skin lesions, taken at the time of diagnosis after written consent was obtained and before any treatment was started, were lysed in Qiagen RLT buffer (Qiagen, Hilden, Germany) and homogenized with silica beads.

    Isolation:

    Article Title: Targeted Mutation of NGN3 Gene Disrupts Pancreatic Endocrine Cell Development in Pigs
    Article Snippet: Pancreas tissue was isolated from fetal pigs at day 60 d.p.c. and immediately placed into RNALater Solution (Ambion). .. Approximately 30 mg of tissue was lysed using Qiagen buffer RLT with Betamercaptoethanol (BME) and polytron benchtop tissue homogenizer.

    Article Title: S100A8 promotes migration and infiltration of inflammatory cells in acute anterior uveitis
    Article Snippet: The eye globe was enucleated and immediately submerged in RNA stabilization reagent (Qiagen, Hilden, Germany). .. Next, the iris-ciliary body tissue was isolated from the stabilized eye, and then homogenized with a rotor- stator homogenizer in buffer RLT (Qiagen). .. Total RNA was extracted using an RNeasy Protect Mini Kit (Qiagen) and treated with RNase- free DNase Set (Qiagen) to remove any residual genomic DNA. cDNA from each sample was obtained by reverse transcription with random hexamers using MultiScribe reverse transcriptase (Thermo Fisher Scientific, Waltham, MA).

    Article Title: Targeting Endothelial Adhesion Molecule Transcription for Treatment of Inflammatory Disease: A Proof-of-Concept Study
    Article Snippet: Paragraph title: 2.3. RNA Isolation, Reverse Transcription, and Polymerase Chain Reaction ... At the conclusion of retinal endothelial cell manipulations, culture medium was replaced with Buffer RLT (Qiagen, Hilden, Germany) with 0.55 mM β -mercaptoethanol (Sigma-Aldrich, St. Louis, MO), and cells were frozen at −80°C prior to RNA extraction.

    Article Title: Allele-specific transcription factor binding to common and rare variants associated with disease and gene expression
    Article Snippet: The B cells were isolated from PBMCs by positive selection using CD19+ B-cell isolation kit (Miltenyi Biotec) according to manufacturer’s instructions. .. The harvested cells were stored in RLT buffer (QIAGEN) at −80 °C.

    Article Title: Gene expression of INPP5F as an independent prognostic marker in fludarabine-based therapy of chronic lymphocytic leukemia
    Article Snippet: CLL samples were positively enriched by magnetic cell sorting using CD19 microbeads and MACS columns (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and resuspended in RLT buffer (Qiagen, Crawley, West Sussex, UK). .. CLL samples were positively enriched by magnetic cell sorting using CD19 microbeads and MACS columns (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and resuspended in RLT buffer (Qiagen, Crawley, West Sussex, UK).

    Article Title: A virosomal respiratory syncytial virus vaccine adjuvanted with monophosphoryl lipid A provides protection against viral challenge without priming for enhanced disease in cotton rats) A virosomal respiratory syncytial virus vaccine adjuvanted with monophosphoryl lipid A provides protection against viral challenge without priming for enhanced disease in cotton rats
    Article Snippet: Paragraph title: Lung RNA isolation ... Lung samples stored in RNA later (Qiagen) were transferred to tubes containing 300 μl RLT buffer (Qiagen) and homogenized using disposable pestles with a pestle motor (VWR, Amsterdam, the Netherlands).

    Article Title: Normalization with Corresponding Naïve Tissue Minimizes Bias Caused by Commercial Reverse Transcription Kits on Quantitative Real-Time PCR Results
    Article Snippet: Paragraph title: RNA isolation, quality control and cDNA synthesis ... For RNA extraction, brain tissue samples were lysed with RLT-buffer (Qiagen, Hilden, Germany) and homogenized with a MM300 mill mixer (Retsch, Haan, Germany) at 30 Hz for 2 minutes.

    Article Title: Versatility of Biofilm Matrix Molecules in Staphylococcus epidermidis Clinical Isolates and Importance of Polysaccharide Intercellular Adhesin Expression during High Shear Stress
    Article Snippet: Biofilms were harvested by removing TSB from the flow cell chamber and resuspending biofilm cells in 900 µl of RLT buffer (Qiagen)–1% β-mercaptoethanol. .. Biofilms were harvested by removing TSB from the flow cell chamber and resuspending biofilm cells in 900 µl of RLT buffer (Qiagen)–1% β-mercaptoethanol.

    Article Title: Suppression of ischemia in arterial occlusive disease by JNK-promoted native collateral artery development
    Article Snippet: Total RNA was extracted with TRIzol (Life Technologies) and was purified using an RNeasy kit (Qiagen). .. RNA from cells and other tissues homogenized in RLT buffer (Qiagen) was isolated using the RNeasy kit. .. We used purified RNA to prepare cDNA using The High Capacity Reverse Transcription Kit (Life Technologies).

    Article Title: Pathogenic Differences between Nipah Virus Bangladesh and Malaysia Strains in Primates: Implications for Antibody Therapy
    Article Snippet: Paragraph title: RNA Isolation ... RNAlater was completely removed, and tissues were homogenized in 600 μl RLT buffer (Qiagen) in a 2-mL cryovial using a tissue lyser (Qiagen) and ceramic beads.

    RNA Extraction:

    Article Title: Targeting Endothelial Adhesion Molecule Transcription for Treatment of Inflammatory Disease: A Proof-of-Concept Study
    Article Snippet: The THP-1 human leukocyte line (American Type Culture Collection, Manassas, VA) was maintained in suspension in RPMI-1640 medium (ThermoFisher-GIBCO, Grand Island, NY) supplemented with 10% FBS and 0.05 mM 2-mercaptoethanol. .. At the conclusion of retinal endothelial cell manipulations, culture medium was replaced with Buffer RLT (Qiagen, Hilden, Germany) with 0.55 mM β -mercaptoethanol (Sigma-Aldrich, St. Louis, MO), and cells were frozen at −80°C prior to RNA extraction. .. Total RNA was extracted using the RNeasy mini kit (Qiagen), according to the manufacturer's instructions and including the optional on-column DNase treatment.

    Article Title: In vivo genome-wide profiling reveals a tissue-specific role for 5-formylcytosine
    Article Snippet: Paragraph title: DNA and RNA extraction ... Embryonic tissues were lysed in RLT plus buffer (Qiagen), homogenised via centrifugation through a QIAshreddred column (Qiagen) at full speed for 2 min in a microcentrifuge.

    Article Title: Gene expression of INPP5F as an independent prognostic marker in fludarabine-based therapy of chronic lymphocytic leukemia
    Article Snippet: CLL samples were positively enriched by magnetic cell sorting using CD19 microbeads and MACS columns (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and resuspended in RLT buffer (Qiagen, Crawley, West Sussex, UK). .. CLL samples were positively enriched by magnetic cell sorting using CD19 microbeads and MACS columns (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and resuspended in RLT buffer (Qiagen, Crawley, West Sussex, UK).

    Article Title: Normalization with Corresponding Naïve Tissue Minimizes Bias Caused by Commercial Reverse Transcription Kits on Quantitative Real-Time PCR Results
    Article Snippet: The right upper regions were collected, frozen in liquid nitrogen and stored at -80°C. .. For RNA extraction, brain tissue samples were lysed with RLT-buffer (Qiagen, Hilden, Germany) and homogenized with a MM300 mill mixer (Retsch, Haan, Germany) at 30 Hz for 2 minutes. .. Then, we isolated the total RNA from the lysed and homogenized cells by using a RNeasy Lipid Tissue Mini Kit (Qiagen) according to the manufacturer's instructions and eluted these with 30 μL to 50 μL RNase-free water.

    Article Title: Pathogenic Differences between Nipah Virus Bangladesh and Malaysia Strains in Primates: Implications for Antibody Therapy
    Article Snippet: Immediately following sampling, 100 μl of blood was added to 600 μl of AVL viral lysis buffer (Qiagen) for RNA extraction. .. RNAlater was completely removed, and tissues were homogenized in 600 μl RLT buffer (Qiagen) in a 2-mL cryovial using a tissue lyser (Qiagen) and ceramic beads.

    Purification:

    Article Title: The transcription factors SOX9 and SOX5/SOX6 cooperate genome-wide through super-enhancers to drive chondrogenesis
    Article Snippet: RCS cells were harvested at 95% confluence in Buffer RLT (Qiagen). .. RCS cells were harvested at 95% confluence in Buffer RLT (Qiagen).

    Article Title: Genetic encoding of DNA nanostructures and their self-assembly in living bacteria
    Article Snippet: The RFP ratio is calculated by dividing the fluorescence with the expression of the RT by that in the absence of the RT (cells containing the same plasmid, including inducible system, but lacking the HIVRT genes). .. Cells were inoculated in 500 μl LB Miller Broth with antibiotics in a 96-well plate covered with a breathable membrane (AeraSeal, Excel Scientific) at 37 °C and 1,000 r.p.m. for 16 h. Overnight cultures were then diluted 1,000-fold by mixing 10 μl of the culture into 10 ml of LB Miller Broth containing the appropriate inducer and incubated at 37 °C and 250 r.p.m. for 18 h. After incubation, the DNA nanostructures were purified using the following protocol: (i) Cells were centrifuged at 5,000g for 7 min at 4 °C; (ii) the supernatant was removed; (iii) cells were resuspended in 200 μl of TE buffer (10 mM Tris-EDTA) containing 3 mg ml−1 of Lysozyme; (iv) 700 μl of RLT buffer (Qiagen, #79216) was added; (v) the resulting solution was centrifuged at 21,130 g for 2 min in order to remove the insoluble materials and the supernatant is transferred to a clean tube; (vi) 500 μl of 100% ethanol was added to the supernatant; (vii) 700 μl of the sample was transferred into a QIAquick Spin Column (Qiagen, #1018215) and centrifuged at 15,000g for 15 s; (viii) step vii was repeated until all the supernatant solution from step vi has passed through the same column tube (the flow through was discarded after each step); (ix) 700 μl of RW1 buffer (Qiagen, # 1053394) was added to the collection tube and centrifuged for 15 s at 15,000g (the flow through was discarded); (x) 500 μl RPE buffer (Qiagen, # 1018013) was pipetted into the column tube and centrifuged for 15 s at 15,000g (the flow through was discarded); (xi) 500 μl Buffer RPE (Mat. .. 1018013 Qiagen) was pipetted into the column tube and centrifuged for 2 min at 15,000g (the flow through was discarded); (xii) the empty column tube was centrifuged for 1 min at 15,000g ; (xiii) the column was placed into a clean tube and 50 μl of water was added; (xiv) the resulting solution was then incubated with RNase A (100 μg ml−1 , Qiagen) in the presence of 150 mM NaCl to recover the DNA–RNA chimera or without salt to recover just the ssDNA.

    Article Title: Allele-specific transcription factor binding to common and rare variants associated with disease and gene expression
    Article Snippet: Peripheral blood mononuclear cells (PBMCs) were purified from healthy donor buffy coats (Department of Transfusion Medicine, Uppsala University Hospital, Sweden) using Ficoll density-gradient centrifugation. .. The harvested cells were stored in RLT buffer (QIAGEN) at −80 °C.

    Article Title: Versatility of Biofilm Matrix Molecules in Staphylococcus epidermidis Clinical Isolates and Importance of Polysaccharide Intercellular Adhesin Expression during High Shear Stress
    Article Snippet: Biofilms were harvested by removing TSB from the flow cell chamber and resuspending biofilm cells in 900 µl of RLT buffer (Qiagen)–1% β-mercaptoethanol. .. Biofilms were harvested by removing TSB from the flow cell chamber and resuspending biofilm cells in 900 µl of RLT buffer (Qiagen)–1% β-mercaptoethanol.

    Article Title: Suppression of ischemia in arterial occlusive disease by JNK-promoted native collateral artery development
    Article Snippet: Total RNA was extracted with TRIzol (Life Technologies) and was purified using an RNeasy kit (Qiagen). .. RNA from cells and other tissues homogenized in RLT buffer (Qiagen) was isolated using the RNeasy kit.

    Sequencing:

    Article Title: PEGylated IL-10 Activates Kupffer Cells to Control Hypercholesterolemia
    Article Snippet: Mouse livers were ground up in Buffer RLT (QIAGEN) with 10 μL β-mercaptoethanol (Sigma-Aldrich) using pestles (VWR), after which RNA was extracted using an RNeasy kit (QIAGEN) according to the manufacturer’s instructions. .. The purified RNA was used as template for RT-PCR using an RT2 First Strand kit (QIAGEN).

    FACS:

    Article Title: Unique cell-type specific patterns of DNA methylation in the root meristem
    Article Snippet: Fluorescent Activated Cell Sorting (FACS) was performed using cell specific GFP lines as described previously . .. Cells used for bisulfite sequencing, mRNA-seq, smRNA-seq were lysed in Buffer AP1 (Qiagen), Buffer RLT (Qiagen), Trizol (Invitrogen).

    Article Title: Gene expression of INPP5F as an independent prognostic marker in fludarabine-based therapy of chronic lymphocytic leukemia
    Article Snippet: Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll density gradient centrifugation. .. CLL samples were positively enriched by magnetic cell sorting using CD19 microbeads and MACS columns (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and resuspended in RLT buffer (Qiagen, Crawley, West Sussex, UK). .. In addition, as a validation set, PBMC samples (n =92) were processed into RLT buffer without CD19 enrichment.

    Blocking Assay:

    Article Title: Suppression of ischemia in arterial occlusive disease by JNK-promoted native collateral artery development
    Article Snippet: Hindlimb adductor and calf skeletal muscles were harvested en block, snap frozen in liquid nitrogen, and then pulverized into a powder using a CryoPREP impactor (Covaris (Woburn, MA)). .. RNA from cells and other tissues homogenized in RLT buffer (Qiagen) was isolated using the RNeasy kit.

    Lysis:

    Article Title: S100A8 promotes migration and infiltration of inflammatory cells in acute anterior uveitis
    Article Snippet: Leukocytes were isolated using SV RNA red blood cell lysis solution (Promega, Madison, WI), and total RNA was extracted using the SV total RNA isolation system (Promega). .. Next, the iris-ciliary body tissue was isolated from the stabilized eye, and then homogenized with a rotor- stator homogenizer in buffer RLT (Qiagen).

    Article Title: Unique cell-type specific patterns of DNA methylation in the root meristem
    Article Snippet: Cells used for bisulfite sequencing, mRNA-seq, smRNA-seq were lysed in Buffer AP1 (Qiagen), Buffer RLT (Qiagen), Trizol (Invitrogen). .. Cells used for bisulfite sequencing, mRNA-seq, smRNA-seq were lysed in Buffer AP1 (Qiagen), Buffer RLT (Qiagen), Trizol (Invitrogen).

    Article Title: Cardiac Non-myocyte Cells Show Enhanced Pharmacological Function Suggestive of Contractile Maturity in Stem Cell Derived Cardiomyocyte Microtissues
    Article Snippet: RNA was harvested following 72 h in culture as per the recommended manufacturer instructions. .. All primary cells were detached with prewarmed Accutase (Sigma, A6964) for 5 min at 37°C, 5% CO2 , centrifuged for 3 min at 1200 ×g before lysis in RLT buffer (Qiagen) and stored at −80 °C until processing. .. Microtissues were pooled and transferred into a falcon tube, centrifuged at 1200 ×g for 2 mins and re-suspended in prewarmed PBS.

    Article Title: Pathogenic Differences between Nipah Virus Bangladesh and Malaysia Strains in Primates: Implications for Antibody Therapy
    Article Snippet: Immediately following sampling, 100 μl of blood was added to 600 μl of AVL viral lysis buffer (Qiagen) for RNA extraction. .. RNAlater was completely removed, and tissues were homogenized in 600 μl RLT buffer (Qiagen) in a 2-mL cryovial using a tissue lyser (Qiagen) and ceramic beads.

    Agarose Gel Electrophoresis:

    Article Title: Targeted Mutation of NGN3 Gene Disrupts Pancreatic Endocrine Cell Development in Pigs
    Article Snippet: Approximately 30 mg of tissue was lysed using Qiagen buffer RLT with Betamercaptoethanol (BME) and polytron benchtop tissue homogenizer. .. Approximately 30 mg of tissue was lysed using Qiagen buffer RLT with Betamercaptoethanol (BME) and polytron benchtop tissue homogenizer.

    Mouse Assay:

    Article Title: Suppression of ischemia in arterial occlusive disease by JNK-promoted native collateral artery development
    Article Snippet: To isolate RNA from tissues, mice were perfusion cleared with PBS via the left ventricle. .. RNA from cells and other tissues homogenized in RLT buffer (Qiagen) was isolated using the RNeasy kit.

    Real-time Polymerase Chain Reaction:

    Article Title: S100A8 promotes migration and infiltration of inflammatory cells in acute anterior uveitis
    Article Snippet: Paragraph title: Real-time PCR ... Next, the iris-ciliary body tissue was isolated from the stabilized eye, and then homogenized with a rotor- stator homogenizer in buffer RLT (Qiagen).

    Article Title: Targeting Endothelial Adhesion Molecule Transcription for Treatment of Inflammatory Disease: A Proof-of-Concept Study
    Article Snippet: At the conclusion of retinal endothelial cell manipulations, culture medium was replaced with Buffer RLT (Qiagen, Hilden, Germany) with 0.55 mM β -mercaptoethanol (Sigma-Aldrich, St. Louis, MO), and cells were frozen at −80°C prior to RNA extraction. .. At the conclusion of retinal endothelial cell manipulations, culture medium was replaced with Buffer RLT (Qiagen, Hilden, Germany) with 0.55 mM β -mercaptoethanol (Sigma-Aldrich, St. Louis, MO), and cells were frozen at −80°C prior to RNA extraction.

    Article Title: PEGylated IL-10 Activates Kupffer Cells to Control Hypercholesterolemia
    Article Snippet: Paragraph title: qPCR analysis ... Mouse livers were ground up in Buffer RLT (QIAGEN) with 10 μL β-mercaptoethanol (Sigma-Aldrich) using pestles (VWR), after which RNA was extracted using an RNeasy kit (QIAGEN) according to the manufacturer’s instructions.

    Article Title: Versatility of Biofilm Matrix Molecules in Staphylococcus epidermidis Clinical Isolates and Importance of Polysaccharide Intercellular Adhesin Expression during High Shear Stress
    Article Snippet: Biofilms were harvested by removing TSB from the flow cell chamber and resuspending biofilm cells in 900 µl of RLT buffer (Qiagen)–1% β-mercaptoethanol. .. Biofilms were harvested by removing TSB from the flow cell chamber and resuspending biofilm cells in 900 µl of RLT buffer (Qiagen)–1% β-mercaptoethanol.

    Negative Control:

    Article Title: Does Caspase-6 Have a Role in Perinatal Brain Injury?
    Article Snippet: Cells were washed with RNAse-free PBS and then lysed with 350-μl buffer RLT (Qiagen; containing β-mercaptoethanol at a concentration of 1:100), harvested and stored at −80°C for gene expression analysis. .. Cells were washed with RNAse-free PBS and then lysed with 350-μl buffer RLT (Qiagen; containing β-mercaptoethanol at a concentration of 1:100), harvested and stored at −80°C for gene expression analysis.

    Selection:

    Article Title: Allele-specific transcription factor binding to common and rare variants associated with disease and gene expression
    Article Snippet: The B cells were isolated from PBMCs by positive selection using CD19+ B-cell isolation kit (Miltenyi Biotec) according to manufacturer’s instructions. .. The harvested cells were stored in RLT buffer (QIAGEN) at −80 °C.

    Sample Prep:

    Article Title: Adaptation of Laser Microdissection Technique for the Study of a Spontaneous Metastatic Mammary Carcinoma Mouse Model by NanoString Technologies
    Article Snippet: To evaluate the LCM workflow and the number of sections required for collection of 100 ng of RNA we conducted a pilot study for RNA yield and quality. .. Following the described above approach to NanoString sample preparation we acquired 5μl lysates from pilot tissues, adjusted their volumes to 350μl with buffer RLT, then extracted RNA and qualified it as described above. .. Our LCM slide preparation, dissection and NanoString lysate preparation protocols effectively preserved RNA in the LCM targets ( ).

    In Vitro:

    Article Title: Does Caspase-6 Have a Role in Perinatal Brain Injury?
    Article Snippet: Paragraph title: In vitro Lipopolysaccharide and Q-VD-OPh Stimulation ... Cells were washed with RNAse-free PBS and then lysed with 350-μl buffer RLT (Qiagen; containing β-mercaptoethanol at a concentration of 1:100), harvested and stored at −80°C for gene expression analysis.

    Electrophoresis:

    Article Title: Targeted Mutation of NGN3 Gene Disrupts Pancreatic Endocrine Cell Development in Pigs
    Article Snippet: Approximately 30 mg of tissue was lysed using Qiagen buffer RLT with Betamercaptoethanol (BME) and polytron benchtop tissue homogenizer. .. Approximately 30 mg of tissue was lysed using Qiagen buffer RLT with Betamercaptoethanol (BME) and polytron benchtop tissue homogenizer.

    Article Title: Genetic encoding of DNA nanostructures and their self-assembly in living bacteria
    Article Snippet: Cells were inoculated in 500 μl LB Miller Broth with antibiotics in a 96-well plate covered with a breathable membrane (AeraSeal, Excel Scientific) at 37 °C and 1,000 r.p.m. for 16 h. Overnight cultures were then diluted 1,000-fold by mixing 10 μl of the culture into 10 ml of LB Miller Broth containing the appropriate inducer and incubated at 37 °C and 250 r.p.m. for 18 h. After incubation, the DNA nanostructures were purified using the following protocol: (i) Cells were centrifuged at 5,000g for 7 min at 4 °C; (ii) the supernatant was removed; (iii) cells were resuspended in 200 μl of TE buffer (10 mM Tris-EDTA) containing 3 mg ml−1 of Lysozyme; (iv) 700 μl of RLT buffer (Qiagen, #79216) was added; (v) the resulting solution was centrifuged at 21,130 g for 2 min in order to remove the insoluble materials and the supernatant is transferred to a clean tube; (vi) 500 μl of 100% ethanol was added to the supernatant; (vii) 700 μl of the sample was transferred into a QIAquick Spin Column (Qiagen, #1018215) and centrifuged at 15,000g for 15 s; (viii) step vii was repeated until all the supernatant solution from step vi has passed through the same column tube (the flow through was discarded after each step); (ix) 700 μl of RW1 buffer (Qiagen, # 1053394) was added to the collection tube and centrifuged for 15 s at 15,000g (the flow through was discarded); (x) 500 μl RPE buffer (Qiagen, # 1018013) was pipetted into the column tube and centrifuged for 15 s at 15,000g (the flow through was discarded); (xi) 500 μl Buffer RPE (Mat. .. Cells were inoculated in 500 μl LB Miller Broth with antibiotics in a 96-well plate covered with a breathable membrane (AeraSeal, Excel Scientific) at 37 °C and 1,000 r.p.m. for 16 h. Overnight cultures were then diluted 1,000-fold by mixing 10 μl of the culture into 10 ml of LB Miller Broth containing the appropriate inducer and incubated at 37 °C and 250 r.p.m. for 18 h. After incubation, the DNA nanostructures were purified using the following protocol: (i) Cells were centrifuged at 5,000g for 7 min at 4 °C; (ii) the supernatant was removed; (iii) cells were resuspended in 200 μl of TE buffer (10 mM Tris-EDTA) containing 3 mg ml−1 of Lysozyme; (iv) 700 μl of RLT buffer (Qiagen, #79216) was added; (v) the resulting solution was centrifuged at 21,130 g for 2 min in order to remove the insoluble materials and the supernatant is transferred to a clean tube; (vi) 500 μl of 100% ethanol was added to the supernatant; (vii) 700 μl of the sample was transferred into a QIAquick Spin Column (Qiagen, #1018215) and centrifuged at 15,000g for 15 s; (viii) step vii was repeated until all the supernatant solution from step vi has passed through the same column tube (the flow through was discarded after each step); (ix) 700 μl of RW1 buffer (Qiagen, # 1053394) was added to the collection tube and centrifuged for 15 s at 15,000g (the flow through was discarded); (x) 500 μl RPE buffer (Qiagen, # 1018013) was pipetted into the column tube and centrifuged for 15 s at 15,000g (the flow through was discarded); (xi) 500 μl Buffer RPE (Mat.

    Article Title: Normalization with Corresponding Naïve Tissue Minimizes Bias Caused by Commercial Reverse Transcription Kits on Quantitative Real-Time PCR Results
    Article Snippet: For RNA extraction, brain tissue samples were lysed with RLT-buffer (Qiagen, Hilden, Germany) and homogenized with a MM300 mill mixer (Retsch, Haan, Germany) at 30 Hz for 2 minutes. .. Then, we isolated the total RNA from the lysed and homogenized cells by using a RNeasy Lipid Tissue Mini Kit (Qiagen) according to the manufacturer's instructions and eluted these with 30 μL to 50 μL RNase-free water.

    Spectrophotometry:

    Article Title: Targeting Endothelial Adhesion Molecule Transcription for Treatment of Inflammatory Disease: A Proof-of-Concept Study
    Article Snippet: At the conclusion of retinal endothelial cell manipulations, culture medium was replaced with Buffer RLT (Qiagen, Hilden, Germany) with 0.55 mM β -mercaptoethanol (Sigma-Aldrich, St. Louis, MO), and cells were frozen at −80°C prior to RNA extraction. .. At the conclusion of retinal endothelial cell manipulations, culture medium was replaced with Buffer RLT (Qiagen, Hilden, Germany) with 0.55 mM β -mercaptoethanol (Sigma-Aldrich, St. Louis, MO), and cells were frozen at −80°C prior to RNA extraction.

    Article Title: Cardiac Non-myocyte Cells Show Enhanced Pharmacological Function Suggestive of Contractile Maturity in Stem Cell Derived Cardiomyocyte Microtissues
    Article Snippet: All primary cells were detached with prewarmed Accutase (Sigma, A6964) for 5 min at 37°C, 5% CO2 , centrifuged for 3 min at 1200 ×g before lysis in RLT buffer (Qiagen) and stored at −80 °C until processing. .. All primary cells were detached with prewarmed Accutase (Sigma, A6964) for 5 min at 37°C, 5% CO2 , centrifuged for 3 min at 1200 ×g before lysis in RLT buffer (Qiagen) and stored at −80 °C until processing.

    Sampling:

    Article Title: Pathogenic Differences between Nipah Virus Bangladesh and Malaysia Strains in Primates: Implications for Antibody Therapy
    Article Snippet: Immediately following sampling, 100 μl of blood was added to 600 μl of AVL viral lysis buffer (Qiagen) for RNA extraction. .. RNAlater was completely removed, and tissues were homogenized in 600 μl RLT buffer (Qiagen) in a 2-mL cryovial using a tissue lyser (Qiagen) and ceramic beads.

    Concentration Assay:

    Article Title: Targeting Endothelial Adhesion Molecule Transcription for Treatment of Inflammatory Disease: A Proof-of-Concept Study
    Article Snippet: At the conclusion of retinal endothelial cell manipulations, culture medium was replaced with Buffer RLT (Qiagen, Hilden, Germany) with 0.55 mM β -mercaptoethanol (Sigma-Aldrich, St. Louis, MO), and cells were frozen at −80°C prior to RNA extraction. .. At the conclusion of retinal endothelial cell manipulations, culture medium was replaced with Buffer RLT (Qiagen, Hilden, Germany) with 0.55 mM β -mercaptoethanol (Sigma-Aldrich, St. Louis, MO), and cells were frozen at −80°C prior to RNA extraction.

    Article Title: Does Caspase-6 Have a Role in Perinatal Brain Injury?
    Article Snippet: The entire supernatant was collected (1 ml) at 6, 24 or 48 h; 300 μl was used immediately for a lactate dehydrogenase assay (Roche, USA) and the remaining supernatant stored at −80°C until further analysis. .. Cells were washed with RNAse-free PBS and then lysed with 350-μl buffer RLT (Qiagen; containing β-mercaptoethanol at a concentration of 1:100), harvested and stored at −80°C for gene expression analysis. .. All time points were run in each independent experiment and all group experiments were performed a minimum of 3-6 independent times.

    Article Title: Allele-specific transcription factor binding to common and rare variants associated with disease and gene expression
    Article Snippet: The cells were stimulated with a phosphorothioate-modified CpG A oligonucleotide ODN2216 (CyberGene) at the concentration of 3 μg/ml and incubated for 5 h at 37 °C with 5 % CO2 . .. The harvested cells were stored in RLT buffer (QIAGEN) at −80 °C.

    Article Title: A virosomal respiratory syncytial virus vaccine adjuvanted with monophosphoryl lipid A provides protection against viral challenge without priming for enhanced disease in cotton rats) A virosomal respiratory syncytial virus vaccine adjuvanted with monophosphoryl lipid A provides protection against viral challenge without priming for enhanced disease in cotton rats
    Article Snippet: Lung samples stored in RNA later (Qiagen) were transferred to tubes containing 300 μl RLT buffer (Qiagen) and homogenized using disposable pestles with a pestle motor (VWR, Amsterdam, the Netherlands). .. Subsequently RNA was isolated with an RNeasy mini kit (Qiagen) according to the manufacturer's protocol.

    Article Title: Normalization with Corresponding Naïve Tissue Minimizes Bias Caused by Commercial Reverse Transcription Kits on Quantitative Real-Time PCR Results
    Article Snippet: For RNA extraction, brain tissue samples were lysed with RLT-buffer (Qiagen, Hilden, Germany) and homogenized with a MM300 mill mixer (Retsch, Haan, Germany) at 30 Hz for 2 minutes. .. RNA quality was confirmed via gel electrophoresis (Experion Automated Electrophoresis System, Bio-Rad, Munich, Germany) for RQI (RNA quality indicator) ≥8 [ ].

    Staining:

    Article Title: Genetic encoding of DNA nanostructures and their self-assembly in living bacteria
    Article Snippet: Cells were inoculated in 500 μl LB Miller Broth with antibiotics in a 96-well plate covered with a breathable membrane (AeraSeal, Excel Scientific) at 37 °C and 1,000 r.p.m. for 16 h. Overnight cultures were then diluted 1,000-fold by mixing 10 μl of the culture into 10 ml of LB Miller Broth containing the appropriate inducer and incubated at 37 °C and 250 r.p.m. for 18 h. After incubation, the DNA nanostructures were purified using the following protocol: (i) Cells were centrifuged at 5,000g for 7 min at 4 °C; (ii) the supernatant was removed; (iii) cells were resuspended in 200 μl of TE buffer (10 mM Tris-EDTA) containing 3 mg ml−1 of Lysozyme; (iv) 700 μl of RLT buffer (Qiagen, #79216) was added; (v) the resulting solution was centrifuged at 21,130 g for 2 min in order to remove the insoluble materials and the supernatant is transferred to a clean tube; (vi) 500 μl of 100% ethanol was added to the supernatant; (vii) 700 μl of the sample was transferred into a QIAquick Spin Column (Qiagen, #1018215) and centrifuged at 15,000g for 15 s; (viii) step vii was repeated until all the supernatant solution from step vi has passed through the same column tube (the flow through was discarded after each step); (ix) 700 μl of RW1 buffer (Qiagen, # 1053394) was added to the collection tube and centrifuged for 15 s at 15,000g (the flow through was discarded); (x) 500 μl RPE buffer (Qiagen, # 1018013) was pipetted into the column tube and centrifuged for 15 s at 15,000g (the flow through was discarded); (xi) 500 μl Buffer RPE (Mat. .. Cells were inoculated in 500 μl LB Miller Broth with antibiotics in a 96-well plate covered with a breathable membrane (AeraSeal, Excel Scientific) at 37 °C and 1,000 r.p.m. for 16 h. Overnight cultures were then diluted 1,000-fold by mixing 10 μl of the culture into 10 ml of LB Miller Broth containing the appropriate inducer and incubated at 37 °C and 250 r.p.m. for 18 h. After incubation, the DNA nanostructures were purified using the following protocol: (i) Cells were centrifuged at 5,000g for 7 min at 4 °C; (ii) the supernatant was removed; (iii) cells were resuspended in 200 μl of TE buffer (10 mM Tris-EDTA) containing 3 mg ml−1 of Lysozyme; (iv) 700 μl of RLT buffer (Qiagen, #79216) was added; (v) the resulting solution was centrifuged at 21,130 g for 2 min in order to remove the insoluble materials and the supernatant is transferred to a clean tube; (vi) 500 μl of 100% ethanol was added to the supernatant; (vii) 700 μl of the sample was transferred into a QIAquick Spin Column (Qiagen, #1018215) and centrifuged at 15,000g for 15 s; (viii) step vii was repeated until all the supernatant solution from step vi has passed through the same column tube (the flow through was discarded after each step); (ix) 700 μl of RW1 buffer (Qiagen, # 1053394) was added to the collection tube and centrifuged for 15 s at 15,000g (the flow through was discarded); (x) 500 μl RPE buffer (Qiagen, # 1018013) was pipetted into the column tube and centrifuged for 15 s at 15,000g (the flow through was discarded); (xi) 500 μl Buffer RPE (Mat.

    Gradient Centrifugation:

    Article Title: Gene expression of INPP5F as an independent prognostic marker in fludarabine-based therapy of chronic lymphocytic leukemia
    Article Snippet: Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll density gradient centrifugation. .. CLL samples were positively enriched by magnetic cell sorting using CD19 microbeads and MACS columns (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and resuspended in RLT buffer (Qiagen, Crawley, West Sussex, UK).

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    Depleting the microbiota limits infant Ccl2 expression and accelerates pneumococcal clearance. (A) Breeding pairs were placed on sterile filtered tap water or water containing 5 antibiotics (Abx) for at least 5 days prior to giving birth, and continued until the infant mice were 10d old. Mice were sacrificed, and nasal lavages obtained with PBS. DNA was isolated from lavage fluid, and qPCR used to measure the number of copies of 16S bacterial ribosomal DNA. (B-C) Adult mice were maintained on sterile filtered tap water or water containing 5 antibiotics for at least 2 weeks. Breeding pairs were provided water as above. Mice were sacrificed, and nasal lavages obtained with <t>RLT</t> lysis buffer. <t>RNA</t> was isolated and reverse-transcribed into cDNA, and qRT-PCR used to measure relative expression of Ccl2 (B) and Ccl7 (C) in the nasopharynx. (D) Infant mice were exposed to tap water or antibiotics as in the previously described protocol. Treatments were continued until infant mice were 6d old, then all mice were given tap water to drink. 24 hrs later, mice were inoculated with pneumococci. At 7 dpi, mice were sacrificed and nasal lavages fixed and stained for flow cytometry to measure the number of macrophages present in the airway lumen. (E-F) Infant mice were treated as in (D). At 14d (E) and 7d (F) post-inoculation, mice were sacrificed. Nasal lavages were obtained and plated on media containing neomycin and catalase to measure pneumococcal density in the URT. (G) Ccl2 expression was measured in the nasopharynx of tap- or antibiotic-water exposed conventionally-reared (Conv), or germ-free infant mice. Data are represented as mean +/- SEM. Horizontal lines indicate median values. n.s., not significant. * = p
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    Depleting the microbiota limits infant Ccl2 expression and accelerates pneumococcal clearance. (A) Breeding pairs were placed on sterile filtered tap water or water containing 5 antibiotics (Abx) for at least 5 days prior to giving birth, and continued until the infant mice were 10d old. Mice were sacrificed, and nasal lavages obtained with PBS. DNA was isolated from lavage fluid, and qPCR used to measure the number of copies of 16S bacterial ribosomal DNA. (B-C) Adult mice were maintained on sterile filtered tap water or water containing 5 antibiotics for at least 2 weeks. Breeding pairs were provided water as above. Mice were sacrificed, and nasal lavages obtained with RLT lysis buffer. RNA was isolated and reverse-transcribed into cDNA, and qRT-PCR used to measure relative expression of Ccl2 (B) and Ccl7 (C) in the nasopharynx. (D) Infant mice were exposed to tap water or antibiotics as in the previously described protocol. Treatments were continued until infant mice were 6d old, then all mice were given tap water to drink. 24 hrs later, mice were inoculated with pneumococci. At 7 dpi, mice were sacrificed and nasal lavages fixed and stained for flow cytometry to measure the number of macrophages present in the airway lumen. (E-F) Infant mice were treated as in (D). At 14d (E) and 7d (F) post-inoculation, mice were sacrificed. Nasal lavages were obtained and plated on media containing neomycin and catalase to measure pneumococcal density in the URT. (G) Ccl2 expression was measured in the nasopharynx of tap- or antibiotic-water exposed conventionally-reared (Conv), or germ-free infant mice. Data are represented as mean +/- SEM. Horizontal lines indicate median values. n.s., not significant. * = p

    Journal: PLoS Pathogens

    Article Title: Clearance of Pneumococcal Colonization in Infants Is Delayed through Altered Macrophage Trafficking

    doi: 10.1371/journal.ppat.1005004

    Figure Lengend Snippet: Depleting the microbiota limits infant Ccl2 expression and accelerates pneumococcal clearance. (A) Breeding pairs were placed on sterile filtered tap water or water containing 5 antibiotics (Abx) for at least 5 days prior to giving birth, and continued until the infant mice were 10d old. Mice were sacrificed, and nasal lavages obtained with PBS. DNA was isolated from lavage fluid, and qPCR used to measure the number of copies of 16S bacterial ribosomal DNA. (B-C) Adult mice were maintained on sterile filtered tap water or water containing 5 antibiotics for at least 2 weeks. Breeding pairs were provided water as above. Mice were sacrificed, and nasal lavages obtained with RLT lysis buffer. RNA was isolated and reverse-transcribed into cDNA, and qRT-PCR used to measure relative expression of Ccl2 (B) and Ccl7 (C) in the nasopharynx. (D) Infant mice were exposed to tap water or antibiotics as in the previously described protocol. Treatments were continued until infant mice were 6d old, then all mice were given tap water to drink. 24 hrs later, mice were inoculated with pneumococci. At 7 dpi, mice were sacrificed and nasal lavages fixed and stained for flow cytometry to measure the number of macrophages present in the airway lumen. (E-F) Infant mice were treated as in (D). At 14d (E) and 7d (F) post-inoculation, mice were sacrificed. Nasal lavages were obtained and plated on media containing neomycin and catalase to measure pneumococcal density in the URT. (G) Ccl2 expression was measured in the nasopharynx of tap- or antibiotic-water exposed conventionally-reared (Conv), or germ-free infant mice. Data are represented as mean +/- SEM. Horizontal lines indicate median values. n.s., not significant. * = p

    Article Snippet: RNA was obtained from URT epithelium by lavage with RLT buffer (Qiagen) with 1% β-mercaptoethanol, or from cultured peritoneal macrophages by lysing cells in RLT buffer with 1% β-mercaptoethanol and frozen at -80°C until used.

    Techniques: Expressing, Mouse Assay, Isolation, Real-time Polymerase Chain Reaction, Lysis, Quantitative RT-PCR, Staining, Flow Cytometry, Cytometry

    Infant mice do not form a gradient of CCL2 expression during colonization. (A) Adult (42d old) and infant (7d old) mice were inoculated with PBS (mock) or pneumococci. At 3d post-inoculation, nasal lavages were obtained with RLT lysis buffer, and RNA was isolated and reverse transcribed to cDNA. qRT-PCR was performed to measure relative expression of Ccl2 . (B) Serum was obtained from adult and infant mice, and ELISA used to measure CCL2 levels. (C-D) Peritoneal macrophages from adult and infant mice were lysed with RLT buffer and RNA isolated. qRT-PCR was used to measure relative expression of Ccl2 (C) and Ccr2 (D). In (C), cultured macrophages were incubated overnight with PBS (Mock) or heat-killed bacterial lysates (Stim) prior to lysis. (E-H) Uncolonized adult and infant mice were sacrificed, and nasal lavages obtained with RLT lysis buffer. RNA was isolated and reverse-transcribed into cDNA. qRT-PCR was used to measure relative expression of Ccl7 , (E) Il6 , (F) Cxcl1 , (G) and Cxcl2 (H). Data are represented as mean +/- SEM. n.s., not significant. * = p

    Journal: PLoS Pathogens

    Article Title: Clearance of Pneumococcal Colonization in Infants Is Delayed through Altered Macrophage Trafficking

    doi: 10.1371/journal.ppat.1005004

    Figure Lengend Snippet: Infant mice do not form a gradient of CCL2 expression during colonization. (A) Adult (42d old) and infant (7d old) mice were inoculated with PBS (mock) or pneumococci. At 3d post-inoculation, nasal lavages were obtained with RLT lysis buffer, and RNA was isolated and reverse transcribed to cDNA. qRT-PCR was performed to measure relative expression of Ccl2 . (B) Serum was obtained from adult and infant mice, and ELISA used to measure CCL2 levels. (C-D) Peritoneal macrophages from adult and infant mice were lysed with RLT buffer and RNA isolated. qRT-PCR was used to measure relative expression of Ccl2 (C) and Ccr2 (D). In (C), cultured macrophages were incubated overnight with PBS (Mock) or heat-killed bacterial lysates (Stim) prior to lysis. (E-H) Uncolonized adult and infant mice were sacrificed, and nasal lavages obtained with RLT lysis buffer. RNA was isolated and reverse-transcribed into cDNA. qRT-PCR was used to measure relative expression of Ccl7 , (E) Il6 , (F) Cxcl1 , (G) and Cxcl2 (H). Data are represented as mean +/- SEM. n.s., not significant. * = p

    Article Snippet: RNA was obtained from URT epithelium by lavage with RLT buffer (Qiagen) with 1% β-mercaptoethanol, or from cultured peritoneal macrophages by lysing cells in RLT buffer with 1% β-mercaptoethanol and frozen at -80°C until used.

    Techniques: Mouse Assay, Expressing, Lysis, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Cell Culture, Incubation

    CCL2 overexpression increases macrophage recruitment and pneumococcal clearance. (A-C) Mice were inoculated with a control (GFP-expressing) or a CCL2-expressing AAV5 vector at 4d of age, followed by pneumococcal colonization at 7d old. Seven and 21 days later, mice were sacrificed and nasal lavages were obtained with RLT lysis buffer. (A) RNA was isolated and cDNA reverse-transcribed, followed by qRT-PCR to measure the relative expression of Ccl2 in the upper respiratory tract, using primers Ccl2 ORF-F and Ccl2 ORF-R. ELISA was used to measure CCL2 protein levels in nasal lavage (B) and serum (C) at 7 and 21 dpi. (D) Mice were inoculated with a control (GFP-expressing) or a CCL2-expressing AAV5 vector at 4d of age, followed by pneumococcal colonization at 7d of age. Seven days later, mice were sacrificed, nasal lavages obtained and flow cytometry used to measure macrophage recruitment. (E) At 21d post-inoculation, nasal lavages were obtained and plated to measure the pneumococcal load in the nasopharynx. Data are represented as mean +/- SEM. n.s., not significant. * = p

    Journal: PLoS Pathogens

    Article Title: Clearance of Pneumococcal Colonization in Infants Is Delayed through Altered Macrophage Trafficking

    doi: 10.1371/journal.ppat.1005004

    Figure Lengend Snippet: CCL2 overexpression increases macrophage recruitment and pneumococcal clearance. (A-C) Mice were inoculated with a control (GFP-expressing) or a CCL2-expressing AAV5 vector at 4d of age, followed by pneumococcal colonization at 7d old. Seven and 21 days later, mice were sacrificed and nasal lavages were obtained with RLT lysis buffer. (A) RNA was isolated and cDNA reverse-transcribed, followed by qRT-PCR to measure the relative expression of Ccl2 in the upper respiratory tract, using primers Ccl2 ORF-F and Ccl2 ORF-R. ELISA was used to measure CCL2 protein levels in nasal lavage (B) and serum (C) at 7 and 21 dpi. (D) Mice were inoculated with a control (GFP-expressing) or a CCL2-expressing AAV5 vector at 4d of age, followed by pneumococcal colonization at 7d of age. Seven days later, mice were sacrificed, nasal lavages obtained and flow cytometry used to measure macrophage recruitment. (E) At 21d post-inoculation, nasal lavages were obtained and plated to measure the pneumococcal load in the nasopharynx. Data are represented as mean +/- SEM. n.s., not significant. * = p

    Article Snippet: RNA was obtained from URT epithelium by lavage with RLT buffer (Qiagen) with 1% β-mercaptoethanol, or from cultured peritoneal macrophages by lysing cells in RLT buffer with 1% β-mercaptoethanol and frozen at -80°C until used.

    Techniques: Over Expression, Mouse Assay, Expressing, Plasmid Preparation, Lysis, Isolation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry

    Genome-wide expression analysis of IL-1R-regulated genes. (A) Schematic diagram of the microarray design used in this study. WT or Il - 1r −/− BMDCs were mock infected or infected with WNV at an MOI of 2.5. Total RNA was extracted at 24 and 48 h p.i. and subjected to Agilent Whole Mouse Genome Microarray analysis. (B) Gene expression levels were determined as fold changes with respect to matched, mock-treated controls. A significant change is defined as a > 1.5-fold increase or decrease with respect to mock treatment, with a BH-adjusted P value of

    Journal: mBio

    Article Title: Interleukin-1β Signaling in Dendritic Cells Induces Antiviral Interferon Responses

    doi: 10.1128/mBio.00342-18

    Figure Lengend Snippet: Genome-wide expression analysis of IL-1R-regulated genes. (A) Schematic diagram of the microarray design used in this study. WT or Il - 1r −/− BMDCs were mock infected or infected with WNV at an MOI of 2.5. Total RNA was extracted at 24 and 48 h p.i. and subjected to Agilent Whole Mouse Genome Microarray analysis. (B) Gene expression levels were determined as fold changes with respect to matched, mock-treated controls. A significant change is defined as a > 1.5-fold increase or decrease with respect to mock treatment, with a BH-adjusted P value of

    Article Snippet: Total RNA was isolated from BMDCs with RNA extraction buffer (RLT; Qiagen) and the RNeasy kit in accordance with the manufacturer’s protocol (Qiagen).

    Techniques: Genome Wide, Expressing, Microarray, Infection

    IL-1β treatment drives expression of IFN-β and ISGs. (A) Schematic diagram of the microarray design used in this study. WT BMDCs were mock treated or treated with IL-1β (100 ng/ml). Total RNA was extracted at 24 and 48 h posttreatment and subjected to Agilent Whole Mouse Genome Microarray analysis. (B) Gene expression levels were determined as fold changes with respect to matched, mock-treated controls. A significant changes is defined as a > 1.5-fold increase or decrease with respect to mock treatment, with a BH-adjusted P value of

    Journal: mBio

    Article Title: Interleukin-1β Signaling in Dendritic Cells Induces Antiviral Interferon Responses

    doi: 10.1128/mBio.00342-18

    Figure Lengend Snippet: IL-1β treatment drives expression of IFN-β and ISGs. (A) Schematic diagram of the microarray design used in this study. WT BMDCs were mock treated or treated with IL-1β (100 ng/ml). Total RNA was extracted at 24 and 48 h posttreatment and subjected to Agilent Whole Mouse Genome Microarray analysis. (B) Gene expression levels were determined as fold changes with respect to matched, mock-treated controls. A significant changes is defined as a > 1.5-fold increase or decrease with respect to mock treatment, with a BH-adjusted P value of

    Article Snippet: Total RNA was isolated from BMDCs with RNA extraction buffer (RLT; Qiagen) and the RNeasy kit in accordance with the manufacturer’s protocol (Qiagen).

    Techniques: Expressing, Microarray

    IL-1 signaling is required for WNV control. BMDCs (A, C) or BMMs (B, D) from WT or Il-1r −/− mice were infected with WNV at an MOI of 2.5 and compared with mock-infected cells. At 24 and 48 h, WNV titers were determined by plaque assay (A, B) and IFN-β levels were measured by ELISA (C, D). (E) IL-1β (0, 10, or 100 ng/ml) was titrated onto WT BMDCs 24 h prior to infection with WNV at an MOI of 2.5. WNV RNA was measured by qRT-PCR at 48 h p.i. The data are averages of three (A to D) or five (E) independent experiments. Asterisks indicate values that are statistically significantly different by Mann-Whitney U test (A, B) or by unpaired t test (C to E) (*, P

    Journal: mBio

    Article Title: Interleukin-1β Signaling in Dendritic Cells Induces Antiviral Interferon Responses

    doi: 10.1128/mBio.00342-18

    Figure Lengend Snippet: IL-1 signaling is required for WNV control. BMDCs (A, C) or BMMs (B, D) from WT or Il-1r −/− mice were infected with WNV at an MOI of 2.5 and compared with mock-infected cells. At 24 and 48 h, WNV titers were determined by plaque assay (A, B) and IFN-β levels were measured by ELISA (C, D). (E) IL-1β (0, 10, or 100 ng/ml) was titrated onto WT BMDCs 24 h prior to infection with WNV at an MOI of 2.5. WNV RNA was measured by qRT-PCR at 48 h p.i. The data are averages of three (A to D) or five (E) independent experiments. Asterisks indicate values that are statistically significantly different by Mann-Whitney U test (A, B) or by unpaired t test (C to E) (*, P

    Article Snippet: Total RNA was isolated from BMDCs with RNA extraction buffer (RLT; Qiagen) and the RNeasy kit in accordance with the manufacturer’s protocol (Qiagen).

    Techniques: Mouse Assay, Infection, Plaque Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, MANN-WHITNEY

    Repressed IL-1 signaling in infants. Five uninfected pups aged 1 week (Infant Mock) and five u ninfected adults aged 8 weeks (Adult Mock) received RLT lavages from which RNA was subjected to qRT-PCR analysis. (A-J) Expression of (A) Il1a , (B) Il1b , (C) Il1r1 , (D) Irak1 , (E) Map3k14 , (F) Mapk3 , (G) Irak2 , (H) Tirap , (I) Capn13 , and (J) Casp1 by qRT-PCR was calculated as fold change in uninfected adults (aged 8 weeks) as compared to uninfected infants (aged 1 week). (K) Il1b expression in uninfected (mock) and serotype 23F infected infants and adults calculated as fold-change compared to mock-infected infants. (L-N) Serotype 23F i.n. colonization of (L) C57BL6 and Il1r -/- and (M) Il1a -/- and Il1b -/- adult mice (aged 8 weeks) and (N) C57BL6 and Il1r -/- pups (aged 1 week). Pneumococcal colonization density in adult and infant URT was determined 3 days post-inoculation. Groups represent n = 4–15 animals with mean ±SEM. Significance is indicated by *, P

    Journal: PLoS Pathogens

    Article Title: Age-related differences in IL-1 signaling and capsule serotype affect persistence of Streptococcus pneumoniae colonization

    doi: 10.1371/journal.ppat.1007396

    Figure Lengend Snippet: Repressed IL-1 signaling in infants. Five uninfected pups aged 1 week (Infant Mock) and five u ninfected adults aged 8 weeks (Adult Mock) received RLT lavages from which RNA was subjected to qRT-PCR analysis. (A-J) Expression of (A) Il1a , (B) Il1b , (C) Il1r1 , (D) Irak1 , (E) Map3k14 , (F) Mapk3 , (G) Irak2 , (H) Tirap , (I) Capn13 , and (J) Casp1 by qRT-PCR was calculated as fold change in uninfected adults (aged 8 weeks) as compared to uninfected infants (aged 1 week). (K) Il1b expression in uninfected (mock) and serotype 23F infected infants and adults calculated as fold-change compared to mock-infected infants. (L-N) Serotype 23F i.n. colonization of (L) C57BL6 and Il1r -/- and (M) Il1a -/- and Il1b -/- adult mice (aged 8 weeks) and (N) C57BL6 and Il1r -/- pups (aged 1 week). Pneumococcal colonization density in adult and infant URT was determined 3 days post-inoculation. Groups represent n = 4–15 animals with mean ±SEM. Significance is indicated by *, P

    Article Snippet: A second URT lavage with 600 μL RLT lysis buffer (Qiagen) + 1% β-mercaptoethanol was performed to obtain host RNA from the URT epithelia for gene expression analyses.

    Techniques: Quantitative RT-PCR, Expressing, Infection, Mouse Assay

    Pathways involved in clearance of pneumococcal colonization. At day 4 of life, pups were i.n. infected with serotype 23F wild-type or ply- and URT was lavaged with RLT to obtain RNA for RNA-seq comparisons. RNA-seq heatmaps show gene expression of the comparison wild-type colonized versus mock controls with relative gene expression in log2 fold change demonstrating increased expression in red and decreased expression in blue. Expression of these genes during ply- colonization is shown for comparison. (A) Heat-map illustrating gene expression in IL-1 related inflammation pathways. qRT-PCR was used to measure expression of (B) Ccl22 and (C) Il1rn that was calculated as fold-change compared to mock-challenged age-controlled animals. (D-E) Heatmaps showing go-term clusters (D) Chemokine-mediated signaling and (E) Phagocytosis that are significantly upregulated following pneumococcal colonization. qRT-PCR was used to measure expression of (F) Cxcl2 , (G) Cd11b , (H), Nos2 , and (I) Fcyr3 at 21 days post-inoculation. Fold-change compared to mock-challenged age-controlled animals. (J) Fold change in Fcyr3 expression in serotype 4 ply- colonized pups following IL-1α and IL-1β dosing as compared to PBS-treated control pups at 4 days post-challenge. (K) Fold change in Fcyr3 expression in 23F colonized C57BL6 and Il1r -/- pups at 21 days post-inoculation. Groups represent n = 7–11 animals with mean ±SEM. Significance is indicated by *, P

    Journal: PLoS Pathogens

    Article Title: Age-related differences in IL-1 signaling and capsule serotype affect persistence of Streptococcus pneumoniae colonization

    doi: 10.1371/journal.ppat.1007396

    Figure Lengend Snippet: Pathways involved in clearance of pneumococcal colonization. At day 4 of life, pups were i.n. infected with serotype 23F wild-type or ply- and URT was lavaged with RLT to obtain RNA for RNA-seq comparisons. RNA-seq heatmaps show gene expression of the comparison wild-type colonized versus mock controls with relative gene expression in log2 fold change demonstrating increased expression in red and decreased expression in blue. Expression of these genes during ply- colonization is shown for comparison. (A) Heat-map illustrating gene expression in IL-1 related inflammation pathways. qRT-PCR was used to measure expression of (B) Ccl22 and (C) Il1rn that was calculated as fold-change compared to mock-challenged age-controlled animals. (D-E) Heatmaps showing go-term clusters (D) Chemokine-mediated signaling and (E) Phagocytosis that are significantly upregulated following pneumococcal colonization. qRT-PCR was used to measure expression of (F) Cxcl2 , (G) Cd11b , (H), Nos2 , and (I) Fcyr3 at 21 days post-inoculation. Fold-change compared to mock-challenged age-controlled animals. (J) Fold change in Fcyr3 expression in serotype 4 ply- colonized pups following IL-1α and IL-1β dosing as compared to PBS-treated control pups at 4 days post-challenge. (K) Fold change in Fcyr3 expression in 23F colonized C57BL6 and Il1r -/- pups at 21 days post-inoculation. Groups represent n = 7–11 animals with mean ±SEM. Significance is indicated by *, P

    Article Snippet: A second URT lavage with 600 μL RLT lysis buffer (Qiagen) + 1% β-mercaptoethanol was performed to obtain host RNA from the URT epithelia for gene expression analyses.

    Techniques: Infection, RNA Sequencing Assay, Expressing, Quantitative RT-PCR

    Fhl2 upregulation induced by BG-enriched compounds is under the control of Dectin-1. (A) Wild-type WT or Clec7a −/− bone marrow-derived macrophages (BMDM) were pre-treated as described in Figure 4 A. Fhl2 expression was determined by RT-qPCR as described for Csf2 . (B) WT or Clec7a −/− BMDM were stimulated with BG-containing compounds for 8 h. At the end of the incubation, BMDM were lysed in RLT lysis buffer. After RNA extraction, cDNA synthesis and RT-qPCR were performed. Data are expressed as the fold change (FC) related to the mock condition value and are representative of two experiments performed in triplicate. (C) WT BMDM were stimulated following the same protocol as in (A) . After incubation, BMDM were lysed in RIPA buffer supplemented with 5 mM EDTA and protease inhibitor (Pierce™). After clarification and protein quantitation, 10 mg of total lysates were separated on a polyacrylamide gel. After transfer, nitrocellulose membrane was subjected to Western blotting with anti-FHL2 (F4B2-B11) and anti-β-actin (C4) mAbs (Santa Cruz Biotechnologies). Blotting was revealed using chemiluminescence. Results are also presented as the relative quantity of FHL2 protein detected in each condition as compared to the reference corresponding to the unstimulated cells. The data are representative of three independent experiments.

    Journal: Frontiers in Immunology

    Article Title: Molecular Analysis of a Short-term Model of β-Glucans-Trained Immunity Highlights the Accessory Contribution of GM-CSF in Priming Mouse Macrophages Response

    doi: 10.3389/fimmu.2017.01089

    Figure Lengend Snippet: Fhl2 upregulation induced by BG-enriched compounds is under the control of Dectin-1. (A) Wild-type WT or Clec7a −/− bone marrow-derived macrophages (BMDM) were pre-treated as described in Figure 4 A. Fhl2 expression was determined by RT-qPCR as described for Csf2 . (B) WT or Clec7a −/− BMDM were stimulated with BG-containing compounds for 8 h. At the end of the incubation, BMDM were lysed in RLT lysis buffer. After RNA extraction, cDNA synthesis and RT-qPCR were performed. Data are expressed as the fold change (FC) related to the mock condition value and are representative of two experiments performed in triplicate. (C) WT BMDM were stimulated following the same protocol as in (A) . After incubation, BMDM were lysed in RIPA buffer supplemented with 5 mM EDTA and protease inhibitor (Pierce™). After clarification and protein quantitation, 10 mg of total lysates were separated on a polyacrylamide gel. After transfer, nitrocellulose membrane was subjected to Western blotting with anti-FHL2 (F4B2-B11) and anti-β-actin (C4) mAbs (Santa Cruz Biotechnologies). Blotting was revealed using chemiluminescence. Results are also presented as the relative quantity of FHL2 protein detected in each condition as compared to the reference corresponding to the unstimulated cells. The data are representative of three independent experiments.

    Article Snippet: After stimulation with Sc BG compounds and then with ultraPure LPS, BMDM were lysed in Buffer RLT (Qiagen, Hilden, Germany) and was subjected to RNA extraction using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.

    Techniques: Derivative Assay, Expressing, Quantitative RT-PCR, Incubation, Lysis, RNA Extraction, Flow Cytometry, Protease Inhibitor, Clarification Assay, Protein Quantitation, Western Blot

    Csf2 upregulation induced by BG-enriched compounds treatment and lipopolysaccharide (LPS) exposure is mainly Dectin-1-dependent. Wild-type or Clec7a −/− bone marrow-derived macrophages (BMDM) were stimulated with 100 µg/mL of the various BG-containing compounds for 8 h. After incubation, cell culture supernatants were removed and 100 ng/mL of ultraPure LPS or medium was added in each well for 8 h (A) or 48 h (B) . (A) Supernatants were discarded and BMDM were lysed in RLT lysis buffer. Total RNA was extracted as described before and retro-transcribed. Csf2 expression was determined by quantitative PCR after normalization with three housekeeping genes ( Sdha, Rpl9 , and Hprt1 ). Fold change (FC) data are expressed as the mean ± SD as compared to the mock condition in three independent experiments. Mean values not sharing the same letter are significantly different according to the Student’s t -tes t ( p

    Journal: Frontiers in Immunology

    Article Title: Molecular Analysis of a Short-term Model of β-Glucans-Trained Immunity Highlights the Accessory Contribution of GM-CSF in Priming Mouse Macrophages Response

    doi: 10.3389/fimmu.2017.01089

    Figure Lengend Snippet: Csf2 upregulation induced by BG-enriched compounds treatment and lipopolysaccharide (LPS) exposure is mainly Dectin-1-dependent. Wild-type or Clec7a −/− bone marrow-derived macrophages (BMDM) were stimulated with 100 µg/mL of the various BG-containing compounds for 8 h. After incubation, cell culture supernatants were removed and 100 ng/mL of ultraPure LPS or medium was added in each well for 8 h (A) or 48 h (B) . (A) Supernatants were discarded and BMDM were lysed in RLT lysis buffer. Total RNA was extracted as described before and retro-transcribed. Csf2 expression was determined by quantitative PCR after normalization with three housekeeping genes ( Sdha, Rpl9 , and Hprt1 ). Fold change (FC) data are expressed as the mean ± SD as compared to the mock condition in three independent experiments. Mean values not sharing the same letter are significantly different according to the Student’s t -tes t ( p

    Article Snippet: After stimulation with Sc BG compounds and then with ultraPure LPS, BMDM were lysed in Buffer RLT (Qiagen, Hilden, Germany) and was subjected to RNA extraction using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.

    Techniques: Derivative Assay, Incubation, Cell Culture, Lysis, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry