mircury lna mirna pcr assay  (Qiagen)

 
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    Name:
    miRCURY LNA miRNA PCR Assay
    Description:
    miRCURY LNA miRNA PCR Assays are individual miRNA PCR primer sets that enable extremely sensitive and specific miRNA quantification with themiRCURY LNA miRNA PCR System Both forward and reverse PCR amplification primers are miRNA specific and are optimized with LNA technology Assays are available in tube or plate format with 200 reactions per tube or well
    Catalog Number:
    339306
    Price:
    None
    Category:
    qPCR Arrays and Assays
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    Structured Review

    Qiagen mircury lna mirna pcr assay
    miRCURY LNA miRNA PCR Assay
    miRCURY LNA miRNA PCR Assays are individual miRNA PCR primer sets that enable extremely sensitive and specific miRNA quantification with themiRCURY LNA miRNA PCR System Both forward and reverse PCR amplification primers are miRNA specific and are optimized with LNA technology Assays are available in tube or plate format with 200 reactions per tube or well
    https://www.bioz.com/result/mircury lna mirna pcr assay/product/Qiagen
    Average 93 stars, based on 21550 article reviews
    Price from $9.99 to $1999.99
    mircury lna mirna pcr assay - by Bioz Stars, 2020-07
    93/100 stars

    Images

    1) Product Images from "MiR-337-3p Promotes Adipocyte Browning by Inhibiting TWIST1"

    Article Title: MiR-337-3p Promotes Adipocyte Browning by Inhibiting TWIST1

    Journal: Cells

    doi: 10.3390/cells9041056

    miR-337-3p is a potential regulator of browning of adipose tissue. ( a ) Representative heatmap showing the results of RT-PCR analysis used to screen a panel of microRNAs (miR) in mouse white adipose tissue (WAT) and brown adipose tissue (BAT): Let-7c, miR-199a-5p, miR-151-3p, miR-145-5p, Let-7b-3p, miR-24-3p, miR-361-5p, miR-335-5p, miR-337-3p, miR-134-5p. Real-time PCR was performed using the miRCURY LNA miR PCR Assay and normalized to U6 RNA and relative to WAT. Differentially expressed miR (fold change ≥0.5) are indicated in yellow, which represents upregulated miRNA in BAT compared to WAT, and in blue are the downregulated miRs (fold change ≤−0.5) in BAT compared to WAT. N = 3/group. ( b ) RT-PCR analysis of transcript abundance of BAT thermogenic and mitochondrial markers Ucp1 , Cs , and Crls1 in BAT compared to WAT. mRNA expression values were normalized to L7 mRNA. N = 8/group. ( c ) RT-PCR analysis of mmu-miR-337-3p expression in mouse WAT and BAT normalized to U6 RNA. N = 8/group ( d ) RT-PCR analysis of mmu-miR-337-3p abundance in BAT, WAT, liver, kidney, small intestine, skeletal muscle, pancreas, brains, and lungs presented as percentage of mmu-miR-337-3p transcript abundance from its expression in BAT. Expression of miR-337-3p in BAT is set to 100%. ( e ) Representative image of the genomic mapping of miR-337-3p in humans and conservation of its seed sequence among species. Data are presented as means ± SEM; individual data points are given by the dot plot. Either Mann–Whitney-U or Independent sample t -test was used to assess differences in expression levels. ### p
    Figure Legend Snippet: miR-337-3p is a potential regulator of browning of adipose tissue. ( a ) Representative heatmap showing the results of RT-PCR analysis used to screen a panel of microRNAs (miR) in mouse white adipose tissue (WAT) and brown adipose tissue (BAT): Let-7c, miR-199a-5p, miR-151-3p, miR-145-5p, Let-7b-3p, miR-24-3p, miR-361-5p, miR-335-5p, miR-337-3p, miR-134-5p. Real-time PCR was performed using the miRCURY LNA miR PCR Assay and normalized to U6 RNA and relative to WAT. Differentially expressed miR (fold change ≥0.5) are indicated in yellow, which represents upregulated miRNA in BAT compared to WAT, and in blue are the downregulated miRs (fold change ≤−0.5) in BAT compared to WAT. N = 3/group. ( b ) RT-PCR analysis of transcript abundance of BAT thermogenic and mitochondrial markers Ucp1 , Cs , and Crls1 in BAT compared to WAT. mRNA expression values were normalized to L7 mRNA. N = 8/group. ( c ) RT-PCR analysis of mmu-miR-337-3p expression in mouse WAT and BAT normalized to U6 RNA. N = 8/group ( d ) RT-PCR analysis of mmu-miR-337-3p abundance in BAT, WAT, liver, kidney, small intestine, skeletal muscle, pancreas, brains, and lungs presented as percentage of mmu-miR-337-3p transcript abundance from its expression in BAT. Expression of miR-337-3p in BAT is set to 100%. ( e ) Representative image of the genomic mapping of miR-337-3p in humans and conservation of its seed sequence among species. Data are presented as means ± SEM; individual data points are given by the dot plot. Either Mann–Whitney-U or Independent sample t -test was used to assess differences in expression levels. ### p

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

    2) Product Images from "Interplay between the Epigenetic Enzyme Lysine (K)-Specific Demethylase 2B and Epstein-Barr Virus Infection"

    Article Title: Interplay between the Epigenetic Enzyme Lysine (K)-Specific Demethylase 2B and Epstein-Barr Virus Infection

    Journal: Journal of Virology

    doi: 10.1128/JVI.00273-19

    EBV also uses LMP1-independent mechanisms to downregulate KDM2B. (A) B cells from two donors were infected with EBV or EBVΔLMP1 or mock infected (MI) and collected at 48 h after infection. Reverse-transcribed RNA samples were analyzed by qPCR for the KDM2B expression level. (B) B cells from two donors were infected with EBV and collected at 12, 24, and 48 h after infection. Cells were processed for RNA extraction and analyzed by qPCR for the expression levels of EBNA1, EBNA2, EBER, LMP1, and KDM2B transcripts. The expression levels of viral gene transcripts were measured relative to their levels in B cells collected 12 h (EBNA2) or 48 h (EBNA1, EBER, LMP1) postinfection. The levels of KDM2B mRNA were measured relative to its levels in mock-infected cells. (C) Different EBV(+) cell lines in latency phase I (BL110, I100, MUTU) or in latency phase III (LCL, Raji, B95) and primary B cells (BC) were collected, processed for RNA extraction and reverse transcription, and analyzed by qPCR for LMP1 (top), EBNA1 (middle), and KDM2B (bottom) mRNA levels. (D and E) RPMI (D) and Louckes (E) cells were transiently transfected with different constructs carrying individual EBV genes (TC) in three independent experiments. At 48 h after transfection, cells were processed for RNA and protein extraction and analyzed for the KDM2B expression level by RT-qPCR analysis. The levels of KDM2B mRNA were measured relative to its levels in mock-infected cells. Western blotting (LMP1, EBNA1, EBNA3A/3B, and EBNALP) or RT-PCR analysis (LMP2A, EBNA2, and EBNA3C) was performed to measure the expression of the different viral latent proteins. (F) RPMI cells were transfected with different EBV gene-carrying constructs. At 44 h after transfection, cells were exposed to the proteasome inhibitor MG132 for 4 h and then collected, processed for total protein extraction, and analyzed for the indicated proteins by immunoblotting. The histogram shows the average phosphorylated IκBα (P-IκBα) signal normalized to the levels of total IκBα. (G) B cells from different donors were untreated or treated with a miRNA-146a-5p inhibitor for 24 h before infection with EBV. At 48 h after infection, cells were collected and processed for RNA extraction and reverse transcription. cDNA samples were analyzed by qPCR for LMP1 and KDM2B expression levels. The levels of KDM2B mRNA were measured relative to its levels in mock-infected cells. The histograms show the average expression levels measured in 2 independent experiments (*, P  
    Figure Legend Snippet: EBV also uses LMP1-independent mechanisms to downregulate KDM2B. (A) B cells from two donors were infected with EBV or EBVΔLMP1 or mock infected (MI) and collected at 48 h after infection. Reverse-transcribed RNA samples were analyzed by qPCR for the KDM2B expression level. (B) B cells from two donors were infected with EBV and collected at 12, 24, and 48 h after infection. Cells were processed for RNA extraction and analyzed by qPCR for the expression levels of EBNA1, EBNA2, EBER, LMP1, and KDM2B transcripts. The expression levels of viral gene transcripts were measured relative to their levels in B cells collected 12 h (EBNA2) or 48 h (EBNA1, EBER, LMP1) postinfection. The levels of KDM2B mRNA were measured relative to its levels in mock-infected cells. (C) Different EBV(+) cell lines in latency phase I (BL110, I100, MUTU) or in latency phase III (LCL, Raji, B95) and primary B cells (BC) were collected, processed for RNA extraction and reverse transcription, and analyzed by qPCR for LMP1 (top), EBNA1 (middle), and KDM2B (bottom) mRNA levels. (D and E) RPMI (D) and Louckes (E) cells were transiently transfected with different constructs carrying individual EBV genes (TC) in three independent experiments. At 48 h after transfection, cells were processed for RNA and protein extraction and analyzed for the KDM2B expression level by RT-qPCR analysis. The levels of KDM2B mRNA were measured relative to its levels in mock-infected cells. Western blotting (LMP1, EBNA1, EBNA3A/3B, and EBNALP) or RT-PCR analysis (LMP2A, EBNA2, and EBNA3C) was performed to measure the expression of the different viral latent proteins. (F) RPMI cells were transfected with different EBV gene-carrying constructs. At 44 h after transfection, cells were exposed to the proteasome inhibitor MG132 for 4 h and then collected, processed for total protein extraction, and analyzed for the indicated proteins by immunoblotting. The histogram shows the average phosphorylated IκBα (P-IκBα) signal normalized to the levels of total IκBα. (G) B cells from different donors were untreated or treated with a miRNA-146a-5p inhibitor for 24 h before infection with EBV. At 48 h after infection, cells were collected and processed for RNA extraction and reverse transcription. cDNA samples were analyzed by qPCR for LMP1 and KDM2B expression levels. The levels of KDM2B mRNA were measured relative to its levels in mock-infected cells. The histograms show the average expression levels measured in 2 independent experiments (*, P  

    Techniques Used: Infection, Real-time Polymerase Chain Reaction, Expressing, RNA Extraction, Transfection, Construct, Protein Extraction, Quantitative RT-PCR, Western Blot, Reverse Transcription Polymerase Chain Reaction

    KDM2B regulates EBV gene expression in latently infected cells. (A to C) LCL were transfected with 1.5 μg of pCDNA3-KDM2B or with pCDNA as a control in three independent experiments. Cells were collected at 24 h after transfection and processed for RNA/DNA and total protein extraction. (A) KDM2B mRNA and protein levels were shown by qPCR (bottom) and immunoblotting (top; the lines corresponding to conditions with 0.5 μg and 1 μg of KDM2B, originally present in the Western blot, are not shown because they were excluded from all the analyses). (B) DNA samples were analyzed by TaqMan PCR to assess the number of EBV genome copies per cell (ns, not significant). (C) The mRNA expression levels of EBV latent and early genes were assessed by qPCR (*, P  
    Figure Legend Snippet: KDM2B regulates EBV gene expression in latently infected cells. (A to C) LCL were transfected with 1.5 μg of pCDNA3-KDM2B or with pCDNA as a control in three independent experiments. Cells were collected at 24 h after transfection and processed for RNA/DNA and total protein extraction. (A) KDM2B mRNA and protein levels were shown by qPCR (bottom) and immunoblotting (top; the lines corresponding to conditions with 0.5 μg and 1 μg of KDM2B, originally present in the Western blot, are not shown because they were excluded from all the analyses). (B) DNA samples were analyzed by TaqMan PCR to assess the number of EBV genome copies per cell (ns, not significant). (C) The mRNA expression levels of EBV latent and early genes were assessed by qPCR (*, P  

    Techniques Used: Expressing, Infection, Transfection, Protein Extraction, Real-time Polymerase Chain Reaction, Western Blot, Polymerase Chain Reaction

    KDM2B deregulation alters EBV gene expression. (A) Louckes cells were transiently transfected with increasing concentrations of the KDM2B expression vector in three independent experiments, collected at 24 h after transfection, and processed for protein and RNA extraction to assess KDM2B levels by immunoblotting (top) or qPCR (bottom). (B) Cells were then infected with EBV, and at 24 h after infection they were collected and processed for FACS analysis and for RNA/DNA extraction. DNA samples were used to measure EBV genome copy number by TaqMan PCR (ns, not significant). (C) Live cells were analyzed by FACS to measure the GFP mean fluorescence intensity (MFI). (D) cDNA samples were analyzed for the expression level of EBV early and late genes by qPCR. The levels of EBV genes transcripts were normalized to the mRNA levels of the housekeeping gene β-globin and calculated relative to their levels in cells transfected with the empty vector (KDM2B, 0 μg). The values shown in the histogram are the average from 3 independent experiments (*, P  
    Figure Legend Snippet: KDM2B deregulation alters EBV gene expression. (A) Louckes cells were transiently transfected with increasing concentrations of the KDM2B expression vector in three independent experiments, collected at 24 h after transfection, and processed for protein and RNA extraction to assess KDM2B levels by immunoblotting (top) or qPCR (bottom). (B) Cells were then infected with EBV, and at 24 h after infection they were collected and processed for FACS analysis and for RNA/DNA extraction. DNA samples were used to measure EBV genome copy number by TaqMan PCR (ns, not significant). (C) Live cells were analyzed by FACS to measure the GFP mean fluorescence intensity (MFI). (D) cDNA samples were analyzed for the expression level of EBV early and late genes by qPCR. The levels of EBV genes transcripts were normalized to the mRNA levels of the housekeeping gene β-globin and calculated relative to their levels in cells transfected with the empty vector (KDM2B, 0 μg). The values shown in the histogram are the average from 3 independent experiments (*, P  

    Techniques Used: Expressing, Transfection, Plasmid Preparation, RNA Extraction, Real-time Polymerase Chain Reaction, Infection, FACS, DNA Extraction, Polymerase Chain Reaction, Fluorescence

    3) Product Images from "Evaluation of extraction kits and RT-qPCR systems adapted to high-throughput platform for circulating miRNAs"

    Article Title: Evaluation of extraction kits and RT-qPCR systems adapted to high-throughput platform for circulating miRNAs

    Journal: Scientific Reports

    doi: 10.1038/srep09430

    miRNA recovery of different extraction kits were evaluated by the C q values for spike-in controls (cel-miR-39 and cel-miR-54) assayed in the (a) TaqMan and (b) miScript RT-qPCR system. Box plots with whiskers show 5–95 percentile of the C q values for spike-in controls and the corresponding CVs are shown. The extent of extraction bias across samples was reflected in the range and CV of C q values for spike-in controls. Higher median C q compared to other extraction kits indicate poorer performance in miRNA recovery. Q, miRNeasy Serum/Plasma kit; E, miRCURY™ RNA Isolation Kit - Biofluids; A, mirVana™ PARIS™ Kit; MN, NucleoSpin® miRNA Plasma; NB, Plasma/Serum Circulating RNA Purification Kit.
    Figure Legend Snippet: miRNA recovery of different extraction kits were evaluated by the C q values for spike-in controls (cel-miR-39 and cel-miR-54) assayed in the (a) TaqMan and (b) miScript RT-qPCR system. Box plots with whiskers show 5–95 percentile of the C q values for spike-in controls and the corresponding CVs are shown. The extent of extraction bias across samples was reflected in the range and CV of C q values for spike-in controls. Higher median C q compared to other extraction kits indicate poorer performance in miRNA recovery. Q, miRNeasy Serum/Plasma kit; E, miRCURY™ RNA Isolation Kit - Biofluids; A, mirVana™ PARIS™ Kit; MN, NucleoSpin® miRNA Plasma; NB, Plasma/Serum Circulating RNA Purification Kit.

    Techniques Used: Quantitative RT-PCR, Isolation, Purification

    Comparison of TaqMan and miScript RT-qPCR systems adapted to the high-throughput platform by performance parameters which include reproducibility, accuracy and sensitivity. Transformation of data to z-score was carried out to facilitate direct comparison. A1, accuracy when measuring fold change of abundant miRNAs; A2, accuracy when measuring fold change of less abundant miRNAs, R1, reproducibility of RT replicates; R2, reproducibility of RT replicates after normalisation; R3, reproducibility of qPCR replicates; S1, sensitivity to detect the presence of plasma miRNAs and S2, sensitivity to detect the presence of miRNAs in titration points.
    Figure Legend Snippet: Comparison of TaqMan and miScript RT-qPCR systems adapted to the high-throughput platform by performance parameters which include reproducibility, accuracy and sensitivity. Transformation of data to z-score was carried out to facilitate direct comparison. A1, accuracy when measuring fold change of abundant miRNAs; A2, accuracy when measuring fold change of less abundant miRNAs, R1, reproducibility of RT replicates; R2, reproducibility of RT replicates after normalisation; R3, reproducibility of qPCR replicates; S1, sensitivity to detect the presence of plasma miRNAs and S2, sensitivity to detect the presence of miRNAs in titration points.

    Techniques Used: Quantitative RT-PCR, High Throughput Screening Assay, Transformation Assay, Real-time Polymerase Chain Reaction, Titration

    Evaluation of consistency between technical replicatesin RT-qPCR. Consistencies among (a) RT replicates in TaqMan system, (b) RT replicates in miScript system, (c) qPCR replicates in TaqMan system and (d) qPCR replicates in miScript system were evaluated by measuring the levels of 11–16 miRNAs in a same set of RNA samples (n = 5–14). Each data point in scatter plot represents the average C q value obtained from the duplicate or triplicate wells of qPCR. Overall, significant correlations (ICC = 0.712–0.996, p
    Figure Legend Snippet: Evaluation of consistency between technical replicatesin RT-qPCR. Consistencies among (a) RT replicates in TaqMan system, (b) RT replicates in miScript system, (c) qPCR replicates in TaqMan system and (d) qPCR replicates in miScript system were evaluated by measuring the levels of 11–16 miRNAs in a same set of RNA samples (n = 5–14). Each data point in scatter plot represents the average C q value obtained from the duplicate or triplicate wells of qPCR. Overall, significant correlations (ICC = 0.712–0.996, p

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

    Comparison of different RT-qPCR systems adapted to the BioMark high-throughput platform. (a) Average C q values for endogeneous plasma miRNAs were depicted in box plot with whiskers showing the 5–95 percentile. Irrespective of extraction kits being used, within the same set of samples, more undetermined C q values were obtained in the miScript system. (b) Correlation of qPCR results from TaqMan and miScript systems may vary among different miRNA assays. Each data point represents the normalised C q value transformed into z-score. Q, miRNeasy Serum/Plasma kit; E, miRCURY™ RNA Isolation Kit - Biofluids; A, mirVana™ PARIS™ Kit; MN, NucleoSpin® miRNA Plasma; NB, Plasma/Serum Circulating RNA Purification Kit.
    Figure Legend Snippet: Comparison of different RT-qPCR systems adapted to the BioMark high-throughput platform. (a) Average C q values for endogeneous plasma miRNAs were depicted in box plot with whiskers showing the 5–95 percentile. Irrespective of extraction kits being used, within the same set of samples, more undetermined C q values were obtained in the miScript system. (b) Correlation of qPCR results from TaqMan and miScript systems may vary among different miRNA assays. Each data point represents the normalised C q value transformed into z-score. Q, miRNeasy Serum/Plasma kit; E, miRCURY™ RNA Isolation Kit - Biofluids; A, mirVana™ PARIS™ Kit; MN, NucleoSpin® miRNA Plasma; NB, Plasma/Serum Circulating RNA Purification Kit.

    Techniques Used: Quantitative RT-PCR, High Throughput Screening Assay, Real-time Polymerase Chain Reaction, Transformation Assay, Isolation, Purification

    4) Product Images from "MiR-337-3p Promotes Adipocyte Browning by Inhibiting TWIST1"

    Article Title: MiR-337-3p Promotes Adipocyte Browning by Inhibiting TWIST1

    Journal: Cells

    doi: 10.3390/cells9041056

    miR-337-3p is a potential regulator of browning of adipose tissue. ( a ) Representative heatmap showing the results of RT-PCR analysis used to screen a panel of microRNAs (miR) in mouse white adipose tissue (WAT) and brown adipose tissue (BAT): Let-7c, miR-199a-5p, miR-151-3p, miR-145-5p, Let-7b-3p, miR-24-3p, miR-361-5p, miR-335-5p, miR-337-3p, miR-134-5p. Real-time PCR was performed using the miRCURY LNA miR PCR Assay and normalized to U6 RNA and relative to WAT. Differentially expressed miR (fold change ≥0.5) are indicated in yellow, which represents upregulated miRNA in BAT compared to WAT, and in blue are the downregulated miRs (fold change ≤−0.5) in BAT compared to WAT. N = 3/group. ( b ) RT-PCR analysis of transcript abundance of BAT thermogenic and mitochondrial markers Ucp1 , Cs , and Crls1 in BAT compared to WAT. mRNA expression values were normalized to L7 mRNA. N = 8/group. ( c ) RT-PCR analysis of mmu-miR-337-3p expression in mouse WAT and BAT normalized to U6 RNA. N = 8/group ( d ) RT-PCR analysis of mmu-miR-337-3p abundance in BAT, WAT, liver, kidney, small intestine, skeletal muscle, pancreas, brains, and lungs presented as percentage of mmu-miR-337-3p transcript abundance from its expression in BAT. Expression of miR-337-3p in BAT is set to 100%. ( e ) Representative image of the genomic mapping of miR-337-3p in humans and conservation of its seed sequence among species. Data are presented as means ± SEM; individual data points are given by the dot plot. Either Mann–Whitney-U or Independent sample t -test was used to assess differences in expression levels. ### p
    Figure Legend Snippet: miR-337-3p is a potential regulator of browning of adipose tissue. ( a ) Representative heatmap showing the results of RT-PCR analysis used to screen a panel of microRNAs (miR) in mouse white adipose tissue (WAT) and brown adipose tissue (BAT): Let-7c, miR-199a-5p, miR-151-3p, miR-145-5p, Let-7b-3p, miR-24-3p, miR-361-5p, miR-335-5p, miR-337-3p, miR-134-5p. Real-time PCR was performed using the miRCURY LNA miR PCR Assay and normalized to U6 RNA and relative to WAT. Differentially expressed miR (fold change ≥0.5) are indicated in yellow, which represents upregulated miRNA in BAT compared to WAT, and in blue are the downregulated miRs (fold change ≤−0.5) in BAT compared to WAT. N = 3/group. ( b ) RT-PCR analysis of transcript abundance of BAT thermogenic and mitochondrial markers Ucp1 , Cs , and Crls1 in BAT compared to WAT. mRNA expression values were normalized to L7 mRNA. N = 8/group. ( c ) RT-PCR analysis of mmu-miR-337-3p expression in mouse WAT and BAT normalized to U6 RNA. N = 8/group ( d ) RT-PCR analysis of mmu-miR-337-3p abundance in BAT, WAT, liver, kidney, small intestine, skeletal muscle, pancreas, brains, and lungs presented as percentage of mmu-miR-337-3p transcript abundance from its expression in BAT. Expression of miR-337-3p in BAT is set to 100%. ( e ) Representative image of the genomic mapping of miR-337-3p in humans and conservation of its seed sequence among species. Data are presented as means ± SEM; individual data points are given by the dot plot. Either Mann–Whitney-U or Independent sample t -test was used to assess differences in expression levels. ### p

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

    5) Product Images from "Stemness, Pluripotentiality, and Wnt Antagonism: sFRP4, a Wnt antagonist Mediates Pluripotency and Stemness in Glioblastoma"

    Article Title: Stemness, Pluripotentiality, and Wnt Antagonism: sFRP4, a Wnt antagonist Mediates Pluripotency and Stemness in Glioblastoma

    Journal: Cancers

    doi: 10.3390/cancers11010025

    ( A – F ). Apoptosis related genes identified in sFRP4 chromatin immunoprecipitation (ChIP) and pull-down sequencing analysis. Schematic 1 represents the sequence of steps of ChIP and downstream sequencing. ChIP DNA resolved on agarose gel indicated a 150 bp DNA band ( A ), ChIP mapping statistics by Burrow-Wheeler Aligner software indicated 5,581,398 mapped reads ( B ); peak calling analysis output using MACS2 software revealed 34,711 peaks related to secreted frizzled-related protein 4 ( sFRP4 ) ( C , left panel), categorization of peak identities represented by pie chart and table, analysis gene list present within 5′UTR (enlarged) indicated the presence of miRNA 885 ( C , right panel), RNA sequencing data of 5′UTR revealed an upregulation of three 5′UTR genes in sFRP4 OE (OE) and downregulation in sFRP4 SI (SI) cells as indicated in the box ( D ), upregulation of active miR-885 in sFRP4 OE cells using an miR-885 5′LNA probe was detected as red fluorescence using a fluorescence microscope (scale bar = 10 μm) ( E ), quantitative RT-PCR indicated an over expression of miR885 in sFRP4 OE and downregulation in sFRP4 SI cells ( F ). Schematic 2 represents a model indicating the mode of action of miR-885 through its target genes CDK2 and MCM5 in cellular homeostasis via activation of p53 . Results are mean ± SD of three independent experiments performed in triplicates (* p value
    Figure Legend Snippet: ( A – F ). Apoptosis related genes identified in sFRP4 chromatin immunoprecipitation (ChIP) and pull-down sequencing analysis. Schematic 1 represents the sequence of steps of ChIP and downstream sequencing. ChIP DNA resolved on agarose gel indicated a 150 bp DNA band ( A ), ChIP mapping statistics by Burrow-Wheeler Aligner software indicated 5,581,398 mapped reads ( B ); peak calling analysis output using MACS2 software revealed 34,711 peaks related to secreted frizzled-related protein 4 ( sFRP4 ) ( C , left panel), categorization of peak identities represented by pie chart and table, analysis gene list present within 5′UTR (enlarged) indicated the presence of miRNA 885 ( C , right panel), RNA sequencing data of 5′UTR revealed an upregulation of three 5′UTR genes in sFRP4 OE (OE) and downregulation in sFRP4 SI (SI) cells as indicated in the box ( D ), upregulation of active miR-885 in sFRP4 OE cells using an miR-885 5′LNA probe was detected as red fluorescence using a fluorescence microscope (scale bar = 10 μm) ( E ), quantitative RT-PCR indicated an over expression of miR885 in sFRP4 OE and downregulation in sFRP4 SI cells ( F ). Schematic 2 represents a model indicating the mode of action of miR-885 through its target genes CDK2 and MCM5 in cellular homeostasis via activation of p53 . Results are mean ± SD of three independent experiments performed in triplicates (* p value

    Techniques Used: Chromatin Immunoprecipitation, Sequencing, Agarose Gel Electrophoresis, Software, RNA Sequencing Assay, Fluorescence, Microscopy, Quantitative RT-PCR, Over Expression, Activation Assay

    6) Product Images from "MiR-337-3p Promotes Adipocyte Browning by Inhibiting TWIST1"

    Article Title: MiR-337-3p Promotes Adipocyte Browning by Inhibiting TWIST1

    Journal: Cells

    doi: 10.3390/cells9041056

    miR-337-3p is a potential regulator of browning of adipose tissue. ( a ) Representative heatmap showing the results of RT-PCR analysis used to screen a panel of microRNAs (miR) in mouse white adipose tissue (WAT) and brown adipose tissue (BAT): Let-7c, miR-199a-5p, miR-151-3p, miR-145-5p, Let-7b-3p, miR-24-3p, miR-361-5p, miR-335-5p, miR-337-3p, miR-134-5p. Real-time PCR was performed using the miRCURY LNA miR PCR Assay and normalized to U6 RNA and relative to WAT. Differentially expressed miR (fold change ≥0.5) are indicated in yellow, which represents upregulated miRNA in BAT compared to WAT, and in blue are the downregulated miRs (fold change ≤−0.5) in BAT compared to WAT. N = 3/group. ( b ) RT-PCR analysis of transcript abundance of BAT thermogenic and mitochondrial markers Ucp1 , Cs , and Crls1 in BAT compared to WAT. mRNA expression values were normalized to L7 mRNA. N = 8/group. ( c ) RT-PCR analysis of mmu-miR-337-3p expression in mouse WAT and BAT normalized to U6 RNA. N = 8/group ( d ) RT-PCR analysis of mmu-miR-337-3p abundance in BAT, WAT, liver, kidney, small intestine, skeletal muscle, pancreas, brains, and lungs presented as percentage of mmu-miR-337-3p transcript abundance from its expression in BAT. Expression of miR-337-3p in BAT is set to 100%. ( e ) Representative image of the genomic mapping of miR-337-3p in humans and conservation of its seed sequence among species. Data are presented as means ± SEM; individual data points are given by the dot plot. Either Mann–Whitney-U or Independent sample t -test was used to assess differences in expression levels. ### p
    Figure Legend Snippet: miR-337-3p is a potential regulator of browning of adipose tissue. ( a ) Representative heatmap showing the results of RT-PCR analysis used to screen a panel of microRNAs (miR) in mouse white adipose tissue (WAT) and brown adipose tissue (BAT): Let-7c, miR-199a-5p, miR-151-3p, miR-145-5p, Let-7b-3p, miR-24-3p, miR-361-5p, miR-335-5p, miR-337-3p, miR-134-5p. Real-time PCR was performed using the miRCURY LNA miR PCR Assay and normalized to U6 RNA and relative to WAT. Differentially expressed miR (fold change ≥0.5) are indicated in yellow, which represents upregulated miRNA in BAT compared to WAT, and in blue are the downregulated miRs (fold change ≤−0.5) in BAT compared to WAT. N = 3/group. ( b ) RT-PCR analysis of transcript abundance of BAT thermogenic and mitochondrial markers Ucp1 , Cs , and Crls1 in BAT compared to WAT. mRNA expression values were normalized to L7 mRNA. N = 8/group. ( c ) RT-PCR analysis of mmu-miR-337-3p expression in mouse WAT and BAT normalized to U6 RNA. N = 8/group ( d ) RT-PCR analysis of mmu-miR-337-3p abundance in BAT, WAT, liver, kidney, small intestine, skeletal muscle, pancreas, brains, and lungs presented as percentage of mmu-miR-337-3p transcript abundance from its expression in BAT. Expression of miR-337-3p in BAT is set to 100%. ( e ) Representative image of the genomic mapping of miR-337-3p in humans and conservation of its seed sequence among species. Data are presented as means ± SEM; individual data points are given by the dot plot. Either Mann–Whitney-U or Independent sample t -test was used to assess differences in expression levels. ### p

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

    7) Product Images from "Interplay between the Epigenetic Enzyme Lysine (K)-Specific Demethylase 2B and Epstein-Barr Virus Infection"

    Article Title: Interplay between the Epigenetic Enzyme Lysine (K)-Specific Demethylase 2B and Epstein-Barr Virus Infection

    Journal: Journal of Virology

    doi: 10.1128/JVI.00273-19

    EBV also uses LMP1-independent mechanisms to downregulate KDM2B. (A) B cells from two donors were infected with EBV or EBVΔLMP1 or mock infected (MI) and collected at 48 h after infection. Reverse-transcribed RNA samples were analyzed by qPCR for the KDM2B expression level. (B) B cells from two donors were infected with EBV and collected at 12, 24, and 48 h after infection. Cells were processed for RNA extraction and analyzed by qPCR for the expression levels of EBNA1, EBNA2, EBER, LMP1, and KDM2B transcripts. The expression levels of viral gene transcripts were measured relative to their levels in B cells collected 12 h (EBNA2) or 48 h (EBNA1, EBER, LMP1) postinfection. The levels of KDM2B mRNA were measured relative to its levels in mock-infected cells. (C) Different EBV(+) cell lines in latency phase I (BL110, I100, MUTU) or in latency phase III (LCL, Raji, B95) and primary B cells (BC) were collected, processed for RNA extraction and reverse transcription, and analyzed by qPCR for LMP1 (top), EBNA1 (middle), and KDM2B (bottom) mRNA levels. (D and E) RPMI (D) and Louckes (E) cells were transiently transfected with different constructs carrying individual EBV genes (TC) in three independent experiments. At 48 h after transfection, cells were processed for RNA and protein extraction and analyzed for the KDM2B expression level by RT-qPCR analysis. The levels of KDM2B mRNA were measured relative to its levels in mock-infected cells. Western blotting (LMP1, EBNA1, EBNA3A/3B, and EBNALP) or RT-PCR analysis (LMP2A, EBNA2, and EBNA3C) was performed to measure the expression of the different viral latent proteins. (F) RPMI cells were transfected with different EBV gene-carrying constructs. At 44 h after transfection, cells were exposed to the proteasome inhibitor MG132 for 4 h and then collected, processed for total protein extraction, and analyzed for the indicated proteins by immunoblotting. The histogram shows the average phosphorylated IκBα (P-IκBα) signal normalized to the levels of total IκBα. (G) B cells from different donors were untreated or treated with a miRNA-146a-5p inhibitor for 24 h before infection with EBV. At 48 h after infection, cells were collected and processed for RNA extraction and reverse transcription. cDNA samples were analyzed by qPCR for LMP1 and KDM2B expression levels. The levels of KDM2B mRNA were measured relative to its levels in mock-infected cells. The histograms show the average expression levels measured in 2 independent experiments (*, P
    Figure Legend Snippet: EBV also uses LMP1-independent mechanisms to downregulate KDM2B. (A) B cells from two donors were infected with EBV or EBVΔLMP1 or mock infected (MI) and collected at 48 h after infection. Reverse-transcribed RNA samples were analyzed by qPCR for the KDM2B expression level. (B) B cells from two donors were infected with EBV and collected at 12, 24, and 48 h after infection. Cells were processed for RNA extraction and analyzed by qPCR for the expression levels of EBNA1, EBNA2, EBER, LMP1, and KDM2B transcripts. The expression levels of viral gene transcripts were measured relative to their levels in B cells collected 12 h (EBNA2) or 48 h (EBNA1, EBER, LMP1) postinfection. The levels of KDM2B mRNA were measured relative to its levels in mock-infected cells. (C) Different EBV(+) cell lines in latency phase I (BL110, I100, MUTU) or in latency phase III (LCL, Raji, B95) and primary B cells (BC) were collected, processed for RNA extraction and reverse transcription, and analyzed by qPCR for LMP1 (top), EBNA1 (middle), and KDM2B (bottom) mRNA levels. (D and E) RPMI (D) and Louckes (E) cells were transiently transfected with different constructs carrying individual EBV genes (TC) in three independent experiments. At 48 h after transfection, cells were processed for RNA and protein extraction and analyzed for the KDM2B expression level by RT-qPCR analysis. The levels of KDM2B mRNA were measured relative to its levels in mock-infected cells. Western blotting (LMP1, EBNA1, EBNA3A/3B, and EBNALP) or RT-PCR analysis (LMP2A, EBNA2, and EBNA3C) was performed to measure the expression of the different viral latent proteins. (F) RPMI cells were transfected with different EBV gene-carrying constructs. At 44 h after transfection, cells were exposed to the proteasome inhibitor MG132 for 4 h and then collected, processed for total protein extraction, and analyzed for the indicated proteins by immunoblotting. The histogram shows the average phosphorylated IκBα (P-IκBα) signal normalized to the levels of total IκBα. (G) B cells from different donors were untreated or treated with a miRNA-146a-5p inhibitor for 24 h before infection with EBV. At 48 h after infection, cells were collected and processed for RNA extraction and reverse transcription. cDNA samples were analyzed by qPCR for LMP1 and KDM2B expression levels. The levels of KDM2B mRNA were measured relative to its levels in mock-infected cells. The histograms show the average expression levels measured in 2 independent experiments (*, P

    Techniques Used: Infection, Real-time Polymerase Chain Reaction, Expressing, RNA Extraction, Transfection, Construct, Protein Extraction, Quantitative RT-PCR, Western Blot, Reverse Transcription Polymerase Chain Reaction

    KDM2B regulates EBV gene expression in latently infected cells. (A to C) LCL were transfected with 1.5 μg of pCDNA3-KDM2B or with pCDNA as a control in three independent experiments. Cells were collected at 24 h after transfection and processed for RNA/DNA and total protein extraction. (A) KDM2B mRNA and protein levels were shown by qPCR (bottom) and immunoblotting (top; the lines corresponding to conditions with 0.5 μg and 1 μg of KDM2B, originally present in the Western blot, are not shown because they were excluded from all the analyses). (B) DNA samples were analyzed by TaqMan PCR to assess the number of EBV genome copies per cell (ns, not significant). (C) The mRNA expression levels of EBV latent and early genes were assessed by qPCR (*, P
    Figure Legend Snippet: KDM2B regulates EBV gene expression in latently infected cells. (A to C) LCL were transfected with 1.5 μg of pCDNA3-KDM2B or with pCDNA as a control in three independent experiments. Cells were collected at 24 h after transfection and processed for RNA/DNA and total protein extraction. (A) KDM2B mRNA and protein levels were shown by qPCR (bottom) and immunoblotting (top; the lines corresponding to conditions with 0.5 μg and 1 μg of KDM2B, originally present in the Western blot, are not shown because they were excluded from all the analyses). (B) DNA samples were analyzed by TaqMan PCR to assess the number of EBV genome copies per cell (ns, not significant). (C) The mRNA expression levels of EBV latent and early genes were assessed by qPCR (*, P

    Techniques Used: Expressing, Infection, Transfection, Protein Extraction, Real-time Polymerase Chain Reaction, Western Blot, Polymerase Chain Reaction

    KDM2B deregulation alters EBV gene expression. (A) Louckes cells were transiently transfected with increasing concentrations of the KDM2B expression vector in three independent experiments, collected at 24 h after transfection, and processed for protein and RNA extraction to assess KDM2B levels by immunoblotting (top) or qPCR (bottom). (B) Cells were then infected with EBV, and at 24 h after infection they were collected and processed for FACS analysis and for RNA/DNA extraction. DNA samples were used to measure EBV genome copy number by TaqMan PCR (ns, not significant). (C) Live cells were analyzed by FACS to measure the GFP mean fluorescence intensity (MFI). (D) cDNA samples were analyzed for the expression level of EBV early and late genes by qPCR. The levels of EBV genes transcripts were normalized to the mRNA levels of the housekeeping gene β-globin and calculated relative to their levels in cells transfected with the empty vector (KDM2B, 0 μg). The values shown in the histogram are the average from 3 independent experiments (*, P
    Figure Legend Snippet: KDM2B deregulation alters EBV gene expression. (A) Louckes cells were transiently transfected with increasing concentrations of the KDM2B expression vector in three independent experiments, collected at 24 h after transfection, and processed for protein and RNA extraction to assess KDM2B levels by immunoblotting (top) or qPCR (bottom). (B) Cells were then infected with EBV, and at 24 h after infection they were collected and processed for FACS analysis and for RNA/DNA extraction. DNA samples were used to measure EBV genome copy number by TaqMan PCR (ns, not significant). (C) Live cells were analyzed by FACS to measure the GFP mean fluorescence intensity (MFI). (D) cDNA samples were analyzed for the expression level of EBV early and late genes by qPCR. The levels of EBV genes transcripts were normalized to the mRNA levels of the housekeeping gene β-globin and calculated relative to their levels in cells transfected with the empty vector (KDM2B, 0 μg). The values shown in the histogram are the average from 3 independent experiments (*, P

    Techniques Used: Expressing, Transfection, Plasmid Preparation, RNA Extraction, Real-time Polymerase Chain Reaction, Infection, FACS, DNA Extraction, Polymerase Chain Reaction, Fluorescence

    8) Product Images from "Microarray and deep sequencing cross-platform analysis of the mirRNome and isomiR variation in response to epidermal growth factor"

    Article Title: Microarray and deep sequencing cross-platform analysis of the mirRNome and isomiR variation in response to epidermal growth factor

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-14-371

    miRNA transcriptome profiles of EGF-treated HeLa cells using Exiqon and Agilent miRNA arrays. A . Shared miRNA genes between Exiqon and Agilent microRNA arrays. Venn diagram showing the unique and named miRNAs genes shared between Agilent and Exiqon microarray platforms. The pool of 346 shared genes was used for all subsequent cross-platform analyses. Numbers outside diagrams indicate the total amount of miRNA genes contained in each platform. In brackets, percentage of miRNA genes contained in each platform over the total miRNA genes contained in all platforms. B . Regulated miRNAs after 6 hours EGF treatment using Exiqon and Agilent miRNA arrays. HeLa cells were serum-starved for 24 h and treated with EGF for 6 h. Total RNAs prepared from cells lysates were hybridized to Exiqon miRCURY LNA microRNA Array V9.2 and Agilent Human miRNA V2 Oligo Microarray. List shows regulated miRNAs identified by SAM analysis (FDR = 0.05) for miRNAs with a minimal expression change of 1.2 fold.
    Figure Legend Snippet: miRNA transcriptome profiles of EGF-treated HeLa cells using Exiqon and Agilent miRNA arrays. A . Shared miRNA genes between Exiqon and Agilent microRNA arrays. Venn diagram showing the unique and named miRNAs genes shared between Agilent and Exiqon microarray platforms. The pool of 346 shared genes was used for all subsequent cross-platform analyses. Numbers outside diagrams indicate the total amount of miRNA genes contained in each platform. In brackets, percentage of miRNA genes contained in each platform over the total miRNA genes contained in all platforms. B . Regulated miRNAs after 6 hours EGF treatment using Exiqon and Agilent miRNA arrays. HeLa cells were serum-starved for 24 h and treated with EGF for 6 h. Total RNAs prepared from cells lysates were hybridized to Exiqon miRCURY LNA microRNA Array V9.2 and Agilent Human miRNA V2 Oligo Microarray. List shows regulated miRNAs identified by SAM analysis (FDR = 0.05) for miRNAs with a minimal expression change of 1.2 fold.

    Techniques Used: Microarray, Expressing

    RT-qPCR validation of EGF-regulated miRNAs. Total RNA prepared from cells lysates were analyzed by quantitative real time PCR using the miRCURY LNA™ microRNA PCR System (Exiqon) for each of the miRNAs as indicated. A . Control treatment without inhibitors: HeLa cells were serum-starved for 24 hours and treated with EGF for 1 and 6 hours. B . Inhibitor treatment. HeLa cells were serum-starved for 24 hours and treated with EGF for 6 hours in the presence or absence of protein kinase inhibitors: AG1470 (EGFR inhibitor), U0126 (MEK inhibitor) and Wortmannin (PI3K inhibitor). In addition, HeLa cells were transfected with a constitutively active form of Ras (RasV12). Effective pathway inhibition was verified by western blotting in parallel samples from the same experiment (see Additional file 8 ).
    Figure Legend Snippet: RT-qPCR validation of EGF-regulated miRNAs. Total RNA prepared from cells lysates were analyzed by quantitative real time PCR using the miRCURY LNA™ microRNA PCR System (Exiqon) for each of the miRNAs as indicated. A . Control treatment without inhibitors: HeLa cells were serum-starved for 24 hours and treated with EGF for 1 and 6 hours. B . Inhibitor treatment. HeLa cells were serum-starved for 24 hours and treated with EGF for 6 hours in the presence or absence of protein kinase inhibitors: AG1470 (EGFR inhibitor), U0126 (MEK inhibitor) and Wortmannin (PI3K inhibitor). In addition, HeLa cells were transfected with a constitutively active form of Ras (RasV12). Effective pathway inhibition was verified by western blotting in parallel samples from the same experiment (see Additional file 8 ).

    Techniques Used: Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Transfection, Inhibition, Western Blot

    9) Product Images from "MicroRNA-215 Regulates Fibroblast Function: Insights from a Human Fibrotic Disease"

    Article Title: MicroRNA-215 Regulates Fibroblast Function: Insights from a Human Fibrotic Disease

    Journal: Cell Cycle

    doi: 10.1080/15384101.2014.998077

    ( A ) Bar chart showing results of the Exiqon microRNA Array. Height of the bars shows the mean normalized log values of miR-215 levels in conjunctiva and pterygium tissues from different patients. ( B ) Bar chart showing individual and overall results from qRT-PCR assay performed on 3 separate pairs of human pterygium and conjunctival tissues. Height of the bars represents the relative fold change of miR-215 levels in pterygium with respect to conjunctival control. Error bars represent standard error of mean. Values are normalized to the 5S rRNA control values. ( C ) miR-215 in situ staining of human pterygium and conjunctival tissues. Diagram on the right showing conjunctival epithelium and some stroma. Pterygium and conjunctival sections hybridized with DIG-labeled miR-215 LNA probes (red). DNA was counterstained with DAPI (blue). Scale bar represents 100 μm. Dashed lines represent position of basement membrane separating epithelium from stroma.
    Figure Legend Snippet: ( A ) Bar chart showing results of the Exiqon microRNA Array. Height of the bars shows the mean normalized log values of miR-215 levels in conjunctiva and pterygium tissues from different patients. ( B ) Bar chart showing individual and overall results from qRT-PCR assay performed on 3 separate pairs of human pterygium and conjunctival tissues. Height of the bars represents the relative fold change of miR-215 levels in pterygium with respect to conjunctival control. Error bars represent standard error of mean. Values are normalized to the 5S rRNA control values. ( C ) miR-215 in situ staining of human pterygium and conjunctival tissues. Diagram on the right showing conjunctival epithelium and some stroma. Pterygium and conjunctival sections hybridized with DIG-labeled miR-215 LNA probes (red). DNA was counterstained with DAPI (blue). Scale bar represents 100 μm. Dashed lines represent position of basement membrane separating epithelium from stroma.

    Techniques Used: Quantitative RT-PCR, In Situ, Staining, Labeling

    10) Product Images from "Olea europaea small RNA with functional homology to human miR34a in cross-kingdom interaction of anti-tumoral response"

    Article Title: Olea europaea small RNA with functional homology to human miR34a in cross-kingdom interaction of anti-tumoral response

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-30718-w

    Effects of oeu -sR pool derived from O . europaea drupe. Relative RT-qPCR expression analysis of some oeu miRNAs contained in the pool of small RNAs extracted from drupes of O. europaea (panel A). The analysis was carried out on three independent biological experiments and expressed as fold change with respect to oeu -miR159 expression previously normalised with a housekeeping gene (5 S rRNA). Flow cytometry analysis of SIRT1 (panels B and C) and BCL2 (panels D and E) proteins intracellular expression in Jurkat 72 hours after oeu -sR pool transfection. One representative dot plot overlay, and histogram overlay of three independent biological experiments is reported (% of SIRT1 and BCL2 positive cells, oeu -sRs vs HF, SIRT1% 2.36 ± 0.36** vs 10.32 ± 0.63; BCL2% 42.63 ± 8.65 vs 70.51 ± 4.62**). Cell viability analysed via Trypan blue (panel F), percentage of apoptotic cells (panel G) after oeu- sRs or oeu-sR 20 analysed via Flow cytometry (means ± SD of three independent biological experiments, p
    Figure Legend Snippet: Effects of oeu -sR pool derived from O . europaea drupe. Relative RT-qPCR expression analysis of some oeu miRNAs contained in the pool of small RNAs extracted from drupes of O. europaea (panel A). The analysis was carried out on three independent biological experiments and expressed as fold change with respect to oeu -miR159 expression previously normalised with a housekeeping gene (5 S rRNA). Flow cytometry analysis of SIRT1 (panels B and C) and BCL2 (panels D and E) proteins intracellular expression in Jurkat 72 hours after oeu -sR pool transfection. One representative dot plot overlay, and histogram overlay of three independent biological experiments is reported (% of SIRT1 and BCL2 positive cells, oeu -sRs vs HF, SIRT1% 2.36 ± 0.36** vs 10.32 ± 0.63; BCL2% 42.63 ± 8.65 vs 70.51 ± 4.62**). Cell viability analysed via Trypan blue (panel F), percentage of apoptotic cells (panel G) after oeu- sRs or oeu-sR 20 analysed via Flow cytometry (means ± SD of three independent biological experiments, p

    Techniques Used: Derivative Assay, Quantitative RT-PCR, Expressing, Flow Cytometry, Cytometry, Transfection

    Bioinformatics analysis and transfection efficiency of synthetic FITC- oeu -sR20, -27 and -34 in Jurkat, THP1 cell lines and in PBMCs from healthy donors. Workflow of the MirCompare analysis used for comparing H . sapiens microRNA and O . europaea putative miRNAs (panel A): Sequences homology comparison (n = 117 oeu-human miRNAs homologous) and distribution analysis. Among the 5 oeu- sRs having more than 50 homologous human miRNAs, oeu -sR20, -27 and -34 (homologous to hsa -miRNA34a) were aligned and the homology percentage was calculated. qRT-PCR expression analysis (ΔΔCT method) of hsa -miR34a in cell lines compared to PBMCs; data are expressed as means ± SD of three independent samples (panel B). Quantification was performed using the threshold cycle (Ct) comparative method and normalized with 18S rRNA. Presence of fluorescinated oeu -sRs in Jurkat, THP-1 cell lines and PBMCs 72 hours after transfection (panel C). The efficiency of oeu -sRs transfection was confirmed by observing the fluorescent cells as they appear at fluorescence microscope in these representative samples (Evos Floid Cells Imaging Station Life Technologies). Localization of oeu -sR20 FITC and calreticulin (ER) in THP1 cell after 72 hours from mimic oeu- sR20 transfection (panel D); Representative histogram obtained by flow cytometric analysis of fluorescinated oeu -sRs positive cells in Jurkat cell line after 72 hours from miRNA transfection (panel E). Red curves represent control cells receiving only HF, the empty curves represent transfected cells. The percentages of FITC-miRNAs positive Jurkat, THP1 cells and PBMCs were assessed via Cytexpert 2.0 software (Beckman Coulter) and showed in graphs (panels F). The results show the means ± SD of three independent experiments and 10 PBMCs samples (mean of percentage FL-1-positive for all three oeu -sR vs HF) 72 hours after from miRNA transfection.
    Figure Legend Snippet: Bioinformatics analysis and transfection efficiency of synthetic FITC- oeu -sR20, -27 and -34 in Jurkat, THP1 cell lines and in PBMCs from healthy donors. Workflow of the MirCompare analysis used for comparing H . sapiens microRNA and O . europaea putative miRNAs (panel A): Sequences homology comparison (n = 117 oeu-human miRNAs homologous) and distribution analysis. Among the 5 oeu- sRs having more than 50 homologous human miRNAs, oeu -sR20, -27 and -34 (homologous to hsa -miRNA34a) were aligned and the homology percentage was calculated. qRT-PCR expression analysis (ΔΔCT method) of hsa -miR34a in cell lines compared to PBMCs; data are expressed as means ± SD of three independent samples (panel B). Quantification was performed using the threshold cycle (Ct) comparative method and normalized with 18S rRNA. Presence of fluorescinated oeu -sRs in Jurkat, THP-1 cell lines and PBMCs 72 hours after transfection (panel C). The efficiency of oeu -sRs transfection was confirmed by observing the fluorescent cells as they appear at fluorescence microscope in these representative samples (Evos Floid Cells Imaging Station Life Technologies). Localization of oeu -sR20 FITC and calreticulin (ER) in THP1 cell after 72 hours from mimic oeu- sR20 transfection (panel D); Representative histogram obtained by flow cytometric analysis of fluorescinated oeu -sRs positive cells in Jurkat cell line after 72 hours from miRNA transfection (panel E). Red curves represent control cells receiving only HF, the empty curves represent transfected cells. The percentages of FITC-miRNAs positive Jurkat, THP1 cells and PBMCs were assessed via Cytexpert 2.0 software (Beckman Coulter) and showed in graphs (panels F). The results show the means ± SD of three independent experiments and 10 PBMCs samples (mean of percentage FL-1-positive for all three oeu -sR vs HF) 72 hours after from miRNA transfection.

    Techniques Used: Transfection, Quantitative RT-PCR, Expressing, Fluorescence, Microscopy, Imaging, Flow Cytometry, Software

    oeu- sR pool transfection effects on SNAIL protein modulation and lipid accumulation in Hepatoma cell lines. Percentage of FITC- oeu -sR20 positive HepG2 (panel A). The efficiency of miRNA transfection was confirmed by observing the fluorescent cells as they appear at fluorescence microscope in panel B. Percentage of viabilty and apoptosis in oeu -sR20, hsa -miR34 and oeu -sR pool transfected Hep-G2 (panel C). One representative western blot image of SNAIL and E-Cadherin (panel D, Figure S4 ) in HepG2 cells after transfection as well as densitometric analysis of three independent biological experiments are reported (panel E and F). The scratch test was used to detect HepG2 migration rates at 0 and 72 h after transfection. The representative microscopy picture (×40) was shown (panel G). FACS analysis of lipid accumulation induced by TGFbeta1 in HEPG2 in the presence or absence of oeu -sR20, oeu -sR pool and hsa -miR34, 72 hours post-transfection (panel H). Results are from at least three independent biological experiments. Histograms represent the mean and the bars ± S.D, **p
    Figure Legend Snippet: oeu- sR pool transfection effects on SNAIL protein modulation and lipid accumulation in Hepatoma cell lines. Percentage of FITC- oeu -sR20 positive HepG2 (panel A). The efficiency of miRNA transfection was confirmed by observing the fluorescent cells as they appear at fluorescence microscope in panel B. Percentage of viabilty and apoptosis in oeu -sR20, hsa -miR34 and oeu -sR pool transfected Hep-G2 (panel C). One representative western blot image of SNAIL and E-Cadherin (panel D, Figure S4 ) in HepG2 cells after transfection as well as densitometric analysis of three independent biological experiments are reported (panel E and F). The scratch test was used to detect HepG2 migration rates at 0 and 72 h after transfection. The representative microscopy picture (×40) was shown (panel G). FACS analysis of lipid accumulation induced by TGFbeta1 in HEPG2 in the presence or absence of oeu -sR20, oeu -sR pool and hsa -miR34, 72 hours post-transfection (panel H). Results are from at least three independent biological experiments. Histograms represent the mean and the bars ± S.D, **p

    Techniques Used: Transfection, Fluorescence, Microscopy, Western Blot, Migration, FACS

    11) Product Images from "Microarray and deep sequencing cross-platform analysis of the mirRNome and isomiR variation in response to epidermal growth factor"

    Article Title: Microarray and deep sequencing cross-platform analysis of the mirRNome and isomiR variation in response to epidermal growth factor

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-14-371

    miRNA transcriptome profiles of EGF-treated HeLa cells using Exiqon and Agilent miRNA arrays. A . Shared miRNA genes between Exiqon and Agilent microRNA arrays. Venn diagram showing the unique and named miRNAs genes shared between Agilent and Exiqon microarray platforms. The pool of 346 shared genes was used for all subsequent cross-platform analyses. Numbers outside diagrams indicate the total amount of miRNA genes contained in each platform. In brackets, percentage of miRNA genes contained in each platform over the total miRNA genes contained in all platforms. B . Regulated miRNAs after 6 hours EGF treatment using Exiqon and Agilent miRNA arrays. HeLa cells were serum-starved for 24 h and treated with EGF for 6 h. Total RNAs prepared from cells lysates were hybridized to Exiqon miRCURY LNA microRNA Array V9.2 and Agilent Human miRNA V2 Oligo Microarray. List shows regulated miRNAs identified by SAM analysis (FDR = 0.05) for miRNAs with a minimal expression change of 1.2 fold.
    Figure Legend Snippet: miRNA transcriptome profiles of EGF-treated HeLa cells using Exiqon and Agilent miRNA arrays. A . Shared miRNA genes between Exiqon and Agilent microRNA arrays. Venn diagram showing the unique and named miRNAs genes shared between Agilent and Exiqon microarray platforms. The pool of 346 shared genes was used for all subsequent cross-platform analyses. Numbers outside diagrams indicate the total amount of miRNA genes contained in each platform. In brackets, percentage of miRNA genes contained in each platform over the total miRNA genes contained in all platforms. B . Regulated miRNAs after 6 hours EGF treatment using Exiqon and Agilent miRNA arrays. HeLa cells were serum-starved for 24 h and treated with EGF for 6 h. Total RNAs prepared from cells lysates were hybridized to Exiqon miRCURY LNA microRNA Array V9.2 and Agilent Human miRNA V2 Oligo Microarray. List shows regulated miRNAs identified by SAM analysis (FDR = 0.05) for miRNAs with a minimal expression change of 1.2 fold.

    Techniques Used: Microarray, Expressing

    RT-qPCR validation of EGF-regulated miRNAs. Total RNA prepared from cells lysates were analyzed by quantitative real time PCR using the miRCURY LNA™ microRNA PCR System (Exiqon) for each of the miRNAs as indicated. A . Control treatment without inhibitors: HeLa cells were serum-starved for 24 hours and treated with EGF for 1 and 6 hours. B . Inhibitor treatment. HeLa cells were serum-starved for 24 hours and treated with EGF for 6 hours in the presence or absence of protein kinase inhibitors: AG1470 (EGFR inhibitor), U0126 (MEK inhibitor) and Wortmannin (PI3K inhibitor). In addition, HeLa cells were transfected with a constitutively active form of Ras (RasV12). Effective pathway inhibition was verified by western blotting in parallel samples from the same experiment (see Additional file 8 ).
    Figure Legend Snippet: RT-qPCR validation of EGF-regulated miRNAs. Total RNA prepared from cells lysates were analyzed by quantitative real time PCR using the miRCURY LNA™ microRNA PCR System (Exiqon) for each of the miRNAs as indicated. A . Control treatment without inhibitors: HeLa cells were serum-starved for 24 hours and treated with EGF for 1 and 6 hours. B . Inhibitor treatment. HeLa cells were serum-starved for 24 hours and treated with EGF for 6 hours in the presence or absence of protein kinase inhibitors: AG1470 (EGFR inhibitor), U0126 (MEK inhibitor) and Wortmannin (PI3K inhibitor). In addition, HeLa cells were transfected with a constitutively active form of Ras (RasV12). Effective pathway inhibition was verified by western blotting in parallel samples from the same experiment (see Additional file 8 ).

    Techniques Used: Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Transfection, Inhibition, Western Blot

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    Article Snippet: .. RT-qPCR Validation In order to validate the miRNAs selected by Rankprod analysis we measured the relative expression of the 8 most significant differentially expressed miRNAs at 1 hour and 6 hours after EGF treatment with the miRCURY LNA™ microRNA PCR System (Exiqon). .. This system uses the LNA technology allowing an accurate and sensitive quantitation of mature microRNAs [ ].

    Article Title: MicroRNA-215 Regulates Fibroblast Function: Insights from a Human Fibrotic Disease
    Article Snippet: .. For quantification of microRNA expression, qRT-PCR was performed using the miRCURY LNA™ PCR system (Exiqon, Vedbaek, Denmark) in accordance with the manufacturer′s instructions. .. Total RNA was diluted to a concentration of 10 ng in 4.5 μl using nuclease-free water and added to reagents from the miRCURY LNA™ First-strand cDNA Synthesis Kit.

    Real-time Polymerase Chain Reaction:

    Article Title: Microarray and deep sequencing cross-platform analysis of the mirRNome and isomiR variation in response to epidermal growth factor
    Article Snippet: .. miRNA RT-qPCR Quantitative real time PCR was performed using the miRCURY LNA™ microRNA PCR System (Exiqon) on total RNA extracted from HeLa cells treated at different times with EGF (with or without protein kinase inhibitors) with miRVana’s isolation kit (Ambion) following the manufacturer’s instructions. .. PCR amplification and detection were performed with the ROCHE LightCycler 480 detector, using 2x SYBR GREEN Master Mix.

    Article Title: Evaluation of extraction kits and RT-qPCR systems adapted to high-throughput platform for circulating miRNAs
    Article Snippet: .. Reverse transcription and quantitative real-time PCR (RT-qPCR) High-throughput RT-qPCR was carried out on three different systems: TaqMan miRNA PCR system (Applied Biosystems, California, USA), miRCURY LNA microRNA PCR system (Exiqon) and miScript miRNA PCR system (Qiagen). .. The RT, preamplification and qPCR reactions were carried out according to previous publication and as indicated in .

    Sequencing:

    Article Title: Stemness, Pluripotentiality, and Wnt Antagonism: sFRP4, a Wnt antagonist Mediates Pluripotency and Stemness in Glioblastoma
    Article Snippet: .. MicroRNA 885 Analysis Total RNA was isolated from U87 treated cells by using the Trizol method. miRCURY LNATM Universal RT microRNA PCR kit (Exiqon, Woburn, MA, USA), hsa-miR-885-5p (Accession ID: MIMAT0004947; Sequence-UCCAUUACACUACCCUGCCUCU) and hsa-miR-885-3p (Accession ID: MIMAT0004948; Sequence-AGGCAGCGGGGUGUAGUGGAUA) were used in the miRNA885 analysis. miRCURY LNA miRNA Detection probe (hsa-miR-885-3p; Exiqon) was used to localize mature miRNA activity in treated U87 cells by using in situ hybridization as previously described [ ]. .. RNA Sequencing Total RNA from U87 cells subjected to either sFRP4 OE or sFRP4 silencing and untreated cells was isolated and sequenced with Illumina HiSeq 2500 System (Illumina Inc.) for the whole transcriptome analysis.

    Polymerase Chain Reaction:

    Article Title: Stemness, Pluripotentiality, and Wnt Antagonism: sFRP4, a Wnt antagonist Mediates Pluripotency and Stemness in Glioblastoma
    Article Snippet: .. MicroRNA 885 Analysis Total RNA was isolated from U87 treated cells by using the Trizol method. miRCURY LNATM Universal RT microRNA PCR kit (Exiqon, Woburn, MA, USA), hsa-miR-885-5p (Accession ID: MIMAT0004947; Sequence-UCCAUUACACUACCCUGCCUCU) and hsa-miR-885-3p (Accession ID: MIMAT0004948; Sequence-AGGCAGCGGGGUGUAGUGGAUA) were used in the miRNA885 analysis. miRCURY LNA miRNA Detection probe (hsa-miR-885-3p; Exiqon) was used to localize mature miRNA activity in treated U87 cells by using in situ hybridization as previously described [ ]. .. RNA Sequencing Total RNA from U87 cells subjected to either sFRP4 OE or sFRP4 silencing and untreated cells was isolated and sequenced with Illumina HiSeq 2500 System (Illumina Inc.) for the whole transcriptome analysis.

    Article Title: Microarray and deep sequencing cross-platform analysis of the mirRNome and isomiR variation in response to epidermal growth factor
    Article Snippet: .. miRNA RT-qPCR Quantitative real time PCR was performed using the miRCURY LNA™ microRNA PCR System (Exiqon) on total RNA extracted from HeLa cells treated at different times with EGF (with or without protein kinase inhibitors) with miRVana’s isolation kit (Ambion) following the manufacturer’s instructions. .. PCR amplification and detection were performed with the ROCHE LightCycler 480 detector, using 2x SYBR GREEN Master Mix.

    Article Title: Evaluation of extraction kits and RT-qPCR systems adapted to high-throughput platform for circulating miRNAs
    Article Snippet: .. Reverse transcription and quantitative real-time PCR (RT-qPCR) High-throughput RT-qPCR was carried out on three different systems: TaqMan miRNA PCR system (Applied Biosystems, California, USA), miRCURY LNA microRNA PCR system (Exiqon) and miScript miRNA PCR system (Qiagen). .. The RT, preamplification and qPCR reactions were carried out according to previous publication and as indicated in .

    Article Title: Evaluation of BRCA1-related molecular features and microRNAs as prognostic factors for triple negative breast cancers
    Article Snippet: .. Tumoral miRNA expression assessment A total of 27 miRNAs were quantified by RT-qPCR in tumors using miRCURY LNA™ Universal RT microRNA PCR assays from Exiqon (Denmark), according to the manufacturer’s instructions. .. Those miRNAs were chosen because: (i) their expression was reported in the literature to be related to the survival of breast cancer patients; (ii) they are known to be expressed in lymphoid cells and to reflect the lymphoid invasion of the tumor; or (iii) they were emphasized in our previous work (unpublished results).

    Article Title: miR-125a-5p Functions as Tumor Suppressor microRNA And Is a Marker of Locoregional Recurrence And Poor prognosis in Head And Neck Cancer
    Article Snippet: .. The cells were then extracted with the TRIzol reagent then quantified using a the miRCURY LNA miRNA PCR System, using the U6 snRNA as an internal, housekeeping control. qRT-PCR reveals efficacy of transfection of miR-125a-5p mimic into HN5 and UM-SCC-22B cell lines, compared to the control miRNA. .. Data were analyzed with the Student’s t -test and are presented as the mean ± standard deviation.

    Article Title: Olea europaea small RNA with functional homology to human miR34a in cross-kingdom interaction of anti-tumoral response
    Article Snippet: .. In brief, cDNA was synthesized using a specific reverse transcription kit for microRNAs (miRCURY LNA Universal RT microRNA PCR, Synthesis Kit II; EXIQON), according to the manufacturer’s guidelines. .. To verify the absence of nucleases in the reaction and to evaluate the efficiency of retro-transcription and RT-qPCR amplification, 108 copies of a synthetic spike-in control miRNA (UniSp6, EXIQON) were added to each RNA sample before conversion to cDNA.

    Article Title: Microarray and deep sequencing cross-platform analysis of the mirRNome and isomiR variation in response to epidermal growth factor
    Article Snippet: .. RT-qPCR Validation In order to validate the miRNAs selected by Rankprod analysis we measured the relative expression of the 8 most significant differentially expressed miRNAs at 1 hour and 6 hours after EGF treatment with the miRCURY LNA™ microRNA PCR System (Exiqon). .. This system uses the LNA technology allowing an accurate and sensitive quantitation of mature microRNAs [ ].

    Article Title: MicroRNA-215 Regulates Fibroblast Function: Insights from a Human Fibrotic Disease
    Article Snippet: .. For quantification of microRNA expression, qRT-PCR was performed using the miRCURY LNA™ PCR system (Exiqon, Vedbaek, Denmark) in accordance with the manufacturer′s instructions. .. Total RNA was diluted to a concentration of 10 ng in 4.5 μl using nuclease-free water and added to reagents from the miRCURY LNA™ First-strand cDNA Synthesis Kit.

    Activity Assay:

    Article Title: Stemness, Pluripotentiality, and Wnt Antagonism: sFRP4, a Wnt antagonist Mediates Pluripotency and Stemness in Glioblastoma
    Article Snippet: .. MicroRNA 885 Analysis Total RNA was isolated from U87 treated cells by using the Trizol method. miRCURY LNATM Universal RT microRNA PCR kit (Exiqon, Woburn, MA, USA), hsa-miR-885-5p (Accession ID: MIMAT0004947; Sequence-UCCAUUACACUACCCUGCCUCU) and hsa-miR-885-3p (Accession ID: MIMAT0004948; Sequence-AGGCAGCGGGGUGUAGUGGAUA) were used in the miRNA885 analysis. miRCURY LNA miRNA Detection probe (hsa-miR-885-3p; Exiqon) was used to localize mature miRNA activity in treated U87 cells by using in situ hybridization as previously described [ ]. .. RNA Sequencing Total RNA from U87 cells subjected to either sFRP4 OE or sFRP4 silencing and untreated cells was isolated and sequenced with Illumina HiSeq 2500 System (Illumina Inc.) for the whole transcriptome analysis.

    Expressing:

    Article Title: Evaluation of BRCA1-related molecular features and microRNAs as prognostic factors for triple negative breast cancers
    Article Snippet: .. Tumoral miRNA expression assessment A total of 27 miRNAs were quantified by RT-qPCR in tumors using miRCURY LNA™ Universal RT microRNA PCR assays from Exiqon (Denmark), according to the manufacturer’s instructions. .. Those miRNAs were chosen because: (i) their expression was reported in the literature to be related to the survival of breast cancer patients; (ii) they are known to be expressed in lymphoid cells and to reflect the lymphoid invasion of the tumor; or (iii) they were emphasized in our previous work (unpublished results).

    Article Title: Microarray and deep sequencing cross-platform analysis of the mirRNome and isomiR variation in response to epidermal growth factor
    Article Snippet: .. RT-qPCR Validation In order to validate the miRNAs selected by Rankprod analysis we measured the relative expression of the 8 most significant differentially expressed miRNAs at 1 hour and 6 hours after EGF treatment with the miRCURY LNA™ microRNA PCR System (Exiqon). .. This system uses the LNA technology allowing an accurate and sensitive quantitation of mature microRNAs [ ].

    Article Title: MicroRNA-215 Regulates Fibroblast Function: Insights from a Human Fibrotic Disease
    Article Snippet: .. For quantification of microRNA expression, qRT-PCR was performed using the miRCURY LNA™ PCR system (Exiqon, Vedbaek, Denmark) in accordance with the manufacturer′s instructions. .. Total RNA was diluted to a concentration of 10 ng in 4.5 μl using nuclease-free water and added to reagents from the miRCURY LNA™ First-strand cDNA Synthesis Kit.

    High Throughput Screening Assay:

    Article Title: Evaluation of extraction kits and RT-qPCR systems adapted to high-throughput platform for circulating miRNAs
    Article Snippet: .. Reverse transcription and quantitative real-time PCR (RT-qPCR) High-throughput RT-qPCR was carried out on three different systems: TaqMan miRNA PCR system (Applied Biosystems, California, USA), miRCURY LNA microRNA PCR system (Exiqon) and miScript miRNA PCR system (Qiagen). .. The RT, preamplification and qPCR reactions were carried out according to previous publication and as indicated in .

    In Situ Hybridization:

    Article Title: Stemness, Pluripotentiality, and Wnt Antagonism: sFRP4, a Wnt antagonist Mediates Pluripotency and Stemness in Glioblastoma
    Article Snippet: .. MicroRNA 885 Analysis Total RNA was isolated from U87 treated cells by using the Trizol method. miRCURY LNATM Universal RT microRNA PCR kit (Exiqon, Woburn, MA, USA), hsa-miR-885-5p (Accession ID: MIMAT0004947; Sequence-UCCAUUACACUACCCUGCCUCU) and hsa-miR-885-3p (Accession ID: MIMAT0004948; Sequence-AGGCAGCGGGGUGUAGUGGAUA) were used in the miRNA885 analysis. miRCURY LNA miRNA Detection probe (hsa-miR-885-3p; Exiqon) was used to localize mature miRNA activity in treated U87 cells by using in situ hybridization as previously described [ ]. .. RNA Sequencing Total RNA from U87 cells subjected to either sFRP4 OE or sFRP4 silencing and untreated cells was isolated and sequenced with Illumina HiSeq 2500 System (Illumina Inc.) for the whole transcriptome analysis.

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    Qiagen mircury lna mirna pcr assay
    miR-337-3p is a potential regulator of browning of adipose tissue. ( a ) Representative heatmap showing the results of <t>RT-PCR</t> analysis used to screen a panel of microRNAs (miR) in mouse white adipose tissue (WAT) and brown adipose tissue (BAT): Let-7c, miR-199a-5p, miR-151-3p, miR-145-5p, Let-7b-3p, miR-24-3p, miR-361-5p, miR-335-5p, miR-337-3p, miR-134-5p. Real-time PCR was performed using the <t>miRCURY</t> <t>LNA</t> miR PCR Assay and normalized to U6 RNA and relative to WAT. Differentially expressed miR (fold change ≥0.5) are indicated in yellow, which represents upregulated <t>miRNA</t> in BAT compared to WAT, and in blue are the downregulated miRs (fold change ≤−0.5) in BAT compared to WAT. N = 3/group. ( b ) RT-PCR analysis of transcript abundance of BAT thermogenic and mitochondrial markers Ucp1 , Cs , and Crls1 in BAT compared to WAT. mRNA expression values were normalized to L7 mRNA. N = 8/group. ( c ) RT-PCR analysis of mmu-miR-337-3p expression in mouse WAT and BAT normalized to U6 RNA. N = 8/group ( d ) RT-PCR analysis of mmu-miR-337-3p abundance in BAT, WAT, liver, kidney, small intestine, skeletal muscle, pancreas, brains, and lungs presented as percentage of mmu-miR-337-3p transcript abundance from its expression in BAT. Expression of miR-337-3p in BAT is set to 100%. ( e ) Representative image of the genomic mapping of miR-337-3p in humans and conservation of its seed sequence among species. Data are presented as means ± SEM; individual data points are given by the dot plot. Either Mann–Whitney-U or Independent sample t -test was used to assess differences in expression levels. ### p
    Mircury Lna Mirna Pcr Assay, supplied by Qiagen, used in various techniques. Bioz Stars score: 93/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    miR-337-3p is a potential regulator of browning of adipose tissue. ( a ) Representative heatmap showing the results of RT-PCR analysis used to screen a panel of microRNAs (miR) in mouse white adipose tissue (WAT) and brown adipose tissue (BAT): Let-7c, miR-199a-5p, miR-151-3p, miR-145-5p, Let-7b-3p, miR-24-3p, miR-361-5p, miR-335-5p, miR-337-3p, miR-134-5p. Real-time PCR was performed using the miRCURY LNA miR PCR Assay and normalized to U6 RNA and relative to WAT. Differentially expressed miR (fold change ≥0.5) are indicated in yellow, which represents upregulated miRNA in BAT compared to WAT, and in blue are the downregulated miRs (fold change ≤−0.5) in BAT compared to WAT. N = 3/group. ( b ) RT-PCR analysis of transcript abundance of BAT thermogenic and mitochondrial markers Ucp1 , Cs , and Crls1 in BAT compared to WAT. mRNA expression values were normalized to L7 mRNA. N = 8/group. ( c ) RT-PCR analysis of mmu-miR-337-3p expression in mouse WAT and BAT normalized to U6 RNA. N = 8/group ( d ) RT-PCR analysis of mmu-miR-337-3p abundance in BAT, WAT, liver, kidney, small intestine, skeletal muscle, pancreas, brains, and lungs presented as percentage of mmu-miR-337-3p transcript abundance from its expression in BAT. Expression of miR-337-3p in BAT is set to 100%. ( e ) Representative image of the genomic mapping of miR-337-3p in humans and conservation of its seed sequence among species. Data are presented as means ± SEM; individual data points are given by the dot plot. Either Mann–Whitney-U or Independent sample t -test was used to assess differences in expression levels. ### p

    Journal: Cells

    Article Title: MiR-337-3p Promotes Adipocyte Browning by Inhibiting TWIST1

    doi: 10.3390/cells9041056

    Figure Lengend Snippet: miR-337-3p is a potential regulator of browning of adipose tissue. ( a ) Representative heatmap showing the results of RT-PCR analysis used to screen a panel of microRNAs (miR) in mouse white adipose tissue (WAT) and brown adipose tissue (BAT): Let-7c, miR-199a-5p, miR-151-3p, miR-145-5p, Let-7b-3p, miR-24-3p, miR-361-5p, miR-335-5p, miR-337-3p, miR-134-5p. Real-time PCR was performed using the miRCURY LNA miR PCR Assay and normalized to U6 RNA and relative to WAT. Differentially expressed miR (fold change ≥0.5) are indicated in yellow, which represents upregulated miRNA in BAT compared to WAT, and in blue are the downregulated miRs (fold change ≤−0.5) in BAT compared to WAT. N = 3/group. ( b ) RT-PCR analysis of transcript abundance of BAT thermogenic and mitochondrial markers Ucp1 , Cs , and Crls1 in BAT compared to WAT. mRNA expression values were normalized to L7 mRNA. N = 8/group. ( c ) RT-PCR analysis of mmu-miR-337-3p expression in mouse WAT and BAT normalized to U6 RNA. N = 8/group ( d ) RT-PCR analysis of mmu-miR-337-3p abundance in BAT, WAT, liver, kidney, small intestine, skeletal muscle, pancreas, brains, and lungs presented as percentage of mmu-miR-337-3p transcript abundance from its expression in BAT. Expression of miR-337-3p in BAT is set to 100%. ( e ) Representative image of the genomic mapping of miR-337-3p in humans and conservation of its seed sequence among species. Data are presented as means ± SEM; individual data points are given by the dot plot. Either Mann–Whitney-U or Independent sample t -test was used to assess differences in expression levels. ### p

    Article Snippet: For validation of transfection efficiency in cell culture experiments, miR-337-3p pre-designed miRCURY LNA Uni RT primer mix (339306, Qiagen) was used.

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

    miRNA recovery of different extraction kits were evaluated by the C q values for spike-in controls (cel-miR-39 and cel-miR-54) assayed in the (a) TaqMan and (b) miScript RT-qPCR system. Box plots with whiskers show 5–95 percentile of the C q values for spike-in controls and the corresponding CVs are shown. The extent of extraction bias across samples was reflected in the range and CV of C q values for spike-in controls. Higher median C q compared to other extraction kits indicate poorer performance in miRNA recovery. Q, miRNeasy Serum/Plasma kit; E, miRCURY™ RNA Isolation Kit - Biofluids; A, mirVana™ PARIS™ Kit; MN, NucleoSpin® miRNA Plasma; NB, Plasma/Serum Circulating RNA Purification Kit.

    Journal: Scientific Reports

    Article Title: Evaluation of extraction kits and RT-qPCR systems adapted to high-throughput platform for circulating miRNAs

    doi: 10.1038/srep09430

    Figure Lengend Snippet: miRNA recovery of different extraction kits were evaluated by the C q values for spike-in controls (cel-miR-39 and cel-miR-54) assayed in the (a) TaqMan and (b) miScript RT-qPCR system. Box plots with whiskers show 5–95 percentile of the C q values for spike-in controls and the corresponding CVs are shown. The extent of extraction bias across samples was reflected in the range and CV of C q values for spike-in controls. Higher median C q compared to other extraction kits indicate poorer performance in miRNA recovery. Q, miRNeasy Serum/Plasma kit; E, miRCURY™ RNA Isolation Kit - Biofluids; A, mirVana™ PARIS™ Kit; MN, NucleoSpin® miRNA Plasma; NB, Plasma/Serum Circulating RNA Purification Kit.

    Article Snippet: Reverse transcription and quantitative real-time PCR (RT-qPCR) High-throughput RT-qPCR was carried out on three different systems: TaqMan miRNA PCR system (Applied Biosystems, California, USA), miRCURY LNA microRNA PCR system (Exiqon) and miScript miRNA PCR system (Qiagen).

    Techniques: Quantitative RT-PCR, Isolation, Purification

    Comparison of TaqMan and miScript RT-qPCR systems adapted to the high-throughput platform by performance parameters which include reproducibility, accuracy and sensitivity. Transformation of data to z-score was carried out to facilitate direct comparison. A1, accuracy when measuring fold change of abundant miRNAs; A2, accuracy when measuring fold change of less abundant miRNAs, R1, reproducibility of RT replicates; R2, reproducibility of RT replicates after normalisation; R3, reproducibility of qPCR replicates; S1, sensitivity to detect the presence of plasma miRNAs and S2, sensitivity to detect the presence of miRNAs in titration points.

    Journal: Scientific Reports

    Article Title: Evaluation of extraction kits and RT-qPCR systems adapted to high-throughput platform for circulating miRNAs

    doi: 10.1038/srep09430

    Figure Lengend Snippet: Comparison of TaqMan and miScript RT-qPCR systems adapted to the high-throughput platform by performance parameters which include reproducibility, accuracy and sensitivity. Transformation of data to z-score was carried out to facilitate direct comparison. A1, accuracy when measuring fold change of abundant miRNAs; A2, accuracy when measuring fold change of less abundant miRNAs, R1, reproducibility of RT replicates; R2, reproducibility of RT replicates after normalisation; R3, reproducibility of qPCR replicates; S1, sensitivity to detect the presence of plasma miRNAs and S2, sensitivity to detect the presence of miRNAs in titration points.

    Article Snippet: Reverse transcription and quantitative real-time PCR (RT-qPCR) High-throughput RT-qPCR was carried out on three different systems: TaqMan miRNA PCR system (Applied Biosystems, California, USA), miRCURY LNA microRNA PCR system (Exiqon) and miScript miRNA PCR system (Qiagen).

    Techniques: Quantitative RT-PCR, High Throughput Screening Assay, Transformation Assay, Real-time Polymerase Chain Reaction, Titration

    Evaluation of consistency between technical replicatesin RT-qPCR. Consistencies among (a) RT replicates in TaqMan system, (b) RT replicates in miScript system, (c) qPCR replicates in TaqMan system and (d) qPCR replicates in miScript system were evaluated by measuring the levels of 11–16 miRNAs in a same set of RNA samples (n = 5–14). Each data point in scatter plot represents the average C q value obtained from the duplicate or triplicate wells of qPCR. Overall, significant correlations (ICC = 0.712–0.996, p

    Journal: Scientific Reports

    Article Title: Evaluation of extraction kits and RT-qPCR systems adapted to high-throughput platform for circulating miRNAs

    doi: 10.1038/srep09430

    Figure Lengend Snippet: Evaluation of consistency between technical replicatesin RT-qPCR. Consistencies among (a) RT replicates in TaqMan system, (b) RT replicates in miScript system, (c) qPCR replicates in TaqMan system and (d) qPCR replicates in miScript system were evaluated by measuring the levels of 11–16 miRNAs in a same set of RNA samples (n = 5–14). Each data point in scatter plot represents the average C q value obtained from the duplicate or triplicate wells of qPCR. Overall, significant correlations (ICC = 0.712–0.996, p

    Article Snippet: Reverse transcription and quantitative real-time PCR (RT-qPCR) High-throughput RT-qPCR was carried out on three different systems: TaqMan miRNA PCR system (Applied Biosystems, California, USA), miRCURY LNA microRNA PCR system (Exiqon) and miScript miRNA PCR system (Qiagen).

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

    Comparison of different RT-qPCR systems adapted to the BioMark high-throughput platform. (a) Average C q values for endogeneous plasma miRNAs were depicted in box plot with whiskers showing the 5–95 percentile. Irrespective of extraction kits being used, within the same set of samples, more undetermined C q values were obtained in the miScript system. (b) Correlation of qPCR results from TaqMan and miScript systems may vary among different miRNA assays. Each data point represents the normalised C q value transformed into z-score. Q, miRNeasy Serum/Plasma kit; E, miRCURY™ RNA Isolation Kit - Biofluids; A, mirVana™ PARIS™ Kit; MN, NucleoSpin® miRNA Plasma; NB, Plasma/Serum Circulating RNA Purification Kit.

    Journal: Scientific Reports

    Article Title: Evaluation of extraction kits and RT-qPCR systems adapted to high-throughput platform for circulating miRNAs

    doi: 10.1038/srep09430

    Figure Lengend Snippet: Comparison of different RT-qPCR systems adapted to the BioMark high-throughput platform. (a) Average C q values for endogeneous plasma miRNAs were depicted in box plot with whiskers showing the 5–95 percentile. Irrespective of extraction kits being used, within the same set of samples, more undetermined C q values were obtained in the miScript system. (b) Correlation of qPCR results from TaqMan and miScript systems may vary among different miRNA assays. Each data point represents the normalised C q value transformed into z-score. Q, miRNeasy Serum/Plasma kit; E, miRCURY™ RNA Isolation Kit - Biofluids; A, mirVana™ PARIS™ Kit; MN, NucleoSpin® miRNA Plasma; NB, Plasma/Serum Circulating RNA Purification Kit.

    Article Snippet: Reverse transcription and quantitative real-time PCR (RT-qPCR) High-throughput RT-qPCR was carried out on three different systems: TaqMan miRNA PCR system (Applied Biosystems, California, USA), miRCURY LNA microRNA PCR system (Exiqon) and miScript miRNA PCR system (Qiagen).

    Techniques: Quantitative RT-PCR, High Throughput Screening Assay, Real-time Polymerase Chain Reaction, Transformation Assay, Isolation, Purification