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  • 99
    Thermo Fisher trizol method
    Plasma from aged Rhesus macaques have lower levels of bioactive IFN compared to adult animals. Plasma samples from adult (Animal #22855, 21551, 20374) and aged (Animal #20969, 22163, 20994) RM at 3 days post infection with CHIKV were serially diluted and used to stimulate Rhesus fibroblasts for 24 hours. Recombinant universal Type-1 IFN stimulated cells (0.1, 1, 10 units/ml) were used as positive controls and untreated cells were used as a negative control. Then total <t>RNA</t> was prepared with <t>Trizol</t> and IFN stimulated genes Mx1 (A) and ISG56 (B) were quantified by qRT-PCR using gene-specific primers. Fold change is determined as the ratio of gene expression treated vs. untreated control samples.
    Trizol Method, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 6790 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    76
    Millipore trizol
    Reciprocal regulation of Sphingosine kinase 1 and Sphingosine kinase 2 on <t>cisplatin</t> treated PKCδ silenced B16F10 melanoma cells (A) B16F10 cells were transfected with PKCδ siRNA followed by cisplatin treatment in a time dependent manner. At different time points the treated cells were collected in <t>TRIZOL</t> for Sphk1 and Sphk2 mRNA expression by semi quantitative RT-PCR analysis. GAPDH was used as a reference. Data are from one of the three representative experiments. At different time points, data were represented as mean ± SD. * P
    Trizol, supplied by Millipore, used in various techniques. Bioz Stars score: 76/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    Qiagen trizol method tri
    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 <t>TRIzol</t> (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.
    Trizol Method Tri, supplied by Qiagen, used in various techniques. Bioz Stars score: 78/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Plasma from aged Rhesus macaques have lower levels of bioactive IFN compared to adult animals. Plasma samples from adult (Animal #22855, 21551, 20374) and aged (Animal #20969, 22163, 20994) RM at 3 days post infection with CHIKV were serially diluted and used to stimulate Rhesus fibroblasts for 24 hours. Recombinant universal Type-1 IFN stimulated cells (0.1, 1, 10 units/ml) were used as positive controls and untreated cells were used as a negative control. Then total RNA was prepared with Trizol and IFN stimulated genes Mx1 (A) and ISG56 (B) were quantified by qRT-PCR using gene-specific primers. Fold change is determined as the ratio of gene expression treated vs. untreated control samples.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Chikungunya Virus Infection Results in Higher and Persistent Viral Replication in Aged Rhesus Macaques Due to Defects in Anti-Viral Immunity

    doi: 10.1371/journal.pntd.0002343

    Figure Lengend Snippet: Plasma from aged Rhesus macaques have lower levels of bioactive IFN compared to adult animals. Plasma samples from adult (Animal #22855, 21551, 20374) and aged (Animal #20969, 22163, 20994) RM at 3 days post infection with CHIKV were serially diluted and used to stimulate Rhesus fibroblasts for 24 hours. Recombinant universal Type-1 IFN stimulated cells (0.1, 1, 10 units/ml) were used as positive controls and untreated cells were used as a negative control. Then total RNA was prepared with Trizol and IFN stimulated genes Mx1 (A) and ISG56 (B) were quantified by qRT-PCR using gene-specific primers. Fold change is determined as the ratio of gene expression treated vs. untreated control samples.

    Article Snippet: Total RNA was isolated from the cells using the Trizol method (Life Technologies).

    Techniques: Infection, Recombinant, Negative Control, Quantitative RT-PCR, Expressing

    CHIKV viremia in blood peaks 1 to 2 days post infection of Rhesus macaques. CHIKV load in plasma and PBMC was quantified by qRT-PCR using virus specific primers and probes. A and C) Virus was detected in RNA prepared from 10 µl of plasma using ZR Viral RNA extraction kit (Zymol). B and D) Virus was detected in 0.1 µg of total RNA prepared with Trizol from 1×10 6 PBMC.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Chikungunya Virus Infection Results in Higher and Persistent Viral Replication in Aged Rhesus Macaques Due to Defects in Anti-Viral Immunity

    doi: 10.1371/journal.pntd.0002343

    Figure Lengend Snippet: CHIKV viremia in blood peaks 1 to 2 days post infection of Rhesus macaques. CHIKV load in plasma and PBMC was quantified by qRT-PCR using virus specific primers and probes. A and C) Virus was detected in RNA prepared from 10 µl of plasma using ZR Viral RNA extraction kit (Zymol). B and D) Virus was detected in 0.1 µg of total RNA prepared with Trizol from 1×10 6 PBMC.

    Article Snippet: Total RNA was isolated from the cells using the Trizol method (Life Technologies).

    Techniques: Infection, Quantitative RT-PCR, RNA Extraction

    CHIKV-LR persists in spleen of aged Rhesus macaques. CHIKV load was quantified in spleen tissue at necropsy (35 dpi) by virus-specific qRT-PCR from total RNA samples prepared using Trizol.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Chikungunya Virus Infection Results in Higher and Persistent Viral Replication in Aged Rhesus Macaques Due to Defects in Anti-Viral Immunity

    doi: 10.1371/journal.pntd.0002343

    Figure Lengend Snippet: CHIKV-LR persists in spleen of aged Rhesus macaques. CHIKV load was quantified in spleen tissue at necropsy (35 dpi) by virus-specific qRT-PCR from total RNA samples prepared using Trizol.

    Article Snippet: Total RNA was isolated from the cells using the Trizol method (Life Technologies).

    Techniques: Quantitative RT-PCR

    Measure of hypoxia in the obese SAT versus blood. A. Hypoxia was measured by RNA expression of HIF-1α in adipocytes (AD) and SVF from the same individuals. AD were sonicated for cell disruption in the presence of TRIzol. The SVF was also resuspended in TRIzol. AD and SVF were from the same individuals. Results show qPCR values (2 -ΔΔCt ) of HIF-1α RNA expression. Mean comparisons between groups were performed by Student’s t test (two-tailed). *p

    Journal: PLoS ONE

    Article Title: Secretion of autoimmune antibodies in the human subcutaneous adipose tissue

    doi: 10.1371/journal.pone.0197472

    Figure Lengend Snippet: Measure of hypoxia in the obese SAT versus blood. A. Hypoxia was measured by RNA expression of HIF-1α in adipocytes (AD) and SVF from the same individuals. AD were sonicated for cell disruption in the presence of TRIzol. The SVF was also resuspended in TRIzol. AD and SVF were from the same individuals. Results show qPCR values (2 -ΔΔCt ) of HIF-1α RNA expression. Mean comparisons between groups were performed by Student’s t test (two-tailed). *p

    Article Snippet: After sorting, cells were resuspended in TRIzol (Ambion) (106 cells/100 μl), then RNA extracted for quantitative (q)PCR.

    Techniques: RNA Expression, Sonication, Real-time Polymerase Chain Reaction, Two Tailed Test

    RNA expression of pro-inflammatory cytokines in the obese SAT versus blood. Adipocytes (AD), SVF and PBMC (blood) were sonicated for cell disruption in the presence of TRIzol to separate the soluble fraction (used for RNA isolation) from lipids and cell debris. AD and SVF were from the same obese individuals. PBMC (blood) were from obese individuals age-, gender- and BMI-matched. Results show qPCR values (2 -ΔΔCt ) of TNF-α and IL-6 RNA expression.

    Journal: PLoS ONE

    Article Title: Secretion of autoimmune antibodies in the human subcutaneous adipose tissue

    doi: 10.1371/journal.pone.0197472

    Figure Lengend Snippet: RNA expression of pro-inflammatory cytokines in the obese SAT versus blood. Adipocytes (AD), SVF and PBMC (blood) were sonicated for cell disruption in the presence of TRIzol to separate the soluble fraction (used for RNA isolation) from lipids and cell debris. AD and SVF were from the same obese individuals. PBMC (blood) were from obese individuals age-, gender- and BMI-matched. Results show qPCR values (2 -ΔΔCt ) of TNF-α and IL-6 RNA expression.

    Article Snippet: After sorting, cells were resuspended in TRIzol (Ambion) (106 cells/100 μl), then RNA extracted for quantitative (q)PCR.

    Techniques: RNA Expression, Sonication, Isolation, Real-time Polymerase Chain Reaction

    Measure of lipolysis in the obese SAT versus blood. A. Adipocytes (AD), SVF and PBMC (blood) were sonicated for cell disruption in the presence of TRIzol to separate the soluble fraction (used for RNA isolation) from lipids and cell debris. AD and SVF were from the same obese individuals. PBMC (blood) were from obese individuals age-, gender- and BMI-matched. Results show qPCR values (2 -ΔΔCt ) of LPL RNA expression. B. Total protein lysates of adipocytes (AD) and SVF (from different obese individuals age-, gender- and BMI-matched) were prepared and run in WB to measure phospho-HSL. A representative WB for the higest and lowest values is shown (top). Mean comparisons between groups were performed by Student’s t test (two-tailed). ***p

    Journal: PLoS ONE

    Article Title: Secretion of autoimmune antibodies in the human subcutaneous adipose tissue

    doi: 10.1371/journal.pone.0197472

    Figure Lengend Snippet: Measure of lipolysis in the obese SAT versus blood. A. Adipocytes (AD), SVF and PBMC (blood) were sonicated for cell disruption in the presence of TRIzol to separate the soluble fraction (used for RNA isolation) from lipids and cell debris. AD and SVF were from the same obese individuals. PBMC (blood) were from obese individuals age-, gender- and BMI-matched. Results show qPCR values (2 -ΔΔCt ) of LPL RNA expression. B. Total protein lysates of adipocytes (AD) and SVF (from different obese individuals age-, gender- and BMI-matched) were prepared and run in WB to measure phospho-HSL. A representative WB for the higest and lowest values is shown (top). Mean comparisons between groups were performed by Student’s t test (two-tailed). ***p

    Article Snippet: After sorting, cells were resuspended in TRIzol (Ambion) (106 cells/100 μl), then RNA extracted for quantitative (q)PCR.

    Techniques: Sonication, Isolation, Real-time Polymerase Chain Reaction, RNA Expression, Western Blot, Two Tailed Test

    RNA expression of IFN-γ and IL-21 in the obese SAT. Adipocytes (AD) were sonicated for cell disruption in the presence of TRIzol. SVF was also resuspended in TRIzol. AD and SVF were from the same individuals. Results show qPCR values (2 -ΔΔCt ) of IFN-γ and IL-21. Mean comparisons between groups were performed by Student’s t test (two-tailed). **p

    Journal: PLoS ONE

    Article Title: Secretion of autoimmune antibodies in the human subcutaneous adipose tissue

    doi: 10.1371/journal.pone.0197472

    Figure Lengend Snippet: RNA expression of IFN-γ and IL-21 in the obese SAT. Adipocytes (AD) were sonicated for cell disruption in the presence of TRIzol. SVF was also resuspended in TRIzol. AD and SVF were from the same individuals. Results show qPCR values (2 -ΔΔCt ) of IFN-γ and IL-21. Mean comparisons between groups were performed by Student’s t test (two-tailed). **p

    Article Snippet: After sorting, cells were resuspended in TRIzol (Ambion) (106 cells/100 μl), then RNA extracted for quantitative (q)PCR.

    Techniques: RNA Expression, Sonication, Real-time Polymerase Chain Reaction, Two Tailed Test

    RNA expression of chemokines in adipocytes and corresponding chemokine receptors in the obese SAT versus blood. Top. Adipocytes (AD) were sonicated for cell disruption in the presence of TRIzol to separate the soluble fraction (used for RNA isolation) from lipids and cell debris. Results show qPCR values (2 -ΔΔCt ) of CXCL10, IL-8, CCL2, CCL5 RNA expression. Bottom. The SVF were resuspended in TRIzol. AD and SVF were from the same obese individuals. PBMC (blood) were from obese individuals age-, gender- and BMI-matched. Results show qPCR values (2 -ΔΔCt ) of CXCR2, CXCR3, CCR2, CCR3 RNA expression. Mean comparisons between groups were performed by Student’s t test (two-tailed). **p

    Journal: PLoS ONE

    Article Title: Secretion of autoimmune antibodies in the human subcutaneous adipose tissue

    doi: 10.1371/journal.pone.0197472

    Figure Lengend Snippet: RNA expression of chemokines in adipocytes and corresponding chemokine receptors in the obese SAT versus blood. Top. Adipocytes (AD) were sonicated for cell disruption in the presence of TRIzol to separate the soluble fraction (used for RNA isolation) from lipids and cell debris. Results show qPCR values (2 -ΔΔCt ) of CXCL10, IL-8, CCL2, CCL5 RNA expression. Bottom. The SVF were resuspended in TRIzol. AD and SVF were from the same obese individuals. PBMC (blood) were from obese individuals age-, gender- and BMI-matched. Results show qPCR values (2 -ΔΔCt ) of CXCR2, CXCR3, CCR2, CCR3 RNA expression. Mean comparisons between groups were performed by Student’s t test (two-tailed). **p

    Article Snippet: After sorting, cells were resuspended in TRIzol (Ambion) (106 cells/100 μl), then RNA extracted for quantitative (q)PCR.

    Techniques: RNA Expression, Sonication, Isolation, Real-time Polymerase Chain Reaction, Two Tailed Test

    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

    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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0034683

    Figure Lengend 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.

    Article Snippet: Genomic DNA from fresh tissue (mouse) and cells (human MCF10A) was extracted using phenol/Chloroform method or TRIzol, following manufacturer's instructions (Invitrogen, CA, USA).

    Techniques: 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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0034683

    Figure Lengend 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.

    Article Snippet: Genomic DNA from fresh tissue (mouse) and cells (human MCF10A) was extracted using phenol/Chloroform method or TRIzol, following manufacturer's instructions (Invitrogen, CA, USA).

    Techniques: 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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0034683

    Figure Lengend 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.

    Article Snippet: Genomic DNA from fresh tissue (mouse) and cells (human MCF10A) was extracted using phenol/Chloroform method or TRIzol, following manufacturer's instructions (Invitrogen, CA, USA).

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

    Effect of endogenous tiRNAs on protein synthesis. (A) Endogenous 5′ and 3′ (lane 5), 5′ (lane 6), or 3′ (lane 7) tiRNAs extracted from angiogenin-treated U2OS cells were transfected into U2OS cells using lipofectamine. After 6 h, cells were pulsed with [ 35 S]methionine-containing medium for 30 min before protein extraction. Total counts per minute (cpm) per microgram of protein was normalized to cells treated with a combination of three PIWI-associated control RNAs (Control 1-2-3, lane 1; Control-1, lane 2; Control 3, lane 3) and expressed as a mean ± SD ( n = 3). *, P = 0.01; **, P = 0.01. (B) U2OS cells were transfected with the indicated control RNAs (lanes 1–5) or endogenous 5′ (lane 7), 3′ (lane 8), or 5′ and 3′ (lane 6) tiRNAs. After 6 h, cells were washed, and Trizol extracts were separated on a 15% TBD-urea gel and stained with CYBR gold. (C) Wild-type (SS) and S51A mutant (AA) MEFs were transfected with control RNAs (lanes 1 and 4) or endogenous 5′ (lanes 2 and 5) or 3′ (lanes 3 and 6) tiRNAs, pulsed with [ 35 S]methionine-containing medium, and extracted. Total cpm per microgram of protein was normalized to that of cells treated with control RNA and expressed as means ± SD ( n = 3). *, P = 0.01; **, P = 0.003. (D) Samples from C were separated by 15% SDS-PAGE, transferred to nitrocellulose, and exposed for autoradiography. Migration of molecular size markers is shown at the right.

    Journal: The Journal of Cell Biology

    Article Title: Angiogenin cleaves tRNA and promotes stress-induced translational repression

    doi: 10.1083/jcb.200811106

    Figure Lengend Snippet: Effect of endogenous tiRNAs on protein synthesis. (A) Endogenous 5′ and 3′ (lane 5), 5′ (lane 6), or 3′ (lane 7) tiRNAs extracted from angiogenin-treated U2OS cells were transfected into U2OS cells using lipofectamine. After 6 h, cells were pulsed with [ 35 S]methionine-containing medium for 30 min before protein extraction. Total counts per minute (cpm) per microgram of protein was normalized to cells treated with a combination of three PIWI-associated control RNAs (Control 1-2-3, lane 1; Control-1, lane 2; Control 3, lane 3) and expressed as a mean ± SD ( n = 3). *, P = 0.01; **, P = 0.01. (B) U2OS cells were transfected with the indicated control RNAs (lanes 1–5) or endogenous 5′ (lane 7), 3′ (lane 8), or 5′ and 3′ (lane 6) tiRNAs. After 6 h, cells were washed, and Trizol extracts were separated on a 15% TBD-urea gel and stained with CYBR gold. (C) Wild-type (SS) and S51A mutant (AA) MEFs were transfected with control RNAs (lanes 1 and 4) or endogenous 5′ (lanes 2 and 5) or 3′ (lanes 3 and 6) tiRNAs, pulsed with [ 35 S]methionine-containing medium, and extracted. Total cpm per microgram of protein was normalized to that of cells treated with control RNA and expressed as means ± SD ( n = 3). *, P = 0.01; **, P = 0.003. (D) Samples from C were separated by 15% SDS-PAGE, transferred to nitrocellulose, and exposed for autoradiography. Migration of molecular size markers is shown at the right.

    Article Snippet: Total RNA was extracted by using Trizol (Invitrogen).

    Techniques: Transfection, Protein Extraction, Staining, Mutagenesis, SDS Page, Autoradiography, Migration

    Effect of RNH1 on tiRNA production. (A) U2OS cells were treated with siRNA targeting RNH1 (lanes 2 and 4) 48 h before culturing cells in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of sodium arsenite (SA, 200 µM) for 2 h. Cells were extracted with Trizol, and RNA was separated by 15% PAGE and stained with SYBR gold. The location of tRNAs is indicated. tiRNAs migrate as small RNAs centered around 30 and 40 nucleotides. The expression of RNH1, phospho-eIF2α, and tubulin were quantified by Western blotting analysis (bottom). (B) U2OS cells were treated with siRNAs targeting RNH1 (lanes 2 and 4), then cultured in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of sodium arsenite (SA, 500 µM) for 1 h. Cells were then processed for gel electrophoresis and autoradiography (top) or TCA precipitation and scintillation counting (mean counts per microgram ± SD, n = 3, are shown over each lane). The expression of RNH1, phospho-eIF2α, and tubulin were quantified by Western blotting analysis (bottom).

    Journal: The Journal of Cell Biology

    Article Title: Angiogenin cleaves tRNA and promotes stress-induced translational repression

    doi: 10.1083/jcb.200811106

    Figure Lengend Snippet: Effect of RNH1 on tiRNA production. (A) U2OS cells were treated with siRNA targeting RNH1 (lanes 2 and 4) 48 h before culturing cells in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of sodium arsenite (SA, 200 µM) for 2 h. Cells were extracted with Trizol, and RNA was separated by 15% PAGE and stained with SYBR gold. The location of tRNAs is indicated. tiRNAs migrate as small RNAs centered around 30 and 40 nucleotides. The expression of RNH1, phospho-eIF2α, and tubulin were quantified by Western blotting analysis (bottom). (B) U2OS cells were treated with siRNAs targeting RNH1 (lanes 2 and 4), then cultured in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of sodium arsenite (SA, 500 µM) for 1 h. Cells were then processed for gel electrophoresis and autoradiography (top) or TCA precipitation and scintillation counting (mean counts per microgram ± SD, n = 3, are shown over each lane). The expression of RNH1, phospho-eIF2α, and tubulin were quantified by Western blotting analysis (bottom).

    Article Snippet: Total RNA was extracted by using Trizol (Invitrogen).

    Techniques: Polyacrylamide Gel Electrophoresis, Staining, Expressing, Western Blot, Cell Culture, Nucleic Acid Electrophoresis, Autoradiography, TCA Precipitation

    Stress-induced production of tiRNA. (A) U2OS cells treated with sodium arsenite (SA; 500 µM, 2 h), heat (42°C, 2 h), or UV irradiation (200 J/m 2 , 12 h) were extracted with Trizol, and total RNA (10 µg) was separated on a 15% TBE-urea gel before processing with SYBR gold. The gel was also transferred to membrane and hybridized to a biotin probe complementary to the 5′ end of tRNA Met (NB). (B) Northern blotting analysis of RNA extracted from U2OS cells cultured in the absence (−) or presence (+) of sodium arsenite (500 µM, 2 h). Blots were hybridized to cDNAs complementary to the 5′ or 3′ fragments of the indicated tRNAs (bottom) or 5S RNA as a loading control (top). (C) MEFs derived from wild type (wt) or eIF2α (S51A) mutant (mut) mice were cultured in the absence (−) or presence (SA) of sodium arsenite (500 µM) for the indicated times before Trizol extraction. tRNA and 5′ tRNA fragments were quantified by Northern blotting (top) and SYBR gold staining (middle). Phospho- and total eIF2α was quantified by immunoblotting (IB; bottom).

    Journal: The Journal of Cell Biology

    Article Title: Angiogenin cleaves tRNA and promotes stress-induced translational repression

    doi: 10.1083/jcb.200811106

    Figure Lengend Snippet: Stress-induced production of tiRNA. (A) U2OS cells treated with sodium arsenite (SA; 500 µM, 2 h), heat (42°C, 2 h), or UV irradiation (200 J/m 2 , 12 h) were extracted with Trizol, and total RNA (10 µg) was separated on a 15% TBE-urea gel before processing with SYBR gold. The gel was also transferred to membrane and hybridized to a biotin probe complementary to the 5′ end of tRNA Met (NB). (B) Northern blotting analysis of RNA extracted from U2OS cells cultured in the absence (−) or presence (+) of sodium arsenite (500 µM, 2 h). Blots were hybridized to cDNAs complementary to the 5′ or 3′ fragments of the indicated tRNAs (bottom) or 5S RNA as a loading control (top). (C) MEFs derived from wild type (wt) or eIF2α (S51A) mutant (mut) mice were cultured in the absence (−) or presence (SA) of sodium arsenite (500 µM) for the indicated times before Trizol extraction. tRNA and 5′ tRNA fragments were quantified by Northern blotting (top) and SYBR gold staining (middle). Phospho- and total eIF2α was quantified by immunoblotting (IB; bottom).

    Article Snippet: Total RNA was extracted by using Trizol (Invitrogen).

    Techniques: Irradiation, Northern Blot, Cell Culture, Derivative Assay, Mutagenesis, Mouse Assay, Staining

    RT-PCR analysis of defensin mRNA expression by primary epithelial cells . Primary epithelial cells were obtained from human nasal turbinates (HNT), as described in Methods. The cells (5 × 10 6 ) were grown in the six well plates for 48 hours. The cells were then exposed to either the latex beads or A. fumigatus organisms for 18 hours. The mRNA was then isolated by TRIzol Reagent and RT-PCR was performed as described above in Materials and Methods. Specific primer pairs (Table 1) were used for RNA amplification. The sizes of amplified products are indicated and were as predicted. The hBD2 and hBD9 products were sequenced and confirmed to be identical to the predicted sequence. GAPDH was uniformly expressed. Cells in a control well were cultivated in the absence of A. fumigatus . One of the three results is shown.

    Journal: BMC Microbiology

    Article Title: Inducible expression of beta defensins by human respiratory epithelial cells exposed to Aspergillus fumigatus organisms

    doi: 10.1186/1471-2180-9-33

    Figure Lengend Snippet: RT-PCR analysis of defensin mRNA expression by primary epithelial cells . Primary epithelial cells were obtained from human nasal turbinates (HNT), as described in Methods. The cells (5 × 10 6 ) were grown in the six well plates for 48 hours. The cells were then exposed to either the latex beads or A. fumigatus organisms for 18 hours. The mRNA was then isolated by TRIzol Reagent and RT-PCR was performed as described above in Materials and Methods. Specific primer pairs (Table 1) were used for RNA amplification. The sizes of amplified products are indicated and were as predicted. The hBD2 and hBD9 products were sequenced and confirmed to be identical to the predicted sequence. GAPDH was uniformly expressed. Cells in a control well were cultivated in the absence of A. fumigatus . One of the three results is shown.

    Article Snippet: Total RNA was isolated with TRIzol Reagent (Invitrogen, Cat N 15596-026) according to the manufacturer's instructions.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation, Amplification, Sequencing

    RT-PCR analysis of defensin expression by 16HBE cells exposed to A. fumigatus organisms in the presence of different serums . 16HBE human epithelial bronchial cells (5 × 10 6 ) were grown in six well plates for 24 hours. The cells were then exposed to the different morphotypes of A. fumigatus or the latex beads in the presence of either Human (HS) or Fetal Calf Serum (FCS), (heated or not at 56°C). After 18 hours of incubation, the cells were washed with PBS, mRNA was isolated by TRIzol Reagent, and RT-PCR was performed as described above in Materials and Methods. Specific primer pairs (Table 1) were used for RNA amplification. The sizes of amplified products are indicated and were as predicted. All products were amplified according to the conditions described in Table 1. Cells were cultivated in a control well in the absence of A. fumigatus . GAPDH was uniformly expressed. One of the four results is shown.

    Journal: BMC Microbiology

    Article Title: Inducible expression of beta defensins by human respiratory epithelial cells exposed to Aspergillus fumigatus organisms

    doi: 10.1186/1471-2180-9-33

    Figure Lengend Snippet: RT-PCR analysis of defensin expression by 16HBE cells exposed to A. fumigatus organisms in the presence of different serums . 16HBE human epithelial bronchial cells (5 × 10 6 ) were grown in six well plates for 24 hours. The cells were then exposed to the different morphotypes of A. fumigatus or the latex beads in the presence of either Human (HS) or Fetal Calf Serum (FCS), (heated or not at 56°C). After 18 hours of incubation, the cells were washed with PBS, mRNA was isolated by TRIzol Reagent, and RT-PCR was performed as described above in Materials and Methods. Specific primer pairs (Table 1) were used for RNA amplification. The sizes of amplified products are indicated and were as predicted. All products were amplified according to the conditions described in Table 1. Cells were cultivated in a control well in the absence of A. fumigatus . GAPDH was uniformly expressed. One of the four results is shown.

    Article Snippet: Total RNA was isolated with TRIzol Reagent (Invitrogen, Cat N 15596-026) according to the manufacturer's instructions.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Incubation, Isolation, Amplification

    RT-PCR analysis of various defensin expression levels in human 16HBE epithelial bronchial cells exposed to A. fumigatus organisms . 16HBE human epithelial tracheal cells (5 × 10 6 ) were grown in six well plates for 24 hours. After exposing the cells to RC, SC, HF or latex beads for 18 hours, the cells were washed with PBS, mRNA was isolated by TRIzol Reagent and RT-PCR was performed as described above in Materials and Methods. Specific primer pairs (Table 1) were used for RNA amplification: hBD1, 273 bp product; hBD2, 199 bp product; hBD8, 176 bp product; hBD9, 174 bp product; hBD18, 400 bp product and human GAPDH, which was used as an internal control, 473-bp product. All products were amplified according to the conditions described in Table 1. Cells were cultivated in a control well in the absence of A. fumigatus . As a positive control for defensin expression, exposure to human Il-1β was used in all experiments. The hBD1, hBD2 and hBD9 products were sequenced and confirmed to be identical to the predicted sequence. GAPDH was uniformly expressed. One of the four experiments is shown. Abbreviations: resting conidia (RC), swollen conidia (SC), hyphal fragments (HF), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), interleukin-1β (Il–1β).

    Journal: BMC Microbiology

    Article Title: Inducible expression of beta defensins by human respiratory epithelial cells exposed to Aspergillus fumigatus organisms

    doi: 10.1186/1471-2180-9-33

    Figure Lengend Snippet: RT-PCR analysis of various defensin expression levels in human 16HBE epithelial bronchial cells exposed to A. fumigatus organisms . 16HBE human epithelial tracheal cells (5 × 10 6 ) were grown in six well plates for 24 hours. After exposing the cells to RC, SC, HF or latex beads for 18 hours, the cells were washed with PBS, mRNA was isolated by TRIzol Reagent and RT-PCR was performed as described above in Materials and Methods. Specific primer pairs (Table 1) were used for RNA amplification: hBD1, 273 bp product; hBD2, 199 bp product; hBD8, 176 bp product; hBD9, 174 bp product; hBD18, 400 bp product and human GAPDH, which was used as an internal control, 473-bp product. All products were amplified according to the conditions described in Table 1. Cells were cultivated in a control well in the absence of A. fumigatus . As a positive control for defensin expression, exposure to human Il-1β was used in all experiments. The hBD1, hBD2 and hBD9 products were sequenced and confirmed to be identical to the predicted sequence. GAPDH was uniformly expressed. One of the four experiments is shown. Abbreviations: resting conidia (RC), swollen conidia (SC), hyphal fragments (HF), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), interleukin-1β (Il–1β).

    Article Snippet: Total RNA was isolated with TRIzol Reagent (Invitrogen, Cat N 15596-026) according to the manufacturer's instructions.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation, Amplification, Positive Control, Sequencing

    Analysis of the defensin expression and its localisation in pneumocytes A549 exposed to live A. fumigatus . A . RT-PCR analysis of defensin mRNA expression by human pneumocyte A549 cells exposed to live A. fumigatus . A549 human epithelial bronchial cells (5 × 10 6 ) were grown in six well plates for 24 hours. The cells were then exposed either to live A. fumigatus conidia or latex beads. After 18 hours of incubation, the cells were washed with PBS, mRNA was isolated by TRIzol Reagent, and RT-PCR was performed as described above in Methods. Specific primer pairs (Table 1) were used for RNA amplification. The size of the amplified product is indicated and was as predicted. Cells were cultivated in a control well in the absence of A. fumigatus . As an additional control, the cells were exposed to 10 6 latex beads for the same period. GAPDH was uniformly expressed. One of the four results is shown. B . Immunofluorescence detection of hBD2 in the A549 exposed to live A. fumigatus conidia. A549 cells were seeded at 5 × 10 5 cells per well in 1 ml of DMEM/F12 on 18-mm-diameter cover slips in 12 well plates in triplicate and grown for 16 h at 37°C. After washing the cover slips with 1%BSA/PBS, the cells were exposed to either latex beads or live A. fumigatus conidia for 18 hours. Il-1β was used as a positive control. Some cells were treated with TNF-α. Following washing with PBS, the cells were fixed with a paraformaldehyde solution for 30 min at 37°C. The slides were then incubated in 1% BSA/PBS, followed by a solution of 10% normal goat serum. After washing, polyclonal rabbit anti-human hBD2 at a dilution of 1:250 was applied as primary antibody overnight at 4°C, followed by incubation with FITC-labelled goat anti-rabbit secondary antibody at a dilution of 1:300 for 4 hours at room temperature. After washing, the cover slips were mounted on slides with ProLong antifade Vectashield. Samples were viewed with a Zeiss fluorescence microscope using ×400 magnification. The arrows indicate the cells stained with anti-hBD2 antibody. The percentage of stained cells was computed from triplicates of four experiments. Means followed by the same letter are not significantly different. +, presence; -, absence of Il-1β, TNF-α, live A. fumigatus organism and latex beads. The punctuated localisation of the signal, which is concentrated adjacent to the nucleus (arrow), was observed. The data shown are representative of four independent experiments.

    Journal: BMC Microbiology

    Article Title: Inducible expression of beta defensins by human respiratory epithelial cells exposed to Aspergillus fumigatus organisms

    doi: 10.1186/1471-2180-9-33

    Figure Lengend Snippet: Analysis of the defensin expression and its localisation in pneumocytes A549 exposed to live A. fumigatus . A . RT-PCR analysis of defensin mRNA expression by human pneumocyte A549 cells exposed to live A. fumigatus . A549 human epithelial bronchial cells (5 × 10 6 ) were grown in six well plates for 24 hours. The cells were then exposed either to live A. fumigatus conidia or latex beads. After 18 hours of incubation, the cells were washed with PBS, mRNA was isolated by TRIzol Reagent, and RT-PCR was performed as described above in Methods. Specific primer pairs (Table 1) were used for RNA amplification. The size of the amplified product is indicated and was as predicted. Cells were cultivated in a control well in the absence of A. fumigatus . As an additional control, the cells were exposed to 10 6 latex beads for the same period. GAPDH was uniformly expressed. One of the four results is shown. B . Immunofluorescence detection of hBD2 in the A549 exposed to live A. fumigatus conidia. A549 cells were seeded at 5 × 10 5 cells per well in 1 ml of DMEM/F12 on 18-mm-diameter cover slips in 12 well plates in triplicate and grown for 16 h at 37°C. After washing the cover slips with 1%BSA/PBS, the cells were exposed to either latex beads or live A. fumigatus conidia for 18 hours. Il-1β was used as a positive control. Some cells were treated with TNF-α. Following washing with PBS, the cells were fixed with a paraformaldehyde solution for 30 min at 37°C. The slides were then incubated in 1% BSA/PBS, followed by a solution of 10% normal goat serum. After washing, polyclonal rabbit anti-human hBD2 at a dilution of 1:250 was applied as primary antibody overnight at 4°C, followed by incubation with FITC-labelled goat anti-rabbit secondary antibody at a dilution of 1:300 for 4 hours at room temperature. After washing, the cover slips were mounted on slides with ProLong antifade Vectashield. Samples were viewed with a Zeiss fluorescence microscope using ×400 magnification. The arrows indicate the cells stained with anti-hBD2 antibody. The percentage of stained cells was computed from triplicates of four experiments. Means followed by the same letter are not significantly different. +, presence; -, absence of Il-1β, TNF-α, live A. fumigatus organism and latex beads. The punctuated localisation of the signal, which is concentrated adjacent to the nucleus (arrow), was observed. The data shown are representative of four independent experiments.

    Article Snippet: Total RNA was isolated with TRIzol Reagent (Invitrogen, Cat N 15596-026) according to the manufacturer's instructions.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation, Isolation, Amplification, Immunofluorescence, Positive Control, Fluorescence, Microscopy, Staining

    Inhibition of HCV1b attachment to DHHs by purified HSPG (A) and Heparin (B). The HCV1b was pre-incubated with varying amounts of HSPG or Heparin for 1 hr on ice prior to adding to day-11 DHHs in 12-well cell culture plates as described in materials and methods . After incubation on ice for 2 hrs, the unbound HCV was removed by washing cells with PBS for three times. The vRNA of the cell-bound HCV was extracted with Trizol reagent (Invitrogen). The levels of HCV1b vRNA were determined using the same real-time RT-qPCR method as in Fig. 1.

    Journal: PLoS ONE

    Article Title: Apolipoprotein E Mediates Attachment of Clinical Hepatitis C Virus to Hepatocytes by Binding to Cell Surface Heparan Sulfate Proteoglycan Receptors

    doi: 10.1371/journal.pone.0067982

    Figure Lengend Snippet: Inhibition of HCV1b attachment to DHHs by purified HSPG (A) and Heparin (B). The HCV1b was pre-incubated with varying amounts of HSPG or Heparin for 1 hr on ice prior to adding to day-11 DHHs in 12-well cell culture plates as described in materials and methods . After incubation on ice for 2 hrs, the unbound HCV was removed by washing cells with PBS for three times. The vRNA of the cell-bound HCV was extracted with Trizol reagent (Invitrogen). The levels of HCV1b vRNA were determined using the same real-time RT-qPCR method as in Fig. 1.

    Article Snippet: The vRNA of the cell-bound HCV was extracted with Trizol Reagent (Invitrogen) and quantified by qRT-PCR using the StepOnePlus real-time PCR system.

    Techniques: Inhibition, Purification, Incubation, Cell Culture, Quantitative RT-PCR

    Blockade of HCV1b cell attachment by apoE monoclonal antibody mAb23. DHHs at day-11 were incubated with HCV1b in the absence (Control) or presence of 10 µg/ml of normal mouse IgG1 (mIgG1) or increasing amounts of apoE mAb23 (0.4, 2, and 10 µg/ml) at 37°C for 2 hrs. The unbound HCV was removed by washing cells with 1x PBS for three times. The vRNA of the cell-bound HCV was extracted with Trizol reagent (Invitrogen). The levels of HCV vRNA were quantified by a real-time RT-PCR method using SuperScript® III Platinum® SYBR® Green One-Step qPCR Kit (Invitrogen). Reactions were run in a StepOnePlus real-time PCR system (Applied Biosystems) using the conditions provided by the qPCR kit. A house-keeping gene GAPDH was used as an internal control, which was quantified using Hu-GAPDH primer/probe mix containing vic-TAMRA (Applied Biosystems). The levels of HCV vRNA were calculated from the average data of three experiments upon normalization with the level of GAPDH.

    Journal: PLoS ONE

    Article Title: Apolipoprotein E Mediates Attachment of Clinical Hepatitis C Virus to Hepatocytes by Binding to Cell Surface Heparan Sulfate Proteoglycan Receptors

    doi: 10.1371/journal.pone.0067982

    Figure Lengend Snippet: Blockade of HCV1b cell attachment by apoE monoclonal antibody mAb23. DHHs at day-11 were incubated with HCV1b in the absence (Control) or presence of 10 µg/ml of normal mouse IgG1 (mIgG1) or increasing amounts of apoE mAb23 (0.4, 2, and 10 µg/ml) at 37°C for 2 hrs. The unbound HCV was removed by washing cells with 1x PBS for three times. The vRNA of the cell-bound HCV was extracted with Trizol reagent (Invitrogen). The levels of HCV vRNA were quantified by a real-time RT-PCR method using SuperScript® III Platinum® SYBR® Green One-Step qPCR Kit (Invitrogen). Reactions were run in a StepOnePlus real-time PCR system (Applied Biosystems) using the conditions provided by the qPCR kit. A house-keeping gene GAPDH was used as an internal control, which was quantified using Hu-GAPDH primer/probe mix containing vic-TAMRA (Applied Biosystems). The levels of HCV vRNA were calculated from the average data of three experiments upon normalization with the level of GAPDH.

    Article Snippet: The vRNA of the cell-bound HCV was extracted with Trizol Reagent (Invitrogen) and quantified by qRT-PCR using the StepOnePlus real-time PCR system.

    Techniques: Cell Attachment Assay, Incubation, Quantitative RT-PCR, SYBR Green Assay, Real-time Polymerase Chain Reaction

    Effect of heparinase treatment on HCV1b attachment to DHHs. The day-11 DHHs in 12-well cell culture plates were incubated with varying concentrations of heparinase I in a buffer containing 20 mM Tris-HCl (pH 6.8), 50 mM NaCl, 4 mM CaCl 2 and 0.01% bovine serum albumin at 37°C for 1 hr [18] . The heparinase-treated DHHs were then incubated with HCV1b on ice for 2 hrs. The unbound HCV was removed and the cells were washed with 1x PBS for three times. The HCV1b vRNA of the cell-bound HCV was extracted with Trizol reagent and quantified by RT-qPCR using the StepOnePlus real-time PCR system same as that in Fig. 1.

    Journal: PLoS ONE

    Article Title: Apolipoprotein E Mediates Attachment of Clinical Hepatitis C Virus to Hepatocytes by Binding to Cell Surface Heparan Sulfate Proteoglycan Receptors

    doi: 10.1371/journal.pone.0067982

    Figure Lengend Snippet: Effect of heparinase treatment on HCV1b attachment to DHHs. The day-11 DHHs in 12-well cell culture plates were incubated with varying concentrations of heparinase I in a buffer containing 20 mM Tris-HCl (pH 6.8), 50 mM NaCl, 4 mM CaCl 2 and 0.01% bovine serum albumin at 37°C for 1 hr [18] . The heparinase-treated DHHs were then incubated with HCV1b on ice for 2 hrs. The unbound HCV was removed and the cells were washed with 1x PBS for three times. The HCV1b vRNA of the cell-bound HCV was extracted with Trizol reagent and quantified by RT-qPCR using the StepOnePlus real-time PCR system same as that in Fig. 1.

    Article Snippet: The vRNA of the cell-bound HCV was extracted with Trizol Reagent (Invitrogen) and quantified by qRT-PCR using the StepOnePlus real-time PCR system.

    Techniques: Cell Culture, Incubation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    Inhibition of HCV1b attachment to DHHs by apoE-derived or HSPG-binding peptide. The day-11 DHHs in 12-well cell culture plates were incubated with HCV1b in the absence or presence of increasing concentrations (0, 6.7, 20, and 60 µM) of peptides on ice for 2 hrs. Upon removal of unbound HCV1b by extensive washing with PBS, the vRNA of the cell-bound HCV1b was extracted with Trizol reagent. The levels of HCV1b vRNA were quantified by a real-time RT-qPCR method. A . Sequences of synthetic peptides. B . Inhibition of HCV1b cell attachment by a peptide derived from the apoE receptor-binding domain. C . Blockade of HCV1b cell attachment by an HSPG-binding peptide 6a-P. The relative levels of HCV1b vRNA are on average of three experiments were converted to percentage of control (%) considering the level of HCV vRNA in the absence of peptide 100%. The relative levels of HCV1b vRNA (y-axis) are plotted against concentrations of peptides (x-axis).

    Journal: PLoS ONE

    Article Title: Apolipoprotein E Mediates Attachment of Clinical Hepatitis C Virus to Hepatocytes by Binding to Cell Surface Heparan Sulfate Proteoglycan Receptors

    doi: 10.1371/journal.pone.0067982

    Figure Lengend Snippet: Inhibition of HCV1b attachment to DHHs by apoE-derived or HSPG-binding peptide. The day-11 DHHs in 12-well cell culture plates were incubated with HCV1b in the absence or presence of increasing concentrations (0, 6.7, 20, and 60 µM) of peptides on ice for 2 hrs. Upon removal of unbound HCV1b by extensive washing with PBS, the vRNA of the cell-bound HCV1b was extracted with Trizol reagent. The levels of HCV1b vRNA were quantified by a real-time RT-qPCR method. A . Sequences of synthetic peptides. B . Inhibition of HCV1b cell attachment by a peptide derived from the apoE receptor-binding domain. C . Blockade of HCV1b cell attachment by an HSPG-binding peptide 6a-P. The relative levels of HCV1b vRNA are on average of three experiments were converted to percentage of control (%) considering the level of HCV vRNA in the absence of peptide 100%. The relative levels of HCV1b vRNA (y-axis) are plotted against concentrations of peptides (x-axis).

    Article Snippet: The vRNA of the cell-bound HCV was extracted with Trizol Reagent (Invitrogen) and quantified by qRT-PCR using the StepOnePlus real-time PCR system.

    Techniques: Inhibition, Derivative Assay, Binding Assay, Cell Culture, Incubation, Quantitative RT-PCR, Cell Attachment Assay

    Effect of camel milk on apoptotic and oxidative stress markers Caspase-3 (a), DR4 (b), and HO-1 (c) mRNA levels in MCF7 cells. MCF7 cells were treated for 6 h with a various concentrations of camel milk (2.5, 5, 10, and 20 mg/mL). Thereafter, total RNA was isolated using TRIzol reagent and the mRNA levels of Caspase-3, DR4, and HO-1 were quantified using RT-PCR normalized to β -actin housekeeping gene as described Section 2 . Duplicate reactions were performed for each experiment, and the values presented are the means ± SEM ( n = 6) of three independent experiments. + P

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Camel Milk Triggers Apoptotic Signaling Pathways in Human Hepatoma HepG2 and Breast Cancer MCF7 Cell Lines through Transcriptional Mechanism

    doi: 10.1155/2012/593195

    Figure Lengend Snippet: Effect of camel milk on apoptotic and oxidative stress markers Caspase-3 (a), DR4 (b), and HO-1 (c) mRNA levels in MCF7 cells. MCF7 cells were treated for 6 h with a various concentrations of camel milk (2.5, 5, 10, and 20 mg/mL). Thereafter, total RNA was isolated using TRIzol reagent and the mRNA levels of Caspase-3, DR4, and HO-1 were quantified using RT-PCR normalized to β -actin housekeeping gene as described Section 2 . Duplicate reactions were performed for each experiment, and the values presented are the means ± SEM ( n = 6) of three independent experiments. + P

    Article Snippet: Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions and quantified by measuring the absorbance at 260 nm.

    Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction

    Effect of camel milk on apoptotic markers Caspase-3 (a), p53 (b), BcL2 (c), and DR4 (d) mRNA levels in HepG2 cells. HepG2 cells were treated for 6 h with a various concentrations of camel milk (2.5, 5, 10, and 20 mg/mL). Thereafter, total RNA was isolated using TRIzol reagent, and the mRNA levels of Caspase-3, p53, BcL2, and DR4 were quantified using RT-PCR normalized to β -actin housekeeping gene as described in Section 2 . Duplicate reactions were performed for each experiment, and the values presented are the means ± SEM ( n = 6) of three independent experiments. + P

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Camel Milk Triggers Apoptotic Signaling Pathways in Human Hepatoma HepG2 and Breast Cancer MCF7 Cell Lines through Transcriptional Mechanism

    doi: 10.1155/2012/593195

    Figure Lengend Snippet: Effect of camel milk on apoptotic markers Caspase-3 (a), p53 (b), BcL2 (c), and DR4 (d) mRNA levels in HepG2 cells. HepG2 cells were treated for 6 h with a various concentrations of camel milk (2.5, 5, 10, and 20 mg/mL). Thereafter, total RNA was isolated using TRIzol reagent, and the mRNA levels of Caspase-3, p53, BcL2, and DR4 were quantified using RT-PCR normalized to β -actin housekeeping gene as described in Section 2 . Duplicate reactions were performed for each experiment, and the values presented are the means ± SEM ( n = 6) of three independent experiments. + P

    Article Snippet: Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions and quantified by measuring the absorbance at 260 nm.

    Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction

    Effect of MAPKs inhibitors on camel milk mediated induction of caspase-3 mRNA levels in HepG2 cells. HepG2 cells were pre-treated with for 2 h with JNK inhibitor, SP600125, p38 inhibitor, SB203580, and ERK inhibitor, U0126, before the addition of camel milk (10 mg/mL) for additional 6 h. Thereafter, total RNA was isolated using TRIzol reagent and the mRNA levels of Caspase-3 were quantified using RT-PCR normalized to β -actin housekeeping gene as described Section 2 . Duplicate reactions were performed for each experiment, and the values presented are the means ± SEM ( n = 6) of three independent experiments. + P

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Camel Milk Triggers Apoptotic Signaling Pathways in Human Hepatoma HepG2 and Breast Cancer MCF7 Cell Lines through Transcriptional Mechanism

    doi: 10.1155/2012/593195

    Figure Lengend Snippet: Effect of MAPKs inhibitors on camel milk mediated induction of caspase-3 mRNA levels in HepG2 cells. HepG2 cells were pre-treated with for 2 h with JNK inhibitor, SP600125, p38 inhibitor, SB203580, and ERK inhibitor, U0126, before the addition of camel milk (10 mg/mL) for additional 6 h. Thereafter, total RNA was isolated using TRIzol reagent and the mRNA levels of Caspase-3 were quantified using RT-PCR normalized to β -actin housekeeping gene as described Section 2 . Duplicate reactions were performed for each experiment, and the values presented are the means ± SEM ( n = 6) of three independent experiments. + P

    Article Snippet: Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions and quantified by measuring the absorbance at 260 nm.

    Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction

    Effect of camel milk on oxidative stress markers HO-1 (a) and NQO1 (b) mRNA levels, and ROS (c) production in HepG2 cells. (a) and (b) HepG2 cells were treated for 6 h with a various concentrations of camel milk (2.5, 5, 10, and 20 mg/mL). Thereafter, total RNA was isolated using TRIzol reagent and the mRNA levels of HO-1 and NQO1 were quantified using RT-PCR normalized to β -actin housekeeping gene as described Section 2 . Duplicate reactions were performed for each experiment, and the values presented are the means ± SEM ( n = 6) of three independent experiments. + P

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Camel Milk Triggers Apoptotic Signaling Pathways in Human Hepatoma HepG2 and Breast Cancer MCF7 Cell Lines through Transcriptional Mechanism

    doi: 10.1155/2012/593195

    Figure Lengend Snippet: Effect of camel milk on oxidative stress markers HO-1 (a) and NQO1 (b) mRNA levels, and ROS (c) production in HepG2 cells. (a) and (b) HepG2 cells were treated for 6 h with a various concentrations of camel milk (2.5, 5, 10, and 20 mg/mL). Thereafter, total RNA was isolated using TRIzol reagent and the mRNA levels of HO-1 and NQO1 were quantified using RT-PCR normalized to β -actin housekeeping gene as described Section 2 . Duplicate reactions were performed for each experiment, and the values presented are the means ± SEM ( n = 6) of three independent experiments. + P

    Article Snippet: Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's instructions and quantified by measuring the absorbance at 260 nm.

    Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction

    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

    p65 inhibited E3 ligase FBW7 degradation. (A B) T24T(shp65-1) vs. T24T(nonsense) or MEF(p65−/−), MEF[p65−/−(p65)] vs. MEF(WT) cells were extracted for total RNA with Trizol reagent. RT-PCR assay was used to determine fbw7 mRNA expression. β-Actin was used as an internal control. (C) The indicated cells were pretreated with proteasome inhibitor MG132 for 10 hours, and the cells were then treated with CHX for the indicated times. Then cell extracts were subjected to Western blot, and GAPDH was used as a protein loading control.

    Journal: Neoplasia (New York, N.Y.)

    Article Title: NF-κB p65 Overexpression Promotes Bladder Cancer Cell Migration via FBW7-Mediated Degradation of RhoGDIα Protein

    doi: 10.1016/j.neo.2017.06.002

    Figure Lengend Snippet: p65 inhibited E3 ligase FBW7 degradation. (A B) T24T(shp65-1) vs. T24T(nonsense) or MEF(p65−/−), MEF[p65−/−(p65)] vs. MEF(WT) cells were extracted for total RNA with Trizol reagent. RT-PCR assay was used to determine fbw7 mRNA expression. β-Actin was used as an internal control. (C) The indicated cells were pretreated with proteasome inhibitor MG132 for 10 hours, and the cells were then treated with CHX for the indicated times. Then cell extracts were subjected to Western blot, and GAPDH was used as a protein loading control.

    Article Snippet: Total RNA was extracted by using the TRIzol reagent (Invitrogen, Grand Island, NY) as described in the manufacturer's instructions.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot

    PTEN was p65 a downstream effector and promoted FBW7 protein degradation and inhibited BC migration. (A) The indicated cell extracts were subjected to Western blot to determine protein expression of PTEN, AKT, p-AKT(Thr308), and p-AKT(Ser473). (B) The indicated cell extracts were subjected to Western blot to determine protein expression of PTEN. GAPDH was used as a protein loading control. (C) MEF(p65−/−), MEF[p65−/−(p65)] vs. MEF(WT) cells were extracted for total RNA with Trizol reagent. RT-PCR assay was used to determine pten mRNA expression. β-Actin was used as an internal control. (D) Human PTEN promoter-driven luciferase activity was evaluated in MEF(p65−/−), MEF[p65−/−(p65)] and MEF(WT) cells. The results were normalized by internal TK activity. Bars represent mean ± SD from three independent experiments. Student's t test was utilized to determine the P value. The symbol (♣) indicates a significant increase as compared with MEF(WT) cells ( ♣ P

    Journal: Neoplasia (New York, N.Y.)

    Article Title: NF-κB p65 Overexpression Promotes Bladder Cancer Cell Migration via FBW7-Mediated Degradation of RhoGDIα Protein

    doi: 10.1016/j.neo.2017.06.002

    Figure Lengend Snippet: PTEN was p65 a downstream effector and promoted FBW7 protein degradation and inhibited BC migration. (A) The indicated cell extracts were subjected to Western blot to determine protein expression of PTEN, AKT, p-AKT(Thr308), and p-AKT(Ser473). (B) The indicated cell extracts were subjected to Western blot to determine protein expression of PTEN. GAPDH was used as a protein loading control. (C) MEF(p65−/−), MEF[p65−/−(p65)] vs. MEF(WT) cells were extracted for total RNA with Trizol reagent. RT-PCR assay was used to determine pten mRNA expression. β-Actin was used as an internal control. (D) Human PTEN promoter-driven luciferase activity was evaluated in MEF(p65−/−), MEF[p65−/−(p65)] and MEF(WT) cells. The results were normalized by internal TK activity. Bars represent mean ± SD from three independent experiments. Student's t test was utilized to determine the P value. The symbol (♣) indicates a significant increase as compared with MEF(WT) cells ( ♣ P

    Article Snippet: Total RNA was extracted by using the TRIzol reagent (Invitrogen, Grand Island, NY) as described in the manufacturer's instructions.

    Techniques: Migration, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Luciferase, Activity Assay

    p65 promoted degradation of RhoGDIα protein. (A B) T24T(shp65-1), T24T(shp65-2) vs. T24T(nonsense) or MEF(p65−/−), MEF[p65−/−(p65)] vs. MEF(WT) cells were extracted for total RNA with Trizol reagent. RT-PCR assay was used to determine RhoGDiα mRNA expression. β-Actin was used as an internal control. (C) The indicated cells were pretreated with proteasome inhibitor MG132 for 10 hours and were then followed by treatment of cells with CHX for the indicated times. The cell extracts were subjected to Western blot, and GAPDH was used as a protein loading control.

    Journal: Neoplasia (New York, N.Y.)

    Article Title: NF-κB p65 Overexpression Promotes Bladder Cancer Cell Migration via FBW7-Mediated Degradation of RhoGDIα Protein

    doi: 10.1016/j.neo.2017.06.002

    Figure Lengend Snippet: p65 promoted degradation of RhoGDIα protein. (A B) T24T(shp65-1), T24T(shp65-2) vs. T24T(nonsense) or MEF(p65−/−), MEF[p65−/−(p65)] vs. MEF(WT) cells were extracted for total RNA with Trizol reagent. RT-PCR assay was used to determine RhoGDiα mRNA expression. β-Actin was used as an internal control. (C) The indicated cells were pretreated with proteasome inhibitor MG132 for 10 hours and were then followed by treatment of cells with CHX for the indicated times. The cell extracts were subjected to Western blot, and GAPDH was used as a protein loading control.

    Article Snippet: Total RNA was extracted by using the TRIzol reagent (Invitrogen, Grand Island, NY) as described in the manufacturer's instructions.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot

    Relative mRNA expression of estrogen-relative genes in uterus from immature female rats exposed to Ginkgo biloba extract (EGb761) alone (A) or the combination of EGb761 and 17β-estradiol (E2) or tamoxifen (TM) (B). The total RNA was extracted using TRIzol in liver obtained from the immature female rats exposed to respective compounds. The mRNA levels of CaBP-9 were measured using RT-PCR along with cytochrome c Oxidase I (CO1) mRNA as the internal standard. The PCR product was identified using a gel documentation and quantified by ImageJ 1.43u. The results are expressed as mean ± SD of three separate experiments for each group. Values significantly different from the control are indicated by an asterisk (*p

    Journal: Environmental Health and Toxicology

    Article Title: Ginkgo biloba extract (EGb761) did not express estrogenic activity in an immature rat uterotrophic assay

    doi: 10.5620/eht.e2018016

    Figure Lengend Snippet: Relative mRNA expression of estrogen-relative genes in uterus from immature female rats exposed to Ginkgo biloba extract (EGb761) alone (A) or the combination of EGb761 and 17β-estradiol (E2) or tamoxifen (TM) (B). The total RNA was extracted using TRIzol in liver obtained from the immature female rats exposed to respective compounds. The mRNA levels of CaBP-9 were measured using RT-PCR along with cytochrome c Oxidase I (CO1) mRNA as the internal standard. The PCR product was identified using a gel documentation and quantified by ImageJ 1.43u. The results are expressed as mean ± SD of three separate experiments for each group. Values significantly different from the control are indicated by an asterisk (*p

    Article Snippet: Total RNA was extracted from uteruses and livers using Trizol Reagent (Gibco BRL, Grand Island, NY, USA) according to the manufacturer’s protocol and stored at -80°C until needed.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    Relative mRNA expression of estrogen-relative genes in liver from immature female rats exposed to Ginkgo biloba extract (EGb761) alone (A) or the combination of EGb761 and 17β-estradiol (E2) or tamoxifen (TM) (B). The total RNA was extracted using TRIzol in liver obtained from the immature female rats exposed to respective compounds. The mRNA levels (IGFBP) were measured using RT-PCR along with cytochrome c Oxidase I (CO1) mRNA as the internal standard. The PCR product was identified using a gel documentation and quantified by ImageJ 1.43u. The results are expressed as mean ± SD of three separate experiments for each group. Values significantly different from the control are indicated by an asterisk (**p

    Journal: Environmental Health and Toxicology

    Article Title: Ginkgo biloba extract (EGb761) did not express estrogenic activity in an immature rat uterotrophic assay

    doi: 10.5620/eht.e2018016

    Figure Lengend Snippet: Relative mRNA expression of estrogen-relative genes in liver from immature female rats exposed to Ginkgo biloba extract (EGb761) alone (A) or the combination of EGb761 and 17β-estradiol (E2) or tamoxifen (TM) (B). The total RNA was extracted using TRIzol in liver obtained from the immature female rats exposed to respective compounds. The mRNA levels (IGFBP) were measured using RT-PCR along with cytochrome c Oxidase I (CO1) mRNA as the internal standard. The PCR product was identified using a gel documentation and quantified by ImageJ 1.43u. The results are expressed as mean ± SD of three separate experiments for each group. Values significantly different from the control are indicated by an asterisk (**p

    Article Snippet: Total RNA was extracted from uteruses and livers using Trizol Reagent (Gibco BRL, Grand Island, NY, USA) according to the manufacturer’s protocol and stored at -80°C until needed.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    Renal expression of AT1 (a) and TGF- β 1 (b) in ZL and ZDF rats, and levels of AT1 (c) and TGF- β 1 (d) mRNAs, and TGF- β protein (e) in rat mesangial cells. In cell culture, SO (SO10: 10; SO20: 20 and SO40: 40 μ g/ml), MA (25 μ M) or telmisartan (Tel. 10 μ M) was added 1 h prior to treatment with angiotensin II (10 −6 M), followed by twenty-four hours incubation. Total RNA was extracted from renal tissues and the mesangial cells using TRIzol, respectively. The relative levels of specific mRNAs were determined by RT–PCR. Results were normalized to β -actin. In a parallel experiment, the medium was collected after mesangial cells were treated with the test agents in combination with angiotensin II. TGF- β protein level was determined by commercial ELISA kit according to the manufacturer's instructions. All values are means ± SEM ( n = 5, each group in vivo ; n = 3, each group in vitro ). * P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: The Ayurvedic Medicine Salacia oblonga Attenuates Diabetic Renal Fibrosis in Rats: Suppression of Angiotensin II/AT1 Signaling

    doi: 10.1093/ecam/nep095

    Figure Lengend Snippet: Renal expression of AT1 (a) and TGF- β 1 (b) in ZL and ZDF rats, and levels of AT1 (c) and TGF- β 1 (d) mRNAs, and TGF- β protein (e) in rat mesangial cells. In cell culture, SO (SO10: 10; SO20: 20 and SO40: 40 μ g/ml), MA (25 μ M) or telmisartan (Tel. 10 μ M) was added 1 h prior to treatment with angiotensin II (10 −6 M), followed by twenty-four hours incubation. Total RNA was extracted from renal tissues and the mesangial cells using TRIzol, respectively. The relative levels of specific mRNAs were determined by RT–PCR. Results were normalized to β -actin. In a parallel experiment, the medium was collected after mesangial cells were treated with the test agents in combination with angiotensin II. TGF- β protein level was determined by commercial ELISA kit according to the manufacturer's instructions. All values are means ± SEM ( n = 5, each group in vivo ; n = 3, each group in vitro ). * P

    Article Snippet: Total RNA was prepared from the kidneys of individual rats or rat mesangial cells using TRIzol reagent (Invitrogen, Australia and Invitrogen, China).

    Techniques: Expressing, Cell Culture, Incubation, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, In Vivo, In Vitro

    Renal expression of PAI-1 (a), uPA (b) mRNAs, and the ratio of uPA to PAI-1 (c) in ZL and ZDF rats, and levels of PAI-1 (d) and uPA (e) mRNAs, and the ratio of uPA to PAI-1 (f) in rat mesangial cells. In cell culture, SO (SO10: 10 and SO40: 40 μ g/ml), MA (25 μ M) or Tel (10 μ M) was added 1 h prior to treatment with angiotensin II (10 −6 M), followed by 24-h incubation. Total RNA was extracted from renal tissues and the mesangial cells using TRIzol, respectively. The relative levels of specific mRNAs were determined by RT–PCR. Results were normalized to β -actin. All values are means ± SEM ( n = 5, each group in vivo ; n = 3, each group in vitro ). * P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: The Ayurvedic Medicine Salacia oblonga Attenuates Diabetic Renal Fibrosis in Rats: Suppression of Angiotensin II/AT1 Signaling

    doi: 10.1093/ecam/nep095

    Figure Lengend Snippet: Renal expression of PAI-1 (a), uPA (b) mRNAs, and the ratio of uPA to PAI-1 (c) in ZL and ZDF rats, and levels of PAI-1 (d) and uPA (e) mRNAs, and the ratio of uPA to PAI-1 (f) in rat mesangial cells. In cell culture, SO (SO10: 10 and SO40: 40 μ g/ml), MA (25 μ M) or Tel (10 μ M) was added 1 h prior to treatment with angiotensin II (10 −6 M), followed by 24-h incubation. Total RNA was extracted from renal tissues and the mesangial cells using TRIzol, respectively. The relative levels of specific mRNAs were determined by RT–PCR. Results were normalized to β -actin. All values are means ± SEM ( n = 5, each group in vivo ; n = 3, each group in vitro ). * P

    Article Snippet: Total RNA was prepared from the kidneys of individual rats or rat mesangial cells using TRIzol reagent (Invitrogen, Australia and Invitrogen, China).

    Techniques: Expressing, Cell Culture, Incubation, Reverse Transcription Polymerase Chain Reaction, In Vivo, In Vitro

    Expression of collagen (Col) I (a), Col IV (b), fibronectin (c) mRNAs and fibronectin protein (d) in rat mesangial cells. SO (SO10: 10; SO20: 20 and SO40: 40 μ g/ml), MA (25 μ M) or telmisartan (Tel. 10 μ M) was added 1 h before mesangial cells were treated with angiotensin II (10 −6 M). Twenty-four hours later total RNA was extracted from the mesangial cells using TRIzol. The relative levels of specific mRNAs were determined by RT-PCR. Results were normalized to β -actin. In a parallel experiment, the medium was collected after mesangial cells were treated with the test agents in combination with angiotensin II. fibronectin protein was determined by commercial ELISA kit according to the manufacturer's instructions. All values are means ± SEM ( n = 3, each group). * P

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    Article Title: The Ayurvedic Medicine Salacia oblonga Attenuates Diabetic Renal Fibrosis in Rats: Suppression of Angiotensin II/AT1 Signaling

    doi: 10.1093/ecam/nep095

    Figure Lengend Snippet: Expression of collagen (Col) I (a), Col IV (b), fibronectin (c) mRNAs and fibronectin protein (d) in rat mesangial cells. SO (SO10: 10; SO20: 20 and SO40: 40 μ g/ml), MA (25 μ M) or telmisartan (Tel. 10 μ M) was added 1 h before mesangial cells were treated with angiotensin II (10 −6 M). Twenty-four hours later total RNA was extracted from the mesangial cells using TRIzol. The relative levels of specific mRNAs were determined by RT-PCR. Results were normalized to β -actin. In a parallel experiment, the medium was collected after mesangial cells were treated with the test agents in combination with angiotensin II. fibronectin protein was determined by commercial ELISA kit according to the manufacturer's instructions. All values are means ± SEM ( n = 3, each group). * P

    Article Snippet: Total RNA was prepared from the kidneys of individual rats or rat mesangial cells using TRIzol reagent (Invitrogen, Australia and Invitrogen, China).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    HuR antagonizes the downregulation of COX-2 expression caused by miR-16 in hepatoma cell lines. WRL68 cell extracts (500 µg per lane) were immunoprecipitated with HuR or IgG antibodies. Bound RNA was harvested with TRIzol reagent, reverse transcriptased, and PCR amplified with COX-2 ( A ) or miR-16 primers ( B ). PCR products were visualized by electrophoresis in SYBR Safe DNA gel stain agarose gels. The abundance of the transcripts present in WRL68 cells after HuR immunoprecipitation was assessed, and fold differences were plotted. Input, total mRNA in cell extract; unbound, unbound mRNA after immunoprecipitation with HuR antibody; bound, bound mRNA after immunoprecipitation with HuR antibody; and control, bound mRNA after immunoprecipitation with IgG antiboby. ( C–D ) WRL68 and Hep3B cell lines were transfected with miR-16 or In-miR-16 (50 nM). COX-2 and HuR protein levels were analyzed by Western Blot. ( E–F ) WRL68 and Hep3B cell lines were cotransfected with miR-16 (50 nM) and pcDNA3-HuR-GFP expression vector (4 µg). COX-2 and HuR protein levels were analyzed by Western Blot. Data are reported as means±SD of three independent experiments. *p

    Journal: PLoS ONE

    Article Title: Cyclooxygenase-2 Is a Target of MicroRNA-16 in Human Hepatoma Cells

    doi: 10.1371/journal.pone.0050935

    Figure Lengend Snippet: HuR antagonizes the downregulation of COX-2 expression caused by miR-16 in hepatoma cell lines. WRL68 cell extracts (500 µg per lane) were immunoprecipitated with HuR or IgG antibodies. Bound RNA was harvested with TRIzol reagent, reverse transcriptased, and PCR amplified with COX-2 ( A ) or miR-16 primers ( B ). PCR products were visualized by electrophoresis in SYBR Safe DNA gel stain agarose gels. The abundance of the transcripts present in WRL68 cells after HuR immunoprecipitation was assessed, and fold differences were plotted. Input, total mRNA in cell extract; unbound, unbound mRNA after immunoprecipitation with HuR antibody; bound, bound mRNA after immunoprecipitation with HuR antibody; and control, bound mRNA after immunoprecipitation with IgG antiboby. ( C–D ) WRL68 and Hep3B cell lines were transfected with miR-16 or In-miR-16 (50 nM). COX-2 and HuR protein levels were analyzed by Western Blot. ( E–F ) WRL68 and Hep3B cell lines were cotransfected with miR-16 (50 nM) and pcDNA3-HuR-GFP expression vector (4 µg). COX-2 and HuR protein levels were analyzed by Western Blot. Data are reported as means±SD of three independent experiments. *p

    Article Snippet: Total RNA from HCC cells or human biopsies was extracted by using TRIzol reagent (Invitrogen, Grand Island, NY, USA).

    Techniques: Expressing, Immunoprecipitation, Polymerase Chain Reaction, Amplification, Electrophoresis, Staining, Transfection, Western Blot, Plasmid Preparation

    miR-16 binds COX-2 mRNA and inhibits its translation. ( A ) WRL68 cell extracts (500 µg per lane) were immunoprecipitated with Ago-2 or IgG antibodies. Bound RNA was harvested with TRIzol reagent, reverse transcriptased, and PCR amplified with COX-2 primers. PCR products were visualized by electrophoresis in SYBR Safe DNA gel stain agarose gels. The presence of COX-2 mRNA in WRL68 cell transfected with miR-16 or Lipofectamine after Ago2 immunoprecipitation was assessed, and fold differences were plotted. Input, total mRNA in cell extract; and control, bound mRNA after immunoprecipitation with IgG antiboby. ( B ) Scheme of pGL3-empty, pGL3-seed and pGL3-mut reporter vectors. In pGL3-seed, the putative binding site of miR-16 on COX-2 mRNA 3′-UTR region (as detected by RNAhybrid software) was introduced downstream luciferase gene. In pGL3-mut this region was mutated in order to avoid the binding between miR-16 and Luc mRNA. ( C–D ) A luciferase assay was carried out on HuH-7 and HepG2 cell lines using pGL3-seed and pGL3-mut reporter vectors. Firefly luciferase activity was evaluated 48 h after co-transfection with pGL3-empty/seed/mut (750 ng), miR-16 (50 nM), In-miR-16 (50 nM) and miR-NC (50 nM) as indicated. Data were normalized against renilla luciferase activity (all samples were co-transfected with 50 ng pRL vector and refer to the positive control, pGL3 empty vector). Data are reported as means±SD of three independent experiments. *p

    Journal: PLoS ONE

    Article Title: Cyclooxygenase-2 Is a Target of MicroRNA-16 in Human Hepatoma Cells

    doi: 10.1371/journal.pone.0050935

    Figure Lengend Snippet: miR-16 binds COX-2 mRNA and inhibits its translation. ( A ) WRL68 cell extracts (500 µg per lane) were immunoprecipitated with Ago-2 or IgG antibodies. Bound RNA was harvested with TRIzol reagent, reverse transcriptased, and PCR amplified with COX-2 primers. PCR products were visualized by electrophoresis in SYBR Safe DNA gel stain agarose gels. The presence of COX-2 mRNA in WRL68 cell transfected with miR-16 or Lipofectamine after Ago2 immunoprecipitation was assessed, and fold differences were plotted. Input, total mRNA in cell extract; and control, bound mRNA after immunoprecipitation with IgG antiboby. ( B ) Scheme of pGL3-empty, pGL3-seed and pGL3-mut reporter vectors. In pGL3-seed, the putative binding site of miR-16 on COX-2 mRNA 3′-UTR region (as detected by RNAhybrid software) was introduced downstream luciferase gene. In pGL3-mut this region was mutated in order to avoid the binding between miR-16 and Luc mRNA. ( C–D ) A luciferase assay was carried out on HuH-7 and HepG2 cell lines using pGL3-seed and pGL3-mut reporter vectors. Firefly luciferase activity was evaluated 48 h after co-transfection with pGL3-empty/seed/mut (750 ng), miR-16 (50 nM), In-miR-16 (50 nM) and miR-NC (50 nM) as indicated. Data were normalized against renilla luciferase activity (all samples were co-transfected with 50 ng pRL vector and refer to the positive control, pGL3 empty vector). Data are reported as means±SD of three independent experiments. *p

    Article Snippet: Total RNA from HCC cells or human biopsies was extracted by using TRIzol reagent (Invitrogen, Grand Island, NY, USA).

    Techniques: Immunoprecipitation, Polymerase Chain Reaction, Amplification, Electrophoresis, Staining, Transfection, Binding Assay, Software, Luciferase, Activity Assay, Cotransfection, Plasmid Preparation, Positive Control

    Effect of miR-16 on COX-2 mRNA and protein stability. Hep3B cells were transfected with 50 nM miR-16 or miR-NC, or 30 nM siCOX-2. 5 µg/ml actinomycin-D (Act D) or 10 µg/ml cycloheximide (CHX) were added after transfection. ( A–B ) COX-2 protein was analyzed by Western blot at different time after actinomycin-D treatment. Corresponding densitometry analysis is shown and the relative expression of each sample is related to sample at 0 h as 1. ( C ) mRNA COX-2 levels were analyzed by real time PCR. COX-2 mRNA amounts were calculated as relative expression and normalized to the expression of 36b4 mRNA. Values represent fold change relative to sample at 0 h. ( D–E ) COX-2 protein levels were analyzed by Western blot in the presence or absence of cycloheximide. Corresponding densitometric analysis is shown and the relative expression of each sample is related to the value at 0 h as 1. F) Hep3B cells were transfected with 50 nM miR-16, miR-16 inhibitor (In-miR-16) or lipofectamine and permeabilized with digitonine to obtain soluble and pellet fractions enriched in PB as described in Methods. RNA was isolated from each fraction with Trizol reagent, reverse transcriptased, and PCR amplified with COX-2, Xrn1 and actin primers. Input, RNA extracted from cells prior to fractionation. PCR products were visualized by electrophoresis in SYBR Safe DNA gel stain agarose gels. G) The presence of COX-2 mRNA in soluble and PB fractions was assessed and fold differences were plotted. Data are reported as means±SD of three independent experiments. **p

    Journal: PLoS ONE

    Article Title: Cyclooxygenase-2 Is a Target of MicroRNA-16 in Human Hepatoma Cells

    doi: 10.1371/journal.pone.0050935

    Figure Lengend Snippet: Effect of miR-16 on COX-2 mRNA and protein stability. Hep3B cells were transfected with 50 nM miR-16 or miR-NC, or 30 nM siCOX-2. 5 µg/ml actinomycin-D (Act D) or 10 µg/ml cycloheximide (CHX) were added after transfection. ( A–B ) COX-2 protein was analyzed by Western blot at different time after actinomycin-D treatment. Corresponding densitometry analysis is shown and the relative expression of each sample is related to sample at 0 h as 1. ( C ) mRNA COX-2 levels were analyzed by real time PCR. COX-2 mRNA amounts were calculated as relative expression and normalized to the expression of 36b4 mRNA. Values represent fold change relative to sample at 0 h. ( D–E ) COX-2 protein levels were analyzed by Western blot in the presence or absence of cycloheximide. Corresponding densitometric analysis is shown and the relative expression of each sample is related to the value at 0 h as 1. F) Hep3B cells were transfected with 50 nM miR-16, miR-16 inhibitor (In-miR-16) or lipofectamine and permeabilized with digitonine to obtain soluble and pellet fractions enriched in PB as described in Methods. RNA was isolated from each fraction with Trizol reagent, reverse transcriptased, and PCR amplified with COX-2, Xrn1 and actin primers. Input, RNA extracted from cells prior to fractionation. PCR products were visualized by electrophoresis in SYBR Safe DNA gel stain agarose gels. G) The presence of COX-2 mRNA in soluble and PB fractions was assessed and fold differences were plotted. Data are reported as means±SD of three independent experiments. **p

    Article Snippet: Total RNA from HCC cells or human biopsies was extracted by using TRIzol reagent (Invitrogen, Grand Island, NY, USA).

    Techniques: Transfection, Activated Clotting Time Assay, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Isolation, Polymerase Chain Reaction, Amplification, Fractionation, Electrophoresis, Staining

    Reciprocal regulation of Sphingosine kinase 1 and Sphingosine kinase 2 on cisplatin treated PKCδ silenced B16F10 melanoma cells (A) B16F10 cells were transfected with PKCδ siRNA followed by cisplatin treatment in a time dependent manner. At different time points the treated cells were collected in TRIZOL for Sphk1 and Sphk2 mRNA expression by semi quantitative RT-PCR analysis. GAPDH was used as a reference. Data are from one of the three representative experiments. At different time points, data were represented as mean ± SD. * P

    Journal: Oncotarget

    Article Title: TNFα mediated ceramide generation triggers cisplatin induced apoptosis in B16F10 melanoma in a PKCδ independent manner

    doi: 10.18632/oncotarget.26478

    Figure Lengend Snippet: Reciprocal regulation of Sphingosine kinase 1 and Sphingosine kinase 2 on cisplatin treated PKCδ silenced B16F10 melanoma cells (A) B16F10 cells were transfected with PKCδ siRNA followed by cisplatin treatment in a time dependent manner. At different time points the treated cells were collected in TRIZOL for Sphk1 and Sphk2 mRNA expression by semi quantitative RT-PCR analysis. GAPDH was used as a reference. Data are from one of the three representative experiments. At different time points, data were represented as mean ± SD. * P

    Article Snippet: DMEM medium, penicillin, streptomycin, L-glutamine, Cisplatin, AKT, Fumonisin B-1 (FB-1), Imipramine and Trizol were purchased from Sigma (St Louis, MO, USA).

    Techniques: Transfection, Expressing, Quantitative RT-PCR

    Cisplatin treatment in PKCδ silenced A375 human melanoma cells induces ceramide mediated apoptosis through IRF1-TNFα axis (A) 2 × 10 6 A375 cells were transfected with PKCδ specific siRNA or control siRNA as mentioned in Materials and Methods and the transfected cells were collected in TRIZOL for mRNA expression of PKCδ by semi quantitative RT-PCR. GAPDH was used as a reference. Data are from one of the three representative experiments. Bar diagram is represented as mean ± SD. * P

    Journal: Oncotarget

    Article Title: TNFα mediated ceramide generation triggers cisplatin induced apoptosis in B16F10 melanoma in a PKCδ independent manner

    doi: 10.18632/oncotarget.26478

    Figure Lengend Snippet: Cisplatin treatment in PKCδ silenced A375 human melanoma cells induces ceramide mediated apoptosis through IRF1-TNFα axis (A) 2 × 10 6 A375 cells were transfected with PKCδ specific siRNA or control siRNA as mentioned in Materials and Methods and the transfected cells were collected in TRIZOL for mRNA expression of PKCδ by semi quantitative RT-PCR. GAPDH was used as a reference. Data are from one of the three representative experiments. Bar diagram is represented as mean ± SD. * P

    Article Snippet: DMEM medium, penicillin, streptomycin, L-glutamine, Cisplatin, AKT, Fumonisin B-1 (FB-1), Imipramine and Trizol were purchased from Sigma (St Louis, MO, USA).

    Techniques: Transfection, Expressing, Quantitative RT-PCR

    Cisplatin induces apoptosis in PKCδ silenced B16F10 cells by TNFα mediated pathway (A) and (B) B16F10 cells were transfected with PKCδ siRNA followed by cisplatin, cisplatin along with impiramine, and C2 ceramide treatment. The treated cells were collected in TRIZOL for mRNA expression analyses of TNF-α, IL-12, IFN-γ, IL-10 and TGF-β by semi quantitative RT-PCR. The cell supernatants were collected for estimation of TNF-α, IL-12, IFN-γ, IL-10 and TGF-β by ELISA. GAPDH was used as a reference. Data are from one of three representative experiments. Bar diagram is represented as mean ± SD. * P

    Journal: Oncotarget

    Article Title: TNFα mediated ceramide generation triggers cisplatin induced apoptosis in B16F10 melanoma in a PKCδ independent manner

    doi: 10.18632/oncotarget.26478

    Figure Lengend Snippet: Cisplatin induces apoptosis in PKCδ silenced B16F10 cells by TNFα mediated pathway (A) and (B) B16F10 cells were transfected with PKCδ siRNA followed by cisplatin, cisplatin along with impiramine, and C2 ceramide treatment. The treated cells were collected in TRIZOL for mRNA expression analyses of TNF-α, IL-12, IFN-γ, IL-10 and TGF-β by semi quantitative RT-PCR. The cell supernatants were collected for estimation of TNF-α, IL-12, IFN-γ, IL-10 and TGF-β by ELISA. GAPDH was used as a reference. Data are from one of three representative experiments. Bar diagram is represented as mean ± SD. * P

    Article Snippet: DMEM medium, penicillin, streptomycin, L-glutamine, Cisplatin, AKT, Fumonisin B-1 (FB-1), Imipramine and Trizol were purchased from Sigma (St Louis, MO, USA).

    Techniques: Transfection, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

    Cisplatin induces IRF-1 dependent TNFα activation for initiation of apoptosis in PKCδ silenced B16F10 melanoma cells (A) and (B) B16F10 cells were transfected with PKCδ siRNA followed by Cisplatin treatment. The treated cells were collected in TRIZOL for mRNA extraction and semi quantitative RT-PCR analyses for IRF 1- 8 were done. The expression IRF 1 was analyzed in whole cell lysates by Western blotting. GAPDH was used as a reference. Data are from one of three representative experiments. Bar diagram is represented as mean ± SD. * P

    Journal: Oncotarget

    Article Title: TNFα mediated ceramide generation triggers cisplatin induced apoptosis in B16F10 melanoma in a PKCδ independent manner

    doi: 10.18632/oncotarget.26478

    Figure Lengend Snippet: Cisplatin induces IRF-1 dependent TNFα activation for initiation of apoptosis in PKCδ silenced B16F10 melanoma cells (A) and (B) B16F10 cells were transfected with PKCδ siRNA followed by Cisplatin treatment. The treated cells were collected in TRIZOL for mRNA extraction and semi quantitative RT-PCR analyses for IRF 1- 8 were done. The expression IRF 1 was analyzed in whole cell lysates by Western blotting. GAPDH was used as a reference. Data are from one of three representative experiments. Bar diagram is represented as mean ± SD. * P

    Article Snippet: DMEM medium, penicillin, streptomycin, L-glutamine, Cisplatin, AKT, Fumonisin B-1 (FB-1), Imipramine and Trizol were purchased from Sigma (St Louis, MO, USA).

    Techniques: Activation Assay, Transfection, Quantitative RT-PCR, Expressing, Western Blot

    Cisplatin inhibits cell cycle progression and induces apoptosis in PKCδ silenced B16F10 cells via ceramide generation (A) 2 × 10 6 B16F10 cells were transfected with PKCδ specific siRNA or control siRNA as mentioned in Materials and Methods. The transfected cells were collected in TRIZOL for mRNA expression of PKCδ by semi quantitative RT-PCR. GAPDH was used as a reference. Data are from one of the three representative experiments. Bar diagram is represented as mean ± SD. * P

    Journal: Oncotarget

    Article Title: TNFα mediated ceramide generation triggers cisplatin induced apoptosis in B16F10 melanoma in a PKCδ independent manner

    doi: 10.18632/oncotarget.26478

    Figure Lengend Snippet: Cisplatin inhibits cell cycle progression and induces apoptosis in PKCδ silenced B16F10 cells via ceramide generation (A) 2 × 10 6 B16F10 cells were transfected with PKCδ specific siRNA or control siRNA as mentioned in Materials and Methods. The transfected cells were collected in TRIZOL for mRNA expression of PKCδ by semi quantitative RT-PCR. GAPDH was used as a reference. Data are from one of the three representative experiments. Bar diagram is represented as mean ± SD. * P

    Article Snippet: DMEM medium, penicillin, streptomycin, L-glutamine, Cisplatin, AKT, Fumonisin B-1 (FB-1), Imipramine and Trizol were purchased from Sigma (St Louis, MO, USA).

    Techniques: Transfection, Expressing, Quantitative RT-PCR

    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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0034683

    Figure Lengend 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.

    Article Snippet: Our analyses demonstrated that the two co-extraction methods tested (optimized TRIzol method (TRI), and Qiagen AllPrep DNA/RNA FFPE kit (QDR)) provided higher yields as well as more reliable material for molecular studies than the separate extraction method (Ambion RecoverAll™ kit (AMB)).

    Techniques: 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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0034683

    Figure Lengend 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.

    Article Snippet: Our analyses demonstrated that the two co-extraction methods tested (optimized TRIzol method (TRI), and Qiagen AllPrep DNA/RNA FFPE kit (QDR)) provided higher yields as well as more reliable material for molecular studies than the separate extraction method (Ambion RecoverAll™ kit (AMB)).

    Techniques: 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.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0034683

    Figure Lengend 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.

    Article Snippet: Our analyses demonstrated that the two co-extraction methods tested (optimized TRIzol method (TRI), and Qiagen AllPrep DNA/RNA FFPE kit (QDR)) provided higher yields as well as more reliable material for molecular studies than the separate extraction method (Ambion RecoverAll™ kit (AMB)).

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