trizol  (Thermo Fisher)


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    Name:
    TRIzol Max Bacterial RNA Isolation Kit
    Description:
    The TRIzol Max Bacterial RNA Isolation Kit provides a simple and reliable method to improve the isolation of intact total RNA from gram-positive and gram-negative bacteria. The kit utilizes both the Max Bacterial Enhancement Reagent and TRIzol Reagent to inactivate endogenous RNases and promote protein denaturing, improving RNA quality and integrity. Isolation of RNA takes about an hour.Key Features of the TRIzol Max Bacterial RNA Isolation Kit:• Formulated for the isolation of RNA from both Gram-positive and Gram-negative bacteria• Uses two technologies for maximum RNA yield and purity• Minimizes time required for RNA isolation by eliminating enzymatic and mechanical lytic stepsTwo Products in One Kit The TRIzol Max Bacterial RNA Isolation Kit combines Max Bacterial Enhancement Reagent with TRIzol Reagent. A 5-minute pre-treatment of bacteria with Max Bacterial Enhancement Reagent, containing chelating agents, detergent and buffer, denatures bacterial proteins and effectively deactivates endogenous RNases. Subsequent bacterial lysis by TRIzol Reagent generates a high-quality, minimally degraded RNA product.Purified RNA is Ideal for Use with a Variety of Applications and Products Bacterial RNA isolated using the TRIzol Max Bacterial RNA Isolation Kit is suitable for downstream applications such as real-time PCR (qPCR), RT-PCR, northern blotting, nuclease protection assays, cDNA synthesis, microarray analysis, and dot blot hybridization. For Research Use Only. Not intended for animal or human diagnostic or therapeutic use.
    Catalog Number:
    16096020
    Price:
    None
    Applications:
    DNA & RNA Purification & Analysis|RNA Extraction|Total RNA Isolation|Total RNA from Bacterial Samples
    Size:
    100 preps
    Category:
    Kits and Assays, DNA⁄RNA Purification Kits & Reagents, DNA⁄RNA Purification Reagents
    Score:
    85
    Quantity:
    1
    Buy from Supplier
    Name:
    TRIzol LS Reagent
    Description:

    Catalog Number:
    10296028
    Price:
    None
    Score:
    85
    Buy from Supplier


    Structured Review

    Thermo Fisher trizol
    Toll-like receptor (TLR) messenger <t>RNA</t> (mRNA) expression is not altered in bone marrow-derived dendritic cells (BM-DCs) from cats infected with feline immunodeficiency virus (FIV). The BM-DCs were cultured for 10 days in the presence of recombinant feline granulocyte–macrophage colony-stimulating factor (100 ng/ml) and then purified for CD11c and major histocompatibility complex class II expression. Purified BM-DCs were lysed with <t>Trizol</t> (Gibco), and the RNA was extracted. Real-time polymerase chain reaction was performed to quantify RNA levels of TLRs 2, 3, 4, 5, 7 and 9. Results are shown as the mean and SD of cells from eight naïve cats and six FIV-infected cats. There were no statistically significant differences in expression levels as determined by Student’s t -test.

    https://www.bioz.com/result/trizol/product/Thermo Fisher
    Average 79 stars, based on 1 article reviews
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    trizol - by Bioz Stars, 2019-11
    79/100 stars

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    1) Product Images from "Altered bone marrow dendritic cell cytokine production to toll-like receptor and CD40 ligation during chronic feline immunodeficiency virus infection"

    Article Title: Altered bone marrow dendritic cell cytokine production to toll-like receptor and CD40 ligation during chronic feline immunodeficiency virus infection

    Journal: Immunology

    doi: 10.1111/j.1365-2567.2008.02907.x

    Toll-like receptor (TLR) messenger RNA (mRNA) expression is not altered in bone marrow-derived dendritic cells (BM-DCs) from cats infected with feline immunodeficiency virus (FIV). The BM-DCs were cultured for 10 days in the presence of recombinant feline granulocyte–macrophage colony-stimulating factor (100 ng/ml) and then purified for CD11c and major histocompatibility complex class II expression. Purified BM-DCs were lysed with Trizol (Gibco), and the RNA was extracted. Real-time polymerase chain reaction was performed to quantify RNA levels of TLRs 2, 3, 4, 5, 7 and 9. Results are shown as the mean and SD of cells from eight naïve cats and six FIV-infected cats. There were no statistically significant differences in expression levels as determined by Student’s t -test.
    Figure Legend Snippet: Toll-like receptor (TLR) messenger RNA (mRNA) expression is not altered in bone marrow-derived dendritic cells (BM-DCs) from cats infected with feline immunodeficiency virus (FIV). The BM-DCs were cultured for 10 days in the presence of recombinant feline granulocyte–macrophage colony-stimulating factor (100 ng/ml) and then purified for CD11c and major histocompatibility complex class II expression. Purified BM-DCs were lysed with Trizol (Gibco), and the RNA was extracted. Real-time polymerase chain reaction was performed to quantify RNA levels of TLRs 2, 3, 4, 5, 7 and 9. Results are shown as the mean and SD of cells from eight naïve cats and six FIV-infected cats. There were no statistically significant differences in expression levels as determined by Student’s t -test.

    Techniques Used: Expressing, Derivative Assay, Infection, Cell Culture, Recombinant, Purification, Real-time Polymerase Chain Reaction

    2) Product Images from "Nonstructural Protein 11 of Porcine Reproductive and Respiratory Syndrome Virus Suppresses Both MAVS and RIG-I Expression as One of the Mechanisms to Antagonize Type I Interferon Production"

    Article Title: Nonstructural Protein 11 of Porcine Reproductive and Respiratory Syndrome Virus Suppresses Both MAVS and RIG-I Expression as One of the Mechanisms to Antagonize Type I Interferon Production

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0168314

    Reduced expression of MAVS and RIG-I by overexpression of nsp11. (A, B) MARC-145 cells were co-transfected with nsp11 and pFlag-MAVS. At 24 h post-transfection, total cell lysates were prepared and subjected to Western blot using anti-Flag MAb (A), or total cellular RNA was extracted for cDNA synthesis using random primers followed by qPCR in the SYBR Green system (B). For (A), lane 1, cells transfected with the empty vector only. Lane 2), cells transfected with the Flagged MAVS plasmid. Lane 3), cells transfected with wild-type Flagged nsp11 plasmid. Lane 4, cells co-transfected with the Flagged nsp11 and Flagged MAVS plasmids. (C) MARC-145 cells were infected with PRRSV or VSV-GFP at an M.O.I. of 0.1. Infected cells and supernatants were harvested at indicated times post-infection. Cells were lysed by Trizol for MAVS mRNA analysis by RT-qPCR. MAVS gene expression was shown as the fold change of copy numbers from infected cells (black bars) relative to that of uninfected cells (white bars). Titers of viruses were determined by TCID 50 using cell culture supernatants. (D) Cell lysates were prepared in parallel and subjected to SDS-PAGE and Western blot using mouse α-MAVS PAb, rabbit α-nsp11 PAb, α-PRRSV swine serum, and α-β-actin MAb. (E) MARC-145 cells were infected with PRRSV at an M.O.I. of 5, and at 24 h post-infection, cells were stained with mouse α-MAVS PAb, rabbit α-PRRSV nsp11 PAb, and DAPI. Blue arrows indicated uninfected cells and white arrows indicated virus-infected cells. (F-I) MARC-145 cells were either transfected with nsp11 (F, G), or infected with PRRSV(H, I). At 24 h post-transfection or D3 post-infection, total cellular RNAs and cell lysates were harvested. For qPCR, cDNA were synthesized using random primers followed by qPCR using the MAVS, RIG-I, p65, and IRF3 specific primers. MAVS mRNA fold changes were calculated based on the copy numbers in nsp11-transfected cell relative to those of mock-transfected cell control. All gene copy numbers was relative to the housing keeping gene GAPDH. (n = 3, *, P
    Figure Legend Snippet: Reduced expression of MAVS and RIG-I by overexpression of nsp11. (A, B) MARC-145 cells were co-transfected with nsp11 and pFlag-MAVS. At 24 h post-transfection, total cell lysates were prepared and subjected to Western blot using anti-Flag MAb (A), or total cellular RNA was extracted for cDNA synthesis using random primers followed by qPCR in the SYBR Green system (B). For (A), lane 1, cells transfected with the empty vector only. Lane 2), cells transfected with the Flagged MAVS plasmid. Lane 3), cells transfected with wild-type Flagged nsp11 plasmid. Lane 4, cells co-transfected with the Flagged nsp11 and Flagged MAVS plasmids. (C) MARC-145 cells were infected with PRRSV or VSV-GFP at an M.O.I. of 0.1. Infected cells and supernatants were harvested at indicated times post-infection. Cells were lysed by Trizol for MAVS mRNA analysis by RT-qPCR. MAVS gene expression was shown as the fold change of copy numbers from infected cells (black bars) relative to that of uninfected cells (white bars). Titers of viruses were determined by TCID 50 using cell culture supernatants. (D) Cell lysates were prepared in parallel and subjected to SDS-PAGE and Western blot using mouse α-MAVS PAb, rabbit α-nsp11 PAb, α-PRRSV swine serum, and α-β-actin MAb. (E) MARC-145 cells were infected with PRRSV at an M.O.I. of 5, and at 24 h post-infection, cells were stained with mouse α-MAVS PAb, rabbit α-PRRSV nsp11 PAb, and DAPI. Blue arrows indicated uninfected cells and white arrows indicated virus-infected cells. (F-I) MARC-145 cells were either transfected with nsp11 (F, G), or infected with PRRSV(H, I). At 24 h post-transfection or D3 post-infection, total cellular RNAs and cell lysates were harvested. For qPCR, cDNA were synthesized using random primers followed by qPCR using the MAVS, RIG-I, p65, and IRF3 specific primers. MAVS mRNA fold changes were calculated based on the copy numbers in nsp11-transfected cell relative to those of mock-transfected cell control. All gene copy numbers was relative to the housing keeping gene GAPDH. (n = 3, *, P

    Techniques Used: Expressing, Over Expression, Transfection, Western Blot, Real-time Polymerase Chain Reaction, SYBR Green Assay, Plasmid Preparation, Infection, Quantitative RT-PCR, Cell Culture, SDS Page, Staining, Synthesized

    3) Product Images from "Neuron-specific ELAV/Hu proteins suppress HuR mRNA during neuronal differentiation by alternative polyadenylation"

    Article Title: Neuron-specific ELAV/Hu proteins suppress HuR mRNA during neuronal differentiation by alternative polyadenylation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr1114

    The 6.0 - kb HuR mRNA isoform is expressed during retinoic acid induced neuronal differentiation of P19 embryonic carcinoma cells. P19 cells were differentiated in the presence (RA) or absence (Ctrl) of 5 µM RA for 4 days, then replated and allowed to differentiate for 2–6 days. At each time point, total RNA was isolated via Trizol. ( A ) Northern Blot showing increasing expression of the 6.0 - kb mRNA isoform with either a neuron-specific probe (HuR NS from B ) or a probe detecting all HuR mRNA isoforms (HuR ORF from B). β-Actin and ribosomal RNA (18S/28S) were used as loading controls. MW = molecular weight as determined by position of rRNA 18S and 28S bands (B) Semiquantitative PCR to confirm results of Northern blots shown in A. PCR products show consistent expression of the HuR ORF but increased expression of the 6.0 - kb HuR neuron-specific mRNA isoform (HuR NS). Neuron-specific mRNAs HuC and Neuroligin 3 (Neuro3) were used to verify the progression of P19 neuronal differentiation. ( C ) Western blot analysis of Hu protein expression showing induction of neuronal-specific HuB and D during P19 differentiation. GapDH served as a loading control.
    Figure Legend Snippet: The 6.0 - kb HuR mRNA isoform is expressed during retinoic acid induced neuronal differentiation of P19 embryonic carcinoma cells. P19 cells were differentiated in the presence (RA) or absence (Ctrl) of 5 µM RA for 4 days, then replated and allowed to differentiate for 2–6 days. At each time point, total RNA was isolated via Trizol. ( A ) Northern Blot showing increasing expression of the 6.0 - kb mRNA isoform with either a neuron-specific probe (HuR NS from B ) or a probe detecting all HuR mRNA isoforms (HuR ORF from B). β-Actin and ribosomal RNA (18S/28S) were used as loading controls. MW = molecular weight as determined by position of rRNA 18S and 28S bands (B) Semiquantitative PCR to confirm results of Northern blots shown in A. PCR products show consistent expression of the HuR ORF but increased expression of the 6.0 - kb HuR neuron-specific mRNA isoform (HuR NS). Neuron-specific mRNAs HuC and Neuroligin 3 (Neuro3) were used to verify the progression of P19 neuronal differentiation. ( C ) Western blot analysis of Hu protein expression showing induction of neuronal-specific HuB and D during P19 differentiation. GapDH served as a loading control.

    Techniques Used: Isolation, Northern Blot, Expressing, Molecular Weight, Polymerase Chain Reaction, Western Blot

    4) Product Images from "Gnotobiotic IL-10−/−; NF-?BEGFP Mice Develop Rapid and Severe Colitis Following Campylobacter jejuni Infection"

    Article Title: Gnotobiotic IL-10−/−; NF-?BEGFP Mice Develop Rapid and Severe Colitis Following Campylobacter jejuni Infection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0007413

    Increased TNFα and IL-12p40 mRNA accumulation in C. jejuni -associated IL-10 −/− ; NF-κB EGFP mice. IL-10 wt/wt ; NF-κB EGFP and IL-10 −/− ; NF-κB EGFP were associated with C. jejuni for 14 days. Colonic sections were lysed in trizol and RNA was extracted, reverse-transcribed (1 µg) and amplified by PCR using specific murine TNFa and IL12p40 primers. GAPDH was used as loading control. WT = IL-10 wt/wt ; NF-κB EGFP , n = 7; KO = IL-10 −/− ; NF-κB EGFP , n = 7; WT C. jejuni associated: n = 12; KO C. jejuni associated: n = 10. (*p = 0.002 for IL12p40 vs. control, *p = 0.001 for TNF vs. control).
    Figure Legend Snippet: Increased TNFα and IL-12p40 mRNA accumulation in C. jejuni -associated IL-10 −/− ; NF-κB EGFP mice. IL-10 wt/wt ; NF-κB EGFP and IL-10 −/− ; NF-κB EGFP were associated with C. jejuni for 14 days. Colonic sections were lysed in trizol and RNA was extracted, reverse-transcribed (1 µg) and amplified by PCR using specific murine TNFa and IL12p40 primers. GAPDH was used as loading control. WT = IL-10 wt/wt ; NF-κB EGFP , n = 7; KO = IL-10 −/− ; NF-κB EGFP , n = 7; WT C. jejuni associated: n = 12; KO C. jejuni associated: n = 10. (*p = 0.002 for IL12p40 vs. control, *p = 0.001 for TNF vs. control).

    Techniques Used: Mouse Assay, Amplification, Polymerase Chain Reaction, Gene Knockout

    NF-κB signaling drives C. jejuni induced cytokine gene expression in BMDC. BMDC were generated from WT mice and treated with Bay 11-7085 (10 µM) and then stimulated with C. jejuni (MOI 50) for 4 h. Cells were lysed in Trizol, total RNA was extracted, reverse transcribed and IL-12p40 (A) and IL-6 (B) mRNA expression was detected using real-time PCR (Applied Biosystems 7700 sequence detection system). Results were normalized to the housekeeping gene GAPDH to ascertain similar loading. Results are the mean +/− SD of triplicates samples and are from one of three independent experiments (*p = 0.011 for IL-12p40 vs. Bay 11-7085-treated cells; *p = 0.005 for IL-6 vs. Bay 11-7085-treated cells).
    Figure Legend Snippet: NF-κB signaling drives C. jejuni induced cytokine gene expression in BMDC. BMDC were generated from WT mice and treated with Bay 11-7085 (10 µM) and then stimulated with C. jejuni (MOI 50) for 4 h. Cells were lysed in Trizol, total RNA was extracted, reverse transcribed and IL-12p40 (A) and IL-6 (B) mRNA expression was detected using real-time PCR (Applied Biosystems 7700 sequence detection system). Results were normalized to the housekeeping gene GAPDH to ascertain similar loading. Results are the mean +/− SD of triplicates samples and are from one of three independent experiments (*p = 0.011 for IL-12p40 vs. Bay 11-7085-treated cells; *p = 0.005 for IL-6 vs. Bay 11-7085-treated cells).

    Techniques Used: Expressing, Generated, Mouse Assay, Real-time Polymerase Chain Reaction, Sequencing

    C. jejuni induces NF-κB-signalling and gene expression in CMT-93 cells. (A) C. jejuni infection induced IκB degradation in CMT93 cells. Cells were infected with C. jejuni (MOI 50) for the indicated time points in presence or absence of cycloheximide (CHX; 25 µg/ml). Total protein was extracted and 20 µg was subjected to SDS-PAGE followed by immunoblotting with IκBα and RelA specific antibodies. (B) C. jejuni infection induced NF-κB transcriptional activity in CMT93 cells. CMT-93 cells were infected with the reporter Ad5NF-κB-LUC adenoviral vector and where indicated co-infected with Ad5IκBAA (MOI: 50) to block the κB-luciferase activity. Cells were then infected with C. jejuni (MOI 50) for 24 h and luciferase activity measured after 16 h using an Lmax microplate reader. Results were normalized for extract protein concentrations (*p = 0.008 control vs. C. jejuni infected cells; p = 0.013 C. jejuni vs. C. jejuni -Ad5IκBAA). (C) Increased IL-6, MIP-2 and NOD2 mRNA accumulation in C. jejuni infected CMT93 cells. CMT-93 cells were infected with Ad5IκBAA and then stimulated with LPS (5 µg/ml) or infected with C. jejuni (MOI 50) for the indicated time points. Cells were lysed in trizol, and total RNA was extracted, reverse-transcribed (1 µg), and amplified by PCR using specific murine IL-6, MIP-2 and NOD2 primers. The housekeeping gene GAPDH was utilized to ascertain similar loading. Results are representative of 3 independent experiments.
    Figure Legend Snippet: C. jejuni induces NF-κB-signalling and gene expression in CMT-93 cells. (A) C. jejuni infection induced IκB degradation in CMT93 cells. Cells were infected with C. jejuni (MOI 50) for the indicated time points in presence or absence of cycloheximide (CHX; 25 µg/ml). Total protein was extracted and 20 µg was subjected to SDS-PAGE followed by immunoblotting with IκBα and RelA specific antibodies. (B) C. jejuni infection induced NF-κB transcriptional activity in CMT93 cells. CMT-93 cells were infected with the reporter Ad5NF-κB-LUC adenoviral vector and where indicated co-infected with Ad5IκBAA (MOI: 50) to block the κB-luciferase activity. Cells were then infected with C. jejuni (MOI 50) for 24 h and luciferase activity measured after 16 h using an Lmax microplate reader. Results were normalized for extract protein concentrations (*p = 0.008 control vs. C. jejuni infected cells; p = 0.013 C. jejuni vs. C. jejuni -Ad5IκBAA). (C) Increased IL-6, MIP-2 and NOD2 mRNA accumulation in C. jejuni infected CMT93 cells. CMT-93 cells were infected with Ad5IκBAA and then stimulated with LPS (5 µg/ml) or infected with C. jejuni (MOI 50) for the indicated time points. Cells were lysed in trizol, and total RNA was extracted, reverse-transcribed (1 µg), and amplified by PCR using specific murine IL-6, MIP-2 and NOD2 primers. The housekeeping gene GAPDH was utilized to ascertain similar loading. Results are representative of 3 independent experiments.

    Techniques Used: Expressing, Infection, SDS Page, Activity Assay, Plasmid Preparation, Blocking Assay, Luciferase, Amplification, Polymerase Chain Reaction

    5) Product Images from "Gnotobiotic IL-10−/−; NF-?BEGFP Mice Develop Rapid and Severe Colitis Following Campylobacter jejuni Infection"

    Article Title: Gnotobiotic IL-10−/−; NF-?BEGFP Mice Develop Rapid and Severe Colitis Following Campylobacter jejuni Infection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0007413

    Increased TNFα and IL-12p40 mRNA accumulation in C. jejuni -associated IL-10 −/− ; NF-κB EGFP mice. IL-10 wt/wt ; NF-κB EGFP and IL-10 −/− ; NF-κB EGFP were associated with C. jejuni for 14 days. Colonic sections were lysed in trizol and RNA was extracted, reverse-transcribed (1 µg) and amplified by PCR using specific murine TNFa and IL12p40 primers. GAPDH was used as loading control. WT = IL-10 wt/wt ; NF-κB EGFP , n = 7; KO = IL-10 −/− ; NF-κB EGFP , n = 7; WT C. jejuni associated: n = 12; KO C. jejuni associated: n = 10. (*p = 0.002 for IL12p40 vs. control, *p = 0.001 for TNF vs. control).
    Figure Legend Snippet: Increased TNFα and IL-12p40 mRNA accumulation in C. jejuni -associated IL-10 −/− ; NF-κB EGFP mice. IL-10 wt/wt ; NF-κB EGFP and IL-10 −/− ; NF-κB EGFP were associated with C. jejuni for 14 days. Colonic sections were lysed in trizol and RNA was extracted, reverse-transcribed (1 µg) and amplified by PCR using specific murine TNFa and IL12p40 primers. GAPDH was used as loading control. WT = IL-10 wt/wt ; NF-κB EGFP , n = 7; KO = IL-10 −/− ; NF-κB EGFP , n = 7; WT C. jejuni associated: n = 12; KO C. jejuni associated: n = 10. (*p = 0.002 for IL12p40 vs. control, *p = 0.001 for TNF vs. control).

    Techniques Used: Mouse Assay, Amplification, Polymerase Chain Reaction, Gene Knockout

    NF-κB signaling drives C. jejuni induced cytokine gene expression in BMDC. BMDC were generated from WT mice and treated with Bay 11-7085 (10 µM) and then stimulated with C. jejuni (MOI 50) for 4 h. Cells were lysed in Trizol, total RNA was extracted, reverse transcribed and IL-12p40 (A) and IL-6 (B) mRNA expression was detected using real-time PCR (Applied Biosystems 7700 sequence detection system). Results were normalized to the housekeeping gene GAPDH to ascertain similar loading. Results are the mean +/− SD of triplicates samples and are from one of three independent experiments (*p = 0.011 for IL-12p40 vs. Bay 11-7085-treated cells; *p = 0.005 for IL-6 vs. Bay 11-7085-treated cells).
    Figure Legend Snippet: NF-κB signaling drives C. jejuni induced cytokine gene expression in BMDC. BMDC were generated from WT mice and treated with Bay 11-7085 (10 µM) and then stimulated with C. jejuni (MOI 50) for 4 h. Cells were lysed in Trizol, total RNA was extracted, reverse transcribed and IL-12p40 (A) and IL-6 (B) mRNA expression was detected using real-time PCR (Applied Biosystems 7700 sequence detection system). Results were normalized to the housekeeping gene GAPDH to ascertain similar loading. Results are the mean +/− SD of triplicates samples and are from one of three independent experiments (*p = 0.011 for IL-12p40 vs. Bay 11-7085-treated cells; *p = 0.005 for IL-6 vs. Bay 11-7085-treated cells).

    Techniques Used: Expressing, Generated, Mouse Assay, Real-time Polymerase Chain Reaction, Sequencing

    C. jejuni induces NF-κB-signalling and gene expression in CMT-93 cells. (A) C. jejuni infection induced IκB degradation in CMT93 cells. Cells were infected with C. jejuni (MOI 50) for the indicated time points in presence or absence of cycloheximide (CHX; 25 µg/ml). Total protein was extracted and 20 µg was subjected to SDS-PAGE followed by immunoblotting with IκBα and RelA specific antibodies. (B) C. jejuni infection induced NF-κB transcriptional activity in CMT93 cells. CMT-93 cells were infected with the reporter Ad5NF-κB-LUC adenoviral vector and where indicated co-infected with Ad5IκBAA (MOI: 50) to block the κB-luciferase activity. Cells were then infected with C. jejuni (MOI 50) for 24 h and luciferase activity measured after 16 h using an Lmax microplate reader. Results were normalized for extract protein concentrations (*p = 0.008 control vs. C. jejuni infected cells; p = 0.013 C. jejuni vs. C. jejuni -Ad5IκBAA). (C) Increased IL-6, MIP-2 and NOD2 mRNA accumulation in C. jejuni infected CMT93 cells. CMT-93 cells were infected with Ad5IκBAA and then stimulated with LPS (5 µg/ml) or infected with C. jejuni (MOI 50) for the indicated time points. Cells were lysed in trizol, and total RNA was extracted, reverse-transcribed (1 µg), and amplified by PCR using specific murine IL-6, MIP-2 and NOD2 primers. The housekeeping gene GAPDH was utilized to ascertain similar loading. Results are representative of 3 independent experiments.
    Figure Legend Snippet: C. jejuni induces NF-κB-signalling and gene expression in CMT-93 cells. (A) C. jejuni infection induced IκB degradation in CMT93 cells. Cells were infected with C. jejuni (MOI 50) for the indicated time points in presence or absence of cycloheximide (CHX; 25 µg/ml). Total protein was extracted and 20 µg was subjected to SDS-PAGE followed by immunoblotting with IκBα and RelA specific antibodies. (B) C. jejuni infection induced NF-κB transcriptional activity in CMT93 cells. CMT-93 cells were infected with the reporter Ad5NF-κB-LUC adenoviral vector and where indicated co-infected with Ad5IκBAA (MOI: 50) to block the κB-luciferase activity. Cells were then infected with C. jejuni (MOI 50) for 24 h and luciferase activity measured after 16 h using an Lmax microplate reader. Results were normalized for extract protein concentrations (*p = 0.008 control vs. C. jejuni infected cells; p = 0.013 C. jejuni vs. C. jejuni -Ad5IκBAA). (C) Increased IL-6, MIP-2 and NOD2 mRNA accumulation in C. jejuni infected CMT93 cells. CMT-93 cells were infected with Ad5IκBAA and then stimulated with LPS (5 µg/ml) or infected with C. jejuni (MOI 50) for the indicated time points. Cells were lysed in trizol, and total RNA was extracted, reverse-transcribed (1 µg), and amplified by PCR using specific murine IL-6, MIP-2 and NOD2 primers. The housekeeping gene GAPDH was utilized to ascertain similar loading. Results are representative of 3 independent experiments.

    Techniques Used: Expressing, Infection, SDS Page, Activity Assay, Plasmid Preparation, Blocking Assay, Luciferase, Amplification, Polymerase Chain Reaction

    6) Product Images from "BCPA {N,N′-1,4-Butanediylbis[3-(2-chlorophenyl)acrylamide]} Inhibits Osteoclast Differentiation through Increased Retention of Peptidyl-Prolyl cis-trans Isomerase Never in Mitosis A-Interacting 1"

    Article Title: BCPA {N,N′-1,4-Butanediylbis[3-(2-chlorophenyl)acrylamide]} Inhibits Osteoclast Differentiation through Increased Retention of Peptidyl-Prolyl cis-trans Isomerase Never in Mitosis A-Interacting 1

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19113436

    BCPA represses mRNA expression of DC-STAMP and OSCAR . ( A ) Mouse bone marrow-derived macrophage cells were seeded in 6-well plates and treated with M-CSF (30 ng/mL), RANKL (50 ng/mL) and BCPA (10 μM) for 4 days. ( B ) Mouse bone marrow-derived macrophage cells were treated with M-CSF (30 ng/mL), RANKL (50 ng/mL) and BCPA (10 μM) and then cells were harvested after 4 days. Total RNA was isolated using the TRIzol reagent. Gene expression was analyzed by quantitative real-time PCR. Data are presented as mean ± SD; * p
    Figure Legend Snippet: BCPA represses mRNA expression of DC-STAMP and OSCAR . ( A ) Mouse bone marrow-derived macrophage cells were seeded in 6-well plates and treated with M-CSF (30 ng/mL), RANKL (50 ng/mL) and BCPA (10 μM) for 4 days. ( B ) Mouse bone marrow-derived macrophage cells were treated with M-CSF (30 ng/mL), RANKL (50 ng/mL) and BCPA (10 μM) and then cells were harvested after 4 days. Total RNA was isolated using the TRIzol reagent. Gene expression was analyzed by quantitative real-time PCR. Data are presented as mean ± SD; * p

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

    7) Product Images from "Capture, Amplification, and Global Profiling of microRNAs from Low Quantities of Whole Cell Lysate"

    Article Title: Capture, Amplification, and Global Profiling of microRNAs from Low Quantities of Whole Cell Lysate

    Journal:

    doi: 10.1039/c7an00670e

    Cell lysis and effect of lysis buffer concentration on the qRT-PCR detection of miR142 (A) BaF3 cells stably expressing mCherry as a fluorescence marker were lysed with 1% Triton buffer in 30 seconds. Representative optical (Phase Contrast) and fluorescence images are shown before and after lysis. (B) BaF3 cells (WT) or BaF3 cells with miR-142 knockout (KO) were subjected to RNA preparation with TRIzol or cell lysis with lysis buffer containing the indicated concentrations of Triton. Synthetic miR-371 was spiked in to control experimental variation. miR-142-3p and miR-371-3p levels were determined via qRT-PCR. The relative detection levels of miR-142-3p are shown in log scale to reflect quantification from the same fraction input. N = 3. Error bars present the standard deviation.
    Figure Legend Snippet: Cell lysis and effect of lysis buffer concentration on the qRT-PCR detection of miR142 (A) BaF3 cells stably expressing mCherry as a fluorescence marker were lysed with 1% Triton buffer in 30 seconds. Representative optical (Phase Contrast) and fluorescence images are shown before and after lysis. (B) BaF3 cells (WT) or BaF3 cells with miR-142 knockout (KO) were subjected to RNA preparation with TRIzol or cell lysis with lysis buffer containing the indicated concentrations of Triton. Synthetic miR-371 was spiked in to control experimental variation. miR-142-3p and miR-371-3p levels were determined via qRT-PCR. The relative detection levels of miR-142-3p are shown in log scale to reflect quantification from the same fraction input. N = 3. Error bars present the standard deviation.

    Techniques Used: Lysis, Concentration Assay, Quantitative RT-PCR, Stable Transfection, Expressing, Fluorescence, Marker, Knock-Out, Gene Knockout, Standard Deviation

    Effect of post-lysis heat treatment on the detection of miR-142 and miR-222 by qRT-PCR (A) BaF3 cells (WT) or BaF3 cells with miR-142 knockout (KO) were subjected to RNA preparation with TRIzol or cell lysis with lysis buffer containing 1% Triton. Synthetic miR-371 was spiked in to control experimental variation. Levels of miR-142-3p, miR-222-3p and miR-371-3p were determined by qRT-PCR after heat treatment post cell lysis. The relative detected levels of miR-142-3p and miR-222-3p are shown in log scale to reflect quantification from the same fraction input. (B) Relative levels of miR-142-3p were determined by qRT-PCR of BaF3 cells (WT) with heat treatment at the indicated temperatures for 5 minutes. N = 3. Error bars present the standard deviation. The level of miR-142-3p is shown in log scale. N = 3. Error bars present the standard deviation.
    Figure Legend Snippet: Effect of post-lysis heat treatment on the detection of miR-142 and miR-222 by qRT-PCR (A) BaF3 cells (WT) or BaF3 cells with miR-142 knockout (KO) were subjected to RNA preparation with TRIzol or cell lysis with lysis buffer containing 1% Triton. Synthetic miR-371 was spiked in to control experimental variation. Levels of miR-142-3p, miR-222-3p and miR-371-3p were determined by qRT-PCR after heat treatment post cell lysis. The relative detected levels of miR-142-3p and miR-222-3p are shown in log scale to reflect quantification from the same fraction input. (B) Relative levels of miR-142-3p were determined by qRT-PCR of BaF3 cells (WT) with heat treatment at the indicated temperatures for 5 minutes. N = 3. Error bars present the standard deviation. The level of miR-142-3p is shown in log scale. N = 3. Error bars present the standard deviation.

    Techniques Used: Lysis, Quantitative RT-PCR, Knock-Out, Gene Knockout, Standard Deviation

    8) Product Images from "A fast and cost-effective microsampling protocol incorporating reduced animal usage for time-series transcriptomics in rodent malaria parasites"

    Article Title: A fast and cost-effective microsampling protocol incorporating reduced animal usage for time-series transcriptomics in rodent malaria parasites

    Journal: Malaria Journal

    doi: 10.1186/s12936-019-2659-4

    Microsampling protocol design and reproducibility. a In terminal blood sampling, at each time point, groups of mice are exsanguinated to obtain 0.5–0.6 mL blood volumes, which are then subject to leukocyte depletion and saponin lysis before TRIzol treatment. Thus, the number of mice increases proportionally with the number of biological replicates and time points in the study design (number of mice per timepoint X number of timepoints; NT). Microsampling involves obtaining sample volumes as low as 20 μL from the same mouse at different time points, thus confining the number of mice to just biological replicates (N) and significantly lowering costs and biological variability. Leukocyte depletion and saponin lysis are also not performed on the low volume samples, thus saving time and manpower. b Heatmap shows pair-wise Pearson correlation coefficients and the inset shows multidimensional scaling to visualize the level of similarity between the P. vinckei microsamples. Microsamples show low degree of variability and are highly reproducible as proved by tight correlations between biological replicates. c High Pearson correlations were observed between normalized gene expression values (shown as logarithm of fragments per kilobase of transcript per million mapped reads) from microsampling (x-axis) and terminal blood sampling (y-axis) methods. d Bioanalyser electrophoregrams of total RNA from Plasmodium vinckei vinckei CY microsamples show that high quality RNA could be extracted consistently from 20 μL microsamples
    Figure Legend Snippet: Microsampling protocol design and reproducibility. a In terminal blood sampling, at each time point, groups of mice are exsanguinated to obtain 0.5–0.6 mL blood volumes, which are then subject to leukocyte depletion and saponin lysis before TRIzol treatment. Thus, the number of mice increases proportionally with the number of biological replicates and time points in the study design (number of mice per timepoint X number of timepoints; NT). Microsampling involves obtaining sample volumes as low as 20 μL from the same mouse at different time points, thus confining the number of mice to just biological replicates (N) and significantly lowering costs and biological variability. Leukocyte depletion and saponin lysis are also not performed on the low volume samples, thus saving time and manpower. b Heatmap shows pair-wise Pearson correlation coefficients and the inset shows multidimensional scaling to visualize the level of similarity between the P. vinckei microsamples. Microsamples show low degree of variability and are highly reproducible as proved by tight correlations between biological replicates. c High Pearson correlations were observed between normalized gene expression values (shown as logarithm of fragments per kilobase of transcript per million mapped reads) from microsampling (x-axis) and terminal blood sampling (y-axis) methods. d Bioanalyser electrophoregrams of total RNA from Plasmodium vinckei vinckei CY microsamples show that high quality RNA could be extracted consistently from 20 μL microsamples

    Techniques Used: Sampling, Mouse Assay, Lysis, Expressing

    9) Product Images from "Selection of reference genes for qPCR in hairy root cultures of peanut"

    Article Title: Selection of reference genes for qPCR in hairy root cultures of peanut

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-4-392

    Comparison of RNA extraction methods . RNA yields under three different extraction methods: TRIzol ® , Maxwell ® and Maxwell ® plus the addition of BME. Three amounts of peanut hairy root were evaluated: 10, 20 and 40 mg DW. Lyophilized tissue of 9-day old root culture was extracted under each method. RNA was quantified by Quant-iT™ RiboGreen ® RNA kit. Data shown represents a single replicate per method at 3 distinct amounts of starting material.
    Figure Legend Snippet: Comparison of RNA extraction methods . RNA yields under three different extraction methods: TRIzol ® , Maxwell ® and Maxwell ® plus the addition of BME. Three amounts of peanut hairy root were evaluated: 10, 20 and 40 mg DW. Lyophilized tissue of 9-day old root culture was extracted under each method. RNA was quantified by Quant-iT™ RiboGreen ® RNA kit. Data shown represents a single replicate per method at 3 distinct amounts of starting material.

    Techniques Used: RNA Extraction

    Comparison of integrity and yields of RNA between lyophilized and frozen peanut hairy roots . ( A ) RNA integrity was compared between lyophilized and frozen material. The two ribosomal subunits (28S and 18S) are indicators of RNA integrity on agarose gel electrophoresis. Peanut hairy roots elicited with NaOAc and non-elicited (control) were also compared. Numbers 1 to 12 represent biological replicates. ( B ) RNA yields were compared between these two conditions. For lyophilized tissue, RNA yields were expressed based on fresh weight (FW). Bars represent the average of three biological replicates and error bars represent the standard deviation. Total RNA from 9-day old root cultures was extracted with TRIzol ® and quantified spectrophotometrically as described in Methods section.
    Figure Legend Snippet: Comparison of integrity and yields of RNA between lyophilized and frozen peanut hairy roots . ( A ) RNA integrity was compared between lyophilized and frozen material. The two ribosomal subunits (28S and 18S) are indicators of RNA integrity on agarose gel electrophoresis. Peanut hairy roots elicited with NaOAc and non-elicited (control) were also compared. Numbers 1 to 12 represent biological replicates. ( B ) RNA yields were compared between these two conditions. For lyophilized tissue, RNA yields were expressed based on fresh weight (FW). Bars represent the average of three biological replicates and error bars represent the standard deviation. Total RNA from 9-day old root cultures was extracted with TRIzol ® and quantified spectrophotometrically as described in Methods section.

    Techniques Used: Agarose Gel Electrophoresis, Standard Deviation

    10) Product Images from "Efficient Delivery of DNA Vaccines using Human Papillomavirus Pseudovirions"

    Article Title: Efficient Delivery of DNA Vaccines using Human Papillomavirus Pseudovirions

    Journal: Gene therapy

    doi: 10.1038/gt.2010.106

    Analysis of cells infected by HPV pseudovirion A) In vitro infection of BMDCs by HPV pseudovirus. BMDCs were generated from bone marrow progenitor cells and infected with HPV16-GFP or HPV16-OVA pseudovirus at day 4 (4 μg L1 protein). After 72 hours, BMDCs were harvested and GFP expression was examined by flow cytometry. B) RT-PCR to demonstrate the expression of GFP mRNA in draining lymph nodes of mice infected with HPV16 pseudovirions containing GFP or OVA. 5–8 weeks old C57BL/6 mice were vaccinated with 10 μg/mouse of HPV16 pseudovirions carrying GFP or OVA DNA subcutaneously. After 72 hours, draining lymph nodes were harvested and total RNA was isolated with TRIzol. RT-PCR was then performed to detect GFP mRNA expression. C) Representative flow cytometry data depicting the percentage of CD11c+ cells and B220+ cells that uptake the FITC-labeled pseudovirions. HPV16-OVA pseudovirus was labeled with FITC. 5–8 weeks old C57BL/6 mice were given 10 μg/mouse of HPV16-OVA or HPV16-OVA-FITC pseudovirus subcutaneously. After 72 hours, draining lymph nodes were harvested, and digested with 0.05 mg/ml Collagenase I, 0.05 mg/ml collagenase IV, 0.025 mg/ml Hyaluronidase IV and 0.25 mg/ml DNase I. The cells were then stained with anti-mouse CD11c-APC and PE-Cy5-conjugated anti-mouse B220 followed by flow cytometry analysis. D) Representative bar graph depicting the percentage of FITC+ CD11c+ cells and FITC+ B220+ cells.
    Figure Legend Snippet: Analysis of cells infected by HPV pseudovirion A) In vitro infection of BMDCs by HPV pseudovirus. BMDCs were generated from bone marrow progenitor cells and infected with HPV16-GFP or HPV16-OVA pseudovirus at day 4 (4 μg L1 protein). After 72 hours, BMDCs were harvested and GFP expression was examined by flow cytometry. B) RT-PCR to demonstrate the expression of GFP mRNA in draining lymph nodes of mice infected with HPV16 pseudovirions containing GFP or OVA. 5–8 weeks old C57BL/6 mice were vaccinated with 10 μg/mouse of HPV16 pseudovirions carrying GFP or OVA DNA subcutaneously. After 72 hours, draining lymph nodes were harvested and total RNA was isolated with TRIzol. RT-PCR was then performed to detect GFP mRNA expression. C) Representative flow cytometry data depicting the percentage of CD11c+ cells and B220+ cells that uptake the FITC-labeled pseudovirions. HPV16-OVA pseudovirus was labeled with FITC. 5–8 weeks old C57BL/6 mice were given 10 μg/mouse of HPV16-OVA or HPV16-OVA-FITC pseudovirus subcutaneously. After 72 hours, draining lymph nodes were harvested, and digested with 0.05 mg/ml Collagenase I, 0.05 mg/ml collagenase IV, 0.025 mg/ml Hyaluronidase IV and 0.25 mg/ml DNase I. The cells were then stained with anti-mouse CD11c-APC and PE-Cy5-conjugated anti-mouse B220 followed by flow cytometry analysis. D) Representative bar graph depicting the percentage of FITC+ CD11c+ cells and FITC+ B220+ cells.

    Techniques Used: Infection, In Vitro, Generated, Expressing, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Isolation, Labeling, Staining

    11) Product Images from "The function and clinical relevance of lncRNA UBE2CP3-001 in human gliomas"

    Article Title: The function and clinical relevance of lncRNA UBE2CP3-001 in human gliomas

    Journal: Archives of Medical Science : AMS

    doi: 10.5114/aoms.2018.79004

    Overexpression of the long non-coding RNA UBE2CP3 in gliomas. A – RNA was extracted with Trizol from human glioma and adjacent tissues. UBE2CP3 expression was assessed in human glioma tissues and adjacent normal tissues using real-time RT-PCR. Bars represent the ratio between expression in glioma and in adjacent normal tissues. B – Statistical analysis shows the expression of UBE2CP3-001 in different grades of gliomas. C – The expression of UBE2CP3-001 in U87 cells compared with normal astrocyte cells was detected using real-time RT-PCR. D – RT-PCR was used to detect lncRNA UBE2CP3-001 expression in U87 stable cells with transfected shRNA 1-6
    Figure Legend Snippet: Overexpression of the long non-coding RNA UBE2CP3 in gliomas. A – RNA was extracted with Trizol from human glioma and adjacent tissues. UBE2CP3 expression was assessed in human glioma tissues and adjacent normal tissues using real-time RT-PCR. Bars represent the ratio between expression in glioma and in adjacent normal tissues. B – Statistical analysis shows the expression of UBE2CP3-001 in different grades of gliomas. C – The expression of UBE2CP3-001 in U87 cells compared with normal astrocyte cells was detected using real-time RT-PCR. D – RT-PCR was used to detect lncRNA UBE2CP3-001 expression in U87 stable cells with transfected shRNA 1-6

    Techniques Used: Over Expression, Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Transfection, shRNA

    12) Product Images from "Altered bone marrow dendritic cell cytokine production to toll-like receptor and CD40 ligation during chronic feline immunodeficiency virus infection"

    Article Title: Altered bone marrow dendritic cell cytokine production to toll-like receptor and CD40 ligation during chronic feline immunodeficiency virus infection

    Journal: Immunology

    doi: 10.1111/j.1365-2567.2008.02907.x

    Toll-like receptor (TLR) messenger RNA (mRNA) expression is not altered in bone marrow-derived dendritic cells (BM-DCs) from cats infected with feline immunodeficiency virus (FIV). The BM-DCs were cultured for 10 days in the presence of recombinant feline granulocyte–macrophage colony-stimulating factor (100 ng/ml) and then purified for CD11c and major histocompatibility complex class II expression. Purified BM-DCs were lysed with Trizol (Gibco), and the RNA was extracted. Real-time polymerase chain reaction was performed to quantify RNA levels of TLRs 2, 3, 4, 5, 7 and 9. Results are shown as the mean and SD of cells from eight naïve cats and six FIV-infected cats. There were no statistically significant differences in expression levels as determined by Student’s t -test.
    Figure Legend Snippet: Toll-like receptor (TLR) messenger RNA (mRNA) expression is not altered in bone marrow-derived dendritic cells (BM-DCs) from cats infected with feline immunodeficiency virus (FIV). The BM-DCs were cultured for 10 days in the presence of recombinant feline granulocyte–macrophage colony-stimulating factor (100 ng/ml) and then purified for CD11c and major histocompatibility complex class II expression. Purified BM-DCs were lysed with Trizol (Gibco), and the RNA was extracted. Real-time polymerase chain reaction was performed to quantify RNA levels of TLRs 2, 3, 4, 5, 7 and 9. Results are shown as the mean and SD of cells from eight naïve cats and six FIV-infected cats. There were no statistically significant differences in expression levels as determined by Student’s t -test.

    Techniques Used: Expressing, Derivative Assay, Infection, Cell Culture, Recombinant, Purification, Real-time Polymerase Chain Reaction

    13) Product Images from "Evaluation of a range of mammalian and mosquito cell lines for use in Chikungunya virus research"

    Article Title: Evaluation of a range of mammalian and mosquito cell lines for use in Chikungunya virus research

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-15269-w

    CHIKV infection of mosquito cell lines. (a) Virus titres. U4.4 and C6/36 cells were infected at an MOI of 1 and 5. At 24 hpi, the media was collected and virus titred by plaque assay (n = 3, experimental replicates) using BHK cells. (b) Corresponding RNA quantification of the infected cells. Intracellular RNA was extracted using TRIzol and quantified by qRT-PCR using primers specific for nsP3 (n = 3). Values in (a) and (b) were derived from the same number of cells and are thus directly comparable. (c) IF of mosquito cell lines infected with nsP3-ZsGreen virus. Both cell lines demonstrated a distinct puncta organisation of nsP3. Representative images shown.
    Figure Legend Snippet: CHIKV infection of mosquito cell lines. (a) Virus titres. U4.4 and C6/36 cells were infected at an MOI of 1 and 5. At 24 hpi, the media was collected and virus titred by plaque assay (n = 3, experimental replicates) using BHK cells. (b) Corresponding RNA quantification of the infected cells. Intracellular RNA was extracted using TRIzol and quantified by qRT-PCR using primers specific for nsP3 (n = 3). Values in (a) and (b) were derived from the same number of cells and are thus directly comparable. (c) IF of mosquito cell lines infected with nsP3-ZsGreen virus. Both cell lines demonstrated a distinct puncta organisation of nsP3. Representative images shown.

    Techniques Used: Infection, Plaque Assay, Quantitative RT-PCR, Derivative Assay

    CHIKV infection of mammalian cell lines. (a) Virus titres. Huh7, C2C12, SVG-A, and dermal fibroblast (D Fibs) cells were infected at MOIs of either 1 or 5. At 24 hpi, the media was collected and virus titred by plaque assay (n = 3, experimental replicates). (b) Corresponding RNA quantification of the infected cells. Intracellular RNA was extracted using TRIzol and quantified by qRT-PCR using primers specific for nsP3 (n = 3). Values in (a) and (b) were derived from the same number of cells and are thus directly comparable. (c) Western blots for nsP3 in infected cells. Cells were infected with wildtype CHIKV at an MOI = 1. Cells were lysed at 24 hpi and western blot conducted for nsP3.
    Figure Legend Snippet: CHIKV infection of mammalian cell lines. (a) Virus titres. Huh7, C2C12, SVG-A, and dermal fibroblast (D Fibs) cells were infected at MOIs of either 1 or 5. At 24 hpi, the media was collected and virus titred by plaque assay (n = 3, experimental replicates). (b) Corresponding RNA quantification of the infected cells. Intracellular RNA was extracted using TRIzol and quantified by qRT-PCR using primers specific for nsP3 (n = 3). Values in (a) and (b) were derived from the same number of cells and are thus directly comparable. (c) Western blots for nsP3 in infected cells. Cells were infected with wildtype CHIKV at an MOI = 1. Cells were lysed at 24 hpi and western blot conducted for nsP3.

    Techniques Used: Infection, Plaque Assay, Quantitative RT-PCR, Derivative Assay, Western Blot

    14) Product Images from "Optimization and analysis of a quantitative real-time PCR-based technique to determine microRNA expression in formalin-fixed paraffin-embedded samples"

    Article Title: Optimization and analysis of a quantitative real-time PCR-based technique to determine microRNA expression in formalin-fixed paraffin-embedded samples

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-10-47

    Comparison of miR abundance in paired fresh-frozen and FFPE tissues, using different RNA extraction methods . Plots show the comparison of Ct values for three normal and three mantle cell lymphoma samples by the three different extraction methods: A) FFPE (RecoverAll) vs. mirVana; B) FFPE (RecoverAll) vs. TRIzol; C) TRIzol vs. mirVana. Red lines delineate the abundance strata for each extraction method, with numbers indicating the average number of miRs per sample in each zone. For example, in the FFPE vs. mirVana plot (Figure 5A), 44 miRs are high abundance by mirVana and medium abundance by FFPE (RecoverAll), but only 4 miRs are high abundance by FFPE (RecoverAll) and medium abundance by mirVana.
    Figure Legend Snippet: Comparison of miR abundance in paired fresh-frozen and FFPE tissues, using different RNA extraction methods . Plots show the comparison of Ct values for three normal and three mantle cell lymphoma samples by the three different extraction methods: A) FFPE (RecoverAll) vs. mirVana; B) FFPE (RecoverAll) vs. TRIzol; C) TRIzol vs. mirVana. Red lines delineate the abundance strata for each extraction method, with numbers indicating the average number of miRs per sample in each zone. For example, in the FFPE vs. mirVana plot (Figure 5A), 44 miRs are high abundance by mirVana and medium abundance by FFPE (RecoverAll), but only 4 miRs are high abundance by FFPE (RecoverAll) and medium abundance by mirVana.

    Techniques Used: Formalin-fixed Paraffin-Embedded, RNA Extraction

    Ct values according to different extraction methods for paired fresh-frozen and FFPE tissues . Comparison of the summary statistics (mean Ct, standard deviation, and overall range of Ct values) using raw Ct scores obtained from the normal lymph nodes and mantle cell lymphoma samples extracted using three separate RNA extraction methods. On comparison of the FFPE summary statistics to those from the mirVana and TRIzol-Qiagen extraction protocols, we see that the mean Ct values obtained from the FFPE samples are significantly higher than those obtained from the mirVana and TRIzol-Qiagen extraction protocols (p
    Figure Legend Snippet: Ct values according to different extraction methods for paired fresh-frozen and FFPE tissues . Comparison of the summary statistics (mean Ct, standard deviation, and overall range of Ct values) using raw Ct scores obtained from the normal lymph nodes and mantle cell lymphoma samples extracted using three separate RNA extraction methods. On comparison of the FFPE summary statistics to those from the mirVana and TRIzol-Qiagen extraction protocols, we see that the mean Ct values obtained from the FFPE samples are significantly higher than those obtained from the mirVana and TRIzol-Qiagen extraction protocols (p

    Techniques Used: Formalin-fixed Paraffin-Embedded, Standard Deviation, RNA Extraction

    15) Product Images from "High-quality RNA extraction from the sea urchin Paracentrotus lividus embryos"

    Article Title: High-quality RNA extraction from the sea urchin Paracentrotus lividus embryos

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0172171

    Bioanalyzer Agilent electrophoresis runs. Examples of representative Bioanalyzer Agilent electrophoresis runs for the five different methods applied for RNA extractions from P . lividus embryos: TRIzol, GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich), RNAqueous® Micro Kit (Ambion from Life Technologies), RNeasy® Micro Kit (Qiagen) and Aurum™ Total RNA Mini Kit (Biorad). Four different numerical amounts of embryos were used for RNA extraction: 500, 1000, 2500 and 5000 embryos. The ladder (L) is reported in the first lane of each run. The green band at the bottom of each panel is the RNA 6000 Nano Marker (Agilent RNA 6000 Nano Kit, Agilent Technologies, Inc.). Red box in the 500 lane of TRIzol indicates that the Bioanalyzer software cannot calculate RIN values (reported as N/A in the Table 1 ) for this sample, because of very low concentration and high level of degradation of the RNA.
    Figure Legend Snippet: Bioanalyzer Agilent electrophoresis runs. Examples of representative Bioanalyzer Agilent electrophoresis runs for the five different methods applied for RNA extractions from P . lividus embryos: TRIzol, GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich), RNAqueous® Micro Kit (Ambion from Life Technologies), RNeasy® Micro Kit (Qiagen) and Aurum™ Total RNA Mini Kit (Biorad). Four different numerical amounts of embryos were used for RNA extraction: 500, 1000, 2500 and 5000 embryos. The ladder (L) is reported in the first lane of each run. The green band at the bottom of each panel is the RNA 6000 Nano Marker (Agilent RNA 6000 Nano Kit, Agilent Technologies, Inc.). Red box in the 500 lane of TRIzol indicates that the Bioanalyzer software cannot calculate RIN values (reported as N/A in the Table 1 ) for this sample, because of very low concentration and high level of degradation of the RNA.

    Techniques Used: Electrophoresis, RNA Extraction, Marker, Software, Concentration Assay

    Agilent Bioanlyzer electropherograms. Examples of representative Agilent Bioanlyzer electropherograms of P . lividus RNA: for TRIzol, GenElute and RNAqueous RNA extraction from 5000 embryos extraction; for RNeasy and Aurum RNA extraction from 2500 embryos (see also Table 1 ). Relative Fluorescent Unit (FU) and seconds of migration (s) of RNA samples isolated according to the five different extraction methods are reported. RIN values are also reported.
    Figure Legend Snippet: Agilent Bioanlyzer electropherograms. Examples of representative Agilent Bioanlyzer electropherograms of P . lividus RNA: for TRIzol, GenElute and RNAqueous RNA extraction from 5000 embryos extraction; for RNeasy and Aurum RNA extraction from 2500 embryos (see also Table 1 ). Relative Fluorescent Unit (FU) and seconds of migration (s) of RNA samples isolated according to the five different extraction methods are reported. RIN values are also reported.

    Techniques Used: RNA Extraction, Migration, Isolation

    16) Product Images from "Genome-wide identification of novel expression signatures reveal distinct patterns and prevalence of binding motifs for p53, nuclear factor-?B and other signal transcription factors in head and neck squamous cell carcinoma"

    Article Title: Genome-wide identification of novel expression signatures reveal distinct patterns and prevalence of binding motifs for p53, nuclear factor-?B and other signal transcription factors in head and neck squamous cell carcinoma

    Journal: Genome Biology

    doi: 10.1186/gb-2007-8-5-r78

    Basal and inducible gene expression, and promoter binding activity were modulated by doxorubicin or TNF-α. (a) Human keratinocyte (HKC) cells were treated with doxorubicin (Dox; 0.5 μg/ml, left panels), and the UM-SCC 6 cell line was treated with tumor necrosis factor (TNF)-α (2000 U/ml, center and right panels) for different periods, as indicated. Total RNA was harvested by Trizol and genes selected from clusters A to C were analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR). The data are presented as the mean plus standard deviation from triplicates with normalization by 18S ribosome RNA. '(C1)', '(C2)', and '(C3)' refer to the three subclusters of cluster C. '(C)' refers to genes in cluster C outside subclusters C1 to C3. (b) Chromatin immunoprecipitation assays were performed in UM-SCC 11A cells using rabbit polyclonal anti-p65 or cRel antibodies with IgG isotype control.
    Figure Legend Snippet: Basal and inducible gene expression, and promoter binding activity were modulated by doxorubicin or TNF-α. (a) Human keratinocyte (HKC) cells were treated with doxorubicin (Dox; 0.5 μg/ml, left panels), and the UM-SCC 6 cell line was treated with tumor necrosis factor (TNF)-α (2000 U/ml, center and right panels) for different periods, as indicated. Total RNA was harvested by Trizol and genes selected from clusters A to C were analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR). The data are presented as the mean plus standard deviation from triplicates with normalization by 18S ribosome RNA. '(C1)', '(C2)', and '(C3)' refer to the three subclusters of cluster C. '(C)' refers to genes in cluster C outside subclusters C1 to C3. (b) Chromatin immunoprecipitation assays were performed in UM-SCC 11A cells using rabbit polyclonal anti-p65 or cRel antibodies with IgG isotype control.

    Techniques Used: Expressing, Binding Assay, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Standard Deviation, Chromatin Immunoprecipitation

    17) Product Images from "Green Tea Extract Provides Extensive Nrf2-Independent Protection Against Lipid Accumulation and NFκB Pro- Inflammatory Responses During Nonalcoholic Steatohepatitis In Mice Fed A High-Fat Diet"

    Article Title: Green Tea Extract Provides Extensive Nrf2-Independent Protection Against Lipid Accumulation and NFκB Pro- Inflammatory Responses During Nonalcoholic Steatohepatitis In Mice Fed A High-Fat Diet

    Journal:

    doi: 10.1002/mnfr.201500814

    Time- and dose-response effects of EGCG on cell cytotoxicity and nuclear accumulation and mRNA expression of Nrf2 in HC-04 human hepatocytes HC-04 were treated with 0-5 μM EGCG for up to 2 h as described under Methods. A) Representative Western blot of Nrf2 from isolated nuclear fractions of HC-04 cells following 2 h incubation with EGCG or 100 μM t-butylhydroperoxide (positive control). B) HC-04 cells were treated for up to 2 h with 0.1-5 μM EGCG before isolating total RNA using Trizol and reverse transcribing for RT-PCR analysis of Nrf2 mRNA expression. C) LDH leakage and D) cell viability were assessed using spectrophotometric kits as described under Methods following treatment of HC-04 for 2 h with 0-5 μM EGCG. Shown are mean responses of experiments performed in triplicate. Data were analyzed by 1- or 2-way ANOVA as appropriate. There were no statistically significant time- or concentration-dependent effects of EGCG. Abbreviations: EGCG, epigallocatechin gallate; LDH, lactate dehydrogenase; Nrf2, nuclear factor erythroid-2-related factor-2.
    Figure Legend Snippet: Time- and dose-response effects of EGCG on cell cytotoxicity and nuclear accumulation and mRNA expression of Nrf2 in HC-04 human hepatocytes HC-04 were treated with 0-5 μM EGCG for up to 2 h as described under Methods. A) Representative Western blot of Nrf2 from isolated nuclear fractions of HC-04 cells following 2 h incubation with EGCG or 100 μM t-butylhydroperoxide (positive control). B) HC-04 cells were treated for up to 2 h with 0.1-5 μM EGCG before isolating total RNA using Trizol and reverse transcribing for RT-PCR analysis of Nrf2 mRNA expression. C) LDH leakage and D) cell viability were assessed using spectrophotometric kits as described under Methods following treatment of HC-04 for 2 h with 0-5 μM EGCG. Shown are mean responses of experiments performed in triplicate. Data were analyzed by 1- or 2-way ANOVA as appropriate. There were no statistically significant time- or concentration-dependent effects of EGCG. Abbreviations: EGCG, epigallocatechin gallate; LDH, lactate dehydrogenase; Nrf2, nuclear factor erythroid-2-related factor-2.

    Techniques Used: Expressing, Western Blot, Isolation, Incubation, Positive Control, Reverse Transcription Polymerase Chain Reaction, Concentration Assay

    Hepatic mRNA expression of Nrf2 (A) and Nqo1 (B) in Nrf2-null and wild-type mice fed a high-fat diet containing GTE at 0 or 2% for 8 wk RNA was isolated using Trizol and reverse transcribed for RT-PCR analysis using the primers described in . Data (means ± SEM, n = 10-12 mice per group) were analyzed by 2-way ANOVA with Newman–Keuls post-test to evaluate main and interactive effects. Groups without a common letter are significantly different ( P < 0.05). Abbreviations: GTE, green tea extract; Nrf2, nuclear factor erythroid-2-related factor-2; Nqo1, NADPH:quinone oxidoreductase 1; WT, wild-type.
    Figure Legend Snippet: Hepatic mRNA expression of Nrf2 (A) and Nqo1 (B) in Nrf2-null and wild-type mice fed a high-fat diet containing GTE at 0 or 2% for 8 wk RNA was isolated using Trizol and reverse transcribed for RT-PCR analysis using the primers described in . Data (means ± SEM, n = 10-12 mice per group) were analyzed by 2-way ANOVA with Newman–Keuls post-test to evaluate main and interactive effects. Groups without a common letter are significantly different ( P < 0.05). Abbreviations: GTE, green tea extract; Nrf2, nuclear factor erythroid-2-related factor-2; Nqo1, NADPH:quinone oxidoreductase 1; WT, wild-type.

    Techniques Used: Expressing, Mouse Assay, Isolation, Reverse Transcription Polymerase Chain Reaction

    18) Product Images from "An optimised protocol for the extraction of non-viral mRNA from human plasma frozen for three years"

    Article Title: An optimised protocol for the extraction of non-viral mRNA from human plasma frozen for three years

    Journal:

    doi: 10.1136/jcp.2003.007880

    Lack of detection of RNA in plasma from patients with colorectal cancer stored for three years without Trizol: (A) lanes 1–10, β catenin mRNA from 10 patients; (B) lanes 1–10,
    Figure Legend Snippet: Lack of detection of RNA in plasma from patients with colorectal cancer stored for three years without Trizol: (A) lanes 1–10, β catenin mRNA from 10 patients; (B) lanes 1–10,

    Techniques Used:

    Detection of plasma RNA in patients 1–3 with colorectal cancer using both methods independently: lanes 1–3, β catenin mRNA extracted by Trizol; lanes 4–6, β catenin mRNA extracted
    Figure Legend Snippet: Detection of plasma RNA in patients 1–3 with colorectal cancer using both methods independently: lanes 1–3, β catenin mRNA extracted by Trizol; lanes 4–6, β catenin mRNA extracted

    Techniques Used:

    19) Product Images from "Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens"

    Article Title: Effective DNA/RNA Co-Extraction for Analysis of MicroRNAs, mRNAs, and Genomic DNA from Formalin-Fixed Paraffin-Embedded Specimens

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0034683

    MicroRNA expression analysis of matched fresh and FFPE RNA from MCF10A cells using different RNA extraction methods. The upper panel displays a graphic representation of quantitative RT-PCR (Taqman® miRNA assays). Measurements obtain for miR-10a, miR-196b, miR-135b, miR-32a and miR-21 using matched fresh and FFPE RNA from MCF10A cells. MiRNAs were quantified using FFPE RNA extracted with TRIzol (TRI), Qiagen AllPrep DNA/RNA FFPE (QDR), AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB) kits and compared to control RNA extracted from fresh cells with TRIzol (TRI-Fr). Results are represented as ΔδC t (δC t target miRNA - δC t miR-10a (least expressed miRNA)). The lower panels show the comparison of global miRNA quantification obtained between fresh and FFPE RNA samples using the Illumina miRNA platform. Comparisons were performed between triplicate RNA extractions obtained from matched fresh (TRI-Fr1, TRI-Fr2, TRI-Fr3) and FFPE (TRI1-3, QDR1-3, and AMB1-3) cells. The correlation coefficient (r) between matched fresh and FFPE RNAs is displayed in each graph.
    Figure Legend Snippet: MicroRNA expression analysis of matched fresh and FFPE RNA from MCF10A cells using different RNA extraction methods. The upper panel displays a graphic representation of quantitative RT-PCR (Taqman® miRNA assays). Measurements obtain for miR-10a, miR-196b, miR-135b, miR-32a and miR-21 using matched fresh and FFPE RNA from MCF10A cells. MiRNAs were quantified using FFPE RNA extracted with TRIzol (TRI), Qiagen AllPrep DNA/RNA FFPE (QDR), AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB) kits and compared to control RNA extracted from fresh cells with TRIzol (TRI-Fr). Results are represented as ΔδC t (δC t target miRNA - δC t miR-10a (least expressed miRNA)). The lower panels show the comparison of global miRNA quantification obtained between fresh and FFPE RNA samples using the Illumina miRNA platform. Comparisons were performed between triplicate RNA extractions obtained from matched fresh (TRI-Fr1, TRI-Fr2, TRI-Fr3) and FFPE (TRI1-3, QDR1-3, and AMB1-3) cells. The correlation coefficient (r) between matched fresh and FFPE RNAs is displayed in each graph.

    Techniques Used: Expressing, Formalin-fixed Paraffin-Embedded, RNA Extraction, Quantitative RT-PCR, Isolation

    Summary of sequential recovery of DNA and RNA from MCF10A Fresh and FFPE samples using different extraction methods. (A) Schematic representation of cell culture and DNA/RNA extraction methods used with matched fresh and 1 month-old formalin-fixed paraffin-embedded (FFPE) human mammary epithelial MCF10A cells. FFPE DNA and RNA extractions (QD, TRI, QDR, AMB) were performed in triplicate using three 10 µm sections for each replicate. (B) Analysis of RNA extracted from matched fresh and FFPE MCF10A cells. Total RNA extracted from fresh cells using TRIzol (TRI-Fr; Lane 2), and total RNA extracted from FFPE cells using TRIzol (TRI; lane 3), Qiagen QIAamp DNA/RNA extraction kit (QDR; lane 4), and AMBion RecoverAll™ Total Nucleic Acid Isolation kit (AMB; lane 5) was analyzed and quantified using an Agilent 2100 Bioanalyzer 6000 Nanochip (size ladder in lane 1). The bar graph placed above the Bioanalyzer image displays total amounts of RNA recovered from three consecutive 10 µm sections, in triplicate experiments, using the three different methods (TRI, QDR, AMB). (C) Analysis of genomic DNA extracted from matched fresh and FFPE MCF10A cells. DNA was extracted from fresh cells using a phenol/chloroform based method (PC-Fr; lane 2), and TRIzol (TRI-Fr lane 3); and from FFPE cells using Qiagen QIAamp DNA FFPE kit (QD; lane 4), TRIzol DNA/RNA extraction method (TRI; lane 5), Qiagen AllPrep DNA/RNA FFPE kit (QDR; lane 6), and AMBion RecoverAll™ Total Nucleic Acid Isolation kit (AMB; lane 7) was analyzed on a 1% agarose gel (size ladder in lane 1). The bar graph placed above the agarose gel displays total amounts of DNA recovered alone (QD), simultaneously with RNA (TRI, QDR), or separately from RNA (AMB), using three consecutive 10 µm sections, in triplicate experiments for each method.
    Figure Legend Snippet: Summary of sequential recovery of DNA and RNA from MCF10A Fresh and FFPE samples using different extraction methods. (A) Schematic representation of cell culture and DNA/RNA extraction methods used with matched fresh and 1 month-old formalin-fixed paraffin-embedded (FFPE) human mammary epithelial MCF10A cells. FFPE DNA and RNA extractions (QD, TRI, QDR, AMB) were performed in triplicate using three 10 µm sections for each replicate. (B) Analysis of RNA extracted from matched fresh and FFPE MCF10A cells. Total RNA extracted from fresh cells using TRIzol (TRI-Fr; Lane 2), and total RNA extracted from FFPE cells using TRIzol (TRI; lane 3), Qiagen QIAamp DNA/RNA extraction kit (QDR; lane 4), and AMBion RecoverAll™ Total Nucleic Acid Isolation kit (AMB; lane 5) was analyzed and quantified using an Agilent 2100 Bioanalyzer 6000 Nanochip (size ladder in lane 1). The bar graph placed above the Bioanalyzer image displays total amounts of RNA recovered from three consecutive 10 µm sections, in triplicate experiments, using the three different methods (TRI, QDR, AMB). (C) Analysis of genomic DNA extracted from matched fresh and FFPE MCF10A cells. DNA was extracted from fresh cells using a phenol/chloroform based method (PC-Fr; lane 2), and TRIzol (TRI-Fr lane 3); and from FFPE cells using Qiagen QIAamp DNA FFPE kit (QD; lane 4), TRIzol DNA/RNA extraction method (TRI; lane 5), Qiagen AllPrep DNA/RNA FFPE kit (QDR; lane 6), and AMBion RecoverAll™ Total Nucleic Acid Isolation kit (AMB; lane 7) was analyzed on a 1% agarose gel (size ladder in lane 1). The bar graph placed above the agarose gel displays total amounts of DNA recovered alone (QD), simultaneously with RNA (TRI, QDR), or separately from RNA (AMB), using three consecutive 10 µm sections, in triplicate experiments for each method.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Cell Culture, RNA Extraction, Isolation, Agarose Gel Electrophoresis

    Messenger RNA expression analysis of matched fresh and FFPE RNA using different RNA extraction methods. The upper panel displays a graphic representation of quantitative RT-PCR (Taqman® mRNA assays) Measurements obtained for ESR1, CCND2 and KRT14 genes using matched fresh and FFPE RNA from MCF10A cells. The three genes were quantified using matched fresh RNA recovered with TRIzol (TRI-Fr), and FFPE RNA recovered with TRIzol (TRI), with Qiagen AllPrep DNA/RNA FFPE (QDR), with AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB) and with the Roche RNA FFPE (Roche) kits. The results are represented as fold changes. The lower panels show the comparison of global mRNA quantifications obtained between fresh and FFPE RNA samples using the Illumina whole-Genome DASL platform. The different panels display comparison between triplicate RNA extractions from matched fresh (TRI-Fr1, TRI-Fr2, TRI-Fr3 (bottom to top panel)) and FFPE (TRI1-3, QDR1-3, AMB1-3 and Roche1-3 (from left to right panel)) cells. The correlation coefficient (r) between matched fresh and FFPE RNAs is displayed in each graph.
    Figure Legend Snippet: Messenger RNA expression analysis of matched fresh and FFPE RNA using different RNA extraction methods. The upper panel displays a graphic representation of quantitative RT-PCR (Taqman® mRNA assays) Measurements obtained for ESR1, CCND2 and KRT14 genes using matched fresh and FFPE RNA from MCF10A cells. The three genes were quantified using matched fresh RNA recovered with TRIzol (TRI-Fr), and FFPE RNA recovered with TRIzol (TRI), with Qiagen AllPrep DNA/RNA FFPE (QDR), with AMBion RecoverAll™ Total Nucleic Acid Isolation (AMB) and with the Roche RNA FFPE (Roche) kits. The results are represented as fold changes. The lower panels show the comparison of global mRNA quantifications obtained between fresh and FFPE RNA samples using the Illumina whole-Genome DASL platform. The different panels display comparison between triplicate RNA extractions from matched fresh (TRI-Fr1, TRI-Fr2, TRI-Fr3 (bottom to top panel)) and FFPE (TRI1-3, QDR1-3, AMB1-3 and Roche1-3 (from left to right panel)) cells. The correlation coefficient (r) between matched fresh and FFPE RNAs is displayed in each graph.

    Techniques Used: RNA Expression, Formalin-fixed Paraffin-Embedded, RNA Extraction, Quantitative RT-PCR, Isolation

    20) Product Images from "The histone deacetylase inhibitor, romidepsin, as a potential treatment for pulmonary fibrosis"

    Article Title: The histone deacetylase inhibitor, romidepsin, as a potential treatment for pulmonary fibrosis

    Journal: Oncotarget

    doi: 10.18632/oncotarget.17114

    Inhibition of fibroblast proliferation by romidepsin: IPF and normal fibroblasts were cultured for up to 144h in DMEM/FBS ± TGF-β1 (5ng/ml) and ± romidepsin Cells were formalin-fixed before being stained with methylene blue. Stained cells were eluted with a 1:1 ratio of 0.1% HCl and ethanol. Absorbance was measured at 630nm using a photometric plate reader. A . Dose response of IPF fibroblasts to romidepsin at 144h in the absence (solid symbols) or presence (open symbols) of TGF-β1. B . Comparison of the romidepsin concentration required to achieve 50% growth inhibition (IC50) of the IPF and normal fibroblasts at 144h. C . At 48h, after culturing as described, RNA was extracted using the Trizol method prior to cDNA synthesis and analysis by RTqPCR. Data were normalized to the housekeeping genes UBC/A2 using the ΔΔCT method. CDKN1A mRNA expression in response to increasing doses response of romidepsin in IPF fibroblasts without (solid bars) or with (open bars) TGF-β1. Data in B . C . are shown as mean + SD ( n = 3; two-way ANOVA with Dunnett's multiple comparisons). *P
    Figure Legend Snippet: Inhibition of fibroblast proliferation by romidepsin: IPF and normal fibroblasts were cultured for up to 144h in DMEM/FBS ± TGF-β1 (5ng/ml) and ± romidepsin Cells were formalin-fixed before being stained with methylene blue. Stained cells were eluted with a 1:1 ratio of 0.1% HCl and ethanol. Absorbance was measured at 630nm using a photometric plate reader. A . Dose response of IPF fibroblasts to romidepsin at 144h in the absence (solid symbols) or presence (open symbols) of TGF-β1. B . Comparison of the romidepsin concentration required to achieve 50% growth inhibition (IC50) of the IPF and normal fibroblasts at 144h. C . At 48h, after culturing as described, RNA was extracted using the Trizol method prior to cDNA synthesis and analysis by RTqPCR. Data were normalized to the housekeeping genes UBC/A2 using the ΔΔCT method. CDKN1A mRNA expression in response to increasing doses response of romidepsin in IPF fibroblasts without (solid bars) or with (open bars) TGF-β1. Data in B . C . are shown as mean + SD ( n = 3; two-way ANOVA with Dunnett's multiple comparisons). *P

    Techniques Used: Inhibition, Cell Culture, Staining, Concentration Assay, Expressing

    Romidepsin suppressed myofibroblast differentiation: Fibroblasts were cultured in DMEM/FBS ± TGF-β1with the indicated concentration of romidepsin for 48 hours and then samples harvested into Trizol for RNA isolation, cDNA synthesis and RTqPCR analysis Figure shows mRNA expression of A . ACTA2 B . HDAC4 and C . COL3A1 mRNA in IPF fibroblasts in response to romidepsin in the absence (solid bars) or presence (open bars) of TGF-β1. D . Inhibition of COL3A1 mRNA expression by 5nM romidepsin ± TGF-β1 in normal or IPF fibroblasts expressed as a percentage of the corresponding untreated control. Data were normalized to the housekeeping genes UBC/A2 using the ΔΔCT method. Data are presented as mean + SD ( n = 3; two-way ANOVA with Dunnett's multiple comparisons). *P
    Figure Legend Snippet: Romidepsin suppressed myofibroblast differentiation: Fibroblasts were cultured in DMEM/FBS ± TGF-β1with the indicated concentration of romidepsin for 48 hours and then samples harvested into Trizol for RNA isolation, cDNA synthesis and RTqPCR analysis Figure shows mRNA expression of A . ACTA2 B . HDAC4 and C . COL3A1 mRNA in IPF fibroblasts in response to romidepsin in the absence (solid bars) or presence (open bars) of TGF-β1. D . Inhibition of COL3A1 mRNA expression by 5nM romidepsin ± TGF-β1 in normal or IPF fibroblasts expressed as a percentage of the corresponding untreated control. Data were normalized to the housekeeping genes UBC/A2 using the ΔΔCT method. Data are presented as mean + SD ( n = 3; two-way ANOVA with Dunnett's multiple comparisons). *P

    Techniques Used: Cell Culture, Concentration Assay, Isolation, Expressing, Inhibition

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    Isolation:

    Article Title: Plasma vesicle miRNAs for therapy response monitoring in Hodgkin lymphoma patients
    Article Snippet: 50 μl exosomes were spiked into 1.5 ml plasma from an EBV-negative healthy donor prior to SEC. Fractions were collected and stored at –80°C until RNA analysis. .. Total RNA was isolated using TRIzol (Thermo Fisher Scientific) according to the manufacturer’s instructions, with some modifications. .. 0.75 ml TRIzol was added to 0.25 ml SEC fractions, mixed properly, and incubated at room temperature for 15 minutes.

    Article Title: Gnotobiotic IL-10−/−; NF-?BEGFP Mice Develop Rapid and Severe Colitis Following Campylobacter jejuni Infection
    Article Snippet: Supernatant and cells were collected for further analysis in 1X Laemmli-buffer for protein analysis and Trizol (Invitrogen, Carlsbad, CA) for RNA analysis. .. RNA was isolated using TRIzol (Invitrogen), reverse transcribed and amplified as previously described using primers specific for murine MIP-2, IL-12p40, TNFα, IL-6, and GAPDH , . .. For PCR analysis, products were subjected to electrophoresis on 2% agarose gels containing GelStar fluorescent dye (Cambrex BioScience Rockland).

    Article Title: Green Tea Extract Provides Extensive Nrf2-Independent Protection Against Lipid Accumulation and NFκB Pro- Inflammatory Responses During Nonalcoholic Steatohepatitis In Mice Fed A High-Fat Diet
    Article Snippet: Nuclear accumulation of Nrf2 protein in response to EGCG (0-5 μM) or the pro-oxidant positive control t-butylhydroperoxide (100 μM) was assessed by Western Blot, as described below, after isolating the nuclear fraction using a kit (Active Motif, Carlsbad, CA). .. Nrf2 mRNA expression was assessed at 0-5 μM EGCG at 0-2 h post-treatment using RT-PCR, as described below, after total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA). .. Cytotoxicity was assessed using the lactate dehydrogenase (LDH) assay (Sigma) and the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS; Promega, Madison, WI) assay at 2 h post-treatment according to the manufacturers' instructions.

    Article Title: The histone deacetylase inhibitor, romidepsin, as a potential treatment for pulmonary fibrosis
    Article Snippet: Fibroblasts (1×105 /well in DMEM/FBS) were pre-treated with medium alone, or with 5 or 10ng/ml TGF-β1 for 24h prior to treatment with Romidepsin. .. At 48h, RNA was isolated using Trizol (Life Technologies, Paisley, UK) and any genomic DNA contamination removed by DNase treatment (Ambion, Warrington, UK) before cDNA synthesis. .. Changes in mRNA expression using the following primers were analyzed by qPCR and normalized to the housekeeping genes ubiquitin C and phospholipase A2 (UBC and A2) using the ΔΔCT method [ ]: CDKN1A (NM_000389), Fwd 5′-TCCAGCCGACCTTCCTCATC-3′ and Rev 5′-GCCTCTACTGCCACCATCT-3′; ACTA2 (NM_001613), Fwd 5′-AAGCACAGAGCAAAAGAGGAAT-3′ and Rev 5′-ATGTCGTCCCAGTTGGTGAT-3′; HDAC4 (NM_006037), Fwd 5′-GCCACCATTCACCTCTGTAAT-3′ and Rev 5′-AAATCCACCCACACAAAACAAG’-3; COL1A1 (NM_000088), Fwd 5′-AGACAGTGATTGAATACAAAACCA-3′ and Rev 5′-GGAGTTTACAGGAAGCAGACA-3′; COL3A1 (NM_000090), Fwd 5′-GTCCCGCTGGCATTCCTG-3′ and Rev 5′-CTCTCCTTTGGCACCATTCTTAC-3′ LOX (NM_002317), Fwd 5′-GATATAGTCTAAATTAGCAAAGCACATAG-3′ and Rev 5′-ATTACGCAGCACAGTCCTTG-3′ .

    Article Title: Induction of Bex genes by curcumin is associated with apoptosis and activation of p53 in N2a neuroblastoma cells
    Article Snippet: DMSO treated and only curcumin treated N2a was used as negative and positive controls of the experiment respectively. .. Total RNA was isolated using Trizol (Invitrogen) as per manufacturer’s instructions and quantified by utilizing Nanovue spectrophotometer (GE healthcare). .. Total RNA was treated with 2 units of amplification grade DNase I (Life technologies, USA) for 15 minutes at room temperature.

    Article Title: Purification and characterisation of dsRNA using ion pair reverse phase chromatography and mass spectrometry
    Article Snippet: Then IPTG was added to the culture to 1 mM final concentration followed by further incubation at 37 °C for 3 h. .. 2.4 The total RNA was extracted from the cells using TRIzol® Max™ Bacterial RNA Isolation Kit with TRIzol® and Max Bacterial Enhancement Reagent (Life technologies) was used to extract total RNA following the manufacturer’s instructions. .. Approximately 108 E. coli cells were suspended in pre-heated 200 μL Max Bacterial Enhancement Reagent and heated at 95 °C for 4 mins.

    Article Title: Aerobic exercise training enhances the in vivo cholesterol trafficking from macrophages to the liver independently of changes in the expression of genes involved in lipid flux in macrophages and aorta
    Article Snippet: Following, mice were transcardially perfused under low-pressure, with a 0.9 % NaCl cold solution and then, aortic arch and liver were excised in the fresh state and preserved in liquid nitrogen as far as analysis. .. RNA was isolated from tissues and macrophages by using Trizol (Invitrogen Life Technologies, Carlsbad, CA, USA). .. The cDNA was synthetized from 100 ng of total RNA using the High Capacity RNA-to-cDNA kit (Applied Biosystems).

    Article Title: Circulating miRNA-24 and its target YKL-40 as potential biomarkers in patients with coronary heart disease and type 2 diabetes mellitus
    Article Snippet: Paragraph title: Isolation of total RNA and miRNA ... Total RNA extraction from blood was performed with TRIzol (Invitrogen, Carlsbad, CA.).

    Size-exclusion Chromatography:

    Article Title: LRP5 knockdown: effect on prostate cancer invasion growth and skeletal metastasis in vitro and in vivo
    Article Snippet: Total cellular RNA from PC-3 cells and cells transfected with vector alone or DN-LRP5 plasmid was extracted using TRIzol (Invitrogen Life Technologies, Burlington, ON) according to the manufacturer's protocol. .. Two microliter of cDNA was used in a 20 μL reaction with SYBR green master mix, 0.5 μmol/L forward and reverse primers.

    Labeling:

    Article Title: Angiogenin cleaves tRNA and promotes stress-induced translational repression
    Article Snippet: Total RNA was extracted by using Trizol (Invitrogen). .. Total RNA was extracted by using Trizol (Invitrogen).

    Mouse Assay:

    Article Title: Green Tea Extract Provides Extensive Nrf2-Independent Protection Against Lipid Accumulation and NFκB Pro- Inflammatory Responses During Nonalcoholic Steatohepatitis In Mice Fed A High-Fat Diet
    Article Snippet: In brief, total RNA was extracted using TRIzol (Invitrogen). .. RT-PCR was performed using a SYBR Green PCR Kit, a Bio-Rad CFX384 instrument (Hercules, CA), and primer sequences purchased from Sigma ( ). mRNA expression of human Nrf2 in HC-04 cells were quantified relative to β-actin using the 2−ΔΔCT method [ ].

    Article Title: Aerobic exercise training enhances the in vivo cholesterol trafficking from macrophages to the liver independently of changes in the expression of genes involved in lipid flux in macrophages and aorta
    Article Snippet: Following, mice were transcardially perfused under low-pressure, with a 0.9 % NaCl cold solution and then, aortic arch and liver were excised in the fresh state and preserved in liquid nitrogen as far as analysis. .. RNA was isolated from tissues and macrophages by using Trizol (Invitrogen Life Technologies, Carlsbad, CA, USA).

    Polymerase Chain Reaction:

    Article Title: Ras/ERK-signalling promotes tRNA synthesis and growth via the RNA polymerase III repressor Maf1 in Drosophila
    Article Snippet: Total RNA was extracted using TRIzol according to manufacturer’s instructions (Invitrogen; 15596–018). .. Total RNA was extracted using TRIzol according to manufacturer’s instructions (Invitrogen; 15596–018).

    Quantitative RT-PCR:

    Article Title: Evaluation of a range of mammalian and mosquito cell lines for use in Chikungunya virus research
    Article Snippet: Paragraph title: qRT-PCR ... Total cell RNA was extracted from cells using TRIzol (ThermoFisher).

    Purification:

    Article Title: Purification and characterisation of dsRNA using ion pair reverse phase chromatography and mass spectrometry
    Article Snippet: Paragraph title: Extraction and purification of RNA ... 2.4 The total RNA was extracted from the cells using TRIzol® Max™ Bacterial RNA Isolation Kit with TRIzol® and Max Bacterial Enhancement Reagent (Life technologies) was used to extract total RNA following the manufacturer’s instructions.

    Plasmid Preparation:

    Article Title: LRP5 knockdown: effect on prostate cancer invasion growth and skeletal metastasis in vitro and in vivo
    Article Snippet: These stably transfected cell lines were pooled to avoid any artifacts associated with transfection of plasmids and used for all further studies. .. Total cellular RNA from PC-3 cells and cells transfected with vector alone or DN-LRP5 plasmid was extracted using TRIzol (Invitrogen Life Technologies, Burlington, ON) according to the manufacturer's protocol. .. Two micrograms of total RNA was used for reverse transcription and amplification.

    Software:

    Article Title: Secretion of autoimmune antibodies in the human subcutaneous adipose tissue
    Article Snippet: After sorting, cells were resuspended in TRIzol (Ambion) (106 cells/100 μl), then RNA extracted for quantitative (q)PCR. .. After sorting, cells were resuspended in TRIzol (Ambion) (106 cells/100 μl), then RNA extracted for quantitative (q)PCR.

    Article Title: LRP5 knockdown: effect on prostate cancer invasion growth and skeletal metastasis in vitro and in vivo
    Article Snippet: Total cellular RNA from PC-3 cells and cells transfected with vector alone or DN-LRP5 plasmid was extracted using TRIzol (Invitrogen Life Technologies, Burlington, ON) according to the manufacturer's protocol. .. Two microliter of cDNA was used in a 20 μL reaction with SYBR green master mix, 0.5 μmol/L forward and reverse primers.

    Dominant Negative Mutation:

    Article Title: LRP5 knockdown: effect on prostate cancer invasion growth and skeletal metastasis in vitro and in vivo
    Article Snippet: These stably transfected cell lines were pooled to avoid any artifacts associated with transfection of plasmids and used for all further studies. .. Total cellular RNA from PC-3 cells and cells transfected with vector alone or DN-LRP5 plasmid was extracted using TRIzol (Invitrogen Life Technologies, Burlington, ON) according to the manufacturer's protocol. .. Two micrograms of total RNA was used for reverse transcription and amplification.

    SYBR Green Assay:

    Article Title: LRP5 knockdown: effect on prostate cancer invasion growth and skeletal metastasis in vitro and in vivo
    Article Snippet: Total cellular RNA from PC-3 cells and cells transfected with vector alone or DN-LRP5 plasmid was extracted using TRIzol (Invitrogen Life Technologies, Burlington, ON) according to the manufacturer's protocol. .. Two micrograms of total RNA was used for reverse transcription and amplification.

    Article Title: Ras/ERK-signalling promotes tRNA synthesis and growth via the RNA polymerase III repressor Maf1 in Drosophila
    Article Snippet: Total RNA was extracted using TRIzol according to manufacturer’s instructions (Invitrogen; 15596–018). .. Total RNA was extracted using TRIzol according to manufacturer’s instructions (Invitrogen; 15596–018).

    RNA Extraction:

    Article Title: Secretion of autoimmune antibodies in the human subcutaneous adipose tissue
    Article Snippet: Paragraph title: RNA extraction and quantitative (q)PCR ... After sorting, cells were resuspended in TRIzol (Ambion) (106 cells/100 μl), then RNA extracted for quantitative (q)PCR.

    Article Title: Gnotobiotic IL-10−/−; NF-?BEGFP Mice Develop Rapid and Severe Colitis Following Campylobacter jejuni Infection
    Article Snippet: Paragraph title: RNA extraction and amplification by RT-PCR ... RNA was isolated using TRIzol (Invitrogen), reverse transcribed and amplified as previously described using primers specific for murine MIP-2, IL-12p40, TNFα, IL-6, and GAPDH , .

    Article Title: The histone deacetylase inhibitor, romidepsin, as a potential treatment for pulmonary fibrosis
    Article Snippet: At 48h, RNA was isolated using Trizol (Life Technologies, Paisley, UK) and any genomic DNA contamination removed by DNase treatment (Ambion, Warrington, UK) before cDNA synthesis. .. At 48h, RNA was isolated using Trizol (Life Technologies, Paisley, UK) and any genomic DNA contamination removed by DNase treatment (Ambion, Warrington, UK) before cDNA synthesis.

    Article Title: Ectopic expressed miR-203 contributes to chronic obstructive pulmonary disease via targeting TAK1 and PIK3CA
    Article Snippet: Paragraph title: RNA extraction and miRNA quantification ... Total RNA was extracted from tissues and cells, using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

    Article Title: Circulating miRNA-24 and its target YKL-40 as potential biomarkers in patients with coronary heart disease and type 2 diabetes mellitus
    Article Snippet: The evolutionary conservation was studied using Multiple EM for Motif Elicitation (MEME) software ( http://meme.nbcr.net/meme/tools/meme ). .. Total RNA extraction from blood was performed with TRIzol (Invitrogen, Carlsbad, CA.). .. Small RNAs, including miRNAs by using the mirVanaTM miRNA Isolation Kit (Ambion, Austin, TX) according to the manufacturer's recommendations.

    In Vitro:

    Article Title: Green Tea Extract Provides Extensive Nrf2-Independent Protection Against Lipid Accumulation and NFκB Pro- Inflammatory Responses During Nonalcoholic Steatohepatitis In Mice Fed A High-Fat Diet
    Article Snippet: Paragraph title: 2.2 In Vitro Study ... Nrf2 mRNA expression was assessed at 0-5 μM EGCG at 0-2 h post-treatment using RT-PCR, as described below, after total RNA was isolated using Trizol (Invitrogen, Carlsbad, CA).

    Spectrophotometry:

    Article Title: Induction of Bex genes by curcumin is associated with apoptosis and activation of p53 in N2a neuroblastoma cells
    Article Snippet: DMSO treated and only curcumin treated N2a was used as negative and positive controls of the experiment respectively. .. Total RNA was isolated using Trizol (Invitrogen) as per manufacturer’s instructions and quantified by utilizing Nanovue spectrophotometer (GE healthcare). .. Total RNA was treated with 2 units of amplification grade DNase I (Life technologies, USA) for 15 minutes at room temperature.

    Concentration Assay:

    Article Title: Induction of Bex genes by curcumin is associated with apoptosis and activation of p53 in N2a neuroblastoma cells
    Article Snippet: Total RNA was isolated using Trizol (Invitrogen) as per manufacturer’s instructions and quantified by utilizing Nanovue spectrophotometer (GE healthcare). .. Total RNA was treated with 2 units of amplification grade DNase I (Life technologies, USA) for 15 minutes at room temperature.

    Staining:

    Article Title: Gnotobiotic IL-10−/−; NF-?BEGFP Mice Develop Rapid and Severe Colitis Following Campylobacter jejuni Infection
    Article Snippet: RNA was isolated using TRIzol (Invitrogen), reverse transcribed and amplified as previously described using primers specific for murine MIP-2, IL-12p40, TNFα, IL-6, and GAPDH , . .. For PCR analysis, products were subjected to electrophoresis on 2% agarose gels containing GelStar fluorescent dye (Cambrex BioScience Rockland).

    Article Title: Ultraviolet Irradiation Induces the Accumulation of Chondroitin Sulfate, but Not Other Glycosaminoglycans, in Human Skin
    Article Snippet: After removal of medium, cells were washed with phosphate-buffered saline and total RNA was extracted by adding 1 ml Trizol (Invitrogen, Carlsbad, CA) directly to the dishes, followed by isopropanol precipitation and 70% ethanol wash. .. After removal of medium, cells were washed with phosphate-buffered saline and total RNA was extracted by adding 1 ml Trizol (Invitrogen, Carlsbad, CA) directly to the dishes, followed by isopropanol precipitation and 70% ethanol wash.

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  • 79
    Thermo Fisher trizol
    Candidate miRNA levels are elevated in EVs of cHL patients compared with healthy controls. RT-PCR analysis of miR127-3p ( A ), miR155-5p ( B ), miR21-5p ( C ), let7a-5p ( D ), miR24-3p ( E ), and miR10b-5p ( F ) in plasma extracellular vesicles (EVs) of healthy individuals ( n = 9) and cHL patients ( n = 20) after size-exclusion chromatography (SEC) and total <t>RNA</t> isolation using <t>TRIzol.</t> For each individual sample, the mean Ct value of SEC fractions 9 and 10 was used. Boxes show the 25%–75% percentile; whiskers show the minimum-maximum; and lines represent the median. * P
    Trizol, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/trizol/product/Thermo Fisher
    Average 79 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    trizol - by Bioz Stars, 2019-11
    79/100 stars
      Buy from Supplier

    99
    Thermo Fisher trizol ls reagent
    Characterization of HBV DNA and <t>RNA</t> in sera of CHB patients. (A and B) Analyses of serum viral DNA from CHB patients by Southern blotting. Viral DNA was extracted from serum samples obtained from forty-five chronic hepatitis B patients (20% of input sample used for protein A/G agarose beads pulldown) and subjected to Southern blot analysis. Alternatively, these samples were first incubated with protein A/G agarose beads, and then viral DNA in the pulldown mixtures was analyzed by Southern blotting. Serum samples selected for further examining are marked with arrows, and samples with SS DNA detection are labeled with asterisks. (C) Protein A/G agarose bead pulldown of viral particles. Sera (25 μl each) from CHB patients 37, 38, 14, and 35 (M1, mixture one) or from patients 17, 21, 42, and 44 (M2, mixture two) were pooled and incubated with protein A/G agarose beads. Viral DNA in input sera, protein A/G bead pulldown mixtures (beads), and the remaining supernatants (sup.) were extracted and subjected to Southern blot analysis. (D) Northern blot detection of serum viral RNA from patients 37, 38, 14, 35, 17, 21, 42, and 44. Total RNA were extracted from serum samples by <t>TRIzol</t> reagent and treated with DNase I before Northern blot analysis. (E to G) Southern blot analyses of viral DNA from selected samples. Viral DNA was separated by electrophoresis through TAE or alkaline agarose gels, followed by Southern blot detection with the indicated riboprobes.
    Trizol Ls Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 318 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/trizol ls reagent/product/Thermo Fisher
    Average 99 stars, based on 318 article reviews
    Price from $9.99 to $1999.99
    trizol ls reagent - by Bioz Stars, 2019-11
    99/100 stars
      Buy from Supplier

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    Candidate miRNA levels are elevated in EVs of cHL patients compared with healthy controls. RT-PCR analysis of miR127-3p ( A ), miR155-5p ( B ), miR21-5p ( C ), let7a-5p ( D ), miR24-3p ( E ), and miR10b-5p ( F ) in plasma extracellular vesicles (EVs) of healthy individuals ( n = 9) and cHL patients ( n = 20) after size-exclusion chromatography (SEC) and total RNA isolation using TRIzol. For each individual sample, the mean Ct value of SEC fractions 9 and 10 was used. Boxes show the 25%–75% percentile; whiskers show the minimum-maximum; and lines represent the median. * P

    Journal: JCI Insight

    Article Title: Plasma vesicle miRNAs for therapy response monitoring in Hodgkin lymphoma patients

    doi: 10.1172/jci.insight.89631

    Figure Lengend Snippet: Candidate miRNA levels are elevated in EVs of cHL patients compared with healthy controls. RT-PCR analysis of miR127-3p ( A ), miR155-5p ( B ), miR21-5p ( C ), let7a-5p ( D ), miR24-3p ( E ), and miR10b-5p ( F ) in plasma extracellular vesicles (EVs) of healthy individuals ( n = 9) and cHL patients ( n = 20) after size-exclusion chromatography (SEC) and total RNA isolation using TRIzol. For each individual sample, the mean Ct value of SEC fractions 9 and 10 was used. Boxes show the 25%–75% percentile; whiskers show the minimum-maximum; and lines represent the median. * P

    Article Snippet: Total RNA was isolated using TRIzol (Thermo Fisher Scientific) according to the manufacturer’s instructions, with some modifications.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Size-exclusion Chromatography, Isolation

    EV outperforms total plasma for monitoring treatment response and corresponds with TARC. ( A ) RT-PCR analysis of miR127-3p in total plasma of cHL patients ( n = 7) before and after treatment, after RNA isolation using TRIzol-LS. ( B ) RT-PCR analysis of miR127-3p in plasma extracellular vesicles (EVs) of the same cHL patients ( n = 7) as in A , after size-exclusion chromatography (SEC) and total RNA isolation. For each individual, the mean Ct value of SEC fractions 9 and 10 is used. Boxes show the 25%–75% percentile; whiskers show the minimum-maximum; and lines represent the median. ( C and D ) As in A and B , but for miR155-5p. ( E and F ) RT-PCR analysis of miR21-5p, miR155-5p, and miR127-3p in total plasma ( E ) and in plasma EVs ( F ) of an individual cHL patient with primary tumor before and after first-line treatment (gray symbols) and a cHL patient with relapsed disease before and after second-line treatment (black symbols). ( G – J ) RT-PCR analysis of miR127-3p ( G ), miR155-5p ( H ), miR21-5p ( I ), and let7a-5p ( J ) in plasma EVs of cHL patients before and after treatment ( n = 7). Each data point is the mean Ct value of the 2 consecutive SEC fractions 9 and 10. ( K ) Serum TARC levels in the same cHL patients as in G–J before and after treatment, as measured by ELISA. Data are shown as paired before and after therapy samples ( E–K ).

    Journal: JCI Insight

    Article Title: Plasma vesicle miRNAs for therapy response monitoring in Hodgkin lymphoma patients

    doi: 10.1172/jci.insight.89631

    Figure Lengend Snippet: EV outperforms total plasma for monitoring treatment response and corresponds with TARC. ( A ) RT-PCR analysis of miR127-3p in total plasma of cHL patients ( n = 7) before and after treatment, after RNA isolation using TRIzol-LS. ( B ) RT-PCR analysis of miR127-3p in plasma extracellular vesicles (EVs) of the same cHL patients ( n = 7) as in A , after size-exclusion chromatography (SEC) and total RNA isolation. For each individual, the mean Ct value of SEC fractions 9 and 10 is used. Boxes show the 25%–75% percentile; whiskers show the minimum-maximum; and lines represent the median. ( C and D ) As in A and B , but for miR155-5p. ( E and F ) RT-PCR analysis of miR21-5p, miR155-5p, and miR127-3p in total plasma ( E ) and in plasma EVs ( F ) of an individual cHL patient with primary tumor before and after first-line treatment (gray symbols) and a cHL patient with relapsed disease before and after second-line treatment (black symbols). ( G – J ) RT-PCR analysis of miR127-3p ( G ), miR155-5p ( H ), miR21-5p ( I ), and let7a-5p ( J ) in plasma EVs of cHL patients before and after treatment ( n = 7). Each data point is the mean Ct value of the 2 consecutive SEC fractions 9 and 10. ( K ) Serum TARC levels in the same cHL patients as in G–J before and after treatment, as measured by ELISA. Data are shown as paired before and after therapy samples ( E–K ).

    Article Snippet: Total RNA was isolated using TRIzol (Thermo Fisher Scientific) according to the manufacturer’s instructions, with some modifications.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Size-exclusion Chromatography, Enzyme-linked Immunosorbent Assay

    Small RNA distribution and recovery in EV fractions 9 and 10. ( A and B ) RNA distribution of miR142-3p, let7a-5p, and vtRNA1-1 ( A ) and miR92a-3p, miR21-5p, and miR451-5p ( B ) in 26 fractions upon size-exclusion chromatography (SEC) of 1.5 ml healthy donor plasma. Total RNA was isolated with TRIzol followed by RT-PCR. Data are depicted as raw Ct values; error bars represent SEM from PCR duplicates. ( C ) Fold enrichment of vtRNA1-1, let7a-5p, and miR142-3p in plasma extracellular vesicles (EVs) (fractions 9 and 10) compared with protein/HDL (fractions 20 and 21). Data are shown as the mean of 2 donors; dots indicate individual samples. ( D ) Fold enrichment of miR92a-3p, miR21-5p, and miR451-5p in protein/HDL (fractions 20 and 21) compared with plasma EVs (fractions 9 and 10). Data are shown as the mean of 2 donors; dots indicate individual samples. ( E ) Fold enrichment of vtRNA1-1 in tumor EV (tEV; fractions 9 and 10) compared with protein/HDL (fractions 20 and 21) after SEC of 1.5 ml B cell culture supernatant. ( F ) SEC of 1.5 ml healthy donor plasma after spike in with 50 μl tumor cell line–derived exosomes. Shown is the fold increase of EBV-miR BHRF1-3 and BART2-5p in EV (fractions 9 and 10) compared with protein/HDL (fractions 20 and 21). Data are shown as the mean of the 2 consecutive SEC fractions; dots represent individual fractions ( E and F ).

    Journal: JCI Insight

    Article Title: Plasma vesicle miRNAs for therapy response monitoring in Hodgkin lymphoma patients

    doi: 10.1172/jci.insight.89631

    Figure Lengend Snippet: Small RNA distribution and recovery in EV fractions 9 and 10. ( A and B ) RNA distribution of miR142-3p, let7a-5p, and vtRNA1-1 ( A ) and miR92a-3p, miR21-5p, and miR451-5p ( B ) in 26 fractions upon size-exclusion chromatography (SEC) of 1.5 ml healthy donor plasma. Total RNA was isolated with TRIzol followed by RT-PCR. Data are depicted as raw Ct values; error bars represent SEM from PCR duplicates. ( C ) Fold enrichment of vtRNA1-1, let7a-5p, and miR142-3p in plasma extracellular vesicles (EVs) (fractions 9 and 10) compared with protein/HDL (fractions 20 and 21). Data are shown as the mean of 2 donors; dots indicate individual samples. ( D ) Fold enrichment of miR92a-3p, miR21-5p, and miR451-5p in protein/HDL (fractions 20 and 21) compared with plasma EVs (fractions 9 and 10). Data are shown as the mean of 2 donors; dots indicate individual samples. ( E ) Fold enrichment of vtRNA1-1 in tumor EV (tEV; fractions 9 and 10) compared with protein/HDL (fractions 20 and 21) after SEC of 1.5 ml B cell culture supernatant. ( F ) SEC of 1.5 ml healthy donor plasma after spike in with 50 μl tumor cell line–derived exosomes. Shown is the fold increase of EBV-miR BHRF1-3 and BART2-5p in EV (fractions 9 and 10) compared with protein/HDL (fractions 20 and 21). Data are shown as the mean of the 2 consecutive SEC fractions; dots represent individual fractions ( E and F ).

    Article Snippet: Total RNA was isolated using TRIzol (Thermo Fisher Scientific) according to the manufacturer’s instructions, with some modifications.

    Techniques: Size-exclusion Chromatography, Isolation, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Cell Culture, Derivative Assay

    miR127-3p EV outperforms total plasma in distinguishing cHL patients from controls. ( A ) RT-PCR analysis of miR127-3p in total plasma of healthy controls ( n = 7) and cHL patients ( n = 8) after RNA isolation using TRIzol-LS. ( B ) RT-PCR analysis of miR127-3p in extracellular vesicle (EV) fractions of the same healthy individuals and cHL patients as in A after size-exclusion chromatography (SEC) and total RNA isolation. For each individual, the mean Ct value of SEC fractions 9 and 10 is used. ( A and B ) Boxes show the 25%–75% percentile; whiskers show the minimum-maximum; and lines represent the median. ** P

    Journal: JCI Insight

    Article Title: Plasma vesicle miRNAs for therapy response monitoring in Hodgkin lymphoma patients

    doi: 10.1172/jci.insight.89631

    Figure Lengend Snippet: miR127-3p EV outperforms total plasma in distinguishing cHL patients from controls. ( A ) RT-PCR analysis of miR127-3p in total plasma of healthy controls ( n = 7) and cHL patients ( n = 8) after RNA isolation using TRIzol-LS. ( B ) RT-PCR analysis of miR127-3p in extracellular vesicle (EV) fractions of the same healthy individuals and cHL patients as in A after size-exclusion chromatography (SEC) and total RNA isolation. For each individual, the mean Ct value of SEC fractions 9 and 10 is used. ( A and B ) Boxes show the 25%–75% percentile; whiskers show the minimum-maximum; and lines represent the median. ** P

    Article Snippet: Total RNA was isolated using TRIzol (Thermo Fisher Scientific) according to the manufacturer’s instructions, with some modifications.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Size-exclusion Chromatography

    Differentially expressed genes in Mkp-1 +/+ and Mkp-1 −/− mice before and following E. coli infection. Mkp-1 +/+ and Mkp-1 −/− mice were either infected i.v. with E. coli at a dose of 2.5 × 10 7 CFU/g of body weight or injected with PBS (controls). Mice were euthanized after 24 h, and total RNA was isolated from the livers of four mice using Trizol for RNA-seq analyses. Volcano plots show the extent of differentially expressed gene (adjusted p value

    Journal: International Journal of Molecular Sciences

    Article Title: Dysregulation of Lipid Metabolism in Mkp-1 Deficient Mice during Gram-Negative Sepsis

    doi: 10.3390/ijms19123904

    Figure Lengend Snippet: Differentially expressed genes in Mkp-1 +/+ and Mkp-1 −/− mice before and following E. coli infection. Mkp-1 +/+ and Mkp-1 −/− mice were either infected i.v. with E. coli at a dose of 2.5 × 10 7 CFU/g of body weight or injected with PBS (controls). Mice were euthanized after 24 h, and total RNA was isolated from the livers of four mice using Trizol for RNA-seq analyses. Volcano plots show the extent of differentially expressed gene (adjusted p value

    Article Snippet: Total RNA was extracted from frozen liver tissues using Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA), solubilized in UltraPure RNase/DNase-free water (Thermo Fisher Scientific), and quantified by using NanoDrop ND-1000 spectrophotometer (Marshall Scientific, Hampton, NH, USA).

    Techniques: Mouse Assay, Infection, Injection, Isolation, RNA Sequencing Assay

    Liver mRNA expression levels of Cd36 in control and E. coli -infected mice. Control and E. coli -infected mice (2.5 × 10 7 CFU/g of body weight, i.v.) were euthanized 24 h post infection. Total RNA was isolated from the livers using Trizol. Cd36 mRNA levels were assessed via qRT-PCR. Expression in un-infected Mkp-1 +/+ mice was set as 1. Values represent means ± S.E. from 4–7 animals in each group. The results were analyzed by two-way ANOVA. Groups marked with distinct letters above the bars indicate significant differences ( p

    Journal: International Journal of Molecular Sciences

    Article Title: Dysregulation of Lipid Metabolism in Mkp-1 Deficient Mice during Gram-Negative Sepsis

    doi: 10.3390/ijms19123904

    Figure Lengend Snippet: Liver mRNA expression levels of Cd36 in control and E. coli -infected mice. Control and E. coli -infected mice (2.5 × 10 7 CFU/g of body weight, i.v.) were euthanized 24 h post infection. Total RNA was isolated from the livers using Trizol. Cd36 mRNA levels were assessed via qRT-PCR. Expression in un-infected Mkp-1 +/+ mice was set as 1. Values represent means ± S.E. from 4–7 animals in each group. The results were analyzed by two-way ANOVA. Groups marked with distinct letters above the bars indicate significant differences ( p

    Article Snippet: Total RNA was extracted from frozen liver tissues using Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA), solubilized in UltraPure RNase/DNase-free water (Thermo Fisher Scientific), and quantified by using NanoDrop ND-1000 spectrophotometer (Marshall Scientific, Hampton, NH, USA).

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

    Hepatic mRNA expression of lipogenesis genes before and after E. coli infection. Mkp-1 +/+ and Mkp-1 −/− mice were either infected i.v. with E. coli at a dose of 2.5 × 10 7 CFU/g of body weight or injected with PBS. Mice were euthanized after 24 h, and total RNA was isolated from the livers using Trizol. mRNA expression for different genes was assessed based on the RNA-seq data set, or quantified via qRT-PCR. Expression in un-infected Mkp-1 +/+ mice was set as 1. Values represent means ± S.E. from 4 animals for RNA-seq and 4–7 animals for qRT-PCR in each group. ( A ) Expression levels of lipogenic regulator genes based on RNA-seq; ( B ) Expression levels of Mtor and Pparg based on qRT-PCR; ( C ) Expression levels of lipogenic genes based on RNA-seq. Values in the graphs were compared by two-way ANOVA. Groups marked with distinct letters above the bars indicate significant differences ( p

    Journal: International Journal of Molecular Sciences

    Article Title: Dysregulation of Lipid Metabolism in Mkp-1 Deficient Mice during Gram-Negative Sepsis

    doi: 10.3390/ijms19123904

    Figure Lengend Snippet: Hepatic mRNA expression of lipogenesis genes before and after E. coli infection. Mkp-1 +/+ and Mkp-1 −/− mice were either infected i.v. with E. coli at a dose of 2.5 × 10 7 CFU/g of body weight or injected with PBS. Mice were euthanized after 24 h, and total RNA was isolated from the livers using Trizol. mRNA expression for different genes was assessed based on the RNA-seq data set, or quantified via qRT-PCR. Expression in un-infected Mkp-1 +/+ mice was set as 1. Values represent means ± S.E. from 4 animals for RNA-seq and 4–7 animals for qRT-PCR in each group. ( A ) Expression levels of lipogenic regulator genes based on RNA-seq; ( B ) Expression levels of Mtor and Pparg based on qRT-PCR; ( C ) Expression levels of lipogenic genes based on RNA-seq. Values in the graphs were compared by two-way ANOVA. Groups marked with distinct letters above the bars indicate significant differences ( p

    Article Snippet: Total RNA was extracted from frozen liver tissues using Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA), solubilized in UltraPure RNase/DNase-free water (Thermo Fisher Scientific), and quantified by using NanoDrop ND-1000 spectrophotometer (Marshall Scientific, Hampton, NH, USA).

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

    CHIKV infection of mosquito cell lines. (a) Virus titres. U4.4 and C6/36 cells were infected at an MOI of 1 and 5. At 24 hpi, the media was collected and virus titred by plaque assay (n = 3, experimental replicates) using BHK cells. (b) Corresponding RNA quantification of the infected cells. Intracellular RNA was extracted using TRIzol and quantified by qRT-PCR using primers specific for nsP3 (n = 3). Values in (a) and (b) were derived from the same number of cells and are thus directly comparable. (c) IF of mosquito cell lines infected with nsP3-ZsGreen virus. Both cell lines demonstrated a distinct puncta organisation of nsP3. Representative images shown.

    Journal: Scientific Reports

    Article Title: Evaluation of a range of mammalian and mosquito cell lines for use in Chikungunya virus research

    doi: 10.1038/s41598-017-15269-w

    Figure Lengend Snippet: CHIKV infection of mosquito cell lines. (a) Virus titres. U4.4 and C6/36 cells were infected at an MOI of 1 and 5. At 24 hpi, the media was collected and virus titred by plaque assay (n = 3, experimental replicates) using BHK cells. (b) Corresponding RNA quantification of the infected cells. Intracellular RNA was extracted using TRIzol and quantified by qRT-PCR using primers specific for nsP3 (n = 3). Values in (a) and (b) were derived from the same number of cells and are thus directly comparable. (c) IF of mosquito cell lines infected with nsP3-ZsGreen virus. Both cell lines demonstrated a distinct puncta organisation of nsP3. Representative images shown.

    Article Snippet: Total cell RNA was extracted from cells using TRIzol (ThermoFisher).

    Techniques: Infection, Plaque Assay, Quantitative RT-PCR, Derivative Assay

    CHIKV infection of mammalian cell lines. (a) Virus titres. Huh7, C2C12, SVG-A, and dermal fibroblast (D Fibs) cells were infected at MOIs of either 1 or 5. At 24 hpi, the media was collected and virus titred by plaque assay (n = 3, experimental replicates). (b) Corresponding RNA quantification of the infected cells. Intracellular RNA was extracted using TRIzol and quantified by qRT-PCR using primers specific for nsP3 (n = 3). Values in (a) and (b) were derived from the same number of cells and are thus directly comparable. (c) Western blots for nsP3 in infected cells. Cells were infected with wildtype CHIKV at an MOI = 1. Cells were lysed at 24 hpi and western blot conducted for nsP3.

    Journal: Scientific Reports

    Article Title: Evaluation of a range of mammalian and mosquito cell lines for use in Chikungunya virus research

    doi: 10.1038/s41598-017-15269-w

    Figure Lengend Snippet: CHIKV infection of mammalian cell lines. (a) Virus titres. Huh7, C2C12, SVG-A, and dermal fibroblast (D Fibs) cells were infected at MOIs of either 1 or 5. At 24 hpi, the media was collected and virus titred by plaque assay (n = 3, experimental replicates). (b) Corresponding RNA quantification of the infected cells. Intracellular RNA was extracted using TRIzol and quantified by qRT-PCR using primers specific for nsP3 (n = 3). Values in (a) and (b) were derived from the same number of cells and are thus directly comparable. (c) Western blots for nsP3 in infected cells. Cells were infected with wildtype CHIKV at an MOI = 1. Cells were lysed at 24 hpi and western blot conducted for nsP3.

    Article Snippet: Total cell RNA was extracted from cells using TRIzol (ThermoFisher).

    Techniques: Infection, Plaque Assay, Quantitative RT-PCR, Derivative Assay, Western Blot

    Characterization of HBV DNA and RNA in sera of CHB patients. (A and B) Analyses of serum viral DNA from CHB patients by Southern blotting. Viral DNA was extracted from serum samples obtained from forty-five chronic hepatitis B patients (20% of input sample used for protein A/G agarose beads pulldown) and subjected to Southern blot analysis. Alternatively, these samples were first incubated with protein A/G agarose beads, and then viral DNA in the pulldown mixtures was analyzed by Southern blotting. Serum samples selected for further examining are marked with arrows, and samples with SS DNA detection are labeled with asterisks. (C) Protein A/G agarose bead pulldown of viral particles. Sera (25 μl each) from CHB patients 37, 38, 14, and 35 (M1, mixture one) or from patients 17, 21, 42, and 44 (M2, mixture two) were pooled and incubated with protein A/G agarose beads. Viral DNA in input sera, protein A/G bead pulldown mixtures (beads), and the remaining supernatants (sup.) were extracted and subjected to Southern blot analysis. (D) Northern blot detection of serum viral RNA from patients 37, 38, 14, 35, 17, 21, 42, and 44. Total RNA were extracted from serum samples by TRIzol reagent and treated with DNase I before Northern blot analysis. (E to G) Southern blot analyses of viral DNA from selected samples. Viral DNA was separated by electrophoresis through TAE or alkaline agarose gels, followed by Southern blot detection with the indicated riboprobes.

    Journal: Journal of Virology

    Article Title: Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients

    doi: 10.1128/JVI.00798-18

    Figure Lengend Snippet: Characterization of HBV DNA and RNA in sera of CHB patients. (A and B) Analyses of serum viral DNA from CHB patients by Southern blotting. Viral DNA was extracted from serum samples obtained from forty-five chronic hepatitis B patients (20% of input sample used for protein A/G agarose beads pulldown) and subjected to Southern blot analysis. Alternatively, these samples were first incubated with protein A/G agarose beads, and then viral DNA in the pulldown mixtures was analyzed by Southern blotting. Serum samples selected for further examining are marked with arrows, and samples with SS DNA detection are labeled with asterisks. (C) Protein A/G agarose bead pulldown of viral particles. Sera (25 μl each) from CHB patients 37, 38, 14, and 35 (M1, mixture one) or from patients 17, 21, 42, and 44 (M2, mixture two) were pooled and incubated with protein A/G agarose beads. Viral DNA in input sera, protein A/G bead pulldown mixtures (beads), and the remaining supernatants (sup.) were extracted and subjected to Southern blot analysis. (D) Northern blot detection of serum viral RNA from patients 37, 38, 14, 35, 17, 21, 42, and 44. Total RNA were extracted from serum samples by TRIzol reagent and treated with DNase I before Northern blot analysis. (E to G) Southern blot analyses of viral DNA from selected samples. Viral DNA was separated by electrophoresis through TAE or alkaline agarose gels, followed by Southern blot detection with the indicated riboprobes.

    Article Snippet: In addition to the SDS-proteinase K method, viral RNA was also extracted with TRIzol LS reagent according to the manufacturer’s instructions (Thermo Fisher Scientific).

    Techniques: Southern Blot, Incubation, Labeling, Northern Blot, Electrophoresis

    Mapping and identifying 3′ ends of extracellular HBV RNAs. (A) Northern blot detection of extracellular HBV RNAs with various riboprobes. Viral RNA from cytoplasmic (C) nucleocapsids (lanes 2, 5, 8, 11, 14, and 17) or culture supernatant (S) (lanes 3, 6, 9, 12, 15, and 18) of HepAD38 cells was extracted with TRIzol reagent and treated with DNase I before Northern blot analysis with plus-strand-specific riboprobes spanning the HBV genome as indicated. pgRNA was used as a reference, and map coordinates were numbered according to the sequence of the HBV genome (genotype D, accession number AJ344117.1 ). (B) Identification of 3′ ends of extracellular HBV RNAs. 3′ Ends of extracellular HBV RNAs were identified by the 3′ RACE method using different HBV-specific anchor primers (the same 5′ primers used for generating templates for producing riboprobes used in panel A, lower). Identified 3′ ends were numbered as described above, and numbers in parentheses indicate the amount of clones with the same 3′ ends. The asterisk indicates unknown nucleic acid copurified with intracellular capsid-associated viral RNA by TRIzol reagent. FL, full-length; Cap, 5′ cap of pregenomic RNA; pA, the polyadenylation site; An, poly(A) tail.

    Journal: Journal of Virology

    Article Title: Extracellular Hepatitis B Virus RNAs Are Heterogeneous in Length and Circulate as Capsid-Antibody Complexes in Addition to Virions in Chronic Hepatitis B Patients

    doi: 10.1128/JVI.00798-18

    Figure Lengend Snippet: Mapping and identifying 3′ ends of extracellular HBV RNAs. (A) Northern blot detection of extracellular HBV RNAs with various riboprobes. Viral RNA from cytoplasmic (C) nucleocapsids (lanes 2, 5, 8, 11, 14, and 17) or culture supernatant (S) (lanes 3, 6, 9, 12, 15, and 18) of HepAD38 cells was extracted with TRIzol reagent and treated with DNase I before Northern blot analysis with plus-strand-specific riboprobes spanning the HBV genome as indicated. pgRNA was used as a reference, and map coordinates were numbered according to the sequence of the HBV genome (genotype D, accession number AJ344117.1 ). (B) Identification of 3′ ends of extracellular HBV RNAs. 3′ Ends of extracellular HBV RNAs were identified by the 3′ RACE method using different HBV-specific anchor primers (the same 5′ primers used for generating templates for producing riboprobes used in panel A, lower). Identified 3′ ends were numbered as described above, and numbers in parentheses indicate the amount of clones with the same 3′ ends. The asterisk indicates unknown nucleic acid copurified with intracellular capsid-associated viral RNA by TRIzol reagent. FL, full-length; Cap, 5′ cap of pregenomic RNA; pA, the polyadenylation site; An, poly(A) tail.

    Article Snippet: In addition to the SDS-proteinase K method, viral RNA was also extracted with TRIzol LS reagent according to the manufacturer’s instructions (Thermo Fisher Scientific).

    Techniques: Northern Blot, Sequencing, Clone Assay