dna methylation analysis genomic dna  (Qiagen)

 
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    AllPrep DNA RNA Mini Kit
    Description:
    For simultaneous purification of DNA and RNA from cells and tissues Kit contents Qiagen AllPrep DNA RNA Mini Kit 50 preps 30mg Sample 100L Elution Volume Silica Technology Spin Column Format Manual Processing Genomic DNA Total RNA Purification 35 min Time Run Ideal for PCR Real time PCR Microarray Blotting For Simultaneous Purification of DNA and RNA from Cells and Tissues Includes AllPrep DNA Spin Columns RNeasy Mini Spin Columns Collection Tubes RNase free Water and Buffers Benefits High quality DNA and RNA from the same sample Maximal yields of DNA and RNA from precious samples Rapid purification with short streamlined protocol Ready to use DNA and RNA for any downstream analysis
    Catalog Number:
    80204
    Price:
    541
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    AllPrep DNA RNA Mini Kit
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    Structured Review

    Qiagen dna methylation analysis genomic dna
    AllPrep DNA RNA Mini Kit
    For simultaneous purification of DNA and RNA from cells and tissues Kit contents Qiagen AllPrep DNA RNA Mini Kit 50 preps 30mg Sample 100L Elution Volume Silica Technology Spin Column Format Manual Processing Genomic DNA Total RNA Purification 35 min Time Run Ideal for PCR Real time PCR Microarray Blotting For Simultaneous Purification of DNA and RNA from Cells and Tissues Includes AllPrep DNA Spin Columns RNeasy Mini Spin Columns Collection Tubes RNase free Water and Buffers Benefits High quality DNA and RNA from the same sample Maximal yields of DNA and RNA from precious samples Rapid purification with short streamlined protocol Ready to use DNA and RNA for any downstream analysis
    https://www.bioz.com/result/dna methylation analysis genomic dna/product/Qiagen
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    Price from $9.99 to $1999.99
    dna methylation analysis genomic dna - by Bioz Stars, 2020-07
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    Images

    1) Product Images from "Maternal overnutrition programs epigenetic changes in the regulatory regions of hypothalamic Pomc in the offspring of rats"

    Article Title: Maternal overnutrition programs epigenetic changes in the regulatory regions of hypothalamic Pomc in the offspring of rats

    Journal: International Journal of Obesity (2005)

    doi: 10.1038/s41366-018-0094-1

    Gene expression and hypothalamic Pomc DNA methylation changes in offspring at weaning. a mRNA expression levels of Pomc , Agrp , Npy , and b Ob-Rb in the ARC. c Relative mRNA levels of Mc4r and Npy1r in the PVN analyzed by qRT-PCR in the 3-week-old offspring of LF- or HF-fed dams (Student’s t -test, n = 8). d Map of the Pro-opiomelanocortin ( Pomc ) gene promoter and enhancer region including functional regulatory elements and CpG dinucleotides (red lines). e Methylation analyzes of hypothalamic Pomc promoter (− 150 bp to transcription start site [TSS]) (Student’s t -test, D-LF, n = 6; D-HF, n = 7) and f , g of neuronal Pomc enhancer region 1 and 2 in the offspring of LF- or HF-fed mothers at 3 weeks of age (Student’s t -test, n = 8). Data are shown as mean ± SEM. * p
    Figure Legend Snippet: Gene expression and hypothalamic Pomc DNA methylation changes in offspring at weaning. a mRNA expression levels of Pomc , Agrp , Npy , and b Ob-Rb in the ARC. c Relative mRNA levels of Mc4r and Npy1r in the PVN analyzed by qRT-PCR in the 3-week-old offspring of LF- or HF-fed dams (Student’s t -test, n = 8). d Map of the Pro-opiomelanocortin ( Pomc ) gene promoter and enhancer region including functional regulatory elements and CpG dinucleotides (red lines). e Methylation analyzes of hypothalamic Pomc promoter (− 150 bp to transcription start site [TSS]) (Student’s t -test, D-LF, n = 6; D-HF, n = 7) and f , g of neuronal Pomc enhancer region 1 and 2 in the offspring of LF- or HF-fed mothers at 3 weeks of age (Student’s t -test, n = 8). Data are shown as mean ± SEM. * p

    Techniques Used: Expressing, DNA Methylation Assay, Quantitative RT-PCR, Functional Assay, Methylation

    2) Product Images from "Generation of minipigs with targeted transgene insertion by recombinase-mediated cassette exchange (RMCE) and somatic cell nuclear transfer (SCNT)"

    Article Title: Generation of minipigs with targeted transgene insertion by recombinase-mediated cassette exchange (RMCE) and somatic cell nuclear transfer (SCNT)

    Journal: Transgenic Research

    doi: 10.1007/s11248-012-9671-6

    Generation of live born PSEN1M146I RMCE piglets. a Piglets generated by RMCE and SCNT. Four of 20 live born piglets are shown. b Southern blot analysis of genomic DNA isolated from 16 RMCE piglets and pig #2772 digested with SpeI . A 670-bp Neo r fragment was used as probe. Lanes 1–16 represent 16 RMCE piglets, lane 17 pig #2772, lane 18 wt pig, lane 19 wt pig DNA mixed with PSEN1M146I plasmid DNA, and lane 20 molecular weight marker. The blue arrow marks the band in pig #2772 absent in RMCE piglets. c Southern blot analyses as in b except for the use of a PSEN1M146I probe. The blue arrow marks the PSEN1M146I transgene present in RMCE piglets. Three other bands present in the wt pig and pig #2772 are marked with black arrows (endogenous PSEN1 ). Positive control band ( lane 19 ) is marked with a black triangle . d Top panel Schematic drawing of the RMCE targeted acceptor locus B. Black arrows indicate positions of primers used to reveal RMCE. f Arrow marks the forward genomic primer upstream of LIR and x marks the reverse primer specific of either PSEN1M146I or GFP. Lower panel PCR on genomic DNA from two RMCE piglets (lanes 1, 2, 5, and 6) and pig #2772 ( lanes 3 and 7 ). Lanes 4 and 8 are water controls. f Primer was used with primer x, PSEN1M146I or GFP, in lanes 1–4 and 5–8, respectively. M is a 1 kb ladder. e Expression of bi-cistronic PSEN1M146I -IRES- Pac mRNA in fibroblasts of five RMCE piglets. Lanes 1–5 PCR on cDNA synthesized from fibroblast RNA, lanes 6–10 control PCR on –RT templates, lane 11 water control (W), and lane 12 positive control (P). M, 0.1 kb ladder
    Figure Legend Snippet: Generation of live born PSEN1M146I RMCE piglets. a Piglets generated by RMCE and SCNT. Four of 20 live born piglets are shown. b Southern blot analysis of genomic DNA isolated from 16 RMCE piglets and pig #2772 digested with SpeI . A 670-bp Neo r fragment was used as probe. Lanes 1–16 represent 16 RMCE piglets, lane 17 pig #2772, lane 18 wt pig, lane 19 wt pig DNA mixed with PSEN1M146I plasmid DNA, and lane 20 molecular weight marker. The blue arrow marks the band in pig #2772 absent in RMCE piglets. c Southern blot analyses as in b except for the use of a PSEN1M146I probe. The blue arrow marks the PSEN1M146I transgene present in RMCE piglets. Three other bands present in the wt pig and pig #2772 are marked with black arrows (endogenous PSEN1 ). Positive control band ( lane 19 ) is marked with a black triangle . d Top panel Schematic drawing of the RMCE targeted acceptor locus B. Black arrows indicate positions of primers used to reveal RMCE. f Arrow marks the forward genomic primer upstream of LIR and x marks the reverse primer specific of either PSEN1M146I or GFP. Lower panel PCR on genomic DNA from two RMCE piglets (lanes 1, 2, 5, and 6) and pig #2772 ( lanes 3 and 7 ). Lanes 4 and 8 are water controls. f Primer was used with primer x, PSEN1M146I or GFP, in lanes 1–4 and 5–8, respectively. M is a 1 kb ladder. e Expression of bi-cistronic PSEN1M146I -IRES- Pac mRNA in fibroblasts of five RMCE piglets. Lanes 1–5 PCR on cDNA synthesized from fibroblast RNA, lanes 6–10 control PCR on –RT templates, lane 11 water control (W), and lane 12 positive control (P). M, 0.1 kb ladder

    Techniques Used: Generated, Southern Blot, Isolation, Plasmid Preparation, Molecular Weight, Marker, Positive Control, Polymerase Chain Reaction, Expressing, Synthesized

    3) Product Images from "Molecular Cloning, Characterization and Predicted Structure of a Putative Copper-Zinc SOD from the Camel, Camelus dromedarius"

    Article Title: Molecular Cloning, Characterization and Predicted Structure of a Putative Copper-Zinc SOD from the Camel, Camelus dromedarius

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms13010879

    Expression of SOD1 using Real time PCR and cDNA from different camel tissues. The results are expressed relative to liver as calibrator and using 18S ribosomal subunit as housekeeping gene.
    Figure Legend Snippet: Expression of SOD1 using Real time PCR and cDNA from different camel tissues. The results are expressed relative to liver as calibrator and using 18S ribosomal subunit as housekeeping gene.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    4) Product Images from "Tissue-Specific Stem Cells Obtained by Reprogramming of Non-Obese Diabetic (NOD) Mouse-Derived Pancreatic Cells Confer Insulin Production in Response to Glucose"

    Article Title: Tissue-Specific Stem Cells Obtained by Reprogramming of Non-Obese Diabetic (NOD) Mouse-Derived Pancreatic Cells Confer Insulin Production in Response to Glucose

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0163580

    Expression analysis in iTS-P. (A) RT-PCR analysis for mRNA expression of pluripotency-related markers (Oct3/4, Sox2, Klf4, c-Myc, Esg1, and Rex1) and the pancreas-related marker (Pdx1) in the pancreatic tissue of NOD mice (Panc), the ES cells (ES), and the iTS-P lines (4–2, 4–3, 4–4, 4–7, 4–9 and 4–11). Abbreviations: Oct3/4, octamer-binding transcription factor 3/4; Sox2, SRY (sex determining region Y)-box 2; Klf4, Kruppel-like factor 4; c-Myc, proto-oncogene for avian myelocytomatosis viral oncogene homolog; Esg1, embryonic stem cell-specific gene 1; Rex1, RNA exonuclease 1 homolog; Pdx1, pancreatic and duodenal homeobox 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B) PCR analysis of genomic DNA isolated from the pancreatic tissue of NOD mice (Panc), ES cells (ES), iTS-P lines (4–2, 4–3, 4–4, 4–7, 4–9, and 4–11) to detect the presence of FUW-OSKM integrated into the chromosomes of iTS-P lines. Primers 2 (O-1), 3 (O-2), and 4 (K) correspond to the cDNA for each protein in FUW-OSKM ( Fig 1A and S1 Table ). When genomic PCR is performed using these primers, the size of the amplified endogenous gene (Endo; shown by open arrowheads) is always larger than that of the cDNA (Tg; shown by solid arrowheads) in FUW-OSKM, since the former products contain intronic sequences. Since primers 1 and 5 are specific to FUW-OSKM, the samples showing amplification with these primers are thought to be the ones carrying FUW-OSKM in their genome. Lane OSKM shows FUW-OSKM plasmid (~10 ng) amplified as a positive control.
    Figure Legend Snippet: Expression analysis in iTS-P. (A) RT-PCR analysis for mRNA expression of pluripotency-related markers (Oct3/4, Sox2, Klf4, c-Myc, Esg1, and Rex1) and the pancreas-related marker (Pdx1) in the pancreatic tissue of NOD mice (Panc), the ES cells (ES), and the iTS-P lines (4–2, 4–3, 4–4, 4–7, 4–9 and 4–11). Abbreviations: Oct3/4, octamer-binding transcription factor 3/4; Sox2, SRY (sex determining region Y)-box 2; Klf4, Kruppel-like factor 4; c-Myc, proto-oncogene for avian myelocytomatosis viral oncogene homolog; Esg1, embryonic stem cell-specific gene 1; Rex1, RNA exonuclease 1 homolog; Pdx1, pancreatic and duodenal homeobox 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B) PCR analysis of genomic DNA isolated from the pancreatic tissue of NOD mice (Panc), ES cells (ES), iTS-P lines (4–2, 4–3, 4–4, 4–7, 4–9, and 4–11) to detect the presence of FUW-OSKM integrated into the chromosomes of iTS-P lines. Primers 2 (O-1), 3 (O-2), and 4 (K) correspond to the cDNA for each protein in FUW-OSKM ( Fig 1A and S1 Table ). When genomic PCR is performed using these primers, the size of the amplified endogenous gene (Endo; shown by open arrowheads) is always larger than that of the cDNA (Tg; shown by solid arrowheads) in FUW-OSKM, since the former products contain intronic sequences. Since primers 1 and 5 are specific to FUW-OSKM, the samples showing amplification with these primers are thought to be the ones carrying FUW-OSKM in their genome. Lane OSKM shows FUW-OSKM plasmid (~10 ng) amplified as a positive control.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Marker, Mouse Assay, Binding Assay, Polymerase Chain Reaction, Isolation, Amplification, Plasmid Preparation, Positive Control

    5) Product Images from "Tissue-Specific Stem Cells Obtained by Reprogramming of Non-Obese Diabetic (NOD) Mouse-Derived Pancreatic Cells Confer Insulin Production in Response to Glucose"

    Article Title: Tissue-Specific Stem Cells Obtained by Reprogramming of Non-Obese Diabetic (NOD) Mouse-Derived Pancreatic Cells Confer Insulin Production in Response to Glucose

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0163580

    Expression analysis in iTS-P. (A) RT-PCR analysis for mRNA expression of pluripotency-related markers (Oct3/4, Sox2, Klf4, c-Myc, Esg1, and Rex1) and the pancreas-related marker (Pdx1) in the pancreatic tissue of NOD mice (Panc), the ES cells (ES), and the iTS-P lines (4–2, 4–3, 4–4, 4–7, 4–9 and 4–11). Abbreviations: Oct3/4, octamer-binding transcription factor 3/4; Sox2, SRY (sex determining region Y)-box 2; Klf4, Kruppel-like factor 4; c-Myc, proto-oncogene for avian myelocytomatosis viral oncogene homolog; Esg1, embryonic stem cell-specific gene 1; Rex1, RNA exonuclease 1 homolog; Pdx1, pancreatic and duodenal homeobox 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B) PCR analysis of genomic DNA isolated from the pancreatic tissue of NOD mice (Panc), ES cells (ES), iTS-P lines (4–2, 4–3, 4–4, 4–7, 4–9, and 4–11) to detect the presence of FUW-OSKM integrated into the chromosomes of iTS-P lines. Primers 2 (O-1), 3 (O-2), and 4 (K) correspond to the cDNA for each protein in FUW-OSKM ( Fig 1A and S1 Table ). When genomic PCR is performed using these primers, the size of the amplified endogenous gene (Endo; shown by open arrowheads) is always larger than that of the cDNA (Tg; shown by solid arrowheads) in FUW-OSKM, since the former products contain intronic sequences. Since primers 1 and 5 are specific to FUW-OSKM, the samples showing amplification with these primers are thought to be the ones carrying FUW-OSKM in their genome. Lane OSKM shows FUW-OSKM plasmid (~10 ng) amplified as a positive control.
    Figure Legend Snippet: Expression analysis in iTS-P. (A) RT-PCR analysis for mRNA expression of pluripotency-related markers (Oct3/4, Sox2, Klf4, c-Myc, Esg1, and Rex1) and the pancreas-related marker (Pdx1) in the pancreatic tissue of NOD mice (Panc), the ES cells (ES), and the iTS-P lines (4–2, 4–3, 4–4, 4–7, 4–9 and 4–11). Abbreviations: Oct3/4, octamer-binding transcription factor 3/4; Sox2, SRY (sex determining region Y)-box 2; Klf4, Kruppel-like factor 4; c-Myc, proto-oncogene for avian myelocytomatosis viral oncogene homolog; Esg1, embryonic stem cell-specific gene 1; Rex1, RNA exonuclease 1 homolog; Pdx1, pancreatic and duodenal homeobox 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B) PCR analysis of genomic DNA isolated from the pancreatic tissue of NOD mice (Panc), ES cells (ES), iTS-P lines (4–2, 4–3, 4–4, 4–7, 4–9, and 4–11) to detect the presence of FUW-OSKM integrated into the chromosomes of iTS-P lines. Primers 2 (O-1), 3 (O-2), and 4 (K) correspond to the cDNA for each protein in FUW-OSKM ( Fig 1A and S1 Table ). When genomic PCR is performed using these primers, the size of the amplified endogenous gene (Endo; shown by open arrowheads) is always larger than that of the cDNA (Tg; shown by solid arrowheads) in FUW-OSKM, since the former products contain intronic sequences. Since primers 1 and 5 are specific to FUW-OSKM, the samples showing amplification with these primers are thought to be the ones carrying FUW-OSKM in their genome. Lane OSKM shows FUW-OSKM plasmid (~10 ng) amplified as a positive control.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Marker, Mouse Assay, Binding Assay, Polymerase Chain Reaction, Isolation, Amplification, Plasmid Preparation, Positive Control

    6) Product Images from "Increasing quality, throughput and speed of sample preparation for strand-specific messenger RNA sequencing"

    Article Title: Increasing quality, throughput and speed of sample preparation for strand-specific messenger RNA sequencing

    Journal: BMC Genomics

    doi: 10.1186/s12864-017-3900-6

    Bead binding time point analysis. a Pre-PCR assessment. Various gDNA input amounts (X-axis) were used and libraries were made where the binding time for each of the bead cleanups was varied. The cumulative effect after all cleanups up to the point of post-ligation cleanups is shown. The purified ligated DNA was measured using a Qubit HS assay. The values from this assay were normalized to that of the 15 min condition. b Post-PCR assessment. As in ( a ) but purified DNA was measured after PCR enrichment. i.e. after additional two post-PCR bead-based purifications. n = 3; error bars = Standard Deviation. * P
    Figure Legend Snippet: Bead binding time point analysis. a Pre-PCR assessment. Various gDNA input amounts (X-axis) were used and libraries were made where the binding time for each of the bead cleanups was varied. The cumulative effect after all cleanups up to the point of post-ligation cleanups is shown. The purified ligated DNA was measured using a Qubit HS assay. The values from this assay were normalized to that of the 15 min condition. b Post-PCR assessment. As in ( a ) but purified DNA was measured after PCR enrichment. i.e. after additional two post-PCR bead-based purifications. n = 3; error bars = Standard Deviation. * P

    Techniques Used: Binding Assay, Polymerase Chain Reaction, Ligation, Purification, Standard Deviation

    7) Product Images from "Chromatin maturation of the HIV-1 provirus in primary resting CD4+ T cells"

    Article Title: Chromatin maturation of the HIV-1 provirus in primary resting CD4+ T cells

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008264

    Primary cells to study long-term HIV-1 latency establishment. ( A ) Schematic of the generation of HIV-1 carrying Bcl2 model cells. ( B ). ddPCR scatterplots of HIV-1 infected Bcl2-cells from three HIV-negative donors with probes against env (y-axis) and gag (x-axis). DNA isolated at 3 dpi. ( C ) Quantification of ddPCR results over three probes ( 5´LTR , gag and env ) in control cell line J-lat 5A8, and primary cells from the three donors in biological duplicate, either with both Bcl2 and HIV-1 or Bcl2-only. The data from individual donors and duplicates are visualized by differently shaped points. ( D ) Ratios of gag / env ddPCR signals to estimate internal 5´and 3´ HIV-1 deletions in the three donors and the control cell line J-lat 5A8. ( E ) Fraction of “rain”, i.e. low env signals (approximately below 10,000 a.u.) reflecting APOBEC3G-induced hypermutations. ( F ) RT-ddPCR results of cell-associated RNA isolated at 3 dpi ( n = 2). Probes were as in previous results. *p
    Figure Legend Snippet: Primary cells to study long-term HIV-1 latency establishment. ( A ) Schematic of the generation of HIV-1 carrying Bcl2 model cells. ( B ). ddPCR scatterplots of HIV-1 infected Bcl2-cells from three HIV-negative donors with probes against env (y-axis) and gag (x-axis). DNA isolated at 3 dpi. ( C ) Quantification of ddPCR results over three probes ( 5´LTR , gag and env ) in control cell line J-lat 5A8, and primary cells from the three donors in biological duplicate, either with both Bcl2 and HIV-1 or Bcl2-only. The data from individual donors and duplicates are visualized by differently shaped points. ( D ) Ratios of gag / env ddPCR signals to estimate internal 5´and 3´ HIV-1 deletions in the three donors and the control cell line J-lat 5A8. ( E ) Fraction of “rain”, i.e. low env signals (approximately below 10,000 a.u.) reflecting APOBEC3G-induced hypermutations. ( F ) RT-ddPCR results of cell-associated RNA isolated at 3 dpi ( n = 2). Probes were as in previous results. *p

    Techniques Used: Infection, Isolation

    8) Product Images from "G9a/GLP Histone Lysine Dimethyltransferase Complex Activity in the Hippocampus and the Entorhinal Cortex is Required for Gene Activation and Silencing during Memory Consolidation"

    Article Title: G9a/GLP Histone Lysine Dimethyltransferase Complex Activity in the Hippocampus and the Entorhinal Cortex is Required for Gene Activation and Silencing during Memory Consolidation

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.0147-12.2012

    G9a/GLP inhibition in the EC alters histone lysine methylation in the EC and area CA1 after fear conditioning
    Figure Legend Snippet: G9a/GLP inhibition in the EC alters histone lysine methylation in the EC and area CA1 after fear conditioning

    Techniques Used: Inhibition, Methylation

    G9a/GLP inhibition in the EC alters gene expression in area CA1 after fear conditioning
    Figure Legend Snippet: G9a/GLP inhibition in the EC alters gene expression in area CA1 after fear conditioning

    Techniques Used: Inhibition, Expressing

    9) Product Images from "Simultaneous purification of DNA and RNA from microbiota in a single colonic mucosal biopsy"

    Article Title: Simultaneous purification of DNA and RNA from microbiota in a single colonic mucosal biopsy

    Journal: BMC Research Notes

    doi: 10.1186/s13104-016-2110-7

    Comparison on taxonomic level of DNA purification using protocol 1 and commonly used DNA purification methods. The 16S sequence comparison bar charts are made using Classifier ( http://rdp.cme.msu.edu/classifier/classifier.jsp ). The significant differences of Firmicutes between paired libraries were calculated by the Library Compare Tool using a confidence threshold of 80 % ( http://rdp.cme.msu.edu/comparison ). Protocol 1: Mechanical pre-treatment only, followed by purification with AllPrep DNA/RNA Mini Kit as described in Additional file 1 ; DNA kit 1, 2 and 3: A combination of mechanical and enzymatic pre-treatments as recommended by Qiagen for lysis of Gram-positive bacteria, followed by purificaton with AllPrep DNA/RNA Mini Kit (kit 1), QIAamp DNA Stool Mini Kit (kit 2) and DNeasy Blood Tissue Kit (kit 3) as described in Additional file 1 . n number of clones sequenced
    Figure Legend Snippet: Comparison on taxonomic level of DNA purification using protocol 1 and commonly used DNA purification methods. The 16S sequence comparison bar charts are made using Classifier ( http://rdp.cme.msu.edu/classifier/classifier.jsp ). The significant differences of Firmicutes between paired libraries were calculated by the Library Compare Tool using a confidence threshold of 80 % ( http://rdp.cme.msu.edu/comparison ). Protocol 1: Mechanical pre-treatment only, followed by purification with AllPrep DNA/RNA Mini Kit as described in Additional file 1 ; DNA kit 1, 2 and 3: A combination of mechanical and enzymatic pre-treatments as recommended by Qiagen for lysis of Gram-positive bacteria, followed by purificaton with AllPrep DNA/RNA Mini Kit (kit 1), QIAamp DNA Stool Mini Kit (kit 2) and DNeasy Blood Tissue Kit (kit 3) as described in Additional file 1 . n number of clones sequenced

    Techniques Used: DNA Purification, Sequencing, Purification, Lysis, Clone Assay

    10) Product Images from "Simultaneous purification of DNA and RNA from microbiota in a single colonic mucosal biopsy"

    Article Title: Simultaneous purification of DNA and RNA from microbiota in a single colonic mucosal biopsy

    Journal: BMC Research Notes

    doi: 10.1186/s13104-016-2110-7

    Comparison on taxonomic level of DNA purification using protocol 1 and commonly used DNA purification methods. The 16S sequence comparison bar charts are made using Classifier ( http://rdp.cme.msu.edu/classifier/classifier.jsp ). The significant differences of Firmicutes between paired libraries were calculated by the Library Compare Tool using a confidence threshold of 80 % ( http://rdp.cme.msu.edu/comparison ). Protocol 1: Mechanical pre-treatment only, followed by purification with AllPrep DNA/RNA Mini Kit as described in Additional file 1 ; DNA kit 1, 2 and 3: A combination of mechanical and enzymatic pre-treatments as recommended by Qiagen for lysis of Gram-positive bacteria, followed by purificaton with AllPrep DNA/RNA Mini Kit (kit 1), QIAamp DNA Stool Mini Kit (kit 2) and DNeasy Blood Tissue Kit (kit 3) as described in Additional file 1 . n number of clones sequenced
    Figure Legend Snippet: Comparison on taxonomic level of DNA purification using protocol 1 and commonly used DNA purification methods. The 16S sequence comparison bar charts are made using Classifier ( http://rdp.cme.msu.edu/classifier/classifier.jsp ). The significant differences of Firmicutes between paired libraries were calculated by the Library Compare Tool using a confidence threshold of 80 % ( http://rdp.cme.msu.edu/comparison ). Protocol 1: Mechanical pre-treatment only, followed by purification with AllPrep DNA/RNA Mini Kit as described in Additional file 1 ; DNA kit 1, 2 and 3: A combination of mechanical and enzymatic pre-treatments as recommended by Qiagen for lysis of Gram-positive bacteria, followed by purificaton with AllPrep DNA/RNA Mini Kit (kit 1), QIAamp DNA Stool Mini Kit (kit 2) and DNeasy Blood Tissue Kit (kit 3) as described in Additional file 1 . n number of clones sequenced

    Techniques Used: DNA Purification, Sequencing, Purification, Lysis, Clone Assay

    11) Product Images from "Simultaneous purification of DNA and RNA from microbiota in a single colonic mucosal biopsy"

    Article Title: Simultaneous purification of DNA and RNA from microbiota in a single colonic mucosal biopsy

    Journal: BMC Research Notes

    doi: 10.1186/s13104-016-2110-7

    Comparison on taxonomic level of DNA purification using protocol 1 and commonly used DNA purification methods. The 16S sequence comparison bar charts are made using Classifier ( http://rdp.cme.msu.edu/classifier/classifier.jsp ). The significant differences of Firmicutes between paired libraries were calculated by the Library Compare Tool using a confidence threshold of 80 % ( http://rdp.cme.msu.edu/comparison ). Protocol 1: Mechanical pre-treatment only, followed by purification with AllPrep DNA/RNA Mini Kit as described in Additional file 1 ; DNA kit 1, 2 and 3: A combination of mechanical and enzymatic pre-treatments as recommended by Qiagen for lysis of Gram-positive bacteria, followed by purificaton with AllPrep DNA/RNA Mini Kit (kit 1), QIAamp DNA Stool Mini Kit (kit 2) and DNeasy Blood Tissue Kit (kit 3) as described in Additional file 1 . n number of clones sequenced
    Figure Legend Snippet: Comparison on taxonomic level of DNA purification using protocol 1 and commonly used DNA purification methods. The 16S sequence comparison bar charts are made using Classifier ( http://rdp.cme.msu.edu/classifier/classifier.jsp ). The significant differences of Firmicutes between paired libraries were calculated by the Library Compare Tool using a confidence threshold of 80 % ( http://rdp.cme.msu.edu/comparison ). Protocol 1: Mechanical pre-treatment only, followed by purification with AllPrep DNA/RNA Mini Kit as described in Additional file 1 ; DNA kit 1, 2 and 3: A combination of mechanical and enzymatic pre-treatments as recommended by Qiagen for lysis of Gram-positive bacteria, followed by purificaton with AllPrep DNA/RNA Mini Kit (kit 1), QIAamp DNA Stool Mini Kit (kit 2) and DNeasy Blood Tissue Kit (kit 3) as described in Additional file 1 . n number of clones sequenced

    Techniques Used: DNA Purification, Sequencing, Purification, Lysis, Clone Assay

    12) Product Images from "Association between TLR-9 polymorphisms and colon cancer susceptibility in Saudi Arabian female patients"

    Article Title: Association between TLR-9 polymorphisms and colon cancer susceptibility in Saudi Arabian female patients

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S106024

    TLR-9 mRNA expression in colon cancer cells and colon cancer tissues. Notes: Total RNA of tissues was extracted from matching normal and colon cancer tissues, reverse-transcribed into cDNA, and then used to measure TLR-9 mRNA expression with specific primers. TLR-9 expression in colon cancer tissues and matching control tissues is shown as mean ± SD. Abbreviations: cDNA, complementary DNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
    Figure Legend Snippet: TLR-9 mRNA expression in colon cancer cells and colon cancer tissues. Notes: Total RNA of tissues was extracted from matching normal and colon cancer tissues, reverse-transcribed into cDNA, and then used to measure TLR-9 mRNA expression with specific primers. TLR-9 expression in colon cancer tissues and matching control tissues is shown as mean ± SD. Abbreviations: cDNA, complementary DNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

    Techniques Used: Expressing

    13) Product Images from "Clinical and Mucosal Immune Correlates of HIV-1 Semen Levels in Antiretroviral-Naive Men"

    Article Title: Clinical and Mucosal Immune Correlates of HIV-1 Semen Levels in Antiretroviral-Naive Men

    Journal: Open Forum Infectious Diseases

    doi: 10.1093/ofid/ofx033

    Reactivation of semen herepesviruses and the semen HIV viral load. Semen HIV RNA levels in ARV-Naive men were associated with levels of (A) semen CMV DNA (left) but not (B) semen EBV DNA (right).
    Figure Legend Snippet: Reactivation of semen herepesviruses and the semen HIV viral load. Semen HIV RNA levels in ARV-Naive men were associated with levels of (A) semen CMV DNA (left) but not (B) semen EBV DNA (right).

    Techniques Used:

    14) Product Images from "Increased HIV-1 transcriptional activity and infectious burden in peripheral blood and gut-associated CD4+ T cells expressing CD30"

    Article Title: Increased HIV-1 transcriptional activity and infectious burden in peripheral blood and gut-associated CD4+ T cells expressing CD30

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006856

    HIV-1 is enriched in CD30 expressing CD4 + T cells. Cell-associated HIV-1 unspliced RNA (A) and DNA (B) in sorted CD4 + T cell populations, in samples from ART suppressed (n = 17) and viremic donors (n = 9) are shown. HIV-1 RNA is significantly enriched in CD30 expressing cells from ART suppressed donors (p = 0.008) and viremic donors (p = 0.007). (C) The contribution of CD30 + CD4 + T cells to total CD4 + T cell population, and the contribution of HIV-1 RNA (D) and DNA (E) from CD30 + CD4 + sorted cells to total HIV-1 RNA and DNA burden in CD4 + T cells, are shown. Despite the rarity of CD30 + T cells, a large contribution of HIV-1 DNA and RNA are attributed to these cells in some individuals. The percentage of total HIV-1 RNA (F) but not DNA (G) found in CD30 + CD4 + T cells was significantly higher in African American (n = 13) than white (n = 12) participants (p = 0.0103). Bars represent mean ± standard deviation; *P
    Figure Legend Snippet: HIV-1 is enriched in CD30 expressing CD4 + T cells. Cell-associated HIV-1 unspliced RNA (A) and DNA (B) in sorted CD4 + T cell populations, in samples from ART suppressed (n = 17) and viremic donors (n = 9) are shown. HIV-1 RNA is significantly enriched in CD30 expressing cells from ART suppressed donors (p = 0.008) and viremic donors (p = 0.007). (C) The contribution of CD30 + CD4 + T cells to total CD4 + T cell population, and the contribution of HIV-1 RNA (D) and DNA (E) from CD30 + CD4 + sorted cells to total HIV-1 RNA and DNA burden in CD4 + T cells, are shown. Despite the rarity of CD30 + T cells, a large contribution of HIV-1 DNA and RNA are attributed to these cells in some individuals. The percentage of total HIV-1 RNA (F) but not DNA (G) found in CD30 + CD4 + T cells was significantly higher in African American (n = 13) than white (n = 12) participants (p = 0.0103). Bars represent mean ± standard deviation; *P

    Techniques Used: Expressing, Standard Deviation

    HIV-1 RNA and DNA quantification in rectal tissue-derived CD4 + T cell subsets. HIV-1 RNA and DNA levels for each CD4 + T cell subset (based on co-expression of CD30 and CD32) are shown in A and B , respectively. ( C ) A significantly higher mean HLA-DR MFI was observed in CD30 + CD32 + cells compared with CD30 - CD32 - CD4 + T cells (P = 0.004 by Friedman test with Dunns correction for multiple comparisons). In contrast, no significant intergroup differences were observed in cell-associated HIV-1 RNA or DNA between cohorts. Bars represent mean ± standard deviation.
    Figure Legend Snippet: HIV-1 RNA and DNA quantification in rectal tissue-derived CD4 + T cell subsets. HIV-1 RNA and DNA levels for each CD4 + T cell subset (based on co-expression of CD30 and CD32) are shown in A and B , respectively. ( C ) A significantly higher mean HLA-DR MFI was observed in CD30 + CD32 + cells compared with CD30 - CD32 - CD4 + T cells (P = 0.004 by Friedman test with Dunns correction for multiple comparisons). In contrast, no significant intergroup differences were observed in cell-associated HIV-1 RNA or DNA between cohorts. Bars represent mean ± standard deviation.

    Techniques Used: Derivative Assay, Expressing, Standard Deviation

    15) Product Images from "Decreased expression of the immediate early protein, ICP4, by deletion of the tegument protein VP22 of equine herpesvirus type 1"

    Article Title: Decreased expression of the immediate early protein, ICP4, by deletion of the tegument protein VP22 of equine herpesvirus type 1

    Journal: The Journal of Veterinary Medical Science

    doi: 10.1292/jvms.17-0380

    mRNA levels of ICP4 (a) and ICP0 (b) in EHV-1 attB-, EHV-1∆VP22- and EHV-1∆VP22R-infected cells. MDBK cells were inoculated with EHV-1 attB, EHV-1∆VP22 and EHV-1∆VP22R at an MOI of 3. After 0 and 1 hrpi, total RNA and DNA was extracted. Quantification of mRNA was carried out by real-time quantitative RT-PCR. Expression levels of ICP4 and ICP0 mRNA were normalized with the GAPDH mRNA levels and genome DNA, and expressed relatively as the ratio against the mRNA levels in EHV-1 attB-infected cells at 0 hrpi. There are no significant (n.s.) differences between EHV-1 attB and EHV-1∆VP22. * means “below the threshold”.
    Figure Legend Snippet: mRNA levels of ICP4 (a) and ICP0 (b) in EHV-1 attB-, EHV-1∆VP22- and EHV-1∆VP22R-infected cells. MDBK cells were inoculated with EHV-1 attB, EHV-1∆VP22 and EHV-1∆VP22R at an MOI of 3. After 0 and 1 hrpi, total RNA and DNA was extracted. Quantification of mRNA was carried out by real-time quantitative RT-PCR. Expression levels of ICP4 and ICP0 mRNA were normalized with the GAPDH mRNA levels and genome DNA, and expressed relatively as the ratio against the mRNA levels in EHV-1 attB-infected cells at 0 hrpi. There are no significant (n.s.) differences between EHV-1 attB and EHV-1∆VP22. * means “below the threshold”.

    Techniques Used: Infection, Quantitative RT-PCR, Expressing

    16) Product Images from "Characterization of the fecal and mucosa-associated microbiota in dogs with colorectal epithelial tumors"

    Article Title: Characterization of the fecal and mucosa-associated microbiota in dogs with colorectal epithelial tumors

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0198342

    A scatterplot showing the ratio of live, potentially active bacteria (RNA/DNA) in tumor vs . non-tumor tissue.
    Figure Legend Snippet: A scatterplot showing the ratio of live, potentially active bacteria (RNA/DNA) in tumor vs . non-tumor tissue.

    Techniques Used:

    17) Product Images from "Seasonal dynamics of active SAR11 ecotypes in the oligotrophic Northwest Mediterranean Sea"

    Article Title: Seasonal dynamics of active SAR11 ecotypes in the oligotrophic Northwest Mediterranean Sea

    Journal: The ISME Journal

    doi: 10.1038/ismej.2014.129

    Phylogenetic relationship and relative abundance of SAR11 OTUs. Only SAR11 OTUs representing > 0.2% of total SAR11 OTU sequences are included. Bubble size is scaled to sequence abundance of OTUs relative to total SAR11 communities. DNA and RNA is 16S rRNA gene copies and 16S rRNA, respectively. High and low diversity sub-groups were empirically defined; see Figure 1 and text for further details.
    Figure Legend Snippet: Phylogenetic relationship and relative abundance of SAR11 OTUs. Only SAR11 OTUs representing > 0.2% of total SAR11 OTU sequences are included. Bubble size is scaled to sequence abundance of OTUs relative to total SAR11 communities. DNA and RNA is 16S rRNA gene copies and 16S rRNA, respectively. High and low diversity sub-groups were empirically defined; see Figure 1 and text for further details.

    Techniques Used: Sequencing

    Leucine incorporation and RNA/DNA ratios in the SAR11 clade . The ratio of ribosomal RNA (rRNA) to ribosomal RNA gene copies (rDNA) were calculated by summing the sequences for all SAR11 clusters. SAR11-active cells were calculated as the percentage of total SAR11 cells assimilating tritiated leucine.
    Figure Legend Snippet: Leucine incorporation and RNA/DNA ratios in the SAR11 clade . The ratio of ribosomal RNA (rRNA) to ribosomal RNA gene copies (rDNA) were calculated by summing the sequences for all SAR11 clusters. SAR11-active cells were calculated as the percentage of total SAR11 cells assimilating tritiated leucine.

    Techniques Used:

    18) Product Images from "Integrating host response and unbiased microbe detection for lower respiratory tract infection diagnosis in critically ill adults"

    Article Title: Integrating host response and unbiased microbe detection for lower respiratory tract infection diagnosis in critically ill adults

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

    doi: 10.1073/pnas.1809700115

    Workflow for distinguishing LRTI pathogens from commensal respiratory microbiota using an algorithmic approach. ( A ) Projection of microbial relative abundance in log reads per million reads sequenced (rpm) by RNA sequencing (RNA-seq) ( x axis) versus DNA sequencing (DNA-seq) ( y axis) for representative cases. In the LRTI +C+M group, pathogens identified by standard clinical microbiology (filled shapes) had higher overall relative abundance compared with other taxa detected by sequencing (open shapes). The largest score differential between ranked microbes (max Δrpm) was used as a threshold to identify high-scoring taxa, distinct from the other microbes based on abundance (line with arrows). Red indicates taxa represented in the reference list of established LRTI pathogens. ( B ) Receiver operating characteristic (ROC) curve demonstrating logistic regression model (LRM) performance for detecting pathogens versus commensal microbiota in both the derivation and validation cohorts. The gray ROC curve and shaded region indicate results from 1,000 rounds of training and testing on randomized sets the derivation cohort. The blue and green lines indicate predictions using leave-one-patient-out cross-validation (LOPO-CV) on the derivation and validation on the validation cohort, respectively. ( C ) Microbes predicted by the LRM to represent putative pathogens. The x axis represents combined RNA-seq and DNA-seq relative abundance, and the y axis indicates pathogen probability. The dashed line reflects the optimized probability threshold for pathogen assignment. Red filled circles: microbes predicted by LRM to represent putative LRTI pathogens that were also identified by conventional microbiologic tests. Blue filled circles: microbes predicted to represent putative LRTI pathogens by LRM only. Blue open circles: microbes identified by NGS but not predicted by the LRM to represent putative pathogens. Red open circles: microbes identified using NGS and by standard microbiologic testing but not predicted to be putative pathogens. Dark red outlined circles: microbes detected as part of a polymicrobial culture.
    Figure Legend Snippet: Workflow for distinguishing LRTI pathogens from commensal respiratory microbiota using an algorithmic approach. ( A ) Projection of microbial relative abundance in log reads per million reads sequenced (rpm) by RNA sequencing (RNA-seq) ( x axis) versus DNA sequencing (DNA-seq) ( y axis) for representative cases. In the LRTI +C+M group, pathogens identified by standard clinical microbiology (filled shapes) had higher overall relative abundance compared with other taxa detected by sequencing (open shapes). The largest score differential between ranked microbes (max Δrpm) was used as a threshold to identify high-scoring taxa, distinct from the other microbes based on abundance (line with arrows). Red indicates taxa represented in the reference list of established LRTI pathogens. ( B ) Receiver operating characteristic (ROC) curve demonstrating logistic regression model (LRM) performance for detecting pathogens versus commensal microbiota in both the derivation and validation cohorts. The gray ROC curve and shaded region indicate results from 1,000 rounds of training and testing on randomized sets the derivation cohort. The blue and green lines indicate predictions using leave-one-patient-out cross-validation (LOPO-CV) on the derivation and validation on the validation cohort, respectively. ( C ) Microbes predicted by the LRM to represent putative pathogens. The x axis represents combined RNA-seq and DNA-seq relative abundance, and the y axis indicates pathogen probability. The dashed line reflects the optimized probability threshold for pathogen assignment. Red filled circles: microbes predicted by LRM to represent putative LRTI pathogens that were also identified by conventional microbiologic tests. Blue filled circles: microbes predicted to represent putative LRTI pathogens by LRM only. Blue open circles: microbes identified by NGS but not predicted by the LRM to represent putative pathogens. Red open circles: microbes identified using NGS and by standard microbiologic testing but not predicted to be putative pathogens. Dark red outlined circles: microbes detected as part of a polymicrobial culture.

    Techniques Used: RNA Sequencing Assay, DNA Sequencing, Sequencing, Next-Generation Sequencing

    Study overview and analysis workflow. Patients with acute respiratory failure were enrolled within 72 h of ICU admission, and TA samples were collected and underwent both RNA sequencing (RNA-seq) and shotgun DNA sequencing (DNA-seq). Post hoc clinical adjudication blinded to mNGS results identified patients with LRTI defined by clinical and microbiologic criteria (LRTI +C+M ); LRTI defined by clinical criteria only (LRTI +C ); patients with noninfectious reasons for acute respiratory failure (no-LRTI); and respiratory failure due to unknown cause (unk-LRTI). The LRTI +C+M and no-LRTI groups were divided into derivation and validation cohorts. To detect pathogens and differentiate them from a background of commensal microbiota, we developed two models: a rules-based model (RBM) and a logistic regression model (LRM). LRTI probability was next evaluated with ( i ) a pathogen metric, ( ii ) a lung microbiome diversity metric, and ( iii ) a 12-gene host transcriptional classifier. Models were then combined and optimized for LRTI rule out.
    Figure Legend Snippet: Study overview and analysis workflow. Patients with acute respiratory failure were enrolled within 72 h of ICU admission, and TA samples were collected and underwent both RNA sequencing (RNA-seq) and shotgun DNA sequencing (DNA-seq). Post hoc clinical adjudication blinded to mNGS results identified patients with LRTI defined by clinical and microbiologic criteria (LRTI +C+M ); LRTI defined by clinical criteria only (LRTI +C ); patients with noninfectious reasons for acute respiratory failure (no-LRTI); and respiratory failure due to unknown cause (unk-LRTI). The LRTI +C+M and no-LRTI groups were divided into derivation and validation cohorts. To detect pathogens and differentiate them from a background of commensal microbiota, we developed two models: a rules-based model (RBM) and a logistic regression model (LRM). LRTI probability was next evaluated with ( i ) a pathogen metric, ( ii ) a lung microbiome diversity metric, and ( iii ) a 12-gene host transcriptional classifier. Models were then combined and optimized for LRTI rule out.

    Techniques Used: RNA Sequencing Assay, DNA Sequencing

    19) Product Images from "Endogenous retroviruses are a source of enhancers with oncogenic potential in acute myeloid leukaemia"

    Article Title: Endogenous retroviruses are a source of enhancers with oncogenic potential in acute myeloid leukaemia

    Journal: bioRxiv

    doi: 10.1101/772954

    APOC1 -LTR2 element regulates APOC1 expression and is essential for cell proliferation. A. Genome browser snapshot for APOC1 -LTR2 element, showing TAL1, STAT5 ChIP-seq tracks for WT; H3K27ac, H3K9me3 ChIP-seq and RNA-seq tracks for no control and CRISPRi K562 cells. B. Schematic of the experimental design to genetically excise APOC1 -LTR2 element. C. qPCR and D. RT-qPCR data from cells with APOC1 -LTR2 excision. The error bars show standard deviation (n≥3 biological replicates, t-test (C) ANOVA with Tukey’s multiple comparison test (D), * p
    Figure Legend Snippet: APOC1 -LTR2 element regulates APOC1 expression and is essential for cell proliferation. A. Genome browser snapshot for APOC1 -LTR2 element, showing TAL1, STAT5 ChIP-seq tracks for WT; H3K27ac, H3K9me3 ChIP-seq and RNA-seq tracks for no control and CRISPRi K562 cells. B. Schematic of the experimental design to genetically excise APOC1 -LTR2 element. C. qPCR and D. RT-qPCR data from cells with APOC1 -LTR2 excision. The error bars show standard deviation (n≥3 biological replicates, t-test (C) ANOVA with Tukey’s multiple comparison test (D), * p

    Techniques Used: Expressing, Chromatin Immunoprecipitation, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Standard Deviation

    20) Product Images from "Molecular Cloning, Characterization and Predicted Structure of a Putative Copper-Zinc SOD from the Camel, Camelus dromedarius"

    Article Title: Molecular Cloning, Characterization and Predicted Structure of a Putative Copper-Zinc SOD from the Camel, Camelus dromedarius

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms13010879

    Expression of SOD1 using Real time PCR and cDNA from different camel tissues. The results are expressed relative to liver as calibrator and using 18S ribosomal subunit as housekeeping gene.
    Figure Legend Snippet: Expression of SOD1 using Real time PCR and cDNA from different camel tissues. The results are expressed relative to liver as calibrator and using 18S ribosomal subunit as housekeeping gene.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    21) Product Images from "Activity of translation regulator eukaryotic elongation factor-2 kinase is increased in Parkinson disease brain and its inhibition reduces alpha synuclein toxicity"

    Article Title: Activity of translation regulator eukaryotic elongation factor-2 kinase is increased in Parkinson disease brain and its inhibition reduces alpha synuclein toxicity

    Journal: Acta Neuropathologica Communications

    doi: 10.1186/s40478-018-0554-9

    Effects of eEF2K inhibition on human AS cytotoxicity in differentiated N2A cells. a - b Western blot analysis of p-eEF2 (T56) levels in N2A cells subsequent to transient overexpression of human wild type or mutant A53T AS, with or without siRNA mediated eEF2K knockdown ( a ), and corresponding densitometry analysis ( b ) ( n = 6–9/group from three independent experiments; One-way ANOVA post-hoc Bonferroni test, * p
    Figure Legend Snippet: Effects of eEF2K inhibition on human AS cytotoxicity in differentiated N2A cells. a - b Western blot analysis of p-eEF2 (T56) levels in N2A cells subsequent to transient overexpression of human wild type or mutant A53T AS, with or without siRNA mediated eEF2K knockdown ( a ), and corresponding densitometry analysis ( b ) ( n = 6–9/group from three independent experiments; One-way ANOVA post-hoc Bonferroni test, * p

    Techniques Used: Inhibition, Western Blot, Over Expression, Mutagenesis

    Effects of eEF2K inhibition on mitochondrial dysfunction and oxidative stress induced by human AS in differentiated N2A cells. a - b Measurements of basal oxygen consumption rate-OCR ( b ) and ATP levels ( c ) in N2A cells subsequent to transient overexpression of human wild type or mutant A53T AS, with or without siRNA mediated eEF2K knockdown ( n = 9–12/group from three independent experiments; Unpaired T-test, * p
    Figure Legend Snippet: Effects of eEF2K inhibition on mitochondrial dysfunction and oxidative stress induced by human AS in differentiated N2A cells. a - b Measurements of basal oxygen consumption rate-OCR ( b ) and ATP levels ( c ) in N2A cells subsequent to transient overexpression of human wild type or mutant A53T AS, with or without siRNA mediated eEF2K knockdown ( n = 9–12/group from three independent experiments; Unpaired T-test, * p

    Techniques Used: Inhibition, Over Expression, Mutagenesis

    22) Product Images from "Decoupling of DNA methylation and activity of intergenic LINE-1 promoters in colorectal cancer"

    Article Title: Decoupling of DNA methylation and activity of intergenic LINE-1 promoters in colorectal cancer

    Journal: Epigenetics

    doi: 10.1080/15592294.2017.1300729

    Relationship between methylation and expression of LCT14 in CRC. (A) Schematic diagram of the LCT14 genomic locus on human chromosome 5 (coordinates: 24,487,209–27,038,689) with indicated positions of the annotated genes CDH10, LOC105374693 , and CDH9 and of the intact intergenic LINE1 (L1) that drives transcription of LCT14. At the bottom is an enlargement of the region including the LINE-1 (L1PA2; chr5:25,378,639–25,384,665) from which LCT14 originates with the regions (black bars) tested by bisulfite or hydroxymethylated DNA (hMeDIP) and chromatin (ChIP) immunoprecipitations and, below these, the LCT14 transcript (chr5: 25,384,485–25,384,958) and the region amplified for expression studies. All coordinates are from hg19 annotations; scale is in kb. (B) Expression of LCT14 measured by real-time RT-PCR and expressed relatively to the geometric mean of 3 reference genes in matched normal (dark gray) and tumor (light gray) tissues from 4 colorectal cancer patients (left panel) and of 5 colorectal cancer cell lines (right panel). (C) Methylation levels measured by bisulfite sequencing in the paired normal and tumor tissues of the 4 patients (left panel) and cell lines (right panel) described in B.
    Figure Legend Snippet: Relationship between methylation and expression of LCT14 in CRC. (A) Schematic diagram of the LCT14 genomic locus on human chromosome 5 (coordinates: 24,487,209–27,038,689) with indicated positions of the annotated genes CDH10, LOC105374693 , and CDH9 and of the intact intergenic LINE1 (L1) that drives transcription of LCT14. At the bottom is an enlargement of the region including the LINE-1 (L1PA2; chr5:25,378,639–25,384,665) from which LCT14 originates with the regions (black bars) tested by bisulfite or hydroxymethylated DNA (hMeDIP) and chromatin (ChIP) immunoprecipitations and, below these, the LCT14 transcript (chr5: 25,384,485–25,384,958) and the region amplified for expression studies. All coordinates are from hg19 annotations; scale is in kb. (B) Expression of LCT14 measured by real-time RT-PCR and expressed relatively to the geometric mean of 3 reference genes in matched normal (dark gray) and tumor (light gray) tissues from 4 colorectal cancer patients (left panel) and of 5 colorectal cancer cell lines (right panel). (C) Methylation levels measured by bisulfite sequencing in the paired normal and tumor tissues of the 4 patients (left panel) and cell lines (right panel) described in B.

    Techniques Used: Methylation, Expressing, Chromatin Immunoprecipitation, Amplification, Quantitative RT-PCR, Methylation Sequencing

    23) Product Images from "Challenges in EGFRvIII Detection in Head and Neck Squamous Cell Carcinoma"

    Article Title: Challenges in EGFRvIII Detection in Head and Neck Squamous Cell Carcinoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0117781

    PCR amplification of EGFR for genomic alterations leading to EGFRvIII transcript. A. Schematic of sequencing primers and areas of interest. Arrows indicate the location of the primers used to detect EGFRvIII in genomic DNA and unspliced RNA. Bars with diamond caps indicate areas amplified for splice donor and acceptor mutations. The shaded area is lost in EGFRvIII. B. Representative PCR amplification of the splice donor/acceptor sites of EGFR exons 1, 2, 7 and 8 in an EGFRvIII positive HNSCC DNA sample. These bands were excised and sequenced for mutations. C. Representative long-range PCR amplification of EGFR intron 1 for a single EGFRvIII positive HNSCC DNA sample. L: base pair marker, W: water control, a-j: primer sets.
    Figure Legend Snippet: PCR amplification of EGFR for genomic alterations leading to EGFRvIII transcript. A. Schematic of sequencing primers and areas of interest. Arrows indicate the location of the primers used to detect EGFRvIII in genomic DNA and unspliced RNA. Bars with diamond caps indicate areas amplified for splice donor and acceptor mutations. The shaded area is lost in EGFRvIII. B. Representative PCR amplification of the splice donor/acceptor sites of EGFR exons 1, 2, 7 and 8 in an EGFRvIII positive HNSCC DNA sample. These bands were excised and sequenced for mutations. C. Representative long-range PCR amplification of EGFR intron 1 for a single EGFRvIII positive HNSCC DNA sample. L: base pair marker, W: water control, a-j: primer sets.

    Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing, Marker

    24) Product Images from "Genome-wide screen for differentially methylated long noncoding RNAs identifies Esrp2 and lncRNA Esrp2-as regulated by enhancer DNA methylation with prognostic relevance for human breast cancer"

    Article Title: Genome-wide screen for differentially methylated long noncoding RNAs identifies Esrp2 and lncRNA Esrp2-as regulated by enhancer DNA methylation with prognostic relevance for human breast cancer

    Journal: Oncogene

    doi: 10.1038/onc.2017.246

    A human homolog of Esrp2-as is overexpressed in human breast cancer and associated with poor prognosis. ( a ) UCSC Genome Browser 40 scheme of the human ESPR2 locus on chr16 depicts the genomic location of the human ESRP2 gene (blue), CpG islands (green), genomic location of ESRP2-AS (red), MCF-7 CAGE-seq peaks for the positive (red) and negative strand (red), positions of RT–qPCR primers to quantify expression of ESRP2-AS (RNA1-4, red), strand-specific RNA-seq signals obtained with MCF7 whole cell lysates, cytosolic and nuclear fraction (black), location of mouse EPITYPER MassArray amplicons (using UCSC genome browser liftover tool, beige), DNA methylation levels for individual CpG sites (indicated by beige lines) assessed by WGBS (downloaded from TCGA), CpG sites covered on the Illumina 450 k methylation array (blue). ( b ) Left: Relative expression of ESRP2-AS (using four primer pairs designated as RNA1-4) and ESRP2 in MCF7 ( n =2), HepG2 ( n =1), MDA-MB231 ( n =1) and MCF10a ( n =1) human (cancer) cell lines, assessed by RT–qPCR. Right: Fold change of ESRP2 and ESRP2-AS levels in MCF7 ( n =2) and MDA-MB231 cells ( n =1) after DAC treatment, relative to DMSO solvent control set as 1. Statistical significance was assessed using the one-sample t-test with * P
    Figure Legend Snippet: A human homolog of Esrp2-as is overexpressed in human breast cancer and associated with poor prognosis. ( a ) UCSC Genome Browser 40 scheme of the human ESPR2 locus on chr16 depicts the genomic location of the human ESRP2 gene (blue), CpG islands (green), genomic location of ESRP2-AS (red), MCF-7 CAGE-seq peaks for the positive (red) and negative strand (red), positions of RT–qPCR primers to quantify expression of ESRP2-AS (RNA1-4, red), strand-specific RNA-seq signals obtained with MCF7 whole cell lysates, cytosolic and nuclear fraction (black), location of mouse EPITYPER MassArray amplicons (using UCSC genome browser liftover tool, beige), DNA methylation levels for individual CpG sites (indicated by beige lines) assessed by WGBS (downloaded from TCGA), CpG sites covered on the Illumina 450 k methylation array (blue). ( b ) Left: Relative expression of ESRP2-AS (using four primer pairs designated as RNA1-4) and ESRP2 in MCF7 ( n =2), HepG2 ( n =1), MDA-MB231 ( n =1) and MCF10a ( n =1) human (cancer) cell lines, assessed by RT–qPCR. Right: Fold change of ESRP2 and ESRP2-AS levels in MCF7 ( n =2) and MDA-MB231 cells ( n =1) after DAC treatment, relative to DMSO solvent control set as 1. Statistical significance was assessed using the one-sample t-test with * P

    Techniques Used: Quantitative RT-PCR, Expressing, RNA Sequencing Assay, DNA Methylation Assay, Methylation, Multiple Displacement Amplification

    25) Product Images from "The genomic landscape of undifferentiated embryonal sarcoma of the liver is typified by C19MC structural rearrangement and overexpression combined with TP53 mutation or loss"

    Article Title: The genomic landscape of undifferentiated embryonal sarcoma of the liver is typified by C19MC structural rearrangement and overexpression combined with TP53 mutation or loss

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1008642

    UESL display aberrant transcriptional activity of the C19MC region. A novel PEG3/ZIM2 -C19MC fusion is identified. A, Read counts of mapped whole transcriptome show high levels of aberrant transcriptional activity in the C19MC region. Note that the abrupt starting location of transcriptional mapping is in different co-ordinates in different UESL samples, suggestive of sample specific fusional events. The genomic position of the experimentally verified fusion in PATWXD is indicated by the red arrow in panel-A and notably corresponds to the start (5’ end) of transcriptional activity in this sample. Hep3B cell line (hepatocellular carcinoma) and normal liver samples are shown at the bottom for comparison and as expected show negligible amounts of RNA mapping to this non-coding region. B, Targeted DNA sequencing of PATWXD UESL tumor showing abrupt end of read mapping near the C19MC start site (left) as well as in the PEG2/ZIM2 gene locus (right, shared gene region). The reads also mark the position of primers designed for gDNA PCR (one primer at 5’ end of reads and another at 3’end of reads for each locus (therefore one set of primers will form a nested primer set). C, Nested multiplex PCR of PATWXD genomic DNA showing amplicon (~550 bp) including the nested product (~450 bp). D, Paired end Sanger sequencing of ~550 bp product from panel-B showing the PEG3/ZIM2 locus fused to C19MC aberrant transcriptional start site.
    Figure Legend Snippet: UESL display aberrant transcriptional activity of the C19MC region. A novel PEG3/ZIM2 -C19MC fusion is identified. A, Read counts of mapped whole transcriptome show high levels of aberrant transcriptional activity in the C19MC region. Note that the abrupt starting location of transcriptional mapping is in different co-ordinates in different UESL samples, suggestive of sample specific fusional events. The genomic position of the experimentally verified fusion in PATWXD is indicated by the red arrow in panel-A and notably corresponds to the start (5’ end) of transcriptional activity in this sample. Hep3B cell line (hepatocellular carcinoma) and normal liver samples are shown at the bottom for comparison and as expected show negligible amounts of RNA mapping to this non-coding region. B, Targeted DNA sequencing of PATWXD UESL tumor showing abrupt end of read mapping near the C19MC start site (left) as well as in the PEG2/ZIM2 gene locus (right, shared gene region). The reads also mark the position of primers designed for gDNA PCR (one primer at 5’ end of reads and another at 3’end of reads for each locus (therefore one set of primers will form a nested primer set). C, Nested multiplex PCR of PATWXD genomic DNA showing amplicon (~550 bp) including the nested product (~450 bp). D, Paired end Sanger sequencing of ~550 bp product from panel-B showing the PEG3/ZIM2 locus fused to C19MC aberrant transcriptional start site.

    Techniques Used: Activity Assay, DNA Sequencing, Polymerase Chain Reaction, Multiplex Assay, Amplification, Sequencing

    26) Product Images from "HIV Maintains an Evolving and Dispersed Population in Multiple Tissues during Suppressive Combined Antiretroviral Therapy in Individuals with Cancer"

    Article Title: HIV Maintains an Evolving and Dispersed Population in Multiple Tissues during Suppressive Combined Antiretroviral Therapy in Individuals with Cancer

    Journal: Journal of Virology

    doi: 10.1128/JVI.00684-16

    Maximum-likelihood trees of the env (left) and nef (right) sequences for patient C02. Branches are drawn in substitutions/site according to the scale at the bottom. Circles are colored by the tissue of origin. Open circles, RNA; filled circles, DNA. Asterisks
    Figure Legend Snippet: Maximum-likelihood trees of the env (left) and nef (right) sequences for patient C02. Branches are drawn in substitutions/site according to the scale at the bottom. Circles are colored by the tissue of origin. Open circles, RNA; filled circles, DNA. Asterisks

    Techniques Used:

    Maximum-likelihood trees of the env (left) and nef (right) sequences for patient C05. Branches are drawn in substitutions/site according to the scale at the bottom. Circles are colored by the tissue of origin. Open circles, RNA; filled circles, DNA. Asterisks
    Figure Legend Snippet: Maximum-likelihood trees of the env (left) and nef (right) sequences for patient C05. Branches are drawn in substitutions/site according to the scale at the bottom. Circles are colored by the tissue of origin. Open circles, RNA; filled circles, DNA. Asterisks

    Techniques Used:

    Maximum-likelihood trees of the env (left) and nef (right) sequences for patient C04. Branches are drawn in substitutions/site according to the scale at the bottom. Circles are colored by the tissue of origin. Open circles, RNA; filled circles, DNA. Asterisks
    Figure Legend Snippet: Maximum-likelihood trees of the env (left) and nef (right) sequences for patient C04. Branches are drawn in substitutions/site according to the scale at the bottom. Circles are colored by the tissue of origin. Open circles, RNA; filled circles, DNA. Asterisks

    Techniques Used:

    HIV RNA and DNA sequences were obtained from tissues for four of the five participants.
    Figure Legend Snippet: HIV RNA and DNA sequences were obtained from tissues for four of the five participants.

    Techniques Used:

    27) Product Images from "Mitochondrial Transcription Factor A (TFAM) Binds to RNA Containing 4-Way Junctions and Mitochondrial tRNA"

    Article Title: Mitochondrial Transcription Factor A (TFAM) Binds to RNA Containing 4-Way Junctions and Mitochondrial tRNA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0142436

    TFAM binding of linear DNA and RNA substrates by EMSA. Increasing amounts of TFAM were bound to 20 fM of each biotinylated substrate as follows; (A) dsDNA control sequence LSP, 0–1.44 μM TFAM with five-fold serial dilutions, apparent K d of 6 nM, (B) dsDNA control sequence scrambled LSP, with serial dilutions of TFAM as in (A), apparent K d of 100 nM, (C) dsRNA:DNA hybrid with 0–3.5 μM TFAM with two-fold serial dilutions, (D) ss poly [rArC] 12 , with TFAM dilutions as in panel (C), (E) poly [rU] 20 , with TFAM dilutions as in (C), (F) dsRNA 5681 , with TFAM dilutions as in panel (C).
    Figure Legend Snippet: TFAM binding of linear DNA and RNA substrates by EMSA. Increasing amounts of TFAM were bound to 20 fM of each biotinylated substrate as follows; (A) dsDNA control sequence LSP, 0–1.44 μM TFAM with five-fold serial dilutions, apparent K d of 6 nM, (B) dsDNA control sequence scrambled LSP, with serial dilutions of TFAM as in (A), apparent K d of 100 nM, (C) dsRNA:DNA hybrid with 0–3.5 μM TFAM with two-fold serial dilutions, (D) ss poly [rArC] 12 , with TFAM dilutions as in panel (C), (E) poly [rU] 20 , with TFAM dilutions as in (C), (F) dsRNA 5681 , with TFAM dilutions as in panel (C).

    Techniques Used: Binding Assay, Sequencing

    Binding kinetics of TFAM to RNA and DNA substrates using surface plasmon resonance. (A-D) Sensograms displaying the TFAM binding and dissociation rates of (A) a DNA 4-way junction, (B) an RNA 4-way junction, (C) linear double-stranded DNA, and (D) purified mitochondrial tRNAs. Individual tracings represent a single value in a range of TFAM concentrations in each of the experiments. (E) Kinetic data derived from these tracings include associate rate constant ( k a ), dissociate rate constant ( k d ) and the apparent dissociation constant ( K d ) for each of these substrates.
    Figure Legend Snippet: Binding kinetics of TFAM to RNA and DNA substrates using surface plasmon resonance. (A-D) Sensograms displaying the TFAM binding and dissociation rates of (A) a DNA 4-way junction, (B) an RNA 4-way junction, (C) linear double-stranded DNA, and (D) purified mitochondrial tRNAs. Individual tracings represent a single value in a range of TFAM concentrations in each of the experiments. (E) Kinetic data derived from these tracings include associate rate constant ( k a ), dissociate rate constant ( k d ) and the apparent dissociation constant ( K d ) for each of these substrates.

    Techniques Used: Binding Assay, SPR Assay, Purification, Derivative Assay

    TFAM-bound mitochondrial tRNAs are processed and have mature ends. (A) Schematic for PCR detection of unprocessed tRNAs showing a tRNA flanked by putative RNA sequences from adjacent genes. PCR primer positions used on cDNAs are shown as arrows. Expected PCR fragments from unprocessed 5’ ends (i), unprocessed 3’ ends (ii) and internal tRNA (iii) are displayed. (B and C) PCR templates in lane 1 from total cellular cDNA, lane 2 templates made excluding reverse transcriptase, lane 3 templates are from TFAM-RNA IP, and lane 4 from TFAM-DNA IP. Samples for lanes 2, 3, and 4 are identical to those used for data obtained in Fig 4 , which further controls for the sample preparation and PCR procedures. (B) PCR amplicons detecting 5’ flanking regions from each tRNA as in (Ai). V (internal) serves as a control for the TFAM-RIP reaction using tRNA internal primers as in (Aiii). (C) PCR amplicons detecting 3’ flanking regions from each tRNA as in (Aii). P(internal) serves as a control for RT-PCR using tRNA internal primers as in (Aiii). (D and E) 3’-end sequence frequency of multiple clones isolated from tRNA Val RNA circularization is pie-graph displayed. (D) tRNA Val sequences isolated from total cellular RNA, n = 104. (E) TFAM-RIP isolated tRNA Val sequences, n = 67.
    Figure Legend Snippet: TFAM-bound mitochondrial tRNAs are processed and have mature ends. (A) Schematic for PCR detection of unprocessed tRNAs showing a tRNA flanked by putative RNA sequences from adjacent genes. PCR primer positions used on cDNAs are shown as arrows. Expected PCR fragments from unprocessed 5’ ends (i), unprocessed 3’ ends (ii) and internal tRNA (iii) are displayed. (B and C) PCR templates in lane 1 from total cellular cDNA, lane 2 templates made excluding reverse transcriptase, lane 3 templates are from TFAM-RNA IP, and lane 4 from TFAM-DNA IP. Samples for lanes 2, 3, and 4 are identical to those used for data obtained in Fig 4 , which further controls for the sample preparation and PCR procedures. (B) PCR amplicons detecting 5’ flanking regions from each tRNA as in (Ai). V (internal) serves as a control for the TFAM-RIP reaction using tRNA internal primers as in (Aiii). (C) PCR amplicons detecting 3’ flanking regions from each tRNA as in (Aii). P(internal) serves as a control for RT-PCR using tRNA internal primers as in (Aiii). (D and E) 3’-end sequence frequency of multiple clones isolated from tRNA Val RNA circularization is pie-graph displayed. (D) tRNA Val sequences isolated from total cellular RNA, n = 104. (E) TFAM-RIP isolated tRNA Val sequences, n = 67.

    Techniques Used: Polymerase Chain Reaction, Sample Prep, Reverse Transcription Polymerase Chain Reaction, Sequencing, Clone Assay, Isolation

    TFAM bound RNA represents a lesser fraction of TFAM bound DNA. (A) Immunofluorescence images of TFAM in untreated cells (i), cells treated with RNase (ii), DNase (iii) or both DNase and RNase (iv). (B) Relative mean fluorescence of TFAM retained during nuclease treatments shown in (A). (C) Levels of TFAM-bound tRNA relative to TFAM-bound to the corresponding mtDNA. Parallel RNA and DNA immunoprecipitations from the same samples were quantified by RT-PCR and PCR, respectively. Relative bound tRNA level is expressed as a percentage of bound mtDNA.
    Figure Legend Snippet: TFAM bound RNA represents a lesser fraction of TFAM bound DNA. (A) Immunofluorescence images of TFAM in untreated cells (i), cells treated with RNase (ii), DNase (iii) or both DNase and RNase (iv). (B) Relative mean fluorescence of TFAM retained during nuclease treatments shown in (A). (C) Levels of TFAM-bound tRNA relative to TFAM-bound to the corresponding mtDNA. Parallel RNA and DNA immunoprecipitations from the same samples were quantified by RT-PCR and PCR, respectively. Relative bound tRNA level is expressed as a percentage of bound mtDNA.

    Techniques Used: Immunofluorescence, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    TFAM binding of complex DNA and RNA substrates by EMSA. Varying amounts of TFAM were bound to 20 fM of each biotinylated substrate with as follows; (A) Stem-loop RNA, 2.4–0.0375 μM TFAM with two-fold serial dilutions, (B) dsRNA with internal 8 nucleotide mismatch loop, TFAM dilutions as in (A), apparent K d of 2.04 μM, (C) Alternating, four arm RNA:DNA 4-way junction, TFAM dilutions as in (A), apparent K d of 299 nM, (D) RNA 4-way junction, 600–9.375 nM TFAM with two-fold serial dilutions, apparent K d of 270 nM, (E) Mixed pairing RNA and DNA 4-way junction, TFAM dilutions as in (A), apparent K d of 63 nM, (F) DNA 4-way junction, TFAM dilutions as in (D), apparent K d of 63 nM. Left lane in each panel is free template without TFAM. Substrate diagrams appear to the right of each panel with RNA depicted in red and DNA in blue.
    Figure Legend Snippet: TFAM binding of complex DNA and RNA substrates by EMSA. Varying amounts of TFAM were bound to 20 fM of each biotinylated substrate with as follows; (A) Stem-loop RNA, 2.4–0.0375 μM TFAM with two-fold serial dilutions, (B) dsRNA with internal 8 nucleotide mismatch loop, TFAM dilutions as in (A), apparent K d of 2.04 μM, (C) Alternating, four arm RNA:DNA 4-way junction, TFAM dilutions as in (A), apparent K d of 299 nM, (D) RNA 4-way junction, 600–9.375 nM TFAM with two-fold serial dilutions, apparent K d of 270 nM, (E) Mixed pairing RNA and DNA 4-way junction, TFAM dilutions as in (A), apparent K d of 63 nM, (F) DNA 4-way junction, TFAM dilutions as in (D), apparent K d of 63 nM. Left lane in each panel is free template without TFAM. Substrate diagrams appear to the right of each panel with RNA depicted in red and DNA in blue.

    Techniques Used: Binding Assay

    28) Product Images from "The Biogeographical Distribution of Benthic Roseobacter Group Members along a Pacific Transect Is Structured by Nutrient Availability within the Sediments and Primary Production in Different Oceanic Provinces"

    Article Title: The Biogeographical Distribution of Benthic Roseobacter Group Members along a Pacific Transect Is Structured by Nutrient Availability within the Sediments and Primary Production in Different Oceanic Provinces

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2017.02550

    Non-metric multidimensional scaling (NMDS) plots of (A) the DNA-based and (B) the RNA-based community compositions of the Roseobacter group. Plots were calculated from 16S rRNA gene and 16S rRNA transcript libraries. Arrows indicate the relation of the Roseobacter group to significant environmental factors ( p ≤ 0.05).
    Figure Legend Snippet: Non-metric multidimensional scaling (NMDS) plots of (A) the DNA-based and (B) the RNA-based community compositions of the Roseobacter group. Plots were calculated from 16S rRNA gene and 16S rRNA transcript libraries. Arrows indicate the relation of the Roseobacter group to significant environmental factors ( p ≤ 0.05).

    Techniques Used:

    Heat maps for the different sampling sites according to the compositions of (A) the DNA-based, (B) the RNA-based community composition of the Roseobacter group and (C) nutrients in sediments and porewaters as well as chlorophyll in the water column. The clustering on the left side reflects the relative frequencies of the different OTUs/nutrient concentrations, while on top the clustering based on the similarity of the sampling sites is shown.
    Figure Legend Snippet: Heat maps for the different sampling sites according to the compositions of (A) the DNA-based, (B) the RNA-based community composition of the Roseobacter group and (C) nutrients in sediments and porewaters as well as chlorophyll in the water column. The clustering on the left side reflects the relative frequencies of the different OTUs/nutrient concentrations, while on top the clustering based on the similarity of the sampling sites is shown.

    Techniques Used: Sampling

    29) Product Images from "Reprogramming the antigen specificity of B cells using genome-editing technologies"

    Article Title: Reprogramming the antigen specificity of B cells using genome-editing technologies

    Journal: eLife

    doi: 10.7554/eLife.42995

    CRISPR/cas9 guide RNA selection. The human reference genome (GenBank: AB019437, AB019439 and AL122127) used to annotate the IGHV gene locus in the International Immunogenetics Information System ( http://www.imgt.org ) was used to design CRISPR/cas9 guide RNAs using the Zhang lab-optimized CRISPR Design online platform ( http://crispr.mit.edu ). Primers were ordered and cloned into the pX330-U6-chimeric_BB-CBh- hSpCas9 vector as described in Materials and methods. Target DNA (genomic sequences to be cleaved by these nucleases) were either synthesized or amplified from 293T or Ramos B-cell gDNA as 250–300 bp products that could be cloned into the pCAG-eGxxFP vector. The pX330 vectors were then co- transfected with their respective target pCAG vectors in 293 T cells as described in Materials and methods. If the target DNA gets cut by the CRISPR/cas9/gRNA complex expressed in the cell, the pCAG vector undergoes homologous recombination to express GFP protein. 2 days after transfection, guide RNAs were scored visually based on GFP expression in the 293 cells according to the sample confocal microscopic images above. Highest scoring guide RNAs which could achieve cutting against the target DNA sequences derived from all three sources were chosen for B-cell engineering experiments to insert PG9 mature HC VDJ genes by homology directed repair.
    Figure Legend Snippet: CRISPR/cas9 guide RNA selection. The human reference genome (GenBank: AB019437, AB019439 and AL122127) used to annotate the IGHV gene locus in the International Immunogenetics Information System ( http://www.imgt.org ) was used to design CRISPR/cas9 guide RNAs using the Zhang lab-optimized CRISPR Design online platform ( http://crispr.mit.edu ). Primers were ordered and cloned into the pX330-U6-chimeric_BB-CBh- hSpCas9 vector as described in Materials and methods. Target DNA (genomic sequences to be cleaved by these nucleases) were either synthesized or amplified from 293T or Ramos B-cell gDNA as 250–300 bp products that could be cloned into the pCAG-eGxxFP vector. The pX330 vectors were then co- transfected with their respective target pCAG vectors in 293 T cells as described in Materials and methods. If the target DNA gets cut by the CRISPR/cas9/gRNA complex expressed in the cell, the pCAG vector undergoes homologous recombination to express GFP protein. 2 days after transfection, guide RNAs were scored visually based on GFP expression in the 293 cells according to the sample confocal microscopic images above. Highest scoring guide RNAs which could achieve cutting against the target DNA sequences derived from all three sources were chosen for B-cell engineering experiments to insert PG9 mature HC VDJ genes by homology directed repair.

    Techniques Used: CRISPR, Selection, Clone Assay, Plasmid Preparation, Genomic Sequencing, Synthesized, Amplification, Transfection, Homologous Recombination, Expressing, Derivative Assay

    30) Product Images from "Manufacture of Clinical-Grade CD19-Specific T Cells Stably Expressing Chimeric Antigen Receptor Using Sleeping Beauty System and Artificial Antigen Presenting Cells"

    Article Title: Manufacture of Clinical-Grade CD19-Specific T Cells Stably Expressing Chimeric Antigen Receptor Using Sleeping Beauty System and Artificial Antigen Presenting Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0064138

    Schematic of the process of generating clinical grade CD19-specific T cells. A MCB (PACT) and WCB (MDACC) were generated for K562-derived aAPC (clone #4). For the generation of CAR + T cells, aAPC were numerically expanded in bags, harvested using the Sepax II system, irradiated (100 Gy), and cryopreserved for later use. CD19-specific T cells were manufactured as follows; PBMC were isolated from normal donor apheresis products using the Sepax II system and cryopreserved. The PBMC were later thawed, electroporated with the SB DNA plasmids (CD19RCD28 CAR transposon, SB11 transposase) using the Nucleofector System, co-cultured with thawed irradiated aAPC along with cytokines (IL-2 and IL-21) for a culture period of 28 days and cryopreserved.
    Figure Legend Snippet: Schematic of the process of generating clinical grade CD19-specific T cells. A MCB (PACT) and WCB (MDACC) were generated for K562-derived aAPC (clone #4). For the generation of CAR + T cells, aAPC were numerically expanded in bags, harvested using the Sepax II system, irradiated (100 Gy), and cryopreserved for later use. CD19-specific T cells were manufactured as follows; PBMC were isolated from normal donor apheresis products using the Sepax II system and cryopreserved. The PBMC were later thawed, electroporated with the SB DNA plasmids (CD19RCD28 CAR transposon, SB11 transposase) using the Nucleofector System, co-cultured with thawed irradiated aAPC along with cytokines (IL-2 and IL-21) for a culture period of 28 days and cryopreserved.

    Techniques Used: Generated, Derivative Assay, Irradiation, Isolation, Cell Culture

    Safety profile associated with the SB system. (A) Telomere length of cells was measured using fluorescence in situ hybridization and flow cytometry (Flow-FISH) assay. Predominant T cell population at day 28 (V1 and V2, CD8 + T cells; V3, CD4 + T cells) was compared to respective miltenyi column purified subset of T cells from day 0. Mean ± SD of triplicates for each validation run is represented. (B) Genomic DNA from CAR + T cells at day 28 was amplified using primers and probes specific for CD19RCD28 CAR. Relative Quantity (RQ) analyses of the CD19RCD28 target copy number was determined using normal donor PBMC as reference and endogenous RNaseP as a normalizer. Mean ± SD of triplicates for each validation run is shown. (C) TCR Vβ analysis of day 28 and day 35 CAR + T cells. Data shows mean ± SD of three validation run CAR + T cells as compared to day 0 unmanipulated controls. (D) A representative genomic PCR showing lack of SB11 transposase integration. Genomic DNA (20 ng) was amplified using SB11 or GAPDH primers. CAR neg control T cells (lane 5) and CAR + T cells (lane 7) amplified using SB11 primers; CAR neg control T cells (lane 6), CAR + T cells (lane 8) and Jurkat stably expressing SB11 (lane 4) amplified using GAPDH primers. Jurkat stably expressing SB11 (Jurkat/SB11-IRES2-EGFP) (lane 3) and the linearized plasmid, pKan-CMV-SB11 (lane 2) amplified using SB11 primers were used as positive controls. (E) G-banded karyotypes of CAR + T cells from the three validation runs reveal no structural or numeric alteration. A representative spread from validation 2 is shown.
    Figure Legend Snippet: Safety profile associated with the SB system. (A) Telomere length of cells was measured using fluorescence in situ hybridization and flow cytometry (Flow-FISH) assay. Predominant T cell population at day 28 (V1 and V2, CD8 + T cells; V3, CD4 + T cells) was compared to respective miltenyi column purified subset of T cells from day 0. Mean ± SD of triplicates for each validation run is represented. (B) Genomic DNA from CAR + T cells at day 28 was amplified using primers and probes specific for CD19RCD28 CAR. Relative Quantity (RQ) analyses of the CD19RCD28 target copy number was determined using normal donor PBMC as reference and endogenous RNaseP as a normalizer. Mean ± SD of triplicates for each validation run is shown. (C) TCR Vβ analysis of day 28 and day 35 CAR + T cells. Data shows mean ± SD of three validation run CAR + T cells as compared to day 0 unmanipulated controls. (D) A representative genomic PCR showing lack of SB11 transposase integration. Genomic DNA (20 ng) was amplified using SB11 or GAPDH primers. CAR neg control T cells (lane 5) and CAR + T cells (lane 7) amplified using SB11 primers; CAR neg control T cells (lane 6), CAR + T cells (lane 8) and Jurkat stably expressing SB11 (lane 4) amplified using GAPDH primers. Jurkat stably expressing SB11 (Jurkat/SB11-IRES2-EGFP) (lane 3) and the linearized plasmid, pKan-CMV-SB11 (lane 2) amplified using SB11 primers were used as positive controls. (E) G-banded karyotypes of CAR + T cells from the three validation runs reveal no structural or numeric alteration. A representative spread from validation 2 is shown.

    Techniques Used: Fluorescence, In Situ Hybridization, Flow Cytometry, Cytometry, Fluorescence In Situ Hybridization, Purification, Amplification, Polymerase Chain Reaction, Stable Transfection, Expressing, Plasmid Preparation

    31) Product Images from "FIV establishes a latent infection in feline peripheral blood CD4+ T lymphocytes in vivo during the asymptomatic phase of infection"

    Article Title: FIV establishes a latent infection in feline peripheral blood CD4+ T lymphocytes in vivo during the asymptomatic phase of infection

    Journal: Retrovirology

    doi: 10.1186/1742-4690-9-12

    CD4+ T cells persistently infected in the absence of detectable plasma viral RNA . PBMCs (a), monocytes (b), CD4+CD25- T cells (c), and CD4+CD25+ T cells (d) isolated from blood from all FIV-infected cats were examined over time PI for viral DNA and vRNA by real-time PCR assays as described in Methods. Data from individual FIV-infected cats are plotted as solid black lines (165), medium-dashed lines (187), short-dashed lines (186) and dotted lines (184).
    Figure Legend Snippet: CD4+ T cells persistently infected in the absence of detectable plasma viral RNA . PBMCs (a), monocytes (b), CD4+CD25- T cells (c), and CD4+CD25+ T cells (d) isolated from blood from all FIV-infected cats were examined over time PI for viral DNA and vRNA by real-time PCR assays as described in Methods. Data from individual FIV-infected cats are plotted as solid black lines (165), medium-dashed lines (187), short-dashed lines (186) and dotted lines (184).

    Techniques Used: Infection, Isolation, Real-time Polymerase Chain Reaction

    32) Product Images from "Identification of Molecular Tumor Markers in Renal Cell Carcinomas with TFE3 Protein Expression by RNA Sequencing 1Identification of Molecular Tumor Markers in Renal Cell Carcinomas with TFE3 Protein Expression by RNA Sequencing 1 2"

    Article Title: Identification of Molecular Tumor Markers in Renal Cell Carcinomas with TFE3 Protein Expression by RNA Sequencing 1Identification of Molecular Tumor Markers in Renal Cell Carcinomas with TFE3 Protein Expression by RNA Sequencing 1 2

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

    doi:

    TMED6-COG8 ( TC ) read-through in RCC. (A) DNA: Schematic representation of the genomic structure of TMED6 and COG8 on the DNA (-) strand. RNA: Three isoforms were expressed with TCv1 being the prominent one. The TaqMan assay used to detect TC levels in
    Figure Legend Snippet: TMED6-COG8 ( TC ) read-through in RCC. (A) DNA: Schematic representation of the genomic structure of TMED6 and COG8 on the DNA (-) strand. RNA: Three isoforms were expressed with TCv1 being the prominent one. The TaqMan assay used to detect TC levels in

    Techniques Used: TaqMan Assay

    33) Product Images from "Determinants of Orofacial Clefting I: Effects of 5-Aza-2′-deoxycytidine on Cellular Processes and Gene Expression during Development of the First Branchial Arch"

    Article Title: Determinants of Orofacial Clefting I: Effects of 5-Aza-2′-deoxycytidine on Cellular Processes and Gene Expression during Development of the First Branchial Arch

    Journal: Reproductive toxicology (Elmsford, N.Y.)

    doi: 10.1016/j.reprotox.2016.11.016

    Photomicrographs of embryos exposed in utero on GD 9.5 to 1 mg/kg AzaD or vehicle, for 6, 9 and 12 hours (A) The region demarcated by the black line was excised from AzaD- or vehicle-exposed embryos for extraction of total RNA and genomic DNA. (B), (D) and (F) represent 6, 9 and 12 hour AzaD-exposed embryos, respectively; (C), (E) and (G) represent 6, 9 and 12 hour vehicle-exposed embryos, respectively. 1-BA = first branchial arch; 2-BA = second branchial arch.
    Figure Legend Snippet: Photomicrographs of embryos exposed in utero on GD 9.5 to 1 mg/kg AzaD or vehicle, for 6, 9 and 12 hours (A) The region demarcated by the black line was excised from AzaD- or vehicle-exposed embryos for extraction of total RNA and genomic DNA. (B), (D) and (F) represent 6, 9 and 12 hour AzaD-exposed embryos, respectively; (C), (E) and (G) represent 6, 9 and 12 hour vehicle-exposed embryos, respectively. 1-BA = first branchial arch; 2-BA = second branchial arch.

    Techniques Used: In Utero

    34) Product Images from "The Effect of Captivity on the Dynamics of Active Bacterial Communities Differs Between Two Deep-Sea Coral Species"

    Article Title: The Effect of Captivity on the Dynamics of Active Bacterial Communities Differs Between Two Deep-Sea Coral Species

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.02565

    Dispersion of the bacterial communities’ beta-diversity at different times of captivity for the DNA and RNA fraction in M. oculata (A) and L. pertusa (B) . The dispersion is expressed as the distance to the centroid for each Bray Curtis values computed between replicates of a same category.
    Figure Legend Snippet: Dispersion of the bacterial communities’ beta-diversity at different times of captivity for the DNA and RNA fraction in M. oculata (A) and L. pertusa (B) . The dispersion is expressed as the distance to the centroid for each Bray Curtis values computed between replicates of a same category.

    Techniques Used:

    Differences between DNA and RNA sample scores on the first CA axes for L. pertusa (A) and M. oculata (B) . Bars represent the standard error of the mean.
    Figure Legend Snippet: Differences between DNA and RNA sample scores on the first CA axes for L. pertusa (A) and M. oculata (B) . Bars represent the standard error of the mean.

    Techniques Used:

    Correspondence analysis of bacterial communities based on 16S rDNA (DNA) (A) and 16S rRNA (RNA) (B) . L. pertusa communities are marked as black open circles, M. oculata as blue open circles, and water as filled circles. The size of the circles corresponds to the time in captivity.
    Figure Legend Snippet: Correspondence analysis of bacterial communities based on 16S rDNA (DNA) (A) and 16S rRNA (RNA) (B) . L. pertusa communities are marked as black open circles, M. oculata as blue open circles, and water as filled circles. The size of the circles corresponds to the time in captivity.

    Techniques Used:

    35) Product Images from "Characterization of the fecal and mucosa-associated microbiota in dogs with colorectal epithelial tumors"

    Article Title: Characterization of the fecal and mucosa-associated microbiota in dogs with colorectal epithelial tumors

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0198342

    A scatterplot showing the ratio of live, potentially active bacteria (RNA/DNA) in tumor vs . non-tumor tissue.
    Figure Legend Snippet: A scatterplot showing the ratio of live, potentially active bacteria (RNA/DNA) in tumor vs . non-tumor tissue.

    Techniques Used:

    36) Product Images from "Giant Hydrogen Sulfide Plume in the Oxygen Minimum Zone off Peru Supports Chemolithoautotrophy"

    Article Title: Giant Hydrogen Sulfide Plume in the Oxygen Minimum Zone off Peru Supports Chemolithoautotrophy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0068661

    Vertical distribution of organisms involved in the sulfur cycle and abundance and taxonomic affiliation of transcripts encoding for cytochrome c oxidases. (A) H 2 S concentration and abundance of DNA sequences affiliated to organisms either capable of oxidizing or reducing inorganic sulfur species. Shown in percent of all DNA sequences (excluding rRNA genes) and summed according to their metabolic potentials. (B) Abundance of RNA sequences affiliated to organisms either capable of oxidizing or reducing inorganic sulfur species. Shown in percent of all RNA sequences (excluding rRNAs) and summed according to their metabolic potentials. (C) O 2 concentrations and transcript abundance of the (low-affinity) cytochrome c oxidase and the (high-affinity) cbb 3 -type cytochrome c oxidase (both EC 1.9.3.1). Shown in percent of all protein-coding RNA sequences. (D) Phylogenetic affiliation of the transcripts encoding for both types of the cytochrome c oxidase.
    Figure Legend Snippet: Vertical distribution of organisms involved in the sulfur cycle and abundance and taxonomic affiliation of transcripts encoding for cytochrome c oxidases. (A) H 2 S concentration and abundance of DNA sequences affiliated to organisms either capable of oxidizing or reducing inorganic sulfur species. Shown in percent of all DNA sequences (excluding rRNA genes) and summed according to their metabolic potentials. (B) Abundance of RNA sequences affiliated to organisms either capable of oxidizing or reducing inorganic sulfur species. Shown in percent of all RNA sequences (excluding rRNAs) and summed according to their metabolic potentials. (C) O 2 concentrations and transcript abundance of the (low-affinity) cytochrome c oxidase and the (high-affinity) cbb 3 -type cytochrome c oxidase (both EC 1.9.3.1). Shown in percent of all protein-coding RNA sequences. (D) Phylogenetic affiliation of the transcripts encoding for both types of the cytochrome c oxidase.

    Techniques Used: Concentration Assay

    Vertical distribution of dominant proteobacterial taxa. Shown in percent of all sequences (excluding rRNA genes and rRNAs). (A) DNA and (B) RNA sequence abundances for γ-proteobacteria. (C) DNA and (D) RNA sequence abundances for ε-proteobacteria. (E) DNA and (F) RNA sequence abundances for δ-proteobacteria.
    Figure Legend Snippet: Vertical distribution of dominant proteobacterial taxa. Shown in percent of all sequences (excluding rRNA genes and rRNAs). (A) DNA and (B) RNA sequence abundances for γ-proteobacteria. (C) DNA and (D) RNA sequence abundances for ε-proteobacteria. (E) DNA and (F) RNA sequence abundances for δ-proteobacteria.

    Techniques Used: Sequencing

    Vertical distribution of nitrogen transformation process rates and abundances of sequences encoding for involved enzymes. Shown in percent of all protein-coding DNA and RNA sequences, respectively. (A) NO 3 − reduction to N 2 (denitrification). (B) NO 3 − reduction to NO 2 − , respiratory nitrate reductase (EC 1.7.99.4). (C) NO 2 − reduction to N 2 , (NO forming) nitrite reductase (EC 1.7.2.1). (D) N 2 O reduction to N 2 , nitrous-oxide reductase (EC 1.7.2.4). (E) NO 2 − reduction to NH 4 + (DNRA), (NH 4 + forming) nitrite reductase (EC 1.7.2.2). (F) NO 2 − +NH 4 + to N 2 (anammox, based on the sole addition of NO 2 − ), hydrazine oxidoreductase (EC 1.7.99.8). Please note that at 40 m only NO 3 − reduction to N 2 (A) and NO 3 − reduction to NO 2 − (B) were measured.
    Figure Legend Snippet: Vertical distribution of nitrogen transformation process rates and abundances of sequences encoding for involved enzymes. Shown in percent of all protein-coding DNA and RNA sequences, respectively. (A) NO 3 − reduction to N 2 (denitrification). (B) NO 3 − reduction to NO 2 − , respiratory nitrate reductase (EC 1.7.99.4). (C) NO 2 − reduction to N 2 , (NO forming) nitrite reductase (EC 1.7.2.1). (D) N 2 O reduction to N 2 , nitrous-oxide reductase (EC 1.7.2.4). (E) NO 2 − reduction to NH 4 + (DNRA), (NH 4 + forming) nitrite reductase (EC 1.7.2.2). (F) NO 2 − +NH 4 + to N 2 (anammox, based on the sole addition of NO 2 − ), hydrazine oxidoreductase (EC 1.7.99.8). Please note that at 40 m only NO 3 − reduction to N 2 (A) and NO 3 − reduction to NO 2 − (B) were measured.

    Techniques Used: Transformation Assay

    37) Product Images from "High-throughput Characterization of HIV-1 Reservoir Reactivation Using a Single-Cell-in-Droplet PCR Assay"

    Article Title: High-throughput Characterization of HIV-1 Reservoir Reactivation Using a Single-Cell-in-Droplet PCR Assay

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2017.05.006

    Schema of the single cell encapsulation, lysis, HIV-1 detection, and rescue of cellular genomic DNA and mRNA. Cells are encapsulated in a master mix including PCR enzymes, primers, probes, and cell lysis agents (Supplementary Table 1) into oil by either brioprinting or microfluidic chip encapsulation (A, B) as described in the methods. Up to 20,000 droplets are then added to each well of a 96 well PCR plate. Cells are lysed within isolated droplet microenvironenments followed by PCR amplification of Tat-Rev spliced or unspliced cell-associated HIV-1 RNA (C). Cells are contained in the hydrophilic droplets which are stabilized by the oil-droplet interface. Droplets containing infected cells can be enumerated using an oil-based commercial flow cytometer or by direct visualization followed by microfluidic sorting of positive droplets (D). HIV-1 RNA positive or negative cells can be isolated using ultrafine needle aspiration, placed into individual microwell tubes or plate wells followed by droplet “cracking” ( i.e. lysing droplets to release nucleic acids) and further characterized. In this study, human or viral genomic DNA and human mRNA from droplets containing a single encapsulated cell or from bulk droplets containing HIV-1 RNA positive cells were quantified or sequenced.
    Figure Legend Snippet: Schema of the single cell encapsulation, lysis, HIV-1 detection, and rescue of cellular genomic DNA and mRNA. Cells are encapsulated in a master mix including PCR enzymes, primers, probes, and cell lysis agents (Supplementary Table 1) into oil by either brioprinting or microfluidic chip encapsulation (A, B) as described in the methods. Up to 20,000 droplets are then added to each well of a 96 well PCR plate. Cells are lysed within isolated droplet microenvironenments followed by PCR amplification of Tat-Rev spliced or unspliced cell-associated HIV-1 RNA (C). Cells are contained in the hydrophilic droplets which are stabilized by the oil-droplet interface. Droplets containing infected cells can be enumerated using an oil-based commercial flow cytometer or by direct visualization followed by microfluidic sorting of positive droplets (D). HIV-1 RNA positive or negative cells can be isolated using ultrafine needle aspiration, placed into individual microwell tubes or plate wells followed by droplet “cracking” ( i.e. lysing droplets to release nucleic acids) and further characterized. In this study, human or viral genomic DNA and human mRNA from droplets containing a single encapsulated cell or from bulk droplets containing HIV-1 RNA positive cells were quantified or sequenced.

    Techniques Used: Lysis, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Isolation, Amplification, Infection, Flow Cytometry, Cytometry

    Isolation of single scdPCR droplets based on HIV-1 usRNA fluorescence and subsequent human gDNA and mRNA characterization. Fluorescent droplets containing single HIV-1 transcriptionally active 8E5 or participant-derived CD4 + T cell lysates obtained by scdPCR were isolated and placed in single-cell DNA or RNA lysis buffers using an ultrafine gauge blunt needle (A). The percentages of individual droplet/cells with detectable CCR5 genomic DNA using real-time PCR or IPO8 mRNA using traditional ddPCR are shown in (B).
    Figure Legend Snippet: Isolation of single scdPCR droplets based on HIV-1 usRNA fluorescence and subsequent human gDNA and mRNA characterization. Fluorescent droplets containing single HIV-1 transcriptionally active 8E5 or participant-derived CD4 + T cell lysates obtained by scdPCR were isolated and placed in single-cell DNA or RNA lysis buffers using an ultrafine gauge blunt needle (A). The percentages of individual droplet/cells with detectable CCR5 genomic DNA using real-time PCR or IPO8 mRNA using traditional ddPCR are shown in (B).

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

    38) Product Images from "Redefining transcriptional regulation of the APOE gene and its association with Alzheimer’s disease"

    Article Title: Redefining transcriptional regulation of the APOE gene and its association with Alzheimer’s disease

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0227667

    Effects of DNA methylation, RNA/protein production, and protein binding in 5-aza-dC treated cell lines HepG2, LN-229, and SH-SY5Y. (A) Comparison of APOE DNA methylation and APOE RNA expression and secreted ApoE levels in 5-aza-dC treated cells. Levels from treated cells are plotted as fold change when compared with their untreated counterparts (set at 1.0, dashed line). The DNA methylation levels at the APOE CGI were quantified using bisulfite pyrosequencing. The expression levels of APOE RNAs (circRNA, full-length RNA, and total RNA) were quantified by qRT-PCR using TaqMan assay. The expression levels of secreted ApoE proteins were quantified by ELISA. (B) Binding of MECP2 protein in three APOE gene regions (promoter, exon 2, and CGI) between 5-aza-dC treated and untreated cells using ChIP-qPCR. Error bars represent three independent assays. *: t-test, p
    Figure Legend Snippet: Effects of DNA methylation, RNA/protein production, and protein binding in 5-aza-dC treated cell lines HepG2, LN-229, and SH-SY5Y. (A) Comparison of APOE DNA methylation and APOE RNA expression and secreted ApoE levels in 5-aza-dC treated cells. Levels from treated cells are plotted as fold change when compared with their untreated counterparts (set at 1.0, dashed line). The DNA methylation levels at the APOE CGI were quantified using bisulfite pyrosequencing. The expression levels of APOE RNAs (circRNA, full-length RNA, and total RNA) were quantified by qRT-PCR using TaqMan assay. The expression levels of secreted ApoE proteins were quantified by ELISA. (B) Binding of MECP2 protein in three APOE gene regions (promoter, exon 2, and CGI) between 5-aza-dC treated and untreated cells using ChIP-qPCR. Error bars represent three independent assays. *: t-test, p

    Techniques Used: DNA Methylation Assay, Protein Binding, RNA Expression, Expressing, Quantitative RT-PCR, TaqMan Assay, Enzyme-linked Immunosorbent Assay, Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Total RNA expression versus DNA methylation levels in frontal lobe. Total APOE RNA ΔCt is plotted against mean methylation across DMR 1 (CpG site #11–37). (A) All frontal lobe samples (includes both AD and control subjects). (B) Plot separating AD (red) from control (blue). Dashed lines and p-values are associated with the respective fitted linear regression models. Note that lower ΔCt values represent higher expression levels. AD: Alzheimer’s disease; Ct: cycle threshold; DMR 1: differentially methylated region 1.
    Figure Legend Snippet: Total RNA expression versus DNA methylation levels in frontal lobe. Total APOE RNA ΔCt is plotted against mean methylation across DMR 1 (CpG site #11–37). (A) All frontal lobe samples (includes both AD and control subjects). (B) Plot separating AD (red) from control (blue). Dashed lines and p-values are associated with the respective fitted linear regression models. Note that lower ΔCt values represent higher expression levels. AD: Alzheimer’s disease; Ct: cycle threshold; DMR 1: differentially methylated region 1.

    Techniques Used: RNA Expression, DNA Methylation Assay, Methylation, Expressing

    39) Product Images from "CTLA-4+PD-1− memory CD4+ T cells critically contribute to viral persistence in antiretroviral therapy-suppressed, SIV-infected rhesus macaques"

    Article Title: CTLA-4+PD-1− memory CD4+ T cells critically contribute to viral persistence in antiretroviral therapy-suppressed, SIV-infected rhesus macaques

    Journal: Immunity

    doi: 10.1016/j.immuni.2017.09.018

    CTLA-4 + PD-1 − memory CD4 + T cells harbor higher amounts of SIV DNA following ART-mediated, viral load suppression ( A )Study design. Ten RMs were infected i.v. with 1000 TCID50 SIVmac251 (day 0), and at 7 weeks post-infection, initiated ART (PMPA, FTC, raltegravir, and ritonavir-boosted darunavir). All animals were maintained on ART regimen until plasma viremia was undetectable for at least 3 months. Peripheral blood (WB), rectal biopsy (Gut), and lymph node (LN) biopsies were collected at the indicated time points and multiple organs were harvested at elective necropsy. Sorting of memory CD4 + T cells by Co-IR expression was performed at two time points during ART: first, at Mid ART (approximately 1 month following undetectable viremia); and second, at necropsy. ( B ) Plasma viral loads are shown for the 10 individual RMs, quantified using the standard qRT-PCR assay (limit of detection, LOD, of 60 SIV RNA copies/mL of plasma represented by the horizontal dotted line). Undetectable measurements are plotted as one-half of the LOD (30 copies/mL). ( C ) Frequencies (of live CD3 + T cells) of CD4 + T cells were longitudinally measured in WB, LN, and gut biopsies. The gray shaded area represents time on ART; Nx represents the measured values from animal necropsy. Repeated-measures analyses were performed using a means model (SAS Mixed Procedure, version 9.4) to determine statistical significance, with indicated tests of significance representing comparison to pre-SIV infection (WB, Gut) or pre-ART initiation (LN). ( D ) Representative SIV DNA quantities in the PBMCs, LN, spleen, and gut tissues for an individual RM (RLr10) after 206 days of viral load suppression (n=9). ( E ) Cell-associated SIV GAG .
    Figure Legend Snippet: CTLA-4 + PD-1 − memory CD4 + T cells harbor higher amounts of SIV DNA following ART-mediated, viral load suppression ( A )Study design. Ten RMs were infected i.v. with 1000 TCID50 SIVmac251 (day 0), and at 7 weeks post-infection, initiated ART (PMPA, FTC, raltegravir, and ritonavir-boosted darunavir). All animals were maintained on ART regimen until plasma viremia was undetectable for at least 3 months. Peripheral blood (WB), rectal biopsy (Gut), and lymph node (LN) biopsies were collected at the indicated time points and multiple organs were harvested at elective necropsy. Sorting of memory CD4 + T cells by Co-IR expression was performed at two time points during ART: first, at Mid ART (approximately 1 month following undetectable viremia); and second, at necropsy. ( B ) Plasma viral loads are shown for the 10 individual RMs, quantified using the standard qRT-PCR assay (limit of detection, LOD, of 60 SIV RNA copies/mL of plasma represented by the horizontal dotted line). Undetectable measurements are plotted as one-half of the LOD (30 copies/mL). ( C ) Frequencies (of live CD3 + T cells) of CD4 + T cells were longitudinally measured in WB, LN, and gut biopsies. The gray shaded area represents time on ART; Nx represents the measured values from animal necropsy. Repeated-measures analyses were performed using a means model (SAS Mixed Procedure, version 9.4) to determine statistical significance, with indicated tests of significance representing comparison to pre-SIV infection (WB, Gut) or pre-ART initiation (LN). ( D ) Representative SIV DNA quantities in the PBMCs, LN, spleen, and gut tissues for an individual RM (RLr10) after 206 days of viral load suppression (n=9). ( E ) Cell-associated SIV GAG .

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

    40) Product Images from "Genome-Scale Oscillations in DNA Methylation during Exit from Pluripotency"

    Article Title: Genome-Scale Oscillations in DNA Methylation during Exit from Pluripotency

    Journal: Cell Systems

    doi: 10.1016/j.cels.2018.06.012

    Oscillatory Dynamics of DNA Methylation during Transition from Naïve to Primed Pluripotency In Vitro Naïve ESCs were transferred to primed conditions in two independent “2i release” experiments. In the initial experiment, triplicate samples were collected at each time point, and BS-seq and RNA-seq libraries were prepared (A–F). In the second, single samples were collected at each time point and AmpBS-seq data are presented in (D). (A) Average DNA methylation at H3K4me1 sites over the time course. For the average, we took into account 50% of enhancers with the highest coverage depth over the time course (n = 10,324). (B) Average methylation at promoter regions (n = 637) and (C) exons (n = 4,990). (D) Average spectral densities for different genomic features calculated from whole genome BS-seq data (see also Figure S4 C and STAR Methods ). Green dots denote significant enrichment of a given period (p
    Figure Legend Snippet: Oscillatory Dynamics of DNA Methylation during Transition from Naïve to Primed Pluripotency In Vitro Naïve ESCs were transferred to primed conditions in two independent “2i release” experiments. In the initial experiment, triplicate samples were collected at each time point, and BS-seq and RNA-seq libraries were prepared (A–F). In the second, single samples were collected at each time point and AmpBS-seq data are presented in (D). (A) Average DNA methylation at H3K4me1 sites over the time course. For the average, we took into account 50% of enhancers with the highest coverage depth over the time course (n = 10,324). (B) Average methylation at promoter regions (n = 637) and (C) exons (n = 4,990). (D) Average spectral densities for different genomic features calculated from whole genome BS-seq data (see also Figure S4 C and STAR Methods ). Green dots denote significant enrichment of a given period (p

    Techniques Used: DNA Methylation Assay, In Vitro, RNA Sequencing Assay, Methylation

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    Qiagen allprep dna rna mini kit
    Comparison on taxonomic level of <t>DNA</t> purification using protocol 1 and commonly used DNA purification methods. The 16S sequence comparison bar charts are made using Classifier ( http://rdp.cme.msu.edu/classifier/classifier.jsp ). The significant differences of Firmicutes between paired libraries were calculated by the Library Compare Tool using a confidence threshold of 80 % ( http://rdp.cme.msu.edu/comparison ). Protocol 1: Mechanical pre-treatment only, followed by purification with <t>AllPrep</t> <t>DNA/RNA</t> Mini Kit as described in Additional file 1 ; DNA kit 1, 2 and 3: A combination of mechanical and enzymatic pre-treatments as recommended by Qiagen for lysis of Gram-positive bacteria, followed by purificaton with AllPrep DNA/RNA Mini Kit (kit 1), QIAamp DNA Stool Mini Kit (kit 2) and DNeasy Blood Tissue Kit (kit 3) as described in Additional file 1 . n number of clones sequenced
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    Comparison on taxonomic level of DNA purification using protocol 1 and commonly used DNA purification methods. The 16S sequence comparison bar charts are made using Classifier ( http://rdp.cme.msu.edu/classifier/classifier.jsp ). The significant differences of Firmicutes between paired libraries were calculated by the Library Compare Tool using a confidence threshold of 80 % ( http://rdp.cme.msu.edu/comparison ). Protocol 1: Mechanical pre-treatment only, followed by purification with AllPrep DNA/RNA Mini Kit as described in Additional file 1 ; DNA kit 1, 2 and 3: A combination of mechanical and enzymatic pre-treatments as recommended by Qiagen for lysis of Gram-positive bacteria, followed by purificaton with AllPrep DNA/RNA Mini Kit (kit 1), QIAamp DNA Stool Mini Kit (kit 2) and DNeasy Blood Tissue Kit (kit 3) as described in Additional file 1 . n number of clones sequenced

    Journal: BMC Research Notes

    Article Title: Simultaneous purification of DNA and RNA from microbiota in a single colonic mucosal biopsy

    doi: 10.1186/s13104-016-2110-7

    Figure Lengend Snippet: Comparison on taxonomic level of DNA purification using protocol 1 and commonly used DNA purification methods. The 16S sequence comparison bar charts are made using Classifier ( http://rdp.cme.msu.edu/classifier/classifier.jsp ). The significant differences of Firmicutes between paired libraries were calculated by the Library Compare Tool using a confidence threshold of 80 % ( http://rdp.cme.msu.edu/comparison ). Protocol 1: Mechanical pre-treatment only, followed by purification with AllPrep DNA/RNA Mini Kit as described in Additional file 1 ; DNA kit 1, 2 and 3: A combination of mechanical and enzymatic pre-treatments as recommended by Qiagen for lysis of Gram-positive bacteria, followed by purificaton with AllPrep DNA/RNA Mini Kit (kit 1), QIAamp DNA Stool Mini Kit (kit 2) and DNeasy Blood Tissue Kit (kit 3) as described in Additional file 1 . n number of clones sequenced

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    Techniques: DNA Purification, Sequencing, Purification, Lysis, Clone Assay

    TLR-9 mRNA expression in colon cancer cells and colon cancer tissues. Notes: Total RNA of tissues was extracted from matching normal and colon cancer tissues, reverse-transcribed into cDNA, and then used to measure TLR-9 mRNA expression with specific primers. TLR-9 expression in colon cancer tissues and matching control tissues is shown as mean ± SD. Abbreviations: cDNA, complementary DNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

    Journal: OncoTargets and therapy

    Article Title: Association between TLR-9 polymorphisms and colon cancer susceptibility in Saudi Arabian female patients

    doi: 10.2147/OTT.S106024

    Figure Lengend Snippet: TLR-9 mRNA expression in colon cancer cells and colon cancer tissues. Notes: Total RNA of tissues was extracted from matching normal and colon cancer tissues, reverse-transcribed into cDNA, and then used to measure TLR-9 mRNA expression with specific primers. TLR-9 expression in colon cancer tissues and matching control tissues is shown as mean ± SD. Abbreviations: cDNA, complementary DNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

    Article Snippet: Total RNA isolation Total RNA was extracted from 40 colon cancer tissues and 40 matched normal colon tissues using an AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol.

    Techniques: Expressing