taqman universal master mix ii  (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:
    TaqMan Universal Master Mix II
    Description:
    Alternative Product Try TaqMan Fast Advanced Master Mix our highest performance probe based master mix With TaqMan Fast Advanced Master Mix we ve taken the best of TaqMan Universal Master Mix II and added additional capabilities for your gene expression analysis TaqMan Universal Master Mix II is a convenient mix of components except primers probes template and water necessary to perform a real time polymerase chain reaction PCR You can use TaqMan Universal Master Mix II to amplify complementary DNA cDNA and DNA targets for a variety of applications including quantitation and genotyping This TaqMan Universal Master Mix II is supplied as a 2X concentrate and contains • AmpliTaq Gold DNA Polymerase UP Ultra Pure • dNTPs with dUTP • ROX Passive Reference • Uracil N glycosylase UNG • Optimized buffer components Note This TaqMan Universal Master Mix II contains Uracil N glycoslyase UNG See a complete listing of all TaqMan Universal Master Mix II products with and without UNG here TaqMan Universal Master Mix II has been optimized for use with primers and TaqMan probes that have been designed according to Life Technologies guidelines The master mix can be used with custom TaqMan assays available from our custom assay service or with pre optimized assays such as • TaqMan Gene Expression Assays • TaqMan MicroRNA Assays • TaqMan Drug Metabolism Genotyping Assays • TaqMan SNP Genotyping Assays For RNA quantitation experiments the TaqMan Universal Master Mix II is used in the second step of a two step reverse transcription polymerase chain reaction RT PCR protocol The cDNA template used with the master mix can be generated in a reverse transcription reaction using cDNA synthesis kits available from Life Technologies
    Catalog Number:
    4440038
    Price:
    None
    Applications:
    Enzymes & Master Mixes for Real-Time PCR|PCR & Real-Time PCR|RNAi, Epigenetics & Non-Coding RNA Research|Real Time PCR (qPCR)|Real Time PCR-Based Gene Expression Profiling|Real Time PCR-Based miRNA Analysis|miRNA & Non-Coding RNA Analysis|Gene Expression Analysis & Genotyping
    Category:
    Kits and Assays
    Buy from Supplier


    Structured Review

    Thermo Fisher taqman universal master mix ii
    Fibrogenic, active LX‐2 demonstrate higher bioenergetic ratio than the less‐active LX‐2. A , Verification of the fibrogenic phenotype of active LX‐2 cells by <t>TaqMan</t> <t>qPCR</t> quantification of ColIα1 , one of the biochemical markers of fibrogenesis. B , Immunoblot showing increased expression of F 1 –F 0 ATPase in active (fibrogenic) LX‐2. TIMP‐2 confirmed the pro‐fibrogenic phenotype of active LX‐2 cells. β‐actin is shown as the loading control. C , The bar graph represents densitometry data. D , Bar graph showing the rate of 3 H‐2‐deoxyglucose uptake in less‐active and active LX‐2 cells.
    Alternative Product Try TaqMan Fast Advanced Master Mix our highest performance probe based master mix With TaqMan Fast Advanced Master Mix we ve taken the best of TaqMan Universal Master Mix II and added additional capabilities for your gene expression analysis TaqMan Universal Master Mix II is a convenient mix of components except primers probes template and water necessary to perform a real time polymerase chain reaction PCR You can use TaqMan Universal Master Mix II to amplify complementary DNA cDNA and DNA targets for a variety of applications including quantitation and genotyping This TaqMan Universal Master Mix II is supplied as a 2X concentrate and contains • AmpliTaq Gold DNA Polymerase UP Ultra Pure • dNTPs with dUTP • ROX Passive Reference • Uracil N glycosylase UNG • Optimized buffer components Note This TaqMan Universal Master Mix II contains Uracil N glycoslyase UNG See a complete listing of all TaqMan Universal Master Mix II products with and without UNG here TaqMan Universal Master Mix II has been optimized for use with primers and TaqMan probes that have been designed according to Life Technologies guidelines The master mix can be used with custom TaqMan assays available from our custom assay service or with pre optimized assays such as • TaqMan Gene Expression Assays • TaqMan MicroRNA Assays • TaqMan Drug Metabolism Genotyping Assays • TaqMan SNP Genotyping Assays For RNA quantitation experiments the TaqMan Universal Master Mix II is used in the second step of a two step reverse transcription polymerase chain reaction RT PCR protocol The cDNA template used with the master mix can be generated in a reverse transcription reaction using cDNA synthesis kits available from Life Technologies
    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

    Images

    1) Product Images from "Elevated mitochondrial activity distinguishes fibrogenic hepatic stellate cells and sensitizes for selective inhibition by mitotropic doxorubicin"

    Article Title: Elevated mitochondrial activity distinguishes fibrogenic hepatic stellate cells and sensitizes for selective inhibition by mitotropic doxorubicin

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.13501

    Fibrogenic, active LX‐2 demonstrate higher bioenergetic ratio than the less‐active LX‐2. A , Verification of the fibrogenic phenotype of active LX‐2 cells by TaqMan qPCR quantification of ColIα1 , one of the biochemical markers of fibrogenesis. B , Immunoblot showing increased expression of F 1 –F 0 ATPase in active (fibrogenic) LX‐2. TIMP‐2 confirmed the pro‐fibrogenic phenotype of active LX‐2 cells. β‐actin is shown as the loading control. C , The bar graph represents densitometry data. D , Bar graph showing the rate of 3 H‐2‐deoxyglucose uptake in less‐active and active LX‐2 cells.
    Figure Legend Snippet: Fibrogenic, active LX‐2 demonstrate higher bioenergetic ratio than the less‐active LX‐2. A , Verification of the fibrogenic phenotype of active LX‐2 cells by TaqMan qPCR quantification of ColIα1 , one of the biochemical markers of fibrogenesis. B , Immunoblot showing increased expression of F 1 –F 0 ATPase in active (fibrogenic) LX‐2. TIMP‐2 confirmed the pro‐fibrogenic phenotype of active LX‐2 cells. β‐actin is shown as the loading control. C , The bar graph represents densitometry data. D , Bar graph showing the rate of 3 H‐2‐deoxyglucose uptake in less‐active and active LX‐2 cells.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    Fibrogenic, active HSCs demonstrate higher bioenergetic ratio than the less‐active HSCs ex vivo . A , TaqMan qPCR quantification of ColIα1 expression verified the fibrogenic phenotype of HSCs ex vivo , from the mouse and rat models of BDL fibrosis. B , TaqMan qPCR data showing increased bioenergetic ratio of fibrogenic HSCs ex vivo , from the mouse and rat models of BDL fibrosis. Data represent Mean ± S.E. ( n = 3).
    Figure Legend Snippet: Fibrogenic, active HSCs demonstrate higher bioenergetic ratio than the less‐active HSCs ex vivo . A , TaqMan qPCR quantification of ColIα1 expression verified the fibrogenic phenotype of HSCs ex vivo , from the mouse and rat models of BDL fibrosis. B , TaqMan qPCR data showing increased bioenergetic ratio of fibrogenic HSCs ex vivo , from the mouse and rat models of BDL fibrosis. Data represent Mean ± S.E. ( n = 3).

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

    2) Product Images from "Prevalence of HMTV in breast carcinomas and unaffected tissue from Mexican women"

    Article Title: Prevalence of HMTV in breast carcinomas and unaffected tissue from Mexican women

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-14-942

    Real-time PCR. The plot shows the amplification obtained using TaqMan probes with the same DNA sources as those used for the nested PCR that yielded positive results. The probes are based in a different region of the env gene, namely from 5943 to 6011 (AF033807). Each line represents a sample, positive control, or negative control. The PCR cycles are shown on the x-axis, and the ΔRn values (representing the fluorescent units) are shown on the y-axis. All of the assays were performed in 48-well plates.
    Figure Legend Snippet: Real-time PCR. The plot shows the amplification obtained using TaqMan probes with the same DNA sources as those used for the nested PCR that yielded positive results. The probes are based in a different region of the env gene, namely from 5943 to 6011 (AF033807). Each line represents a sample, positive control, or negative control. The PCR cycles are shown on the x-axis, and the ΔRn values (representing the fluorescent units) are shown on the y-axis. All of the assays were performed in 48-well plates.

    Techniques Used: Real-time Polymerase Chain Reaction, Amplification, Nested PCR, Positive Control, Negative Control, Polymerase Chain Reaction

    3) Product Images from "Cholangiocarcinoma therapy with nanoparticles that combine downregulation of MicroRNA-210 with inhibition of cancer cell invasiveness"

    Article Title: Cholangiocarcinoma therapy with nanoparticles that combine downregulation of MicroRNA-210 with inhibition of cancer cell invasiveness

    Journal: Theranostics

    doi: 10.7150/thno.26506

    Effective delivery of anti-miRNA to CCA cells. (A) Confocal microscopy images of Mz-ChA-1 cells incubated with AF647-PCX/FAM-anti-miRNA for 4 h. Scale bar 20 µm. (B) Flow cytometry analysis of cells treated with AF647-PCX/FAM-anti-miRNA for 4 h. (C) Intracellular trafficking of AF647-PCX/anti-miRNA in cells after 4 h of incubation. Scale bar 20 µm. (D) MiR-210 expression in normoxic and hypoxic conditions measured by TaqMan qRT-PCR in Mz-ChA-1 cells after treatment with PCX/anti-miRNA for 48 h. Data are shown as mean ± SD (n = 3). ** P
    Figure Legend Snippet: Effective delivery of anti-miRNA to CCA cells. (A) Confocal microscopy images of Mz-ChA-1 cells incubated with AF647-PCX/FAM-anti-miRNA for 4 h. Scale bar 20 µm. (B) Flow cytometry analysis of cells treated with AF647-PCX/FAM-anti-miRNA for 4 h. (C) Intracellular trafficking of AF647-PCX/anti-miRNA in cells after 4 h of incubation. Scale bar 20 µm. (D) MiR-210 expression in normoxic and hypoxic conditions measured by TaqMan qRT-PCR in Mz-ChA-1 cells after treatment with PCX/anti-miRNA for 48 h. Data are shown as mean ± SD (n = 3). ** P

    Techniques Used: Confocal Microscopy, Incubation, Flow Cytometry, Cytometry, Expressing, Quantitative RT-PCR

    4) Product Images from "miR-182 Regulates Slit2-Mediated Axon Guidance by Modulating the Local Translation of a Specific mRNA"

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

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2016.12.093

    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 .
    Figure Legend 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 .

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

    5) Product Images from "Dual Action of miR-125b As a Tumor Suppressor and OncomiR-22 Promotes Prostate Cancer Tumorigenesis"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0142373

    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.
    Figure Legend 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.

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

    6) Product Images from "MicroRNA-1 inhibits tumorigenicity of esophageal squamous cell carcinoma and enhances sensitivity to gefitinib"

    Article Title: MicroRNA-1 inhibits tumorigenicity of esophageal squamous cell carcinoma and enhances sensitivity to gefitinib

    Journal: Oncology Letters

    doi: 10.3892/ol.2017.7378

    Inverse correlation between the levels of miR-1 expression and PIK3CA expression in ESCC tissues. (A) The relative level of miR-1 expression was analyzed by TaqMan RT-qPCR in 74 ESCC tissues and corresponding non-tumor tissues. (B) The level of PIK3CA mRNA expression was analyzed by RT-qPCR in 74 ESCC tissues and corresponding non-tumor tissues. (C) Correlation between the levels of miR-1 and PIK3CA mRNA expression in ESCC tissues. Data are presented as the mean ± standard error from three independent experiments. **P
    Figure Legend Snippet: Inverse correlation between the levels of miR-1 expression and PIK3CA expression in ESCC tissues. (A) The relative level of miR-1 expression was analyzed by TaqMan RT-qPCR in 74 ESCC tissues and corresponding non-tumor tissues. (B) The level of PIK3CA mRNA expression was analyzed by RT-qPCR in 74 ESCC tissues and corresponding non-tumor tissues. (C) Correlation between the levels of miR-1 and PIK3CA mRNA expression in ESCC tissues. Data are presented as the mean ± standard error from three independent experiments. **P

    Techniques Used: Expressing, Quantitative RT-PCR

    Expression of miR-1 and PIK3CA in transfected cells. TE-1 cells were transiently transfected with miR-1 mimics (50 nM) or the negative control. The cells were obtained after 48 h for analysis. (A) TaqMan RT-qPCR detection of miR-1 expression levels in TE-1 cells. (B) The level of PIK3CA mRNA expression was detected by RT-qPCR in TE-1 cells. (C) Cell lysates were prepared and used for western blotting with antibodies specific for PIK3CA, total Akt, p-Akt and survivin. Data are presented as the mean ± standard error from three independent experiments. **P
    Figure Legend Snippet: Expression of miR-1 and PIK3CA in transfected cells. TE-1 cells were transiently transfected with miR-1 mimics (50 nM) or the negative control. The cells were obtained after 48 h for analysis. (A) TaqMan RT-qPCR detection of miR-1 expression levels in TE-1 cells. (B) The level of PIK3CA mRNA expression was detected by RT-qPCR in TE-1 cells. (C) Cell lysates were prepared and used for western blotting with antibodies specific for PIK3CA, total Akt, p-Akt and survivin. Data are presented as the mean ± standard error from three independent experiments. **P

    Techniques Used: Expressing, Transfection, Negative Control, Quantitative RT-PCR, Western Blot

    7) Product Images from "Chloroquine-containing DMAEMA copolymers as efficient anti-miRNA delivery vectors with improved endosomal escape and anti-migratory activity in cancer cells"

    Article Title: Chloroquine-containing DMAEMA copolymers as efficient anti-miRNA delivery vectors with improved endosomal escape and anti-migratory activity in cancer cells

    Journal: Macromolecular bioscience

    doi: 10.1002/mabi.201700194

    Transfection activity of polyplexes (N:P=10, anti-miRNA 100 nM). MiR-210 level was detected by TaqMan qRT-PCR in MDA-MB 231 cells. Data are shown as mean ± SD (n = 3). **P
    Figure Legend Snippet: Transfection activity of polyplexes (N:P=10, anti-miRNA 100 nM). MiR-210 level was detected by TaqMan qRT-PCR in MDA-MB 231 cells. Data are shown as mean ± SD (n = 3). **P

    Techniques Used: Transfection, Activity Assay, Quantitative RT-PCR, Multiple Displacement Amplification

    8) Product Images from "microRNA regulatory circuits in a mouse model of inherited retinal degeneration"

    Article Title: microRNA regulatory circuits in a mouse model of inherited retinal degeneration

    Journal: Scientific Reports

    doi: 10.1038/srep31431

    In vivo Rac1-miR-CATCH. ( a ) Schematic representation of miR-CATCH 24 . First, active mRNA:miRISC complexes are cross-linked using formaldehyde fixation. Cells are lysed and capture oligonucleotide probes (complexed with metal beads) are hybridized to target mRNAs of interest. Next, captured mRNAs with bound miRISC complexes are pulled down using magnetic separation. Unbound mRNAs are washed away resulting in enrichment of target mRNA:miRISCs complexes. Finally, cross-links are reversed and capture oligonucleotides removed enabling evaluation of target mRNA and the captured targeting miRNAs. ( b ) Rac1-miR-CATCH was performed using C9 plus C10 capture oligonucleotides (Capture; n = 3) or scrambled control oligonucleotide (Scrambled; n = 3). Total RNA was purified from the samples and Rac1, Ttc21b, Folr1, Tmed5 and Plxna4 mRNAs were quantified by RT-qPCR (n = 3); note that the y-axis is in log scale. ( c ) Expression of miR-96 and miR-182 was analysed using Exiqon rodent miRNA PCR panel (PCR Panel; n = 2) and Applied Biosystem TaqMan microRNA Assays (TaqMan; n = 3). As miR-96 and miR-182 target the same site (TargetScan) 4 , the combined miR-96 and miR-182 levels are also given (miR-96/182 target site). *p
    Figure Legend Snippet: In vivo Rac1-miR-CATCH. ( a ) Schematic representation of miR-CATCH 24 . First, active mRNA:miRISC complexes are cross-linked using formaldehyde fixation. Cells are lysed and capture oligonucleotide probes (complexed with metal beads) are hybridized to target mRNAs of interest. Next, captured mRNAs with bound miRISC complexes are pulled down using magnetic separation. Unbound mRNAs are washed away resulting in enrichment of target mRNA:miRISCs complexes. Finally, cross-links are reversed and capture oligonucleotides removed enabling evaluation of target mRNA and the captured targeting miRNAs. ( b ) Rac1-miR-CATCH was performed using C9 plus C10 capture oligonucleotides (Capture; n = 3) or scrambled control oligonucleotide (Scrambled; n = 3). Total RNA was purified from the samples and Rac1, Ttc21b, Folr1, Tmed5 and Plxna4 mRNAs were quantified by RT-qPCR (n = 3); note that the y-axis is in log scale. ( c ) Expression of miR-96 and miR-182 was analysed using Exiqon rodent miRNA PCR panel (PCR Panel; n = 2) and Applied Biosystem TaqMan microRNA Assays (TaqMan; n = 3). As miR-96 and miR-182 target the same site (TargetScan) 4 , the combined miR-96 and miR-182 levels are also given (miR-96/182 target site). *p

    Techniques Used: In Vivo, Purification, Quantitative RT-PCR, Expressing, Polymerase Chain Reaction

    9) Product Images from "CIC protein instability contributes to tumorigenesis in glioblastoma"

    Article Title: CIC protein instability contributes to tumorigenesis in glioblastoma

    Journal: Nature Communications

    doi: 10.1038/s41467-018-08087-9

    Expression of CIC and its targets in human GBM tumors and cells. Human operative GBM samples or normal derived brains (NB) were lysed and a immunoblotted with indicated antibodies b or total RNA extracted and quantitative real-time PCR analysis was carried out using TaqMan gene expression assays. The graph depicts fold changes in CIC expression relative to normal brain. c Nuclear or cytoplasmic lysates were isolated from human operative GBM samples or normal brain and were immunoblotted with indicated antibodies. d Human operative astrocytoma samples were lysed and immunoblotted with indicated antibodies. Human-derived GBM cell lines or normal human astrocytes (NHA) were lysed and e protein lysates were immunoblotted with indicated antibodies ( f ) or total RNA extracted and quantitative real-time PCR analysis was carried out using TaqMan gene expression assays. The graph depicts fold changes in CIC expression relative to NHA. Data represent mean ± s.e.m. of three independent experiments performed in triplicate. * P
    Figure Legend Snippet: Expression of CIC and its targets in human GBM tumors and cells. Human operative GBM samples or normal derived brains (NB) were lysed and a immunoblotted with indicated antibodies b or total RNA extracted and quantitative real-time PCR analysis was carried out using TaqMan gene expression assays. The graph depicts fold changes in CIC expression relative to normal brain. c Nuclear or cytoplasmic lysates were isolated from human operative GBM samples or normal brain and were immunoblotted with indicated antibodies. d Human operative astrocytoma samples were lysed and immunoblotted with indicated antibodies. Human-derived GBM cell lines or normal human astrocytes (NHA) were lysed and e protein lysates were immunoblotted with indicated antibodies ( f ) or total RNA extracted and quantitative real-time PCR analysis was carried out using TaqMan gene expression assays. The graph depicts fold changes in CIC expression relative to NHA. Data represent mean ± s.e.m. of three independent experiments performed in triplicate. * P

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

    10) Product Images from "The Potential of TaqMan Array Cards for Detection of Multiple Biological Agents by Real-Time PCR"

    Article Title: The Potential of TaqMan Array Cards for Detection of Multiple Biological Agents by Real-Time PCR

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0035971

    Quantification of B. anthracis (Ames) DNA by pXO2 MGB PCR replicates incorporated within the TaqMan Array Cards. Trendline and R2 value generated from mean CT values by Microsoft Excel.
    Figure Legend Snippet: Quantification of B. anthracis (Ames) DNA by pXO2 MGB PCR replicates incorporated within the TaqMan Array Cards. Trendline and R2 value generated from mean CT values by Microsoft Excel.

    Techniques Used: Polymerase Chain Reaction, Generated

    11) Product Images from "Mapping the Pax6 3’ untranslated region microRNA regulatory landscape"

    Article Title: Mapping the Pax6 3’ untranslated region microRNA regulatory landscape

    Journal: BMC Genomics

    doi: 10.1186/s12864-018-5212-x

    miTRAP as a strategy to purify Pax6 3’UTR-associated miRNAs. a Schematic of the Pax6 3’UTR affinity purification approach. (i) Plasmid vectors expressing GFP tagged with the MS2 RNA sequence motif followed by the SV40 polyadenylation signal are introduced into pancreatic αTC1–6 cells via transient transfection. (ii) MS2 coat protein fused to maltose binding (MS2-MBP) is used to purify GFP transcripts with bound miRNAs from αTC1–6 cell lysate. (iii) Real-time quantitative PCR (RT-qPCR) is used to detect GFP transcript and bound miRNAs. Schematic of the Pax6 3’UTR shows the location of the highly conserved miR-375 target site located at 3’UTR position 201 and miR-375 target site mutation. b Validation of the MS2-mediated affinity purification strategy by RT-qPCR quantification of GFP transcripts with and without the MS2 RNA motif. Fold change was calculated using Pfaffl’s method [ 61 ]. qPCR results for GFP with the MS2 motif (grey bar) were expressed relative to data without the MS2 motif (unfilled bar). Data represents 5 independent samples, p = 0.0079. c Affinity purification of miR-375 with the Pax6 3’UTR in αTC1–6 cells using TaqMan individual qPCR assays. Normalized relative quantity was calculated using Pfaffl’s method, and a normalized relative quantity greater than 1 indicates that more target miRNA is purified with the Pax6 3’UTR (grey bar) than the control lacking a Pax6 3’UTR (unfilled bar). Data represents six independent samples, p = 0.013. d Disruption of miR-375 binding to the Pax6 3’UTR following mutation of the miR-375 target site. Target miR-375 values were normalized to GFP as a reference gene, then normalized values for the mutant Pax6 3’UTR samples (grey bar), and are presented relative to the wt 3’UTR (unfilled bar). Data represents six independent wt 3’UTR samples and three miR-375 target site mutant 3’UTR samples, p = 0.0476. Error bars represent 95% confidence intervals, and p -values were calculated using the Mann Whitney test. Note scale bar differences between the graphs
    Figure Legend Snippet: miTRAP as a strategy to purify Pax6 3’UTR-associated miRNAs. a Schematic of the Pax6 3’UTR affinity purification approach. (i) Plasmid vectors expressing GFP tagged with the MS2 RNA sequence motif followed by the SV40 polyadenylation signal are introduced into pancreatic αTC1–6 cells via transient transfection. (ii) MS2 coat protein fused to maltose binding (MS2-MBP) is used to purify GFP transcripts with bound miRNAs from αTC1–6 cell lysate. (iii) Real-time quantitative PCR (RT-qPCR) is used to detect GFP transcript and bound miRNAs. Schematic of the Pax6 3’UTR shows the location of the highly conserved miR-375 target site located at 3’UTR position 201 and miR-375 target site mutation. b Validation of the MS2-mediated affinity purification strategy by RT-qPCR quantification of GFP transcripts with and without the MS2 RNA motif. Fold change was calculated using Pfaffl’s method [ 61 ]. qPCR results for GFP with the MS2 motif (grey bar) were expressed relative to data without the MS2 motif (unfilled bar). Data represents 5 independent samples, p = 0.0079. c Affinity purification of miR-375 with the Pax6 3’UTR in αTC1–6 cells using TaqMan individual qPCR assays. Normalized relative quantity was calculated using Pfaffl’s method, and a normalized relative quantity greater than 1 indicates that more target miRNA is purified with the Pax6 3’UTR (grey bar) than the control lacking a Pax6 3’UTR (unfilled bar). Data represents six independent samples, p = 0.013. d Disruption of miR-375 binding to the Pax6 3’UTR following mutation of the miR-375 target site. Target miR-375 values were normalized to GFP as a reference gene, then normalized values for the mutant Pax6 3’UTR samples (grey bar), and are presented relative to the wt 3’UTR (unfilled bar). Data represents six independent wt 3’UTR samples and three miR-375 target site mutant 3’UTR samples, p = 0.0476. Error bars represent 95% confidence intervals, and p -values were calculated using the Mann Whitney test. Note scale bar differences between the graphs

    Techniques Used: Affinity Purification, Plasmid Preparation, Expressing, Sequencing, Transfection, Binding Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Mutagenesis, Purification, MANN-WHITNEY

    Characterization of miRNAs bound to the Pax6 3’UTR in pancreatic α cells. a Identification of miRNAs associated with the Pax6 3’UTR in αTC1–6 cells using TaqMan multiplex qPCR arrays. Normalized relative quantity (NRQ) greater than 1 indicates more target miRNA was purified with the Pax6 3’UTR than the control lacking a Pax6 3’UTR. Some miRNAs expressed in α cells that also have predicted MREs in the Pax6 3’UTR did not associate with the Pax6 3’UTR-containing transcript: miR-101, 1187, 144, 15a, 196b, 335-3p, 362-3p, 365, 410, 466f, 466 k, 495, 501–3p, 96. Results represent four independent experiments. Geometric mean +/− 95% confidence intervals are shown. b Landscape of Pax6 3’UTR miRNA interaction in αTC1–6 cells. Average NRQ value for each interacting miRNA is indicated as a peak at the 3’UTR position(s) of the predicted MRE(s). Predicted MREs for interacting miRNAs are labeled in black, and non-interacting miRNAs in grey. Each peak has a 25-nucleotide width on either side of the MRE position. Overlap between interaction peaks spaced 8–50 nucleotides apart is indicated in red, and these interacting miRNAs may be capable of mediating cooperative regulation of Pax6. MREs for Pax6 3’UTR-interacting miRNAs were found to cluster into three regions, i-iii (orange boxes), located approximately at nucleotide positions 250–350, 420–550 and 600–810. Conservation of the Pax6 3’UTR sequence between orthologous placental mammal sequences is shown for the 876-nucleotide length. Secondary, poorly conserved MREs for interacting miRNAs having well conserved predicted MREs are not shown. c miRNA interaction with the Pax6 3’UTR is not directly related to miRNA abundance in αTC1–6 cells. miTRAP ratio and relative miRNA level for αTC1–6-interacting miRNAs is shown. miTRAP ratio was calculated by dividing the relative abundance of each miRNA with the Pax6 3’UTR by the relative abundance in αTC1–6 cells. Larger miTRAP values indicated greater enrichment of the miRNA with the Pax6 3’UTR relative to cellular abundance
    Figure Legend Snippet: Characterization of miRNAs bound to the Pax6 3’UTR in pancreatic α cells. a Identification of miRNAs associated with the Pax6 3’UTR in αTC1–6 cells using TaqMan multiplex qPCR arrays. Normalized relative quantity (NRQ) greater than 1 indicates more target miRNA was purified with the Pax6 3’UTR than the control lacking a Pax6 3’UTR. Some miRNAs expressed in α cells that also have predicted MREs in the Pax6 3’UTR did not associate with the Pax6 3’UTR-containing transcript: miR-101, 1187, 144, 15a, 196b, 335-3p, 362-3p, 365, 410, 466f, 466 k, 495, 501–3p, 96. Results represent four independent experiments. Geometric mean +/− 95% confidence intervals are shown. b Landscape of Pax6 3’UTR miRNA interaction in αTC1–6 cells. Average NRQ value for each interacting miRNA is indicated as a peak at the 3’UTR position(s) of the predicted MRE(s). Predicted MREs for interacting miRNAs are labeled in black, and non-interacting miRNAs in grey. Each peak has a 25-nucleotide width on either side of the MRE position. Overlap between interaction peaks spaced 8–50 nucleotides apart is indicated in red, and these interacting miRNAs may be capable of mediating cooperative regulation of Pax6. MREs for Pax6 3’UTR-interacting miRNAs were found to cluster into three regions, i-iii (orange boxes), located approximately at nucleotide positions 250–350, 420–550 and 600–810. Conservation of the Pax6 3’UTR sequence between orthologous placental mammal sequences is shown for the 876-nucleotide length. Secondary, poorly conserved MREs for interacting miRNAs having well conserved predicted MREs are not shown. c miRNA interaction with the Pax6 3’UTR is not directly related to miRNA abundance in αTC1–6 cells. miTRAP ratio and relative miRNA level for αTC1–6-interacting miRNAs is shown. miTRAP ratio was calculated by dividing the relative abundance of each miRNA with the Pax6 3’UTR by the relative abundance in αTC1–6 cells. Larger miTRAP values indicated greater enrichment of the miRNA with the Pax6 3’UTR relative to cellular abundance

    Techniques Used: Multiplex Assay, Real-time Polymerase Chain Reaction, Purification, Labeling, Sequencing

    Expression profile of miRNAs predicted to target the mouse Pax6 3’UTR. a Expression profile of miRNAs having predicted target sites in the mouse Pax6 3’UTR in various Pax6 -expressing cells and tissues. miRNAs assayed for include those identified by our prediction analysis (Fig. 3a ) as well as others, as described in the results. miRNAs were assayed for using TaqMan multiplex qPCR array cards. Data represents a total of 3 replicates per tissue or cell type, with a miRNA considered to be expressed only if the cycle threshold was less than 40 for all three replicates. b Relative miRNA level in mouse cultured pancreatic α cell line, αTC1–6 and a cultured β cell line, βTC6. c Relative miRNA level in mouse E12.5 retina, adult retina, and adult lens. Heat map indicates relative miRNA expression as a percentage of an internal control, snRNA U6. Data represents the geometric mean of three independent samples. Note the scale differences between the two heat maps
    Figure Legend Snippet: Expression profile of miRNAs predicted to target the mouse Pax6 3’UTR. a Expression profile of miRNAs having predicted target sites in the mouse Pax6 3’UTR in various Pax6 -expressing cells and tissues. miRNAs assayed for include those identified by our prediction analysis (Fig. 3a ) as well as others, as described in the results. miRNAs were assayed for using TaqMan multiplex qPCR array cards. Data represents a total of 3 replicates per tissue or cell type, with a miRNA considered to be expressed only if the cycle threshold was less than 40 for all three replicates. b Relative miRNA level in mouse cultured pancreatic α cell line, αTC1–6 and a cultured β cell line, βTC6. c Relative miRNA level in mouse E12.5 retina, adult retina, and adult lens. Heat map indicates relative miRNA expression as a percentage of an internal control, snRNA U6. Data represents the geometric mean of three independent samples. Note the scale differences between the two heat maps

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

    12) Product Images from "Genome-wide mRNA and miRNA expression profiling reveal multiple regulatory networks in colorectal cancer"

    Article Title: Genome-wide mRNA and miRNA expression profiling reveal multiple regulatory networks in colorectal cancer

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2014.556

    miRNA expression profiling in CRC. ( a ) Hierarchical clustering of 13 colon cancer and 13 normal tissue samples based on miRNA expression levels. Each column represents a sample and each row represents a transcript. Expression level of each miRNA in a single sample is depicted according to the color scale. ( b ) Pie chart illustrating the distribution of the top 20 pathway designations for predicted targets (TargetScan) for the downregulated miRNAs in colon cancer. The pie size corresponds to the number of matched entities. ( c ) Venn diagram depicting the overlap between the predicted gene targets for the downregulated miRNAs (based on TargetScan) versus the differentially upregulated genes in CRC identified in the current study. ( d ) Expression levels of selected miRNAs (hsa-miR-145-5p, hsa-miR-26a-5p, and hsa-miR-30-5p) based on microarray data and validation of those miRNAs using Taqman qRT-PCR (duplicate). ** P
    Figure Legend Snippet: miRNA expression profiling in CRC. ( a ) Hierarchical clustering of 13 colon cancer and 13 normal tissue samples based on miRNA expression levels. Each column represents a sample and each row represents a transcript. Expression level of each miRNA in a single sample is depicted according to the color scale. ( b ) Pie chart illustrating the distribution of the top 20 pathway designations for predicted targets (TargetScan) for the downregulated miRNAs in colon cancer. The pie size corresponds to the number of matched entities. ( c ) Venn diagram depicting the overlap between the predicted gene targets for the downregulated miRNAs (based on TargetScan) versus the differentially upregulated genes in CRC identified in the current study. ( d ) Expression levels of selected miRNAs (hsa-miR-145-5p, hsa-miR-26a-5p, and hsa-miR-30-5p) based on microarray data and validation of those miRNAs using Taqman qRT-PCR (duplicate). ** P

    Techniques Used: Expressing, Microarray, Quantitative RT-PCR

    13) Product Images from "Participation of the miR-22-HDAC4-DLCO Axis in Patients with COPD by Tobacco and Biomass"

    Article Title: Participation of the miR-22-HDAC4-DLCO Axis in Patients with COPD by Tobacco and Biomass

    Journal: Biomolecules

    doi: 10.3390/biom9120837

    miRNA-22 is upregulated in chronic obstructive pulmonary disease (COPD) by biomass related to COPD by tobacco. The cDNAs were obtained using the RT kit and TaqMan Universal Master Mix II with UNG (Uracil-N-glycosylate; Applied Biosystems-Thermo Fisher Scientific). The miRNA validated by RT-qPCR was miR-22-3p, downregulated in COPD-BS compared to COPD-TS ( n = 25), ( p
    Figure Legend Snippet: miRNA-22 is upregulated in chronic obstructive pulmonary disease (COPD) by biomass related to COPD by tobacco. The cDNAs were obtained using the RT kit and TaqMan Universal Master Mix II with UNG (Uracil-N-glycosylate; Applied Biosystems-Thermo Fisher Scientific). The miRNA validated by RT-qPCR was miR-22-3p, downregulated in COPD-BS compared to COPD-TS ( n = 25), ( p

    Techniques Used: Quantitative RT-PCR

    14) Product Images from "BLV-CoCoMo-qPCR: a useful tool for evaluating bovine leukemia virus infection status"

    Article Title: BLV-CoCoMo-qPCR: a useful tool for evaluating bovine leukemia virus infection status

    Journal: BMC Veterinary Research

    doi: 10.1186/1746-6148-8-167

    Sensitivity and reproducibility of each real-time PCR method using a pBLV-IF. The copy number of pBLV-IF was determined by calculation and TaKaRa Cycleave PCR. One hundred copy of pBLV-IF was diluted 2-fold to construct the standard curve. Threshold values (Ct) were plotted against corresponding pBLV-IF copy numbers and the correlation coefficient (R 2 ) was determined. The experiments were run in duplicate and independently repeated three times with the same dilutions. pBLV-IF standard curves were generated by using the results of CoCoMo-qPCR ( A ), the TaqMan MGB assay developed by Lew et al. ( B ), and TaKaRa Cycleave PCR ( C ).
    Figure Legend Snippet: Sensitivity and reproducibility of each real-time PCR method using a pBLV-IF. The copy number of pBLV-IF was determined by calculation and TaKaRa Cycleave PCR. One hundred copy of pBLV-IF was diluted 2-fold to construct the standard curve. Threshold values (Ct) were plotted against corresponding pBLV-IF copy numbers and the correlation coefficient (R 2 ) was determined. The experiments were run in duplicate and independently repeated three times with the same dilutions. pBLV-IF standard curves were generated by using the results of CoCoMo-qPCR ( A ), the TaqMan MGB assay developed by Lew et al. ( B ), and TaKaRa Cycleave PCR ( C ).

    Techniques Used: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Construct, Generated

    15) Product Images from "16S rRNA gene pyrosequencing of reference and clinical samples and investigation of the temperature stability of microbiome profiles"

    Article Title: 16S rRNA gene pyrosequencing of reference and clinical samples and investigation of the temperature stability of microbiome profiles

    Journal: Microbiome

    doi: 10.1186/2049-2618-2-31

    Normalization of PCR amplification by choosing PCR cycle number close to the Ct value. Throat swabs from healthy volunteers were extracted with Qiagen DNeasy (D) or MO BIO PowerSoil (M). DNA extracts were subjected to 16S gene TaqMan qPCR and subsequent PCR using a cycle number of 20, 25, or 30 based on individual sample's qPCR Ct value. PCR amplicons were quantified by PicoGreen dsDNA assay. DNA concentrations for DNA extracts ( blue markers and axis ) and PCR amplicons ( red markers and axis ) are shown in 16S gene copies/μl in the same scale. DNA sample name, 08S1M as an example, depicts the subject number (08), swab number (S1), and extraction method (M).
    Figure Legend Snippet: Normalization of PCR amplification by choosing PCR cycle number close to the Ct value. Throat swabs from healthy volunteers were extracted with Qiagen DNeasy (D) or MO BIO PowerSoil (M). DNA extracts were subjected to 16S gene TaqMan qPCR and subsequent PCR using a cycle number of 20, 25, or 30 based on individual sample's qPCR Ct value. PCR amplicons were quantified by PicoGreen dsDNA assay. DNA concentrations for DNA extracts ( blue markers and axis ) and PCR amplicons ( red markers and axis ) are shown in 16S gene copies/μl in the same scale. DNA sample name, 08S1M as an example, depicts the subject number (08), swab number (S1), and extraction method (M).

    Techniques Used: Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction, Picogreen Assay

    16) Product Images from "Mapping the Pax6 3’ untranslated region microRNA regulatory landscape"

    Article Title: Mapping the Pax6 3’ untranslated region microRNA regulatory landscape

    Journal: BMC Genomics

    doi: 10.1186/s12864-018-5212-x

    miTRAP as a strategy to purify Pax6 3’UTR-associated miRNAs. a Schematic of the Pax6 3’UTR affinity purification approach. (i) Plasmid vectors expressing GFP tagged with the MS2 RNA sequence motif followed by the SV40 polyadenylation signal are introduced into pancreatic αTC1–6 cells via transient transfection. (ii) MS2 coat protein fused to maltose binding (MS2-MBP) is used to purify GFP transcripts with bound miRNAs from αTC1–6 cell lysate. (iii) Real-time quantitative PCR (RT-qPCR) is used to detect GFP transcript and bound miRNAs. Schematic of the Pax6 3’UTR shows the location of the highly conserved miR-375 target site located at 3’UTR position 201 and miR-375 target site mutation. b Validation of the MS2-mediated affinity purification strategy by RT-qPCR quantification of GFP transcripts with and without the MS2 RNA motif. Fold change was calculated using Pfaffl’s method [ 61 ]. qPCR results for GFP with the MS2 motif (grey bar) were expressed relative to data without the MS2 motif (unfilled bar). Data represents 5 independent samples, p = 0.0079. c Affinity purification of miR-375 with the Pax6 3’UTR in αTC1–6 cells using TaqMan individual qPCR assays. Normalized relative quantity was calculated using Pfaffl’s method, and a normalized relative quantity greater than 1 indicates that more target miRNA is purified with the Pax6 3’UTR (grey bar) than the control lacking a Pax6 3’UTR (unfilled bar). Data represents six independent samples, p = 0.013. d Disruption of miR-375 binding to the Pax6 3’UTR following mutation of the miR-375 target site. Target miR-375 values were normalized to GFP as a reference gene, then normalized values for the mutant Pax6 3’UTR samples (grey bar), and are presented relative to the wt 3’UTR (unfilled bar). Data represents six independent wt 3’UTR samples and three miR-375 target site mutant 3’UTR samples, p = 0.0476. Error bars represent 95% confidence intervals, and p -values were calculated using the Mann Whitney test. Note scale bar differences between the graphs
    Figure Legend Snippet: miTRAP as a strategy to purify Pax6 3’UTR-associated miRNAs. a Schematic of the Pax6 3’UTR affinity purification approach. (i) Plasmid vectors expressing GFP tagged with the MS2 RNA sequence motif followed by the SV40 polyadenylation signal are introduced into pancreatic αTC1–6 cells via transient transfection. (ii) MS2 coat protein fused to maltose binding (MS2-MBP) is used to purify GFP transcripts with bound miRNAs from αTC1–6 cell lysate. (iii) Real-time quantitative PCR (RT-qPCR) is used to detect GFP transcript and bound miRNAs. Schematic of the Pax6 3’UTR shows the location of the highly conserved miR-375 target site located at 3’UTR position 201 and miR-375 target site mutation. b Validation of the MS2-mediated affinity purification strategy by RT-qPCR quantification of GFP transcripts with and without the MS2 RNA motif. Fold change was calculated using Pfaffl’s method [ 61 ]. qPCR results for GFP with the MS2 motif (grey bar) were expressed relative to data without the MS2 motif (unfilled bar). Data represents 5 independent samples, p = 0.0079. c Affinity purification of miR-375 with the Pax6 3’UTR in αTC1–6 cells using TaqMan individual qPCR assays. Normalized relative quantity was calculated using Pfaffl’s method, and a normalized relative quantity greater than 1 indicates that more target miRNA is purified with the Pax6 3’UTR (grey bar) than the control lacking a Pax6 3’UTR (unfilled bar). Data represents six independent samples, p = 0.013. d Disruption of miR-375 binding to the Pax6 3’UTR following mutation of the miR-375 target site. Target miR-375 values were normalized to GFP as a reference gene, then normalized values for the mutant Pax6 3’UTR samples (grey bar), and are presented relative to the wt 3’UTR (unfilled bar). Data represents six independent wt 3’UTR samples and three miR-375 target site mutant 3’UTR samples, p = 0.0476. Error bars represent 95% confidence intervals, and p -values were calculated using the Mann Whitney test. Note scale bar differences between the graphs

    Techniques Used: Affinity Purification, Plasmid Preparation, Expressing, Sequencing, Transfection, Binding Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Mutagenesis, Purification, MANN-WHITNEY

    Characterization of miRNAs bound to the Pax6 3’UTR in pancreatic α cells. a Identification of miRNAs associated with the Pax6 3’UTR in αTC1–6 cells using TaqMan multiplex qPCR arrays. Normalized relative quantity (NRQ) greater than 1 indicates more target miRNA was purified with the Pax6 3’UTR than the control lacking a Pax6 3’UTR. Some miRNAs expressed in α cells that also have predicted MREs in the Pax6 3’UTR did not associate with the Pax6 3’UTR-containing transcript: miR-101, 1187, 144, 15a, 196b, 335-3p, 362-3p, 365, 410, 466f, 466 k, 495, 501–3p, 96. Results represent four independent experiments. Geometric mean +/− 95% confidence intervals are shown. b Landscape of Pax6 3’UTR miRNA interaction in αTC1–6 cells. Average NRQ value for each interacting miRNA is indicated as a peak at the 3’UTR position(s) of the predicted MRE(s). Predicted MREs for interacting miRNAs are labeled in black, and non-interacting miRNAs in grey. Each peak has a 25-nucleotide width on either side of the MRE position. Overlap between interaction peaks spaced 8–50 nucleotides apart is indicated in red, and these interacting miRNAs may be capable of mediating cooperative regulation of Pax6. MREs for Pax6 3’UTR-interacting miRNAs were found to cluster into three regions, i-iii (orange boxes), located approximately at nucleotide positions 250–350, 420–550 and 600–810. Conservation of the Pax6 3’UTR sequence between orthologous placental mammal sequences is shown for the 876-nucleotide length. Secondary, poorly conserved MREs for interacting miRNAs having well conserved predicted MREs are not shown. c miRNA interaction with the Pax6 3’UTR is not directly related to miRNA abundance in αTC1–6 cells. miTRAP ratio and relative miRNA level for αTC1–6-interacting miRNAs is shown. miTRAP ratio was calculated by dividing the relative abundance of each miRNA with the Pax6 3’UTR by the relative abundance in αTC1–6 cells. Larger miTRAP values indicated greater enrichment of the miRNA with the Pax6 3’UTR relative to cellular abundance
    Figure Legend Snippet: Characterization of miRNAs bound to the Pax6 3’UTR in pancreatic α cells. a Identification of miRNAs associated with the Pax6 3’UTR in αTC1–6 cells using TaqMan multiplex qPCR arrays. Normalized relative quantity (NRQ) greater than 1 indicates more target miRNA was purified with the Pax6 3’UTR than the control lacking a Pax6 3’UTR. Some miRNAs expressed in α cells that also have predicted MREs in the Pax6 3’UTR did not associate with the Pax6 3’UTR-containing transcript: miR-101, 1187, 144, 15a, 196b, 335-3p, 362-3p, 365, 410, 466f, 466 k, 495, 501–3p, 96. Results represent four independent experiments. Geometric mean +/− 95% confidence intervals are shown. b Landscape of Pax6 3’UTR miRNA interaction in αTC1–6 cells. Average NRQ value for each interacting miRNA is indicated as a peak at the 3’UTR position(s) of the predicted MRE(s). Predicted MREs for interacting miRNAs are labeled in black, and non-interacting miRNAs in grey. Each peak has a 25-nucleotide width on either side of the MRE position. Overlap between interaction peaks spaced 8–50 nucleotides apart is indicated in red, and these interacting miRNAs may be capable of mediating cooperative regulation of Pax6. MREs for Pax6 3’UTR-interacting miRNAs were found to cluster into three regions, i-iii (orange boxes), located approximately at nucleotide positions 250–350, 420–550 and 600–810. Conservation of the Pax6 3’UTR sequence between orthologous placental mammal sequences is shown for the 876-nucleotide length. Secondary, poorly conserved MREs for interacting miRNAs having well conserved predicted MREs are not shown. c miRNA interaction with the Pax6 3’UTR is not directly related to miRNA abundance in αTC1–6 cells. miTRAP ratio and relative miRNA level for αTC1–6-interacting miRNAs is shown. miTRAP ratio was calculated by dividing the relative abundance of each miRNA with the Pax6 3’UTR by the relative abundance in αTC1–6 cells. Larger miTRAP values indicated greater enrichment of the miRNA with the Pax6 3’UTR relative to cellular abundance

    Techniques Used: Multiplex Assay, Real-time Polymerase Chain Reaction, Purification, Labeling, Sequencing

    Expression profile of miRNAs predicted to target the mouse Pax6 3’UTR. a Expression profile of miRNAs having predicted target sites in the mouse Pax6 3’UTR in various Pax6 -expressing cells and tissues. miRNAs assayed for include those identified by our prediction analysis (Fig. 3a ) as well as others, as described in the results. miRNAs were assayed for using TaqMan multiplex qPCR array cards. Data represents a total of 3 replicates per tissue or cell type, with a miRNA considered to be expressed only if the cycle threshold was less than 40 for all three replicates. b Relative miRNA level in mouse cultured pancreatic α cell line, αTC1–6 and a cultured β cell line, βTC6. c Relative miRNA level in mouse E12.5 retina, adult retina, and adult lens. Heat map indicates relative miRNA expression as a percentage of an internal control, snRNA U6. Data represents the geometric mean of three independent samples. Note the scale differences between the two heat maps
    Figure Legend Snippet: Expression profile of miRNAs predicted to target the mouse Pax6 3’UTR. a Expression profile of miRNAs having predicted target sites in the mouse Pax6 3’UTR in various Pax6 -expressing cells and tissues. miRNAs assayed for include those identified by our prediction analysis (Fig. 3a ) as well as others, as described in the results. miRNAs were assayed for using TaqMan multiplex qPCR array cards. Data represents a total of 3 replicates per tissue or cell type, with a miRNA considered to be expressed only if the cycle threshold was less than 40 for all three replicates. b Relative miRNA level in mouse cultured pancreatic α cell line, αTC1–6 and a cultured β cell line, βTC6. c Relative miRNA level in mouse E12.5 retina, adult retina, and adult lens. Heat map indicates relative miRNA expression as a percentage of an internal control, snRNA U6. Data represents the geometric mean of three independent samples. Note the scale differences between the two heat maps

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

    17) Product Images from "CIC protein instability contributes to tumorigenesis in glioblastoma"

    Article Title: CIC protein instability contributes to tumorigenesis in glioblastoma

    Journal: Nature Communications

    doi: 10.1038/s41467-018-08087-9

    Expression of CIC and its targets in human GBM tumors and cells. Human operative GBM samples or normal derived brains (NB) were lysed and a immunoblotted with indicated antibodies b or total RNA extracted and quantitative real-time PCR analysis was carried out using TaqMan gene expression assays. The graph depicts fold changes in CIC expression relative to normal brain. c Nuclear or cytoplasmic lysates were isolated from human operative GBM samples or normal brain and were immunoblotted with indicated antibodies. d Human operative astrocytoma samples were lysed and immunoblotted with indicated antibodies. Human-derived GBM cell lines or normal human astrocytes (NHA) were lysed and e protein lysates were immunoblotted with indicated antibodies ( f ) or total RNA extracted and quantitative real-time PCR analysis was carried out using TaqMan gene expression assays. The graph depicts fold changes in CIC expression relative to NHA. Data represent mean ± s.e.m. of three independent experiments performed in triplicate. * P
    Figure Legend Snippet: Expression of CIC and its targets in human GBM tumors and cells. Human operative GBM samples or normal derived brains (NB) were lysed and a immunoblotted with indicated antibodies b or total RNA extracted and quantitative real-time PCR analysis was carried out using TaqMan gene expression assays. The graph depicts fold changes in CIC expression relative to normal brain. c Nuclear or cytoplasmic lysates were isolated from human operative GBM samples or normal brain and were immunoblotted with indicated antibodies. d Human operative astrocytoma samples were lysed and immunoblotted with indicated antibodies. Human-derived GBM cell lines or normal human astrocytes (NHA) were lysed and e protein lysates were immunoblotted with indicated antibodies ( f ) or total RNA extracted and quantitative real-time PCR analysis was carried out using TaqMan gene expression assays. The graph depicts fold changes in CIC expression relative to NHA. Data represent mean ± s.e.m. of three independent experiments performed in triplicate. * P

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

    18) Product Images from "Identification of miR-16 as an endogenous reference gene for the normalization of urinary exosomal miRNA expression data from CKD patients"

    Article Title: Identification of miR-16 as an endogenous reference gene for the normalization of urinary exosomal miRNA expression data from CKD patients

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0183435

    Expression distribution and stability of normalization candidate mature miRNAs. All four candidate miRNAs are abundantly expressed in urinary exosomes of CKD patients and the normal group. Exosomal RNA was isolated with the Norgen urinary exosome preparation and RNA isolation kit. Expression data was generated by qRT-PCR using Taqman® miRNA Assays. The raw Ct-values were normalized against RNA input and an inter-run calibrator (A). MiR-92a is the most stable candidate normalizer within the data set, as revealed by NormFinder software. The best combination is miR-92a/16 with miR-16 as the most stable one, when input data is split into CKD patients and normal group. Error bars = SD (B).
    Figure Legend Snippet: Expression distribution and stability of normalization candidate mature miRNAs. All four candidate miRNAs are abundantly expressed in urinary exosomes of CKD patients and the normal group. Exosomal RNA was isolated with the Norgen urinary exosome preparation and RNA isolation kit. Expression data was generated by qRT-PCR using Taqman® miRNA Assays. The raw Ct-values were normalized against RNA input and an inter-run calibrator (A). MiR-92a is the most stable candidate normalizer within the data set, as revealed by NormFinder software. The best combination is miR-92a/16 with miR-16 as the most stable one, when input data is split into CKD patients and normal group. Error bars = SD (B).

    Techniques Used: Expressing, Isolation, Generated, Quantitative RT-PCR, Software

    19) Product Images from "Hypoxia-inducible factor-1 alpha as a therapeutic target for primary effusion lymphoma"

    Article Title: Hypoxia-inducible factor-1 alpha as a therapeutic target for primary effusion lymphoma

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006628

    Effect of HIF-1α knockdown on glycolysis and lipid biogenesis. ( A) Levels of extracellular lactate produced after 24 hours in normoxia (N) or hypoxia (H), expressed as fold change relative to cells containing shScr under normoxia. ( B) Protein levels of HIF-1α regulated glycolytic genes after 48 hours in normoxia or hypoxia as assessed by Western blot. β-actin is shown as a loading control. ( C) mRNA levels of HIF-1α regulated glycolytic genes measured by RT-qPCR after 48 hours in N or H. mRNA levels are normalized to that of 18S ribosomal RNA and are expressed as fold change relative to cells containing shScr under N. ( D ) Levels of miR210 measured by taqman RT-qPCR assay. RNA levels are normalized to that of internal control RNU43 and expressed as fold change relative to shScr cells in normoxia. ( E ) Levels of phospholipids produced in shHIF-1 cells relative to shScr cells in normoxia. ( F) mRNA levels of SIAH2 in BCBL-1 cells normalized to 18S and expressed as fold change relative to shScr cells under N. ( G) Protein levels of OGDH2 after 48 hours in N or H. Numbers below the blot for OGDH2 represent protein levels normalized to β-actin and expressed as fold change relative to shScr cells in N. Western blots were done on lysates from three independent experiments and representative blots are shown. Error bars represent standard deviations from at least 3 independent experiments. Statistically significant differences between shScr and shHIF-1 cells are indicated. * P ≤0.05, ** P ≤ 0.01 (2-tailed t-test, paired).
    Figure Legend Snippet: Effect of HIF-1α knockdown on glycolysis and lipid biogenesis. ( A) Levels of extracellular lactate produced after 24 hours in normoxia (N) or hypoxia (H), expressed as fold change relative to cells containing shScr under normoxia. ( B) Protein levels of HIF-1α regulated glycolytic genes after 48 hours in normoxia or hypoxia as assessed by Western blot. β-actin is shown as a loading control. ( C) mRNA levels of HIF-1α regulated glycolytic genes measured by RT-qPCR after 48 hours in N or H. mRNA levels are normalized to that of 18S ribosomal RNA and are expressed as fold change relative to cells containing shScr under N. ( D ) Levels of miR210 measured by taqman RT-qPCR assay. RNA levels are normalized to that of internal control RNU43 and expressed as fold change relative to shScr cells in normoxia. ( E ) Levels of phospholipids produced in shHIF-1 cells relative to shScr cells in normoxia. ( F) mRNA levels of SIAH2 in BCBL-1 cells normalized to 18S and expressed as fold change relative to shScr cells under N. ( G) Protein levels of OGDH2 after 48 hours in N or H. Numbers below the blot for OGDH2 represent protein levels normalized to β-actin and expressed as fold change relative to shScr cells in N. Western blots were done on lysates from three independent experiments and representative blots are shown. Error bars represent standard deviations from at least 3 independent experiments. Statistically significant differences between shScr and shHIF-1 cells are indicated. * P ≤0.05, ** P ≤ 0.01 (2-tailed t-test, paired).

    Techniques Used: Produced, Western Blot, Quantitative RT-PCR

    Effect of HIF-1α knockdown on the expression of KSHV miRNAs. (A) Level of primary miRNA transcript as measured by RT-qPCR and normalized to 18S mRNA. (B) Levels of mature miRNAs measured using taqman assays and normalized to that of RNU43 miRNA. Error bars represent standard deviations from at least 3 independent experiments. Statistically significant differences between shScr and shHIF-1 cells are indicated. * P ≤0.05 (2-tailed t-test).
    Figure Legend Snippet: Effect of HIF-1α knockdown on the expression of KSHV miRNAs. (A) Level of primary miRNA transcript as measured by RT-qPCR and normalized to 18S mRNA. (B) Levels of mature miRNAs measured using taqman assays and normalized to that of RNU43 miRNA. Error bars represent standard deviations from at least 3 independent experiments. Statistically significant differences between shScr and shHIF-1 cells are indicated. * P ≤0.05 (2-tailed t-test).

    Techniques Used: Expressing, Quantitative RT-PCR

    20) Product Images from "MicroRNA in diagnosis and therapy monitoring of early-stage triple-negative breast cancer"

    Article Title: MicroRNA in diagnosis and therapy monitoring of early-stage triple-negative breast cancer

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-29917-2

    TaqMan RT-qPCR validation results for miR34a show a highly significant (p
    Figure Legend Snippet: TaqMan RT-qPCR validation results for miR34a show a highly significant (p

    Techniques Used: Quantitative RT-PCR

    21) Product Images from "MicroRNA in diagnosis and therapy monitoring of early-stage triple-negative breast cancer"

    Article Title: MicroRNA in diagnosis and therapy monitoring of early-stage triple-negative breast cancer

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-29917-2

    TaqMan RT-qPCR validation results for miR34a show a highly significant (p
    Figure Legend Snippet: TaqMan RT-qPCR validation results for miR34a show a highly significant (p

    Techniques Used: Quantitative RT-PCR

    22) Product Images from "Variation of Genes Encoding Tryptophan Catabolites Pathway Enzymes in Stroke"

    Article Title: Variation of Genes Encoding Tryptophan Catabolites Pathway Enzymes in Stroke

    Journal: Journal of Clinical Medicine

    doi: 10.3390/jcm8122133

    Allelic discrimination X-Y scatter-plot of the c.804-7C > A SNP (rs1799913) of the TPH1 gene. The TaqMan ® SNP Genotyping Assay (ID: C___2645661_10) was used for genotyping of this SNP. The χ -axis represents the relative fluorescent emission for the A allele-specific probe labelled with 6-carboxyfluorescein (FAM), and the Ɣ -axis represents the emission for the C allele-specific probe labelled with 2’-chloro-7’-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC). Circles: homozygous A/A; squares: homozygous C/C; triangles: heterozygous C/A.
    Figure Legend Snippet: Allelic discrimination X-Y scatter-plot of the c.804-7C > A SNP (rs1799913) of the TPH1 gene. The TaqMan ® SNP Genotyping Assay (ID: C___2645661_10) was used for genotyping of this SNP. The χ -axis represents the relative fluorescent emission for the A allele-specific probe labelled with 6-carboxyfluorescein (FAM), and the Ɣ -axis represents the emission for the C allele-specific probe labelled with 2’-chloro-7’-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC). Circles: homozygous A/A; squares: homozygous C/C; triangles: heterozygous C/A.

    Techniques Used: TaqMan SNP Genotyping Assay

    23) Product Images from "Targeting KRAS Oncogene in Colon Cancer Cells with 7-Carboxylate Indolo[3,2-b]quinoline Tri-Alkylamine Derivatives"

    Article Title: Targeting KRAS Oncogene in Colon Cancer Cells with 7-Carboxylate Indolo[3,2-b]quinoline Tri-Alkylamine Derivatives

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0126891

    Exposure to IQ3A compounds decreases HSP90 and KRAS mRNA, protein expression and KRAS transcription. A. KRAS and HSP90 protein steady-state expression evaluated by immunoblot relative to DMSO (vehicle control), after 72 h exposure to equitoxic (IC 50 ) concentrations of 5-FU, TMPyP4 and IQ3A treatment; B. KRAS mRNA steady-state expression was evaluated by Taqman Real-time RT-PCR using specific Taqman Assays for KRAS and β-Actin for normalization. KRAS mRNA steady-state expression levels were calculated by the ΔΔCt method, using DMSO (vehicle control) for calibration; and C. HEK293T cells were co-transfected with pGL3-basic vector (empty vector control), or with KRAS promoter luciferase reporter construct PGL-Ras0.5, or PGL-Ras2.0, together with pRL-TK. Twenty-four hours later, cells were replated in 96-well plates, at 5.000 cells per well. Subsequently, 24 h after replating, cells were exposed to IC 50 equitoxic concentration of test compounds IQ3A, TMPyP4 and vehicle (DMSO); D. HCT116, SW620 and HEK293T cells were co-transfected with pGL3-basic vector (empty vector control), or with KRAS promoter luciferase reporter construct PGL-Ras0.5, together with pRL-TK. Twenty-four hours later, cells were replated in 96-well plates, at 5,000 cells per well and exposed to IC 50 equitoxic concentration of test compounds IQ3A, TMPyP4 and vehicle (DMSO). KRAS promoter activity levels were evaluated by Dual-Luciferase assay 72 h ( C. ) or 24 h ( D. ) after compound exposure. Results are expressed as the luciferase signal ratio of pGL-Ras2.0 or pGL-Ras0.5 to pGL3-basic vector transfected cells, after normalization with Renilla Luciferase. Results are expressed as mean ± SEM of at least three independent experiments; *p
    Figure Legend Snippet: Exposure to IQ3A compounds decreases HSP90 and KRAS mRNA, protein expression and KRAS transcription. A. KRAS and HSP90 protein steady-state expression evaluated by immunoblot relative to DMSO (vehicle control), after 72 h exposure to equitoxic (IC 50 ) concentrations of 5-FU, TMPyP4 and IQ3A treatment; B. KRAS mRNA steady-state expression was evaluated by Taqman Real-time RT-PCR using specific Taqman Assays for KRAS and β-Actin for normalization. KRAS mRNA steady-state expression levels were calculated by the ΔΔCt method, using DMSO (vehicle control) for calibration; and C. HEK293T cells were co-transfected with pGL3-basic vector (empty vector control), or with KRAS promoter luciferase reporter construct PGL-Ras0.5, or PGL-Ras2.0, together with pRL-TK. Twenty-four hours later, cells were replated in 96-well plates, at 5.000 cells per well. Subsequently, 24 h after replating, cells were exposed to IC 50 equitoxic concentration of test compounds IQ3A, TMPyP4 and vehicle (DMSO); D. HCT116, SW620 and HEK293T cells were co-transfected with pGL3-basic vector (empty vector control), or with KRAS promoter luciferase reporter construct PGL-Ras0.5, together with pRL-TK. Twenty-four hours later, cells were replated in 96-well plates, at 5,000 cells per well and exposed to IC 50 equitoxic concentration of test compounds IQ3A, TMPyP4 and vehicle (DMSO). KRAS promoter activity levels were evaluated by Dual-Luciferase assay 72 h ( C. ) or 24 h ( D. ) after compound exposure. Results are expressed as the luciferase signal ratio of pGL-Ras2.0 or pGL-Ras0.5 to pGL3-basic vector transfected cells, after normalization with Renilla Luciferase. Results are expressed as mean ± SEM of at least three independent experiments; *p

    Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Luciferase, Construct, Concentration Assay, Activity Assay

    24) Product Images from "The Rtr1p CTD phosphatase autoregulates its mRNA through a degradation pathway involving the REX exonucleases"

    Article Title: The Rtr1p CTD phosphatase autoregulates its mRNA through a degradation pathway involving the REX exonucleases

    Journal: RNA

    doi: 10.1261/rna.055723.115

    Association of 3X-Flag tagged Rtr1p with the RTR1 3′UTR –containing mRNP complex and tagged Dhh1p. ( A ) RNA immunoprecipitation (RIP) assay performed using the endogenously tagged 3X-Flag- RTR1 strain. Cultures were grown in SD-TRP to maintain either the GFP-TEF1 3′UTR/TER , GFP-RTR1 3′UTR/TER , or GFP-RTR1 3′ UTR , Δ BS plasmids. A qPCR was performed on the reverse-transcribed RNA input and IP samples with the GFP-FAM Taqman assay. A WT strain with the WT GFP-RTR1 3′UTR/TER plasmid was used as a negative control and a fold enrichment from this negative control was calculated for all other samples. All samples were normalized to the input Ct. ( B ) Coimmunoprecipitation assay performed utilizing the 3X-Flag- RTR1 strain and the tagged Dhh1p expressed from the BG1805 vector (Yeast ORF collection from Dharmacon). Lysed samples were immunoprecipitated with protein A sepharose beads in the presence of Protease 3C. Western blotting was performed with the anti-HA primary antibody to detect the tagged Dhh1. ( C ) Co-IP assay performed the same as in B , except, RNase A was added into the lysate in the indicated samples.
    Figure Legend Snippet: Association of 3X-Flag tagged Rtr1p with the RTR1 3′UTR –containing mRNP complex and tagged Dhh1p. ( A ) RNA immunoprecipitation (RIP) assay performed using the endogenously tagged 3X-Flag- RTR1 strain. Cultures were grown in SD-TRP to maintain either the GFP-TEF1 3′UTR/TER , GFP-RTR1 3′UTR/TER , or GFP-RTR1 3′ UTR , Δ BS plasmids. A qPCR was performed on the reverse-transcribed RNA input and IP samples with the GFP-FAM Taqman assay. A WT strain with the WT GFP-RTR1 3′UTR/TER plasmid was used as a negative control and a fold enrichment from this negative control was calculated for all other samples. All samples were normalized to the input Ct. ( B ) Coimmunoprecipitation assay performed utilizing the 3X-Flag- RTR1 strain and the tagged Dhh1p expressed from the BG1805 vector (Yeast ORF collection from Dharmacon). Lysed samples were immunoprecipitated with protein A sepharose beads in the presence of Protease 3C. Western blotting was performed with the anti-HA primary antibody to detect the tagged Dhh1. ( C ) Co-IP assay performed the same as in B , except, RNase A was added into the lysate in the indicated samples.

    Techniques Used: Immunoprecipitation, Real-time Polymerase Chain Reaction, TaqMan Assay, Plasmid Preparation, Negative Control, Co-Immunoprecipitation Assay, Western Blot

    25) Product Images from "Biphasic regulation of autophagy by miR-96 in prostate cancer cells under hypoxia"

    Article Title: Biphasic regulation of autophagy by miR-96 in prostate cancer cells under hypoxia

    Journal: Oncotarget

    doi:

    miR-96 expression level in prostate cancer tissues is correlated with clinical parameters A, miR-96 expression in the 20 prostate cancer tissues was determined by TaqMan qPCR analysis and grouped according to WHO I (n=8), II (n=7) and III (n=5) grade. Boxes represent the lower and the upper quartiles with medians; whiskers illustrate the 10 to 90 percentiles of the samples. B, Normalized miR-96 expression in the 10 prostate cancer (PCa) tissues was aligned with MTOR and ATG7 protein levels determined by immunoblots. C, Quantitative analysis of the correlations between miR-96 expression and ATG7 (r=0.82) or MTOR (r=0.67) expression in the above 10 prostate cancer tissues. * p
    Figure Legend Snippet: miR-96 expression level in prostate cancer tissues is correlated with clinical parameters A, miR-96 expression in the 20 prostate cancer tissues was determined by TaqMan qPCR analysis and grouped according to WHO I (n=8), II (n=7) and III (n=5) grade. Boxes represent the lower and the upper quartiles with medians; whiskers illustrate the 10 to 90 percentiles of the samples. B, Normalized miR-96 expression in the 10 prostate cancer (PCa) tissues was aligned with MTOR and ATG7 protein levels determined by immunoblots. C, Quantitative analysis of the correlations between miR-96 expression and ATG7 (r=0.82) or MTOR (r=0.67) expression in the above 10 prostate cancer tissues. * p

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

    26) Product Images from "miR-27 Negatively Regulates Pluripotency-Associated Genes in Human Embryonal Carcinoma Cells"

    Article Title: miR-27 Negatively Regulates Pluripotency-Associated Genes in Human Embryonal Carcinoma Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0111637

    miR-27 inhibits OCT4 and LIN28 expression at both the transcriptional and translational level in embryonal carcinoma cells (NCCIT). (A) Analysis of miR-27 expression was carried out for miR-27a and miR-27b using TaqMan-based PCR on total RNA samples isolated from NCCIT cells undergoing RA stimulated neuronal differentiation for seven days or by blocking TGFßR2 with SB431542 for seven days and normalized to the DMSO-treated control. (B) qRT-PCR of selected genes (log2-fold change relative to the negative control) was validated for NCCIT cells transfected once with miR-27 or treated with SB431542 for 48 h. NCCIT cells transfected with a scrambled miRNA mimic was used for normalization. (C) Relative OCT4 and LIN28 expression in NCCIT cells transfected with scrambled negative control miRNA mimics, let-7a, miR-125b, miR-27a, miR-200c or treated with SB431542 for 72 h and validated by qRT-PCR. (D) Western Blot analysis of OCT4 and LIN28 expression in NCCIT cells treated as described in (C). (E) Normalized densitometric-derived ratios of Western Blot presented in (D).
    Figure Legend Snippet: miR-27 inhibits OCT4 and LIN28 expression at both the transcriptional and translational level in embryonal carcinoma cells (NCCIT). (A) Analysis of miR-27 expression was carried out for miR-27a and miR-27b using TaqMan-based PCR on total RNA samples isolated from NCCIT cells undergoing RA stimulated neuronal differentiation for seven days or by blocking TGFßR2 with SB431542 for seven days and normalized to the DMSO-treated control. (B) qRT-PCR of selected genes (log2-fold change relative to the negative control) was validated for NCCIT cells transfected once with miR-27 or treated with SB431542 for 48 h. NCCIT cells transfected with a scrambled miRNA mimic was used for normalization. (C) Relative OCT4 and LIN28 expression in NCCIT cells transfected with scrambled negative control miRNA mimics, let-7a, miR-125b, miR-27a, miR-200c or treated with SB431542 for 72 h and validated by qRT-PCR. (D) Western Blot analysis of OCT4 and LIN28 expression in NCCIT cells treated as described in (C). (E) Normalized densitometric-derived ratios of Western Blot presented in (D).

    Techniques Used: Expressing, Polymerase Chain Reaction, Isolation, Blocking Assay, Quantitative RT-PCR, Negative Control, Transfection, Western Blot, Derivative Assay

    OCT4 knockdown in the hESC line H1 leads to activation of miR-27a and miR-27b expression. Successful OCT4 knockdown in hESC cells transfected twice with siRNA targeting either OCT4 or EGFP 72 h post transfection and confirmed by real-time PCR (A) and Western Blotting (B). (A) Relative OCT4 and NANOG expression of three biological OCT4 knockdown samples (siOCT4#1-3) normalized to the siGFP knockdown control. (B) Western Blot analysis of OCT4 protein levels carried out for sample (siOCT4#1) and siGFP control sample with densitometric quantification (OCT4/GAPDH) (C) Relative expression of pluripotency-associated genes validated by real-time PCR for sample siOCT4#1 normalized to the siGFP knockdown control. (D) miR-27 expression was carried out using TaqMan-based PCR for all three biological siOCT4 samples and normalized to the siGFP control sample.
    Figure Legend Snippet: OCT4 knockdown in the hESC line H1 leads to activation of miR-27a and miR-27b expression. Successful OCT4 knockdown in hESC cells transfected twice with siRNA targeting either OCT4 or EGFP 72 h post transfection and confirmed by real-time PCR (A) and Western Blotting (B). (A) Relative OCT4 and NANOG expression of three biological OCT4 knockdown samples (siOCT4#1-3) normalized to the siGFP knockdown control. (B) Western Blot analysis of OCT4 protein levels carried out for sample (siOCT4#1) and siGFP control sample with densitometric quantification (OCT4/GAPDH) (C) Relative expression of pluripotency-associated genes validated by real-time PCR for sample siOCT4#1 normalized to the siGFP knockdown control. (D) miR-27 expression was carried out using TaqMan-based PCR for all three biological siOCT4 samples and normalized to the siGFP control sample.

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

    27) Product Images from "Epstein-Barr Virus Latent Membrane Protein 1 Regulates Host B Cell MicroRNA-155 and Its Target FOXO3a via PI3K p110α Activation"

    Article Title: Epstein-Barr Virus Latent Membrane Protein 1 Regulates Host B Cell MicroRNA-155 and Its Target FOXO3a via PI3K p110α Activation

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2019.02692

    Tumor variants of LMP1 differentially regulate miR-155 and miR-193b from the B95.8 lab strain of LMP1. Two million EBV− BL41 cells expressing B95.8 lab strain or natural variant NGFR.LMP1 molecules were activated for 12 h prior to RNA isolation with the miRVana miRNA isolation kit. Relative expression of specific microRNAs was determined by quantitative PCR (qPCR) using TaqMan MicroRNA Assays. Target-specific cDNA was generated from 10 ng of total RNA using the TaqMan MicroRNA Reverse Transcription Kit and pre-amplified using the TaqMan PreAmp Master Mix. Finally, qPCR assays were performed using the TaqMan Universal Master Mix II, No AmpERASE UNG. The relative expression of (A) miR-155 and (B) miR-193b was calculated by first normalizing to the endogenous control U47 (ΔC t ) and then to unactivated samples (ΔΔ C t ). Fold-induction (2 −ΔΔ C t ) of each miR is shown. Each point represents an experimental replicate; two different lines expressing B95.8 NGFR.LMP1 were used. *** p ≤ 0.001, **** p ≤ 0.0001 by one-way ANOVA with post hoc multiple comparisons to activate B95.8 lab strain NGFR.LMP1.
    Figure Legend Snippet: Tumor variants of LMP1 differentially regulate miR-155 and miR-193b from the B95.8 lab strain of LMP1. Two million EBV− BL41 cells expressing B95.8 lab strain or natural variant NGFR.LMP1 molecules were activated for 12 h prior to RNA isolation with the miRVana miRNA isolation kit. Relative expression of specific microRNAs was determined by quantitative PCR (qPCR) using TaqMan MicroRNA Assays. Target-specific cDNA was generated from 10 ng of total RNA using the TaqMan MicroRNA Reverse Transcription Kit and pre-amplified using the TaqMan PreAmp Master Mix. Finally, qPCR assays were performed using the TaqMan Universal Master Mix II, No AmpERASE UNG. The relative expression of (A) miR-155 and (B) miR-193b was calculated by first normalizing to the endogenous control U47 (ΔC t ) and then to unactivated samples (ΔΔ C t ). Fold-induction (2 −ΔΔ C t ) of each miR is shown. Each point represents an experimental replicate; two different lines expressing B95.8 NGFR.LMP1 were used. *** p ≤ 0.001, **** p ≤ 0.0001 by one-way ANOVA with post hoc multiple comparisons to activate B95.8 lab strain NGFR.LMP1.

    Techniques Used: Expressing, Variant Assay, Isolation, Real-time Polymerase Chain Reaction, Generated, Amplification

    28) Product Images from "Exosomes from bulk and stem cells from human prostate cancer have a differential microRNA content that contributes cooperatively over local and pre-metastatic niche"

    Article Title: Exosomes from bulk and stem cells from human prostate cancer have a differential microRNA content that contributes cooperatively over local and pre-metastatic niche

    Journal: Oncotarget

    doi: 10.18632/oncotarget.6540

    RT-qPCR on selected miRNAs in bulk and CSCs cells and exosomes Expression was assessed by TaqMan miRNA assays. Cp values from each assay are compared between samples ( n = 3).
    Figure Legend Snippet: RT-qPCR on selected miRNAs in bulk and CSCs cells and exosomes Expression was assessed by TaqMan miRNA assays. Cp values from each assay are compared between samples ( n = 3).

    Techniques Used: Quantitative RT-PCR, Expressing

    29) Product Images from "miR-509 suppresses brain metastasis of breast cancer cells by modulating RhoC and TNF α"

    Article Title: miR-509 suppresses brain metastasis of breast cancer cells by modulating RhoC and TNF α

    Journal: Oncogene

    doi: 10.1038/onc.2014.412

    miR-509 directly targets RhoC (a) Schematic representation of the RhoC 3’-UTR with a miR-509 binding site. (b) 231BrM (left panel) and CN34BrM (right panel) were infected with lentivirus expressing pre-miR-509 or vector only, and the expression of RhoC was examined by Western blot. (c) MDA231 (left panel) and MCF7 (middle panel) were transfected with miR-509 LNA (50pM) or scramble LNA, and the expression of RhoC was examined by Western blot. MCF10A (right panel) was transfected with various doses of miR-509 LNA, and the expression of RhoC was examined by Western blot. (d) Constructs carrying RhoC 3’ UTR luciferase reporter (WT) or deletion mutant of miR-509 binding site (MT) were transfected to 293TN cells with the miR-509 expression plasmid or vector plasmid. Cells were harvested and luciferase activities were measured after 24hrs transfection. (e) Expression of miR-509-5p and TNFα were measured by TaqMan PCR in brain metastasis samples after micro-dissection. The relative expression level of miR-509-5p to RNU48 and the expression level of RhoC to ACTB were plotted (n=7). (f) RhoC mRNA expression was measured between the primary tumors with or without brain metastasis in a combined GEO data bases (GSE12276, GSE2034, GSE2603, GSE5327, and GSE14020). * indicates p
    Figure Legend Snippet: miR-509 directly targets RhoC (a) Schematic representation of the RhoC 3’-UTR with a miR-509 binding site. (b) 231BrM (left panel) and CN34BrM (right panel) were infected with lentivirus expressing pre-miR-509 or vector only, and the expression of RhoC was examined by Western blot. (c) MDA231 (left panel) and MCF7 (middle panel) were transfected with miR-509 LNA (50pM) or scramble LNA, and the expression of RhoC was examined by Western blot. MCF10A (right panel) was transfected with various doses of miR-509 LNA, and the expression of RhoC was examined by Western blot. (d) Constructs carrying RhoC 3’ UTR luciferase reporter (WT) or deletion mutant of miR-509 binding site (MT) were transfected to 293TN cells with the miR-509 expression plasmid or vector plasmid. Cells were harvested and luciferase activities were measured after 24hrs transfection. (e) Expression of miR-509-5p and TNFα were measured by TaqMan PCR in brain metastasis samples after micro-dissection. The relative expression level of miR-509-5p to RNU48 and the expression level of RhoC to ACTB were plotted (n=7). (f) RhoC mRNA expression was measured between the primary tumors with or without brain metastasis in a combined GEO data bases (GSE12276, GSE2034, GSE2603, GSE5327, and GSE14020). * indicates p

    Techniques Used: Binding Assay, Infection, Expressing, Plasmid Preparation, Western Blot, Transfection, Construct, Luciferase, Mutagenesis, Polymerase Chain Reaction, Dissection

    30) Product Images from "Modulation of the Unfolded Protein Response During Hepatocyte and Cardiomyocyte Apoptosis In Trauma/Hemorrhagic Shock"

    Article Title: Modulation of the Unfolded Protein Response During Hepatocyte and Cardiomyocyte Apoptosis In Trauma/Hemorrhagic Shock

    Journal: Scientific Reports

    doi: 10.1038/srep01187

    Q-RT-PCR using TaqMan® (Life Technologies) for (A) heat shock protein 70 (Hsp70; Hspa1a) and (B) heat shock protein 40 (Hsp40; Dnajb1) performed on whole liver samples from Sham (n = 6), trauma with hemorrhagic shock (T/HS, n = 4), and T/HS animals resuscitated with IL-6 (T/HS-IL6, n = 4). Transcript values reported as relative quantification (RQ) in comparison to a normal rat liver. Values expressed as mean RQ ± SEM. “*”, “**” indicate group comparisons which are statistically different (p
    Figure Legend Snippet: Q-RT-PCR using TaqMan® (Life Technologies) for (A) heat shock protein 70 (Hsp70; Hspa1a) and (B) heat shock protein 40 (Hsp40; Dnajb1) performed on whole liver samples from Sham (n = 6), trauma with hemorrhagic shock (T/HS, n = 4), and T/HS animals resuscitated with IL-6 (T/HS-IL6, n = 4). Transcript values reported as relative quantification (RQ) in comparison to a normal rat liver. Values expressed as mean RQ ± SEM. “*”, “**” indicate group comparisons which are statistically different (p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    31) Product Images from "MicroRNAs in Salivary Exosome as Potential Biomarkers of Aging"

    Article Title: MicroRNAs in Salivary Exosome as Potential Biomarkers of Aging

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms160921294

    Comparison of relative expression rates of candidate miRNAs for aging biomarker between young and old groups. We performed TaqMan RT-qPCR to calculate the relative expression rates of three candidate miRNAs between young and old groups. We used miR-4739 as internal control miRNA. Circle plots represent relative expression rates of each sample. The horizontal lines within each box represent the 25, 50 and 75th percentiles. The p value was calculated by the Pair Wise Fixed Reallocation Randomisation Test using REST 2009 software. * p value
    Figure Legend Snippet: Comparison of relative expression rates of candidate miRNAs for aging biomarker between young and old groups. We performed TaqMan RT-qPCR to calculate the relative expression rates of three candidate miRNAs between young and old groups. We used miR-4739 as internal control miRNA. Circle plots represent relative expression rates of each sample. The horizontal lines within each box represent the 25, 50 and 75th percentiles. The p value was calculated by the Pair Wise Fixed Reallocation Randomisation Test using REST 2009 software. * p value

    Techniques Used: Expressing, Biomarker Assay, Quantitative RT-PCR, Software

    32) Product Images from "Selective Upregulation of microRNA Expression in Peripheral Blood Leukocytes in IL-10−/− Mice Precedes Expression in the Colon"

    Article Title: Selective Upregulation of microRNA Expression in Peripheral Blood Leukocytes in IL-10−/− Mice Precedes Expression in the Colon

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

    doi: 10.4049/jimmunol.1100922

    Total RNA from IL-10 −/− and BALB/c colon were used for TaqMan qRT-PCR analysis of eleven miRNAs in ( A ) colonic tissues and ( B ) PBLs from normal mice (score 0) and IL-10 −/− mice with low pathology (score 1) or high pathology
    Figure Legend Snippet: Total RNA from IL-10 −/− and BALB/c colon were used for TaqMan qRT-PCR analysis of eleven miRNAs in ( A ) colonic tissues and ( B ) PBLs from normal mice (score 0) and IL-10 −/− mice with low pathology (score 1) or high pathology

    Techniques Used: Quantitative RT-PCR, Mouse Assay

    33) Product Images from "E-Cadherin and FGFR1 Expression in Mouse Osteoblastogenesis in Normoxic Cultures"

    Article Title: E-Cadherin and FGFR1 Expression in Mouse Osteoblastogenesis in Normoxic Cultures

    Journal: International Journal of Biomedical Science : IJBS

    doi:

    Mouse primary osteoblastogenesis. Primary osteoblasts were differentiated over 4 weeks in osteogenic medium. Panel A shows significant increases in the ALP activity, an osteoblast differentiation marker, during osteoblastogenesis on day 14 and 21. Panel B shows significant increases in the alizarin red staining, an osteoblasts maturation marker, during osteoblastogenesis. Panel C shows electrophoresis of total mRNA isolated from cells and RT-PCR of GAPDH mRNA during osteoblastogenesis. The relative expression of osteoblast marker genes; COL1A2 (panel D), Runx2 (panel E) and osteocalcin (OCN) (panel F) expression during osteoblastogenesis were analyzed using TaqMan.
    Figure Legend Snippet: Mouse primary osteoblastogenesis. Primary osteoblasts were differentiated over 4 weeks in osteogenic medium. Panel A shows significant increases in the ALP activity, an osteoblast differentiation marker, during osteoblastogenesis on day 14 and 21. Panel B shows significant increases in the alizarin red staining, an osteoblasts maturation marker, during osteoblastogenesis. Panel C shows electrophoresis of total mRNA isolated from cells and RT-PCR of GAPDH mRNA during osteoblastogenesis. The relative expression of osteoblast marker genes; COL1A2 (panel D), Runx2 (panel E) and osteocalcin (OCN) (panel F) expression during osteoblastogenesis were analyzed using TaqMan.

    Techniques Used: ALP Assay, Activity Assay, Marker, Staining, Electrophoresis, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing

    Expression of E-cadherin and FGFR1 during osteoblastogenesis. Primary osteoblasts were differentiated over 4 weeks in osteogenic medium. Panel A shows the expression of E-cadherin and FGFR1 mRNA during osteoblastogenesis. Panel B shows the expression of E-cadherin protein in immature (day 7) and mature (day 28) osteoblasts using western blotting. Panel C shows the expression of FGFR1 protein in immature (day 7) and mature (day 28) osteoblasts using western blotting. Panel D shows the relative expression of E-cadherin during osteoblastogenesis using TaqMan. Panel E shows the relative expression of FGFR1 during osteoblastogenesis using TaqMan.
    Figure Legend Snippet: Expression of E-cadherin and FGFR1 during osteoblastogenesis. Primary osteoblasts were differentiated over 4 weeks in osteogenic medium. Panel A shows the expression of E-cadherin and FGFR1 mRNA during osteoblastogenesis. Panel B shows the expression of E-cadherin protein in immature (day 7) and mature (day 28) osteoblasts using western blotting. Panel C shows the expression of FGFR1 protein in immature (day 7) and mature (day 28) osteoblasts using western blotting. Panel D shows the relative expression of E-cadherin during osteoblastogenesis using TaqMan. Panel E shows the relative expression of FGFR1 during osteoblastogenesis using TaqMan.

    Techniques Used: Expressing, Western Blot

    34) Product Images from "Bone marker gene expression in calvarial bones: different bone microenvironments"

    Article Title: Bone marker gene expression in calvarial bones: different bone microenvironments

    Journal: Journal of Biological Research

    doi: 10.1186/s40709-017-0066-y

    TaqMan analysis of adhesion molecules: N-cadherin and E-cadherin expression in calvariae of C57BL/KalwRiJHsD mice. RNA was extracted with cDNA synthesized from frontal (F), parietal (P) and interparietal (IP) bones. The relative expression of adhesion molecule was adjusted to the relative expression of a housekeeping gene, β-actin. There were no differences in the relative expression of N-cadherin ( a ) and E-cadherin ( b ) between frontal, parietal and interparietal bones
    Figure Legend Snippet: TaqMan analysis of adhesion molecules: N-cadherin and E-cadherin expression in calvariae of C57BL/KalwRiJHsD mice. RNA was extracted with cDNA synthesized from frontal (F), parietal (P) and interparietal (IP) bones. The relative expression of adhesion molecule was adjusted to the relative expression of a housekeeping gene, β-actin. There were no differences in the relative expression of N-cadherin ( a ) and E-cadherin ( b ) between frontal, parietal and interparietal bones

    Techniques Used: Expressing, Mouse Assay, Synthesized

    TaqMan analysis of skeletogenesis markers: FGF2 and FGFR1 expression in calvariae of C57BL/KalwRiJHsD mice. RNA was extracted with cDNA synthesized from frontal (F), parietal (P) and interparietal (IP) bones. The relative expression of skeletogenesis marker genes was adjusted to the relative expression of a housekeeping gene, β-actin. There was a significant increase in the relative expression of FGF2 ( a ) in frontal and parietal bones compared to interparietal bone. In addition, there was no difference in the relative expression of FGF2 between frontal and parietal bones. On the other hand, the relative expression of FGFR1 ( b ) genes was significantly expressed in parietal bone compared to frontal and interparietal bones
    Figure Legend Snippet: TaqMan analysis of skeletogenesis markers: FGF2 and FGFR1 expression in calvariae of C57BL/KalwRiJHsD mice. RNA was extracted with cDNA synthesized from frontal (F), parietal (P) and interparietal (IP) bones. The relative expression of skeletogenesis marker genes was adjusted to the relative expression of a housekeeping gene, β-actin. There was a significant increase in the relative expression of FGF2 ( a ) in frontal and parietal bones compared to interparietal bone. In addition, there was no difference in the relative expression of FGF2 between frontal and parietal bones. On the other hand, the relative expression of FGFR1 ( b ) genes was significantly expressed in parietal bone compared to frontal and interparietal bones

    Techniques Used: Expressing, Mouse Assay, Synthesized, Marker

    35) Product Images from "3C-digital PCR for quantification of chromatin interactions"

    Article Title: 3C-digital PCR for quantification of chromatin interactions

    Journal: BMC Molecular Biology

    doi: 10.1186/s12867-016-0076-6

    3C-dPCR workflow. A TaqMan probe and primer design. The locations of the two possible interaction fragments ( a and b ) are shown ( black rectangle ). Restriction sites used in the 3C assay are depicted as small vertical bars in black . The relative positions of anchor primer ( black arrow ), the TaqMan probe (F-Z-Q) and test primers ( grey arrows ) are also depicted. F fluorophore, Z internal quencher, Q quencher. B Three essential steps of 3C assays: 1. Interacting chromatin segments are cross-linked by formaldehyde. 2. Cross-linked chromatins are digested by a selected restriction enzyme. 3. Cross-linked fragments undergo intra-molecular ligation. C Principle of 3C-dPCR. The reaction mixture containing 3C DNA is prepared and partitioned into thousands of reaction wells. Due to significant dilution, each reaction well receives 0–1 target ligation products. After PCR amplification, the fluorescence signals are imaged and copy numbers of target ligations are reported as copies/μL. In the 3C-dPCR reaction and partition steps, curved lines in blue , red , black , grey and purple curve in the circle represent the different DNA molecules, including ligation products in 3C libraries. In the amplification step, the blue dot (well) shows target amplification signal; the red dots (wells) indicate the genome copy number signal; the green dot (well) displays the overlap of target and genome copy number signals
    Figure Legend Snippet: 3C-dPCR workflow. A TaqMan probe and primer design. The locations of the two possible interaction fragments ( a and b ) are shown ( black rectangle ). Restriction sites used in the 3C assay are depicted as small vertical bars in black . The relative positions of anchor primer ( black arrow ), the TaqMan probe (F-Z-Q) and test primers ( grey arrows ) are also depicted. F fluorophore, Z internal quencher, Q quencher. B Three essential steps of 3C assays: 1. Interacting chromatin segments are cross-linked by formaldehyde. 2. Cross-linked chromatins are digested by a selected restriction enzyme. 3. Cross-linked fragments undergo intra-molecular ligation. C Principle of 3C-dPCR. The reaction mixture containing 3C DNA is prepared and partitioned into thousands of reaction wells. Due to significant dilution, each reaction well receives 0–1 target ligation products. After PCR amplification, the fluorescence signals are imaged and copy numbers of target ligations are reported as copies/μL. In the 3C-dPCR reaction and partition steps, curved lines in blue , red , black , grey and purple curve in the circle represent the different DNA molecules, including ligation products in 3C libraries. In the amplification step, the blue dot (well) shows target amplification signal; the red dots (wells) indicate the genome copy number signal; the green dot (well) displays the overlap of target and genome copy number signals

    Techniques Used: Digital PCR, Ligation, Polymerase Chain Reaction, Amplification, Fluorescence

    36) Product Images from "Knock-out of a mitochondrial sirtuin protects neurons from degeneration in Caenorhabditis elegans"

    Article Title: Knock-out of a mitochondrial sirtuin protects neurons from degeneration in Caenorhabditis elegans

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1006965

    SIR-2.1 does not mediate the protective effects of sir-2 . 3 knock-out. A: Quantitative real-time PCR (qRT-PCR) from total RNA was conducted using Taqman gene expression assays in order to quantify the levels of mRNA for sirtuin genes. mRNA percentage of each target gene was calculated by comparing the cycle threshold of the target gene to that of the housekeeping gene pmp-3 . The wild-type N2 (Bristol) strain was used as the calibrator. All conditions had three technical replicates. B-E : Proportion of animals with 0, 1, 2 and 3 alive touch neurons in mec-4(d) , mec-4(d);sir-2 . 3 and mec-4(d);sir-2 . 1;sir-2 . 3 mutants. Data are expressed as mean +/- SE, N = 3. P was from left to right and from top to bottom: 0.003, 0.0029, 0.0019, 0.0005, 0.000013 and 0.00009 by ANOVA with Bonferroni correction.
    Figure Legend Snippet: SIR-2.1 does not mediate the protective effects of sir-2 . 3 knock-out. A: Quantitative real-time PCR (qRT-PCR) from total RNA was conducted using Taqman gene expression assays in order to quantify the levels of mRNA for sirtuin genes. mRNA percentage of each target gene was calculated by comparing the cycle threshold of the target gene to that of the housekeeping gene pmp-3 . The wild-type N2 (Bristol) strain was used as the calibrator. All conditions had three technical replicates. B-E : Proportion of animals with 0, 1, 2 and 3 alive touch neurons in mec-4(d) , mec-4(d);sir-2 . 3 and mec-4(d);sir-2 . 1;sir-2 . 3 mutants. Data are expressed as mean +/- SE, N = 3. P was from left to right and from top to bottom: 0.003, 0.0029, 0.0019, 0.0005, 0.000013 and 0.00009 by ANOVA with Bonferroni correction.

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

    37) Product Images from "Genome-wide mRNA and miRNA expression profiling reveal multiple regulatory networks in colorectal cancer"

    Article Title: Genome-wide mRNA and miRNA expression profiling reveal multiple regulatory networks in colorectal cancer

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2014.556

    miRNA expression profiling in CRC. ( a ) Hierarchical clustering of 13 colon cancer and 13 normal tissue samples based on miRNA expression levels. Each column represents a sample and each row represents a transcript. Expression level of each miRNA in a single sample is depicted according to the color scale. ( b ) Pie chart illustrating the distribution of the top 20 pathway designations for predicted targets (TargetScan) for the downregulated miRNAs in colon cancer. The pie size corresponds to the number of matched entities. ( c ) Venn diagram depicting the overlap between the predicted gene targets for the downregulated miRNAs (based on TargetScan) versus the differentially upregulated genes in CRC identified in the current study. ( d ) Expression levels of selected miRNAs (hsa-miR-145-5p, hsa-miR-26a-5p, and hsa-miR-30-5p) based on microarray data and validation of those miRNAs using Taqman qRT-PCR (duplicate). ** P
    Figure Legend Snippet: miRNA expression profiling in CRC. ( a ) Hierarchical clustering of 13 colon cancer and 13 normal tissue samples based on miRNA expression levels. Each column represents a sample and each row represents a transcript. Expression level of each miRNA in a single sample is depicted according to the color scale. ( b ) Pie chart illustrating the distribution of the top 20 pathway designations for predicted targets (TargetScan) for the downregulated miRNAs in colon cancer. The pie size corresponds to the number of matched entities. ( c ) Venn diagram depicting the overlap between the predicted gene targets for the downregulated miRNAs (based on TargetScan) versus the differentially upregulated genes in CRC identified in the current study. ( d ) Expression levels of selected miRNAs (hsa-miR-145-5p, hsa-miR-26a-5p, and hsa-miR-30-5p) based on microarray data and validation of those miRNAs using Taqman qRT-PCR (duplicate). ** P

    Techniques Used: Expressing, Microarray, Quantitative RT-PCR

    38) Product Images from "Mapping the Pax6 3’ untranslated region microRNA regulatory landscape"

    Article Title: Mapping the Pax6 3’ untranslated region microRNA regulatory landscape

    Journal: BMC Genomics

    doi: 10.1186/s12864-018-5212-x

    miTRAP as a strategy to purify Pax6 3’UTR-associated miRNAs. a Schematic of the Pax6 3’UTR affinity purification approach. (i) Plasmid vectors expressing GFP tagged with the MS2 RNA sequence motif followed by the SV40 polyadenylation signal are introduced into pancreatic αTC1–6 cells via transient transfection. (ii) MS2 coat protein fused to maltose binding (MS2-MBP) is used to purify GFP transcripts with bound miRNAs from αTC1–6 cell lysate. (iii) Real-time quantitative PCR (RT-qPCR) is used to detect GFP transcript and bound miRNAs. Schematic of the Pax6 3’UTR shows the location of the highly conserved miR-375 target site located at 3’UTR position 201 and miR-375 target site mutation. b ]. qPCR results for GFP with the MS2 motif (grey bar) were expressed relative to data without the MS2 motif (unfilled bar). Data represents 5 independent samples, p = 0.0079. c Affinity purification of miR-375 with the Pax6 3’UTR in αTC1–6 cells using TaqMan individual qPCR assays. Normalized relative quantity was calculated using Pfaffl’s method, and a normalized relative quantity greater than 1 indicates that more target miRNA is purified with the Pax6 3’UTR (grey bar) than the control lacking a Pax6 3’UTR (unfilled bar). Data represents six independent samples, p = 0.013. d Disruption of miR-375 binding to the Pax6 3’UTR following mutation of the miR-375 target site. Target miR-375 values were normalized to GFP as a reference gene, then normalized values for the mutant Pax6 3’UTR samples (grey bar), and are presented relative to the wt 3’UTR (unfilled bar). Data represents six independent wt 3’UTR samples and three miR-375 target site mutant 3’UTR samples, p = 0.0476. Error bars represent 95% confidence intervals, and p -values were calculated using the Mann Whitney test. Note scale bar differences between the graphs
    Figure Legend Snippet: miTRAP as a strategy to purify Pax6 3’UTR-associated miRNAs. a Schematic of the Pax6 3’UTR affinity purification approach. (i) Plasmid vectors expressing GFP tagged with the MS2 RNA sequence motif followed by the SV40 polyadenylation signal are introduced into pancreatic αTC1–6 cells via transient transfection. (ii) MS2 coat protein fused to maltose binding (MS2-MBP) is used to purify GFP transcripts with bound miRNAs from αTC1–6 cell lysate. (iii) Real-time quantitative PCR (RT-qPCR) is used to detect GFP transcript and bound miRNAs. Schematic of the Pax6 3’UTR shows the location of the highly conserved miR-375 target site located at 3’UTR position 201 and miR-375 target site mutation. b ]. qPCR results for GFP with the MS2 motif (grey bar) were expressed relative to data without the MS2 motif (unfilled bar). Data represents 5 independent samples, p = 0.0079. c Affinity purification of miR-375 with the Pax6 3’UTR in αTC1–6 cells using TaqMan individual qPCR assays. Normalized relative quantity was calculated using Pfaffl’s method, and a normalized relative quantity greater than 1 indicates that more target miRNA is purified with the Pax6 3’UTR (grey bar) than the control lacking a Pax6 3’UTR (unfilled bar). Data represents six independent samples, p = 0.013. d Disruption of miR-375 binding to the Pax6 3’UTR following mutation of the miR-375 target site. Target miR-375 values were normalized to GFP as a reference gene, then normalized values for the mutant Pax6 3’UTR samples (grey bar), and are presented relative to the wt 3’UTR (unfilled bar). Data represents six independent wt 3’UTR samples and three miR-375 target site mutant 3’UTR samples, p = 0.0476. Error bars represent 95% confidence intervals, and p -values were calculated using the Mann Whitney test. Note scale bar differences between the graphs

    Techniques Used: Affinity Purification, Plasmid Preparation, Expressing, Sequencing, Transfection, Binding Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Mutagenesis, Purification, MANN-WHITNEY

    39) Product Images from "Mapping the Pax6 3’ untranslated region microRNA regulatory landscape"

    Article Title: Mapping the Pax6 3’ untranslated region microRNA regulatory landscape

    Journal: BMC Genomics

    doi: 10.1186/s12864-018-5212-x

    miTRAP as a strategy to purify Pax6 3’UTR-associated miRNAs. a Schematic of the Pax6 3’UTR affinity purification approach. (i) Plasmid vectors expressing GFP tagged with the MS2 RNA sequence motif followed by the SV40 polyadenylation signal are introduced into pancreatic αTC1–6 cells via transient transfection. (ii) MS2 coat protein fused to maltose binding (MS2-MBP) is used to purify GFP transcripts with bound miRNAs from αTC1–6 cell lysate. (iii) Real-time quantitative PCR (RT-qPCR) is used to detect GFP transcript and bound miRNAs. Schematic of the Pax6 3’UTR shows the location of the highly conserved miR-375 target site located at 3’UTR position 201 and miR-375 target site mutation. b ]. qPCR results for GFP with the MS2 motif (grey bar) were expressed relative to data without the MS2 motif (unfilled bar). Data represents 5 independent samples, p = 0.0079. c Affinity purification of miR-375 with the Pax6 3’UTR in αTC1–6 cells using TaqMan individual qPCR assays. Normalized relative quantity was calculated using Pfaffl’s method, and a normalized relative quantity greater than 1 indicates that more target miRNA is purified with the Pax6 3’UTR (grey bar) than the control lacking a Pax6 3’UTR (unfilled bar). Data represents six independent samples, p = 0.013. d Disruption of miR-375 binding to the Pax6 3’UTR following mutation of the miR-375 target site. Target miR-375 values were normalized to GFP as a reference gene, then normalized values for the mutant Pax6 3’UTR samples (grey bar), and are presented relative to the wt 3’UTR (unfilled bar). Data represents six independent wt 3’UTR samples and three miR-375 target site mutant 3’UTR samples, p = 0.0476. Error bars represent 95% confidence intervals, and p -values were calculated using the Mann Whitney test. Note scale bar differences between the graphs
    Figure Legend Snippet: miTRAP as a strategy to purify Pax6 3’UTR-associated miRNAs. a Schematic of the Pax6 3’UTR affinity purification approach. (i) Plasmid vectors expressing GFP tagged with the MS2 RNA sequence motif followed by the SV40 polyadenylation signal are introduced into pancreatic αTC1–6 cells via transient transfection. (ii) MS2 coat protein fused to maltose binding (MS2-MBP) is used to purify GFP transcripts with bound miRNAs from αTC1–6 cell lysate. (iii) Real-time quantitative PCR (RT-qPCR) is used to detect GFP transcript and bound miRNAs. Schematic of the Pax6 3’UTR shows the location of the highly conserved miR-375 target site located at 3’UTR position 201 and miR-375 target site mutation. b ]. qPCR results for GFP with the MS2 motif (grey bar) were expressed relative to data without the MS2 motif (unfilled bar). Data represents 5 independent samples, p = 0.0079. c Affinity purification of miR-375 with the Pax6 3’UTR in αTC1–6 cells using TaqMan individual qPCR assays. Normalized relative quantity was calculated using Pfaffl’s method, and a normalized relative quantity greater than 1 indicates that more target miRNA is purified with the Pax6 3’UTR (grey bar) than the control lacking a Pax6 3’UTR (unfilled bar). Data represents six independent samples, p = 0.013. d Disruption of miR-375 binding to the Pax6 3’UTR following mutation of the miR-375 target site. Target miR-375 values were normalized to GFP as a reference gene, then normalized values for the mutant Pax6 3’UTR samples (grey bar), and are presented relative to the wt 3’UTR (unfilled bar). Data represents six independent wt 3’UTR samples and three miR-375 target site mutant 3’UTR samples, p = 0.0476. Error bars represent 95% confidence intervals, and p -values were calculated using the Mann Whitney test. Note scale bar differences between the graphs

    Techniques Used: Affinity Purification, Plasmid Preparation, Expressing, Sequencing, Transfection, Binding Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Mutagenesis, Purification, MANN-WHITNEY

    40) Product Images from "Alterations of plant architecture and phase transition by the phytoplasma virulence factor SAP11"

    Article Title: Alterations of plant architecture and phase transition by the phytoplasma virulence factor SAP11

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/ery318

    Regulation of the miR156/SPLs module by phytoplasma SAP11 AYWB . (A) miR156 expression levels in 35S::SAP11 transgenic Arabidopsis plants, examined by qRT–PCR using a TaqMan miRNA assay. The relative expression levels of miR156 in vector-only transgenic plants were set to 1 after normalization to the small non-coding RNA SnoR85. (B) Transcript levels of miR156-targeted SPL genes in 35S::SAP11 transgenic plants, examined by qRT–PCR and normalized to Actin2 . The relative expression level of each gene in vector-only transgenic plants was set to 1. Statistically significant differences were determined using Student’s t -test (* P
    Figure Legend Snippet: Regulation of the miR156/SPLs module by phytoplasma SAP11 AYWB . (A) miR156 expression levels in 35S::SAP11 transgenic Arabidopsis plants, examined by qRT–PCR using a TaqMan miRNA assay. The relative expression levels of miR156 in vector-only transgenic plants were set to 1 after normalization to the small non-coding RNA SnoR85. (B) Transcript levels of miR156-targeted SPL genes in 35S::SAP11 transgenic plants, examined by qRT–PCR and normalized to Actin2 . The relative expression level of each gene in vector-only transgenic plants was set to 1. Statistically significant differences were determined using Student’s t -test (* P

    Techniques Used: Expressing, Transgenic Assay, Quantitative RT-PCR, TaqMan microRNA Assay, Plasmid Preparation

    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: NMDA receptor-dependent regulation of miRNA expression and association with Argonaute during LTP in vivo
    Article Snippet: .. After reverse transcription and pre-amplification, qPCR was analyzed in a 25 μ l reaction volume using 2× TaqMan® Universal PCR Master Mix II with no uracil-N-glycosylase (Applied Biosystems). .. PCR quantification was performed in triplicate using the relative standard curve method to determine gene expression levels.

    Article Title: Elevated mitochondrial activity distinguishes fibrogenic hepatic stellate cells and sensitizes for selective inhibition by mitotropic doxorubicin
    Article Snippet: .. TaqMan real‐time polymerase chain reaction (qPCR) Gene expression analysis was performed with TaqMan Universal Master Mix II with UNG (Applied Biosystems, MA, USA) in a QuantStudio 12K Flex Real‐Time PCR System (Applied Biosystems). .. In brief, total RNA was extracted using Trizol reagent (Thermo Fisher Scientific) followed by RNA clean‐up using the RNeasy kit (Qiagen, Valencia, CA, USA).

    Polymerase Chain Reaction:

    Article Title: NMDA receptor-dependent regulation of miRNA expression and association with Argonaute during LTP in vivo
    Article Snippet: .. After reverse transcription and pre-amplification, qPCR was analyzed in a 25 μ l reaction volume using 2× TaqMan® Universal PCR Master Mix II with no uracil-N-glycosylase (Applied Biosystems). .. PCR quantification was performed in triplicate using the relative standard curve method to determine gene expression levels.

    Article Title: Reduced cohesin destabilizes high-level gene amplification by disrupting pre-replication complex bindings in human cancers with chromosomal instability
    Article Snippet: .. Each 20 μl PCR reaction contained 20 ng gDNA and TaqMan probe/primer mix in TaqMan Universal Master Mix, and was amplified using StepOnePlus (Applied Biosystems). .. Real-time data were collected by CopyCaller v2.0 software.

    Amplification:

    Article Title: Reduced cohesin destabilizes high-level gene amplification by disrupting pre-replication complex bindings in human cancers with chromosomal instability
    Article Snippet: .. Each 20 μl PCR reaction contained 20 ng gDNA and TaqMan probe/primer mix in TaqMan Universal Master Mix, and was amplified using StepOnePlus (Applied Biosystems). .. Real-time data were collected by CopyCaller v2.0 software.

    Quantitative RT-PCR:

    Article Title: Cholangiocarcinoma therapy with nanoparticles that combine downregulation of MicroRNA-210 with inhibition of cancer cell invasiveness
    Article Snippet: .. 10 ng of total RNA was converted into cDNA using specific primers for miR-210 (or the internal control Z30 (Applied Biosystems, Foster City, CA)) and the TaqMan microRNA reverse transcription kit (Applied Biosystems). qRT-PCR was performed using TaqMan Universal Master Mix II, No AmpErase UNG (2×) and specific primers for miR-210 or Z30 (Applied Biosystems, Foster City, CA) on a Rotor-Gene Q instrument (QIAGEN) according to the manufacturer's instructions. .. MiRNA expression levels were expressed relative to the internal control according to the comparative threshold cycle (Ct) method.

    Article Title: Chloroquine-containing DMAEMA copolymers as efficient anti-miRNA delivery vectors with improved endosomal escape and anti-migratory activity in cancer cells
    Article Snippet: .. 10 ng of total RNA was converted into cDNA using specific primers for miR-210 (or the internal control Z30 (Applied Biosystems, Foster City, CA)) and the TaqMan microRNA reverse transcription kit (Applied Biosystems). qRT-PCR was performed using TaqMan Universal Master Mix II, No AmpErase UNG (2×) and specific primers for miR-210 or Z30 (Applied Biosystems, Foster City, CA) on a Rotor-Gene Q instrument (QIAGEN) according to the manufacturer’s instructions. .. MiRNA expression levels were expressed relative to the internal control according to the comparative threshold cycle (Ct) method.

    Expressing:

    Article Title: Elevated mitochondrial activity distinguishes fibrogenic hepatic stellate cells and sensitizes for selective inhibition by mitotropic doxorubicin
    Article Snippet: .. TaqMan real‐time polymerase chain reaction (qPCR) Gene expression analysis was performed with TaqMan Universal Master Mix II with UNG (Applied Biosystems, MA, USA) in a QuantStudio 12K Flex Real‐Time PCR System (Applied Biosystems). .. In brief, total RNA was extracted using Trizol reagent (Thermo Fisher Scientific) followed by RNA clean‐up using the RNeasy kit (Qiagen, Valencia, 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 taqman universal pcr master mix ii
    Quantitative real-time <t>PCR</t> analyses comparing miRNA expression patterns in tissues using different extraction methods. Fold abundance of miRNAs quantitated using <t>TaqMan</t> 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
    Taqman Universal Pcr Master Mix Ii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 106 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taqman universal pcr master mix ii/product/Thermo Fisher
    Average 99 stars, based on 106 article reviews
    Price from $9.99 to $1999.99
    taqman universal pcr master mix ii - 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

    miTRAP as a strategy to purify Pax6 3’UTR-associated miRNAs. a Schematic of the Pax6 3’UTR affinity purification approach. (i) Plasmid vectors expressing GFP tagged with the MS2 RNA sequence motif followed by the SV40 polyadenylation signal are introduced into pancreatic αTC1–6 cells via transient transfection. (ii) MS2 coat protein fused to maltose binding (MS2-MBP) is used to purify GFP transcripts with bound miRNAs from αTC1–6 cell lysate. (iii) Real-time quantitative PCR (RT-qPCR) is used to detect GFP transcript and bound miRNAs. Schematic of the Pax6 3’UTR shows the location of the highly conserved miR-375 target site located at 3’UTR position 201 and miR-375 target site mutation. b Validation of the MS2-mediated affinity purification strategy by RT-qPCR quantification of GFP transcripts with and without the MS2 RNA motif. Fold change was calculated using Pfaffl’s method [ 61 ]. qPCR results for GFP with the MS2 motif (grey bar) were expressed relative to data without the MS2 motif (unfilled bar). Data represents 5 independent samples, p = 0.0079. c Affinity purification of miR-375 with the Pax6 3’UTR in αTC1–6 cells using TaqMan individual qPCR assays. Normalized relative quantity was calculated using Pfaffl’s method, and a normalized relative quantity greater than 1 indicates that more target miRNA is purified with the Pax6 3’UTR (grey bar) than the control lacking a Pax6 3’UTR (unfilled bar). Data represents six independent samples, p = 0.013. d Disruption of miR-375 binding to the Pax6 3’UTR following mutation of the miR-375 target site. Target miR-375 values were normalized to GFP as a reference gene, then normalized values for the mutant Pax6 3’UTR samples (grey bar), and are presented relative to the wt 3’UTR (unfilled bar). Data represents six independent wt 3’UTR samples and three miR-375 target site mutant 3’UTR samples, p = 0.0476. Error bars represent 95% confidence intervals, and p -values were calculated using the Mann Whitney test. Note scale bar differences between the graphs

    Journal: BMC Genomics

    Article Title: Mapping the Pax6 3’ untranslated region microRNA regulatory landscape

    doi: 10.1186/s12864-018-5212-x

    Figure Lengend Snippet: miTRAP as a strategy to purify Pax6 3’UTR-associated miRNAs. a Schematic of the Pax6 3’UTR affinity purification approach. (i) Plasmid vectors expressing GFP tagged with the MS2 RNA sequence motif followed by the SV40 polyadenylation signal are introduced into pancreatic αTC1–6 cells via transient transfection. (ii) MS2 coat protein fused to maltose binding (MS2-MBP) is used to purify GFP transcripts with bound miRNAs from αTC1–6 cell lysate. (iii) Real-time quantitative PCR (RT-qPCR) is used to detect GFP transcript and bound miRNAs. Schematic of the Pax6 3’UTR shows the location of the highly conserved miR-375 target site located at 3’UTR position 201 and miR-375 target site mutation. b Validation of the MS2-mediated affinity purification strategy by RT-qPCR quantification of GFP transcripts with and without the MS2 RNA motif. Fold change was calculated using Pfaffl’s method [ 61 ]. qPCR results for GFP with the MS2 motif (grey bar) were expressed relative to data without the MS2 motif (unfilled bar). Data represents 5 independent samples, p = 0.0079. c Affinity purification of miR-375 with the Pax6 3’UTR in αTC1–6 cells using TaqMan individual qPCR assays. Normalized relative quantity was calculated using Pfaffl’s method, and a normalized relative quantity greater than 1 indicates that more target miRNA is purified with the Pax6 3’UTR (grey bar) than the control lacking a Pax6 3’UTR (unfilled bar). Data represents six independent samples, p = 0.013. d Disruption of miR-375 binding to the Pax6 3’UTR following mutation of the miR-375 target site. Target miR-375 values were normalized to GFP as a reference gene, then normalized values for the mutant Pax6 3’UTR samples (grey bar), and are presented relative to the wt 3’UTR (unfilled bar). Data represents six independent wt 3’UTR samples and three miR-375 target site mutant 3’UTR samples, p = 0.0476. Error bars represent 95% confidence intervals, and p -values were calculated using the Mann Whitney test. Note scale bar differences between the graphs

    Article Snippet: The qPCR reactions for miRNA profiling experiments and miTRAP experiments were prepared using TaqMan Universal Master Mix II with UNG (ThermoFisher, 4,440,038), and custom miRNA microfluidic cards were run on an Applied Biosystems 7900 HT Fast Real Time PCR System fitted with the 384-well block. qPCR reactions for miR-375 were prepared using Universal Master Mix II with UNG and TaqMan small RNA assay for miR-375 (ThermoFisher, 4,427,975, 000564), and were run in MicroAmp fast 96-well reaction plates (0.1 ml, Applied Biosystems, 4,346,907) covered with optical adhesive covers (Applied Biosystems, 4,360,954) using the 7900 HT Fast Real Time PCR System fitted with the 96-well block.

    Techniques: Affinity Purification, Plasmid Preparation, Expressing, Sequencing, Transfection, Binding Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Mutagenesis, Purification, MANN-WHITNEY

    Characterization of miRNAs bound to the Pax6 3’UTR in pancreatic α cells. a Identification of miRNAs associated with the Pax6 3’UTR in αTC1–6 cells using TaqMan multiplex qPCR arrays. Normalized relative quantity (NRQ) greater than 1 indicates more target miRNA was purified with the Pax6 3’UTR than the control lacking a Pax6 3’UTR. Some miRNAs expressed in α cells that also have predicted MREs in the Pax6 3’UTR did not associate with the Pax6 3’UTR-containing transcript: miR-101, 1187, 144, 15a, 196b, 335-3p, 362-3p, 365, 410, 466f, 466 k, 495, 501–3p, 96. Results represent four independent experiments. Geometric mean +/− 95% confidence intervals are shown. b Landscape of Pax6 3’UTR miRNA interaction in αTC1–6 cells. Average NRQ value for each interacting miRNA is indicated as a peak at the 3’UTR position(s) of the predicted MRE(s). Predicted MREs for interacting miRNAs are labeled in black, and non-interacting miRNAs in grey. Each peak has a 25-nucleotide width on either side of the MRE position. Overlap between interaction peaks spaced 8–50 nucleotides apart is indicated in red, and these interacting miRNAs may be capable of mediating cooperative regulation of Pax6. MREs for Pax6 3’UTR-interacting miRNAs were found to cluster into three regions, i-iii (orange boxes), located approximately at nucleotide positions 250–350, 420–550 and 600–810. Conservation of the Pax6 3’UTR sequence between orthologous placental mammal sequences is shown for the 876-nucleotide length. Secondary, poorly conserved MREs for interacting miRNAs having well conserved predicted MREs are not shown. c miRNA interaction with the Pax6 3’UTR is not directly related to miRNA abundance in αTC1–6 cells. miTRAP ratio and relative miRNA level for αTC1–6-interacting miRNAs is shown. miTRAP ratio was calculated by dividing the relative abundance of each miRNA with the Pax6 3’UTR by the relative abundance in αTC1–6 cells. Larger miTRAP values indicated greater enrichment of the miRNA with the Pax6 3’UTR relative to cellular abundance

    Journal: BMC Genomics

    Article Title: Mapping the Pax6 3’ untranslated region microRNA regulatory landscape

    doi: 10.1186/s12864-018-5212-x

    Figure Lengend Snippet: Characterization of miRNAs bound to the Pax6 3’UTR in pancreatic α cells. a Identification of miRNAs associated with the Pax6 3’UTR in αTC1–6 cells using TaqMan multiplex qPCR arrays. Normalized relative quantity (NRQ) greater than 1 indicates more target miRNA was purified with the Pax6 3’UTR than the control lacking a Pax6 3’UTR. Some miRNAs expressed in α cells that also have predicted MREs in the Pax6 3’UTR did not associate with the Pax6 3’UTR-containing transcript: miR-101, 1187, 144, 15a, 196b, 335-3p, 362-3p, 365, 410, 466f, 466 k, 495, 501–3p, 96. Results represent four independent experiments. Geometric mean +/− 95% confidence intervals are shown. b Landscape of Pax6 3’UTR miRNA interaction in αTC1–6 cells. Average NRQ value for each interacting miRNA is indicated as a peak at the 3’UTR position(s) of the predicted MRE(s). Predicted MREs for interacting miRNAs are labeled in black, and non-interacting miRNAs in grey. Each peak has a 25-nucleotide width on either side of the MRE position. Overlap between interaction peaks spaced 8–50 nucleotides apart is indicated in red, and these interacting miRNAs may be capable of mediating cooperative regulation of Pax6. MREs for Pax6 3’UTR-interacting miRNAs were found to cluster into three regions, i-iii (orange boxes), located approximately at nucleotide positions 250–350, 420–550 and 600–810. Conservation of the Pax6 3’UTR sequence between orthologous placental mammal sequences is shown for the 876-nucleotide length. Secondary, poorly conserved MREs for interacting miRNAs having well conserved predicted MREs are not shown. c miRNA interaction with the Pax6 3’UTR is not directly related to miRNA abundance in αTC1–6 cells. miTRAP ratio and relative miRNA level for αTC1–6-interacting miRNAs is shown. miTRAP ratio was calculated by dividing the relative abundance of each miRNA with the Pax6 3’UTR by the relative abundance in αTC1–6 cells. Larger miTRAP values indicated greater enrichment of the miRNA with the Pax6 3’UTR relative to cellular abundance

    Article Snippet: The qPCR reactions for miRNA profiling experiments and miTRAP experiments were prepared using TaqMan Universal Master Mix II with UNG (ThermoFisher, 4,440,038), and custom miRNA microfluidic cards were run on an Applied Biosystems 7900 HT Fast Real Time PCR System fitted with the 384-well block. qPCR reactions for miR-375 were prepared using Universal Master Mix II with UNG and TaqMan small RNA assay for miR-375 (ThermoFisher, 4,427,975, 000564), and were run in MicroAmp fast 96-well reaction plates (0.1 ml, Applied Biosystems, 4,346,907) covered with optical adhesive covers (Applied Biosystems, 4,360,954) using the 7900 HT Fast Real Time PCR System fitted with the 96-well block.

    Techniques: Multiplex Assay, Real-time Polymerase Chain Reaction, Purification, Labeling, Sequencing

    Expression profile of miRNAs predicted to target the mouse Pax6 3’UTR. a Expression profile of miRNAs having predicted target sites in the mouse Pax6 3’UTR in various Pax6 -expressing cells and tissues. miRNAs assayed for include those identified by our prediction analysis (Fig. 3a ) as well as others, as described in the results. miRNAs were assayed for using TaqMan multiplex qPCR array cards. Data represents a total of 3 replicates per tissue or cell type, with a miRNA considered to be expressed only if the cycle threshold was less than 40 for all three replicates. b Relative miRNA level in mouse cultured pancreatic α cell line, αTC1–6 and a cultured β cell line, βTC6. c Relative miRNA level in mouse E12.5 retina, adult retina, and adult lens. Heat map indicates relative miRNA expression as a percentage of an internal control, snRNA U6. Data represents the geometric mean of three independent samples. Note the scale differences between the two heat maps

    Journal: BMC Genomics

    Article Title: Mapping the Pax6 3’ untranslated region microRNA regulatory landscape

    doi: 10.1186/s12864-018-5212-x

    Figure Lengend Snippet: Expression profile of miRNAs predicted to target the mouse Pax6 3’UTR. a Expression profile of miRNAs having predicted target sites in the mouse Pax6 3’UTR in various Pax6 -expressing cells and tissues. miRNAs assayed for include those identified by our prediction analysis (Fig. 3a ) as well as others, as described in the results. miRNAs were assayed for using TaqMan multiplex qPCR array cards. Data represents a total of 3 replicates per tissue or cell type, with a miRNA considered to be expressed only if the cycle threshold was less than 40 for all three replicates. b Relative miRNA level in mouse cultured pancreatic α cell line, αTC1–6 and a cultured β cell line, βTC6. c Relative miRNA level in mouse E12.5 retina, adult retina, and adult lens. Heat map indicates relative miRNA expression as a percentage of an internal control, snRNA U6. Data represents the geometric mean of three independent samples. Note the scale differences between the two heat maps

    Article Snippet: The qPCR reactions for miRNA profiling experiments and miTRAP experiments were prepared using TaqMan Universal Master Mix II with UNG (ThermoFisher, 4,440,038), and custom miRNA microfluidic cards were run on an Applied Biosystems 7900 HT Fast Real Time PCR System fitted with the 384-well block. qPCR reactions for miR-375 were prepared using Universal Master Mix II with UNG and TaqMan small RNA assay for miR-375 (ThermoFisher, 4,427,975, 000564), and were run in MicroAmp fast 96-well reaction plates (0.1 ml, Applied Biosystems, 4,346,907) covered with optical adhesive covers (Applied Biosystems, 4,360,954) using the 7900 HT Fast Real Time PCR System fitted with the 96-well block.

    Techniques: Expressing, Multiplex Assay, Real-time Polymerase Chain Reaction, Cell Culture

    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