allprep dna rna mirna universal kit protocol  (Qiagen)


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    AllPrep DNA RNA miRNA Universal Kit
    Description:
    For simultaneous purification of genomic DNA and total RNA including miRNA from cells and difficult to lyse tissue Kit contents Qiagen AllPrep DNA RNA miRNA Universal Kit 50 preps Developed for Difficult to lyse Samples High Yields of DNA RNA and miRNA from the Same Sample Delivers High quality Nucleic Acids Ready for Downstream use For Simultaneous Purification of Genomic DNA and Total RNA Including miRNA from Cells and Difficult to lyse Tissue Includes 50 AllPrep DNA Mini Spin Columns 50 RNeasy Mini Spin Columns Collection Tubes and Buffers Benefits High yields of DNA RNA and miRNA from the same sample Developed for difficult to lyse samples e g fiber or lipid rich tissue Delivers high quality nucleic acids ready for downstream use Preoptimized protocols for various sample types Convenient procedure that does not require use of pheno
    Catalog Number:
    80224
    Price:
    641
    Category:
    AllPrep DNA RNA miRNA Universal Kit
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    Structured Review

    Qiagen allprep dna rna mirna universal kit protocol
    AllPrep DNA RNA miRNA Universal Kit
    For simultaneous purification of genomic DNA and total RNA including miRNA from cells and difficult to lyse tissue Kit contents Qiagen AllPrep DNA RNA miRNA Universal Kit 50 preps Developed for Difficult to lyse Samples High Yields of DNA RNA and miRNA from the Same Sample Delivers High quality Nucleic Acids Ready for Downstream use For Simultaneous Purification of Genomic DNA and Total RNA Including miRNA from Cells and Difficult to lyse Tissue Includes 50 AllPrep DNA Mini Spin Columns 50 RNeasy Mini Spin Columns Collection Tubes and Buffers Benefits High yields of DNA RNA and miRNA from the same sample Developed for difficult to lyse samples e g fiber or lipid rich tissue Delivers high quality nucleic acids ready for downstream use Preoptimized protocols for various sample types Convenient procedure that does not require use of pheno
    https://www.bioz.com/result/allprep dna rna mirna universal kit protocol/product/Qiagen
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    allprep dna rna mirna universal kit protocol - by Bioz Stars, 2020-04
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    Images

    1) Product Images from "RNA Preservation Agents and Nucleic Acid Extraction Method Bias Perceived Bacterial Community Composition"

    Article Title: RNA Preservation Agents and Nucleic Acid Extraction Method Bias Perceived Bacterial Community Composition

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0121659

    Comparison of DNA/RNA yields and RNA quality (inset). (A) Comparison between the AllPrep standard protocol (APS; described below figure), enzymatic lysis (Enz), enzymatic/bead beating (Bead), and Mirvana extraction protocols (Mirvana). (B) Comparison between different iterations of the AP protocol, examining the effect of preservation method {P}, lysis conditions {L}, and other extraction modification steps {E}. The data presents changes in yield relative to a control, where each column shows the effect of one factor relative to a control that differed in that factor only. The control was extracted using either the AP standard protocol or a modification as indicated by the X-axis labels below the horizontal lines. The factor that was changed is indicated just below the data column. The dotted line indicates no change relative to control. All data represent averages of three extractions, with error bars indicating the 95% confidence interval, except for the effect of lysozyme, which averaged the relative yield effects from multiple samples (from DL, LH, and HR samples, three replicate extractions each). Inset: RIN = RNA integrity number quality (0 (poor)- 10 (high) range) assessment by Bioanalyzer; NT = no preservation treatment.
    Figure Legend Snippet: Comparison of DNA/RNA yields and RNA quality (inset). (A) Comparison between the AllPrep standard protocol (APS; described below figure), enzymatic lysis (Enz), enzymatic/bead beating (Bead), and Mirvana extraction protocols (Mirvana). (B) Comparison between different iterations of the AP protocol, examining the effect of preservation method {P}, lysis conditions {L}, and other extraction modification steps {E}. The data presents changes in yield relative to a control, where each column shows the effect of one factor relative to a control that differed in that factor only. The control was extracted using either the AP standard protocol or a modification as indicated by the X-axis labels below the horizontal lines. The factor that was changed is indicated just below the data column. The dotted line indicates no change relative to control. All data represent averages of three extractions, with error bars indicating the 95% confidence interval, except for the effect of lysozyme, which averaged the relative yield effects from multiple samples (from DL, LH, and HR samples, three replicate extractions each). Inset: RIN = RNA integrity number quality (0 (poor)- 10 (high) range) assessment by Bioanalyzer; NT = no preservation treatment.

    Techniques Used: Lysis, Preserving, Modification

    2) Product Images from "Comparison of Automated and Manual DNA Isolation Methods of Liquid-Based Cytology Samples"

    Article Title: Comparison of Automated and Manual DNA Isolation Methods of Liquid-Based Cytology Samples

    Journal: Biopreservation and Biobanking

    doi: 10.1089/bio.2018.0148

    Schematic representation of DNA procedure from LBC samples. Left side : manual procedure; middle : outline procedure; right side : automated procedure. RT = room temperature; EB = elution buffer; AW1 and AW2 = buffer concentrate provided from AllPrep DNA/RNA/miRNA Universal Kit. LBC, liquid-based cytology.
    Figure Legend Snippet: Schematic representation of DNA procedure from LBC samples. Left side : manual procedure; middle : outline procedure; right side : automated procedure. RT = room temperature; EB = elution buffer; AW1 and AW2 = buffer concentrate provided from AllPrep DNA/RNA/miRNA Universal Kit. LBC, liquid-based cytology.

    Techniques Used:

    3) Product Images from "Establishment of urinary exosome-like vesicles isolation protocol for FHHNC patients and evaluation of different exosomal RNA extraction methods"

    Article Title: Establishment of urinary exosome-like vesicles isolation protocol for FHHNC patients and evaluation of different exosomal RNA extraction methods

    Journal: Journal of Translational Medicine

    doi: 10.1186/s12967-018-1651-z

    RNA quantification. a Bar graph of total RNA quantified from FHHNC uEVs by Bioanalyzer—Picochip using the five different extraction methods. MirCURY kit followed by TRIzol LS were the most efficient methods. b A representative electropherogram shows that uEVs contain small RNA, including microRNAs (10–40 nt). As expected, small amounts of rRNA were detected. c miRNA profiling by RT-qPCR of RNA extracted from FHHNC uEVs. miRNA expression pattern is consistent independently of the RNA extraction method
    Figure Legend Snippet: RNA quantification. a Bar graph of total RNA quantified from FHHNC uEVs by Bioanalyzer—Picochip using the five different extraction methods. MirCURY kit followed by TRIzol LS were the most efficient methods. b A representative electropherogram shows that uEVs contain small RNA, including microRNAs (10–40 nt). As expected, small amounts of rRNA were detected. c miRNA profiling by RT-qPCR of RNA extracted from FHHNC uEVs. miRNA expression pattern is consistent independently of the RNA extraction method

    Techniques Used: Quantitative RT-PCR, Expressing, RNA Extraction

    4) Product Images from "Age-related epigenome-wide DNA methylation and hydroxymethylation in longitudinal mouse blood"

    Article Title: Age-related epigenome-wide DNA methylation and hydroxymethylation in longitudinal mouse blood

    Journal: Epigenetics

    doi: 10.1080/15592294.2018.1507198

    Sequencing data collection and analysis workflow. Genomic DNA was isolated from matched offspring blood samples at 2 months (n = 6), 4 months (n = 6), and 10 months of age (n = 6). DNA samples were then split for separate processing steps specific to the ERRBS and HMeDIP-seq methods. All processed samples were amplified and sequenced on an Illumina HiSeq 4000 sequencer using single-end, 50 nt read length. Separate bioinformatics pipelines were used to analyze ERRBS and HMeDIP-seq data. Differentially methylated CpGs (DMCs) were compared to differentially hydroxymethylated regions (DHMRs) for regions of chromosomal overlap. Regions of overlap were then annotated and visualized in the genome browser. The Nfic gene was validated using RT-qPCR on available RNA from the blood samples.
    Figure Legend Snippet: Sequencing data collection and analysis workflow. Genomic DNA was isolated from matched offspring blood samples at 2 months (n = 6), 4 months (n = 6), and 10 months of age (n = 6). DNA samples were then split for separate processing steps specific to the ERRBS and HMeDIP-seq methods. All processed samples were amplified and sequenced on an Illumina HiSeq 4000 sequencer using single-end, 50 nt read length. Separate bioinformatics pipelines were used to analyze ERRBS and HMeDIP-seq data. Differentially methylated CpGs (DMCs) were compared to differentially hydroxymethylated regions (DHMRs) for regions of chromosomal overlap. Regions of overlap were then annotated and visualized in the genome browser. The Nfic gene was validated using RT-qPCR on available RNA from the blood samples.

    Techniques Used: Sequencing, Isolation, Amplification, Methylation, Quantitative RT-PCR

    5) Product Images from "Integrative microRNA and mRNA deep-sequencing expression profiling in endemic Burkitt lymphoma"

    Article Title: Integrative microRNA and mRNA deep-sequencing expression profiling in endemic Burkitt lymphoma

    Journal: BMC Cancer

    doi: 10.1186/s12885-017-3711-9

    Aberrant transcriptome expression pivotal to eBL lymphomagenesis. a Schematic illustration of the aberrant gene expression and miRNA mediated regulatory changes that would initiate lymphomagenesis as a result of DNA damage. Combined loss of p53 function due to small interfering RNA-mediated regulation of ATM and NLK together with upregulation of TFAP4, would facilitate survival of cells with the c-myc-Igh chromosomal translocation and MYC induced cell cycle progression initiating eBL tumor development. ATM checkpoint kinase, transduces genomic stress signals to halt cell cycle progression in response to DNA damage. It is critical in the regulation of apoptosis and lymphomagenesis in c-myc induced lymphomas. ATM is downregulated in eBL and it is targeted by 4 miRs that are Upregulated in eBL. NLK is required for the upregulation of P53 expression in response to DNA damage. It interacts with P53 to enhance its stability and activity by abrogating MDM2 mediated degradation. NLK is downregulated in eBL tumor cells and also targeted by 2 miRs that are upregulated in eBL tumor cells. TFAP4/AP4 is a central mediator of cell cycle progression in response to c-MYC activation. b RNA seq. Expression counts of MYC , TFAP4 , ATM and NLK in eBL tumor cells and GC B cells. c Hierarchical clustering of eBL and GC B cells based on the expression profiles of MYC , TFAP4 , ATM and NLK also revealed a clear separation of the two groups. d . miRNA seq. Expression counts of hsa-miR-26a-5p, hsa-miR-27b-3p, hsa-miR-30b-5p, miR-17~92-cluster members (hsa-miR-19b-3p, and hsa-miR-92a-3p), and let-7-family miRs (hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7d-5p, hsa-let-7e-5p, and hsa-let-7 g-5p) in eBL tumor cells and GC B cells
    Figure Legend Snippet: Aberrant transcriptome expression pivotal to eBL lymphomagenesis. a Schematic illustration of the aberrant gene expression and miRNA mediated regulatory changes that would initiate lymphomagenesis as a result of DNA damage. Combined loss of p53 function due to small interfering RNA-mediated regulation of ATM and NLK together with upregulation of TFAP4, would facilitate survival of cells with the c-myc-Igh chromosomal translocation and MYC induced cell cycle progression initiating eBL tumor development. ATM checkpoint kinase, transduces genomic stress signals to halt cell cycle progression in response to DNA damage. It is critical in the regulation of apoptosis and lymphomagenesis in c-myc induced lymphomas. ATM is downregulated in eBL and it is targeted by 4 miRs that are Upregulated in eBL. NLK is required for the upregulation of P53 expression in response to DNA damage. It interacts with P53 to enhance its stability and activity by abrogating MDM2 mediated degradation. NLK is downregulated in eBL tumor cells and also targeted by 2 miRs that are upregulated in eBL tumor cells. TFAP4/AP4 is a central mediator of cell cycle progression in response to c-MYC activation. b RNA seq. Expression counts of MYC , TFAP4 , ATM and NLK in eBL tumor cells and GC B cells. c Hierarchical clustering of eBL and GC B cells based on the expression profiles of MYC , TFAP4 , ATM and NLK also revealed a clear separation of the two groups. d . miRNA seq. Expression counts of hsa-miR-26a-5p, hsa-miR-27b-3p, hsa-miR-30b-5p, miR-17~92-cluster members (hsa-miR-19b-3p, and hsa-miR-92a-3p), and let-7-family miRs (hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7d-5p, hsa-let-7e-5p, and hsa-let-7 g-5p) in eBL tumor cells and GC B cells

    Techniques Used: Expressing, Small Interfering RNA, Translocation Assay, Activity Assay, Activation Assay, RNA Sequencing Assay

    6) Product Images from "Establishment of urinary exosome-like vesicles isolation protocol for FHHNC patients and evaluation of different exosomal RNA extraction methods"

    Article Title: Establishment of urinary exosome-like vesicles isolation protocol for FHHNC patients and evaluation of different exosomal RNA extraction methods

    Journal: Journal of Translational Medicine

    doi: 10.1186/s12967-018-1651-z

    RNA quantification. a Bar graph of total RNA quantified from FHHNC uEVs by Bioanalyzer—Picochip using the five different extraction methods. MirCURY kit followed by TRIzol LS were the most efficient methods. b A representative electropherogram shows that uEVs contain small RNA, including microRNAs (10–40 nt). As expected, small amounts of rRNA were detected. c miRNA profiling by RT-qPCR of RNA extracted from FHHNC uEVs. miRNA expression pattern is consistent independently of the RNA extraction method
    Figure Legend Snippet: RNA quantification. a Bar graph of total RNA quantified from FHHNC uEVs by Bioanalyzer—Picochip using the five different extraction methods. MirCURY kit followed by TRIzol LS were the most efficient methods. b A representative electropherogram shows that uEVs contain small RNA, including microRNAs (10–40 nt). As expected, small amounts of rRNA were detected. c miRNA profiling by RT-qPCR of RNA extracted from FHHNC uEVs. miRNA expression pattern is consistent independently of the RNA extraction method

    Techniques Used: Quantitative RT-PCR, Expressing, RNA Extraction

    7) Product Images from "MicroRNA-30 modulates metabolic inflammation by regulating notch signaling in adipose tissue macrophages"

    Article Title: MicroRNA-30 modulates metabolic inflammation by regulating notch signaling in adipose tissue macrophages

    Journal: International journal of obesity (2005)

    doi: 10.1038/s41366-018-0114-1

    DLL4 Expression is Elevated in Obese ATMs HFD-induced obesity was studied as described in Fig 1 legend. At 22-weeks-old, visceral adipose tissue and adipose SVF were analyzed for DLL4 expression. ( A C ) Immunofluorescent staining of whole-mount epididymal fat. Scale bar = 20 μm. Presented are representative confocal micrographs of Notch1 ( A ) or DLL4 ( C ). ( B D ) Image quantification of Notch1 ( B ) or DLL4 ( D ) in adipose tissue. The values are shown as mean ± SEM and are from a single experiment representative of 2 independent experiments with 5 mice per experimental group. ( E ) Flow cytometry dot plots of DLL4 + ATMs in epididymal fat. CD11b int are denoted as infiltrating (“Inf”) and CD11b hi are denoted as resident (“Res”). ( F ) Quantification of DLL4 + ATM cell counts represented per mouse and per gram fat. Values are presented as mean ± SEM and are from a single experiment representative of 2 independent experiments with 4 biological replicates (pools of 1–6 mice) per experimental group. Statistical significance was determined by Student’s t-test. *p
    Figure Legend Snippet: DLL4 Expression is Elevated in Obese ATMs HFD-induced obesity was studied as described in Fig 1 legend. At 22-weeks-old, visceral adipose tissue and adipose SVF were analyzed for DLL4 expression. ( A C ) Immunofluorescent staining of whole-mount epididymal fat. Scale bar = 20 μm. Presented are representative confocal micrographs of Notch1 ( A ) or DLL4 ( C ). ( B D ) Image quantification of Notch1 ( B ) or DLL4 ( D ) in adipose tissue. The values are shown as mean ± SEM and are from a single experiment representative of 2 independent experiments with 5 mice per experimental group. ( E ) Flow cytometry dot plots of DLL4 + ATMs in epididymal fat. CD11b int are denoted as infiltrating (“Inf”) and CD11b hi are denoted as resident (“Res”). ( F ) Quantification of DLL4 + ATM cell counts represented per mouse and per gram fat. Values are presented as mean ± SEM and are from a single experiment representative of 2 independent experiments with 4 biological replicates (pools of 1–6 mice) per experimental group. Statistical significance was determined by Student’s t-test. *p

    Techniques Used: Expressing, Staining, Mouse Assay, Flow Cytometry, Cytometry

    HFD-Induced Obesity Stimulates ATM Inflammation and miRNA Dysregulation To study HFD-induced obesity, male C57BL/6J mice were fed NCD or HFD for 16 weeks until 22-weeks-old. ( A ) Representative photo of mice after 16 weeks of NCD or HFD feeding. ( B ) Weekly measurements of body weight growth. ( C ) DEXA body composition after 16 weeks of diet. ( D ) Oral glucose tolerance test (GTT) after 16 weeks of diet. ( E ) Area under the curve (A.U.C.) for GTT. Represented are A.U.C. above baseline (ABV BL) or total A.U.C. ( F ) Flow cytometry dot plots of F4/80 + /CD11b + /CD11c + ATMs in the epididymal fat stromal vascular fraction (SVF) of NCD- or HFD-fed mice. ( G ) Fold percentage increase quantification of F4/80 + /CD11b + cells in the SVF (denoted as “ATMs”) and CD11c + ATMs. Lean mice were fed either NCD or 10% low-fat diet (LFD). Obese mice were fed 60% HFD. ( H–J ) Pooled F4/80+ ATMs from epididymal fat were used for transcriptome and miRNA microarrays. ( H ) Transcriptome microarray heatmap of differentially expressed mRNAs in ATMs related to macrophage polarization. ( I ) MicroRNA heatmap of differentially expressed miRNAs in ATMs. ( J ) MicroRNA array volcano plot depicting linear fold change (FC) vs. ANOVA p-value significance. ( K–M ) qRT-PCR expression validation of miRs 30a-5p, 30c-5p, and 30e-5p. For (A–F), the values are shown as mean ± SEM and are from a single experiment representative of at least 3 independent experiments with 5 mice per experimental group. For (G) data shown as mean ± SEM of 4 independent experiments with 5 mice per experimental group. For (H–M), the data shown are mean ± SEM and are from 3–4 independent experiments with 20 pooled NCD mice and 10 pooled HFD mice per experiment. Statistical differences were determined by using Student’s t-test. *p
    Figure Legend Snippet: HFD-Induced Obesity Stimulates ATM Inflammation and miRNA Dysregulation To study HFD-induced obesity, male C57BL/6J mice were fed NCD or HFD for 16 weeks until 22-weeks-old. ( A ) Representative photo of mice after 16 weeks of NCD or HFD feeding. ( B ) Weekly measurements of body weight growth. ( C ) DEXA body composition after 16 weeks of diet. ( D ) Oral glucose tolerance test (GTT) after 16 weeks of diet. ( E ) Area under the curve (A.U.C.) for GTT. Represented are A.U.C. above baseline (ABV BL) or total A.U.C. ( F ) Flow cytometry dot plots of F4/80 + /CD11b + /CD11c + ATMs in the epididymal fat stromal vascular fraction (SVF) of NCD- or HFD-fed mice. ( G ) Fold percentage increase quantification of F4/80 + /CD11b + cells in the SVF (denoted as “ATMs”) and CD11c + ATMs. Lean mice were fed either NCD or 10% low-fat diet (LFD). Obese mice were fed 60% HFD. ( H–J ) Pooled F4/80+ ATMs from epididymal fat were used for transcriptome and miRNA microarrays. ( H ) Transcriptome microarray heatmap of differentially expressed mRNAs in ATMs related to macrophage polarization. ( I ) MicroRNA heatmap of differentially expressed miRNAs in ATMs. ( J ) MicroRNA array volcano plot depicting linear fold change (FC) vs. ANOVA p-value significance. ( K–M ) qRT-PCR expression validation of miRs 30a-5p, 30c-5p, and 30e-5p. For (A–F), the values are shown as mean ± SEM and are from a single experiment representative of at least 3 independent experiments with 5 mice per experimental group. For (G) data shown as mean ± SEM of 4 independent experiments with 5 mice per experimental group. For (H–M), the data shown are mean ± SEM and are from 3–4 independent experiments with 20 pooled NCD mice and 10 pooled HFD mice per experiment. Statistical differences were determined by using Student’s t-test. *p

    Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Microarray, Quantitative RT-PCR, Expressing

    DNA Methylation-Dependent Regulation of miR-30 As shown in Figure 1 , F4/80+ ATMs were isolated from epididymal fat of 22-week-old HFD-induced obese and lean mice. Gene expression and DNA methylation were evaluated in ATMs. ( A ) Volcano plot displaying linear FC of genes encoding epigenetic modification enzymes and factors. Fold change and p value observations were extracted from transcriptome microarrays (See Figure 1 and Figure S1 ). ( B ) IGB visualization of meDIP-seq peak intensity of DNA methylation in the Nfyc promoter CpG island. ( C ) Methylation-specific PCR quantification of DNA methylation (DNAme) in the Nfyc promoter CpG island. For (A and C), values presented are representative of 3 independent experiments with 20 pooled NCD mice and 10 pooled HFD mice per experiment. For (B), data are representative of one experiment of 60 pooled LFD and 30 pooled HFD mice. Statistical differences were determined by using Student’s t-test. *p
    Figure Legend Snippet: DNA Methylation-Dependent Regulation of miR-30 As shown in Figure 1 , F4/80+ ATMs were isolated from epididymal fat of 22-week-old HFD-induced obese and lean mice. Gene expression and DNA methylation were evaluated in ATMs. ( A ) Volcano plot displaying linear FC of genes encoding epigenetic modification enzymes and factors. Fold change and p value observations were extracted from transcriptome microarrays (See Figure 1 and Figure S1 ). ( B ) IGB visualization of meDIP-seq peak intensity of DNA methylation in the Nfyc promoter CpG island. ( C ) Methylation-specific PCR quantification of DNA methylation (DNAme) in the Nfyc promoter CpG island. For (A and C), values presented are representative of 3 independent experiments with 20 pooled NCD mice and 10 pooled HFD mice per experiment. For (B), data are representative of one experiment of 60 pooled LFD and 30 pooled HFD mice. Statistical differences were determined by using Student’s t-test. *p

    Techniques Used: DNA Methylation Assay, Isolation, Mouse Assay, Expressing, Modification, Methylated DNA Immunoprecipitation, Methylation, Polymerase Chain Reaction

    8) Product Images from "Hepatic Tumor Formation in Adult Mice Developmentally Exposed to Organotin"

    Article Title: Hepatic Tumor Formation in Adult Mice Developmentally Exposed to Organotin

    Journal: Environmental Health Perspectives

    doi: 10.1289/EHP5414

    Body weight, white adipose tissue weight, adipose magnetic resonance imaging (MRI), and liver lipid of male and female mice developmentally exposed to tributyltin or vehicle. (A) Female mice were fed a phytoestrogen-free diet and treated with tributyltin (TBT) in their drinking water beginning 2 weeks prior to breeding lasting through gestation and lactation. Offspring were then weaned at 3 weeks of age and fed the same phytoestrogen-free diet until euthanasia at 14, 20, or 45 weeks of age. (B) Body weights, white adipose tissue depot [either inguinal (iWAT) or scapular (scWAT)], and brown adipose tissue (BAT) wet weights in 14-week-old animals developmentally exposed to TBT or VEH are displayed; VEH-treated females, n = 4 (from two litters with two pups per litter); VEH-treated males, n = 3 (from two litters); TBT-treated females, n = 3 (from two litters); TBT-treated males, n = 4 (from two litters, two pups per litter). (C) Representative full-body MRI scans are presented on the left and quantitation of surface area is presented on the right for 20-week-old animals: for VEH-treated females, n = 2 (from one litter); for TBT-treated females, n = 4 (from two litters with two pups per litter); for VEH-treated males, n = 2 ; and for TBT-treated males, n = 4 . (D) Macroscopic images of liver tissue at 20 weeks, and Oil Red O stained, left liver lobes are presented on the left with quantitation of Oil Red O staining on the right, n = 6 for VEH-treated males, n = 10 for TBT-treated males, n = 7 for VEH-treated females, and n = 9 for TBT-treated females. Scale bars: 100 μ m . In all graphs, VEH bars are hatched and T BT bars are solid, all bars represent the mean ± standard error of the mean (SEM) and t -tests were performed to ascertain significance (* p
    Figure Legend Snippet: Body weight, white adipose tissue weight, adipose magnetic resonance imaging (MRI), and liver lipid of male and female mice developmentally exposed to tributyltin or vehicle. (A) Female mice were fed a phytoestrogen-free diet and treated with tributyltin (TBT) in their drinking water beginning 2 weeks prior to breeding lasting through gestation and lactation. Offspring were then weaned at 3 weeks of age and fed the same phytoestrogen-free diet until euthanasia at 14, 20, or 45 weeks of age. (B) Body weights, white adipose tissue depot [either inguinal (iWAT) or scapular (scWAT)], and brown adipose tissue (BAT) wet weights in 14-week-old animals developmentally exposed to TBT or VEH are displayed; VEH-treated females, n = 4 (from two litters with two pups per litter); VEH-treated males, n = 3 (from two litters); TBT-treated females, n = 3 (from two litters); TBT-treated males, n = 4 (from two litters, two pups per litter). (C) Representative full-body MRI scans are presented on the left and quantitation of surface area is presented on the right for 20-week-old animals: for VEH-treated females, n = 2 (from one litter); for TBT-treated females, n = 4 (from two litters with two pups per litter); for VEH-treated males, n = 2 ; and for TBT-treated males, n = 4 . (D) Macroscopic images of liver tissue at 20 weeks, and Oil Red O stained, left liver lobes are presented on the left with quantitation of Oil Red O staining on the right, n = 6 for VEH-treated males, n = 10 for TBT-treated males, n = 7 for VEH-treated females, and n = 9 for TBT-treated females. Scale bars: 100 μ m . In all graphs, VEH bars are hatched and T BT bars are solid, all bars represent the mean ± standard error of the mean (SEM) and t -tests were performed to ascertain significance (* p

    Techniques Used: Magnetic Resonance Imaging, Mouse Assay, Quantitation Assay, Staining

    Overrepresentation analysis (ORA) using publicly available mouse and human liver disease data sets. An ORA was conducted using the genes differentially expressed in the tumors from TBT-exposed males with publicly available mouse and human RNA-seq data sets in distinct liver disease states (GSE14520, LIHC TCGA, GSE48452, GSE83596, GSE59930). ORA for (A) 45 week adenoma over TBT liver, (B) 45 week adenoma over VEH liver, and (C) 20 week TBT liver over VEH liver. Significant enrichment was detected in all data sets. Closed bars represent all overlapping genes in log 2 scale, whereas open bars and gray bars represent the subset of all genes that are also present in the growth hormone receptor (GHR) signature. Note: HCC, hepatocellular carcinoma; LIHC, liver hepatocellular carcinoma; NASH, nonalcoholic steatohepatitis.
    Figure Legend Snippet: Overrepresentation analysis (ORA) using publicly available mouse and human liver disease data sets. An ORA was conducted using the genes differentially expressed in the tumors from TBT-exposed males with publicly available mouse and human RNA-seq data sets in distinct liver disease states (GSE14520, LIHC TCGA, GSE48452, GSE83596, GSE59930). ORA for (A) 45 week adenoma over TBT liver, (B) 45 week adenoma over VEH liver, and (C) 20 week TBT liver over VEH liver. Significant enrichment was detected in all data sets. Closed bars represent all overlapping genes in log 2 scale, whereas open bars and gray bars represent the subset of all genes that are also present in the growth hormone receptor (GHR) signature. Note: HCC, hepatocellular carcinoma; LIHC, liver hepatocellular carcinoma; NASH, nonalcoholic steatohepatitis.

    Techniques Used: RNA Sequencing Assay

    Histology of tributyltin (TBT)-treated liver adenomas in male mice. (A) At 45 weeks of age, liver adenomas were observed in many lobes of the liver in male mice. One vehicle (VEH)-exposed liver and two representative tumors are displayed with hematoxylin and eosin and Ki67 immunohistochemistry staining (left panels). The right panel shows a quantitation of Ki67 immunostaining [VEH-exposed liver (V), hatched bar, n = 6 ; TBT-exposed liver (T), n = 6 ; Adenoma (A), n = 7 ]. Bars represent mean ± standard error of the mean (SEM). *** p
    Figure Legend Snippet: Histology of tributyltin (TBT)-treated liver adenomas in male mice. (A) At 45 weeks of age, liver adenomas were observed in many lobes of the liver in male mice. One vehicle (VEH)-exposed liver and two representative tumors are displayed with hematoxylin and eosin and Ki67 immunohistochemistry staining (left panels). The right panel shows a quantitation of Ki67 immunostaining [VEH-exposed liver (V), hatched bar, n = 6 ; TBT-exposed liver (T), n = 6 ; Adenoma (A), n = 7 ]. Bars represent mean ± standard error of the mean (SEM). *** p

    Techniques Used: Mouse Assay, Immunohistochemistry, Staining, Quantitation Assay, Immunostaining

    Expression of members of the growth hormone receptor (GHR)/STAT5 signaling pathway in male mice developmentally exposed to tributyltin. (A) Representative images of immunohistochemical staining of major urinary protein 1 (MUP1) protein in vehicle (VEH)-treated liver and adenomas from tributyltin (TBT)-treated 45-week-old male mice ( n = 5 ; scale bars: 100 μ m ). (B) Immunoblot analysis of growth hormone receptor (GHR) protein levels in liver tissue from VEH- ( n = 6 ) and TBT-exposed ( n = 5 ) males and adenoma tissue from TBT-exposed males ( n = 7 ) at 45 weeks of age (left panels) with quantitation of the immunoblots (right panels). GHR level was normalized to GAPDH protein level in each lysate. Ponceau stain is displayed beneath each blot. Bars represent the mean ± standard error of the mean ( SEM ) . * p
    Figure Legend Snippet: Expression of members of the growth hormone receptor (GHR)/STAT5 signaling pathway in male mice developmentally exposed to tributyltin. (A) Representative images of immunohistochemical staining of major urinary protein 1 (MUP1) protein in vehicle (VEH)-treated liver and adenomas from tributyltin (TBT)-treated 45-week-old male mice ( n = 5 ; scale bars: 100 μ m ). (B) Immunoblot analysis of growth hormone receptor (GHR) protein levels in liver tissue from VEH- ( n = 6 ) and TBT-exposed ( n = 5 ) males and adenoma tissue from TBT-exposed males ( n = 7 ) at 45 weeks of age (left panels) with quantitation of the immunoblots (right panels). GHR level was normalized to GAPDH protein level in each lysate. Ponceau stain is displayed beneath each blot. Bars represent the mean ± standard error of the mean ( SEM ) . * p

    Techniques Used: Expressing, Mouse Assay, Immunohistochemistry, Staining, Quantitation Assay, Western Blot

    9) Product Images from "Epigenetic regulation of lateralized fetal spinal gene expression underlies hemispheric asymmetries"

    Article Title: Epigenetic regulation of lateralized fetal spinal gene expression underlies hemispheric asymmetries

    Journal: eLife

    doi: 10.7554/eLife.22784

    Epigenetic regulation of gene expression asymmetries in human fetal spinal cord. ( A ) Asymmetrically expressed miRNA transcripts at 8, 10 and 12 weeks PC. The extent of expression asymmetries is measured in log 2 (fold change). Red bars show rightward asymmetrically expressed microRNA transcripts, blue bars show leftward asymmetrically expressed miRNA transcripts. ( B ) Number of CpG sites showing differential DNA methylation per chromosome, compared between the left and right spinal cord for 8 and 10 weeks PC. Depicted are only CpG sites with methylation asymmetries in both samples. Red bars represent the number of CpG sites that showed significantly higher DNA methylation on the right side, blue bars show the number of CpG sites that showed significantly more DNA methylation on the left side. ( C ) Percentage of differential DNA methylation in leftward (blue) and rightward (red) asymmetrically methylated CpG sites as a function of p-value. ( D ) Percentage of gene expression asymmetries on each chromosome at 8 weeks PC that can be explained by regulation via asymmetrically expressed miRNAs or asymmetric DNA methylation of CpG sites within and 1500 nucleotides upstream of the expressed genes. The source files of asymmetrically expressed miRNAs, asymmetrically expressed targets of miRNAs, enriched KEGG pathways and differentially methylated CpG sites are available in Figure 3—source data 1 , Figure 3—source data 2 , Figure 3—source data 3 , and Figure 3—source data 4 respectively. DOI: http://dx.doi.org/10.7554/eLife.22784.006 10.7554/eLife.22784.007 Asymmetrically expressed miRNAs per week. DOI: http://dx.doi.org/10.7554/eLife.22784.007 10.7554/eLife.22784.008 Asymmetrically expressed RNA targets of asymmetrically expressed miRNAs per week. DOI: http://dx.doi.org/10.7554/eLife.22784.008 10.7554/eLife.22784.009 Enriched KEGG pathways per week and side of the spinal cord. DOI: http://dx.doi.org/10.7554/eLife.22784.009 10.7554/eLife.22784.010 Asymmetrically methylated CpG sites per week and side of the spinal cord. DOI: http://dx.doi.org/10.7554/eLife.22784.010
    Figure Legend Snippet: Epigenetic regulation of gene expression asymmetries in human fetal spinal cord. ( A ) Asymmetrically expressed miRNA transcripts at 8, 10 and 12 weeks PC. The extent of expression asymmetries is measured in log 2 (fold change). Red bars show rightward asymmetrically expressed microRNA transcripts, blue bars show leftward asymmetrically expressed miRNA transcripts. ( B ) Number of CpG sites showing differential DNA methylation per chromosome, compared between the left and right spinal cord for 8 and 10 weeks PC. Depicted are only CpG sites with methylation asymmetries in both samples. Red bars represent the number of CpG sites that showed significantly higher DNA methylation on the right side, blue bars show the number of CpG sites that showed significantly more DNA methylation on the left side. ( C ) Percentage of differential DNA methylation in leftward (blue) and rightward (red) asymmetrically methylated CpG sites as a function of p-value. ( D ) Percentage of gene expression asymmetries on each chromosome at 8 weeks PC that can be explained by regulation via asymmetrically expressed miRNAs or asymmetric DNA methylation of CpG sites within and 1500 nucleotides upstream of the expressed genes. The source files of asymmetrically expressed miRNAs, asymmetrically expressed targets of miRNAs, enriched KEGG pathways and differentially methylated CpG sites are available in Figure 3—source data 1 , Figure 3—source data 2 , Figure 3—source data 3 , and Figure 3—source data 4 respectively. DOI: http://dx.doi.org/10.7554/eLife.22784.006 10.7554/eLife.22784.007 Asymmetrically expressed miRNAs per week. DOI: http://dx.doi.org/10.7554/eLife.22784.007 10.7554/eLife.22784.008 Asymmetrically expressed RNA targets of asymmetrically expressed miRNAs per week. DOI: http://dx.doi.org/10.7554/eLife.22784.008 10.7554/eLife.22784.009 Enriched KEGG pathways per week and side of the spinal cord. DOI: http://dx.doi.org/10.7554/eLife.22784.009 10.7554/eLife.22784.010 Asymmetrically methylated CpG sites per week and side of the spinal cord. DOI: http://dx.doi.org/10.7554/eLife.22784.010

    Techniques Used: Expressing, DNA Methylation Assay, Methylation

    10) Product Images from "MicroRNA-30 modulates metabolic inflammation by regulating notch signaling in adipose tissue macrophages"

    Article Title: MicroRNA-30 modulates metabolic inflammation by regulating notch signaling in adipose tissue macrophages

    Journal: International journal of obesity (2005)

    doi: 10.1038/s41366-018-0114-1

    DNA Methylation-Dependent Regulation of miR-30 As shown in Figure 1 , F4/80+ ATMs were isolated from epididymal fat of 22-week-old HFD-induced obese and lean mice. Gene expression and DNA methylation were evaluated in ATMs. ( A ) Volcano plot displaying linear FC of genes encoding epigenetic modification enzymes and factors. Fold change and p value observations were extracted from transcriptome microarrays (See Figure 1 and Figure S1 ). ( B ) IGB visualization of meDIP-seq peak intensity of DNA methylation in the Nfyc promoter CpG island. ( C ) Methylation-specific PCR quantification of DNA methylation (DNAme) in the Nfyc promoter CpG island. For (A and C), values presented are representative of 3 independent experiments with 20 pooled NCD mice and 10 pooled HFD mice per experiment. For (B), data are representative of one experiment of 60 pooled LFD and 30 pooled HFD mice. Statistical differences were determined by using Student’s t-test. *p
    Figure Legend Snippet: DNA Methylation-Dependent Regulation of miR-30 As shown in Figure 1 , F4/80+ ATMs were isolated from epididymal fat of 22-week-old HFD-induced obese and lean mice. Gene expression and DNA methylation were evaluated in ATMs. ( A ) Volcano plot displaying linear FC of genes encoding epigenetic modification enzymes and factors. Fold change and p value observations were extracted from transcriptome microarrays (See Figure 1 and Figure S1 ). ( B ) IGB visualization of meDIP-seq peak intensity of DNA methylation in the Nfyc promoter CpG island. ( C ) Methylation-specific PCR quantification of DNA methylation (DNAme) in the Nfyc promoter CpG island. For (A and C), values presented are representative of 3 independent experiments with 20 pooled NCD mice and 10 pooled HFD mice per experiment. For (B), data are representative of one experiment of 60 pooled LFD and 30 pooled HFD mice. Statistical differences were determined by using Student’s t-test. *p

    Techniques Used: DNA Methylation Assay, Isolation, Mouse Assay, Expressing, Modification, Methylated DNA Immunoprecipitation, Methylation, Polymerase Chain Reaction

    11) Product Images from "CpG promoter methylation of the ALKBH3 alkylation repair gene in breast cancer"

    Article Title: CpG promoter methylation of the ALKBH3 alkylation repair gene in breast cancer

    Journal: BMC Cancer

    doi: 10.1186/s12885-017-3453-8

    The ALKBH3 gene promoter region. a The ALKBH3 gene promoter is transcriptionally active in variant human mammary epithelial cells (vHMEC) based on available data from the Roadmap Epigenetic Consortium (Roadmap Epigenomics Consortium, Nature 2015). Data for vHMEC are shown here for markers associated with active transcription (H3K4me3 and H3K36me3) together with repressive markers (H3K27me3, H3K9me3, H3K4me1 and 5-methylcytosine (DNA methyl)). Additionally, DNAse and RNA sequencing results from vHMEC cells are shown – reflecting chromatin accessibility and mRNA expression, respectively. b The ALKBH3 gene promoter region is shown with respect to the FANTOM5 transcription start site (TSS) as arrows p1 and p2 along with the 1st exon and the promoter-associated CpG island (UCSC defined). The CpG methylation assay for ALKBH3 was designed to include CpG sites proximal to the TSS and the regions selected is indicated by a black box (labelled R) covering three closely spaced CpG dinucleotides found -50, −53 and -58 bp upstream of the major TSS (p1 region in FANTOM5). Additionally, the region where statistically significant associations were revealed between CpG methylation and loss of expression for the ALKBH3 gene in tumors is marked out and labelled for expression as “Xprs” (see further information in Additional file 1 )
    Figure Legend Snippet: The ALKBH3 gene promoter region. a The ALKBH3 gene promoter is transcriptionally active in variant human mammary epithelial cells (vHMEC) based on available data from the Roadmap Epigenetic Consortium (Roadmap Epigenomics Consortium, Nature 2015). Data for vHMEC are shown here for markers associated with active transcription (H3K4me3 and H3K36me3) together with repressive markers (H3K27me3, H3K9me3, H3K4me1 and 5-methylcytosine (DNA methyl)). Additionally, DNAse and RNA sequencing results from vHMEC cells are shown – reflecting chromatin accessibility and mRNA expression, respectively. b The ALKBH3 gene promoter region is shown with respect to the FANTOM5 transcription start site (TSS) as arrows p1 and p2 along with the 1st exon and the promoter-associated CpG island (UCSC defined). The CpG methylation assay for ALKBH3 was designed to include CpG sites proximal to the TSS and the regions selected is indicated by a black box (labelled R) covering three closely spaced CpG dinucleotides found -50, −53 and -58 bp upstream of the major TSS (p1 region in FANTOM5). Additionally, the region where statistically significant associations were revealed between CpG methylation and loss of expression for the ALKBH3 gene in tumors is marked out and labelled for expression as “Xprs” (see further information in Additional file 1 )

    Techniques Used: Variant Assay, RNA Sequencing Assay, Expressing, CpG Methylation Assay

    12) Product Images from "The HDAC inhibitor valproate induces a bivalent status of the CD20 promoter in CLL patients suggesting distinct epigenetic regulation of CD20 expression in CLL in vivo"

    Article Title: The HDAC inhibitor valproate induces a bivalent status of the CD20 promoter in CLL patients suggesting distinct epigenetic regulation of CD20 expression in CLL in vivo

    Journal: Oncotarget

    doi: 10.18632/oncotarget.16964

    Global H3K27me3 is increased by valproate in CLL patients and in the I83-E95 CLL cell line I83-E95 cells and circulating untreated CLL cells from patients were incubated with or without 1000 μM of valproate. After 48 hours total protein extracts were prepared and global H3K27me3 was analysed by Western blot. β-actin was used as equal loading control. One out of two independent experiments is shown. For experiment number 2, please see Supplementary Figure 1 .
    Figure Legend Snippet: Global H3K27me3 is increased by valproate in CLL patients and in the I83-E95 CLL cell line I83-E95 cells and circulating untreated CLL cells from patients were incubated with or without 1000 μM of valproate. After 48 hours total protein extracts were prepared and global H3K27me3 was analysed by Western blot. β-actin was used as equal loading control. One out of two independent experiments is shown. For experiment number 2, please see Supplementary Figure 1 .

    Techniques Used: Incubation, Western Blot

    Valproate transiently recruits the transcriptional repressor EZH2 to the CD20 promoter in vivo but not in vitro I83-E95 cells were incubated with or without valproate (VPA) at 1000 μM for 72 hours and subjected to ChIP with anti-EZH2 antibody ( A ). Patient samples collected after 0 and 72 hours of valproate-treatment from cycle 1 ( C ) and cycle 2 ( E ) of the PREVAIL study were subjected to ChIP with anti-EZH2 antibody. Levels of EZH2 on the CD20 promoter were evaluated by densitometry analysis and expressed in graphs ( B , D , F ). (Mean values, bars ± s.e.m, n = 3; ns: not significant). Due to sample limitation, experiments C–F were performed once.
    Figure Legend Snippet: Valproate transiently recruits the transcriptional repressor EZH2 to the CD20 promoter in vivo but not in vitro I83-E95 cells were incubated with or without valproate (VPA) at 1000 μM for 72 hours and subjected to ChIP with anti-EZH2 antibody ( A ). Patient samples collected after 0 and 72 hours of valproate-treatment from cycle 1 ( C ) and cycle 2 ( E ) of the PREVAIL study were subjected to ChIP with anti-EZH2 antibody. Levels of EZH2 on the CD20 promoter were evaluated by densitometry analysis and expressed in graphs ( B , D , F ). (Mean values, bars ± s.e.m, n = 3; ns: not significant). Due to sample limitation, experiments C–F were performed once.

    Techniques Used: In Vivo, In Vitro, Incubation, Chromatin Immunoprecipitation

    Valproate induces simultaneous H3K9ac and H3K27me3 in the CD20 promoter in CLL patients in vivo but not in the I83-E95 CLL cell line I83-E95 cells were incubated with or without valproate (VPA) at 1000 μM for 72 hours and subjected to ChIP with anti-H3K9ac ( A , top) or anti-H3K27me3 (A, bottom) antibodies. Patient samples collected after 0 and 72 hours of valproate-treatment from cycle 1 ( C ) and cycle 2 ( E ) of the PREVAIL study were subjected to ChIP with anti-H3K9ac and anti-H3K27me3 antibodies. Levels of H3K9ac and H3K27me3 on the CD20 promoter were evaluated by densitometry analysis and expressed in graphs ( B , D , F ). (Mean values, bars ± s.e.m, n = 3). Stars indicate statistical significance (* p
    Figure Legend Snippet: Valproate induces simultaneous H3K9ac and H3K27me3 in the CD20 promoter in CLL patients in vivo but not in the I83-E95 CLL cell line I83-E95 cells were incubated with or without valproate (VPA) at 1000 μM for 72 hours and subjected to ChIP with anti-H3K9ac ( A , top) or anti-H3K27me3 (A, bottom) antibodies. Patient samples collected after 0 and 72 hours of valproate-treatment from cycle 1 ( C ) and cycle 2 ( E ) of the PREVAIL study were subjected to ChIP with anti-H3K9ac and anti-H3K27me3 antibodies. Levels of H3K9ac and H3K27me3 on the CD20 promoter were evaluated by densitometry analysis and expressed in graphs ( B , D , F ). (Mean values, bars ± s.e.m, n = 3). Stars indicate statistical significance (* p

    Techniques Used: In Vivo, Incubation, Chromatin Immunoprecipitation

    Valproate induces expression of the transcriptional repressor EZH2 in CLL patients I83-E95 cells and circulating untreated CLL cells from patients were incubated with or without 1000 μM of valproate. After 48 hours’ levels of EZH2 mRNA in I83-E95 cells ( A , mean values, bars ± s.e.m, n = 4, * p
    Figure Legend Snippet: Valproate induces expression of the transcriptional repressor EZH2 in CLL patients I83-E95 cells and circulating untreated CLL cells from patients were incubated with or without 1000 μM of valproate. After 48 hours’ levels of EZH2 mRNA in I83-E95 cells ( A , mean values, bars ± s.e.m, n = 4, * p

    Techniques Used: Expressing, Incubation

    CD20 expression is induced by valproate in the I83-E95 CLL cell line I83-E95 cells were treated for 0h or 72h with valproate (VPA) at 1000 μM. CD20 expression was analysed by qPCR ( A ), FACS ( B ), and Western blot ( C ). (Mean values, bars ± s.e.m, n = 3). Global acetylation of H3K9 was assessed by Western blot (C) and β-actin was used as equal loading control. Stars indicate statistical significance (* p
    Figure Legend Snippet: CD20 expression is induced by valproate in the I83-E95 CLL cell line I83-E95 cells were treated for 0h or 72h with valproate (VPA) at 1000 μM. CD20 expression was analysed by qPCR ( A ), FACS ( B ), and Western blot ( C ). (Mean values, bars ± s.e.m, n = 3). Global acetylation of H3K9 was assessed by Western blot (C) and β-actin was used as equal loading control. Stars indicate statistical significance (* p

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

    13) Product Images from "Age-related epigenome-wide DNA methylation and hydroxymethylation in longitudinal mouse blood"

    Article Title: Age-related epigenome-wide DNA methylation and hydroxymethylation in longitudinal mouse blood

    Journal: Epigenetics

    doi: 10.1080/15592294.2018.1507198

    Sequencing data collection and analysis workflow. Genomic DNA was isolated from matched offspring blood samples at 2 months (n = 6), 4 months (n = 6), and 10 months of age (n = 6). DNA samples were then split for separate processing steps specific to the ERRBS and HMeDIP-seq methods. All processed samples were amplified and sequenced on an Illumina HiSeq 4000 sequencer using single-end, 50 nt read length. Separate bioinformatics pipelines were used to analyze ERRBS and HMeDIP-seq data. Differentially methylated CpGs (DMCs) were compared to differentially hydroxymethylated regions (DHMRs) for regions of chromosomal overlap. Regions of overlap were then annotated and visualized in the genome browser. The Nfic gene was validated using RT-qPCR on available RNA from the blood samples.
    Figure Legend Snippet: Sequencing data collection and analysis workflow. Genomic DNA was isolated from matched offspring blood samples at 2 months (n = 6), 4 months (n = 6), and 10 months of age (n = 6). DNA samples were then split for separate processing steps specific to the ERRBS and HMeDIP-seq methods. All processed samples were amplified and sequenced on an Illumina HiSeq 4000 sequencer using single-end, 50 nt read length. Separate bioinformatics pipelines were used to analyze ERRBS and HMeDIP-seq data. Differentially methylated CpGs (DMCs) were compared to differentially hydroxymethylated regions (DHMRs) for regions of chromosomal overlap. Regions of overlap were then annotated and visualized in the genome browser. The Nfic gene was validated using RT-qPCR on available RNA from the blood samples.

    Techniques Used: Sequencing, Isolation, Amplification, Methylation, Quantitative RT-PCR

    14) Product Images from "Establishment of urinary exosome-like vesicles isolation protocol for FHHNC patients and evaluation of different exosomal RNA extraction methods"

    Article Title: Establishment of urinary exosome-like vesicles isolation protocol for FHHNC patients and evaluation of different exosomal RNA extraction methods

    Journal: Journal of Translational Medicine

    doi: 10.1186/s12967-018-1651-z

    RNA quantification. a Bar graph of total RNA quantified from FHHNC uEVs by Bioanalyzer—Picochip using the five different extraction methods. MirCURY kit followed by TRIzol LS were the most efficient methods. b A representative electropherogram shows that uEVs contain small RNA, including microRNAs (10–40 nt). As expected, small amounts of rRNA were detected. c miRNA profiling by RT-qPCR of RNA extracted from FHHNC uEVs. miRNA expression pattern is consistent independently of the RNA extraction method
    Figure Legend Snippet: RNA quantification. a Bar graph of total RNA quantified from FHHNC uEVs by Bioanalyzer—Picochip using the five different extraction methods. MirCURY kit followed by TRIzol LS were the most efficient methods. b A representative electropherogram shows that uEVs contain small RNA, including microRNAs (10–40 nt). As expected, small amounts of rRNA were detected. c miRNA profiling by RT-qPCR of RNA extracted from FHHNC uEVs. miRNA expression pattern is consistent independently of the RNA extraction method

    Techniques Used: Quantitative RT-PCR, Expressing, RNA Extraction

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    Size-exclusion Chromatography:

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    Purification:

    Article Title: Exercise training alters the genomic response to acute exercise in human adipose tissue
    Article Snippet: .. Genomic DNA and total RNAs were purified from adipose tissue biopsies with the AllPrep DNA/RNA/miRNA Universal Kit (Qiagen, Hilden, Germany). .. The quality of recovered DNA and RNA was assessed by the 260/280 ratio, determined by spectrophotometry with a NanoDrop 2000 (Thermo Fisher Scientific, Roskilde, Denmark), as well as using the Agilent RNA 6000 Nano Kit (Agilent Technologies, Glostrup, Denmark) for RNA.

    Article Title: Comparison of Automated and Manual DNA Isolation Methods of Liquid-Based Cytology Samples
    Article Snippet: Each sample was extracted by either a manual or an automated (Qiacube™; Qiagen, Germantown, MD) method available at the time, following in both cases the protocol of AllPrep DNA/RNA/microRNA Universal Kit™ (Qiagen) as indicated in , using 600 μL of homogenized lysate as a starting material. .. Purified nucleic acids were eluted in 100 μL initially, and a second backup elution with an extra 50 μL from the final step of the protocol was biobanked in 1 mL special DNAse and RNAse free cryotubes (Nunc™; Thermo® , Waltham, MA).

    Article Title: House Fly (Musca domestica L.) Attraction to Insect Honeydew
    Article Snippet: .. Fusarium sp. from the citrus mealybug colony was handled similarly as above except the DNA was extracted using an AllPrep DNA/RNA/miRNA Universal Kit (Cat. No. 80224, Qiagen Sciences, Germantown, MD, USA) and purified using Diffinity RapidTip (Cat. No. RT025-008, Diffinity Genomics, Inc., West Henrietta, NY, USA). .. The resulting amplicons were sequenced at the University of California, Riverside, Genomics Core sequencing facility using the ITS1 primer.

    Polymerase Chain Reaction:

    Article Title: Dwarfism and Altered Craniofacial Development in Rabbits Is Caused by a 12.1 kb Deletion at the HMGA2 Locus
    Article Snippet: Genotypes were obtained using the PCR reactions described above from DNA extracted from the tip of the left hind leg. .. Before library preparation, the entire embryo was homogenized and the RNA was extracted using the Allprep DNA/RNA/miRNA Universal kit (QIAGEN, Valencia, CA).

    Article Title: House Fly (Musca domestica L.) Attraction to Insect Honeydew
    Article Snippet: The resulting amplicons were sequenced in both directions using the same primers used for the PCR reactions. .. Fusarium sp. from the citrus mealybug colony was handled similarly as above except the DNA was extracted using an AllPrep DNA/RNA/miRNA Universal Kit (Cat. No. 80224, Qiagen Sciences, Germantown, MD, USA) and purified using Diffinity RapidTip (Cat. No. RT025-008, Diffinity Genomics, Inc., West Henrietta, NY, USA).

    SYBR Green Assay:

    Article Title: Tryptophan Metabolism through the Kynurenine Pathway is Associated with Endoscopic Inflammation in Ulcerative Colitis
    Article Snippet: Tissue was homogenized with the use of a bullet blender (Next Advance) and extracted with an AllPrep DNA/RNA/ miRNA Universal Kit (Qiagen). .. Real-time quantitative polymerase chain reaction (qPCR) was performed with Fast SYBR Green Master Mix (Thermo Fisher Scientific) using primers from IDT in a Roche 480 LightCycler.

    RNA Extraction:

    Article Title: Mutant p53R270H drives altered metabolism and increased invasion in pancreatic ductal adenocarcinoma
    Article Snippet: Tissue from subcutaneous tumors was collected in lysis buffer for RNA extraction. .. RNA was isolated using the QIAGEN AllPrep DNA/RNA/miRNA Universal Kit according to the manufacturer’s instructions.

    Article Title: RNA Preservation Agents and Nucleic Acid Extraction Method Bias Perceived Bacterial Community Composition
    Article Snippet: Paragraph title: DNA and RNA extraction ... The combined extraction method was based on the AllPrep DNA/RNA/miRNA Universal kit protocol (Qiagen).

    RNA Expression:

    Article Title: Single-walled and multi-walled carbon nanotubes induce sequence-specific epigenetic alterations in 16 HBE cells
    Article Snippet: Global and sequence specific methylation were studied in all the concentrations (2.5, 5 and 25 μg/ml), while miRNA expression and RNA expression were studied for a single concentration (25 μg/ml). .. DNA, RNA and miRNA were extracted using AllPrep DNA/RNA/miRNA Universal Kit (QIAGEN, Belgium).

    Sample Prep:

    Article Title: Dwarfism and Altered Craniofacial Development in Rabbits Is Caused by a 12.1 kb Deletion at the HMGA2 Locus
    Article Snippet: Before library preparation, the entire embryo was homogenized and the RNA was extracted using the Allprep DNA/RNA/miRNA Universal kit (QIAGEN, Valencia, CA). .. Libraries were then prepared using the TruSeq Stranded mRNA (messenger RNA) Sample Preparation Kit (Illumina), and sequenced using 125 bp paired-end reads on an Illumina instrument.

    Spectrophotometry:

    Article Title: Epigenetic regulation of lateralized fetal spinal gene expression underlies hemispheric asymmetries
    Article Snippet: Assessment of RNA and DNA Total RNA including miRNA and DNA was extracted using the AllPrep DNA/RNA/miRNA Universal Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. .. Concentration and purity of RNA and DNA were determined photometrically (NanoDrop ND-1000 Spectrophotometer, Thermo Scientific, Waltham, Massachusetts).

    Article Title: Integrative microRNA and mRNA deep-sequencing expression profiling in endemic Burkitt lymphoma
    Article Snippet: RNA and small RNA isolation Total RNA and Small RNA molecules were extracted from eBL FNA samples in RNAlater using the AllPrep DNA/RNA/miRNA Universal kit (Qiagen) according to manufacturer’s instructions. .. Small RNA abundance and integrity were determined after isolation using Nanodrop-ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA), respectively.

    Article Title: Exercise training alters the genomic response to acute exercise in human adipose tissue
    Article Snippet: Genomic DNA and total RNAs were purified from adipose tissue biopsies with the AllPrep DNA/RNA/miRNA Universal Kit (Qiagen, Hilden, Germany). .. The quality of recovered DNA and RNA was assessed by the 260/280 ratio, determined by spectrophotometry with a NanoDrop 2000 (Thermo Fisher Scientific, Roskilde, Denmark), as well as using the Agilent RNA 6000 Nano Kit (Agilent Technologies, Glostrup, Denmark) for RNA.

    Concentration Assay:

    Article Title: Single-walled and multi-walled carbon nanotubes induce sequence-specific epigenetic alterations in 16 HBE cells
    Article Snippet: Global and sequence specific methylation were studied in all the concentrations (2.5, 5 and 25 μg/ml), while miRNA expression and RNA expression were studied for a single concentration (25 μg/ml). .. DNA, RNA and miRNA were extracted using AllPrep DNA/RNA/miRNA Universal Kit (QIAGEN, Belgium).

    Article Title: Epigenetic regulation of lateralized fetal spinal gene expression underlies hemispheric asymmetries
    Article Snippet: Assessment of RNA and DNA Total RNA including miRNA and DNA was extracted using the AllPrep DNA/RNA/miRNA Universal Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. .. Concentration and purity of RNA and DNA were determined photometrically (NanoDrop ND-1000 Spectrophotometer, Thermo Scientific, Waltham, Massachusetts).

    Article Title: A MBD-seq protocol for large-scale methylome-wide studies with (very) low amounts of DNA
    Article Snippet: Genomic DNA was extracted from the homogenate using the AllPrep DNA/RNA/miRNA Universal Kit (Qiagen, # 80224). .. Purity of extracted DNA was assessed by NanoDrop and concentration was measured using Qubit dsDNA HS Assay.

    Article Title: Integrative microRNA and mRNA deep-sequencing expression profiling in endemic Burkitt lymphoma
    Article Snippet: RNA and small RNA isolation Total RNA and Small RNA molecules were extracted from eBL FNA samples in RNAlater using the AllPrep DNA/RNA/miRNA Universal kit (Qiagen) according to manufacturer’s instructions. .. Only samples with a miRNA concentration > 10 ng/μl and total RNA RIN (RNA integrity number) > 8.0 were considered for small RNA library preparations and sequencing, as a result, of the 28 samples only 17 were considered for miRNA library preparation.

    Lysis:

    Article Title: Mutant p53R270H drives altered metabolism and increased invasion in pancreatic ductal adenocarcinoma
    Article Snippet: Tissue from subcutaneous tumors was collected in lysis buffer for RNA extraction. .. RNA was isolated using the QIAGEN AllPrep DNA/RNA/miRNA Universal Kit according to the manufacturer’s instructions.

    Article Title: RNA Preservation Agents and Nucleic Acid Extraction Method Bias Perceived Bacterial Community Composition
    Article Snippet: The combined extraction method was based on the AllPrep DNA/RNA/miRNA Universal kit protocol (Qiagen). .. A segment of the 142 mm filter was cut into < 0.5 cm2 pieces and added to a 2 ml centrifuge tube to which the lysis buffer was added, mixed and incubated at 37°C for 1 hr.

    Article Title: Comparison of Automated and Manual DNA Isolation Methods of Liquid-Based Cytology Samples
    Article Snippet: The pelleted cells were lysed by adding 600 μL of lysis buffer RLT Plus (Qiagen® , Hilden, Germany) (cells were counted to establish the amount of RLT Plus buffer) and homogenized for 30 seconds with a TissueRuptor™ (Qiagen) at medium velocity. .. Each sample was extracted by either a manual or an automated (Qiacube™; Qiagen, Germantown, MD) method available at the time, following in both cases the protocol of AllPrep DNA/RNA/microRNA Universal Kit™ (Qiagen) as indicated in , using 600 μL of homogenized lysate as a starting material.

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    Qiagen allprep dna rna mirna universal kit protocol
    Comparison of <t>DNA/RNA</t> yields and RNA quality (inset). (A) Comparison between the <t>AllPrep</t> standard protocol (APS; described below figure), enzymatic lysis (Enz), enzymatic/bead beating (Bead), and Mirvana extraction protocols (Mirvana). (B) Comparison between different iterations of the AP protocol, examining the effect of preservation method {P}, lysis conditions {L}, and other extraction modification steps {E}. The data presents changes in yield relative to a control, where each column shows the effect of one factor relative to a control that differed in that factor only. The control was extracted using either the AP standard protocol or a modification as indicated by the X-axis labels below the horizontal lines. The factor that was changed is indicated just below the data column. The dotted line indicates no change relative to control. All data represent averages of three extractions, with error bars indicating the 95% confidence interval, except for the effect of lysozyme, which averaged the relative yield effects from multiple samples (from DL, LH, and HR samples, three replicate extractions each). Inset: RIN = RNA integrity number quality (0 (poor)- 10 (high) range) assessment by Bioanalyzer; NT = no preservation treatment.
    Allprep Dna Rna Mirna Universal Kit Protocol, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Comparison of DNA/RNA yields and RNA quality (inset). (A) Comparison between the AllPrep standard protocol (APS; described below figure), enzymatic lysis (Enz), enzymatic/bead beating (Bead), and Mirvana extraction protocols (Mirvana). (B) Comparison between different iterations of the AP protocol, examining the effect of preservation method {P}, lysis conditions {L}, and other extraction modification steps {E}. The data presents changes in yield relative to a control, where each column shows the effect of one factor relative to a control that differed in that factor only. The control was extracted using either the AP standard protocol or a modification as indicated by the X-axis labels below the horizontal lines. The factor that was changed is indicated just below the data column. The dotted line indicates no change relative to control. All data represent averages of three extractions, with error bars indicating the 95% confidence interval, except for the effect of lysozyme, which averaged the relative yield effects from multiple samples (from DL, LH, and HR samples, three replicate extractions each). Inset: RIN = RNA integrity number quality (0 (poor)- 10 (high) range) assessment by Bioanalyzer; NT = no preservation treatment.

    Journal: PLoS ONE

    Article Title: RNA Preservation Agents and Nucleic Acid Extraction Method Bias Perceived Bacterial Community Composition

    doi: 10.1371/journal.pone.0121659

    Figure Lengend Snippet: Comparison of DNA/RNA yields and RNA quality (inset). (A) Comparison between the AllPrep standard protocol (APS; described below figure), enzymatic lysis (Enz), enzymatic/bead beating (Bead), and Mirvana extraction protocols (Mirvana). (B) Comparison between different iterations of the AP protocol, examining the effect of preservation method {P}, lysis conditions {L}, and other extraction modification steps {E}. The data presents changes in yield relative to a control, where each column shows the effect of one factor relative to a control that differed in that factor only. The control was extracted using either the AP standard protocol or a modification as indicated by the X-axis labels below the horizontal lines. The factor that was changed is indicated just below the data column. The dotted line indicates no change relative to control. All data represent averages of three extractions, with error bars indicating the 95% confidence interval, except for the effect of lysozyme, which averaged the relative yield effects from multiple samples (from DL, LH, and HR samples, three replicate extractions each). Inset: RIN = RNA integrity number quality (0 (poor)- 10 (high) range) assessment by Bioanalyzer; NT = no preservation treatment.

    Article Snippet: The combined extraction method was based on the AllPrep DNA/RNA/miRNA Universal kit protocol (Qiagen).

    Techniques: Lysis, Preserving, Modification

    Schematic representation of DNA procedure from LBC samples. Left side : manual procedure; middle : outline procedure; right side : automated procedure. RT = room temperature; EB = elution buffer; AW1 and AW2 = buffer concentrate provided from AllPrep DNA/RNA/miRNA Universal Kit. LBC, liquid-based cytology.

    Journal: Biopreservation and Biobanking

    Article Title: Comparison of Automated and Manual DNA Isolation Methods of Liquid-Based Cytology Samples

    doi: 10.1089/bio.2018.0148

    Figure Lengend Snippet: Schematic representation of DNA procedure from LBC samples. Left side : manual procedure; middle : outline procedure; right side : automated procedure. RT = room temperature; EB = elution buffer; AW1 and AW2 = buffer concentrate provided from AllPrep DNA/RNA/miRNA Universal Kit. LBC, liquid-based cytology.

    Article Snippet: Each sample was extracted by either a manual or an automated (Qiacube™; Qiagen, Germantown, MD) method available at the time, following in both cases the protocol of AllPrep DNA/RNA/microRNA Universal Kit™ (Qiagen) as indicated in , using 600 μL of homogenized lysate as a starting material.

    Techniques:

    Aberrant transcriptome expression pivotal to eBL lymphomagenesis. a Schematic illustration of the aberrant gene expression and miRNA mediated regulatory changes that would initiate lymphomagenesis as a result of DNA damage. Combined loss of p53 function due to small interfering RNA-mediated regulation of ATM and NLK together with upregulation of TFAP4, would facilitate survival of cells with the c-myc-Igh chromosomal translocation and MYC induced cell cycle progression initiating eBL tumor development. ATM checkpoint kinase, transduces genomic stress signals to halt cell cycle progression in response to DNA damage. It is critical in the regulation of apoptosis and lymphomagenesis in c-myc induced lymphomas. ATM is downregulated in eBL and it is targeted by 4 miRs that are Upregulated in eBL. NLK is required for the upregulation of P53 expression in response to DNA damage. It interacts with P53 to enhance its stability and activity by abrogating MDM2 mediated degradation. NLK is downregulated in eBL tumor cells and also targeted by 2 miRs that are upregulated in eBL tumor cells. TFAP4/AP4 is a central mediator of cell cycle progression in response to c-MYC activation. b RNA seq. Expression counts of MYC , TFAP4 , ATM and NLK in eBL tumor cells and GC B cells. c Hierarchical clustering of eBL and GC B cells based on the expression profiles of MYC , TFAP4 , ATM and NLK also revealed a clear separation of the two groups. d . miRNA seq. Expression counts of hsa-miR-26a-5p, hsa-miR-27b-3p, hsa-miR-30b-5p, miR-17~92-cluster members (hsa-miR-19b-3p, and hsa-miR-92a-3p), and let-7-family miRs (hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7d-5p, hsa-let-7e-5p, and hsa-let-7 g-5p) in eBL tumor cells and GC B cells

    Journal: BMC Cancer

    Article Title: Integrative microRNA and mRNA deep-sequencing expression profiling in endemic Burkitt lymphoma

    doi: 10.1186/s12885-017-3711-9

    Figure Lengend Snippet: Aberrant transcriptome expression pivotal to eBL lymphomagenesis. a Schematic illustration of the aberrant gene expression and miRNA mediated regulatory changes that would initiate lymphomagenesis as a result of DNA damage. Combined loss of p53 function due to small interfering RNA-mediated regulation of ATM and NLK together with upregulation of TFAP4, would facilitate survival of cells with the c-myc-Igh chromosomal translocation and MYC induced cell cycle progression initiating eBL tumor development. ATM checkpoint kinase, transduces genomic stress signals to halt cell cycle progression in response to DNA damage. It is critical in the regulation of apoptosis and lymphomagenesis in c-myc induced lymphomas. ATM is downregulated in eBL and it is targeted by 4 miRs that are Upregulated in eBL. NLK is required for the upregulation of P53 expression in response to DNA damage. It interacts with P53 to enhance its stability and activity by abrogating MDM2 mediated degradation. NLK is downregulated in eBL tumor cells and also targeted by 2 miRs that are upregulated in eBL tumor cells. TFAP4/AP4 is a central mediator of cell cycle progression in response to c-MYC activation. b RNA seq. Expression counts of MYC , TFAP4 , ATM and NLK in eBL tumor cells and GC B cells. c Hierarchical clustering of eBL and GC B cells based on the expression profiles of MYC , TFAP4 , ATM and NLK also revealed a clear separation of the two groups. d . miRNA seq. Expression counts of hsa-miR-26a-5p, hsa-miR-27b-3p, hsa-miR-30b-5p, miR-17~92-cluster members (hsa-miR-19b-3p, and hsa-miR-92a-3p), and let-7-family miRs (hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7d-5p, hsa-let-7e-5p, and hsa-let-7 g-5p) in eBL tumor cells and GC B cells

    Article Snippet: RNA and small RNA isolation Total RNA and Small RNA molecules were extracted from eBL FNA samples in RNAlater using the AllPrep DNA/RNA/miRNA Universal kit (Qiagen) according to manufacturer’s instructions.

    Techniques: Expressing, Small Interfering RNA, Translocation Assay, Activity Assay, Activation Assay, RNA Sequencing Assay

    Epigenetic regulation of gene expression asymmetries in human fetal spinal cord. ( A ) Asymmetrically expressed miRNA transcripts at 8, 10 and 12 weeks PC. The extent of expression asymmetries is measured in log 2 (fold change). Red bars show rightward asymmetrically expressed microRNA transcripts, blue bars show leftward asymmetrically expressed miRNA transcripts. ( B ) Number of CpG sites showing differential DNA methylation per chromosome, compared between the left and right spinal cord for 8 and 10 weeks PC. Depicted are only CpG sites with methylation asymmetries in both samples. Red bars represent the number of CpG sites that showed significantly higher DNA methylation on the right side, blue bars show the number of CpG sites that showed significantly more DNA methylation on the left side. ( C ) Percentage of differential DNA methylation in leftward (blue) and rightward (red) asymmetrically methylated CpG sites as a function of p-value. ( D ) Percentage of gene expression asymmetries on each chromosome at 8 weeks PC that can be explained by regulation via asymmetrically expressed miRNAs or asymmetric DNA methylation of CpG sites within and 1500 nucleotides upstream of the expressed genes. The source files of asymmetrically expressed miRNAs, asymmetrically expressed targets of miRNAs, enriched KEGG pathways and differentially methylated CpG sites are available in Figure 3—source data 1 , Figure 3—source data 2 , Figure 3—source data 3 , and Figure 3—source data 4 respectively. DOI: http://dx.doi.org/10.7554/eLife.22784.006 10.7554/eLife.22784.007 Asymmetrically expressed miRNAs per week. DOI: http://dx.doi.org/10.7554/eLife.22784.007 10.7554/eLife.22784.008 Asymmetrically expressed RNA targets of asymmetrically expressed miRNAs per week. DOI: http://dx.doi.org/10.7554/eLife.22784.008 10.7554/eLife.22784.009 Enriched KEGG pathways per week and side of the spinal cord. DOI: http://dx.doi.org/10.7554/eLife.22784.009 10.7554/eLife.22784.010 Asymmetrically methylated CpG sites per week and side of the spinal cord. DOI: http://dx.doi.org/10.7554/eLife.22784.010

    Journal: eLife

    Article Title: Epigenetic regulation of lateralized fetal spinal gene expression underlies hemispheric asymmetries

    doi: 10.7554/eLife.22784

    Figure Lengend Snippet: Epigenetic regulation of gene expression asymmetries in human fetal spinal cord. ( A ) Asymmetrically expressed miRNA transcripts at 8, 10 and 12 weeks PC. The extent of expression asymmetries is measured in log 2 (fold change). Red bars show rightward asymmetrically expressed microRNA transcripts, blue bars show leftward asymmetrically expressed miRNA transcripts. ( B ) Number of CpG sites showing differential DNA methylation per chromosome, compared between the left and right spinal cord for 8 and 10 weeks PC. Depicted are only CpG sites with methylation asymmetries in both samples. Red bars represent the number of CpG sites that showed significantly higher DNA methylation on the right side, blue bars show the number of CpG sites that showed significantly more DNA methylation on the left side. ( C ) Percentage of differential DNA methylation in leftward (blue) and rightward (red) asymmetrically methylated CpG sites as a function of p-value. ( D ) Percentage of gene expression asymmetries on each chromosome at 8 weeks PC that can be explained by regulation via asymmetrically expressed miRNAs or asymmetric DNA methylation of CpG sites within and 1500 nucleotides upstream of the expressed genes. The source files of asymmetrically expressed miRNAs, asymmetrically expressed targets of miRNAs, enriched KEGG pathways and differentially methylated CpG sites are available in Figure 3—source data 1 , Figure 3—source data 2 , Figure 3—source data 3 , and Figure 3—source data 4 respectively. DOI: http://dx.doi.org/10.7554/eLife.22784.006 10.7554/eLife.22784.007 Asymmetrically expressed miRNAs per week. DOI: http://dx.doi.org/10.7554/eLife.22784.007 10.7554/eLife.22784.008 Asymmetrically expressed RNA targets of asymmetrically expressed miRNAs per week. DOI: http://dx.doi.org/10.7554/eLife.22784.008 10.7554/eLife.22784.009 Enriched KEGG pathways per week and side of the spinal cord. DOI: http://dx.doi.org/10.7554/eLife.22784.009 10.7554/eLife.22784.010 Asymmetrically methylated CpG sites per week and side of the spinal cord. DOI: http://dx.doi.org/10.7554/eLife.22784.010

    Article Snippet: Assessment of RNA and DNA Total RNA including miRNA and DNA was extracted using the AllPrep DNA/RNA/miRNA Universal Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.

    Techniques: Expressing, DNA Methylation Assay, Methylation