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:
    158
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    Buffer RLT
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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 1897 article reviews
    Price from $9.99 to $1999.99
    rlt buffer - by Bioz Stars, 2020-07
    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 "A distinct subpopulation of CD25− T-follicular regulatory cells localizes in the germinal centers"

    Article Title: A distinct subpopulation of CD25− T-follicular regulatory cells localizes in the germinal centers

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1705551114

    RNA-Seq of CD25 − Tfr cells. Mice were vaccinated with NP-Ova in alum, and dLNs were taken at day 7. A total of 1 × 10 4 cells were sorted by Becton Dickinson (BD) FACSAria-SORP before RNA-Seq. RNA was extracted using RLT buffer (Qiagen), and then subjected to library preparation using the Quartz-Seq protocol and sequenced by Ion Proton (Life Technologies). Heat maps, principal component analysis, and Euclidean distance analysis were produced using R software. Differential gene expression analysis was performed in R by TCC/DEseq2. Genes with a false discovery rate of
    Figure Legend Snippet: RNA-Seq of CD25 − Tfr cells. Mice were vaccinated with NP-Ova in alum, and dLNs were taken at day 7. A total of 1 × 10 4 cells were sorted by Becton Dickinson (BD) FACSAria-SORP before RNA-Seq. RNA was extracted using RLT buffer (Qiagen), and then subjected to library preparation using the Quartz-Seq protocol and sequenced by Ion Proton (Life Technologies). Heat maps, principal component analysis, and Euclidean distance analysis were produced using R software. Differential gene expression analysis was performed in R by TCC/DEseq2. Genes with a false discovery rate of

    Techniques Used: RNA Sequencing Assay, Mouse Assay, Produced, Software, Expressing

    3) Product Images from "Effective methods for the inactivation of Francisella tularensis"

    Article Title: Effective methods for the inactivation of Francisella tularensis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0225177

    The viability of F . tularensis SCHU P9 after treatments of commercial products. Bacterial viability was evaluated after the treatment using Cell Lysis Buffer (Cell Signaling Technology) and the RLT buffer supplied by RNeasy mini kit (Qiagen Ltd.,). (A) Bacteria suspended in Cell Lysis Buffer and CDM (control) were incubated at 4°C for the indicated time. (B) Bacterial pellets after the centrifugation at 12,000 × g for 2 min at 4°C were suspended in RLT buffer alone, the mixture of RLT buffer and 70% ethanol, and CDM (control). The samples were incubated at room temperature for 10 min. All incubated samples were centrifuged at 12,000 × g for 2 min at 4°C and the pellets were suspended in CDM. the mean CFU ± SD of control and the treatment samples are shown. Statistical significance was determined by two-way ANOVA (A) and one-way ANOVA (B) with the post hoc test (*** p
    Figure Legend Snippet: The viability of F . tularensis SCHU P9 after treatments of commercial products. Bacterial viability was evaluated after the treatment using Cell Lysis Buffer (Cell Signaling Technology) and the RLT buffer supplied by RNeasy mini kit (Qiagen Ltd.,). (A) Bacteria suspended in Cell Lysis Buffer and CDM (control) were incubated at 4°C for the indicated time. (B) Bacterial pellets after the centrifugation at 12,000 × g for 2 min at 4°C were suspended in RLT buffer alone, the mixture of RLT buffer and 70% ethanol, and CDM (control). The samples were incubated at room temperature for 10 min. All incubated samples were centrifuged at 12,000 × g for 2 min at 4°C and the pellets were suspended in CDM. the mean CFU ± SD of control and the treatment samples are shown. Statistical significance was determined by two-way ANOVA (A) and one-way ANOVA (B) with the post hoc test (*** p

    Techniques Used: Lysis, Incubation, Centrifugation

    4) 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: Cell host & microbe

    doi: 10.1016/j.chom.2014.06.005

    Sialylated airway mucins are required for influenza-induced pneumococcal growth (A) Mouse heads were obtained after 7 days of mock or influenza infection, fixed, decalcified and sectioned. URT sections were stained with alcian blue and nuclear fast red. Scale bars, 50 μm. (B) Nasal lavages were obtained from mice infected with influenza or PBS for 7 days, then analyzed by Western blot for the presence of Muc5ac. (C) Nasal lavages with RLT RNA lysis buffer were obtained from mice influenza or mock-infected for 7 days. qRT-PCR was performed, and relative expression of Muc5ac measured. (D) Mice of indicated genotype were mock or influenza infected for 7 days, followed by inoculation with CFSE-labeled pneumococci for 8 hrs. Nasal lavages were obtained and plated for quantitative culture. (E) Lavages were also fixed, stained for pneumococcal capsule, and flow cytometry performed to measure bacterial replication (division index). (F-G) Wildtype mice were infected with influenza or mock, followed by daily treatment for 7 days with vehicle (PBS) or 0.5 M N-acetylcysteine (NAc). CFSE-labeled pneumococci were inoculated for 8 hrs, then nasal lavages obtained and used for quantitative culture (F) and flow cytometric analysis of cell division (G). (H) Total sialic acid content was measured by thiobarbituric acid assay on samples from D-G. Data are represented as mean +/− SD. Horizontal lines indicate median values. * = p
    Figure Legend Snippet: Sialylated airway mucins are required for influenza-induced pneumococcal growth (A) Mouse heads were obtained after 7 days of mock or influenza infection, fixed, decalcified and sectioned. URT sections were stained with alcian blue and nuclear fast red. Scale bars, 50 μm. (B) Nasal lavages were obtained from mice infected with influenza or PBS for 7 days, then analyzed by Western blot for the presence of Muc5ac. (C) Nasal lavages with RLT RNA lysis buffer were obtained from mice influenza or mock-infected for 7 days. qRT-PCR was performed, and relative expression of Muc5ac measured. (D) Mice of indicated genotype were mock or influenza infected for 7 days, followed by inoculation with CFSE-labeled pneumococci for 8 hrs. Nasal lavages were obtained and plated for quantitative culture. (E) Lavages were also fixed, stained for pneumococcal capsule, and flow cytometry performed to measure bacterial replication (division index). (F-G) Wildtype mice were infected with influenza or mock, followed by daily treatment for 7 days with vehicle (PBS) or 0.5 M N-acetylcysteine (NAc). CFSE-labeled pneumococci were inoculated for 8 hrs, then nasal lavages obtained and used for quantitative culture (F) and flow cytometric analysis of cell division (G). (H) Total sialic acid content was measured by thiobarbituric acid assay on samples from D-G. Data are represented as mean +/− SD. Horizontal lines indicate median values. * = p

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

    Sialic acid catabolism contributes to pneumococcal colonization in the absence of influenza (A) Mice were colonized with pneumococci for the indicated number of days. Sialic acid concentrations in lavages were measured by thiobarbituric acid assay, with or without acid hydrolysis to measure total (solid bars) and free sialic acid (open bars), respectively. (B) WT and Δ satABC pneumococci were competed in vivo by colonizing naive mice with mixed inocula. Nasal lavages were plated for quantitative culture and colonies from the inocula and lavages patched onto antibiotic-selective media to determine the relative advantage of sialic acid catabolism in vivo and calculate competitive indices. CI > 1 indicates WT outcompeted Δ satABC pneumococci. (C) Nasal lavages were obtained with RLT RNA lysis buffer from mice colonized with pneumococci for the indicated number of days. qRT-PCR was performed to measure relative expression of Muc5ac . Data are represented as mean +/− SD. ND = not detected (below the limit of detection, indicated with a dotted line), * = p
    Figure Legend Snippet: Sialic acid catabolism contributes to pneumococcal colonization in the absence of influenza (A) Mice were colonized with pneumococci for the indicated number of days. Sialic acid concentrations in lavages were measured by thiobarbituric acid assay, with or without acid hydrolysis to measure total (solid bars) and free sialic acid (open bars), respectively. (B) WT and Δ satABC pneumococci were competed in vivo by colonizing naive mice with mixed inocula. Nasal lavages were plated for quantitative culture and colonies from the inocula and lavages patched onto antibiotic-selective media to determine the relative advantage of sialic acid catabolism in vivo and calculate competitive indices. CI > 1 indicates WT outcompeted Δ satABC pneumococci. (C) Nasal lavages were obtained with RLT RNA lysis buffer from mice colonized with pneumococci for the indicated number of days. qRT-PCR was performed to measure relative expression of Muc5ac . Data are represented as mean +/− SD. ND = not detected (below the limit of detection, indicated with a dotted line), * = p

    Techniques Used: Mouse Assay, Acid Assay, In Vivo, Lysis, Quantitative RT-PCR, Expressing

    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
    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

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

    5) 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 with Mass Spectroscopy

    Related Articles

    RNA Extraction:

    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
    Article Snippet: .. Microarray Analysis 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. .. RNA was quantified with a NanoDrop® 1000 spectrophotometer and NanoDrop 1000.3.7 software.

    RNA Sequencing Assay:

    Article Title: Age-related differences in IL-1 signaling and capsule serotype affect persistence of Streptococcus pneumoniae colonization
    Article Snippet: .. RNA-sequencing Uninfected infants, serotype 23F wild-type and ply- mutant infected pups (aged 1 week) were euthanized 3 days post-challenge and a URT lavage with RLT lysis buffer was obtained. .. RNA was extracted according to manufacturer’s directions (RNeasy kit, Qiagen) and five replicates per age group were subjected to RNA sequencing (RNA-seq) using Hi-seq and the raw fastq reads were aligned to mm10 mouse reference genome using STAR aligner [ ].

    Mutagenesis:

    Article Title: Age-related differences in IL-1 signaling and capsule serotype affect persistence of Streptococcus pneumoniae colonization
    Article Snippet: .. RNA-sequencing Uninfected infants, serotype 23F wild-type and ply- mutant infected pups (aged 1 week) were euthanized 3 days post-challenge and a URT lavage with RLT lysis buffer was obtained. .. RNA was extracted according to manufacturer’s directions (RNeasy kit, Qiagen) and five replicates per age group were subjected to RNA sequencing (RNA-seq) using Hi-seq and the raw fastq reads were aligned to mm10 mouse reference genome using STAR aligner [ ].

    Isolation:

    Article Title: Pneumococcal quorum sensing drives an asymmetric owner-intruder competitive strategy during carriage via the competence regulon
    Article Snippet: .. To obtain host RNA, nasal lavages with RLT lysis buffer (Qiagen) were performed and RNA isolated with the RNeasy minikit (Qiagen) as previously described . cDNA was reverse transcribed using a high-capacity cDNA kit (Applied Biosystems) following the manufacturer’s instructions. .. Real-time, quantitative reverse transcriptase PCR was carried out with Power SYBR Green Master Mix (Applied Biosystems) using 10–50 ng cDNA and 0.5 μM primers per reaction.

    Centrifugation:

    Article Title: Nucleic acids enrichment of fungal pathogens to study host-pathogen interactions
    Article Snippet: .. In addition, using qPCR and RT-qPCR, we demonstrated that after cell lysis with Buffer RLT, both fungal DNA and RNA can be further enriched by means of a centrifugation step that effectively reduced human nucleic acids. .. However, we observed that pellets of fungal cells were not strongly attached at the bottom of the tube, which may be due to the presence of large amounts of (human) DNA in the supernatant, which may increase viscosity, and interfere with the centrifugation process.

    Infection:

    Article Title: Age-related differences in IL-1 signaling and capsule serotype affect persistence of Streptococcus pneumoniae colonization
    Article Snippet: .. RNA-sequencing Uninfected infants, serotype 23F wild-type and ply- mutant infected pups (aged 1 week) were euthanized 3 days post-challenge and a URT lavage with RLT lysis buffer was obtained. .. RNA was extracted according to manufacturer’s directions (RNeasy kit, Qiagen) and five replicates per age group were subjected to RNA sequencing (RNA-seq) using Hi-seq and the raw fastq reads were aligned to mm10 mouse reference genome using STAR aligner [ ].

    Real-time Polymerase Chain Reaction:

    Article Title: Nucleic acids enrichment of fungal pathogens to study host-pathogen interactions
    Article Snippet: .. In addition, using qPCR and RT-qPCR, we demonstrated that after cell lysis with Buffer RLT, both fungal DNA and RNA can be further enriched by means of a centrifugation step that effectively reduced human nucleic acids. .. However, we observed that pellets of fungal cells were not strongly attached at the bottom of the tube, which may be due to the presence of large amounts of (human) DNA in the supernatant, which may increase viscosity, and interfere with the centrifugation process.

    Microarray:

    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
    Article Snippet: .. Microarray Analysis 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. .. RNA was quantified with a NanoDrop® 1000 spectrophotometer and NanoDrop 1000.3.7 software.

    other:

    Article Title: Nucleic acids enrichment of fungal pathogens to study host-pathogen interactions
    Article Snippet: We concluded that enrichment with Buffer RLT confers high-quality RNA which can be used in transcriptomic analysis.

    Cell Culture:

    Article Title: Clearance of Pneumococcal Colonization in Infants Is Delayed through Altered Macrophage Trafficking
    Article Snippet: .. qRT-PCR 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. .. An RNeasy kit (Qiagen) was used to isolate RNA, and cDNA reverse transcribed by the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). qRT-PCR reactions were performed with Sybr Green (Applied Biosystems) with 10 ng cDNA and 0.5 μM primers.

    Quantitative RT-PCR:

    Article Title: Clearance of Pneumococcal Colonization in Infants Is Delayed through Altered Macrophage Trafficking
    Article Snippet: .. qRT-PCR 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. .. An RNeasy kit (Qiagen) was used to isolate RNA, and cDNA reverse transcribed by the High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). qRT-PCR reactions were performed with Sybr Green (Applied Biosystems) with 10 ng cDNA and 0.5 μM primers.

    Article Title: Nucleic acids enrichment of fungal pathogens to study host-pathogen interactions
    Article Snippet: .. In addition, using qPCR and RT-qPCR, we demonstrated that after cell lysis with Buffer RLT, both fungal DNA and RNA can be further enriched by means of a centrifugation step that effectively reduced human nucleic acids. .. However, we observed that pellets of fungal cells were not strongly attached at the bottom of the tube, which may be due to the presence of large amounts of (human) DNA in the supernatant, which may increase viscosity, and interfere with the centrifugation process.

    Lysis:

    Article Title: Age-related differences in IL-1 signaling and capsule serotype affect persistence of Streptococcus pneumoniae colonization
    Article Snippet: .. RNA-sequencing Uninfected infants, serotype 23F wild-type and ply- mutant infected pups (aged 1 week) were euthanized 3 days post-challenge and a URT lavage with RLT lysis buffer was obtained. .. RNA was extracted according to manufacturer’s directions (RNeasy kit, Qiagen) and five replicates per age group were subjected to RNA sequencing (RNA-seq) using Hi-seq and the raw fastq reads were aligned to mm10 mouse reference genome using STAR aligner [ ].

    Article Title: Pneumococcal quorum sensing drives an asymmetric owner-intruder competitive strategy during carriage via the competence regulon
    Article Snippet: .. To obtain host RNA, nasal lavages with RLT lysis buffer (Qiagen) were performed and RNA isolated with the RNeasy minikit (Qiagen) as previously described . cDNA was reverse transcribed using a high-capacity cDNA kit (Applied Biosystems) following the manufacturer’s instructions. .. Real-time, quantitative reverse transcriptase PCR was carried out with Power SYBR Green Master Mix (Applied Biosystems) using 10–50 ng cDNA and 0.5 μM primers per reaction.

    Article Title: Nucleic acids enrichment of fungal pathogens to study host-pathogen interactions
    Article Snippet: .. In addition, using qPCR and RT-qPCR, we demonstrated that after cell lysis with Buffer RLT, both fungal DNA and RNA can be further enriched by means of a centrifugation step that effectively reduced human nucleic acids. .. However, we observed that pellets of fungal cells were not strongly attached at the bottom of the tube, which may be due to the presence of large amounts of (human) DNA in the supernatant, which may increase viscosity, and interfere with the centrifugation process.

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

    Journal: Nature microbiology

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

    doi: 10.1038/s41564-018-0314-4

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

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

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

    RNA integrity of some RNA samples after enrichment with buffer RLT, Triton X-100 and bead beating. Electropherogram profiles were determined with a Fragment Analyzer. Quality for Fragment Analyzer is shown as the RQN value. Reported on a scale of 1 to 10, with higher values indicating a better quality of total RNA. Values above 7 are considered to represent high quality and non-degraded RNA.

    Journal: Scientific Reports

    Article Title: Nucleic acids enrichment of fungal pathogens to study host-pathogen interactions

    doi: 10.1038/s41598-019-54608-x

    Figure Lengend Snippet: RNA integrity of some RNA samples after enrichment with buffer RLT, Triton X-100 and bead beating. Electropherogram profiles were determined with a Fragment Analyzer. Quality for Fragment Analyzer is shown as the RQN value. Reported on a scale of 1 to 10, with higher values indicating a better quality of total RNA. Values above 7 are considered to represent high quality and non-degraded RNA.

    Article Snippet: In addition, using qPCR and RT-qPCR, we demonstrated that after cell lysis with Buffer RLT, both fungal DNA and RNA can be further enriched by means of a centrifugation step that effectively reduced human nucleic acids.

    Techniques:

    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: qRT-PCR 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: qRT-PCR 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: qRT-PCR 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

    RNA-Seq of CD25 − Tfr cells. Mice were vaccinated with NP-Ova in alum, and dLNs were taken at day 7. A total of 1 × 10 4 cells were sorted by Becton Dickinson (BD) FACSAria-SORP before RNA-Seq. RNA was extracted using RLT buffer (Qiagen), and then subjected to library preparation using the Quartz-Seq protocol and sequenced by Ion Proton (Life Technologies). Heat maps, principal component analysis, and Euclidean distance analysis were produced using R software. Differential gene expression analysis was performed in R by TCC/DEseq2. Genes with a false discovery rate of

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: A distinct subpopulation of CD25− T-follicular regulatory cells localizes in the germinal centers

    doi: 10.1073/pnas.1705551114

    Figure Lengend Snippet: RNA-Seq of CD25 − Tfr cells. Mice were vaccinated with NP-Ova in alum, and dLNs were taken at day 7. A total of 1 × 10 4 cells were sorted by Becton Dickinson (BD) FACSAria-SORP before RNA-Seq. RNA was extracted using RLT buffer (Qiagen), and then subjected to library preparation using the Quartz-Seq protocol and sequenced by Ion Proton (Life Technologies). Heat maps, principal component analysis, and Euclidean distance analysis were produced using R software. Differential gene expression analysis was performed in R by TCC/DEseq2. Genes with a false discovery rate of

    Article Snippet: RNA was extracted using RLT buffer (Qiagen), subjected to library preparation using the Quartz-Seq protocol , and sequenced by Ion Proton (Life Technologies).

    Techniques: RNA Sequencing Assay, Mouse Assay, Produced, Software, Expressing