universal pcr master mix  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    PCR Master Mix 2X
    Description:
    Thermo Scientific PCR Master Mix is a 2X concentrated solution of Taq DNA Polymerase dNTPs and all of the components required for PCR except DNA template and primers This pre mixed formulation saves time and reduces contamination due to a reduced number of pipetting steps required for PCR set up The mix is optimized for efficient and reproducible PCR Highlights• Convenient ready to use mix• Thermostable half life is more than 40 min at 95°C• Generates PCR products with 3 dA overhangs• Incorporates modified nucleotides e g biotin digoxigenin fluorescently labeled nucleotides Applications• Routine PCR amplification of DNA fragments up to 5 kb• High throughput PCR• DNA labelingNote• The error rate of Taq DNA Polymerase in PCR is 2 2 x 10 5 errors per nt per cycle as determined by a modified method described in Accordingly the accuracy of PCR is 4 5 x 104 Accuracy is an inverse of the error rate and shows an average number of correct nucleotides incorporated before an error occurs
    Catalog Number:
    k0171
    Price:
    None
    Applications:
    PCR|PCR & Real-Time PCR|Routine PCR
    Category:
    Proteins Enzymes Peptides
    Buy from Supplier


    Structured Review

    Thermo Fisher universal pcr master mix
    Functional effects of <t>RGS2</t> RNA interference. A : RGS2 and RGS5 mRNA (real-time <t>PCR,</t> n = 3 each) 72 h after short interfering RNA (siRNA) transfection (75 nM). * P
    Thermo Scientific PCR Master Mix is a 2X concentrated solution of Taq DNA Polymerase dNTPs and all of the components required for PCR except DNA template and primers This pre mixed formulation saves time and reduces contamination due to a reduced number of pipetting steps required for PCR set up The mix is optimized for efficient and reproducible PCR Highlights• Convenient ready to use mix• Thermostable half life is more than 40 min at 95°C• Generates PCR products with 3 dA overhangs• Incorporates modified nucleotides e g biotin digoxigenin fluorescently labeled nucleotides Applications• Routine PCR amplification of DNA fragments up to 5 kb• High throughput PCR• DNA labelingNote• The error rate of Taq DNA Polymerase in PCR is 2 2 x 10 5 errors per nt per cycle as determined by a modified method described in Accordingly the accuracy of PCR is 4 5 x 104 Accuracy is an inverse of the error rate and shows an average number of correct nucleotides incorporated before an error occurs
    https://www.bioz.com/result/universal pcr master mix/product/Thermo Fisher
    Average 99 stars, based on 300 article reviews
    Price from $9.99 to $1999.99
    universal pcr master mix - by Bioz Stars, 2020-08
    99/100 stars

    Images

    1) Product Images from "Regulator of G protein signaling 2 is a functionally important negative regulator of angiotensin II-induced cardiac fibroblast responses"

    Article Title: Regulator of G protein signaling 2 is a functionally important negative regulator of angiotensin II-induced cardiac fibroblast responses

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    doi: 10.1152/ajpheart.00026.2011

    Functional effects of RGS2 RNA interference. A : RGS2 and RGS5 mRNA (real-time PCR, n = 3 each) 72 h after short interfering RNA (siRNA) transfection (75 nM). * P
    Figure Legend Snippet: Functional effects of RGS2 RNA interference. A : RGS2 and RGS5 mRNA (real-time PCR, n = 3 each) 72 h after short interfering RNA (siRNA) transfection (75 nM). * P

    Techniques Used: Functional Assay, Real-time Polymerase Chain Reaction, Small Interfering RNA, Transfection

    Transient and selective regulator of G protein signaling 2 (RGS2) mRNA upregulation in response to short-term stimulation with ANG II. A : RT-PCR analysis of RGS2 expression in P0–P2 fibroblasts and comparison with rat brain (Br) and adult cardiac
    Figure Legend Snippet: Transient and selective regulator of G protein signaling 2 (RGS2) mRNA upregulation in response to short-term stimulation with ANG II. A : RT-PCR analysis of RGS2 expression in P0–P2 fibroblasts and comparison with rat brain (Br) and adult cardiac

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing

    Selective RGS2 downregulation in response to prolonged ANG II stimulation in vivo. A : real-time PCR analysis of RGS2 expression in freshly isolated cardiac fibroblasts from rats subjected to subcutaneous ANG II infusion (555 ng·kg −1 ·min
    Figure Legend Snippet: Selective RGS2 downregulation in response to prolonged ANG II stimulation in vivo. A : real-time PCR analysis of RGS2 expression in freshly isolated cardiac fibroblasts from rats subjected to subcutaneous ANG II infusion (555 ng·kg −1 ·min

    Techniques Used: In Vivo, Real-time Polymerase Chain Reaction, Expressing, Isolation

    2) Product Images from "Endogenous angiotensinergic system in neurons of rat and human trigeminal ganglia"

    Article Title: Endogenous angiotensinergic system in neurons of rat and human trigeminal ganglia

    Journal: Regulatory peptides

    doi: 10.1016/j.regpep.2009.02.002

    Determination of relative mRNA levels in rat trigeminal ganglia. Reverse transcribed total RNA extracts of rat trigeminal ganglia from different rats were tested by qRT-PCR for the presence of Ang-N mRNA (a), for renin mRNA (b), for ACE mRNA (c) and cathepsin
    Figure Legend Snippet: Determination of relative mRNA levels in rat trigeminal ganglia. Reverse transcribed total RNA extracts of rat trigeminal ganglia from different rats were tested by qRT-PCR for the presence of Ang-N mRNA (a), for renin mRNA (b), for ACE mRNA (c) and cathepsin

    Techniques Used: Quantitative RT-PCR

    3) Product Images from "ING1b-inducible microRNA203 inhibits cell proliferation"

    Article Title: ING1b-inducible microRNA203 inhibits cell proliferation

    Journal: British Journal of Cancer

    doi: 10.1038/bjc.2013.50

    Real-time PCR confirmation of miRNA expression in response to ING1b. The expression of miR-203, miR-375, miR-449b and miR-200c in Hs68 cells in response to ING1b overexpression for 48 h, was determined by quantitative real-time PCR assays. Bars represent the mean±s.e.m (** P
    Figure Legend Snippet: Real-time PCR confirmation of miRNA expression in response to ING1b. The expression of miR-203, miR-375, miR-449b and miR-200c in Hs68 cells in response to ING1b overexpression for 48 h, was determined by quantitative real-time PCR assays. Bars represent the mean±s.e.m (** P

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Over Expression

    4) Product Images from "The 68 kDa subunit of mammalian cleavage factor I interacts with the U7 small nuclear ribonucleoprotein and participates in 3?-end processing of animal histone mRNAs"

    Article Title: The 68 kDa subunit of mammalian cleavage factor I interacts with the U7 small nuclear ribonucleoprotein and participates in 3?-end processing of animal histone mRNAs

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq613

    Effects of CF I m 68 depletion/overexpression or CF I m 25 depletion on apparent histone RNA 3′-end processing in vivo . ( A ) Schematic representation of quantitative (real-time) reverse transcription–PCR (qRT–PCR) assay. Total mRNA (tot) of histone H3 C is measured with a primer-probe set spanning the translation start codon. To measure the unprocessed precursors (pre), the primer-probe set spans the 3′-cleavage site. See ‘Materials and Methods’ section for further details. ( B ) H3 C RNA 3′-end processing efficiency in Hela cells depleted of CF I m 68, CF I m 25 or Lsm11. The western blots show endogenous CF I m 68 or CF I m 25 detected with corresponding antibodies. SmB/B′ and β-actin served as loading controls. The RT–PCR shows Lsm11 mRNA detected in total RNA. ( C ) H3 C RNA 3′-end processing efficiency in Hela cells overexpressing CF I m 68. The western blots show endogenous and HA-tagged CF I m 68 detected with anti-CF I m 68 antibody. SmB/B′ served as loading control. The relative in vivo processing efficiency was calculated as ratio of pre-mRNA to total mRNA and then normalized with respect to the ratio obtained in cells treated with either control siRNA, Tcrβ-specific shRNA (B) or pcDNA-puro (C). The data shown represent means ± standard deviations. Number of independent measurements (CF I m 68 depletion, 5; CF I m 25 depletion, 3; Lsm11 depletion, 3; CF I m 68 overexpression, 4) p, significance values determined by Student’s t -test. ( D ) Northern blot detecting H3 C mRNA. Nuclear RNA from control cells or cells overexpressing HA-tagged CF I m 68 was separated by polyacrylamide electrophoresis and transferred to positively charged nylon membranes. Histone H3 C mRNA was detected with probes either hybridizing to the 5′ or the 3′-end of the mRNA. 18 S rRNA served as loading control. ( E ) Quantitation of mRNA levels (relative to U6 snRNA) for CF I m 68 (dark columns) or CF I m 25 (light columns) from cells depleted of CF I m 68 or CF I m 25 (number of depletions: 3). ( F ) Western blot detecting endogenous CF I m 68 or CF I m 25 from cells transfected with a control siRNA or depleted of CF I m 68 or CF I m 25. β-actin served as loading control. ( G ) Quantitation of CF I m 68 (dark columns) and CF I m 25 (light columns) protein levels, relative to β-actin, in cells depleted of CF I m 68 or CF I m 25 (number of evaluated blots: 2).
    Figure Legend Snippet: Effects of CF I m 68 depletion/overexpression or CF I m 25 depletion on apparent histone RNA 3′-end processing in vivo . ( A ) Schematic representation of quantitative (real-time) reverse transcription–PCR (qRT–PCR) assay. Total mRNA (tot) of histone H3 C is measured with a primer-probe set spanning the translation start codon. To measure the unprocessed precursors (pre), the primer-probe set spans the 3′-cleavage site. See ‘Materials and Methods’ section for further details. ( B ) H3 C RNA 3′-end processing efficiency in Hela cells depleted of CF I m 68, CF I m 25 or Lsm11. The western blots show endogenous CF I m 68 or CF I m 25 detected with corresponding antibodies. SmB/B′ and β-actin served as loading controls. The RT–PCR shows Lsm11 mRNA detected in total RNA. ( C ) H3 C RNA 3′-end processing efficiency in Hela cells overexpressing CF I m 68. The western blots show endogenous and HA-tagged CF I m 68 detected with anti-CF I m 68 antibody. SmB/B′ served as loading control. The relative in vivo processing efficiency was calculated as ratio of pre-mRNA to total mRNA and then normalized with respect to the ratio obtained in cells treated with either control siRNA, Tcrβ-specific shRNA (B) or pcDNA-puro (C). The data shown represent means ± standard deviations. Number of independent measurements (CF I m 68 depletion, 5; CF I m 25 depletion, 3; Lsm11 depletion, 3; CF I m 68 overexpression, 4) p, significance values determined by Student’s t -test. ( D ) Northern blot detecting H3 C mRNA. Nuclear RNA from control cells or cells overexpressing HA-tagged CF I m 68 was separated by polyacrylamide electrophoresis and transferred to positively charged nylon membranes. Histone H3 C mRNA was detected with probes either hybridizing to the 5′ or the 3′-end of the mRNA. 18 S rRNA served as loading control. ( E ) Quantitation of mRNA levels (relative to U6 snRNA) for CF I m 68 (dark columns) or CF I m 25 (light columns) from cells depleted of CF I m 68 or CF I m 25 (number of depletions: 3). ( F ) Western blot detecting endogenous CF I m 68 or CF I m 25 from cells transfected with a control siRNA or depleted of CF I m 68 or CF I m 25. β-actin served as loading control. ( G ) Quantitation of CF I m 68 (dark columns) and CF I m 25 (light columns) protein levels, relative to β-actin, in cells depleted of CF I m 68 or CF I m 25 (number of evaluated blots: 2).

    Techniques Used: Over Expression, In Vivo, Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Reverse Transcription Polymerase Chain Reaction, shRNA, Northern Blot, Electrophoresis, Quantitation Assay, Transfection

    ( A ) Co-immunoprecipitation of histone H3 C pre-mRNA and total RNA with CF I m 68. The graph shows the ratio between pre-mRNA and total histone H3 C RNAs either in the input sample (light columns) or after co-immunprecipitation with anti-CF I m 68 (dark columns) as determined in three independent experiments. In Experiments 1 and 3 no RNA contamination was detected in the negative controls (BSA-coated beads), whereas for Experiment 2 the contamination is shown as grey column. ( B ) Co-immunoprecipitation of histone H3 C pre-mRNA (light columns), β-actin pre-mRNA (dark columns) and CF I m 68 mRNA with CF I m 25. An antibody against β-actin served as negative control. The cells were fixed with formaldehyde prior to total cell extract preparation and immunprecipitation. All precipitated transcripts were quantitated by qRT–PCR (number of experiments: two).
    Figure Legend Snippet: ( A ) Co-immunoprecipitation of histone H3 C pre-mRNA and total RNA with CF I m 68. The graph shows the ratio between pre-mRNA and total histone H3 C RNAs either in the input sample (light columns) or after co-immunprecipitation with anti-CF I m 68 (dark columns) as determined in three independent experiments. In Experiments 1 and 3 no RNA contamination was detected in the negative controls (BSA-coated beads), whereas for Experiment 2 the contamination is shown as grey column. ( B ) Co-immunoprecipitation of histone H3 C pre-mRNA (light columns), β-actin pre-mRNA (dark columns) and CF I m 68 mRNA with CF I m 25. An antibody against β-actin served as negative control. The cells were fixed with formaldehyde prior to total cell extract preparation and immunprecipitation. All precipitated transcripts were quantitated by qRT–PCR (number of experiments: two).

    Techniques Used: Immunoprecipitation, Negative Control, Quantitative RT-PCR

    5) Product Images from "A Neoglycoconjugate Containing the Human Milk Sugar LNFPIII Drives Anti-Inflammatory Activation of Antigen Presenting Cells in a CD14 Dependent Pathway"

    Article Title: A Neoglycoconjugate Containing the Human Milk Sugar LNFPIII Drives Anti-Inflammatory Activation of Antigen Presenting Cells in a CD14 Dependent Pathway

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0137495

    LNFPIII-NGC drives anti-inflammatory chemokine/cytokine production. a-b) BMDCs from WT and CD14-/- mice were stimulated with LPS (100ng/ml), LNFPIII-NGC (50μg/ml), Dex (50μg/ml) or nothing for 24 hrs at 37C. Supernatant from each well was collected and sent for RBM analysis. Statistical analysis was performed using GraphPad student’s unpaired t test method. c) BMDMs were treated with MEK1/2 inhibitor, U0126 or control inhibitor (U0124) for 30 minutes followed by stimulation with LPS (100ng/ml), LNFPIII-NGC (50μg/ml), Dex (50μg/ml) or nothing for 4 Hrs at 37°C. Cells were harvested, total RNA isolated and qRT-PCR was performed using respective primers (as mentioned). Statistical significance was measured using GraphPad student’s unpaired t test method.
    Figure Legend Snippet: LNFPIII-NGC drives anti-inflammatory chemokine/cytokine production. a-b) BMDCs from WT and CD14-/- mice were stimulated with LPS (100ng/ml), LNFPIII-NGC (50μg/ml), Dex (50μg/ml) or nothing for 24 hrs at 37C. Supernatant from each well was collected and sent for RBM analysis. Statistical analysis was performed using GraphPad student’s unpaired t test method. c) BMDMs were treated with MEK1/2 inhibitor, U0126 or control inhibitor (U0124) for 30 minutes followed by stimulation with LPS (100ng/ml), LNFPIII-NGC (50μg/ml), Dex (50μg/ml) or nothing for 4 Hrs at 37°C. Cells were harvested, total RNA isolated and qRT-PCR was performed using respective primers (as mentioned). Statistical significance was measured using GraphPad student’s unpaired t test method.

    Techniques Used: Mouse Assay, Isolation, Quantitative RT-PCR

    6) Product Images from "Expression Profiling of a Genetic Animal Model of Depression Reveals Novel Molecular Pathways Underlying Depressive-Like Behaviours"

    Article Title: Expression Profiling of a Genetic Animal Model of Depression Reveals Novel Molecular Pathways Underlying Depressive-Like Behaviours

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0012596

    Examples of real-time PCR data. Group Least Squares Means were calculated in Array Studio and expressed as copy number/10ng of total RNA for FRL cohort 1 (FRL1), FRL cohort 2 (FRL2), FSL cohort 1 (FSL1) and FSL cohort 2 (FSL2) in P/FC and HIP. Data included for TMEM176A (A), Pex11b (B), CA3 (C) and invariant gene PHPT1 (D). Error bars represent 95% confidence intervals.
    Figure Legend Snippet: Examples of real-time PCR data. Group Least Squares Means were calculated in Array Studio and expressed as copy number/10ng of total RNA for FRL cohort 1 (FRL1), FRL cohort 2 (FRL2), FSL cohort 1 (FSL1) and FSL cohort 2 (FSL2) in P/FC and HIP. Data included for TMEM176A (A), Pex11b (B), CA3 (C) and invariant gene PHPT1 (D). Error bars represent 95% confidence intervals.

    Techniques Used: Real-time Polymerase Chain Reaction

    7) Product Images from "Near Completely Humanized Liver in Mice Shows Human-Type Metabolic Responses to Drugs"

    Article Title: Near Completely Humanized Liver in Mice Shows Human-Type Metabolic Responses to Drugs

    Journal: The American Journal of Pathology

    doi:

    Demonstration of mouse liver chimerism. Histological serial sections were prepared from six liver lobes of each of 6 chimeric mice (donor, 12YM) and 19 chimeric mice (donor, 9MM), and stained with human genomic probes and anti-hCK8/18 antibodies. A: In situ hybridization of chimeric liver sections with human genomic DNA probes. Regions that contain hepatocytes with positive and negative nuclei are defined as human (H) and mouse (M) areas, respectively, the boundary being indicated by a dashed line . The RI of this chimeric mouse liver, which is calculated as the frequency of positive regions relative to that of the entire examined area in the sections, is 75%. B: CK8/18 immunostaining of a serial section of A . H and M indicate the regions that contain hepatocytes that are immunologically positive and negative, respectively, the boundary being indicated by a dashed line . The RI of this chimeric mouse liver, which is calculated as the frequency of immunopositive regions relative to that of the entire examined area in the sections, is given as RIImmunology = 77%, which is almost identical to the RI value calculated in A . C: High magnification of the region enclosed by the square in B . Bile duct cells, which are indicated with arrows , were negative for immunoreactivity. D: Chimerism determined by PCR. The hAlu sequence was amplified successfully from the liver genomic DNAs of human individuals (h, lane 6 ), and of chimeric mice with RI = 35% ( lane 3 ), RI = 53% ( lane 4 ), and RI = 77% ( lane 5 ), but not from the control uPA −/− /SCID +/+ mouse (cm −/− ; lane 1 ) or the control uPA +/+ /SCID +/+ mouse (cm +/+ ; lane 2 ). PCR products for mc-mos were amplified from the mouse livers ( lanes 1 and 2 ), but not from the human liver ( lane 6 ). Scale bars, 100 μm.
    Figure Legend Snippet: Demonstration of mouse liver chimerism. Histological serial sections were prepared from six liver lobes of each of 6 chimeric mice (donor, 12YM) and 19 chimeric mice (donor, 9MM), and stained with human genomic probes and anti-hCK8/18 antibodies. A: In situ hybridization of chimeric liver sections with human genomic DNA probes. Regions that contain hepatocytes with positive and negative nuclei are defined as human (H) and mouse (M) areas, respectively, the boundary being indicated by a dashed line . The RI of this chimeric mouse liver, which is calculated as the frequency of positive regions relative to that of the entire examined area in the sections, is 75%. B: CK8/18 immunostaining of a serial section of A . H and M indicate the regions that contain hepatocytes that are immunologically positive and negative, respectively, the boundary being indicated by a dashed line . The RI of this chimeric mouse liver, which is calculated as the frequency of immunopositive regions relative to that of the entire examined area in the sections, is given as RIImmunology = 77%, which is almost identical to the RI value calculated in A . C: High magnification of the region enclosed by the square in B . Bile duct cells, which are indicated with arrows , were negative for immunoreactivity. D: Chimerism determined by PCR. The hAlu sequence was amplified successfully from the liver genomic DNAs of human individuals (h, lane 6 ), and of chimeric mice with RI = 35% ( lane 3 ), RI = 53% ( lane 4 ), and RI = 77% ( lane 5 ), but not from the control uPA −/− /SCID +/+ mouse (cm −/− ; lane 1 ) or the control uPA +/+ /SCID +/+ mouse (cm +/+ ; lane 2 ). PCR products for mc-mos were amplified from the mouse livers ( lanes 1 and 2 ), but not from the human liver ( lane 6 ). Scale bars, 100 μm.

    Techniques Used: Mouse Assay, Staining, In Situ Hybridization, Immunostaining, Polymerase Chain Reaction, Sequencing, Amplification

    8) Product Images from "Uncovering Direct Targets of MiR-19a Involved in Lung Cancer Progression"

    Article Title: Uncovering Direct Targets of MiR-19a Involved in Lung Cancer Progression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0137887

    Pull-down assay using biotinylated miRNA. (A) Overview of the in vitro pull-down assay. The biotinylated double-stranded miR-19a or control random RNA was incubated in cell extract (step 1) to yield a complex of the biotinylated miRNA single strand with target mRNA and RISC (step 2). The biotinylated miRNA/target mRNA complex was incubated with streptavidin beads and pulled down (step 3). The complex was treated by DNase I and reverse-transcribed (step 4). (B) The expression of miR-19a target candidate mRNAs in HEK293 cells. PTEN , known as one of miR-19a target genes, was used as a positive control. The gene, whose sequence did not match with miR-19a seed sequences, was used as the negative control. (C) Confirmation of AGO2 protein in biotinylated miRNA/target mRNA complex by western blotting with the AGO2 antibody. Biotinylated miR-19a (lane 1), biotinylated control random RNA (lane 2), and biotin (lane 3) were used for the pull-down assay and subjected to SDS-polyacrylamide gel electrophoresis and western blotting. Total cell extract was used as the positive control (lane 4). (D) Detection of target mRNAs in biotinylated miRNA/target mRNA complex by real-time RT-PCR. The relative level of each target mRNA in the complex pulled down by using biotinylated miR-19a was compared to that of the complex pulled down by using the biotinylated control random RNA. **, p
    Figure Legend Snippet: Pull-down assay using biotinylated miRNA. (A) Overview of the in vitro pull-down assay. The biotinylated double-stranded miR-19a or control random RNA was incubated in cell extract (step 1) to yield a complex of the biotinylated miRNA single strand with target mRNA and RISC (step 2). The biotinylated miRNA/target mRNA complex was incubated with streptavidin beads and pulled down (step 3). The complex was treated by DNase I and reverse-transcribed (step 4). (B) The expression of miR-19a target candidate mRNAs in HEK293 cells. PTEN , known as one of miR-19a target genes, was used as a positive control. The gene, whose sequence did not match with miR-19a seed sequences, was used as the negative control. (C) Confirmation of AGO2 protein in biotinylated miRNA/target mRNA complex by western blotting with the AGO2 antibody. Biotinylated miR-19a (lane 1), biotinylated control random RNA (lane 2), and biotin (lane 3) were used for the pull-down assay and subjected to SDS-polyacrylamide gel electrophoresis and western blotting. Total cell extract was used as the positive control (lane 4). (D) Detection of target mRNAs in biotinylated miRNA/target mRNA complex by real-time RT-PCR. The relative level of each target mRNA in the complex pulled down by using biotinylated miR-19a was compared to that of the complex pulled down by using the biotinylated control random RNA. **, p

    Techniques Used: Pull Down Assay, In Vitro, Incubation, Expressing, Positive Control, Sequencing, Negative Control, Western Blot, Polyacrylamide Gel Electrophoresis, Quantitative RT-PCR

    9) Product Images from "Pathogenesis of Hepatitis E Virus and Hepatitis C Virus in Chimpanzees: Similarities and Differences ▿Pathogenesis of Hepatitis E Virus and Hepatitis C Virus in Chimpanzees: Similarities and Differences ▿ †"

    Article Title: Pathogenesis of Hepatitis E Virus and Hepatitis C Virus in Chimpanzees: Similarities and Differences ▿Pathogenesis of Hepatitis E Virus and Hepatitis C Virus in Chimpanzees: Similarities and Differences ▿ †

    Journal: Journal of Virology

    doi: 10.1128/JVI.01205-10

    qRT-PCR of IFN-γ in chimpanzees infected with HEV or HCV. IFN-γ was tested by primer- and probe-specific TaqMan qPCR (see Table S1 in the supplemental material). Total RNA from liver biopsies was converted to cDNA, amplified, and tested
    Figure Legend Snippet: qRT-PCR of IFN-γ in chimpanzees infected with HEV or HCV. IFN-γ was tested by primer- and probe-specific TaqMan qPCR (see Table S1 in the supplemental material). Total RNA from liver biopsies was converted to cDNA, amplified, and tested

    Techniques Used: Quantitative RT-PCR, Infection, Real-time Polymerase Chain Reaction, Amplification

    10) Product Images from "Liver and Biliary Tract Physiology/Pathophysiology: Intestinal bile acid sequestration improves glucose control by stimulating hepatic miR-182-5p in type 2 diabetes"

    Article Title: Liver and Biliary Tract Physiology/Pathophysiology: Intestinal bile acid sequestration improves glucose control by stimulating hepatic miR-182-5p in type 2 diabetes

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    doi: 10.1152/ajpgi.00238.2018

    The microRNA (miR)-96/182/183 cluster is upregulated in the livers of colesevelam-treated Zucker diabetic fatty (ZDF) rats. A : volcano plot depicting significantly altered miRNAs between colesevelam (Col) and vehicle-treated ZDF rat livers ( left ) and expression (reads per million total reads) of the miR-96/182/183 cluster ( left ); n = 6. B–D : liver expression of the miR-96/182/183 cluster by real-time PCR; n = 9–12. E : liver gene (mRNA) expression changes of Srebf2 and its target genes Hmgcr , Ldlr , and Sqle ; n = 8–12. For sequencing data, unpaired t -tests were used. For comparisons between 2 groups, Mann-Whitney nonparametric tests were used; n.d., not detectable.
    Figure Legend Snippet: The microRNA (miR)-96/182/183 cluster is upregulated in the livers of colesevelam-treated Zucker diabetic fatty (ZDF) rats. A : volcano plot depicting significantly altered miRNAs between colesevelam (Col) and vehicle-treated ZDF rat livers ( left ) and expression (reads per million total reads) of the miR-96/182/183 cluster ( left ); n = 6. B–D : liver expression of the miR-96/182/183 cluster by real-time PCR; n = 9–12. E : liver gene (mRNA) expression changes of Srebf2 and its target genes Hmgcr , Ldlr , and Sqle ; n = 8–12. For sequencing data, unpaired t -tests were used. For comparisons between 2 groups, Mann-Whitney nonparametric tests were used; n.d., not detectable.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Sequencing, MANN-WHITNEY

    Mediator complex subunit 1 (Med1) is regulated by microRNA (miR)-96/182/183 in mice. A : expression of Med1 by real-time PCR in mouse primary hepatocytes transfected with 50 nM of Med1 or scramble siRNA; n = 3 B : Western blot of Med1 and β-actin (loading control) from protein samples of primary hepatocytes transfected with Med1 or scramble siRNA. Ratio of Med1/β-actin densitometry is indicated between the blots and graphed on the right ; n = 3. A.U., arbitrary units. C : hepatic Med1 mRNA levels in db/db mice treated with vehicle or 2% colesevelam (Col)-supplemented diet and PBS or locked-nucleic acid (LNA)-182-5p; real-time PCR; n = 8–9. D : hepatic Med1 and β-actin (loading control) protein levels from 3 representative liver protein samples for Chow + PBS, Col + PBS, and Col + LNA-182. Western blotting. Ratio of Med1/β-actin densitometry is indicated between the blots. E : densitometry quantification of Med1 protein levels normalized to β-actin; n = 8–9. F : expression of miR-96-5p, miR-182-5p, and miR-183-5p in mouse primary hepatocytes transfected with 50 nM miR-96-5p, miR-182-5p, miR-183-5p mimics individually or in combination; real-time PCR; n = 3. G : Med1 mRNA levels in mouse primary hepatocytes transfected with 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination; real-time PCR; n = 3. H : protein levels of Med1 and β-actin (loading control) from protein samples of primary hepatocytes transfected with mock or 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination. Ratio of Med1/β-actin densitometry is indicated between the blots and graphed on the right ; n = 3. For comparisons between 2 groups, Students’ t- tests were used, and for more than 2 groups, Kruskal-Wallis one-way ANOVA with Dunn’s posttest was used (α = 0.05) or one-way ANOVA with Bonferonni’s posttest for comparison to mock group only was used (α = 0.05).
    Figure Legend Snippet: Mediator complex subunit 1 (Med1) is regulated by microRNA (miR)-96/182/183 in mice. A : expression of Med1 by real-time PCR in mouse primary hepatocytes transfected with 50 nM of Med1 or scramble siRNA; n = 3 B : Western blot of Med1 and β-actin (loading control) from protein samples of primary hepatocytes transfected with Med1 or scramble siRNA. Ratio of Med1/β-actin densitometry is indicated between the blots and graphed on the right ; n = 3. A.U., arbitrary units. C : hepatic Med1 mRNA levels in db/db mice treated with vehicle or 2% colesevelam (Col)-supplemented diet and PBS or locked-nucleic acid (LNA)-182-5p; real-time PCR; n = 8–9. D : hepatic Med1 and β-actin (loading control) protein levels from 3 representative liver protein samples for Chow + PBS, Col + PBS, and Col + LNA-182. Western blotting. Ratio of Med1/β-actin densitometry is indicated between the blots. E : densitometry quantification of Med1 protein levels normalized to β-actin; n = 8–9. F : expression of miR-96-5p, miR-182-5p, and miR-183-5p in mouse primary hepatocytes transfected with 50 nM miR-96-5p, miR-182-5p, miR-183-5p mimics individually or in combination; real-time PCR; n = 3. G : Med1 mRNA levels in mouse primary hepatocytes transfected with 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination; real-time PCR; n = 3. H : protein levels of Med1 and β-actin (loading control) from protein samples of primary hepatocytes transfected with mock or 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination. Ratio of Med1/β-actin densitometry is indicated between the blots and graphed on the right ; n = 3. For comparisons between 2 groups, Students’ t- tests were used, and for more than 2 groups, Kruskal-Wallis one-way ANOVA with Dunn’s posttest was used (α = 0.05) or one-way ANOVA with Bonferonni’s posttest for comparison to mock group only was used (α = 0.05).

    Techniques Used: Mouse Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Western Blot

    Mediator complex subunit 1 (MED1) is a direct target of microRNA (miR)-182-5p, miR-183-5p, and miR-96-5p in humans. A : schematic of human MED1 mRNA and the 3 putative miR-182/183/96 target sites in the 3′-untranslated region (3′-UTR). B : predicted MED1 3′-UTR target sites. Bases highlighted in dark gray represent mutations for gene reporter (luciferase) assays. C : normalized luciferase activity in HEK293 cells after dual transfection of miR-96/182/183 mimics and gene (luciferase) reporters harboring putative target sites for miR-96-5p, miR-182-5p, and/or miR-183-5p; n = 4. D : miR-96-5p, miR-182-5p, and miR-183-5p levels in Huh7 cells transfected with 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination; real-time PCR; n = 6. E : MED1 mRNA levels in Huh7 cells transfected with 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination; real-time PCR; n = 6. F : miR-96-5p, miR-182-5p, and miR-183-5p levels in Huh7 cells transfected with mock or 50 nM locked-nucleic acids (LNAs) against miR-182-5p and/or miR-183-5p; real-time PCR; n = 6. G : MED1 mRNA levels in Huh7 cells transfected mock or 50 nM LNAs against miR-182-5p and/or miR-183-5p; real-time PCR; n = 6. H : protein levels of Med1 and β-actin (loading control) from protein lysates of Huh7 cells transfected with mock or 50 nM miR-96, miR-182, and miR-183 LNAs. Ratio of Med1/β-actin densitometry is indicated between the blots and graphed on the right ; n = 6. A.U. arbitrary units. For comparisons between more than 2 groups to mock only, one-way ANOVA with Bonferonni’s posttest was used, α = 0.05. For comparisons between 2 groups, Mann-Whitney nonparametric tests were used.
    Figure Legend Snippet: Mediator complex subunit 1 (MED1) is a direct target of microRNA (miR)-182-5p, miR-183-5p, and miR-96-5p in humans. A : schematic of human MED1 mRNA and the 3 putative miR-182/183/96 target sites in the 3′-untranslated region (3′-UTR). B : predicted MED1 3′-UTR target sites. Bases highlighted in dark gray represent mutations for gene reporter (luciferase) assays. C : normalized luciferase activity in HEK293 cells after dual transfection of miR-96/182/183 mimics and gene (luciferase) reporters harboring putative target sites for miR-96-5p, miR-182-5p, and/or miR-183-5p; n = 4. D : miR-96-5p, miR-182-5p, and miR-183-5p levels in Huh7 cells transfected with 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination; real-time PCR; n = 6. E : MED1 mRNA levels in Huh7 cells transfected with 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination; real-time PCR; n = 6. F : miR-96-5p, miR-182-5p, and miR-183-5p levels in Huh7 cells transfected with mock or 50 nM locked-nucleic acids (LNAs) against miR-182-5p and/or miR-183-5p; real-time PCR; n = 6. G : MED1 mRNA levels in Huh7 cells transfected mock or 50 nM LNAs against miR-182-5p and/or miR-183-5p; real-time PCR; n = 6. H : protein levels of Med1 and β-actin (loading control) from protein lysates of Huh7 cells transfected with mock or 50 nM miR-96, miR-182, and miR-183 LNAs. Ratio of Med1/β-actin densitometry is indicated between the blots and graphed on the right ; n = 6. A.U. arbitrary units. For comparisons between more than 2 groups to mock only, one-way ANOVA with Bonferonni’s posttest was used, α = 0.05. For comparisons between 2 groups, Mann-Whitney nonparametric tests were used.

    Techniques Used: Luciferase, Activity Assay, Transfection, Real-time Polymerase Chain Reaction, MANN-WHITNEY

    11) Product Images from "Curcumin modulates dopaminergic receptor, CREB and phospholipase c gene expression in the cerebral cortex and cerebellum of streptozotocin induced diabetic rats"

    Article Title: Curcumin modulates dopaminergic receptor, CREB and phospholipase c gene expression in the cerebral cortex and cerebellum of streptozotocin induced diabetic rats

    Journal: Journal of Biomedical Science

    doi: 10.1186/1423-0127-17-43

    Real Time PCR amplification of CREB mRNA from the cerebral cortex of control and experimental rats . Values are mean ± S.D of 4-6 separate experiments. Each group consist of 6-8 rats Relative Quantification values and standard deviations are shown in the table. The relative ratios of mRNA levels were calculated using the ΔΔ CT method normalized with β-actin CT value as the internal control and Control CT value as the calibrator. a p
    Figure Legend Snippet: Real Time PCR amplification of CREB mRNA from the cerebral cortex of control and experimental rats . Values are mean ± S.D of 4-6 separate experiments. Each group consist of 6-8 rats Relative Quantification values and standard deviations are shown in the table. The relative ratios of mRNA levels were calculated using the ΔΔ CT method normalized with β-actin CT value as the internal control and Control CT value as the calibrator. a p

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification

    Real Time PCR amplification of phospholipase C mRNA from the cerebellumof control and experimental rats . Values are mean ± S.D of 4-6 separate experiments. Each group consist of 6-8 rats Relative Quantification values and standard deviations are shown in the table. The relative ratios of mRNA levels were calculated using the ΔΔ CT method normalized with β-actin CT value as the internal control and Control CT value as the calibrator. a p
    Figure Legend Snippet: Real Time PCR amplification of phospholipase C mRNA from the cerebellumof control and experimental rats . Values are mean ± S.D of 4-6 separate experiments. Each group consist of 6-8 rats Relative Quantification values and standard deviations are shown in the table. The relative ratios of mRNA levels were calculated using the ΔΔ CT method normalized with β-actin CT value as the internal control and Control CT value as the calibrator. a p

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification

    Real Time PCR amplification of phospholipase C mRNA from the cerebral cortex of control and experimental rats . Values are mean ± S.D of 4-6 separate experiments. Each group consist of 6-8 rats Relative Quantification values and standard deviations are shown in the table. The relative ratios of mRNA levels were calculated using the ΔΔ CT method normalized with β-actin CT value as the internal control and Control CT value as the calibrator. a p
    Figure Legend Snippet: Real Time PCR amplification of phospholipase C mRNA from the cerebral cortex of control and experimental rats . Values are mean ± S.D of 4-6 separate experiments. Each group consist of 6-8 rats Relative Quantification values and standard deviations are shown in the table. The relative ratios of mRNA levels were calculated using the ΔΔ CT method normalized with β-actin CT value as the internal control and Control CT value as the calibrator. a p

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification

    Real Time PCR amplification of dopamine D1 mRNA from the cerebellum of control and experimental rats . Values are mean ± S.D of 4-6 separate experiments. Each group consist of 6-8 rats Relative Quantification values and standard deviations are shown in the table. The relative ratios of mRNA levels were calculated using the ΔΔ CT method normalized with β-actin CT value as the internal control and Control CT value as the calibrator. a p
    Figure Legend Snippet: Real Time PCR amplification of dopamine D1 mRNA from the cerebellum of control and experimental rats . Values are mean ± S.D of 4-6 separate experiments. Each group consist of 6-8 rats Relative Quantification values and standard deviations are shown in the table. The relative ratios of mRNA levels were calculated using the ΔΔ CT method normalized with β-actin CT value as the internal control and Control CT value as the calibrator. a p

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification

    Real Time PCR amplification of dopamine D2 mRNA from the cerebral cortex of control and experimental rats . Values are mean ± S.D of 4-6 separate experiments. Each group consist of 6-8 rats Relative Quantification values and standard deviations are shown in the table. The relative ratios of mRNA levels were calculated using the ΔΔ CT method normalized with β-actin CT value as the internal control and Control CT value as the calibrator. a p
    Figure Legend Snippet: Real Time PCR amplification of dopamine D2 mRNA from the cerebral cortex of control and experimental rats . Values are mean ± S.D of 4-6 separate experiments. Each group consist of 6-8 rats Relative Quantification values and standard deviations are shown in the table. The relative ratios of mRNA levels were calculated using the ΔΔ CT method normalized with β-actin CT value as the internal control and Control CT value as the calibrator. a p

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification

    Real Time PCR amplification of dopamine D2 mRNA from the cerebellum of control and experimental rats . Values are mean ± S.D of 4-6 separate experiments. Each group consist of 6-8 rats Relative Quantification values and standard deviations are shown in the table. The relative ratios of mRNA levels were calculated using the ΔΔ CT method normalized with β-actin CT value as the internal control and Control CT value as the calibrator. a p
    Figure Legend Snippet: Real Time PCR amplification of dopamine D2 mRNA from the cerebellum of control and experimental rats . Values are mean ± S.D of 4-6 separate experiments. Each group consist of 6-8 rats Relative Quantification values and standard deviations are shown in the table. The relative ratios of mRNA levels were calculated using the ΔΔ CT method normalized with β-actin CT value as the internal control and Control CT value as the calibrator. a p

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification

    Real Time PCR amplification of CREB mRNA from the cerebellum of control and experimental rats . Values are mean ± S.D of 4-6 separate experiments. Each group consist of 6-8 rats Relative Quantification values and standard deviations are shown in the table. The relative ratios of mRNA levels were calculated using the ΔΔ CT method normalized with β-actin CT value as the internal control and Control CT value as the calibrator. a p
    Figure Legend Snippet: Real Time PCR amplification of CREB mRNA from the cerebellum of control and experimental rats . Values are mean ± S.D of 4-6 separate experiments. Each group consist of 6-8 rats Relative Quantification values and standard deviations are shown in the table. The relative ratios of mRNA levels were calculated using the ΔΔ CT method normalized with β-actin CT value as the internal control and Control CT value as the calibrator. a p

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification

    Real Time PCR amplification of dopamine D1 receptor mRNA from the cerebral cortex of control and experimental rats . Values are mean ± S.D of 4-6 separate experiments. Each group consist of 6-8 rats Relative Quantification values and standard deviations are shown in the table. The relative ratios of mRNA levels were calculated using the ΔΔ CT method normalized with β-actin CT value as the internal control and Control CT value as the calibrator. a p
    Figure Legend Snippet: Real Time PCR amplification of dopamine D1 receptor mRNA from the cerebral cortex of control and experimental rats . Values are mean ± S.D of 4-6 separate experiments. Each group consist of 6-8 rats Relative Quantification values and standard deviations are shown in the table. The relative ratios of mRNA levels were calculated using the ΔΔ CT method normalized with β-actin CT value as the internal control and Control CT value as the calibrator. a p

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification

    12) Product Images from "Differentiation Therapy for Hepatocellular Carcinoma: multifaceted effects of miR-148a on tumor growth and phenotype and liver fibrosis"

    Article Title: Differentiation Therapy for Hepatocellular Carcinoma: multifaceted effects of miR-148a on tumor growth and phenotype and liver fibrosis

    Journal: Hepatology (Baltimore, Md.)

    doi: 10.1002/hep.28367

    Overexpression of miR-148a accelerates hepatocytic differentiation of HepaRG cells. (A) Expression of selected miRNAs was determined by quantitative PCR in HepaRG-Empty and individual HepaRG-miRNA clones (miR-18a, miR-29c, miR-93, miR-101, miR-148a, miR-150
    Figure Legend Snippet: Overexpression of miR-148a accelerates hepatocytic differentiation of HepaRG cells. (A) Expression of selected miRNAs was determined by quantitative PCR in HepaRG-Empty and individual HepaRG-miRNA clones (miR-18a, miR-29c, miR-93, miR-101, miR-148a, miR-150

    Techniques Used: Over Expression, Expressing, Real-time Polymerase Chain Reaction, Clone Assay

    13) Product Images from "Suppression of microRNA-31 increases sensitivity to 5-FU at an early stage, and affects cell migration and invasion in HCT-116 colon cancer cells"

    Article Title: Suppression of microRNA-31 increases sensitivity to 5-FU at an early stage, and affects cell migration and invasion in HCT-116 colon cancer cells

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-10-616

    Suppression of miR-31 expression by anti-miR-31 as detected by TaqMan real-time PCR . Compared with negative control, miR-31 was reduced to 44.1% in HCT-116 p53+/+ ( p = 0.042) and 67.8% in HCT-116 p53-/- ( p = 0.046) cell line.
    Figure Legend Snippet: Suppression of miR-31 expression by anti-miR-31 as detected by TaqMan real-time PCR . Compared with negative control, miR-31 was reduced to 44.1% in HCT-116 p53+/+ ( p = 0.042) and 67.8% in HCT-116 p53-/- ( p = 0.046) cell line.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Negative Control

    14) Product Images from "A Role for RUNX3 in Inflammation-Induced Expression of IL23A in Gastric Epithelial Cells"

    Article Title: A Role for RUNX3 in Inflammation-Induced Expression of IL23A in Gastric Epithelial Cells

    Journal: Cell reports

    doi: 10.1016/j.celrep.2014.06.003

    IL23A Is Transcriptionally Regulated by RUNX3 in Gastric Epithelial Cells (A) IL23A mRNA expression was induced by exogenous RUNX3 in multiple RUNX3-negative gastric cancer cell lines. GFP-positive transfected cells were enriched by FACS at 24 hr and 48 hr posttransfection and analyzed by qRT-PCR. Normalized IL23A levels are expressed relative to untransfected control values. (B) RUNX3 specifically induced IL23A in gastric epithelial cells. AGS cells transduced with the indicated viruses were analyzed by qRT-PCR for the expression of the IL-12 family of cytokine genes. Normalized data are presented relative to the Lenti-control sample (mean ± SEM; n = 3) (u.d., undetected). (C) RUNX3 mediates its effect through the proximal RUNX sites B, C, and D of the IL23A promoter. Mutation and deletion variants of the IL23A-1200 reporter construct were transiently transfected into KATOIII cells together with either control or RUNX3 expression vectors. Normalized luciferase activities are expressed relative to the values of control samples for each construct. Data presented are derived from independent biological triplicates (mean ± SEM). (D) Physical occupancy of RUNX3 on the IL23A promoter in Lenti-RUNX3-transduced AGS cells was detected by ChIP analysis using polyclonal or monoclonal RUNX3-specific antibodies. Enrichment of the indicated genomic fragments was detected by qPCR ( Table S1 ) and expressed relative to input DNA. Nonspecific immunoglobulin G from rabbit (rIgG) and mouse (mIgG) served as negative controls; H3K9Ace antibody was used as a positive control. NS, nonspecific region; pAb, polyclonal antibody; mAbs, monoclonal antibodies. *p
    Figure Legend Snippet: IL23A Is Transcriptionally Regulated by RUNX3 in Gastric Epithelial Cells (A) IL23A mRNA expression was induced by exogenous RUNX3 in multiple RUNX3-negative gastric cancer cell lines. GFP-positive transfected cells were enriched by FACS at 24 hr and 48 hr posttransfection and analyzed by qRT-PCR. Normalized IL23A levels are expressed relative to untransfected control values. (B) RUNX3 specifically induced IL23A in gastric epithelial cells. AGS cells transduced with the indicated viruses were analyzed by qRT-PCR for the expression of the IL-12 family of cytokine genes. Normalized data are presented relative to the Lenti-control sample (mean ± SEM; n = 3) (u.d., undetected). (C) RUNX3 mediates its effect through the proximal RUNX sites B, C, and D of the IL23A promoter. Mutation and deletion variants of the IL23A-1200 reporter construct were transiently transfected into KATOIII cells together with either control or RUNX3 expression vectors. Normalized luciferase activities are expressed relative to the values of control samples for each construct. Data presented are derived from independent biological triplicates (mean ± SEM). (D) Physical occupancy of RUNX3 on the IL23A promoter in Lenti-RUNX3-transduced AGS cells was detected by ChIP analysis using polyclonal or monoclonal RUNX3-specific antibodies. Enrichment of the indicated genomic fragments was detected by qPCR ( Table S1 ) and expressed relative to input DNA. Nonspecific immunoglobulin G from rabbit (rIgG) and mouse (mIgG) served as negative controls; H3K9Ace antibody was used as a positive control. NS, nonspecific region; pAb, polyclonal antibody; mAbs, monoclonal antibodies. *p

    Techniques Used: Expressing, Transfection, FACS, Quantitative RT-PCR, Transduction, Mutagenesis, Construct, Luciferase, Derivative Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control

    15) Product Images from "Grainyhead-like 2 Promotes Tumor Growth and is Associated with Poor Prognosis in Colorectal Cancer"

    Article Title: Grainyhead-like 2 Promotes Tumor Growth and is Associated with Poor Prognosis in Colorectal Cancer

    Journal: Journal of Cancer

    doi: 10.7150/jca.10969

    GRHL2 expression was increased in colorectal cancer (CRC) tissues and varied in CRC cell lines. (A) GRHL2-negative normal mucosa epithelium adjacent to carcinoma. (B) Nuclear staining of GRHL2 in normal epithelium adjacent to cancer. (C) GRHL2-negative cancer tissue. (D) Moderate positive nuclear stain for GRHL2 in cancer tissue. (E-F) Strong nuclear staining for GRHL2 in CRC tissue. The scale bar indicates 100 um. Original Magnification: × 200 (A-D, F), × 100 (E). (G) The expression of GRHL2 gene transcripts in a panel of human CRC cell lines were detected by qRT-PCR. The relative amount of mRNA was normalized using GAPDH as endogenous control. (H) GRHL2 protein levels were detected by western blot, with GAPDH as control.
    Figure Legend Snippet: GRHL2 expression was increased in colorectal cancer (CRC) tissues and varied in CRC cell lines. (A) GRHL2-negative normal mucosa epithelium adjacent to carcinoma. (B) Nuclear staining of GRHL2 in normal epithelium adjacent to cancer. (C) GRHL2-negative cancer tissue. (D) Moderate positive nuclear stain for GRHL2 in cancer tissue. (E-F) Strong nuclear staining for GRHL2 in CRC tissue. The scale bar indicates 100 um. Original Magnification: × 200 (A-D, F), × 100 (E). (G) The expression of GRHL2 gene transcripts in a panel of human CRC cell lines were detected by qRT-PCR. The relative amount of mRNA was normalized using GAPDH as endogenous control. (H) GRHL2 protein levels were detected by western blot, with GAPDH as control.

    Techniques Used: Expressing, Staining, Quantitative RT-PCR, Western Blot

    Over-expression of GRHL2 promoted proliferation of CRC cell lines in vitro . (A) A qRT-PCR analysis of GRHL2 gene expression revealed a striking up-regulation of exogenous GRHL2 expression in HT29 (HT29/GRHL2+) and SW620 (SW620/GRHL2+) cells compared to Parental and Vector cells. (B) In HT29/GRHL+ and SW620/GRHL2 cells, there were a marked increase in the expression of GRHL2, verified by western blot. Immunoblotting results are typical blots from 3 experiments. Densitometric analysis was expressed relative to the loading control, GAPDH. (C) Effect of GRHL2 on cellular proliferation was measured by CCK-8 assay. HT29/GRHL+ and SW620/GRHL2 cells showed higher proliferation than control cells. (D) GRHL2 over-expression resulted in a significant increase in colony formation in GRHL2+ cell. (E) Representative charts for cell-cycle distribution in Vector and GRHL2 cells. Results are mean ± SD (3-5 experiments). *Relative to Vector control; #relative to Parental control. * p
    Figure Legend Snippet: Over-expression of GRHL2 promoted proliferation of CRC cell lines in vitro . (A) A qRT-PCR analysis of GRHL2 gene expression revealed a striking up-regulation of exogenous GRHL2 expression in HT29 (HT29/GRHL2+) and SW620 (SW620/GRHL2+) cells compared to Parental and Vector cells. (B) In HT29/GRHL+ and SW620/GRHL2 cells, there were a marked increase in the expression of GRHL2, verified by western blot. Immunoblotting results are typical blots from 3 experiments. Densitometric analysis was expressed relative to the loading control, GAPDH. (C) Effect of GRHL2 on cellular proliferation was measured by CCK-8 assay. HT29/GRHL+ and SW620/GRHL2 cells showed higher proliferation than control cells. (D) GRHL2 over-expression resulted in a significant increase in colony formation in GRHL2+ cell. (E) Representative charts for cell-cycle distribution in Vector and GRHL2 cells. Results are mean ± SD (3-5 experiments). *Relative to Vector control; #relative to Parental control. * p

    Techniques Used: Over Expression, In Vitro, Quantitative RT-PCR, Expressing, Plasmid Preparation, Western Blot, CCK-8 Assay

    16) Product Images from "Combination Effects of Salvianolic Acid B with Low Dose Celecoxib on Inhibition of Head and Neck Squamous Cell Carcinoma Growth in vitro and in vivo"

    Article Title: Combination Effects of Salvianolic Acid B with Low Dose Celecoxib on Inhibition of Head and Neck Squamous Cell Carcinoma Growth in vitro and in vivo

    Journal: Cancer prevention research (Philadelphia, Pa.)

    doi: 10.1158/1940-6207.CAPR-09-0243

    Sal-B combined with celecoxib on inhibition COX-2 expression Fig. 4A, the top images is COX-2 protein were evaluated by Western Blot after treatment of JHU-013 and JHU-022 cells with Sal-B and celecoxib, alone or in combination. The bottom images is COX-2 messages RNA expression were measured by RT-PCR. Fig. 4B, COX-2 protein levels were analyzed by flow cytometry with FITC-conjugated COX-2 antibody after treatment of JHU-013 cells with Sal-B and celecoxib, alone or in combination. Fig. 4C, An enzyme immunoassay was used to determine the levels of PGE2 in JHU-013 cells treated with Sal-B (100 μM), celecoxib (10 μM) and combination (50 μM of Sal-B plus 5 μM of celecoxib). The p value was compared to untreated group. (*p
    Figure Legend Snippet: Sal-B combined with celecoxib on inhibition COX-2 expression Fig. 4A, the top images is COX-2 protein were evaluated by Western Blot after treatment of JHU-013 and JHU-022 cells with Sal-B and celecoxib, alone or in combination. The bottom images is COX-2 messages RNA expression were measured by RT-PCR. Fig. 4B, COX-2 protein levels were analyzed by flow cytometry with FITC-conjugated COX-2 antibody after treatment of JHU-013 cells with Sal-B and celecoxib, alone or in combination. Fig. 4C, An enzyme immunoassay was used to determine the levels of PGE2 in JHU-013 cells treated with Sal-B (100 μM), celecoxib (10 μM) and combination (50 μM of Sal-B plus 5 μM of celecoxib). The p value was compared to untreated group. (*p

    Techniques Used: Inhibition, Expressing, Western Blot, RNA Expression, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    17) Product Images from "Cell Size Correlates with Phenotype and Proliferative Capacity in Human Corneal Epithelial Cells"

    Article Title: Cell Size Correlates with Phenotype and Proliferative Capacity in Human Corneal Epithelial Cells

    Journal: Stem cells (Dayton, Ohio)

    doi: 10.1634/stemcells.2005-0148

    (A): Representative semiquantitative reverse transcription–polymerase chain reaction (RT-PCR) profiles showing mRNA expression of ΔNp63, ABCG2, involucrin, K12, and K3, with GAPDH as an internal control in the size-selected populations (A-D) of primary cultured human corneal epithelial cells sorted from the smallest (A) to the largest (D) cells. (B, C) : Real-time PCR analysis showing the relatively quantitative expression of ABCG2, ΔNp63, K3, K12, and Involucrin mRNA in the size-selected populations (A-D) of primary cultured human corneal epithelial cells sorted from the smallest (A) to the largest (D) cells. The comparative C T method was used to determine the fold of the targeted gene expression normalized by internal control GAPDH. Data are the mean ± standard deviation of results with mRNA levels in population A as a reference (equal to 1) in three experiments. * p
    Figure Legend Snippet: (A): Representative semiquantitative reverse transcription–polymerase chain reaction (RT-PCR) profiles showing mRNA expression of ΔNp63, ABCG2, involucrin, K12, and K3, with GAPDH as an internal control in the size-selected populations (A-D) of primary cultured human corneal epithelial cells sorted from the smallest (A) to the largest (D) cells. (B, C) : Real-time PCR analysis showing the relatively quantitative expression of ABCG2, ΔNp63, K3, K12, and Involucrin mRNA in the size-selected populations (A-D) of primary cultured human corneal epithelial cells sorted from the smallest (A) to the largest (D) cells. The comparative C T method was used to determine the fold of the targeted gene expression normalized by internal control GAPDH. Data are the mean ± standard deviation of results with mRNA levels in population A as a reference (equal to 1) in three experiments. * p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Standard Deviation

    18) Product Images from "Glucocorticoids and Tumor Necrosis Factor Alpha Cooperatively Regulate Toll-Like Receptor 2 Gene Expression"

    Article Title: Glucocorticoids and Tumor Necrosis Factor Alpha Cooperatively Regulate Toll-Like Receptor 2 Gene Expression

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.11.4743-4756.2004

    Dex induces MKP-1. (A) MKP-1 mRNA was measured by real-time quantitative reverse transcription-PCR in A549 cells after 8 h of treatment with the indicated concentrations of Dex. Asterisks denote a significant increase between untreated and Dex-treated cultures as determined by Tukey-Kramer pair comparison analysis ( P
    Figure Legend Snippet: Dex induces MKP-1. (A) MKP-1 mRNA was measured by real-time quantitative reverse transcription-PCR in A549 cells after 8 h of treatment with the indicated concentrations of Dex. Asterisks denote a significant increase between untreated and Dex-treated cultures as determined by Tukey-Kramer pair comparison analysis ( P

    Techniques Used: Polymerase Chain Reaction

    19) Product Images from "A Novel Microplate 3D Bioprinting Platform for the Engineering of Muscle and Tendon Tissues"

    Article Title: A Novel Microplate 3D Bioprinting Platform for the Engineering of Muscle and Tendon Tissues

    Journal: Slas Technology

    doi: 10.1177/2472630318776594

    Marker gene analysis of dumbbell-shaped standard muscle and tendon monoculture tissue model differentiation. ( A–D ) Muscle models and ( E ) tendon models were cultured in proliferation medium (PM, gray bars) or differentiation medium (DM, black bars) and were analyzed for corresponding marker gene expressions at different time points as indicated in the figure by quantitative PCR. In graphs A to D , data points at day 7 of proliferation models are not available. Bars at day 0 in graph E have a relative expression of 1 and are not visible in the graph. Three tissue models were analyzed per time point ( n = 3), except in day 4 muscle proliferation models ( n = 2) in graphs A to D . Shown are relative expression mean ± standard error of the mean (SEM).
    Figure Legend Snippet: Marker gene analysis of dumbbell-shaped standard muscle and tendon monoculture tissue model differentiation. ( A–D ) Muscle models and ( E ) tendon models were cultured in proliferation medium (PM, gray bars) or differentiation medium (DM, black bars) and were analyzed for corresponding marker gene expressions at different time points as indicated in the figure by quantitative PCR. In graphs A to D , data points at day 7 of proliferation models are not available. Bars at day 0 in graph E have a relative expression of 1 and are not visible in the graph. Three tissue models were analyzed per time point ( n = 3), except in day 4 muscle proliferation models ( n = 2) in graphs A to D . Shown are relative expression mean ± standard error of the mean (SEM).

    Techniques Used: Marker, Cell Culture, Real-time Polymerase Chain Reaction, Expressing

    20) Product Images from "A Novel Microplate 3D Bioprinting Platform for the Engineering of Muscle and Tendon Tissues"

    Article Title: A Novel Microplate 3D Bioprinting Platform for the Engineering of Muscle and Tendon Tissues

    Journal: Slas Technology

    doi: 10.1177/2472630318776594

    Marker gene analysis of dumbbell-shaped standard muscle and tendon monoculture tissue model differentiation. ( A–D ) Muscle models and ( E ) tendon models were cultured in proliferation medium (PM, gray bars) or differentiation medium (DM, black bars) and were analyzed for corresponding marker gene expressions at different time points as indicated in the figure by quantitative PCR. In graphs A to D , data points at day 7 of proliferation models are not available. Bars at day 0 in graph E have a relative expression of 1 and are not visible in the graph. Three tissue models were analyzed per time point ( n = 3), except in day 4 muscle proliferation models ( n = 2) in graphs A to D . Shown are relative expression mean ± standard error of the mean (SEM).
    Figure Legend Snippet: Marker gene analysis of dumbbell-shaped standard muscle and tendon monoculture tissue model differentiation. ( A–D ) Muscle models and ( E ) tendon models were cultured in proliferation medium (PM, gray bars) or differentiation medium (DM, black bars) and were analyzed for corresponding marker gene expressions at different time points as indicated in the figure by quantitative PCR. In graphs A to D , data points at day 7 of proliferation models are not available. Bars at day 0 in graph E have a relative expression of 1 and are not visible in the graph. Three tissue models were analyzed per time point ( n = 3), except in day 4 muscle proliferation models ( n = 2) in graphs A to D . Shown are relative expression mean ± standard error of the mean (SEM).

    Techniques Used: Marker, Cell Culture, Real-time Polymerase Chain Reaction, Expressing

    21) Product Images from "Thyroid hormone receptor ? is a molecular switch of cardiac function between fetal and postnatal life"

    Article Title: Thyroid hormone receptor ? is a molecular switch of cardiac function between fetal and postnatal life

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

    doi: 10.1073/pnas.0401843101

    Changes in expression of genes in the hearts of WT and mutant mice. ( A – F ) Quantification of transcripts in the heart of WT (plain bars) and TRα 0/0 -mutant (hatched bars) mice. The amounts of HCN2 mRNA ( A ), TR β mRNA ( B ), KCNE1 mRNA ( C ), KCNB1 mRNA ( D ), KCNA5 mRNA ( E ), and KCNQ1 mRNA ( F ) were estimated by quantitative RT-PCR, as described in Methods , and normalized to the corresponding Arbp mRNA levels. Each value represents the mean ± SD obtained from several independent animals (two for E15.5 WT fetuses, seven for E15.5 mutants, three for P18 WT, three for P18 mutants, two for A11 WT, and two for A11 mutants). ( G ) Changes in the levels of expression of genes in the heart as a result of TR α knockout. Bars represent the percentages of variation in TRα 0/0 mutants vs. WT and were calculated from the values presented in A – F . Data are presented as positive when values for TRα 0/0 were superior to WT and negative when inferior to WT.
    Figure Legend Snippet: Changes in expression of genes in the hearts of WT and mutant mice. ( A – F ) Quantification of transcripts in the heart of WT (plain bars) and TRα 0/0 -mutant (hatched bars) mice. The amounts of HCN2 mRNA ( A ), TR β mRNA ( B ), KCNE1 mRNA ( C ), KCNB1 mRNA ( D ), KCNA5 mRNA ( E ), and KCNQ1 mRNA ( F ) were estimated by quantitative RT-PCR, as described in Methods , and normalized to the corresponding Arbp mRNA levels. Each value represents the mean ± SD obtained from several independent animals (two for E15.5 WT fetuses, seven for E15.5 mutants, three for P18 WT, three for P18 mutants, two for A11 WT, and two for A11 mutants). ( G ) Changes in the levels of expression of genes in the heart as a result of TR α knockout. Bars represent the percentages of variation in TRα 0/0 mutants vs. WT and were calculated from the values presented in A – F . Data are presented as positive when values for TRα 0/0 were superior to WT and negative when inferior to WT.

    Techniques Used: Expressing, Mutagenesis, Mouse Assay, Quantitative RT-PCR, Knock-Out

    22) Product Images from "Liver and Biliary Tract Physiology/Pathophysiology: Intestinal bile acid sequestration improves glucose control by stimulating hepatic miR-182-5p in type 2 diabetes"

    Article Title: Liver and Biliary Tract Physiology/Pathophysiology: Intestinal bile acid sequestration improves glucose control by stimulating hepatic miR-182-5p in type 2 diabetes

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    doi: 10.1152/ajpgi.00238.2018

    The microRNA (miR)-96/182/183 cluster is upregulated in the livers of colesevelam-treated Zucker diabetic fatty (ZDF) rats. A : volcano plot depicting significantly altered miRNAs between colesevelam (Col) and vehicle-treated ZDF rat livers ( left ) and expression (reads per million total reads) of the miR-96/182/183 cluster ( left ); n = 6. B–D : liver expression of the miR-96/182/183 cluster by real-time PCR; n = 9–12. E : liver gene (mRNA) expression changes of Srebf2 and its target genes Hmgcr , Ldlr , and Sqle ; n = 8–12. For sequencing data, unpaired t -tests were used. For comparisons between 2 groups, Mann-Whitney nonparametric tests were used; n.d., not detectable.
    Figure Legend Snippet: The microRNA (miR)-96/182/183 cluster is upregulated in the livers of colesevelam-treated Zucker diabetic fatty (ZDF) rats. A : volcano plot depicting significantly altered miRNAs between colesevelam (Col) and vehicle-treated ZDF rat livers ( left ) and expression (reads per million total reads) of the miR-96/182/183 cluster ( left ); n = 6. B–D : liver expression of the miR-96/182/183 cluster by real-time PCR; n = 9–12. E : liver gene (mRNA) expression changes of Srebf2 and its target genes Hmgcr , Ldlr , and Sqle ; n = 8–12. For sequencing data, unpaired t -tests were used. For comparisons between 2 groups, Mann-Whitney nonparametric tests were used; n.d., not detectable.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Sequencing, MANN-WHITNEY

    Mediator complex subunit 1 (Med1) is regulated by microRNA (miR)-96/182/183 in mice. A : expression of Med1 by real-time PCR in mouse primary hepatocytes transfected with 50 nM of Med1 or scramble siRNA; n = 3 B : Western blot of Med1 and β-actin (loading control) from protein samples of primary hepatocytes transfected with Med1 or scramble siRNA. Ratio of Med1/β-actin densitometry is indicated between the blots and graphed on the right ; n = 3. A.U., arbitrary units. C : hepatic Med1 mRNA levels in db/db mice treated with vehicle or 2% colesevelam (Col)-supplemented diet and PBS or locked-nucleic acid (LNA)-182-5p; real-time PCR; n = 8–9. D : hepatic Med1 and β-actin (loading control) protein levels from 3 representative liver protein samples for Chow + PBS, Col + PBS, and Col + LNA-182. Western blotting. Ratio of Med1/β-actin densitometry is indicated between the blots. E : densitometry quantification of Med1 protein levels normalized to β-actin; n = 8–9. F : expression of miR-96-5p, miR-182-5p, and miR-183-5p in mouse primary hepatocytes transfected with 50 nM miR-96-5p, miR-182-5p, miR-183-5p mimics individually or in combination; real-time PCR; n = 3. G : Med1 mRNA levels in mouse primary hepatocytes transfected with 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination; real-time PCR; n = 3. H : protein levels of Med1 and β-actin (loading control) from protein samples of primary hepatocytes transfected with mock or 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination. Ratio of Med1/β-actin densitometry is indicated between the blots and graphed on the right ; n = 3. For comparisons between 2 groups, Students’ t- tests were used, and for more than 2 groups, Kruskal-Wallis one-way ANOVA with Dunn’s posttest was used (α = 0.05) or one-way ANOVA with Bonferonni’s posttest for comparison to mock group only was used (α = 0.05).
    Figure Legend Snippet: Mediator complex subunit 1 (Med1) is regulated by microRNA (miR)-96/182/183 in mice. A : expression of Med1 by real-time PCR in mouse primary hepatocytes transfected with 50 nM of Med1 or scramble siRNA; n = 3 B : Western blot of Med1 and β-actin (loading control) from protein samples of primary hepatocytes transfected with Med1 or scramble siRNA. Ratio of Med1/β-actin densitometry is indicated between the blots and graphed on the right ; n = 3. A.U., arbitrary units. C : hepatic Med1 mRNA levels in db/db mice treated with vehicle or 2% colesevelam (Col)-supplemented diet and PBS or locked-nucleic acid (LNA)-182-5p; real-time PCR; n = 8–9. D : hepatic Med1 and β-actin (loading control) protein levels from 3 representative liver protein samples for Chow + PBS, Col + PBS, and Col + LNA-182. Western blotting. Ratio of Med1/β-actin densitometry is indicated between the blots. E : densitometry quantification of Med1 protein levels normalized to β-actin; n = 8–9. F : expression of miR-96-5p, miR-182-5p, and miR-183-5p in mouse primary hepatocytes transfected with 50 nM miR-96-5p, miR-182-5p, miR-183-5p mimics individually or in combination; real-time PCR; n = 3. G : Med1 mRNA levels in mouse primary hepatocytes transfected with 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination; real-time PCR; n = 3. H : protein levels of Med1 and β-actin (loading control) from protein samples of primary hepatocytes transfected with mock or 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination. Ratio of Med1/β-actin densitometry is indicated between the blots and graphed on the right ; n = 3. For comparisons between 2 groups, Students’ t- tests were used, and for more than 2 groups, Kruskal-Wallis one-way ANOVA with Dunn’s posttest was used (α = 0.05) or one-way ANOVA with Bonferonni’s posttest for comparison to mock group only was used (α = 0.05).

    Techniques Used: Mouse Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Western Blot

    Mediator complex subunit 1 (MED1) is a direct target of microRNA (miR)-182-5p, miR-183-5p, and miR-96-5p in humans. A : schematic of human MED1 mRNA and the 3 putative miR-182/183/96 target sites in the 3′-untranslated region (3′-UTR). B : predicted MED1 3′-UTR target sites. Bases highlighted in dark gray represent mutations for gene reporter (luciferase) assays. C : normalized luciferase activity in HEK293 cells after dual transfection of miR-96/182/183 mimics and gene (luciferase) reporters harboring putative target sites for miR-96-5p, miR-182-5p, and/or miR-183-5p; n = 4. D : miR-96-5p, miR-182-5p, and miR-183-5p levels in Huh7 cells transfected with 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination; real-time PCR; n = 6. E : MED1 mRNA levels in Huh7 cells transfected with 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination; real-time PCR; n = 6. F : miR-96-5p, miR-182-5p, and miR-183-5p levels in Huh7 cells transfected with mock or 50 nM locked-nucleic acids (LNAs) against miR-182-5p and/or miR-183-5p; real-time PCR; n = 6. G : MED1 mRNA levels in Huh7 cells transfected mock or 50 nM LNAs against miR-182-5p and/or miR-183-5p; real-time PCR; n = 6. H : protein levels of Med1 and β-actin (loading control) from protein lysates of Huh7 cells transfected with mock or 50 nM miR-96, miR-182, and miR-183 LNAs. Ratio of Med1/β-actin densitometry is indicated between the blots and graphed on the right ; n = 6. A.U. arbitrary units. For comparisons between more than 2 groups to mock only, one-way ANOVA with Bonferonni’s posttest was used, α = 0.05. For comparisons between 2 groups, Mann-Whitney nonparametric tests were used.
    Figure Legend Snippet: Mediator complex subunit 1 (MED1) is a direct target of microRNA (miR)-182-5p, miR-183-5p, and miR-96-5p in humans. A : schematic of human MED1 mRNA and the 3 putative miR-182/183/96 target sites in the 3′-untranslated region (3′-UTR). B : predicted MED1 3′-UTR target sites. Bases highlighted in dark gray represent mutations for gene reporter (luciferase) assays. C : normalized luciferase activity in HEK293 cells after dual transfection of miR-96/182/183 mimics and gene (luciferase) reporters harboring putative target sites for miR-96-5p, miR-182-5p, and/or miR-183-5p; n = 4. D : miR-96-5p, miR-182-5p, and miR-183-5p levels in Huh7 cells transfected with 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination; real-time PCR; n = 6. E : MED1 mRNA levels in Huh7 cells transfected with 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination; real-time PCR; n = 6. F : miR-96-5p, miR-182-5p, and miR-183-5p levels in Huh7 cells transfected with mock or 50 nM locked-nucleic acids (LNAs) against miR-182-5p and/or miR-183-5p; real-time PCR; n = 6. G : MED1 mRNA levels in Huh7 cells transfected mock or 50 nM LNAs against miR-182-5p and/or miR-183-5p; real-time PCR; n = 6. H : protein levels of Med1 and β-actin (loading control) from protein lysates of Huh7 cells transfected with mock or 50 nM miR-96, miR-182, and miR-183 LNAs. Ratio of Med1/β-actin densitometry is indicated between the blots and graphed on the right ; n = 6. A.U. arbitrary units. For comparisons between more than 2 groups to mock only, one-way ANOVA with Bonferonni’s posttest was used, α = 0.05. For comparisons between 2 groups, Mann-Whitney nonparametric tests were used.

    Techniques Used: Luciferase, Activity Assay, Transfection, Real-time Polymerase Chain Reaction, MANN-WHITNEY

    23) Product Images from "PIK3CA-mediated PI3-kinase signalling is essential for HPV-induced transformation in vitro"

    Article Title: PIK3CA-mediated PI3-kinase signalling is essential for HPV-induced transformation in vitro

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-10-71

    Relation between PI3-kinase activity and HPV oncogene expression . (A) HPV16E7 mRNA expression in FK16A cells at immortal (p36) and anchorage independent (p99) stage as determined by qRT-PCR. (B) HPV16 mRNA expression in anchorage independent FK16A cells (p122) after PI3-kinase inhibition determined by Northern blot using an HPV16 radio-labeled probe. (C) HPV16E7 mRNA expression determined by qRT-PCR after PI3-kinase inhibition relative to mock treatment (p189). * Indicates a statistical significant difference. (D) hTERT mRNA expression determined by qRT-PCR in anchorage independent FK16A cells (p118) silenced for PIK3CA compared to FK16A cells transfected with non-targeting siRNAs. P: passage.
    Figure Legend Snippet: Relation between PI3-kinase activity and HPV oncogene expression . (A) HPV16E7 mRNA expression in FK16A cells at immortal (p36) and anchorage independent (p99) stage as determined by qRT-PCR. (B) HPV16 mRNA expression in anchorage independent FK16A cells (p122) after PI3-kinase inhibition determined by Northern blot using an HPV16 radio-labeled probe. (C) HPV16E7 mRNA expression determined by qRT-PCR after PI3-kinase inhibition relative to mock treatment (p189). * Indicates a statistical significant difference. (D) hTERT mRNA expression determined by qRT-PCR in anchorage independent FK16A cells (p118) silenced for PIK3CA compared to FK16A cells transfected with non-targeting siRNAs. P: passage.

    Techniques Used: Activity Assay, Expressing, Quantitative RT-PCR, Inhibition, Northern Blot, Labeling, Transfection

    24) Product Images from "Distinct Epigenetic Effects of Tobacco Smoking in Whole Blood and among Leukocyte Subtypes"

    Article Title: Distinct Epigenetic Effects of Tobacco Smoking in Whole Blood and among Leukocyte Subtypes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0166486

    Comparison of smoking-associated CpG methylation (A-C) and gene expression (D-F) differences between cell lineages. Methylation levels were measured on 450K arrays and averages for nonsmokers and smokers are displayed (A) cg05575921 ( AHRR ), (B) cg19859270 ( GPR15 ) and (C) cg03636183 ( F2RL3 ). Gene expression level (D) AHRR , (E) GPR15 and (F) F2RL3 for each cell type was measured by RT-PCR and averages are shown based on smoking status. Expression is represented by fold change difference between smokers relative to the average of nonsmokers after normalization to β-actin. Bar = Mean ± Standard Error; * p ≤0.05, ** p ≤0.001, Student’s t-test.
    Figure Legend Snippet: Comparison of smoking-associated CpG methylation (A-C) and gene expression (D-F) differences between cell lineages. Methylation levels were measured on 450K arrays and averages for nonsmokers and smokers are displayed (A) cg05575921 ( AHRR ), (B) cg19859270 ( GPR15 ) and (C) cg03636183 ( F2RL3 ). Gene expression level (D) AHRR , (E) GPR15 and (F) F2RL3 for each cell type was measured by RT-PCR and averages are shown based on smoking status. Expression is represented by fold change difference between smokers relative to the average of nonsmokers after normalization to β-actin. Bar = Mean ± Standard Error; * p ≤0.05, ** p ≤0.001, Student’s t-test.

    Techniques Used: CpG Methylation Assay, Expressing, Methylation, Reverse Transcription Polymerase Chain Reaction

    25) Product Images from "Tryptophan 2,3-dioxygenase is a key modulator of physiological neurogenesis and anxiety-related behavior in mice"

    Article Title: Tryptophan 2,3-dioxygenase is a key modulator of physiological neurogenesis and anxiety-related behavior in mice

    Journal: Molecular Brain

    doi: 10.1186/1756-6606-2-8

    Generation of tdo -deficient ( Tdo -/- ) mice . (A) Schema of the Trp metabolic pathways. (a) the Kyn pathway. Over 95% of the dietary Trp is metabolized along this pathway. (b) the serotonin pathway. (c) the transamination pathway. (B) A targeting strategy for tdo gene disruption. Exons are represented as numbered boxes (coding regions; black boxes ). The probe for Southern blot analysis is indicated by a solid bar . Apa I, A; Pvu II, P; Hind III, H; Eco RI, E; Xba I, X; Neo , PGK-neomycin resistant cassette; DT-A , diphtheria toxin-A. (C) Southern blot analysis of representative progeny. Tail genomic DNA was digested with Pvu II for hybridization with a specific probe against the intron sequence between exons 3 and 4 of tdo . The expected sizes of the hybridized DNA fragment for Tdo +/+ (+/+) and Tdo -/- (-/-) mice are 6.1 kb and 4.9 kb, respectively. (D) Quantitative real-time RT-PCR for tdo mRNA expression in adult liver. Mouse tdo/gapdh of adult liver in Tdo +/+ mice was arbitrarily given a value of 100%. Values are means ± S.D. (E) Western blot analysis. Total liver homogenates were immunoblotted with TDO-specific antiserum. (F) Assay for TDO enzyme activity, determined in total liver lysates from 10-week-old animals of each genotype. Values represent means ± S.D.
    Figure Legend Snippet: Generation of tdo -deficient ( Tdo -/- ) mice . (A) Schema of the Trp metabolic pathways. (a) the Kyn pathway. Over 95% of the dietary Trp is metabolized along this pathway. (b) the serotonin pathway. (c) the transamination pathway. (B) A targeting strategy for tdo gene disruption. Exons are represented as numbered boxes (coding regions; black boxes ). The probe for Southern blot analysis is indicated by a solid bar . Apa I, A; Pvu II, P; Hind III, H; Eco RI, E; Xba I, X; Neo , PGK-neomycin resistant cassette; DT-A , diphtheria toxin-A. (C) Southern blot analysis of representative progeny. Tail genomic DNA was digested with Pvu II for hybridization with a specific probe against the intron sequence between exons 3 and 4 of tdo . The expected sizes of the hybridized DNA fragment for Tdo +/+ (+/+) and Tdo -/- (-/-) mice are 6.1 kb and 4.9 kb, respectively. (D) Quantitative real-time RT-PCR for tdo mRNA expression in adult liver. Mouse tdo/gapdh of adult liver in Tdo +/+ mice was arbitrarily given a value of 100%. Values are means ± S.D. (E) Western blot analysis. Total liver homogenates were immunoblotted with TDO-specific antiserum. (F) Assay for TDO enzyme activity, determined in total liver lysates from 10-week-old animals of each genotype. Values represent means ± S.D.

    Techniques Used: Mouse Assay, Southern Blot, Hybridization, Sequencing, Quantitative RT-PCR, Expressing, Western Blot, Activity Assay

    26) Product Images from "Integrative computational analysis of transcriptional and epigenetic alterations implicates DTX1 as a putative tumor suppressor gene in HNSCC"

    Article Title: Integrative computational analysis of transcriptional and epigenetic alterations implicates DTX1 as a putative tumor suppressor gene in HNSCC

    Journal: Oncotarget

    doi: 10.18632/oncotarget.14856

    DTX1 blocks HNSCC invasiveness Migration assay was performed using UM-SCC-047 ( A ) or UM-SCC-22B ( B ) cells using transient transfection. The image of cells that had invaded through matrigel ( Supplementary Figure 5 ) was processed and quantified in Photoshop. Both UM-SCC-047 and UM-SCC-22B cells had similar 60% invasion when treated with control constructs (empty vector for ectopic expression or non-targeting siRNA pool for RNAi). The migration of each cell was dysregulated significantly by ectopic DTX1 overexpression ( a , UM-SCC-047 cells) or by transient DTX1 downregulation ( b , UM-SCC-22B cells). P-value were calculated by t-test for experiments performed in triplicate. Transfection efficiency for each experiment was confirmed by qRT-PCR ( Supplementary Figure 4 ).
    Figure Legend Snippet: DTX1 blocks HNSCC invasiveness Migration assay was performed using UM-SCC-047 ( A ) or UM-SCC-22B ( B ) cells using transient transfection. The image of cells that had invaded through matrigel ( Supplementary Figure 5 ) was processed and quantified in Photoshop. Both UM-SCC-047 and UM-SCC-22B cells had similar 60% invasion when treated with control constructs (empty vector for ectopic expression or non-targeting siRNA pool for RNAi). The migration of each cell was dysregulated significantly by ectopic DTX1 overexpression ( a , UM-SCC-047 cells) or by transient DTX1 downregulation ( b , UM-SCC-22B cells). P-value were calculated by t-test for experiments performed in triplicate. Transfection efficiency for each experiment was confirmed by qRT-PCR ( Supplementary Figure 4 ).

    Techniques Used: Migration, Transfection, Construct, Plasmid Preparation, Expressing, Over Expression, Quantitative RT-PCR

    27) Product Images from "The Nitric Oxide Pathway Provides Innate Antiviral Protection in Conjunction with the Type I Interferon Pathway in Fibroblasts"

    Article Title: The Nitric Oxide Pathway Provides Innate Antiviral Protection in Conjunction with the Type I Interferon Pathway in Fibroblasts

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0031688

    NF-κB contributes to iNOS induction and subsequent antiviral protection against HSV-1 in IRF3 −/− 9 −/− MEFs. (A) The effective concentration of Bay 11-7082 to inhibit NF-κB translocation to the nucleus after treatment with poly I∶C was determined. The effect of NF-κB inhibition by Bay 11-7082 on iNOS mRNA accumulation was measured by qRT-PCR in poly I∶C-treated WT (B) and IRF3 −/− 9 −/− (C) MEFs. Initiation of HSV-1gfp replication was quantified in WT (D) and IRF3 −/− 9 −/− (E) MEFs following a 5 hour poly I∶C treatment in the presence or absence of Bay 11-7082 and fluorescence compared to untreated monolayers, where fluorescence was set at 100% in each experiment. A 1-way ANOVA with a Tukey post-test was performed to compare efficacy of a range of NF-κB inhibitor concentrations. An unpaired t-test was performed to compare the antiviral response in cells that received poly I∶C treatment with and without Bay 11-7082; *, p
    Figure Legend Snippet: NF-κB contributes to iNOS induction and subsequent antiviral protection against HSV-1 in IRF3 −/− 9 −/− MEFs. (A) The effective concentration of Bay 11-7082 to inhibit NF-κB translocation to the nucleus after treatment with poly I∶C was determined. The effect of NF-κB inhibition by Bay 11-7082 on iNOS mRNA accumulation was measured by qRT-PCR in poly I∶C-treated WT (B) and IRF3 −/− 9 −/− (C) MEFs. Initiation of HSV-1gfp replication was quantified in WT (D) and IRF3 −/− 9 −/− (E) MEFs following a 5 hour poly I∶C treatment in the presence or absence of Bay 11-7082 and fluorescence compared to untreated monolayers, where fluorescence was set at 100% in each experiment. A 1-way ANOVA with a Tukey post-test was performed to compare efficacy of a range of NF-κB inhibitor concentrations. An unpaired t-test was performed to compare the antiviral response in cells that received poly I∶C treatment with and without Bay 11-7082; *, p

    Techniques Used: Concentration Assay, Translocation Assay, Inhibition, Quantitative RT-PCR, Fluorescence

    Nitric oxide is synthesized by iNOS in response to dsRNA in MEFs. (A) Nitric oxide was produced following treatment with poly I∶C in MEFs as determined by Griess assay. (B) DETA-NO, a nitric oxide donor, induced nitric oxide production in MEFs. Nitric oxide produced via DETA-NO limited initiation of replication of HSV-1gfp in WT (C) and IRF3 −/− 9 −/− (D) MEFs, as measured by GFP fluorescence, where fluorescence levels in mock treated cultures were set to 100%. DETA alone did not induce nitric oxide production and failed to reduce initiation of HSV-1 replication. (E) The relative abundance of iNOS mRNA, measured by qRT-PCR, in WT and IRF3 −/− 9 −/− MEFs treated with poly I∶C was found to be significantly higher in MEFs deficient for IRF3 and IRF9 after 5 hours of poly I∶C treatment in comparison to WT counterparts. Fold changes in transcript levels were compared relative to the housekeeping gene, Gapdh . *, p
    Figure Legend Snippet: Nitric oxide is synthesized by iNOS in response to dsRNA in MEFs. (A) Nitric oxide was produced following treatment with poly I∶C in MEFs as determined by Griess assay. (B) DETA-NO, a nitric oxide donor, induced nitric oxide production in MEFs. Nitric oxide produced via DETA-NO limited initiation of replication of HSV-1gfp in WT (C) and IRF3 −/− 9 −/− (D) MEFs, as measured by GFP fluorescence, where fluorescence levels in mock treated cultures were set to 100%. DETA alone did not induce nitric oxide production and failed to reduce initiation of HSV-1 replication. (E) The relative abundance of iNOS mRNA, measured by qRT-PCR, in WT and IRF3 −/− 9 −/− MEFs treated with poly I∶C was found to be significantly higher in MEFs deficient for IRF3 and IRF9 after 5 hours of poly I∶C treatment in comparison to WT counterparts. Fold changes in transcript levels were compared relative to the housekeeping gene, Gapdh . *, p

    Techniques Used: Synthesized, Produced, Griess Assay, Fluorescence, Quantitative RT-PCR

    28) Product Images from "Osteoarthritis-associated basic calcium phosphate crystals activate membrane proximal kinases in human innate immune cells"

    Article Title: Osteoarthritis-associated basic calcium phosphate crystals activate membrane proximal kinases in human innate immune cells

    Journal: Arthritis Research & Therapy

    doi: 10.1186/s13075-017-1225-0

    BCP crystals induce expression of damage-associated molecules and MMPs in primary human macrophages in a Syk- and PI3K-dependent manner. Human macrophages (0.5 × 10 6 cells/well) were stimulated with BCP crystals (50 μg/ml) for 3, 6, and 24 h, and mRNA levels of a S100A8, b S100A12, c MMP1, d MMP2, e MMP9, and f TIMP1 were analysed by real-time PCR. Alternatively, supernatants were harvested; whole cell lysates (WCL) were prepared and both were analysed for the presence of g S100A8, h S100A12, and i MMP1 protein by immunoblotting. Representative blots of three independent experiments are shown. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001
    Figure Legend Snippet: BCP crystals induce expression of damage-associated molecules and MMPs in primary human macrophages in a Syk- and PI3K-dependent manner. Human macrophages (0.5 × 10 6 cells/well) were stimulated with BCP crystals (50 μg/ml) for 3, 6, and 24 h, and mRNA levels of a S100A8, b S100A12, c MMP1, d MMP2, e MMP9, and f TIMP1 were analysed by real-time PCR. Alternatively, supernatants were harvested; whole cell lysates (WCL) were prepared and both were analysed for the presence of g S100A8, h S100A12, and i MMP1 protein by immunoblotting. Representative blots of three independent experiments are shown. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    BCP crystals exert synergistic effects with OA synovial fluid on primary macrophages. Human macrophages (0.5 × 10 6 cells/well) were pre-treated with R788 (2.5 μM) prior to treatment with BCP crystals alone, synovial fluid from one of three OA patients ( a , b or c ), or OA synovial fluid and BCP crystals together for 24 h. Expression of ( upper panel ) S100A8 and ( lower panel ) MMP1 were analysed by real-time PCR. Results indicate mean (± SEM) of at least three healthy macrophage donors treated with synovial fluid from one OA patient. * P ≤ 0.05, ** P ≤ 0.01
    Figure Legend Snippet: BCP crystals exert synergistic effects with OA synovial fluid on primary macrophages. Human macrophages (0.5 × 10 6 cells/well) were pre-treated with R788 (2.5 μM) prior to treatment with BCP crystals alone, synovial fluid from one of three OA patients ( a , b or c ), or OA synovial fluid and BCP crystals together for 24 h. Expression of ( upper panel ) S100A8 and ( lower panel ) MMP1 were analysed by real-time PCR. Results indicate mean (± SEM) of at least three healthy macrophage donors treated with synovial fluid from one OA patient. * P ≤ 0.05, ** P ≤ 0.01

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    BCP crystals induce expression of S100A8, S100A12, and MMP1 in primary macrophages in a Syk-, PI3K-, and ERK-dependent manner. Human macrophages were pre-treated with a piceatannol (50 μM, 100 μM), b LY294002 (25 μM, 50 μM), or c PD98059 (5 μM, 10 μM) for 30 mins prior to stimulation with BCP crystals for 24 h. mRNA levels of S100A8, S100A12, MMP1, and TIMP1 were analysed by real-time PCR. Results indicate mean (± SEM) of three independent experiments. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001
    Figure Legend Snippet: BCP crystals induce expression of S100A8, S100A12, and MMP1 in primary macrophages in a Syk-, PI3K-, and ERK-dependent manner. Human macrophages were pre-treated with a piceatannol (50 μM, 100 μM), b LY294002 (25 μM, 50 μM), or c PD98059 (5 μM, 10 μM) for 30 mins prior to stimulation with BCP crystals for 24 h. mRNA levels of S100A8, S100A12, MMP1, and TIMP1 were analysed by real-time PCR. Results indicate mean (± SEM) of three independent experiments. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    29) Product Images from "miR-132 enhances HIV-1 replication"

    Article Title: miR-132 enhances HIV-1 replication

    Journal: Virology

    doi: 10.1016/j.virol.2012.12.016

    MiR-132 is upregulated in activated CD4 + T cells A) Resting CD4 + T cells were isolated from four healthy blood donors, and a portion of the cells was activated with PHA. Activated CD4 + T cells were harvested two days later, and total RNA was isolated for use in microRNA microarray analysis. Columns represent fold-change in miR-132 expression following activation. B) Resting CD4 + T cells were isolated from five healthy blood donors, and a portion of the cells was activated with anti-CD3/28 beads. Two days later, total RNA was isolated and quantitative real-time PCR was performed to measure miR-132 levels relative to U6 snRNA; values presented were normalized relative to miR-132 levels in the resting cells of Donor 5. MiR-132 levels were found to be significantly higher following CD4 + T cell activation (Student’s t-test, p
    Figure Legend Snippet: MiR-132 is upregulated in activated CD4 + T cells A) Resting CD4 + T cells were isolated from four healthy blood donors, and a portion of the cells was activated with PHA. Activated CD4 + T cells were harvested two days later, and total RNA was isolated for use in microRNA microarray analysis. Columns represent fold-change in miR-132 expression following activation. B) Resting CD4 + T cells were isolated from five healthy blood donors, and a portion of the cells was activated with anti-CD3/28 beads. Two days later, total RNA was isolated and quantitative real-time PCR was performed to measure miR-132 levels relative to U6 snRNA; values presented were normalized relative to miR-132 levels in the resting cells of Donor 5. MiR-132 levels were found to be significantly higher following CD4 + T cell activation (Student’s t-test, p

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

    30) Product Images from "Stratifying risk of recurrence in stage II colorectal cancer using deregulated stromal and epithelial microRNAs"

    Article Title: Stratifying risk of recurrence in stage II colorectal cancer using deregulated stromal and epithelial microRNAs

    Journal: Oncotarget

    doi:

    Validation of stromal miRNA candidates deregulated in fresh-frozen CRC vs. paired normal colonic tissue by Taqman®qRT-PCR (* p
    Figure Legend Snippet: Validation of stromal miRNA candidates deregulated in fresh-frozen CRC vs. paired normal colonic tissue by Taqman®qRT-PCR (* p

    Techniques Used: Quantitative RT-PCR

    31) Product Images from "Efficient downregulation of immunoglobulin ? mRNA with premature translation-termination codons requires the 5?-half of the VDJ exon"

    Article Title: Efficient downregulation of immunoglobulin ? mRNA with premature translation-termination codons requires the 5?-half of the VDJ exon

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkh651

    Testing the ‘50 nucleotides boundary rule’ for NMD of Ig-μ minigenes. ( A ) PTCs were introduced into exon C3 of the Ig-μ minigene at the amino acid positions indicated above the diagram. The numbers below depict the distance of these PTCs from the 3′-most 5′ splice site. The ‘50 nucleotides boundary’ is marked by the dashed line. ( B ) RNA of polyclonal HeLa cell pools stably transfected with the indicated Ig-μ minigenes under control of the human β-actin promotor were analyzed by real-time RT–PCR. The indicated relative Ig-μ mRNA levels were normalized to endogenous GAPDH mRNA. Average values of three real-time PCR runs of a typical experiment are shown. Error bars indicate standard deviations. ( C ) Northern blot analysis of RNA harvested 48 h post transfection with a 32 P-labeled probe for Ig-μ mRNA. As a loading control, the 18S rRNA band from the ethidium bromide-stained gel before blotting is shown in the lower panel. ( D ) RT–PCR analysis of the RNA used in (B) to check for potential cryptic splicing between exon C3 and exon C4. Correct joining of exon C3 to exon C4 is predicted to give a PCR product of 163 bp.
    Figure Legend Snippet: Testing the ‘50 nucleotides boundary rule’ for NMD of Ig-μ minigenes. ( A ) PTCs were introduced into exon C3 of the Ig-μ minigene at the amino acid positions indicated above the diagram. The numbers below depict the distance of these PTCs from the 3′-most 5′ splice site. The ‘50 nucleotides boundary’ is marked by the dashed line. ( B ) RNA of polyclonal HeLa cell pools stably transfected with the indicated Ig-μ minigenes under control of the human β-actin promotor were analyzed by real-time RT–PCR. The indicated relative Ig-μ mRNA levels were normalized to endogenous GAPDH mRNA. Average values of three real-time PCR runs of a typical experiment are shown. Error bars indicate standard deviations. ( C ) Northern blot analysis of RNA harvested 48 h post transfection with a 32 P-labeled probe for Ig-μ mRNA. As a loading control, the 18S rRNA band from the ethidium bromide-stained gel before blotting is shown in the lower panel. ( D ) RT–PCR analysis of the RNA used in (B) to check for potential cryptic splicing between exon C3 and exon C4. Correct joining of exon C3 to exon C4 is predicted to give a PCR product of 163 bp.

    Techniques Used: Stable Transfection, Transfection, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Northern Blot, Labeling, Staining, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    The 5′ half of the Ig-μ minigene VDJ exon is necessary for efficient NMD of Ig-μ/ Sxl hybrid transcripts. ( A ) Relative hybrid mRNA levels of HeLa cells stably expressing the constructs schematically represented on the left were determined by real-time RT–PCR and are shown in the right panel. For each pair of hybrid constructs, the PTC− mRNA level (white bars) was set as 100% and the PTC+ mRNA level (gray bars) was calculated relative to it. The hybrid mRNA values were normalized to endogenous GAPDH mRNA. Average values of three real-time PCR runs of a typical cell pool are shown, and error bars indicate standard deviations. In the left panel, exons originating from Ig-μ are depicted in black and Sxl exons are depicted in gray. The position of the PTC is indicated by an asterisk and corresponds to ter310 in miniμ or ter43 in Sxl , respectively. The white regions labeled with Δ mark deletions. ( B ) Northern blot analysis of 15 μg of total cellular RNA isolated from HeLa cells stably transfected with the indicated hybrid constructs. As a loading control, the 18S rRNA band from the ethidium bromide-stained gel before blotting is shown in the lower panel.
    Figure Legend Snippet: The 5′ half of the Ig-μ minigene VDJ exon is necessary for efficient NMD of Ig-μ/ Sxl hybrid transcripts. ( A ) Relative hybrid mRNA levels of HeLa cells stably expressing the constructs schematically represented on the left were determined by real-time RT–PCR and are shown in the right panel. For each pair of hybrid constructs, the PTC− mRNA level (white bars) was set as 100% and the PTC+ mRNA level (gray bars) was calculated relative to it. The hybrid mRNA values were normalized to endogenous GAPDH mRNA. Average values of three real-time PCR runs of a typical cell pool are shown, and error bars indicate standard deviations. In the left panel, exons originating from Ig-μ are depicted in black and Sxl exons are depicted in gray. The position of the PTC is indicated by an asterisk and corresponds to ter310 in miniμ or ter43 in Sxl , respectively. The white regions labeled with Δ mark deletions. ( B ) Northern blot analysis of 15 μg of total cellular RNA isolated from HeLa cells stably transfected with the indicated hybrid constructs. As a loading control, the 18S rRNA band from the ethidium bromide-stained gel before blotting is shown in the lower panel.

    Techniques Used: Stable Transfection, Expressing, Construct, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Labeling, Northern Blot, Isolation, Transfection, Staining

    Efficiency of NMD increases with 5′ to 3′ polarity. ( A ). The PTC in cell line N89, N114, X54 and U30 is located at amino acid positions 3, 73, 325 and 335, respectively. ( B ) Relative Ig-μ mRNA levels of HeLa cells transiently transfected with the indicated Ig-μ minigenes under control of the human β-actin promotor were analyzed 48 h post transfection by real-time RT–PCR. The indicated relative Ig-μ mRNA levels were normalized to neomycin mRNA encoded on the transfected plasmid. ‘wt’ denotes the PTC-free Ig-μ minigene, the various PTC+ constructs are named ‘Ter’ (for termination) followed by a number indicating the amino acid position of the PTC. ( C ) Analysis of the same RNA used in (B) by northern blotting using a 32 P-labeled probe for Ig-μ mRNA. As a loading control, the 18S rRNA band from the ethidium bromide-stained gel before blotting is shown in the lower panel. ( D ) HeLa cells were transfected with the same Ig-μ minigene constructs as in (B) and stably transfected polyclonal cell pools were generated by selection with Geneticin. Relative Ig-μ mRNA levels were determined by real-time RT–PCR as in (B) and normalized to relative endogenous GAPDH mRNA levels. ( E ) RNA of the parental hybridoma cell line Sp6, encoding a productively rearranged full-length Ig-μ mRNA, and of five Sp6-derived, mutated cell clones was analyzed. X10 has deleted the entire Ig-μ gene and serves as a negative control. Relative Ig-μ mRNA levels were determined by real-time RT–PCR as in (B) and normalized to relative 18S ribosomal RNA levels. In (B), (D) and (E), average values of three real-time PCR runs with cDNAs of one representative experiment are shown. Error bars indicate standard deviations.
    Figure Legend Snippet: Efficiency of NMD increases with 5′ to 3′ polarity. ( A ). The PTC in cell line N89, N114, X54 and U30 is located at amino acid positions 3, 73, 325 and 335, respectively. ( B ) Relative Ig-μ mRNA levels of HeLa cells transiently transfected with the indicated Ig-μ minigenes under control of the human β-actin promotor were analyzed 48 h post transfection by real-time RT–PCR. The indicated relative Ig-μ mRNA levels were normalized to neomycin mRNA encoded on the transfected plasmid. ‘wt’ denotes the PTC-free Ig-μ minigene, the various PTC+ constructs are named ‘Ter’ (for termination) followed by a number indicating the amino acid position of the PTC. ( C ) Analysis of the same RNA used in (B) by northern blotting using a 32 P-labeled probe for Ig-μ mRNA. As a loading control, the 18S rRNA band from the ethidium bromide-stained gel before blotting is shown in the lower panel. ( D ) HeLa cells were transfected with the same Ig-μ minigene constructs as in (B) and stably transfected polyclonal cell pools were generated by selection with Geneticin. Relative Ig-μ mRNA levels were determined by real-time RT–PCR as in (B) and normalized to relative endogenous GAPDH mRNA levels. ( E ) RNA of the parental hybridoma cell line Sp6, encoding a productively rearranged full-length Ig-μ mRNA, and of five Sp6-derived, mutated cell clones was analyzed. X10 has deleted the entire Ig-μ gene and serves as a negative control. Relative Ig-μ mRNA levels were determined by real-time RT–PCR as in (B) and normalized to relative 18S ribosomal RNA levels. In (B), (D) and (E), average values of three real-time PCR runs with cDNAs of one representative experiment are shown. Error bars indicate standard deviations.

    Techniques Used: Transfection, Quantitative RT-PCR, Plasmid Preparation, Construct, Northern Blot, Labeling, Staining, Stable Transfection, Generated, Selection, Derivative Assay, Clone Assay, Negative Control, Real-time Polymerase Chain Reaction

    32) Product Images from "Inhibition of microRNA-155 alleviates lipopolysaccharide-induced kidney injury in mice"

    Article Title: Inhibition of microRNA-155 alleviates lipopolysaccharide-induced kidney injury in mice

    Journal: International Journal of Clinical and Experimental Pathology

    doi:

    Inhibition of miR-155 increases the expression of SOCS1 and suppresses the expression of JAK, STAT1, STAT3 and phospho-STAT3 (p-STAT3). The mRNA levels of Socs1 and Stat1 were determined by RT-PCR at indicated time point after LPS treatment, and the protein levels of SOCS1, JAK2, STAT3, p-STAT3 were detected at 24 h after LPS administration by western blotting (n=6 per group). A: Socs1 mRNA; B: Stat1 mRNA; C: The protein levels of SOCS1, JAK2, STAT3, p-STAT3. *indicates P
    Figure Legend Snippet: Inhibition of miR-155 increases the expression of SOCS1 and suppresses the expression of JAK, STAT1, STAT3 and phospho-STAT3 (p-STAT3). The mRNA levels of Socs1 and Stat1 were determined by RT-PCR at indicated time point after LPS treatment, and the protein levels of SOCS1, JAK2, STAT3, p-STAT3 were detected at 24 h after LPS administration by western blotting (n=6 per group). A: Socs1 mRNA; B: Stat1 mRNA; C: The protein levels of SOCS1, JAK2, STAT3, p-STAT3. *indicates P

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

    33) Product Images from "Circulating microRNAs levels in Chinese heart failure patients caused by dilated cardiomyopathy"

    Article Title: Circulating microRNAs levels in Chinese heart failure patients caused by dilated cardiomyopathy

    Journal: Indian Heart Journal

    doi: 10.1016/j.ihj.2012.12.022

    Circulating miRNAs in research cohorts. Expression of miRNAs in plasma was obtained from patients with DCM ( n = 45) and control subjects ( n = 39), as determined by TaqMan qRT-PCR. Values were normalized to cel-miR-39. Abbreviations:
    Figure Legend Snippet: Circulating miRNAs in research cohorts. Expression of miRNAs in plasma was obtained from patients with DCM ( n = 45) and control subjects ( n = 39), as determined by TaqMan qRT-PCR. Values were normalized to cel-miR-39. Abbreviations:

    Techniques Used: Expressing, Quantitative RT-PCR

    34) Product Images from "Chikungunya Virus Exploits miR-146a to Regulate NF-κB Pathway in Human Synovial Fibroblasts"

    Article Title: Chikungunya Virus Exploits miR-146a to Regulate NF-κB Pathway in Human Synovial Fibroblasts

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0103624

    miR-146a overexpression decreases TRAF6 protein expression levels in primary human synovial fibroblasts. Overexpression of miR-146a suppresses protein expression levels of TRAF6. ( A ) miR-146a expression levels were determined by qRT-PCR with TaqMan primers and probes specific to miR-146a. Scramble and miR-146a transfected primary human synovial fibroblasts were harvested 48 hours post transfection and fold change of miR-146a in overexpressed cells were significantly higher with respect to scrambled miR-146a (*p
    Figure Legend Snippet: miR-146a overexpression decreases TRAF6 protein expression levels in primary human synovial fibroblasts. Overexpression of miR-146a suppresses protein expression levels of TRAF6. ( A ) miR-146a expression levels were determined by qRT-PCR with TaqMan primers and probes specific to miR-146a. Scramble and miR-146a transfected primary human synovial fibroblasts were harvested 48 hours post transfection and fold change of miR-146a in overexpressed cells were significantly higher with respect to scrambled miR-146a (*p

    Techniques Used: Over Expression, Expressing, Quantitative RT-PCR, Transfection

    CHIKV infection increases miR-146a expression and decreases TRAF6, IRAK1, and IRAK2 in primary human synovial fibroblasts. Expression of miR-146a was increased upon CHIKV infection. (A). Human primary synovial fibroblasts were infected with CHIKV at MOI of 2 and cells were harvested 32 hours post infection for RNA isolation and protein lysate preparation. Expression levels of miR-146a were determined by qRT-PCR with TaqMan primers and probes specific for miR-146a. Expression level of RNU 24, an endogenous control, has been used as normalizer and the results are shown as fold changes compared to controls. (B) Western blot analysis showing decrease in the protein expression levels of TRAF6 in CHIKV infected primary human synovial fibroblasts. (C) Graph bars are showing densitometry analysis of TRAF6 normalized with housekeeping gene β-tubulin by ImageJ software. (D). Western blot analysis showing decrease in protein expression levels of IRAK 1 and IRAK 2 in CHIKV infected primary human synovial fibroblasts compared to uninfected controls. (E) Graph bars representing densitometry analysis for expression levels of IRAK1 and IRAK2 normalized with β-tubulin. All the experiments were independently repeated three times and shown as mean ± SEM. * above bars are representing the p value≤0.05 as level of significance, n = 3 (*for p value≤0.05).
    Figure Legend Snippet: CHIKV infection increases miR-146a expression and decreases TRAF6, IRAK1, and IRAK2 in primary human synovial fibroblasts. Expression of miR-146a was increased upon CHIKV infection. (A). Human primary synovial fibroblasts were infected with CHIKV at MOI of 2 and cells were harvested 32 hours post infection for RNA isolation and protein lysate preparation. Expression levels of miR-146a were determined by qRT-PCR with TaqMan primers and probes specific for miR-146a. Expression level of RNU 24, an endogenous control, has been used as normalizer and the results are shown as fold changes compared to controls. (B) Western blot analysis showing decrease in the protein expression levels of TRAF6 in CHIKV infected primary human synovial fibroblasts. (C) Graph bars are showing densitometry analysis of TRAF6 normalized with housekeeping gene β-tubulin by ImageJ software. (D). Western blot analysis showing decrease in protein expression levels of IRAK 1 and IRAK 2 in CHIKV infected primary human synovial fibroblasts compared to uninfected controls. (E) Graph bars representing densitometry analysis for expression levels of IRAK1 and IRAK2 normalized with β-tubulin. All the experiments were independently repeated three times and shown as mean ± SEM. * above bars are representing the p value≤0.05 as level of significance, n = 3 (*for p value≤0.05).

    Techniques Used: Infection, Expressing, Isolation, Quantitative RT-PCR, Western Blot, Software

    35) Product Images from "HIF-1 α promotes autophagic proteolysis of Dicer and enhances tumor metastasis"

    Article Title: HIF-1 α promotes autophagic proteolysis of Dicer and enhances tumor metastasis

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI89212

    HIF-1α–enhanced Dicer proteolysis in miRNA biogenesis. ( A – C ) Effects of HIF-1α on Dicer-dependent miRNA maturation. HCT116 cells overexpressing HIF-1α or HLH domain–truncated HIF-1α ( A ), knockdown of HIF-1α ( B ), or restoration of Dicer ( C ) were subjected to quantitative reverse-transcription PCR (qRT-PCR) for analyzing the expression of miRNAs, including let-7 family members (let-7a, let-7b, let-7d), miR-200b, and miR-451. ( A . ( D – F ) Effects of HIF-1α–regulated Dicer on the expression of miR-200b and let-7b–targeted genes. HCT116 cells were transfected with ZEB1 , LIN41 , or AURKB 3′ UTR luciferase reporter. The expression of luciferase was assayed in HIF-1α–overexpressing cells with or without Dicer restoration ( D ). miR-200b ( E ) or let-7b ( F ) was restored in HIF-1α–expressing cells. Aurora B, let-7b–targeted gene was analyzed by Western blot. The immunoblots presented were derived from replicate samples run on parallel gels ( E ). Data are presented as mean ± SD, with at least n = 3 per group. Unpaired, independent groups of 2 were analyzed by 2-tailed Student’s t test. Multigroup comparisons were analyzed by 1-way ANOVA with Tukey’s post hoc test.
    Figure Legend Snippet: HIF-1α–enhanced Dicer proteolysis in miRNA biogenesis. ( A – C ) Effects of HIF-1α on Dicer-dependent miRNA maturation. HCT116 cells overexpressing HIF-1α or HLH domain–truncated HIF-1α ( A ), knockdown of HIF-1α ( B ), or restoration of Dicer ( C ) were subjected to quantitative reverse-transcription PCR (qRT-PCR) for analyzing the expression of miRNAs, including let-7 family members (let-7a, let-7b, let-7d), miR-200b, and miR-451. ( A . ( D – F ) Effects of HIF-1α–regulated Dicer on the expression of miR-200b and let-7b–targeted genes. HCT116 cells were transfected with ZEB1 , LIN41 , or AURKB 3′ UTR luciferase reporter. The expression of luciferase was assayed in HIF-1α–overexpressing cells with or without Dicer restoration ( D ). miR-200b ( E ) or let-7b ( F ) was restored in HIF-1α–expressing cells. Aurora B, let-7b–targeted gene was analyzed by Western blot. The immunoblots presented were derived from replicate samples run on parallel gels ( E ). Data are presented as mean ± SD, with at least n = 3 per group. Unpaired, independent groups of 2 were analyzed by 2-tailed Student’s t test. Multigroup comparisons were analyzed by 1-way ANOVA with Tukey’s post hoc test.

    Techniques Used: Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Transfection, Luciferase, Western Blot, Derivative Assay

    36) Product Images from "Combining BET and HDAC inhibitors synergistically induces apoptosis of melanoma and suppresses AKT and YAP signaling"

    Article Title: Combining BET and HDAC inhibitors synergistically induces apoptosis of melanoma and suppresses AKT and YAP signaling

    Journal: Oncotarget

    doi:

    Combination-induced apoptosis requires expression of pro-apoptotic BIM A. Induction of BIM mRNA expression level in Patient-1-post and Me1007 cells was determined by qRT-PCR. Mean expression levels (± SEM) of n = 2 experiments are presented. B. Knockdown of BIM protein expression by siRNA was performed in Patient-1-post and Me1007 cells. After 24 h, transfected cells were treated with drugs as described before and incubated for a further 48 h. Expression of the BIM EL isoform is shown and α-Tubulin served as internal control. C. Knockdown of BIM reduces apoptosis in combination-treated in both cell lines. Mean (± SEM) of n = 3 experiments is shown. D. FOXO3a and BIM mRNA expression level analyzed by qRT-PCR are reduced upon siRNA-mediated knockdown of FOXO3a in Me1007 cells. E. Knockdown of FOXO3a reduces percentage of apoptosis in combination-treated Me1007 cells.
    Figure Legend Snippet: Combination-induced apoptosis requires expression of pro-apoptotic BIM A. Induction of BIM mRNA expression level in Patient-1-post and Me1007 cells was determined by qRT-PCR. Mean expression levels (± SEM) of n = 2 experiments are presented. B. Knockdown of BIM protein expression by siRNA was performed in Patient-1-post and Me1007 cells. After 24 h, transfected cells were treated with drugs as described before and incubated for a further 48 h. Expression of the BIM EL isoform is shown and α-Tubulin served as internal control. C. Knockdown of BIM reduces apoptosis in combination-treated in both cell lines. Mean (± SEM) of n = 3 experiments is shown. D. FOXO3a and BIM mRNA expression level analyzed by qRT-PCR are reduced upon siRNA-mediated knockdown of FOXO3a in Me1007 cells. E. Knockdown of FOXO3a reduces percentage of apoptosis in combination-treated Me1007 cells.

    Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Incubation

    37) Product Images from "Circulating miRNAs reflect early myocardial injury and recovery after heart transplantation"

    Article Title: Circulating miRNAs reflect early myocardial injury and recovery after heart transplantation

    Journal: Journal of Cardiothoracic Surgery

    doi: 10.1186/1749-8090-8-165

    Standard curves of miR-133a, miR-208a, and miR-133b for TaqMan qRT-PCR assays. Standard curves were generated for each miRNAs assay using a dilution series of known input amounts of synthetic miRNAs oligonucleotide corresponding to the target of the assay (Table 1 ). Data points not on the trend line are those for which the measured Ct was interpreted to be below the linear range of the assay, and were not used for derivation of the trend line. A , Standard curve of miR-133a; B , Standard curve of miR-208a. C , Standard curve of miR-133b.
    Figure Legend Snippet: Standard curves of miR-133a, miR-208a, and miR-133b for TaqMan qRT-PCR assays. Standard curves were generated for each miRNAs assay using a dilution series of known input amounts of synthetic miRNAs oligonucleotide corresponding to the target of the assay (Table 1 ). Data points not on the trend line are those for which the measured Ct was interpreted to be below the linear range of the assay, and were not used for derivation of the trend line. A , Standard curve of miR-133a; B , Standard curve of miR-208a. C , Standard curve of miR-133b.

    Techniques Used: Quantitative RT-PCR, Generated

    38) Product Images from "Cysteinyl Leukotrienes Are Autocrine and Paracrine Regulators of Fibrocyte Function"

    Article Title: Cysteinyl Leukotrienes Are Autocrine and Paracrine Regulators of Fibrocyte Function

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi:

    Murine and human fibrocytes express CysLT1 and CysLT2. A, Murine fibrocytes were purified from C57BL/6 lung digest cultures, and total RNA was prepared and analyzed for the expression of CysLT1 and CysLT2 by real-time RT-PCR. The expression level of each gene was first normalized to β -actin and next compared with the expression level of CysLT1 in AMs. This data are representative of two independent experiments. B, Murine fibrocytes and fibroblasts were purified from lung digest cultures. AMs were isolated from BAL. Four micrograms of protein from each lysate was run and analyzed by Western blotting with anti-human CysLT1 or anti-murine CysLT2 receptor Abs. Each lane represents cells from a single animal. Blots were stripped and probed for β -actin as a housekeeping control. C, Human fibrocytes were isolated from buffy coats of two normal volunteers. Western blotting was performed on 4 μ g of cell lysates as above.
    Figure Legend Snippet: Murine and human fibrocytes express CysLT1 and CysLT2. A, Murine fibrocytes were purified from C57BL/6 lung digest cultures, and total RNA was prepared and analyzed for the expression of CysLT1 and CysLT2 by real-time RT-PCR. The expression level of each gene was first normalized to β -actin and next compared with the expression level of CysLT1 in AMs. This data are representative of two independent experiments. B, Murine fibrocytes and fibroblasts were purified from lung digest cultures. AMs were isolated from BAL. Four micrograms of protein from each lysate was run and analyzed by Western blotting with anti-human CysLT1 or anti-murine CysLT2 receptor Abs. Each lane represents cells from a single animal. Blots were stripped and probed for β -actin as a housekeeping control. C, Human fibrocytes were isolated from buffy coats of two normal volunteers. Western blotting was performed on 4 μ g of cell lysates as above.

    Techniques Used: Purification, Expressing, Quantitative RT-PCR, Affinity Magnetic Separation, Isolation, Western Blot

    39) Product Images from "Lymphatic Dissemination of Simian Immunodeficiency Virus after Penile Inoculation"

    Article Title: Lymphatic Dissemination of Simian Immunodeficiency Virus after Penile Inoculation

    Journal: Journal of Virology

    doi: 10.1128/JVI.02947-15

    SIV RNA levels in plasma of rhesus macaques after penile SIVmac251 inoculation. Total vRNA levels in plasma were determined by RT-PCR. The last blood sample was collected at necropsy. The animal number associated with each symbol is indicated.
    Figure Legend Snippet: SIV RNA levels in plasma of rhesus macaques after penile SIVmac251 inoculation. Total vRNA levels in plasma were determined by RT-PCR. The last blood sample was collected at necropsy. The animal number associated with each symbol is indicated.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    40) Product Images from "Lipopolysaccharide and platelet-activating factor stimulate expression of platelet-activating factor acetylhydrolase via distinct signaling pathways"

    Article Title: Lipopolysaccharide and platelet-activating factor stimulate expression of platelet-activating factor acetylhydrolase via distinct signaling pathways

    Journal: Inflammation research : official journal of the European Histamine Research Society ... [et al.]

    doi: 10.1007/s00011-011-0326-5

    LPS-stimulated PAF receptor expression. Total RNA (5 μg) isolated from LPS- (200 ng/ml) treated MM6 cells cultured in serum-containing media at times ranging from 0 to 72 h was analyzed by qRT-PCR using TaqMan primers specific to the human PAF
    Figure Legend Snippet: LPS-stimulated PAF receptor expression. Total RNA (5 μg) isolated from LPS- (200 ng/ml) treated MM6 cells cultured in serum-containing media at times ranging from 0 to 72 h was analyzed by qRT-PCR using TaqMan primers specific to the human PAF

    Techniques Used: Expressing, Isolation, Cell Culture, Quantitative RT-PCR

    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: Transcriptional and Phenotypic Responses of Listeria monocytogenes to Chlorine Dioxide
    Article Snippet: .. Quantitative reverse transcriptase PCR (qRT-PCR) was performed as outlined ( ) with TaqMan One-Step RT-PCR Master Mix reagent and MultiScribe RT for single-tube reverse transcription and amplification using a StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA). ..

    Amplification:

    Article Title: Transcriptional and Phenotypic Responses of Listeria monocytogenes to Chlorine Dioxide
    Article Snippet: .. Quantitative reverse transcriptase PCR (qRT-PCR) was performed as outlined ( ) with TaqMan One-Step RT-PCR Master Mix reagent and MultiScribe RT for single-tube reverse transcription and amplification using a StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA). ..

    Article Title: Lipid-like Nanoparticles for Small Interfering RNA Delivery to Endothelial Cells
    Article Snippet: .. The synthesized cDNA was amplified by PCR with human specific primers (SHP-1, KDR/Flk-1, eNOS, and β-actin) using Platinum PCR master mix (Invitrogen). .. The amplification conditions followed several steps; 5 min at 95°C, followed by 25–35 cycles of denaturing (94°C, 30 sec), annealing (55–62°C, 30 sec), and extension (72°C, 45 sec) with a final extension at 72°C for 7 min.

    Synthesized:

    Article Title: Lipid-like Nanoparticles for Small Interfering RNA Delivery to Endothelial Cells
    Article Snippet: .. The synthesized cDNA was amplified by PCR with human specific primers (SHP-1, KDR/Flk-1, eNOS, and β-actin) using Platinum PCR master mix (Invitrogen). .. The amplification conditions followed several steps; 5 min at 95°C, followed by 25–35 cycles of denaturing (94°C, 30 sec), annealing (55–62°C, 30 sec), and extension (72°C, 45 sec) with a final extension at 72°C for 7 min.

    Quantitative RT-PCR:

    Article Title: Multiplexed Digital mRNA Profiling of the Inflammatory Response in the West Nile Swiss Webster Mouse Model
    Article Snippet: .. A volume of 20 µl qRT-PCR master mix was prepared per manufacturer instructions (AgPath-ID One-Step RT-PCR Kit, Life Technologies, Foster City, CA). ..

    Article Title: Transcriptional and Phenotypic Responses of Listeria monocytogenes to Chlorine Dioxide
    Article Snippet: .. Quantitative reverse transcriptase PCR (qRT-PCR) was performed as outlined ( ) with TaqMan One-Step RT-PCR Master Mix reagent and MultiScribe RT for single-tube reverse transcription and amplification using a StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA). ..

    Article Title: A Neoglycoconjugate Containing the Human Milk Sugar LNFPIII Drives Anti-Inflammatory Activation of Antigen Presenting Cells in a CD14 Dependent Pathway
    Article Snippet: .. One microgram (1μg) of RNA was used to perform RT-PCR followed by qRT-PCR using primers (Applied Biosystems, Foster city, CA) against specific genes, Universal PCR master mix and run on an ABI StepOne Plus (Applied biosystems, Foster city, CA). .. Expression was normalized to GAPDH endogenous control and fold expression was measured by comparing to the expression of the uninfected controls.

    Article Title: Molecular evolution and functional divergence of zebrafish (Danio rerio) cryptochrome genes
    Article Snippet: .. Quantitative RT-PCR was performed with an ABI StepOnePlus system and SYBR Green qRT-PCR Master Mix (Invitrogen). ..

    SYBR Green Assay:

    Article Title: Molecular evolution and functional divergence of zebrafish (Danio rerio) cryptochrome genes
    Article Snippet: .. Quantitative RT-PCR was performed with an ABI StepOnePlus system and SYBR Green qRT-PCR Master Mix (Invitrogen). ..

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Multiplexed Digital mRNA Profiling of the Inflammatory Response in the West Nile Swiss Webster Mouse Model
    Article Snippet: .. A volume of 20 µl qRT-PCR master mix was prepared per manufacturer instructions (AgPath-ID One-Step RT-PCR Kit, Life Technologies, Foster City, CA). ..

    Article Title: Transcriptional and Phenotypic Responses of Listeria monocytogenes to Chlorine Dioxide
    Article Snippet: .. Quantitative reverse transcriptase PCR (qRT-PCR) was performed as outlined ( ) with TaqMan One-Step RT-PCR Master Mix reagent and MultiScribe RT for single-tube reverse transcription and amplification using a StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA). ..

    Article Title: Up-Regulation of Hepatitis C Virus Replication and Production by Inhibition of MEK/ERK Signaling
    Article Snippet: .. Both RNA were quantitated in triplicates using a TaqMan® OneStep RT-PCR Master Mix Reagents Kit (Applied Biosystems, Foster City, CA) and the primers and probes described previously . .. The reactions were performed on a Mx3000P QPCR system (Stratagene, La Jolla, CA) with a program of 48°C for 30 min, 95°C for 10 min, and then 50 cycles at 95°C for 15 sec and 60°C for 1 min.

    Article Title: Extensive and coordinated transcription of noncoding RNAs within cell cycle promoters
    Article Snippet: .. RT-PCR using 50-250 ng of total RNA was performed using the One-Step RT-PCR Master Mix (Applied Biosystems) using Taqman Gene Expression Assays and normalized to GAPDH. .. Strand specific RT-PCR for PANDA was performed using the One Step RT-PCR Master Mix SYBR Green (Stratagene)).

    Article Title: A Neoglycoconjugate Containing the Human Milk Sugar LNFPIII Drives Anti-Inflammatory Activation of Antigen Presenting Cells in a CD14 Dependent Pathway
    Article Snippet: .. One microgram (1μg) of RNA was used to perform RT-PCR followed by qRT-PCR using primers (Applied Biosystems, Foster city, CA) against specific genes, Universal PCR master mix and run on an ABI StepOne Plus (Applied biosystems, Foster city, CA). .. Expression was normalized to GAPDH endogenous control and fold expression was measured by comparing to the expression of the uninfected controls.

    Expressing:

    Article Title: Extensive and coordinated transcription of noncoding RNAs within cell cycle promoters
    Article Snippet: .. RT-PCR using 50-250 ng of total RNA was performed using the One-Step RT-PCR Master Mix (Applied Biosystems) using Taqman Gene Expression Assays and normalized to GAPDH. .. Strand specific RT-PCR for PANDA was performed using the One Step RT-PCR Master Mix SYBR Green (Stratagene)).

    Polymerase Chain Reaction:

    Article Title: Transcriptional and Phenotypic Responses of Listeria monocytogenes to Chlorine Dioxide
    Article Snippet: .. Quantitative reverse transcriptase PCR (qRT-PCR) was performed as outlined ( ) with TaqMan One-Step RT-PCR Master Mix reagent and MultiScribe RT for single-tube reverse transcription and amplification using a StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA). ..

    Article Title: Lipid-like Nanoparticles for Small Interfering RNA Delivery to Endothelial Cells
    Article Snippet: .. The synthesized cDNA was amplified by PCR with human specific primers (SHP-1, KDR/Flk-1, eNOS, and β-actin) using Platinum PCR master mix (Invitrogen). .. The amplification conditions followed several steps; 5 min at 95°C, followed by 25–35 cycles of denaturing (94°C, 30 sec), annealing (55–62°C, 30 sec), and extension (72°C, 45 sec) with a final extension at 72°C for 7 min.

    Article Title: A Neoglycoconjugate Containing the Human Milk Sugar LNFPIII Drives Anti-Inflammatory Activation of Antigen Presenting Cells in a CD14 Dependent Pathway
    Article Snippet: .. One microgram (1μg) of RNA was used to perform RT-PCR followed by qRT-PCR using primers (Applied Biosystems, Foster city, CA) against specific genes, Universal PCR master mix and run on an ABI StepOne Plus (Applied biosystems, Foster city, CA). .. Expression was normalized to GAPDH endogenous control and fold expression was measured by comparing to the expression of the uninfected controls.

    Article Title: AAV2-mediated follistatin overexpression induces ovine primary myoblasts proliferation
    Article Snippet: .. The PCR reaction was carried out in a final volume of 20 μl containing the DNA template and 10 μl of commercially available PCR master mix (PyroStart™ Fast PCR Master Mix (2×), Fermentas, Life Sciences, Thermo Fisher). .. After brief centrifugation, the PCR amplification was carried out in an ABI 9700 PCR system (Applied Biosystems, Foster City, CA, USA).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher taqman universal master mix ii
    miR-182 Is Localized in RGC Axons (A) Heatmap representing the average expression of mature miRNAs from two axonal small <t>RNA-sequencing</t> (sRNA-seq) libraries prepared from stage 37/38 retinal cultures. The figure is sorted by decreasing axonal average values. (B) Fluorescent ISH on stage 35/36 RGC GCs cultured in vitro for 24 hr. (C) <t>TaqMan</t> qPCR performed on RNA extracted from laser-captured stage 37/38 RGC axons. U6 snRNA was used as positive control, because it is found in developing axons ( Natera-Naranjo et al., 2010 , Zhang et al., 2013 , Hancock et al., 2014 ). RT−, no template negative control; snRNAU6, U6 snRNA. Scale bar, 5 μm (B). See also Figure S1 and Table S1 .
    Taqman Universal Master Mix Ii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 611 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taqman universal master mix ii/product/Thermo Fisher
    Average 99 stars, based on 611 article reviews
    Price from $9.99 to $1999.99
    taqman universal master mix ii - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher universal pcr master mix
    The microRNA (miR)-96/182/183 cluster is upregulated in the livers of colesevelam-treated Zucker diabetic fatty (ZDF) rats. A : volcano plot depicting significantly altered miRNAs between colesevelam (Col) and vehicle-treated ZDF rat livers ( left ) and expression (reads per million total reads) of the miR-96/182/183 cluster ( left ); n = 6. B–D : liver expression of the miR-96/182/183 cluster by real-time <t>PCR;</t> n = 9–12. E : liver gene (mRNA) expression changes of Srebf2 and its target genes Hmgcr , Ldlr , and Sqle ; n = 8–12. For sequencing data, unpaired t -tests were used. For comparisons between 2 groups, Mann-Whitney nonparametric tests were used; n.d., not detectable.
    Universal Pcr Master Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 406 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/universal pcr master mix/product/Thermo Fisher
    Average 99 stars, based on 406 article reviews
    Price from $9.99 to $1999.99
    universal pcr master mix - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    Image Search Results


    miR-182 Is Localized in RGC Axons (A) Heatmap representing the average expression of mature miRNAs from two axonal small RNA-sequencing (sRNA-seq) libraries prepared from stage 37/38 retinal cultures. The figure is sorted by decreasing axonal average values. (B) Fluorescent ISH on stage 35/36 RGC GCs cultured in vitro for 24 hr. (C) TaqMan qPCR performed on RNA extracted from laser-captured stage 37/38 RGC axons. U6 snRNA was used as positive control, because it is found in developing axons ( Natera-Naranjo et al., 2010 , Zhang et al., 2013 , Hancock et al., 2014 ). RT−, no template negative control; snRNAU6, U6 snRNA. Scale bar, 5 μm (B). See also Figure S1 and Table S1 .

    Journal: Cell Reports

    Article Title: miR-182 Regulates Slit2-Mediated Axon Guidance by Modulating the Local Translation of a Specific mRNA

    doi: 10.1016/j.celrep.2016.12.093

    Figure Lengend Snippet: miR-182 Is Localized in RGC Axons (A) Heatmap representing the average expression of mature miRNAs from two axonal small RNA-sequencing (sRNA-seq) libraries prepared from stage 37/38 retinal cultures. The figure is sorted by decreasing axonal average values. (B) Fluorescent ISH on stage 35/36 RGC GCs cultured in vitro for 24 hr. (C) TaqMan qPCR performed on RNA extracted from laser-captured stage 37/38 RGC axons. U6 snRNA was used as positive control, because it is found in developing axons ( Natera-Naranjo et al., 2010 , Zhang et al., 2013 , Hancock et al., 2014 ). RT−, no template negative control; snRNAU6, U6 snRNA. Scale bar, 5 μm (B). See also Figure S1 and Table S1 .

    Article Snippet: The cDNA obtained was used for the TaqMan Micro RNA assay using xtr-miR-182-5p and U6 snRNA-specific primers and probes and the TaqMan Universal Master Mix II (MMIX II) no AmpErase Uracil N-Glycosylase (UNG) (all Thermo Fisher).

    Techniques: Expressing, RNA Sequencing Assay, In Situ Hybridization, Cell Culture, In Vitro, Real-time Polymerase Chain Reaction, Positive Control, Negative Control

    Dysregulation of miR-125b and miR-22 expression in RNA extracted from prostate tissue samples by LCM. (A) Frozen prostate samples were dissected via LCM into benign glandular epithelial versus tumor. Total RNA was extracted using the PicoPure RNA extraction method, as described in Materials and Methods. qRT-PCR was performed with specific primers against miR-125b and miR-22 using the TaqMan ® Universal Master Mix II. C T values were normalized to RNU48 and reported as fold differences compared to benign tissue using the ΔΔCT method of Livak et al. [ 23 ]. Results are the average from frozen tissue samples (n = 4) repeated in 3 technical replicates. (B) From 10 successive slides of a single FFPE prostate sample, miR-125b was quantitated by qRT-PCR as described in panel A for RNA extracted from stroma, benign glandular tissue, BPH, PIN and tumor of stage G4.

    Journal: PLoS ONE

    Article Title: Dual Action of miR-125b As a Tumor Suppressor and OncomiR-22 Promotes Prostate Cancer Tumorigenesis

    doi: 10.1371/journal.pone.0142373

    Figure Lengend Snippet: Dysregulation of miR-125b and miR-22 expression in RNA extracted from prostate tissue samples by LCM. (A) Frozen prostate samples were dissected via LCM into benign glandular epithelial versus tumor. Total RNA was extracted using the PicoPure RNA extraction method, as described in Materials and Methods. qRT-PCR was performed with specific primers against miR-125b and miR-22 using the TaqMan ® Universal Master Mix II. C T values were normalized to RNU48 and reported as fold differences compared to benign tissue using the ΔΔCT method of Livak et al. [ 23 ]. Results are the average from frozen tissue samples (n = 4) repeated in 3 technical replicates. (B) From 10 successive slides of a single FFPE prostate sample, miR-125b was quantitated by qRT-PCR as described in panel A for RNA extracted from stroma, benign glandular tissue, BPH, PIN and tumor of stage G4.

    Article Snippet: Each individual assay was performed in a 20 μl reaction volume with 1.33 μl of cDNA, 1.0 μl specific miR assay, 10 μl TaqMan™ Universal PCR Master Mix II with no AmpErase UNG and 7.67 μl of nuclease free water.

    Techniques: Expressing, Laser Capture Microdissection, RNA Extraction, Quantitative RT-PCR, Formalin-fixed Paraffin-Embedded

    The microRNA (miR)-96/182/183 cluster is upregulated in the livers of colesevelam-treated Zucker diabetic fatty (ZDF) rats. A : volcano plot depicting significantly altered miRNAs between colesevelam (Col) and vehicle-treated ZDF rat livers ( left ) and expression (reads per million total reads) of the miR-96/182/183 cluster ( left ); n = 6. B–D : liver expression of the miR-96/182/183 cluster by real-time PCR; n = 9–12. E : liver gene (mRNA) expression changes of Srebf2 and its target genes Hmgcr , Ldlr , and Sqle ; n = 8–12. For sequencing data, unpaired t -tests were used. For comparisons between 2 groups, Mann-Whitney nonparametric tests were used; n.d., not detectable.

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    Article Title: Liver and Biliary Tract Physiology/Pathophysiology: Intestinal bile acid sequestration improves glucose control by stimulating hepatic miR-182-5p in type 2 diabetes

    doi: 10.1152/ajpgi.00238.2018

    Figure Lengend Snippet: The microRNA (miR)-96/182/183 cluster is upregulated in the livers of colesevelam-treated Zucker diabetic fatty (ZDF) rats. A : volcano plot depicting significantly altered miRNAs between colesevelam (Col) and vehicle-treated ZDF rat livers ( left ) and expression (reads per million total reads) of the miR-96/182/183 cluster ( left ); n = 6. B–D : liver expression of the miR-96/182/183 cluster by real-time PCR; n = 9–12. E : liver gene (mRNA) expression changes of Srebf2 and its target genes Hmgcr , Ldlr , and Sqle ; n = 8–12. For sequencing data, unpaired t -tests were used. For comparisons between 2 groups, Mann-Whitney nonparametric tests were used; n.d., not detectable.

    Article Snippet: Real-time PCR was completed with Universal PCR Master Mix and TaqMan mRNA and miRNA assays (ThermoFisher).

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Sequencing, MANN-WHITNEY

    Mediator complex subunit 1 (Med1) is regulated by microRNA (miR)-96/182/183 in mice. A : expression of Med1 by real-time PCR in mouse primary hepatocytes transfected with 50 nM of Med1 or scramble siRNA; n = 3 B : Western blot of Med1 and β-actin (loading control) from protein samples of primary hepatocytes transfected with Med1 or scramble siRNA. Ratio of Med1/β-actin densitometry is indicated between the blots and graphed on the right ; n = 3. A.U., arbitrary units. C : hepatic Med1 mRNA levels in db/db mice treated with vehicle or 2% colesevelam (Col)-supplemented diet and PBS or locked-nucleic acid (LNA)-182-5p; real-time PCR; n = 8–9. D : hepatic Med1 and β-actin (loading control) protein levels from 3 representative liver protein samples for Chow + PBS, Col + PBS, and Col + LNA-182. Western blotting. Ratio of Med1/β-actin densitometry is indicated between the blots. E : densitometry quantification of Med1 protein levels normalized to β-actin; n = 8–9. F : expression of miR-96-5p, miR-182-5p, and miR-183-5p in mouse primary hepatocytes transfected with 50 nM miR-96-5p, miR-182-5p, miR-183-5p mimics individually or in combination; real-time PCR; n = 3. G : Med1 mRNA levels in mouse primary hepatocytes transfected with 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination; real-time PCR; n = 3. H : protein levels of Med1 and β-actin (loading control) from protein samples of primary hepatocytes transfected with mock or 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination. Ratio of Med1/β-actin densitometry is indicated between the blots and graphed on the right ; n = 3. For comparisons between 2 groups, Students’ t- tests were used, and for more than 2 groups, Kruskal-Wallis one-way ANOVA with Dunn’s posttest was used (α = 0.05) or one-way ANOVA with Bonferonni’s posttest for comparison to mock group only was used (α = 0.05).

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    Article Title: Liver and Biliary Tract Physiology/Pathophysiology: Intestinal bile acid sequestration improves glucose control by stimulating hepatic miR-182-5p in type 2 diabetes

    doi: 10.1152/ajpgi.00238.2018

    Figure Lengend Snippet: Mediator complex subunit 1 (Med1) is regulated by microRNA (miR)-96/182/183 in mice. A : expression of Med1 by real-time PCR in mouse primary hepatocytes transfected with 50 nM of Med1 or scramble siRNA; n = 3 B : Western blot of Med1 and β-actin (loading control) from protein samples of primary hepatocytes transfected with Med1 or scramble siRNA. Ratio of Med1/β-actin densitometry is indicated between the blots and graphed on the right ; n = 3. A.U., arbitrary units. C : hepatic Med1 mRNA levels in db/db mice treated with vehicle or 2% colesevelam (Col)-supplemented diet and PBS or locked-nucleic acid (LNA)-182-5p; real-time PCR; n = 8–9. D : hepatic Med1 and β-actin (loading control) protein levels from 3 representative liver protein samples for Chow + PBS, Col + PBS, and Col + LNA-182. Western blotting. Ratio of Med1/β-actin densitometry is indicated between the blots. E : densitometry quantification of Med1 protein levels normalized to β-actin; n = 8–9. F : expression of miR-96-5p, miR-182-5p, and miR-183-5p in mouse primary hepatocytes transfected with 50 nM miR-96-5p, miR-182-5p, miR-183-5p mimics individually or in combination; real-time PCR; n = 3. G : Med1 mRNA levels in mouse primary hepatocytes transfected with 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination; real-time PCR; n = 3. H : protein levels of Med1 and β-actin (loading control) from protein samples of primary hepatocytes transfected with mock or 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination. Ratio of Med1/β-actin densitometry is indicated between the blots and graphed on the right ; n = 3. For comparisons between 2 groups, Students’ t- tests were used, and for more than 2 groups, Kruskal-Wallis one-way ANOVA with Dunn’s posttest was used (α = 0.05) or one-way ANOVA with Bonferonni’s posttest for comparison to mock group only was used (α = 0.05).

    Article Snippet: Real-time PCR was completed with Universal PCR Master Mix and TaqMan mRNA and miRNA assays (ThermoFisher).

    Techniques: Mouse Assay, Expressing, Real-time Polymerase Chain Reaction, Transfection, Western Blot

    Mediator complex subunit 1 (MED1) is a direct target of microRNA (miR)-182-5p, miR-183-5p, and miR-96-5p in humans. A : schematic of human MED1 mRNA and the 3 putative miR-182/183/96 target sites in the 3′-untranslated region (3′-UTR). B : predicted MED1 3′-UTR target sites. Bases highlighted in dark gray represent mutations for gene reporter (luciferase) assays. C : normalized luciferase activity in HEK293 cells after dual transfection of miR-96/182/183 mimics and gene (luciferase) reporters harboring putative target sites for miR-96-5p, miR-182-5p, and/or miR-183-5p; n = 4. D : miR-96-5p, miR-182-5p, and miR-183-5p levels in Huh7 cells transfected with 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination; real-time PCR; n = 6. E : MED1 mRNA levels in Huh7 cells transfected with 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination; real-time PCR; n = 6. F : miR-96-5p, miR-182-5p, and miR-183-5p levels in Huh7 cells transfected with mock or 50 nM locked-nucleic acids (LNAs) against miR-182-5p and/or miR-183-5p; real-time PCR; n = 6. G : MED1 mRNA levels in Huh7 cells transfected mock or 50 nM LNAs against miR-182-5p and/or miR-183-5p; real-time PCR; n = 6. H : protein levels of Med1 and β-actin (loading control) from protein lysates of Huh7 cells transfected with mock or 50 nM miR-96, miR-182, and miR-183 LNAs. Ratio of Med1/β-actin densitometry is indicated between the blots and graphed on the right ; n = 6. A.U. arbitrary units. For comparisons between more than 2 groups to mock only, one-way ANOVA with Bonferonni’s posttest was used, α = 0.05. For comparisons between 2 groups, Mann-Whitney nonparametric tests were used.

    Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

    Article Title: Liver and Biliary Tract Physiology/Pathophysiology: Intestinal bile acid sequestration improves glucose control by stimulating hepatic miR-182-5p in type 2 diabetes

    doi: 10.1152/ajpgi.00238.2018

    Figure Lengend Snippet: Mediator complex subunit 1 (MED1) is a direct target of microRNA (miR)-182-5p, miR-183-5p, and miR-96-5p in humans. A : schematic of human MED1 mRNA and the 3 putative miR-182/183/96 target sites in the 3′-untranslated region (3′-UTR). B : predicted MED1 3′-UTR target sites. Bases highlighted in dark gray represent mutations for gene reporter (luciferase) assays. C : normalized luciferase activity in HEK293 cells after dual transfection of miR-96/182/183 mimics and gene (luciferase) reporters harboring putative target sites for miR-96-5p, miR-182-5p, and/or miR-183-5p; n = 4. D : miR-96-5p, miR-182-5p, and miR-183-5p levels in Huh7 cells transfected with 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination; real-time PCR; n = 6. E : MED1 mRNA levels in Huh7 cells transfected with 50 nM miR-96-5p, miR-182-5p, and miR-183-5p mimics individually or in combination; real-time PCR; n = 6. F : miR-96-5p, miR-182-5p, and miR-183-5p levels in Huh7 cells transfected with mock or 50 nM locked-nucleic acids (LNAs) against miR-182-5p and/or miR-183-5p; real-time PCR; n = 6. G : MED1 mRNA levels in Huh7 cells transfected mock or 50 nM LNAs against miR-182-5p and/or miR-183-5p; real-time PCR; n = 6. H : protein levels of Med1 and β-actin (loading control) from protein lysates of Huh7 cells transfected with mock or 50 nM miR-96, miR-182, and miR-183 LNAs. Ratio of Med1/β-actin densitometry is indicated between the blots and graphed on the right ; n = 6. A.U. arbitrary units. For comparisons between more than 2 groups to mock only, one-way ANOVA with Bonferonni’s posttest was used, α = 0.05. For comparisons between 2 groups, Mann-Whitney nonparametric tests were used.

    Article Snippet: Real-time PCR was completed with Universal PCR Master Mix and TaqMan mRNA and miRNA assays (ThermoFisher).

    Techniques: Luciferase, Activity Assay, Transfection, Real-time Polymerase Chain Reaction, MANN-WHITNEY

    Quantitative real-time PCR analyses comparing miRNA expression patterns in tissues using different extraction methods. Fold abundance of miRNAs quantitated using TaqMan MicroRNA Assays via qRT-PCR from brain ( a ), liver ( b ) and lung ( c ) tissues. miRNA expression was normalised using sno202 as a reference gene. Values indicate the average normalised miRNA expression in different tissues on a Log2 scale with SEM error bars. * p

    Journal: BMC Biotechnology

    Article Title: Total RNA extraction from tissues for microRNA and target gene expression analysis: not all kits are created equal

    doi: 10.1186/s12896-018-0421-6

    Figure Lengend Snippet: Quantitative real-time PCR analyses comparing miRNA expression patterns in tissues using different extraction methods. Fold abundance of miRNAs quantitated using TaqMan MicroRNA Assays via qRT-PCR from brain ( a ), liver ( b ) and lung ( c ) tissues. miRNA expression was normalised using sno202 as a reference gene. Values indicate the average normalised miRNA expression in different tissues on a Log2 scale with SEM error bars. * p

    Article Snippet: Reverse transcription products were diluted to 50 μl and 5 μl of diluted sample used in single qPCR reactions, with a total volume of 20 μl. qRT-PCR was performed using Taqman microRNA assays (Thermo Fisher Scientific) and TaqMan universal PCR master mix II, no UNG (Thermo Fisher Scientific) on a ViiA7 Real-Time PCR system (Applied Biosystems/Thermo Fisher Scientific), using recommended PCR cycling conditions.

    Techniques: Real-time Polymerase Chain Reaction, Expressing, Quantitative RT-PCR

    Quantitative real-time PCR analyses of 6 miRNAs comparing different RNA extraction methods. RNA was extracted from murine brain ( a ), liver ( b ) and lung ( c ) tissues using the five indicated methods and endogenous miRNA expression quantitated using TaqMan MicroRNA Assays via qRT-PCR. miRNA expression was normalised using sno202 as a reference gene. Values indicate the average normalised miRNA expression (technical duplicates from triplicate biological isolations) in different tissues on a Log2 scale with SEM error bars. * p

    Journal: BMC Biotechnology

    Article Title: Total RNA extraction from tissues for microRNA and target gene expression analysis: not all kits are created equal

    doi: 10.1186/s12896-018-0421-6

    Figure Lengend Snippet: Quantitative real-time PCR analyses of 6 miRNAs comparing different RNA extraction methods. RNA was extracted from murine brain ( a ), liver ( b ) and lung ( c ) tissues using the five indicated methods and endogenous miRNA expression quantitated using TaqMan MicroRNA Assays via qRT-PCR. miRNA expression was normalised using sno202 as a reference gene. Values indicate the average normalised miRNA expression (technical duplicates from triplicate biological isolations) in different tissues on a Log2 scale with SEM error bars. * p

    Article Snippet: Reverse transcription products were diluted to 50 μl and 5 μl of diluted sample used in single qPCR reactions, with a total volume of 20 μl. qRT-PCR was performed using Taqman microRNA assays (Thermo Fisher Scientific) and TaqMan universal PCR master mix II, no UNG (Thermo Fisher Scientific) on a ViiA7 Real-Time PCR system (Applied Biosystems/Thermo Fisher Scientific), using recommended PCR cycling conditions.

    Techniques: Real-time Polymerase Chain Reaction, RNA Extraction, Expressing, Quantitative RT-PCR