rneasy kit  (Qiagen)

 
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    RNeasy Plus Mini Kit
    Description:
    For phenol free total RNA extraction from cells tissues with removal of genomic DNA contamination Kit contents Qiagen RNeasy Plus Mini Kit 50 preps 30mg Sample 30 to 50L Elution Volume Tissue Cells Sample Total RNA Purification Silica Technology Spin Column Format 25 min Time Run Ideal for PCR Northern Dot and Slot Blotting Array Analysis Sequencing For Purification of Up to 100g Total RNA from Cells tissues Using gDNA Eliminator Columns Includes RNeasy Mini Spin Columns gDNA Eliminator Spin Columns Collection Tubes RNase free Water and Buffers Benefits Efficient gDNA removal with unique gDNA Eliminator columns no need for DNase High quality total RNA in minutes using fast and simple extraction protocols Phenol free RNA isolation Ideal for sensitive applications such as real time RT PCR and RNA Seq
    Catalog Number:
    74134
    Price:
    360
    Category:
    RNeasy Plus Micro and Mini Kits
    Buy from Supplier


    Structured Review

    Qiagen rneasy kit
    RNeasy Plus Mini Kit
    For phenol free total RNA extraction from cells tissues with removal of genomic DNA contamination Kit contents Qiagen RNeasy Plus Mini Kit 50 preps 30mg Sample 30 to 50L Elution Volume Tissue Cells Sample Total RNA Purification Silica Technology Spin Column Format 25 min Time Run Ideal for PCR Northern Dot and Slot Blotting Array Analysis Sequencing For Purification of Up to 100g Total RNA from Cells tissues Using gDNA Eliminator Columns Includes RNeasy Mini Spin Columns gDNA Eliminator Spin Columns Collection Tubes RNase free Water and Buffers Benefits Efficient gDNA removal with unique gDNA Eliminator columns no need for DNase High quality total RNA in minutes using fast and simple extraction protocols Phenol free RNA isolation Ideal for sensitive applications such as real time RT PCR and RNA Seq
    https://www.bioz.com/result/rneasy kit/product/Qiagen
    Average 99 stars, based on 2846 article reviews
    Price from $9.99 to $1999.99
    rneasy kit - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "2?-O Methylation of Internal Adenosine by Flavivirus NS5 Methyltransferase"

    Article Title: 2?-O Methylation of Internal Adenosine by Flavivirus NS5 Methyltransferase

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002642

    2′- O methylation of internal adenosine. (A) Incorporation of 3 H-methyl into polyA. Homopolymer RNAs (1 µg) were incubated with 2 µg of DENV-4 MTase in the presence of [ 3 H-methyl]-SAM. After the methylation reaction, the un-incorporated [ 3 H-methyl]-SAM was removed by RNeasy kit. The amount of 3 H-methyl incorporation was measured by a MicroBeta counting. (B) SPA-based methylation analysis of oligo (A) 12 , (Am) 12 , and (m 6 ,m 6 A) 12 . All three RNA oligos were 3′-end biotinylated to facilitate SPA analysis. Am indicates that the 2′-OH of adenosine is methylated. m 6 ,m 6 A indicates that the amino N 6 position of adenosine is double methylated. (C) SPA-based methylation analysis of DENV-1 RNA. pppA-RNAs, representing the 5′ 211 nt of DENV-1 genome, were in vitro transcribed using biotinylated-CTP plus unmodified ATP, 2′- O -methyladenosine triphosphate (AmTP), or N 6 methyl adenosine triphosphate (m 6 ATP). The transcription reactions generated pppA-RNA, ppp(Am)-RNA, and ppp(m 6 A)RNA, respectively. The RNAs were then subjected to SPA-based internal methylation analysis. Average results and standard deviations from three independent experiments are presented.
    Figure Legend Snippet: 2′- O methylation of internal adenosine. (A) Incorporation of 3 H-methyl into polyA. Homopolymer RNAs (1 µg) were incubated with 2 µg of DENV-4 MTase in the presence of [ 3 H-methyl]-SAM. After the methylation reaction, the un-incorporated [ 3 H-methyl]-SAM was removed by RNeasy kit. The amount of 3 H-methyl incorporation was measured by a MicroBeta counting. (B) SPA-based methylation analysis of oligo (A) 12 , (Am) 12 , and (m 6 ,m 6 A) 12 . All three RNA oligos were 3′-end biotinylated to facilitate SPA analysis. Am indicates that the 2′-OH of adenosine is methylated. m 6 ,m 6 A indicates that the amino N 6 position of adenosine is double methylated. (C) SPA-based methylation analysis of DENV-1 RNA. pppA-RNAs, representing the 5′ 211 nt of DENV-1 genome, were in vitro transcribed using biotinylated-CTP plus unmodified ATP, 2′- O -methyladenosine triphosphate (AmTP), or N 6 methyl adenosine triphosphate (m 6 ATP). The transcription reactions generated pppA-RNA, ppp(Am)-RNA, and ppp(m 6 A)RNA, respectively. The RNAs were then subjected to SPA-based internal methylation analysis. Average results and standard deviations from three independent experiments are presented.

    Techniques Used: Methylation, Incubation, In Vitro, Generated

    2) Product Images from "Therapeutic effects of calcium dobesilate on diabetic nephropathy mediated through reduction of expression of PAI-1"

    Article Title: Therapeutic effects of calcium dobesilate on diabetic nephropathy mediated through reduction of expression of PAI-1

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2012.755

    Quantitative RT-PCR analysis of PAI-1 mRNA levels in patients. The total RNA was harvested from the peripheral blood mononuclear cells using an RNeasy kit according to the manufacturer’s instructions. The RT-PCR experiments were repeated at least 3 times. RNA was reverse transcribed into cDNA using random primers in a Reverse Transcription II system according to the manufacturer’s instructions. The expression of PAI-1 mRNA was quantified by quantitative PCR using an ABI Prism Sequence Detection System. Template-negative and RT-negative conditions were used as controls. Amplification of the endogenous GAPDH cDNA was monitored. The levels (mean value) of PAI-1 transcripts in patients were calculated. RT-PCR, reverse-transcription-PCR, PAI-1, plasminogen activator inhibitor-1.
    Figure Legend Snippet: Quantitative RT-PCR analysis of PAI-1 mRNA levels in patients. The total RNA was harvested from the peripheral blood mononuclear cells using an RNeasy kit according to the manufacturer’s instructions. The RT-PCR experiments were repeated at least 3 times. RNA was reverse transcribed into cDNA using random primers in a Reverse Transcription II system according to the manufacturer’s instructions. The expression of PAI-1 mRNA was quantified by quantitative PCR using an ABI Prism Sequence Detection System. Template-negative and RT-negative conditions were used as controls. Amplification of the endogenous GAPDH cDNA was monitored. The levels (mean value) of PAI-1 transcripts in patients were calculated. RT-PCR, reverse-transcription-PCR, PAI-1, plasminogen activator inhibitor-1.

    Techniques Used: Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Expressing, Real-time Polymerase Chain Reaction, Sequencing, Amplification, Polymerase Chain Reaction

    3) Product Images from "The Tumor Suppressive Role of eIF3f and Its Function in Translation Inhibition and rRNA Degradation"

    Article Title: The Tumor Suppressive Role of eIF3f and Its Function in Translation Inhibition and rRNA Degradation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0034194

    eIF3f induced rRNA degradation in pancreatic cells. (A) Restoration of eIF3f expression in pancreatic cancer cells decreased the rRNA level. MiaPaCa-2 cells were transfected with pcDNA3-eIF3f or pcDNA3. Relative fold changes of rRNA and eIF3f levels were quantified by real time RT-PCR and normalized to GAPDH mRNA. (B)(C) Silencing of eIF3f expression increased the rRNA level during apoptosis. Control and eIF3f-silenced HPDE cells were treated with DMSO vehicle control or etoposide (10 µM) for indicated time to induce apoptosis. Cells were harvested and total RNA was isolated using RNeasy Kit. Relative fold changes of 28S (B) and 18S (C) rRNA levels were quantified by real time RT-PCR after DNase treatment. (D) Time course of cell survival after etoposide treatment. HPDE cells were treated with DMSO or etoposide (10 µM) for indicated time. Cell survival was measured using MTT assay. Relative percentages of survived cells compared to DMSO-treated cells were shown. (E) Silencing of eIF3f expression attenuated rRNA degradation. Control and eIF3f-silenced HPDE cells were treated with actinomycin D (0.5 µg/ml) to block transcription for 3, 6 and 8 h before harvest. Total RNA was isolated, treated with DNase, and relative 28S and 18S rRNAs fold changes compared to control were quantified by real time RT-PCR and normalized to GAPDH mRNA.
    Figure Legend Snippet: eIF3f induced rRNA degradation in pancreatic cells. (A) Restoration of eIF3f expression in pancreatic cancer cells decreased the rRNA level. MiaPaCa-2 cells were transfected with pcDNA3-eIF3f or pcDNA3. Relative fold changes of rRNA and eIF3f levels were quantified by real time RT-PCR and normalized to GAPDH mRNA. (B)(C) Silencing of eIF3f expression increased the rRNA level during apoptosis. Control and eIF3f-silenced HPDE cells were treated with DMSO vehicle control or etoposide (10 µM) for indicated time to induce apoptosis. Cells were harvested and total RNA was isolated using RNeasy Kit. Relative fold changes of 28S (B) and 18S (C) rRNA levels were quantified by real time RT-PCR after DNase treatment. (D) Time course of cell survival after etoposide treatment. HPDE cells were treated with DMSO or etoposide (10 µM) for indicated time. Cell survival was measured using MTT assay. Relative percentages of survived cells compared to DMSO-treated cells were shown. (E) Silencing of eIF3f expression attenuated rRNA degradation. Control and eIF3f-silenced HPDE cells were treated with actinomycin D (0.5 µg/ml) to block transcription for 3, 6 and 8 h before harvest. Total RNA was isolated, treated with DNase, and relative 28S and 18S rRNAs fold changes compared to control were quantified by real time RT-PCR and normalized to GAPDH mRNA.

    Techniques Used: Expressing, Transfection, Quantitative RT-PCR, Isolation, MTT Assay, Blocking Assay

    4) Product Images from "Research Resource: A Genome-Wide Study Identifies Potential New Target Genes for POU1F1"

    Article Title: Research Resource: A Genome-Wide Study Identifies Potential New Target Genes for POU1F1

    Journal: Molecular Endocrinology

    doi: 10.1210/me.2011-1308

    A, Effect of POU1F1 R271W on Icer and Crem mRNA expression in GH4C1 cells. Total RNA from cells infected with the lentiviral vectors (with or without the transgene) was isolated by using the RNeasy Kit (QIAGEN), and reverse transcription was performed
    Figure Legend Snippet: A, Effect of POU1F1 R271W on Icer and Crem mRNA expression in GH4C1 cells. Total RNA from cells infected with the lentiviral vectors (with or without the transgene) was isolated by using the RNeasy Kit (QIAGEN), and reverse transcription was performed

    Techniques Used: Expressing, Infection, Isolation

    5) Product Images from "Mechanisms by which Porphyromonas gingivalis evades innate immunity"

    Article Title: Mechanisms by which Porphyromonas gingivalis evades innate immunity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0182164

    Inflammatory cytokine production from DCs is extinguished in the presence of P . gingivalis . ( A ) DCs from B10.A-Rag2 -/- mice were either unstimulated (medium) or stimulated on the sixth day with 5x10 7 bacteria in a 24-well plate for 16h at 37°C. Cells were harvested, stained with directly labeled monoclonal antibodies and analyzed by FACS analysis. Cells were gated for the CD11c positive population and analyzed for the costimulatory molecules B7.1, B7.2 and CD40, and the antigen-presenting molecule, MHC class II. Numbers indicate the percentage of the population in the gate or quadrant ( B ) Same as (A) Culture CSN were analyzed for the presence of various cytokines using Searchlight protein arrays. ( C ) Same as (B) except the co-culture period was 6h, at which time we isolated total RNA using the RNeasy kit and analyzed it by Real-time RT-PCR. The data are expressed as the mean ± SD of three independent experiments.
    Figure Legend Snippet: Inflammatory cytokine production from DCs is extinguished in the presence of P . gingivalis . ( A ) DCs from B10.A-Rag2 -/- mice were either unstimulated (medium) or stimulated on the sixth day with 5x10 7 bacteria in a 24-well plate for 16h at 37°C. Cells were harvested, stained with directly labeled monoclonal antibodies and analyzed by FACS analysis. Cells were gated for the CD11c positive population and analyzed for the costimulatory molecules B7.1, B7.2 and CD40, and the antigen-presenting molecule, MHC class II. Numbers indicate the percentage of the population in the gate or quadrant ( B ) Same as (A) Culture CSN were analyzed for the presence of various cytokines using Searchlight protein arrays. ( C ) Same as (B) except the co-culture period was 6h, at which time we isolated total RNA using the RNeasy kit and analyzed it by Real-time RT-PCR. The data are expressed as the mean ± SD of three independent experiments.

    Techniques Used: Mouse Assay, Staining, Labeling, FACS, Co-Culture Assay, Isolation, Quantitative RT-PCR

    6) Product Images from "Techniques for the Isolation of High-Quality RNA from Cells Encapsulated in Chitosan Hydrogels"

    Article Title: Techniques for the Isolation of High-Quality RNA from Cells Encapsulated in Chitosan Hydrogels

    Journal: Tissue Engineering. Part C, Methods

    doi: 10.1089/ten.tec.2012.0693

    Representative end-point RT-PCR gene expression results for (a) 18S and (b) TfR from RNA isolated using the freeze grind+CTAB+RNeasy ® (FCR) method, the mince+CTAB+RNeasy ® (MCR) method, and the lysozyme+CTAB+RNeasy ® (LCR) method. Genomic contamination was detected following agarose gel electrophoresis in all minus-RT controls. In addition, as shown in the TfR results, where the primers were designed to span an intron–exon boundary, two products were formed during the PCR, corresponding to a genomic product size of 270 bp and an mRNA product size of 62 bp.
    Figure Legend Snippet: Representative end-point RT-PCR gene expression results for (a) 18S and (b) TfR from RNA isolated using the freeze grind+CTAB+RNeasy ® (FCR) method, the mince+CTAB+RNeasy ® (MCR) method, and the lysozyme+CTAB+RNeasy ® (LCR) method. Genomic contamination was detected following agarose gel electrophoresis in all minus-RT controls. In addition, as shown in the TfR results, where the primers were designed to span an intron–exon boundary, two products were formed during the PCR, corresponding to a genomic product size of 270 bp and an mRNA product size of 62 bp.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation, Agarose Gel Electrophoresis, Polymerase Chain Reaction

    7) Product Images from "Mkp1 is a c-Jun target gene that antagonizes JNK-dependent apoptosis in sympathetic neurons"

    Article Title: Mkp1 is a c-Jun target gene that antagonizes JNK-dependent apoptosis in sympathetic neurons

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    doi: 10.1523/JNEUROSCI.2824-10.2010

    Mkp1 mRNA levels increase in sympathetic neurons after NGF withdrawal and this requires MLK activity. Sympathetic neurons from 1 day-old rats were cultured for 6 days in vitro and then rinsed and refed with medium containing NGF (+NGF), or lacking NGF (−NGF), or lacking NGF but containing 400 nM CEP-11004 (−NGF+CEP-11004) for 16 hours. RNA was then isolated using an RNeasy mini kit. A, Affymetrix Rat Exon 1.0ST microarray analysis of gene expression in sympathetic neurons at the whole gene level. c-jun , bim , dp5 and atf3 are induced after NGF withdrawal and this is reduced by CEP-11004 validating them as known targets of the MLK-JNK-c-Jun pathway. mkp1 induction after NGF withdrawal (4.51-fold) is reduced by CEP-11004 to 1.42-fold suggesting that it too may be a transcriptional target of the JNK pathway. B, c-jun , bim and mkp1 mRNA levels were measured by real time PCR and normalised to the housekeeping genes gapdh and hprt1 . Similar results were obtained in both cases. The data shown represents the average of 3 independent experiments ± SEM. Statistical comparisons were made between +NGF and −NGF or −NGF and −NGF+CEP for each gene. # P
    Figure Legend Snippet: Mkp1 mRNA levels increase in sympathetic neurons after NGF withdrawal and this requires MLK activity. Sympathetic neurons from 1 day-old rats were cultured for 6 days in vitro and then rinsed and refed with medium containing NGF (+NGF), or lacking NGF (−NGF), or lacking NGF but containing 400 nM CEP-11004 (−NGF+CEP-11004) for 16 hours. RNA was then isolated using an RNeasy mini kit. A, Affymetrix Rat Exon 1.0ST microarray analysis of gene expression in sympathetic neurons at the whole gene level. c-jun , bim , dp5 and atf3 are induced after NGF withdrawal and this is reduced by CEP-11004 validating them as known targets of the MLK-JNK-c-Jun pathway. mkp1 induction after NGF withdrawal (4.51-fold) is reduced by CEP-11004 to 1.42-fold suggesting that it too may be a transcriptional target of the JNK pathway. B, c-jun , bim and mkp1 mRNA levels were measured by real time PCR and normalised to the housekeeping genes gapdh and hprt1 . Similar results were obtained in both cases. The data shown represents the average of 3 independent experiments ± SEM. Statistical comparisons were made between +NGF and −NGF or −NGF and −NGF+CEP for each gene. # P

    Techniques Used: Activity Assay, Cell Culture, In Vitro, Isolation, Microarray, Expressing, Real-time Polymerase Chain Reaction

    8) Product Images from "Clostridium difficile Modulates the Gut Microbiota by Inducing the Production of Indole, an Interkingdom Signaling and Antimicrobial Molecule"

    Article Title: Clostridium difficile Modulates the Gut Microbiota by Inducing the Production of Indole, an Interkingdom Signaling and Antimicrobial Molecule

    Journal: mSystems

    doi: 10.1128/mSystems.00346-18

    C. difficile induces tnaA overexpression in enterotoxigenic E. coli . (A) E. coli strains H10407 and 25922 were incubated anaerobically for 6 h at 37°C in fresh BHI medium containing 25% 0.2-µm-filtered cell-free C. difficile R20291 supernatant and 5 mM l -tryptophan. Total RNA was isolated using an RNeasy kit (Qiagen), and this was followed by cDNA synthesis by reverse transcription using a ProtoScript AMV First Strand cDNA synthesis kit (New England BioLabs) with 1 µg of the isolated total RNA. Quantitative PCR was performed using primers specific for tnaA and rpoB genes (control). Known quantities of tnaA DNA were used as standards. Mann-Whitney U test showed a significant difference ( P > 0.001) in the tnaA transcript levels detected in both E. coli strains cultured in the presence and absence of the C. difficile but no significant difference ( P = 0.401) between the two E. coli strains. Samples of the RNA preparation processed without the reverse transcription step uniformly yielded no detectable SYBR green signal. Error bars represent the standard deviations from three independent experiments. (B) Effect of the C. difficile Agr1 quorum signaling system on indole production in E. coli . (I) E. coli H10407 cells were cocultured with and without C. difficile wild-type R20291, agr1 mutant ( agr1 Mut), and the complemented agr1 mutant (Comp agr1 Mut) for 24 h anaerobically. (II) E. coli H10407 cells were cocultured with and without purified Agr1 quorum signaling autoinducing peptide (TI signal) for 6 h aerobically at 37°C. The culture supernatants were tested for indole using the hydroxylamine indole assay ( 15 ). (C) Effect of indole on growth of anaerobes. The anaerobes were cultured anaerobically for 24 h at 37°C in the presence of different indole amounts (0 to 6 mM). Concentration of indole that completely inhibited bacterial growth was recorded. Data shown represent three independent replicates.
    Figure Legend Snippet: C. difficile induces tnaA overexpression in enterotoxigenic E. coli . (A) E. coli strains H10407 and 25922 were incubated anaerobically for 6 h at 37°C in fresh BHI medium containing 25% 0.2-µm-filtered cell-free C. difficile R20291 supernatant and 5 mM l -tryptophan. Total RNA was isolated using an RNeasy kit (Qiagen), and this was followed by cDNA synthesis by reverse transcription using a ProtoScript AMV First Strand cDNA synthesis kit (New England BioLabs) with 1 µg of the isolated total RNA. Quantitative PCR was performed using primers specific for tnaA and rpoB genes (control). Known quantities of tnaA DNA were used as standards. Mann-Whitney U test showed a significant difference ( P > 0.001) in the tnaA transcript levels detected in both E. coli strains cultured in the presence and absence of the C. difficile but no significant difference ( P = 0.401) between the two E. coli strains. Samples of the RNA preparation processed without the reverse transcription step uniformly yielded no detectable SYBR green signal. Error bars represent the standard deviations from three independent experiments. (B) Effect of the C. difficile Agr1 quorum signaling system on indole production in E. coli . (I) E. coli H10407 cells were cocultured with and without C. difficile wild-type R20291, agr1 mutant ( agr1 Mut), and the complemented agr1 mutant (Comp agr1 Mut) for 24 h anaerobically. (II) E. coli H10407 cells were cocultured with and without purified Agr1 quorum signaling autoinducing peptide (TI signal) for 6 h aerobically at 37°C. The culture supernatants were tested for indole using the hydroxylamine indole assay ( 15 ). (C) Effect of indole on growth of anaerobes. The anaerobes were cultured anaerobically for 24 h at 37°C in the presence of different indole amounts (0 to 6 mM). Concentration of indole that completely inhibited bacterial growth was recorded. Data shown represent three independent replicates.

    Techniques Used: Over Expression, Incubation, Isolation, Real-time Polymerase Chain Reaction, MANN-WHITNEY, Cell Culture, SYBR Green Assay, Mutagenesis, Purification, Concentration Assay

    9) Product Images from "Modulation of Innate Immune Responses via Covalently Linked TLR Agonists"

    Article Title: Modulation of Innate Immune Responses via Covalently Linked TLR Agonists

    Journal: ACS Central Science

    doi: 10.1021/acscentsci.5b00274

    BMDC gene expression profile data. (a–d) BMDC gene expression profile illustrating second main trend observed, where Indole contributed to a decrease in CpG immune activity exhibited by Indole_Lox_CpG. BMDCs were incubated with each compound for 18 h at 37 °C. Total RNA was then isolated using RNeasy kit (Qiagen) and analyzed using NanoString Technology. Each figure illustrates the fold change of the specified agonist compared to the no agonist control and is the result of three independent experiments, where * p
    Figure Legend Snippet: BMDC gene expression profile data. (a–d) BMDC gene expression profile illustrating second main trend observed, where Indole contributed to a decrease in CpG immune activity exhibited by Indole_Lox_CpG. BMDCs were incubated with each compound for 18 h at 37 °C. Total RNA was then isolated using RNeasy kit (Qiagen) and analyzed using NanoString Technology. Each figure illustrates the fold change of the specified agonist compared to the no agonist control and is the result of three independent experiments, where * p

    Techniques Used: Expressing, Activity Assay, Incubation, Isolation

    BMDC gene expression profile data. (a) Heat map of immune function related genes. Each figure represents the average of three independent experiments. BMDCs were incubated with each compound for 18 h at 37 °C. Total RNA was then isolated using RNeasy kit (Qiagen) and analyzed using NanoString Technology. (b) Graph illustrating T H 1/T H 2 gene expression profile comparing the gene transcription level of Indole_Lox_CpG to Indole/Lox/CpG. (c) BMDC gene profile illustrating the main trend: Indole_Lox_CpG treated cells elicited the most upregulation in a subset of gene expression. Each figure illustrates the fold change of the specified agonist compared to the no agonist control and is the result of three independent experiments, where * p
    Figure Legend Snippet: BMDC gene expression profile data. (a) Heat map of immune function related genes. Each figure represents the average of three independent experiments. BMDCs were incubated with each compound for 18 h at 37 °C. Total RNA was then isolated using RNeasy kit (Qiagen) and analyzed using NanoString Technology. (b) Graph illustrating T H 1/T H 2 gene expression profile comparing the gene transcription level of Indole_Lox_CpG to Indole/Lox/CpG. (c) BMDC gene profile illustrating the main trend: Indole_Lox_CpG treated cells elicited the most upregulation in a subset of gene expression. Each figure illustrates the fold change of the specified agonist compared to the no agonist control and is the result of three independent experiments, where * p

    Techniques Used: Expressing, Incubation, Isolation

    10) Product Images from "Regulation of the Cellulosomal celS (cel48A) Gene of Clostridium thermocellum Is Growth Rate Dependent"

    Article Title: Regulation of the Cellulosomal celS (cel48A) Gene of Clostridium thermocellum Is Growth Rate Dependent

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.185.10.3042-3048.2003

    RPA for celS -initiated mRNA derived from cellulose-grown C . thermocellum cells. RPAs were performed using RNA from exponential- or stationary-phase cultures grown on crystalline cellulose. Cell samples were harvested and snap-frozen in liquid nitrogen, and total RNA was extracted using the RNeasy kit from Qiagen. Different amounts of RNA (indicated in micrograms above each lane) were hybridized overnight with 50,000 cpm of 32 P-labeled 428-nt antisense celS probe, and then digested with RNase A-RNase T 1 (Promega) for 30 min. The protected RNAs were placed directly in a scintillation counter for quantification and then separated on a 5% polyacrylamide gel containing 7 M urea. The radiolabeled bands were visualized using a phosphorimager system. The expected size of the protected products was 380 nt. (Top) Representative autoradiograph of the protected products subjected to phosphorimager analysis. (Bottom) Correlation between the amount of total RNA used in the assay and the counts obtained for the protected products. The negative control (lane C) contained yeast RNA instead of C. thermocellum RNA. The full-length probe was used in lane P. The graphs represent the average values of at least three separate experiments, and the experimental error was ±15%.
    Figure Legend Snippet: RPA for celS -initiated mRNA derived from cellulose-grown C . thermocellum cells. RPAs were performed using RNA from exponential- or stationary-phase cultures grown on crystalline cellulose. Cell samples were harvested and snap-frozen in liquid nitrogen, and total RNA was extracted using the RNeasy kit from Qiagen. Different amounts of RNA (indicated in micrograms above each lane) were hybridized overnight with 50,000 cpm of 32 P-labeled 428-nt antisense celS probe, and then digested with RNase A-RNase T 1 (Promega) for 30 min. The protected RNAs were placed directly in a scintillation counter for quantification and then separated on a 5% polyacrylamide gel containing 7 M urea. The radiolabeled bands were visualized using a phosphorimager system. The expected size of the protected products was 380 nt. (Top) Representative autoradiograph of the protected products subjected to phosphorimager analysis. (Bottom) Correlation between the amount of total RNA used in the assay and the counts obtained for the protected products. The negative control (lane C) contained yeast RNA instead of C. thermocellum RNA. The full-length probe was used in lane P. The graphs represent the average values of at least three separate experiments, and the experimental error was ±15%.

    Techniques Used: Recombinase Polymerase Amplification, Derivative Assay, Labeling, Autoradiography, Negative Control

    11) Product Images from "2?-O Methylation of Internal Adenosine by Flavivirus NS5 Methyltransferase"

    Article Title: 2?-O Methylation of Internal Adenosine by Flavivirus NS5 Methyltransferase

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002642

    2′- O methylation of internal adenosine. (A) Incorporation of 3 H-methyl into polyA. Homopolymer RNAs (1 µg) were incubated with 2 µg of DENV-4 MTase in the presence of [ 3 H-methyl]-SAM. After the methylation reaction, the un-incorporated [ 3 H-methyl]-SAM was removed by RNeasy kit. The amount of 3 H-methyl incorporation was measured by a MicroBeta counting. (B) SPA-based methylation analysis of oligo (A) 12 , (Am) 12 , and (m 6 ,m 6 A) 12 . All three RNA oligos were 3′-end biotinylated to facilitate SPA analysis. Am indicates that the 2′-OH of adenosine is methylated. m 6 ,m 6 A indicates that the amino N 6 position of adenosine is double methylated. (C) SPA-based methylation analysis of DENV-1 RNA. pppA-RNAs, representing the 5′ 211 nt of DENV-1 genome, were in vitro transcribed using biotinylated-CTP plus unmodified ATP, 2′- O -methyladenosine triphosphate (AmTP), or N 6 methyl adenosine triphosphate (m 6 ATP). The transcription reactions generated pppA-RNA, ppp(Am)-RNA, and ppp(m 6 A)RNA, respectively. The RNAs were then subjected to SPA-based internal methylation analysis. Average results and standard deviations from three independent experiments are presented.
    Figure Legend Snippet: 2′- O methylation of internal adenosine. (A) Incorporation of 3 H-methyl into polyA. Homopolymer RNAs (1 µg) were incubated with 2 µg of DENV-4 MTase in the presence of [ 3 H-methyl]-SAM. After the methylation reaction, the un-incorporated [ 3 H-methyl]-SAM was removed by RNeasy kit. The amount of 3 H-methyl incorporation was measured by a MicroBeta counting. (B) SPA-based methylation analysis of oligo (A) 12 , (Am) 12 , and (m 6 ,m 6 A) 12 . All three RNA oligos were 3′-end biotinylated to facilitate SPA analysis. Am indicates that the 2′-OH of adenosine is methylated. m 6 ,m 6 A indicates that the amino N 6 position of adenosine is double methylated. (C) SPA-based methylation analysis of DENV-1 RNA. pppA-RNAs, representing the 5′ 211 nt of DENV-1 genome, were in vitro transcribed using biotinylated-CTP plus unmodified ATP, 2′- O -methyladenosine triphosphate (AmTP), or N 6 methyl adenosine triphosphate (m 6 ATP). The transcription reactions generated pppA-RNA, ppp(Am)-RNA, and ppp(m 6 A)RNA, respectively. The RNAs were then subjected to SPA-based internal methylation analysis. Average results and standard deviations from three independent experiments are presented.

    Techniques Used: Methylation, Incubation, In Vitro, Generated

    Comparison of internal methylation efficiencies between DENV RNAs and host ribosomal RNAs. (A) Full-length (FL) and 3′ truncated RNAs of DENV-1. pppAG-RNAs, representing the FL and a set of 3′ terminally truncated DENV-1 RNAs, were in vitro synthesized. Numbers indicate nucleoside positions of DENV-1 genome (GenBank accession number U88535). (B) Internal methylation analysis. An equal mass (0.5 µg) of FL and truncated DENV-1 RNAs, and human ribosomal 18 S and 28 S RNAs was treated with DENV MTase in the presence of [ 3 H-methyl]-SAM. The reactions were purified through an RNeasy column to remove unincorporated [ 3 H-methyl]-SAM. The purified RNAs were quantified for internal methylation by a MicroBeta counter. Average results from three experiments are shown; error bars represent standard deviations.
    Figure Legend Snippet: Comparison of internal methylation efficiencies between DENV RNAs and host ribosomal RNAs. (A) Full-length (FL) and 3′ truncated RNAs of DENV-1. pppAG-RNAs, representing the FL and a set of 3′ terminally truncated DENV-1 RNAs, were in vitro synthesized. Numbers indicate nucleoside positions of DENV-1 genome (GenBank accession number U88535). (B) Internal methylation analysis. An equal mass (0.5 µg) of FL and truncated DENV-1 RNAs, and human ribosomal 18 S and 28 S RNAs was treated with DENV MTase in the presence of [ 3 H-methyl]-SAM. The reactions were purified through an RNeasy column to remove unincorporated [ 3 H-methyl]-SAM. The purified RNAs were quantified for internal methylation by a MicroBeta counter. Average results from three experiments are shown; error bars represent standard deviations.

    Techniques Used: Methylation, In Vitro, Synthesized, Purification

    12) Product Images from "Techniques for the Isolation of High-Quality RNA from Cells Encapsulated in Chitosan Hydrogels"

    Article Title: Techniques for the Isolation of High-Quality RNA from Cells Encapsulated in Chitosan Hydrogels

    Journal: Tissue Engineering. Part C, Methods

    doi: 10.1089/ten.tec.2012.0693

    Representative end-point RT-PCR gene expression results for (a) 18S and (b) TfR from RNA isolated using the freeze grind+CTAB+RNeasy ® (FCR) method, the mince+CTAB+RNeasy ® (MCR) method, and the lysozyme+CTAB+RNeasy ® (LCR) method. Genomic contamination was detected following agarose gel electrophoresis in all minus-RT controls. In addition, as shown in the TfR results, where the primers were designed to span an intron–exon boundary, two products were formed during the PCR, corresponding to a genomic product size of 270 bp and an mRNA product size of 62 bp.
    Figure Legend Snippet: Representative end-point RT-PCR gene expression results for (a) 18S and (b) TfR from RNA isolated using the freeze grind+CTAB+RNeasy ® (FCR) method, the mince+CTAB+RNeasy ® (MCR) method, and the lysozyme+CTAB+RNeasy ® (LCR) method. Genomic contamination was detected following agarose gel electrophoresis in all minus-RT controls. In addition, as shown in the TfR results, where the primers were designed to span an intron–exon boundary, two products were formed during the PCR, corresponding to a genomic product size of 270 bp and an mRNA product size of 62 bp.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation, Agarose Gel Electrophoresis, Polymerase Chain Reaction

    13) Product Images from "Quality Control of RNA Preservation and Extraction from Paraffin-Embedded Tissue: Implications for RT-PCR and Microarray Analysis"

    Article Title: Quality Control of RNA Preservation and Extraction from Paraffin-Embedded Tissue: Implications for RT-PCR and Microarray Analysis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0070714

    Fixation has strong influence on RNA fragment size and impairs qRT-PCR results. Electropherograms show different RNA molecule size distribution in cryopreserved and FFPE/TFPE liver tissue samples. (A) In contrast to CRYO, ribosomal RNA peaks are absent and fragmentation of RNA has occurred in all fixed tissues. (B) RNA cleanup by Qiagen RNeasy Kit removes small (
    Figure Legend Snippet: Fixation has strong influence on RNA fragment size and impairs qRT-PCR results. Electropherograms show different RNA molecule size distribution in cryopreserved and FFPE/TFPE liver tissue samples. (A) In contrast to CRYO, ribosomal RNA peaks are absent and fragmentation of RNA has occurred in all fixed tissues. (B) RNA cleanup by Qiagen RNeasy Kit removes small (

    Techniques Used: Quantitative RT-PCR, Formalin-fixed Paraffin-Embedded

    14) Product Images from "Techniques for the Isolation of High-Quality RNA from Cells Encapsulated in Chitosan Hydrogels"

    Article Title: Techniques for the Isolation of High-Quality RNA from Cells Encapsulated in Chitosan Hydrogels

    Journal: Tissue Engineering. Part C, Methods

    doi: 10.1089/ten.tec.2012.0693

    Representative end-point RT-PCR gene expression results for (a) 18S and (b) TfR from RNA isolated using the freeze grind+CTAB+RNeasy ® (FCR) method, the mince+CTAB+RNeasy ® (MCR) method, and the lysozyme+CTAB+RNeasy ® (LCR) method. Genomic contamination was detected following agarose gel electrophoresis in all minus-RT controls. In addition, as shown in the TfR results, where the primers were designed to span an intron–exon boundary, two products were formed during the PCR, corresponding to a genomic product size of 270 bp and an mRNA product size of 62 bp.
    Figure Legend Snippet: Representative end-point RT-PCR gene expression results for (a) 18S and (b) TfR from RNA isolated using the freeze grind+CTAB+RNeasy ® (FCR) method, the mince+CTAB+RNeasy ® (MCR) method, and the lysozyme+CTAB+RNeasy ® (LCR) method. Genomic contamination was detected following agarose gel electrophoresis in all minus-RT controls. In addition, as shown in the TfR results, where the primers were designed to span an intron–exon boundary, two products were formed during the PCR, corresponding to a genomic product size of 270 bp and an mRNA product size of 62 bp.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation, Agarose Gel Electrophoresis, Polymerase Chain Reaction

    15) Product Images from "Accessory Gene Regulator-1 Locus Is Essential for Virulence and Pathogenesis of Clostridium difficile"

    Article Title: Accessory Gene Regulator-1 Locus Is Essential for Virulence and Pathogenesis of Clostridium difficile

    Journal: mBio

    doi: 10.1128/mBio.01237-16

    Analysis of agr mutants for transcription of the toxin genes ( tcdA and tcdB ) in BHI and TY media. The 630 wild-type, agrB1D1 mutant, and complemented agrB1D1 mutant strains were cultured in either BHI (A) or TY (B) medium. The R20291 wild-type, agrB1D1 mutant, complemented agrB1D1 mutant, and agrB2D2 mutant strains were also cultured in either BHI (C) or TY (D) medium. The cultures were incubated for 16 h anaerobically at 37°C. Total RNA was isolated with the RNeasy kit (Qiagen), and this was followed by cDNA synthesis by reverse transcription using the ProtoScript AMV First Strand cDNA synthesis kit (New England Biolabs, Ipswich, MA) with 1 µg of the isolated total RNA. Quantitative PCR was performed with primers specific for tcdA , tcdB , and the cDNA used as the template. Known quantities of tcdA and tcdB DNA were used as standards. The difference between the tcdA and tcdB transcript levels detected in the wild-type and agrB1B1 mutant strains was significant ( P = 0.0001), but there was no significant difference ( P = 0.235) between the two culture media. Error bars represent the standard deviations of three independent experiments.
    Figure Legend Snippet: Analysis of agr mutants for transcription of the toxin genes ( tcdA and tcdB ) in BHI and TY media. The 630 wild-type, agrB1D1 mutant, and complemented agrB1D1 mutant strains were cultured in either BHI (A) or TY (B) medium. The R20291 wild-type, agrB1D1 mutant, complemented agrB1D1 mutant, and agrB2D2 mutant strains were also cultured in either BHI (C) or TY (D) medium. The cultures were incubated for 16 h anaerobically at 37°C. Total RNA was isolated with the RNeasy kit (Qiagen), and this was followed by cDNA synthesis by reverse transcription using the ProtoScript AMV First Strand cDNA synthesis kit (New England Biolabs, Ipswich, MA) with 1 µg of the isolated total RNA. Quantitative PCR was performed with primers specific for tcdA , tcdB , and the cDNA used as the template. Known quantities of tcdA and tcdB DNA were used as standards. The difference between the tcdA and tcdB transcript levels detected in the wild-type and agrB1B1 mutant strains was significant ( P = 0.0001), but there was no significant difference ( P = 0.235) between the two culture media. Error bars represent the standard deviations of three independent experiments.

    Techniques Used: Mutagenesis, Cell Culture, Incubation, Isolation, Real-time Polymerase Chain Reaction

    16) Product Images from "The highly expressed 5’isomiR of hsa-miR-140-3p contributes to the tumor-suppressive effects of miR-140 by reducing breast cancer proliferation and migration"

    Article Title: The highly expressed 5’isomiR of hsa-miR-140-3p contributes to the tumor-suppressive effects of miR-140 by reducing breast cancer proliferation and migration

    Journal: BMC Genomics

    doi: 10.1186/s12864-016-2869-x

    Validation of direct targeting by 3’ UTR luciferase assay and Taqman qRT-PCR. a Venn diagram containing genes that were found to be significantly downregulated in both MCF10A and MDA-MB-231 cells upon overexpression of has-miR-140-3p or 5’isomiR-140-3p to at least 65 % of the expression in control transfected cells. b and c MCF7 cells were transfected with the miRNA mimics and wildtype ( b ) or mutated ( c ) psiCHECK2 3’ UTR reporter plasmids as indicated. 72 h later, cells were lysed and the activity of renilla (480 nm) and firefly (560 nm) luciferase were measured. Renilla measurements were normalized to firefly and values were normalized to the negative control (mimic-ctrl2). Afterwards, values were normalized to the empty psiCHECK2 transfected with 5’ isomiR-140-3p or hsa-miR-140-3p. Bars represent the average of 6 biological replicates ± standard deviation indicated as error bars. d MCF10A and MDA-MB-231 cells were transfected with hsa-miR-140-3p, 5’ isomiR-140-3p or miRNA mimic negative control (mimic-ctrl2). 72 h later, cells were lysed and total mRNA was isolated and purified using RNeasy kit (Qiagen). The mRNA expression levels of the candidate genes were then assessed by Taqman qRT-PCR. Gene expression was normalized to HPRT and GAPDH housekeeping genes. Normalized gene expression is depicted as relative expression to cells transfected with mimic-ctrl2. Values represent the mean of three biological replicates (*** P ≤ 0.001, ** P ≤ 0.01, ns = non-significant compared to control, unpaired t test)
    Figure Legend Snippet: Validation of direct targeting by 3’ UTR luciferase assay and Taqman qRT-PCR. a Venn diagram containing genes that were found to be significantly downregulated in both MCF10A and MDA-MB-231 cells upon overexpression of has-miR-140-3p or 5’isomiR-140-3p to at least 65 % of the expression in control transfected cells. b and c MCF7 cells were transfected with the miRNA mimics and wildtype ( b ) or mutated ( c ) psiCHECK2 3’ UTR reporter plasmids as indicated. 72 h later, cells were lysed and the activity of renilla (480 nm) and firefly (560 nm) luciferase were measured. Renilla measurements were normalized to firefly and values were normalized to the negative control (mimic-ctrl2). Afterwards, values were normalized to the empty psiCHECK2 transfected with 5’ isomiR-140-3p or hsa-miR-140-3p. Bars represent the average of 6 biological replicates ± standard deviation indicated as error bars. d MCF10A and MDA-MB-231 cells were transfected with hsa-miR-140-3p, 5’ isomiR-140-3p or miRNA mimic negative control (mimic-ctrl2). 72 h later, cells were lysed and total mRNA was isolated and purified using RNeasy kit (Qiagen). The mRNA expression levels of the candidate genes were then assessed by Taqman qRT-PCR. Gene expression was normalized to HPRT and GAPDH housekeeping genes. Normalized gene expression is depicted as relative expression to cells transfected with mimic-ctrl2. Values represent the mean of three biological replicates (*** P ≤ 0.001, ** P ≤ 0.01, ns = non-significant compared to control, unpaired t test)

    Techniques Used: Luciferase, Quantitative RT-PCR, Multiple Displacement Amplification, Over Expression, Expressing, Transfection, Activity Assay, Negative Control, Standard Deviation, Isolation, Purification

    17) Product Images from "Quantification of cellular poly(ADP-ribosyl)ation by stable isotope dilution mass spectrometry reveals tissue- and drug-dependent stress response dynamics"

    Article Title: Quantification of cellular poly(ADP-ribosyl)ation by stable isotope dilution mass spectrometry reveals tissue- and drug-dependent stress response dynamics

    Journal: ACS chemical biology

    doi: 10.1021/cb400170b

    Development of an LC-MS/MS-based method for the quantification of cellular PAR ( A ) PAR extraction from untreated or H 2 O 2 -stimulated (5 mM) COPF5 cells using phenol/chloroform (Ph/Chl) extraction, dihydroxyboryl Biorex (DHBB) affinity chromatography (DHBB), or commercially available RNA extraction kits, i.e. , Roche High pure microRNA isolation kit (R), PeqLab PeqGold RNA pure kit (PL), Qiagen RNeasy kit (Q). Extracted PAR samples were immobilized on a nylon membrane and detected using the PAR-specific mAB 10H. ( B ) Densitometric quantification of the slot-blot shown in (A). ( C ) LC-MS/MS quantification of cellular PAR levels extracted from COPF5 cells via classical DHBB chromatography or kit (R). Data represent means from two independent experiments. ( D ) LC-MS/MS chromatograms of cellular ribosyl-adenosine (blue) and corresponding internal standard (red) from digested PAR isolated from 1 × 10 7 untreated or H 2 O 2 -stimulated (100 μM) freshly isolated human PBMCs. ( E ) Evaluation of method robustness as assessed in independent experiments with PBMCs isolated from one donor (technical variability). Cells were treated with 100 μM H 2 O 2 for 5 min. ( F ) Analysis of inter-donor variability (biological variability). PBMCs were isolated from different donors and stimulated with 100 μM H 2 O 2 for 5 min. Data represent means ± SEM. *** P
    Figure Legend Snippet: Development of an LC-MS/MS-based method for the quantification of cellular PAR ( A ) PAR extraction from untreated or H 2 O 2 -stimulated (5 mM) COPF5 cells using phenol/chloroform (Ph/Chl) extraction, dihydroxyboryl Biorex (DHBB) affinity chromatography (DHBB), or commercially available RNA extraction kits, i.e. , Roche High pure microRNA isolation kit (R), PeqLab PeqGold RNA pure kit (PL), Qiagen RNeasy kit (Q). Extracted PAR samples were immobilized on a nylon membrane and detected using the PAR-specific mAB 10H. ( B ) Densitometric quantification of the slot-blot shown in (A). ( C ) LC-MS/MS quantification of cellular PAR levels extracted from COPF5 cells via classical DHBB chromatography or kit (R). Data represent means from two independent experiments. ( D ) LC-MS/MS chromatograms of cellular ribosyl-adenosine (blue) and corresponding internal standard (red) from digested PAR isolated from 1 × 10 7 untreated or H 2 O 2 -stimulated (100 μM) freshly isolated human PBMCs. ( E ) Evaluation of method robustness as assessed in independent experiments with PBMCs isolated from one donor (technical variability). Cells were treated with 100 μM H 2 O 2 for 5 min. ( F ) Analysis of inter-donor variability (biological variability). PBMCs were isolated from different donors and stimulated with 100 μM H 2 O 2 for 5 min. Data represent means ± SEM. *** P

    Techniques Used: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Affinity Chromatography, RNA Extraction, Isolation, Dot Blot, Chromatography

    18) Product Images from "LEDGF1-326 Decreases P23H and Wild Type Rhodopsin Aggregates and P23H Rhodopsin Mediated Cell Damage in Human Retinal Pigment Epithelial Cells"

    Article Title: LEDGF1-326 Decreases P23H and Wild Type Rhodopsin Aggregates and P23H Rhodopsin Mediated Cell Damage in Human Retinal Pigment Epithelial Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0024616

    LEDGF 1-326 does not alter the transcription level of P23H rhodopsin. mRNA was isolated from transfected ARPE-19 cells using the RNeasy kit. To remove contaminating genomic DNA, 10 µg RNA from each sample was treated with DNase using the Turbo DNA free kit. First strand synthesis was done using the high capacity RNA to DNA. PCR was performed to amplify the DNA on an ABI 7500 PCR machine. The threshold thermal cycle was used to calculate the mRNA level of rhodopsin and was normalized to GAPDH mRNA level. Data is expressed as mean ± S.D. for N = 3. Data was considered significant at p
    Figure Legend Snippet: LEDGF 1-326 does not alter the transcription level of P23H rhodopsin. mRNA was isolated from transfected ARPE-19 cells using the RNeasy kit. To remove contaminating genomic DNA, 10 µg RNA from each sample was treated with DNase using the Turbo DNA free kit. First strand synthesis was done using the high capacity RNA to DNA. PCR was performed to amplify the DNA on an ABI 7500 PCR machine. The threshold thermal cycle was used to calculate the mRNA level of rhodopsin and was normalized to GAPDH mRNA level. Data is expressed as mean ± S.D. for N = 3. Data was considered significant at p

    Techniques Used: Isolation, Transfection, Polymerase Chain Reaction

    19) Product Images from "A novel 6C assay uncovers Polycomb-mediated higher order chromatin conformations"

    Article Title: A novel 6C assay uncovers Polycomb-mediated higher order chromatin conformations

    Journal: Genome Research

    doi: 10.1101/gr.073452.107

    The genes flanking the interacting chromatin regions are up-regulated in Tera-2 cells depleted for EZH2 and in the ATRA differentiated cells. The loss of pairing between 6C partners induced by EZH2 knockdown, or by ATRA-induced cellular differentiation of the Tera-2 cells toward a neuronal lineage, is concomitant with the up-regulation of flanking genes residing in the interacting chromatin regions. Total RNA was prepared from NTC and EZH2 siRNA-treated Tera-2 cells 72 h post-transfection using RNeasy Kit (QIAGEN), followed by cDNA synthesis and real-time RT-PCR for a total of 19 genes flanking the interacting chromatin region in each 6C partner for the five clones that we studied. The relative mRNA levels of the chosen genes in NTC vs. EZH2 siRNA-treated Tera-2 cells are shown for clone 1 ( A ), clone 2 ( B ), clone 3 ( C ), clone 4 ( D ), and clone 5 ( E ). PCR of GAPDH mRNA was used to normalize values across different samples.
    Figure Legend Snippet: The genes flanking the interacting chromatin regions are up-regulated in Tera-2 cells depleted for EZH2 and in the ATRA differentiated cells. The loss of pairing between 6C partners induced by EZH2 knockdown, or by ATRA-induced cellular differentiation of the Tera-2 cells toward a neuronal lineage, is concomitant with the up-regulation of flanking genes residing in the interacting chromatin regions. Total RNA was prepared from NTC and EZH2 siRNA-treated Tera-2 cells 72 h post-transfection using RNeasy Kit (QIAGEN), followed by cDNA synthesis and real-time RT-PCR for a total of 19 genes flanking the interacting chromatin region in each 6C partner for the five clones that we studied. The relative mRNA levels of the chosen genes in NTC vs. EZH2 siRNA-treated Tera-2 cells are shown for clone 1 ( A ), clone 2 ( B ), clone 3 ( C ), clone 4 ( D ), and clone 5 ( E ). PCR of GAPDH mRNA was used to normalize values across different samples.

    Techniques Used: Cell Differentiation, Transfection, Quantitative RT-PCR, Clone Assay, Polymerase Chain Reaction

    20) Product Images from "Initiation Factor eIF2-independent Mode of c-Src mRNA Translation Occurs via an Internal Ribosome Entry Site *"

    Article Title: Initiation Factor eIF2-independent Mode of c-Src mRNA Translation Occurs via an Internal Ribosome Entry Site *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.029462

    Effect of deletion mutations on the c-Src sequence-controlled translation of reporter RNAs. A , organization of in vitro transcribed uncapped wild type reporter RNA ( 5′Src-RFLuc ) and the mutants containing various lengths of deletions (Δ) in the c-Src sequences are shown; dashed line , extent of deletion. AUG , initiator codon is underlined. B , RNAs were translated for 1.5 h in HeLa lysates in the presence of [ 35 S]methionine, and the FLuc protein bands were visualized by autoradiography after SDS-PAGE. Lane M , 14 C-labeled protein markers. C , Stability of the reporter RNAs in HeLa translation lysates. The 32 P-labeled reporter RNAs (∼1 × 10 6 dpm) were added to a standard HeLa translation mixture for 1.5 h, and total RNAs were isolated by Qiagen RNeasy column method. Half of the eluted RNA samples (20 μl) were subjected to formaldehyde-agarose gel electrophoresis. The gel was photographed after ethidium bromide staining for detection of ribosomal RNAs ( lower panel ), dried, and autoradiographed ( upper panel ). Reporter RNAs are as indicated for each lane. D , comparison of the kinetics of translation between wild type 5′Src-FLuc and a deletion mutant ( 5′Src Δ 2-FLuc ). The RNAs were translated in a standard HeLa lysates mixture, and FLuc activities were assayed with an aliquot (2 μl) of the reaction at various time points. Control , translation lysates without exogenous RNA. E , transfection of uncapped monocistronic c-Src mutant RNAs. The Huh7 cells (80% confluent in 60-mm culture plates) were transfected in triplicate with uncapped RNAs as indicated, and FLuc activities were assayed 3 h post-transfection in the total lysates. F , relative stability of mutant reporter RNAs in the transfected cells. The 32 P-labeled mutant RNAs (as indicated) were transfected as above. The total RNAs were isolated, and the labeled RNAs were detected by agarose gel electrophoresis followed by autoradiography of the dried gel ( upper panel ). Lower panel , the same gel showing 18 S rRNA in each lane.
    Figure Legend Snippet: Effect of deletion mutations on the c-Src sequence-controlled translation of reporter RNAs. A , organization of in vitro transcribed uncapped wild type reporter RNA ( 5′Src-RFLuc ) and the mutants containing various lengths of deletions (Δ) in the c-Src sequences are shown; dashed line , extent of deletion. AUG , initiator codon is underlined. B , RNAs were translated for 1.5 h in HeLa lysates in the presence of [ 35 S]methionine, and the FLuc protein bands were visualized by autoradiography after SDS-PAGE. Lane M , 14 C-labeled protein markers. C , Stability of the reporter RNAs in HeLa translation lysates. The 32 P-labeled reporter RNAs (∼1 × 10 6 dpm) were added to a standard HeLa translation mixture for 1.5 h, and total RNAs were isolated by Qiagen RNeasy column method. Half of the eluted RNA samples (20 μl) were subjected to formaldehyde-agarose gel electrophoresis. The gel was photographed after ethidium bromide staining for detection of ribosomal RNAs ( lower panel ), dried, and autoradiographed ( upper panel ). Reporter RNAs are as indicated for each lane. D , comparison of the kinetics of translation between wild type 5′Src-FLuc and a deletion mutant ( 5′Src Δ 2-FLuc ). The RNAs were translated in a standard HeLa lysates mixture, and FLuc activities were assayed with an aliquot (2 μl) of the reaction at various time points. Control , translation lysates without exogenous RNA. E , transfection of uncapped monocistronic c-Src mutant RNAs. The Huh7 cells (80% confluent in 60-mm culture plates) were transfected in triplicate with uncapped RNAs as indicated, and FLuc activities were assayed 3 h post-transfection in the total lysates. F , relative stability of mutant reporter RNAs in the transfected cells. The 32 P-labeled mutant RNAs (as indicated) were transfected as above. The total RNAs were isolated, and the labeled RNAs were detected by agarose gel electrophoresis followed by autoradiography of the dried gel ( upper panel ). Lower panel , the same gel showing 18 S rRNA in each lane.

    Techniques Used: Sequencing, In Vitro, Autoradiography, SDS Page, Labeling, Isolation, Agarose Gel Electrophoresis, Staining, Mutagenesis, Transfection

    21) Product Images from "Ethyl Caffeate Ameliorates Collagen-Induced Arthritis by Suppressing Th1 Immune Response"

    Article Title: Ethyl Caffeate Ameliorates Collagen-Induced Arthritis by Suppressing Th1 Immune Response

    Journal: Journal of Immunology Research

    doi: 10.1155/2017/7416792

    ECF suppresses IFN- γ -related pathway in TCR engagement-mediated T lymphocyte activation. Purified CD4 + T cells were stimulated with anti-CD3 (5 μ g/ml) and anti-CD28 (2 μ g/ml) for 16 h. Total RNA was isolated using RNeasy kits, and 1 μ g of total RNA was used to synthesis cDNA. Real-time quantitative PCR assay was carried out using SYBR Premix Ex Taq II kit, and relative quantification of mRNA expression was calculated as the fold increase using the delta-delta Ct; the housekeeping gene is β -actin. Results presented are mean ± s.e.m., n = 3. ∗ P
    Figure Legend Snippet: ECF suppresses IFN- γ -related pathway in TCR engagement-mediated T lymphocyte activation. Purified CD4 + T cells were stimulated with anti-CD3 (5 μ g/ml) and anti-CD28 (2 μ g/ml) for 16 h. Total RNA was isolated using RNeasy kits, and 1 μ g of total RNA was used to synthesis cDNA. Real-time quantitative PCR assay was carried out using SYBR Premix Ex Taq II kit, and relative quantification of mRNA expression was calculated as the fold increase using the delta-delta Ct; the housekeeping gene is β -actin. Results presented are mean ± s.e.m., n = 3. ∗ P

    Techniques Used: Activation Assay, Purification, Isolation, Real-time Polymerase Chain Reaction, Expressing

    22) Product Images from ""

    Article Title:

    Journal: Molecular Pharmacology

    doi: 10.1124/mol.116.107409

    Restoration of site-specific p53-175 mutant protein transactivational function by ZMC1, ZMC2, and ZMC3, but not 3-AP. (A) TOV112D and SKOV3 cells were treated with ZMC1, ZMC2, ZMC3, or 3-AP, and the gene expression of p21 and PUMA was analyzed by quantitative reverse-transcription polymerase chain reaction (RT-PCR). RNA was extracted from the cells using Qiagen RNeasy kit, and the gene expression level was measured by quantitative RT-PCR using TaqMan gene expression assays. β -actin was used as the internal control. Data represent an average ± S.D. performed in triplicate, repeated in three experiments. * P
    Figure Legend Snippet: Restoration of site-specific p53-175 mutant protein transactivational function by ZMC1, ZMC2, and ZMC3, but not 3-AP. (A) TOV112D and SKOV3 cells were treated with ZMC1, ZMC2, ZMC3, or 3-AP, and the gene expression of p21 and PUMA was analyzed by quantitative reverse-transcription polymerase chain reaction (RT-PCR). RNA was extracted from the cells using Qiagen RNeasy kit, and the gene expression level was measured by quantitative RT-PCR using TaqMan gene expression assays. β -actin was used as the internal control. Data represent an average ± S.D. performed in triplicate, repeated in three experiments. * P

    Techniques Used: Mutagenesis, Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

    Related Articles

    Transfection:

    Article Title: The Tumor Suppressive Role of eIF3f and Its Function in Translation Inhibition and rRNA Degradation
    Article Snippet: .. Cells were harvested at 4, 8 and 16 h after transfection and total RNA was isolated using RNeasy kit (QIAGEN). rRNAs (2.0 µg) were separated on an agarose gel and visualized by UV light (B). ..

    Agarose Gel Electrophoresis:

    Article Title: The Tumor Suppressive Role of eIF3f and Its Function in Translation Inhibition and rRNA Degradation
    Article Snippet: .. Cells were harvested at 4, 8 and 16 h after transfection and total RNA was isolated using RNeasy kit (QIAGEN). rRNAs (2.0 µg) were separated on an agarose gel and visualized by UV light (B). ..

    Isolation:

    Article Title: Research Resource: A Genome-Wide Study Identifies Potential New Target Genes for POU1F1
    Article Snippet: .. Total RNA from cells infected with the lentiviral vectors was isolated by using the RNeasy Kit (Qiagen, Hilden, Germany), including on-column DNase I digestion according to the manufacturer's recommendations. .. The quality of isolated RNA was controlled by the Lab-on-Chip-System Bioanalyser 2100 (Agilent Technologies, Inc., Palo Alto, CA).

    Article Title: The Tumor Suppressive Role of eIF3f and Its Function in Translation Inhibition and rRNA Degradation
    Article Snippet: .. Cells were harvested at 4, 8 and 16 h after transfection and total RNA was isolated using RNeasy kit (QIAGEN). rRNAs (2.0 µg) were separated on an agarose gel and visualized by UV light (B). ..

    Article Title: The embryonic type of SPP1 transcriptional regulation is re-activated in glioblastoma
    Article Snippet: .. Real- time PCR Total RNA was isolated from glioma cells using Qiagen RNeasy kit, 1 μg was used as a template. .. Amplifications were performed in duplicates in 20 μl containing 2× SYBR PCR MasterMix (Applied Biosystems) and a set of primers (human SPP1, NANOG and POU5F1_1_SG/OCT4 primers were from QuantiTect Primer Assays: QT01008798, QT01844808 and QT00210840, respectively, Qiagen).

    Article Title: Mechanisms by which Porphyromonas gingivalis evades innate immunity
    Article Snippet: .. Quantitative RT-PCR BMDCs were cultured in a 24-well plate for 6 h with 108 bacteria/well and RNA was isolated from the cells by RNeasy kit (Qiagen). .. RNA was treated to remove DNA contamination (Invitrogen) and the cDNA was synthesized using random primers, and reverse transcribed using Superscript III (Invitrogen).

    Article Title: Identification and analysis of proton-translocating pyrophosphatases in the methanogenic archaeon Methanosarcina mazei
    Article Snippet: .. Total cellular RNA was prepared from methanol-grown M. mazei Gö1 (harvested in the mid-log phase) with an RNA isolation kit (RNeasy, Qiagen) according to the manufacturer’s instructions. .. Northern blot analysis was performed as described previously ( ).

    Quantitative RT-PCR:

    Article Title: Mechanisms by which Porphyromonas gingivalis evades innate immunity
    Article Snippet: .. Quantitative RT-PCR BMDCs were cultured in a 24-well plate for 6 h with 108 bacteria/well and RNA was isolated from the cells by RNeasy kit (Qiagen). .. RNA was treated to remove DNA contamination (Invitrogen) and the cDNA was synthesized using random primers, and reverse transcribed using Superscript III (Invitrogen).

    Purification:

    Article Title: Techniques for the Isolation of High-Quality RNA from Cells Encapsulated in Chitosan Hydrogels
    Article Snippet: .. The protocol developed by Wang et al. uses a combination of mechanical disruption, extraction with CTAB buffer and purification with the RNeasy® kit. .. To test the efficacy of this approach with our specific material and cell source, the hydrogels were either (1) cryo-pulverized with a mortar and pestle into a fine powder or (2) finely minced with sharp surgical scissors and then disrupted using an ultrasonic dismembrator (Fisher Scientific Model 100) with three 5-s bursts at a setting of 4 with intervals of cooling on ice in 600 μL of CTAB buffer [2% CTAB, 2% poly(vinylpyrrolidone) (PVP), 1.4 M NaCl, 100 mM Tris-HCl, 20 mM EDTA, and 1% beta-mercaptoethanol in diethylpyrocarbonate (DEPC)-treated water] prewarmed to 65°C.

    Real-time Polymerase Chain Reaction:

    Article Title: The embryonic type of SPP1 transcriptional regulation is re-activated in glioblastoma
    Article Snippet: .. Real- time PCR Total RNA was isolated from glioma cells using Qiagen RNeasy kit, 1 μg was used as a template. .. Amplifications were performed in duplicates in 20 μl containing 2× SYBR PCR MasterMix (Applied Biosystems) and a set of primers (human SPP1, NANOG and POU5F1_1_SG/OCT4 primers were from QuantiTect Primer Assays: QT01008798, QT01844808 and QT00210840, respectively, Qiagen).

    Infection:

    Article Title: Research Resource: A Genome-Wide Study Identifies Potential New Target Genes for POU1F1
    Article Snippet: .. Total RNA from cells infected with the lentiviral vectors was isolated by using the RNeasy Kit (Qiagen, Hilden, Germany), including on-column DNase I digestion according to the manufacturer's recommendations. .. The quality of isolated RNA was controlled by the Lab-on-Chip-System Bioanalyser 2100 (Agilent Technologies, Inc., Palo Alto, CA).

    Cell Culture:

    Article Title: Mechanisms by which Porphyromonas gingivalis evades innate immunity
    Article Snippet: .. Quantitative RT-PCR BMDCs were cultured in a 24-well plate for 6 h with 108 bacteria/well and RNA was isolated from the cells by RNeasy kit (Qiagen). .. RNA was treated to remove DNA contamination (Invitrogen) and the cDNA was synthesized using random primers, and reverse transcribed using Superscript III (Invitrogen).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Qiagen rneasy mini kit
    Reverse transcription polymerase chain reaction amplification of RNA extracted from VSV‐IND and VSV‐NJ spiked bovine lymph nodes by <t>Qiagen</t> <t>RNeasy</t> mini kit. Lane 1: 100 bp marker; lanes 2–6: IND and NJ RNA with NJ Primer set; lanes 7–11: IND and NJ RNA with IND Primer set; lane 12–16: IND and NJ RNA with both IND and NJ primer sets; lane 17: Negative extraction control; lane 18: Negative PCR control; lane 19: 100 bp marker.
    Rneasy Mini Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 33751 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rneasy mini kit/product/Qiagen
    Average 99 stars, based on 33751 article reviews
    Price from $9.99 to $1999.99
    rneasy mini kit - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    Image Search Results


    Reverse transcription polymerase chain reaction amplification of RNA extracted from VSV‐IND and VSV‐NJ spiked bovine lymph nodes by Qiagen RNeasy mini kit. Lane 1: 100 bp marker; lanes 2–6: IND and NJ RNA with NJ Primer set; lanes 7–11: IND and NJ RNA with IND Primer set; lane 12–16: IND and NJ RNA with both IND and NJ primer sets; lane 17: Negative extraction control; lane 18: Negative PCR control; lane 19: 100 bp marker.

    Journal: Journal of Clinical Laboratory Analysis

    Article Title: Comparison of RNA extraction methods to augment the sensitivity for the differentiation of vesicular stomatitis virus Indiana1 and New Jersey

    doi: 10.1002/jcla.20439

    Figure Lengend Snippet: Reverse transcription polymerase chain reaction amplification of RNA extracted from VSV‐IND and VSV‐NJ spiked bovine lymph nodes by Qiagen RNeasy mini kit. Lane 1: 100 bp marker; lanes 2–6: IND and NJ RNA with NJ Primer set; lanes 7–11: IND and NJ RNA with IND Primer set; lane 12–16: IND and NJ RNA with both IND and NJ primer sets; lane 17: Negative extraction control; lane 18: Negative PCR control; lane 19: 100 bp marker.

    Article Snippet: RNA from various dilutions of each VSV type was extracted by using Qiagen RNeasy mini kit as per manufacturer's instructions (Qiagen, Valencia, CA).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Marker, Polymerase Chain Reaction

    Multiplex RT‐PCR sensitivity and specificity assay for VSV‐IND and VSV‐NJ detection. RNA extracted from serial dilutions of both types of VSV with titers of 10 3.625 TCID 50 /25 µl and 10 4.375 TCID 50 /25 µl, for IND and NJ, respectively, using Qiagen RNeasy mini kit. Lanes 2–5: typical positive VSV‐IND reactions; lane 6: negative VSV‐IND reaction; lane 7–10: positive VSV‐NJ reactions; lane 11: negative VSV‐NJ reaction; lanes 12–15: VSV‐IND and VSV‐NJ positive reactions utilizing both primer sets; lane 16: negative for VSV‐IND NJ reaction; CE: negative extraction control; CP: negative PCR control; M: DNA marker.

    Journal: Journal of Clinical Laboratory Analysis

    Article Title: Comparison of RNA extraction methods to augment the sensitivity for the differentiation of vesicular stomatitis virus Indiana1 and New Jersey

    doi: 10.1002/jcla.20439

    Figure Lengend Snippet: Multiplex RT‐PCR sensitivity and specificity assay for VSV‐IND and VSV‐NJ detection. RNA extracted from serial dilutions of both types of VSV with titers of 10 3.625 TCID 50 /25 µl and 10 4.375 TCID 50 /25 µl, for IND and NJ, respectively, using Qiagen RNeasy mini kit. Lanes 2–5: typical positive VSV‐IND reactions; lane 6: negative VSV‐IND reaction; lane 7–10: positive VSV‐NJ reactions; lane 11: negative VSV‐NJ reaction; lanes 12–15: VSV‐IND and VSV‐NJ positive reactions utilizing both primer sets; lane 16: negative for VSV‐IND NJ reaction; CE: negative extraction control; CP: negative PCR control; M: DNA marker.

    Article Snippet: RNA from various dilutions of each VSV type was extracted by using Qiagen RNeasy mini kit as per manufacturer's instructions (Qiagen, Valencia, CA).

    Techniques: Multiplex Assay, Polymerase Chain Reaction, Marker

    Reverse transcription polymerase chain reaction amplification of RNA extracted from spiked BHK‐21 cell suspensions by using Qiagen RNeasy mini kit. Lane 1: 100 bp marker; lane 2–6: IND and NJ RNA with IND Primer set; lane 7–11: IND and NJ RNA with NJ Primer set; lane 12: Negative extraction control (IND primer set); lane 13–17: IND and NJ RNA with both IND and NJ primer sets; lane 18: Negative extraction control (NJ primer set); lane 19: Negative extraction control (IND and NJ primer sets); lane 20: Negative PCR control.

    Journal: Journal of Clinical Laboratory Analysis

    Article Title: Comparison of RNA extraction methods to augment the sensitivity for the differentiation of vesicular stomatitis virus Indiana1 and New Jersey

    doi: 10.1002/jcla.20439

    Figure Lengend Snippet: Reverse transcription polymerase chain reaction amplification of RNA extracted from spiked BHK‐21 cell suspensions by using Qiagen RNeasy mini kit. Lane 1: 100 bp marker; lane 2–6: IND and NJ RNA with IND Primer set; lane 7–11: IND and NJ RNA with NJ Primer set; lane 12: Negative extraction control (IND primer set); lane 13–17: IND and NJ RNA with both IND and NJ primer sets; lane 18: Negative extraction control (NJ primer set); lane 19: Negative extraction control (IND and NJ primer sets); lane 20: Negative PCR control.

    Article Snippet: RNA from various dilutions of each VSV type was extracted by using Qiagen RNeasy mini kit as per manufacturer's instructions (Qiagen, Valencia, CA).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Marker, Polymerase Chain Reaction

    Comparison of sera exosomal RNA using four different RNA extraction methods. (A) Total RNA yield from ultracentrifugation (UC) and ExoQuick (EQ) treated samples using the RNeasy Mini Kit combined with TRIzol LS, the RNeasy Mini Kit alone, conventional RNA precipitation, and AllPrep DNA/RNA Mini Kit. (B) Demonstration of RNA quality measured by OD 260 /OD 280 in EQ and UC treated samples. Data are shown as the mean ± SD from six independent patient samples. *** P

    Journal: PLoS ONE

    Article Title: Optimizing exosomal RNA isolation for RNA-Seq analyses of archival sera specimens

    doi: 10.1371/journal.pone.0196913

    Figure Lengend Snippet: Comparison of sera exosomal RNA using four different RNA extraction methods. (A) Total RNA yield from ultracentrifugation (UC) and ExoQuick (EQ) treated samples using the RNeasy Mini Kit combined with TRIzol LS, the RNeasy Mini Kit alone, conventional RNA precipitation, and AllPrep DNA/RNA Mini Kit. (B) Demonstration of RNA quality measured by OD 260 /OD 280 in EQ and UC treated samples. Data are shown as the mean ± SD from six independent patient samples. *** P

    Article Snippet: RNA was isolated from each ExoQuick prepared sample using the RNeasy Mini Kit combined with TRIzol LS.

    Techniques: RNA Extraction

    Exosomal RNAs are stable over two decades. Dot plots of total RNA yield and OD 260 /OD 280 from 105 EQ-treated archival patient samples using the RNeasy Mini Kit combined with TRIzol LS. Storage time for each sample is indicated on the x-axis. The Spearman’s rank correlations for RNA yield versus storing time and OD 260 /OD 280 versus storage time are 0.185 ( P = 0.06) and 0.04 ( P = 0.70), respectively.

    Journal: PLoS ONE

    Article Title: Optimizing exosomal RNA isolation for RNA-Seq analyses of archival sera specimens

    doi: 10.1371/journal.pone.0196913

    Figure Lengend Snippet: Exosomal RNAs are stable over two decades. Dot plots of total RNA yield and OD 260 /OD 280 from 105 EQ-treated archival patient samples using the RNeasy Mini Kit combined with TRIzol LS. Storage time for each sample is indicated on the x-axis. The Spearman’s rank correlations for RNA yield versus storing time and OD 260 /OD 280 versus storage time are 0.185 ( P = 0.06) and 0.04 ( P = 0.70), respectively.

    Article Snippet: RNA was isolated from each ExoQuick prepared sample using the RNeasy Mini Kit combined with TRIzol LS.

    Techniques:

    FCM detection of lipid bodies (LBs) in control C57BL/6 DLs and C57BL/6 DLs hosting Ds Red2 L. am amastigotes. Panel A. Representative analysis of the fluorescence of BODIPY 493/503 in MHC II + DLs. A1, A2. Control C57BL/6 DL cultures –i.e. not exposed to L. am amastigotes- were incubated or not at 34°C with 200 µM oleate for 21 hours. A3, A4. Ds Red2- L. am were added to DL cultures at a ratio of 5 Ds Red2- L. am per DL (+ L. am ). Oleic acid (200 µM) was added 3 hours post inoculation, (A4: +Oleate) or not (A3: −Oleate) for a further 21 hours at 34°C. Control and L. am -loaded DLs were then detached. LBs were stained with BODIPY 493/503 2 µg/ml in PBS for 30 minutes at 34°C. The cells were then incubated with anti MHC II- PE-Cy5 mAb to analyze only the MHC class II-positive DLs. FCM histograms show BODIPY 493/503 fluorescence for Ctrl (A1, A2: white histograms) and Ds Red2 L. am -hosting DLs (A3, A4) ( Ds Red2 + , black histograms) and amastigotes-free DLs ( Ds Red2 − , grey histograms). Mean fluorescence intensity was indicated for each histogram. Panel B: Statistical analysis of the BODIPY 493/503 MFI values. Mean BODIPY 493/503 fluorescence by MHC II + DLs was determined for n = 7 independent experiments. MFI in L. am -loaded cultures is shown for Ds Red2 + and Ds Red2 − DLs as black and grey bars, respectively. Statistical analyses were performed by the Mann Whitney test.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Reprogramming Neutral Lipid Metabolism in Mouse Dendritic Leucocytes Hosting Live Leishmania amazonensis Amastigotes

    doi: 10.1371/journal.pntd.0002276

    Figure Lengend Snippet: FCM detection of lipid bodies (LBs) in control C57BL/6 DLs and C57BL/6 DLs hosting Ds Red2 L. am amastigotes. Panel A. Representative analysis of the fluorescence of BODIPY 493/503 in MHC II + DLs. A1, A2. Control C57BL/6 DL cultures –i.e. not exposed to L. am amastigotes- were incubated or not at 34°C with 200 µM oleate for 21 hours. A3, A4. Ds Red2- L. am were added to DL cultures at a ratio of 5 Ds Red2- L. am per DL (+ L. am ). Oleic acid (200 µM) was added 3 hours post inoculation, (A4: +Oleate) or not (A3: −Oleate) for a further 21 hours at 34°C. Control and L. am -loaded DLs were then detached. LBs were stained with BODIPY 493/503 2 µg/ml in PBS for 30 minutes at 34°C. The cells were then incubated with anti MHC II- PE-Cy5 mAb to analyze only the MHC class II-positive DLs. FCM histograms show BODIPY 493/503 fluorescence for Ctrl (A1, A2: white histograms) and Ds Red2 L. am -hosting DLs (A3, A4) ( Ds Red2 + , black histograms) and amastigotes-free DLs ( Ds Red2 − , grey histograms). Mean fluorescence intensity was indicated for each histogram. Panel B: Statistical analysis of the BODIPY 493/503 MFI values. Mean BODIPY 493/503 fluorescence by MHC II + DLs was determined for n = 7 independent experiments. MFI in L. am -loaded cultures is shown for Ds Red2 + and Ds Red2 − DLs as black and grey bars, respectively. Statistical analyses were performed by the Mann Whitney test.

    Article Snippet: DL extracted RNA integrity control Total RNA was extracted from MHC II+ DLs (RNeasy+ Mini-Kit, Qiagen) and its quality and concentration was determined using a NanoDrop ND-1000 micro-spectrophotometer (Kisker, http://www.kisker-biotech.com ) and an Agilent-2100 Bioanalyzer (Agilent, http://www.chem.agilent.com ).

    Techniques: Fluorescence, Incubation, Staining, MANN-WHITNEY

    Flow cytometric analysis of CD36 expression in C57BL/6 DLs hosting live L. am amastigotes. L. am amastigotes were added to DL cultures at a ratio of 5 amastigotes per DL. Twenty-four hours later, DLs were detached and stained successively with anti- MHC II- PE-Cy5 and CD36-PE conjugated mAbs. As the fluorescence of transgenic Ds Red2 amastigotes was quashed by the fixation step, we used 2A3-26 mAb generated in our laboratory to image intact amastigotes inside their parasitophorous vacuoles. Thus, after fixation, the DLs were first permeabilized then labelled with alexafluor480-conjugated 2A3-26 mAbs before being analysed by FCM. This analysis was performed on gated MHC II + DLs. The central dot plot shows CD36 expression and the presence of intracellular parasites. The profiles of CD36 expression and mean CD36 fluorescence value are shown for 2A3-26 + DLs (red gate) and 2A3-26 − DLs (black gate).

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Reprogramming Neutral Lipid Metabolism in Mouse Dendritic Leucocytes Hosting Live Leishmania amazonensis Amastigotes

    doi: 10.1371/journal.pntd.0002276

    Figure Lengend Snippet: Flow cytometric analysis of CD36 expression in C57BL/6 DLs hosting live L. am amastigotes. L. am amastigotes were added to DL cultures at a ratio of 5 amastigotes per DL. Twenty-four hours later, DLs were detached and stained successively with anti- MHC II- PE-Cy5 and CD36-PE conjugated mAbs. As the fluorescence of transgenic Ds Red2 amastigotes was quashed by the fixation step, we used 2A3-26 mAb generated in our laboratory to image intact amastigotes inside their parasitophorous vacuoles. Thus, after fixation, the DLs were first permeabilized then labelled with alexafluor480-conjugated 2A3-26 mAbs before being analysed by FCM. This analysis was performed on gated MHC II + DLs. The central dot plot shows CD36 expression and the presence of intracellular parasites. The profiles of CD36 expression and mean CD36 fluorescence value are shown for 2A3-26 + DLs (red gate) and 2A3-26 − DLs (black gate).

    Article Snippet: DL extracted RNA integrity control Total RNA was extracted from MHC II+ DLs (RNeasy+ Mini-Kit, Qiagen) and its quality and concentration was determined using a NanoDrop ND-1000 micro-spectrophotometer (Kisker, http://www.kisker-biotech.com ) and an Agilent-2100 Bioanalyzer (Agilent, http://www.chem.agilent.com ).

    Techniques: Flow Cytometry, Expressing, Staining, Fluorescence, Transgenic Assay, Generated