dna methylation analysis genomic dna  (Qiagen)


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    AllPrep DNA RNA Mini Kit
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    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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    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 "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

    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 "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:

    13) 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

    14) 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

    15) 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:

    16) 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

    17) Product Images from "Loss of spermatogonia and wide-spread DNA methylation defects in newborn male mice deficient in DNMT3L"

    Article Title: Loss of spermatogonia and wide-spread DNA methylation defects in newborn male mice deficient in DNMT3L

    Journal: BMC Developmental Biology

    doi: 10.1186/1471-213X-7-104

    Dynamic expression of Dnmt3L in male germ cells . A) Relative quantification of Dnmt3L expression in postnatal male germ cells. Real-time RT-PCR was used to determine the expression profile of Dnmt3L in primitive type A (PA), type A (A) and type B (B) spermatogonia, preleptotene (PL), leptotene/zygotene (L/Z), prepubertal pachytene (PP) and pachytene (P) spermatocytes, as well as round spermatids (RS) and residual bodies/elongating spermatids (RB). Expression was determined in triplicate in each of the two series of germ cells and normalized to 18S expression; normalized results were calibrated to expression in pachytene spermatocytes. Shown here are the mean expression results obtained for one series. Mean ± SD. B) Relative quantification of Dnmt3L expression in prenatal male germ cells. Quantitative RT-PCR was used to measure the expression levels of Dnmt3L in total RNA extracted from E13.5, E15.5 and E18.5 prospermatogonia and 6 dpp primitive type A spermatogonia (PA). Expression of Dnmt3L was determined in triplicate in each of the two series of germ cells and normalized to 18S expression; normalized values were calibrated to the expression found in E13.5 gonocytes. Shown here are the mean expression results obtained for one series. Mean ± SD.
    Figure Legend Snippet: Dynamic expression of Dnmt3L in male germ cells . A) Relative quantification of Dnmt3L expression in postnatal male germ cells. Real-time RT-PCR was used to determine the expression profile of Dnmt3L in primitive type A (PA), type A (A) and type B (B) spermatogonia, preleptotene (PL), leptotene/zygotene (L/Z), prepubertal pachytene (PP) and pachytene (P) spermatocytes, as well as round spermatids (RS) and residual bodies/elongating spermatids (RB). Expression was determined in triplicate in each of the two series of germ cells and normalized to 18S expression; normalized results were calibrated to expression in pachytene spermatocytes. Shown here are the mean expression results obtained for one series. Mean ± SD. B) Relative quantification of Dnmt3L expression in prenatal male germ cells. Quantitative RT-PCR was used to measure the expression levels of Dnmt3L in total RNA extracted from E13.5, E15.5 and E18.5 prospermatogonia and 6 dpp primitive type A spermatogonia (PA). Expression of Dnmt3L was determined in triplicate in each of the two series of germ cells and normalized to 18S expression; normalized values were calibrated to the expression found in E13.5 gonocytes. Shown here are the mean expression results obtained for one series. Mean ± SD.

    Techniques Used: Expressing, Quantitative RT-PCR

    18) Product Images from "Loss of spermatogonia and wide-spread DNA methylation defects in newborn male mice deficient in DNMT3L"

    Article Title: Loss of spermatogonia and wide-spread DNA methylation defects in newborn male mice deficient in DNMT3L

    Journal: BMC Developmental Biology

    doi: 10.1186/1471-213X-7-104

    Dynamic expression of Dnmt3L in male germ cells . A) Relative quantification of Dnmt3L expression in postnatal male germ cells. Real-time RT-PCR was used to determine the expression profile of Dnmt3L in primitive type A (PA), type A (A) and type B (B) spermatogonia, preleptotene (PL), leptotene/zygotene (L/Z), prepubertal pachytene (PP) and pachytene (P) spermatocytes, as well as round spermatids (RS) and residual bodies/elongating spermatids (RB). Expression was determined in triplicate in each of the two series of germ cells and normalized to 18S expression; normalized results were calibrated to expression in pachytene spermatocytes. Shown here are the mean expression results obtained for one series. Mean ± SD. B) Relative quantification of Dnmt3L expression in prenatal male germ cells. Quantitative RT-PCR was used to measure the expression levels of Dnmt3L in total RNA extracted from E13.5, E15.5 and E18.5 prospermatogonia and 6 dpp primitive type A spermatogonia (PA). Expression of Dnmt3L was determined in triplicate in each of the two series of germ cells and normalized to 18S expression; normalized values were calibrated to the expression found in E13.5 gonocytes. Shown here are the mean expression results obtained for one series. Mean ± SD.
    Figure Legend Snippet: Dynamic expression of Dnmt3L in male germ cells . A) Relative quantification of Dnmt3L expression in postnatal male germ cells. Real-time RT-PCR was used to determine the expression profile of Dnmt3L in primitive type A (PA), type A (A) and type B (B) spermatogonia, preleptotene (PL), leptotene/zygotene (L/Z), prepubertal pachytene (PP) and pachytene (P) spermatocytes, as well as round spermatids (RS) and residual bodies/elongating spermatids (RB). Expression was determined in triplicate in each of the two series of germ cells and normalized to 18S expression; normalized results were calibrated to expression in pachytene spermatocytes. Shown here are the mean expression results obtained for one series. Mean ± SD. B) Relative quantification of Dnmt3L expression in prenatal male germ cells. Quantitative RT-PCR was used to measure the expression levels of Dnmt3L in total RNA extracted from E13.5, E15.5 and E18.5 prospermatogonia and 6 dpp primitive type A spermatogonia (PA). Expression of Dnmt3L was determined in triplicate in each of the two series of germ cells and normalized to 18S expression; normalized values were calibrated to the expression found in E13.5 gonocytes. Shown here are the mean expression results obtained for one series. Mean ± SD.

    Techniques Used: Expressing, Quantitative RT-PCR

    19) 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

    20) 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

    21) 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

    22) 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:

    23) Product Images from "Multiscale, multimodal analysis of tumor heterogeneity in IDH1 mutant vs wild-type diffuse gliomas"

    Article Title: Multiscale, multimodal analysis of tumor heterogeneity in IDH1 mutant vs wild-type diffuse gliomas

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0219724

    Comprehensive rendering of multiscale measurements in gliomas. Multiscale modalities depicted include: 1) clinical information (red), 2) IDH1 mutational status (blue), 3) MRI derived variables (green), 4) RNA expression level of genes involved in the “inducing angiogenesis” hallmark (black), and 5) MxIF angiogenesis markers or cell clusters (magenta). The data is binned in low, medium and high categories. Across the treatment-naïve gliomas (a) and the recurrent (post-treatment) glioblastoma* (b) cohorts, it can be observed that IDHmt patients have low angiogenesis according to RNA expression levels and expression of S100A4 and VEGRF. Those subjects also have high fraction of cells in clusters 1 and 2 (low angiogenesis markers), and low fraction of cells in cluster 4 and 5 (high angiogenesis markers ( S8 Fig , cluster profiles of angiogenesis clusters). Moreover, MR Images for the same subjects have lower normalized enhancing cores volumes and measure higher intensities on T1 post contrast. *Recurrent GBM (5 subjects are not shown since they were missing MxIF).
    Figure Legend Snippet: Comprehensive rendering of multiscale measurements in gliomas. Multiscale modalities depicted include: 1) clinical information (red), 2) IDH1 mutational status (blue), 3) MRI derived variables (green), 4) RNA expression level of genes involved in the “inducing angiogenesis” hallmark (black), and 5) MxIF angiogenesis markers or cell clusters (magenta). The data is binned in low, medium and high categories. Across the treatment-naïve gliomas (a) and the recurrent (post-treatment) glioblastoma* (b) cohorts, it can be observed that IDHmt patients have low angiogenesis according to RNA expression levels and expression of S100A4 and VEGRF. Those subjects also have high fraction of cells in clusters 1 and 2 (low angiogenesis markers), and low fraction of cells in cluster 4 and 5 (high angiogenesis markers ( S8 Fig , cluster profiles of angiogenesis clusters). Moreover, MR Images for the same subjects have lower normalized enhancing cores volumes and measure higher intensities on T1 post contrast. *Recurrent GBM (5 subjects are not shown since they were missing MxIF).

    Techniques Used: Magnetic Resonance Imaging, Derivative Assay, RNA Expression, Expressing

    24) 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

    25) 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

    26) 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

    27) 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

    28) 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:

    29) 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

    30) 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

    31) 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

    32) 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

    33) Product Images from "Prognostic significance of frequent CLDN18-ARHGAP26/6 fusion in gastric signet-ring cell cancer"

    Article Title: Prognostic significance of frequent CLDN18-ARHGAP26/6 fusion in gastric signet-ring cell cancer

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04907-0

    Somatic copy number variations and structure variation in HSRCC. a Somatic structure variations of all patients were combined and illustrated with CIRCOS plot. Translocations between CLDN18 and ARHGAP26/6 were highlighted in red line. Recurrent mutated genes (SNV/INDEL only) were indicated in the outlier of rim and the SMGs were labeled in red (including ARHGAPs and ARHGEFs ). Cytoband was illustrated in the inner ring, followed by illustration of copy number alteration (orange represent gain and green represent loss). Structure variations were shown inside of the CIRCOS plot, red lines represent the recurrent CLDN18-ARHGAP26/6 fusions, green and black lines represent inter-chromosomal and intra-chromosomal translocations. b Illustration of breakpoint of CLDN18 and ARHGAP26/6 in DNA level (upper arrows) and RNA level (lower arrows). Fusions of CLDN18 with exon 10 of ARHGAP26 , exon 12, and ARHGAP6 were indicated with green, purple and orange upper arrows in DNA, respectively. The junctions of CLDN18 and ARHGAPs in RNA level were indicated with red, yellow, and blue lower arrows or dashed lines in the gene map demonstration and Sanger sequencing graphs, respectively
    Figure Legend Snippet: Somatic copy number variations and structure variation in HSRCC. a Somatic structure variations of all patients were combined and illustrated with CIRCOS plot. Translocations between CLDN18 and ARHGAP26/6 were highlighted in red line. Recurrent mutated genes (SNV/INDEL only) were indicated in the outlier of rim and the SMGs were labeled in red (including ARHGAPs and ARHGEFs ). Cytoband was illustrated in the inner ring, followed by illustration of copy number alteration (orange represent gain and green represent loss). Structure variations were shown inside of the CIRCOS plot, red lines represent the recurrent CLDN18-ARHGAP26/6 fusions, green and black lines represent inter-chromosomal and intra-chromosomal translocations. b Illustration of breakpoint of CLDN18 and ARHGAP26/6 in DNA level (upper arrows) and RNA level (lower arrows). Fusions of CLDN18 with exon 10 of ARHGAP26 , exon 12, and ARHGAP6 were indicated with green, purple and orange upper arrows in DNA, respectively. The junctions of CLDN18 and ARHGAPs in RNA level were indicated with red, yellow, and blue lower arrows or dashed lines in the gene map demonstration and Sanger sequencing graphs, respectively

    Techniques Used: Labeling, Sequencing

    34) 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

    35) 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

    36) 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

    37) Product Images from "Conventional Chemotherapy and Oncogenic Pathway Targeting in Ovarian Carcinosarcoma Using a Patient-Derived Tumorgraft"

    Article Title: Conventional Chemotherapy and Oncogenic Pathway Targeting in Ovarian Carcinosarcoma Using a Patient-Derived Tumorgraft

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0126867

    Potential targets of novel therapies. (A) Receptor tyrosine kinase (RTK) dot blot array. Goat IgG and phosphate buffered saline (PBS) are background controls. Each RTK is blotted in duplicate. (B) The proportion of signal contributed by select RTKs in serum free media (SFM), complete media (CM), and in tissues harvested after engraftment in a mouse ( in vivo ). (C) Microarray analysis for expression of EGFR, VEGFC, and IRS1 in model PH003 (red shape) and 35 other ovarian patient-derived tumorgraft models (black shapes). (D) Array comparative genomic hybridization showing chromosome 7. Red dots represent probes from the tumor genome and Green dots represent probes from the patient’s germline DNA. A balance of Red and Green probes signifies equal representation of tumor and germline DNA in the q arm. The p arm shows an unbalanced representation of tumor genomic DNA (log2 ratio 0.399 compared to germline). The bold vertical jagged line shows the average log2 ratio of tumor and germline DNA along chromosome 7.
    Figure Legend Snippet: Potential targets of novel therapies. (A) Receptor tyrosine kinase (RTK) dot blot array. Goat IgG and phosphate buffered saline (PBS) are background controls. Each RTK is blotted in duplicate. (B) The proportion of signal contributed by select RTKs in serum free media (SFM), complete media (CM), and in tissues harvested after engraftment in a mouse ( in vivo ). (C) Microarray analysis for expression of EGFR, VEGFC, and IRS1 in model PH003 (red shape) and 35 other ovarian patient-derived tumorgraft models (black shapes). (D) Array comparative genomic hybridization showing chromosome 7. Red dots represent probes from the tumor genome and Green dots represent probes from the patient’s germline DNA. A balance of Red and Green probes signifies equal representation of tumor and germline DNA in the q arm. The p arm shows an unbalanced representation of tumor genomic DNA (log2 ratio 0.399 compared to germline). The bold vertical jagged line shows the average log2 ratio of tumor and germline DNA along chromosome 7.

    Techniques Used: Dot Blot, In Vivo, Microarray, Expressing, Derivative Assay, Hybridization

    38) Product Images from "Integrative analysis of methylome and transcriptome in human blood identifies extensive sex- and immune cell-specific differentially methylated regions"

    Article Title: Integrative analysis of methylome and transcriptome in human blood identifies extensive sex- and immune cell-specific differentially methylated regions

    Journal: Epigenetics

    doi: 10.1080/15592294.2015.1084462

    Validation of cell specific genes confirmed BeadChip results with no significant difference between males and females. Heat plots presenting average RNA expression ( A ) and DNA methylation ( B ) with gene names on the Y-axis and cell subsets on the X-axis.
    Figure Legend Snippet: Validation of cell specific genes confirmed BeadChip results with no significant difference between males and females. Heat plots presenting average RNA expression ( A ) and DNA methylation ( B ) with gene names on the Y-axis and cell subsets on the X-axis.

    Techniques Used: RNA Expression, DNA Methylation Assay

    39) Product Images from "A Next-Generation Sequencing-Based Molecular Approach to Characterize a Tick Vector in Lyme Disease"

    Article Title: A Next-Generation Sequencing-Based Molecular Approach to Characterize a Tick Vector in Lyme Disease

    Journal: OMICS : a Journal of Integrative Biology

    doi: 10.1089/omi.2018.0089

    Workflow for analysis of next-generation sequencing data. Workflow employed for analysis of (A) whole-genome and (B) RNA-seq sequencing data. Genomic DNA and RNA sequences for Ixodida (taxonomy ID: txid6935) and bacterial genomes were downloaded from
    Figure Legend Snippet: Workflow for analysis of next-generation sequencing data. Workflow employed for analysis of (A) whole-genome and (B) RNA-seq sequencing data. Genomic DNA and RNA sequences for Ixodida (taxonomy ID: txid6935) and bacterial genomes were downloaded from

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

    40) 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

    41) Product Images from "Multiplexed Spliced-Leader Sequencing: A high-throughput, selective method for RNA-seq in Trypanosomatids"

    Article Title: Multiplexed Spliced-Leader Sequencing: A high-throughput, selective method for RNA-seq in Trypanosomatids

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-03987-0

    Graphical summary of the SL-seq protocol. (A) cDNA generation with Superscript III and a primer that is partially random (7 nucleotides, grey), and partially fixed (yellow). Consequent degradation of the RNA strand with RNAse H, leaving a single stranded DNA molecule. (B) Second strand synthesis of SL-containing DNA molecules with Klenow fragment and a primer that is complementary with the SL (dark blue). (C + D) PCR for amplification and addition of adapter motives (red and purple) making the library compatible with the Nextera XT index kit (Illumina). (E + F) Final PCR adapter extension, indexing (orange and light blue) and amplification of the library fragments with the primers of the Nextera XT index kit. Dark Blue: SL/ complementary with SL, light grey: RNA, dark grey: DNA, green: poly-A tail. Other colors: primer and adapters sequences.
    Figure Legend Snippet: Graphical summary of the SL-seq protocol. (A) cDNA generation with Superscript III and a primer that is partially random (7 nucleotides, grey), and partially fixed (yellow). Consequent degradation of the RNA strand with RNAse H, leaving a single stranded DNA molecule. (B) Second strand synthesis of SL-containing DNA molecules with Klenow fragment and a primer that is complementary with the SL (dark blue). (C + D) PCR for amplification and addition of adapter motives (red and purple) making the library compatible with the Nextera XT index kit (Illumina). (E + F) Final PCR adapter extension, indexing (orange and light blue) and amplification of the library fragments with the primers of the Nextera XT index kit. Dark Blue: SL/ complementary with SL, light grey: RNA, dark grey: DNA, green: poly-A tail. Other colors: primer and adapters sequences.

    Techniques Used: Polymerase Chain Reaction, Amplification

    42) Product Images from "The loss of CCR6+ and CD161+ CD4+ T cell homeostasis contributes to disease progression in SIV-infected rhesus macaques"

    Article Title: The loss of CCR6+ and CD161+ CD4+ T cell homeostasis contributes to disease progression in SIV-infected rhesus macaques

    Journal: Mucosal immunology

    doi: 10.1038/mi.2016.116

    CD161+CCR6− CD4+ T cells harbor the highest levels of SIV RNA in lymphoid tissues (A) Levels of cell-associated SIV gag DNA were quantified from CCR6 and CD161− sorted subsets in the blood and LN of 10 SIV-infected RMs. Representative SIV RNA levels in the PBMCs (B) and LN (C) for 3 RMs during chronic SIV infection. Cumulative data for SIV RNA levels in the CCR6 and CD161 memory CD4+ T cell subsets is shown in the scatter plots. DNA and RNA measurements were excluded for samples in which
    Figure Legend Snippet: CD161+CCR6− CD4+ T cells harbor the highest levels of SIV RNA in lymphoid tissues (A) Levels of cell-associated SIV gag DNA were quantified from CCR6 and CD161− sorted subsets in the blood and LN of 10 SIV-infected RMs. Representative SIV RNA levels in the PBMCs (B) and LN (C) for 3 RMs during chronic SIV infection. Cumulative data for SIV RNA levels in the CCR6 and CD161 memory CD4+ T cell subsets is shown in the scatter plots. DNA and RNA measurements were excluded for samples in which

    Techniques Used: Infection

    43) Product Images from "Functional characterization of enhancer evolution in the primate lineage"

    Article Title: Functional characterization of enhancer evolution in the primate lineage

    Journal: Genome Biology

    doi: 10.1186/s13059-018-1473-6

    Schematic of Experimental Design. a We identified potential hominoid-specific enhancers by intersecting hominoid-specific ChIP-seq predicted enhancers from primary human liver with ChromHMM-predicted strong enhancers in HepG2 cells (screenshot from http://genome.ucsc.edu ) [ 54 ]. We then tiled across each candidate enhancer using 194 nt sequences and identified 697 tiles that were active in the STARR-seq reporter assay in HepG2 cells. b We located orthologous sequences in 11 primates and computationally reconstructed 9 ancestral sequences for 348 of the active tiles, using New World monkeys as an outgroup. c We then cloned all 20 present-day or ancestral orthologs per tile and performed STARR-seq again in HepG2 cells. After collecting DNA and RNA from cells, we calculated enrichment scores as the log 2 ratio of RNA to DNA for each ortholog. Each shade of red represents a different ortholog tested
    Figure Legend Snippet: Schematic of Experimental Design. a We identified potential hominoid-specific enhancers by intersecting hominoid-specific ChIP-seq predicted enhancers from primary human liver with ChromHMM-predicted strong enhancers in HepG2 cells (screenshot from http://genome.ucsc.edu ) [ 54 ]. We then tiled across each candidate enhancer using 194 nt sequences and identified 697 tiles that were active in the STARR-seq reporter assay in HepG2 cells. b We located orthologous sequences in 11 primates and computationally reconstructed 9 ancestral sequences for 348 of the active tiles, using New World monkeys as an outgroup. c We then cloned all 20 present-day or ancestral orthologs per tile and performed STARR-seq again in HepG2 cells. After collecting DNA and RNA from cells, we calculated enrichment scores as the log 2 ratio of RNA to DNA for each ortholog. Each shade of red represents a different ortholog tested

    Techniques Used: Chromatin Immunoprecipitation, Reporter Assay, Clone Assay

    44) Product Images from "The prevalence and transcriptional activity of the mucosal microbiota of ulcerative colitis patients"

    Article Title: The prevalence and transcriptional activity of the mucosal microbiota of ulcerative colitis patients

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-35243-4

    Average log transformed abundances of bacterial families in the total and active microbiota. The diagonal line indicates identical abundances in the DNA and RNA datasets. The colour intensity shows how much the abundance of the bacterial families correlate.
    Figure Legend Snippet: Average log transformed abundances of bacterial families in the total and active microbiota. The diagonal line indicates identical abundances in the DNA and RNA datasets. The colour intensity shows how much the abundance of the bacterial families correlate.

    Techniques Used: Transformation Assay

    Alpha diversity in inflamed and non-inflamed tissue. Distribution of alpha diversity in the inflamed and non-inflamed samples from UC patients and control patients, showing ( a ) the DNA and ( b ) the RNA datasets, and ( c ) a scatterplot of the alpha diversity in the paired RNA and DNA samples, with the average alpha diversity on the x-axis and the difference on the y-axis.
    Figure Legend Snippet: Alpha diversity in inflamed and non-inflamed tissue. Distribution of alpha diversity in the inflamed and non-inflamed samples from UC patients and control patients, showing ( a ) the DNA and ( b ) the RNA datasets, and ( c ) a scatterplot of the alpha diversity in the paired RNA and DNA samples, with the average alpha diversity on the x-axis and the difference on the y-axis.

    Techniques Used:

    UniFrac distances between different diagnosis groups and datasets. PCoA plot for (a ) the DNA dataset and ( b) the RNA dataset. Each sample is coloured according to the disease status and inflammation status. The black lines connect the samples from the same patient. c) PCoA plot of a combined analysis from both the DNA and RNA dataset. Paired RNA-DNA samples are connected with black lines.
    Figure Legend Snippet: UniFrac distances between different diagnosis groups and datasets. PCoA plot for (a ) the DNA dataset and ( b) the RNA dataset. Each sample is coloured according to the disease status and inflammation status. The black lines connect the samples from the same patient. c) PCoA plot of a combined analysis from both the DNA and RNA dataset. Paired RNA-DNA samples are connected with black lines.

    Techniques Used:

    45) 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

    46) Product Images from "Allele-specific gene expression patterns in primary leukemic cells reveal regulation of gene expression by CpG site methylation"

    Article Title: Allele-specific gene expression patterns in primary leukemic cells reveal regulation of gene expression by CpG site methylation

    Journal: Genome Research

    doi: 10.1101/gr.083931.108

    Genotyping by the NS-12 BeadChips to detect allele-specific gene expression. ( A ) Correlation between the allele fractions determined in replicate DNA samples for 3531 expressed SNPs in one ALL sample. The median correlation between the allele fraction obtained in replicate assays in all 197 samples was 0.9969 (range 0.9934–0.9986). ( B ) Correlation between the allele fractions determined by genotyping the same 3531 SNPs in replicate RNA samples from the same sample as in A . The median correlation between the allele fraction obtained in replicate assays in all 197 samples was 0.9956 (range 0.9779–0.9984). ( C ) Average allele fractions from triplicate assays of 3531 SNPs in RNA and DNA from the same sample as above. The red dots represent the allele fraction in RNA for SNPs that display allele-specific expression, i.e., SNPs that are heterozygous in DNA and show a significant difference ( P
    Figure Legend Snippet: Genotyping by the NS-12 BeadChips to detect allele-specific gene expression. ( A ) Correlation between the allele fractions determined in replicate DNA samples for 3531 expressed SNPs in one ALL sample. The median correlation between the allele fraction obtained in replicate assays in all 197 samples was 0.9969 (range 0.9934–0.9986). ( B ) Correlation between the allele fractions determined by genotyping the same 3531 SNPs in replicate RNA samples from the same sample as in A . The median correlation between the allele fraction obtained in replicate assays in all 197 samples was 0.9956 (range 0.9779–0.9984). ( C ) Average allele fractions from triplicate assays of 3531 SNPs in RNA and DNA from the same sample as above. The red dots represent the allele fraction in RNA for SNPs that display allele-specific expression, i.e., SNPs that are heterozygous in DNA and show a significant difference ( P

    Techniques Used: Expressing

    47) Product Images from "Constitutively active androgen receptor splice variants AR-V3, AR-V7 and AR-V9 are co-expressed in castration-resistant prostate cancer metastases"

    Article Title: Constitutively active androgen receptor splice variants AR-V3, AR-V7 and AR-V9 are co-expressed in castration-resistant prostate cancer metastases

    Journal: British Journal of Cancer

    doi: 10.1038/s41416-018-0172-0

    Combined DNA and RNA sequencing data from sample set 2 assayed by targeted SureSelect AR sequencing. AR mutations, copy-number alterations, summed score of AR-regulated gene expression and AR-V expression level as a fraction of AR transcript are shown. AR-V fractions are shown as CI95 lower bound values. Additionally, FOXA1 and SPOP mutation status is included
    Figure Legend Snippet: Combined DNA and RNA sequencing data from sample set 2 assayed by targeted SureSelect AR sequencing. AR mutations, copy-number alterations, summed score of AR-regulated gene expression and AR-V expression level as a fraction of AR transcript are shown. AR-V fractions are shown as CI95 lower bound values. Additionally, FOXA1 and SPOP mutation status is included

    Techniques Used: RNA Sequencing Assay, Sequencing, Expressing, Mutagenesis

    48) Product Images from "Novel PDGFRB rearrangement in multifocal infantile myofibromatosis is tumorigenic and sensitive to imatinib"

    Article Title: Novel PDGFRB rearrangement in multifocal infantile myofibromatosis is tumorigenic and sensitive to imatinib

    Journal: Cold Spring Harbor Molecular Case Studies

    doi: 10.1101/mcs.a004440

    Sequencing and mapping of the gene rearrangement. ( A ) Complex PDGFRB rearrangement is detected by next-generation sequencing (NGS). The rearrangement was detected in DNA and RNA by NGS but is difficult to ascertain because of event complexity. ( B ) PDGFRB rearrangement predicted by NGS is confirmed by Sanger sequencing of genomic DNA (gDNA) and RNA (cDNA) from tumor. ( C ) PDGFRB is highly expressed in RNA-seq data from the patient's tumor (circled) compared to other cases of IM with and without activating PDGFRB point mutations. ( D ) Rearrangement results in the replacement of a portion of the juxtamembrane domain of PDGFRβ by a novel amino acid sequence derived from a portion of exon 15 read out-of-frame. The wild-type kinase domain is retained.
    Figure Legend Snippet: Sequencing and mapping of the gene rearrangement. ( A ) Complex PDGFRB rearrangement is detected by next-generation sequencing (NGS). The rearrangement was detected in DNA and RNA by NGS but is difficult to ascertain because of event complexity. ( B ) PDGFRB rearrangement predicted by NGS is confirmed by Sanger sequencing of genomic DNA (gDNA) and RNA (cDNA) from tumor. ( C ) PDGFRB is highly expressed in RNA-seq data from the patient's tumor (circled) compared to other cases of IM with and without activating PDGFRB point mutations. ( D ) Rearrangement results in the replacement of a portion of the juxtamembrane domain of PDGFRβ by a novel amino acid sequence derived from a portion of exon 15 read out-of-frame. The wild-type kinase domain is retained.

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

    49) Product Images from "Assessing DNA methylation in the developing human intestinal epithelium: potential link to inflammatory bowel disease"

    Article Title: Assessing DNA methylation in the developing human intestinal epithelium: potential link to inflammatory bowel disease

    Journal: Mucosal Immunology

    doi: 10.1038/mi.2015.88

    'Genome-wide gene expression analysis was performed on simultaneously extracted RNA from purified human intestinal epithelium. ( a ) Multidimensional scaling analysis (MDS) plot displaying clustering of samples according to developmental age in the first dimension, whereas the second dimension separates epithelial cells according to gut segment. ( b ) Overview of the algorithm used to identify regulatory differentially methylated regions (rDMRs) by integrating both genome-wide DNA and gene expression data. In total, we identified 259 rDMRs associated with 214 genes. ( c ) Summary plots of selected rDMRs displaying DNA methylation and gene expression of PIGR (polymeric immunoglobulin receptor), TLR3 , TET1 (tet methylcytosine dioxygenase 1), MUC2 , and IL6R . The top panel indicates the level of DNA methylation across the DMR according to the methylation β -value ( y axis). Each point represents one sample and the line indicating the average methylation value. Additional panels indicate relation of DMR to the reference human genome. Inset displays corresponding gene expression plotted as log 2 average expression of each sample. ( d ) Distribution of all DMRs (i.e., including DMRs for which associated genes do not display expression changes) vs. rDMRs according to their genomic location. rDMRs are found more frequently to be located either fully inside or overlapping with transcription start site (TSS). DMP, differentially methylated position; FDG, fetal distal gut ( n =3); FPG, fetal proximal gut ( n =3); PAC, pediatric ascending colon ( n =3); PSC, pediatric sigmoid colon ( n =3); PTI, pediatric terminal ileum ( n =3).
    Figure Legend Snippet: 'Genome-wide gene expression analysis was performed on simultaneously extracted RNA from purified human intestinal epithelium. ( a ) Multidimensional scaling analysis (MDS) plot displaying clustering of samples according to developmental age in the first dimension, whereas the second dimension separates epithelial cells according to gut segment. ( b ) Overview of the algorithm used to identify regulatory differentially methylated regions (rDMRs) by integrating both genome-wide DNA and gene expression data. In total, we identified 259 rDMRs associated with 214 genes. ( c ) Summary plots of selected rDMRs displaying DNA methylation and gene expression of PIGR (polymeric immunoglobulin receptor), TLR3 , TET1 (tet methylcytosine dioxygenase 1), MUC2 , and IL6R . The top panel indicates the level of DNA methylation across the DMR according to the methylation β -value ( y axis). Each point represents one sample and the line indicating the average methylation value. Additional panels indicate relation of DMR to the reference human genome. Inset displays corresponding gene expression plotted as log 2 average expression of each sample. ( d ) Distribution of all DMRs (i.e., including DMRs for which associated genes do not display expression changes) vs. rDMRs according to their genomic location. rDMRs are found more frequently to be located either fully inside or overlapping with transcription start site (TSS). DMP, differentially methylated position; FDG, fetal distal gut ( n =3); FPG, fetal proximal gut ( n =3); PAC, pediatric ascending colon ( n =3); PSC, pediatric sigmoid colon ( n =3); PTI, pediatric terminal ileum ( n =3).

    Techniques Used: Genome Wide, Expressing, Purification, Methylation, DNA Methylation Assay

    50) Product Images from "A conditional knockout mouse model reveals endothelial cells as the principal and possibly exclusive source of plasma factor VIII"

    Article Title: A conditional knockout mouse model reveals endothelial cells as the principal and possibly exclusive source of plasma factor VIII

    Journal: Blood

    doi: 10.1182/blood-2014-02-555151

    Analysis of tissue gDNA and mRNA. Tissue samples were collected from males following whole-animal saline perfusion. DNA and total RNA were isolated from the same tissue sample and analyzed by allele-specific PCR. Except for the F8 KO/y mouse, all animals
    Figure Legend Snippet: Analysis of tissue gDNA and mRNA. Tissue samples were collected from males following whole-animal saline perfusion. DNA and total RNA were isolated from the same tissue sample and analyzed by allele-specific PCR. Except for the F8 KO/y mouse, all animals

    Techniques Used: Isolation, Polymerase Chain Reaction

    51) Product Images from "Relationship of HIV Reservoir Characteristics with Immune Status and Viral Rebound Kinetics in an HIV Therapeutic Vaccine Study"

    Article Title: Relationship of HIV Reservoir Characteristics with Immune Status and Viral Rebound Kinetics in an HIV Therapeutic Vaccine Study

    Journal: AIDS (London, England)

    doi: 10.1097/QAD.0000000000000478

    Therapeutic HIV vaccination was not associated with changes in CA-RNA or CA-DNA levels (A) CA-RNA and (B) CA-DNA levels in participants of A5197 pre- (wk 0) and post-vaccination (wk 8 and 38). Bars represent median values. CA-RNA, cell-associated HIV RNA; CA-DNA, cell-associated HIV DNA.
    Figure Legend Snippet: Therapeutic HIV vaccination was not associated with changes in CA-RNA or CA-DNA levels (A) CA-RNA and (B) CA-DNA levels in participants of A5197 pre- (wk 0) and post-vaccination (wk 8 and 38). Bars represent median values. CA-RNA, cell-associated HIV RNA; CA-DNA, cell-associated HIV DNA.

    Techniques Used:

    Association between HIV-specific CD4+ activity and HIV reservoir levels at baseline (A) CA-RNA and (B) CA-DNA at baseline are associated with the number of HIV-specific CD4+ IFN-γ-producing cells. CA-RNA, cell-associated HIV RNA; CA-DNA, cell-associated HIV DNA.
    Figure Legend Snippet: Association between HIV-specific CD4+ activity and HIV reservoir levels at baseline (A) CA-RNA and (B) CA-DNA at baseline are associated with the number of HIV-specific CD4+ IFN-γ-producing cells. CA-RNA, cell-associated HIV RNA; CA-DNA, cell-associated HIV DNA.

    Techniques Used: Activity Assay

    Baseline HIV reservoir levels differ by HLA group (A) Residual viremia by single-copy assay, (B) CA-RNA, and (C) CA-DNA levels in PBMC in participants of A5197 stratified by the presence of unfavorable, neutral, or protective HLA alleles. SCA, single-copy assay; CA-RNA, cell-associated HIV RNA; CA-DNA, cell-associated HIV DNA; HLA, human leukocyte antigen; PBMC, peripheral blood mononuclear cell.
    Figure Legend Snippet: Baseline HIV reservoir levels differ by HLA group (A) Residual viremia by single-copy assay, (B) CA-RNA, and (C) CA-DNA levels in PBMC in participants of A5197 stratified by the presence of unfavorable, neutral, or protective HLA alleles. SCA, single-copy assay; CA-RNA, cell-associated HIV RNA; CA-DNA, cell-associated HIV DNA; HLA, human leukocyte antigen; PBMC, peripheral blood mononuclear cell.

    Techniques Used:

    Pre-ATI CA-RNA and CA-DNA predict viral load set point Pre-ATI week 38 (A) CA-RNA and (B) CA-DNA levels were both significantly correlated with post-ATI viral load set point. ATI, analytic treatment interruption; CA-RNA, cell-associated HIV RNA; CA-DNA, cell-associated HIV DNA.
    Figure Legend Snippet: Pre-ATI CA-RNA and CA-DNA predict viral load set point Pre-ATI week 38 (A) CA-RNA and (B) CA-DNA levels were both significantly correlated with post-ATI viral load set point. ATI, analytic treatment interruption; CA-RNA, cell-associated HIV RNA; CA-DNA, cell-associated HIV DNA.

    Techniques Used:

    52) Product Images from "Diversity and potential activity patterns of planktonic eukaryotic microbes in a mesoeutrophic coastal area (eastern English Channel)"

    Article Title: Diversity and potential activity patterns of planktonic eukaryotic microbes in a mesoeutrophic coastal area (eastern English Channel)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0196987

    Cluster-based microbial community structure in the EEC. (a) Cluster diagram based on Bray–Curtis dissimilarities calculated based on the non-transformed number of reads for all OTUs found during the study. Red lines in the dendrogram indicate significant differences (p > 0.05) between bifurcations, based on the SIMPROF significance test. ◌ DNA samples, Δ RNA samples. Red dots indicate grouping of samples from the same sampling date (b) Venn diagram of shared diversity between three clusters. The total richness for all groups was 868 OTUs. The number of species shared between the three clusters was 199, corresponding to 23% of the total richness. Similar Bray-Curtis cluster diagrams were constructed from rDNA-based (c) and rRNA-based (d) number of reads.
    Figure Legend Snippet: Cluster-based microbial community structure in the EEC. (a) Cluster diagram based on Bray–Curtis dissimilarities calculated based on the non-transformed number of reads for all OTUs found during the study. Red lines in the dendrogram indicate significant differences (p > 0.05) between bifurcations, based on the SIMPROF significance test. ◌ DNA samples, Δ RNA samples. Red dots indicate grouping of samples from the same sampling date (b) Venn diagram of shared diversity between three clusters. The total richness for all groups was 868 OTUs. The number of species shared between the three clusters was 199, corresponding to 23% of the total richness. Similar Bray-Curtis cluster diagrams were constructed from rDNA-based (c) and rRNA-based (d) number of reads.

    Techniques Used: Transformation Assay, Sampling, Construct

    53) Product Images from "Complex regulation of ADAR-mediated RNA-editing across tissues"

    Article Title: Complex regulation of ADAR-mediated RNA-editing across tissues

    Journal: BMC Genomics

    doi: 10.1186/s12864-015-2291-9

    Experimental design ( a ) and sequencing processing workflow to call RNA-DNA differences (RDDs) ( b ). Tissues were harvested from an 8 week old male C57BL/6J mouse. RNA was isolated from 9 tissues and individually used for 100 bp paired-end RNA-sequencing. After alignment to the mm9 genome the number of uniquely mapped reads (umr) was between 28 million and 77 million for each tissue sample. DNA was extracted from the brain and spleen tissues, and sent for 75 bp paired end whole genome sequencing, to average depths of 31X and 23X coverage (586 and 445 million uniquely mapped reads, respectively)
    Figure Legend Snippet: Experimental design ( a ) and sequencing processing workflow to call RNA-DNA differences (RDDs) ( b ). Tissues were harvested from an 8 week old male C57BL/6J mouse. RNA was isolated from 9 tissues and individually used for 100 bp paired-end RNA-sequencing. After alignment to the mm9 genome the number of uniquely mapped reads (umr) was between 28 million and 77 million for each tissue sample. DNA was extracted from the brain and spleen tissues, and sent for 75 bp paired end whole genome sequencing, to average depths of 31X and 23X coverage (586 and 445 million uniquely mapped reads, respectively)

    Techniques Used: Sequencing, Isolation, RNA Sequencing Assay

    The total number of A-to-G RNA-DNA differences (RDDs) called in each sample, versus the number or analyzed base pairs (number of uniquely mapped reads × length of reads) ( a ). The nine samples with the largest number of analyzed base pairs correspond with the high coverage samples generated in this study. The lower coverage samples were derived from previously published datasets. The high coverage brain sample in this study is remarkable for having a higher amount of RDDs. However, when all RDDs that overlap an intron are discarded the high coverage brain sample is no longer a strong outlier compared to the other high coverage tissues ( b )
    Figure Legend Snippet: The total number of A-to-G RNA-DNA differences (RDDs) called in each sample, versus the number or analyzed base pairs (number of uniquely mapped reads × length of reads) ( a ). The nine samples with the largest number of analyzed base pairs correspond with the high coverage samples generated in this study. The lower coverage samples were derived from previously published datasets. The high coverage brain sample in this study is remarkable for having a higher amount of RDDs. However, when all RDDs that overlap an intron are discarded the high coverage brain sample is no longer a strong outlier compared to the other high coverage tissues ( b )

    Techniques Used: Generated, Derivative Assay

    54) Product Images from "Long Noncoding RNA MEG3 Is an Epigenetic Determinant of Oncogenic Signaling in Functional Pancreatic Neuroendocrine Tumor Cells"

    Article Title: Long Noncoding RNA MEG3 Is an Epigenetic Determinant of Oncogenic Signaling in Functional Pancreatic Neuroendocrine Tumor Cells

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00278-17

    ChIRP-Seq reveals m-Meg3 enrichment at multiple m-c-Met loci. (A) Representative agarose gel image showing the specificity of the m-Meg3 ChIRP probes. RNA was isolated after m-Meg3 ChIRP from the V3 (vector) and the M5 (stable MIN6-4N cells stably expressing the m-Meg3-3 isoform which lacks exon 4) cell lines. The RNA was then used for RT-PCR. Input corresponds to the RT-PCR product using RNA isolated before m-Meg3 ChIRP-Seq. Odd and even correspond to RT-PCR using RNA after m-Meg3 ChIRP with probes located at odd and even locations on the m-Meg3 RNA. The gel image represents products of the RT-PCR performed with primers 1F/1R (flanking exon 7 and exon 8) that recognize all m-Meg3 isoforms. gapdh served as the negative control. cDNAs from three replicates of V3 and M5 ChIRP-Seq were pooled due to low yields and sequenced. (B) m-Meg3 ChIRP-PCR in a stable cell line expressing full-length m-Meg3. RNA was isolated after m-Meg3 ChIRP from the 9V (vector) and the 14M (stable MIN6-4N cells with the m-Meg3-1 isoform, which encompasses all 10 exons) cell lines. The RNA was then used for RT-PCR. Input corresponds to RT-PCR from RNA isolated before m-Meg3 ChIRP. RT-PCR was performed with primers 1F/1R (flanking exons 7 and 8) that recognize all m-Meg3 isoforms and further confirmed with the ex3F/ex4R primer pair (flanking exons 3 and 4), specific for the m-Meg3-1 isoform. gapdh served as the negative control. (C) m-Meg3 enrichment patterns at discrete m-c-Met genomic regions in different m-Meg3 stable cell lines. DNA was isolated after m-Meg3 ChIRP from two different m-Meg-3 stable MIN6-4N cell lines and their respective vector controls. The DNA was then subjected to whole-genome amplification (WGA) and subsequent purification. The purified WGA DNA was then used to set up qPCRs in duplicate with primers specific for m-c-Met genomic regions identified by m-Meg3 ChIRP-Seq, namely, the m-c-Met upstream region, the m-c-Met exon 18 region, the m-c-Met exon 20 region, and also the previously identified kb +63 enhancer. The qPCR data are represented as percent input of DNA.
    Figure Legend Snippet: ChIRP-Seq reveals m-Meg3 enrichment at multiple m-c-Met loci. (A) Representative agarose gel image showing the specificity of the m-Meg3 ChIRP probes. RNA was isolated after m-Meg3 ChIRP from the V3 (vector) and the M5 (stable MIN6-4N cells stably expressing the m-Meg3-3 isoform which lacks exon 4) cell lines. The RNA was then used for RT-PCR. Input corresponds to the RT-PCR product using RNA isolated before m-Meg3 ChIRP-Seq. Odd and even correspond to RT-PCR using RNA after m-Meg3 ChIRP with probes located at odd and even locations on the m-Meg3 RNA. The gel image represents products of the RT-PCR performed with primers 1F/1R (flanking exon 7 and exon 8) that recognize all m-Meg3 isoforms. gapdh served as the negative control. cDNAs from three replicates of V3 and M5 ChIRP-Seq were pooled due to low yields and sequenced. (B) m-Meg3 ChIRP-PCR in a stable cell line expressing full-length m-Meg3. RNA was isolated after m-Meg3 ChIRP from the 9V (vector) and the 14M (stable MIN6-4N cells with the m-Meg3-1 isoform, which encompasses all 10 exons) cell lines. The RNA was then used for RT-PCR. Input corresponds to RT-PCR from RNA isolated before m-Meg3 ChIRP. RT-PCR was performed with primers 1F/1R (flanking exons 7 and 8) that recognize all m-Meg3 isoforms and further confirmed with the ex3F/ex4R primer pair (flanking exons 3 and 4), specific for the m-Meg3-1 isoform. gapdh served as the negative control. (C) m-Meg3 enrichment patterns at discrete m-c-Met genomic regions in different m-Meg3 stable cell lines. DNA was isolated after m-Meg3 ChIRP from two different m-Meg-3 stable MIN6-4N cell lines and their respective vector controls. The DNA was then subjected to whole-genome amplification (WGA) and subsequent purification. The purified WGA DNA was then used to set up qPCRs in duplicate with primers specific for m-c-Met genomic regions identified by m-Meg3 ChIRP-Seq, namely, the m-c-Met upstream region, the m-c-Met exon 18 region, the m-c-Met exon 20 region, and also the previously identified kb +63 enhancer. The qPCR data are represented as percent input of DNA.

    Techniques Used: Agarose Gel Electrophoresis, Isolation, Plasmid Preparation, Stable Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction, Negative Control, Polymerase Chain Reaction, Whole Genome Amplification, Purification, Real-time Polymerase Chain Reaction

    m-Meg3 TFO-9 regulates the m-c-Met transcript. (A) Schematic of full-length m-Meg3-1 exon structure showing the TFO-9 coordinates. Full-length m-Meg3-1 is depicted, with exons 1 to 10 numbered (top) and the sequence length in base pairs (bottom). TFO-9, a GA- and GT-rich 16-mer sequence, is shown mapping to the C-terminal portion of exon 10 in m-Meg3-1. TFO-9, spanning bp 1822 to 1838, was predicted by Triplexator to form triplexes with double-stranded DNA. (B) Effect of TFO-9 on the expression of m-Meg3 and m-c-Met transcripts. RNA was isolated at 48 h and 96 h posttransfection from MIN6-4N cells transiently transfected with TFO RNA oligonucleotides. Purified RNA converted to cDNA was subjected to qPCR analyses with primers specific for m-Meg3-1 or m-c-Met. The data represent an average from three independent experiments and multiple technical replicates (mean ± SD) *, P ≤ 0.05.
    Figure Legend Snippet: m-Meg3 TFO-9 regulates the m-c-Met transcript. (A) Schematic of full-length m-Meg3-1 exon structure showing the TFO-9 coordinates. Full-length m-Meg3-1 is depicted, with exons 1 to 10 numbered (top) and the sequence length in base pairs (bottom). TFO-9, a GA- and GT-rich 16-mer sequence, is shown mapping to the C-terminal portion of exon 10 in m-Meg3-1. TFO-9, spanning bp 1822 to 1838, was predicted by Triplexator to form triplexes with double-stranded DNA. (B) Effect of TFO-9 on the expression of m-Meg3 and m-c-Met transcripts. RNA was isolated at 48 h and 96 h posttransfection from MIN6-4N cells transiently transfected with TFO RNA oligonucleotides. Purified RNA converted to cDNA was subjected to qPCR analyses with primers specific for m-Meg3-1 or m-c-Met. The data represent an average from three independent experiments and multiple technical replicates (mean ± SD) *, P ≤ 0.05.

    Techniques Used: Sequencing, Expressing, Isolation, Transfection, Purification, Real-time Polymerase Chain Reaction

    m-Meg3 associates with PRC2 components in mouse insulinoma cell line models. (A and B) Association of m-Meg3 RNA with EZH2 and H3K27me3. RNA-ChIP in Men1 cells (Meg3 proficient) was performed using the antibodies directed toward EZH2 (A) and H3K27me3 (B). qPCR was performed on cDNA obtained from RNA isolated after RNA-ChIP. qPCR data were calculated as percent RNA input and are shown as the average from two independent biological replicates and multiple technical replicates (mean ± SD). IgG served as the negative control for the RNA-ChIP assay. (C and D) EZH2 and H3K27me3 enrichment at the m-c-Met regions identified by m-Meg3 ChIRP-Seq. ChIP assay was performed in Men1 cells using the indicated antibodies. DNA isolated after ChIP was used for qPCR, with primers specific for the m-c-Met upstream region, the +63-kb enhancer, the m-c-Met exon 18 region, and the m-c-Met exon 20 region. EZH2 (C) and H3K27me3 (D) enrichment was calculated as percent chromatin DNA input and constitutes the average from three independent biological replicates and multiple technical replicates (mean ± SD). (E and F) Enhancer-signature histone modifications at the m-c-Met loci identified by m-Meg3 ChIRP-Seq. MIN6-4N cells were subjected to ChIP assays and qPCR analyses to detect the enrichment of H3K27Ac (E) and H3K4me1 (F) at the m-c-Met upstream region, the kb +63 enhancer, the m-c-Met exon 18 region, and the m-c-Met exon 20 region. The data represent an average from two independent biological replicates and multiple technical replicates (mean ± SD).
    Figure Legend Snippet: m-Meg3 associates with PRC2 components in mouse insulinoma cell line models. (A and B) Association of m-Meg3 RNA with EZH2 and H3K27me3. RNA-ChIP in Men1 cells (Meg3 proficient) was performed using the antibodies directed toward EZH2 (A) and H3K27me3 (B). qPCR was performed on cDNA obtained from RNA isolated after RNA-ChIP. qPCR data were calculated as percent RNA input and are shown as the average from two independent biological replicates and multiple technical replicates (mean ± SD). IgG served as the negative control for the RNA-ChIP assay. (C and D) EZH2 and H3K27me3 enrichment at the m-c-Met regions identified by m-Meg3 ChIRP-Seq. ChIP assay was performed in Men1 cells using the indicated antibodies. DNA isolated after ChIP was used for qPCR, with primers specific for the m-c-Met upstream region, the +63-kb enhancer, the m-c-Met exon 18 region, and the m-c-Met exon 20 region. EZH2 (C) and H3K27me3 (D) enrichment was calculated as percent chromatin DNA input and constitutes the average from three independent biological replicates and multiple technical replicates (mean ± SD). (E and F) Enhancer-signature histone modifications at the m-c-Met loci identified by m-Meg3 ChIRP-Seq. MIN6-4N cells were subjected to ChIP assays and qPCR analyses to detect the enrichment of H3K27Ac (E) and H3K4me1 (F) at the m-c-Met upstream region, the kb +63 enhancer, the m-c-Met exon 18 region, and the m-c-Met exon 20 region. The data represent an average from two independent biological replicates and multiple technical replicates (mean ± SD).

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Isolation, Negative Control

    55) Product Images from "Creation and implantation of acellular rat renal ECM-based scaffolds"

    Article Title: Creation and implantation of acellular rat renal ECM-based scaffolds

    Journal: Organogenesis

    doi: 10.1080/15476278.2015.1072661

    Laminin evaluation and comparison between native, TNX-100-flushed and acellular renal scaffold Images show laminin preservation during the decellularization process. First row illustrates laminin assay (upper left), DAPI staining (upper central) and merged picture (upper right) on native kidney Second row shows laminin assay (middle left), DAPI staining (middle central) and merged picture (middle right) performed on renal organ perfused with TNX only. Third row displays laminin assay (lower left), DAPI staining (lower central) and merged pictured executed on final acellular kidney scaffold. At the end of the process laminin was well conserved whereas, interestingly, DAPI staining during the process looks gradually decreasing. This qualitative data suggest that our protocol is soft enough to preserve laminin but, at the same time, can progressively remove DNA material.
    Figure Legend Snippet: Laminin evaluation and comparison between native, TNX-100-flushed and acellular renal scaffold Images show laminin preservation during the decellularization process. First row illustrates laminin assay (upper left), DAPI staining (upper central) and merged picture (upper right) on native kidney Second row shows laminin assay (middle left), DAPI staining (middle central) and merged picture (middle right) performed on renal organ perfused with TNX only. Third row displays laminin assay (lower left), DAPI staining (lower central) and merged pictured executed on final acellular kidney scaffold. At the end of the process laminin was well conserved whereas, interestingly, DAPI staining during the process looks gradually decreasing. This qualitative data suggest that our protocol is soft enough to preserve laminin but, at the same time, can progressively remove DNA material.

    Techniques Used: Preserving, Staining

    56) Product Images from "Broad activation of latent HIV-1 in vivo"

    Article Title: Broad activation of latent HIV-1 in vivo

    Journal: Nature Communications

    doi: 10.1038/ncomms12731

    Panobinostat and vorinostat non-selectively activate transcription from latent HIV-1 proviruses. ( a ) Representative phylogenetic trees of HIV-1 sequences from HIV-infected participants on suppressive ART who received panobinostat (Pan18) or vorinostat (Vor16) showing the genetic relationship of sequences from each time point. For participant Pan18, the plasma samples were collected ∼1 year and 6 months before initiation of antiretroviral therapy and 14 days following the analytical treatment interruption. Peripheral blood samples were collected at baseline, 2 h after the first dose of panobinostat (TP1), 32 days after the first dose of panobinostat (TP2) and 38 days after the final panobinostat dose. Intestinal lamina propria mononuclear cells were collected at baseline (1 week before the first panobinostat dose) and during week 4 of the panobinostat trial. For participant Vor16, peripheral blood samples were collected at baseline, 7 days after the first dose of vorinostat (TP1), 14 days after the first dose of vorinostat (TP2) and 7 days after the final vorinostat dose. ( b ) Average pairwise distance of cell-associated DNA (Pan n =12, Vor n =12) before and DNA (Pan n =12, Vor n =14) and cell-associated RNA (Pan n = 8, Vor n =3) during vorinostat and panobinostat administration, as well as the plasma HIV-1 RNA following an ATI for the panobinostat trial ( n =7). Each data point represents the group mean±s.e.m. The Wilcoxon signed rank test was used to generate the P values. * P ≤0.05.
    Figure Legend Snippet: Panobinostat and vorinostat non-selectively activate transcription from latent HIV-1 proviruses. ( a ) Representative phylogenetic trees of HIV-1 sequences from HIV-infected participants on suppressive ART who received panobinostat (Pan18) or vorinostat (Vor16) showing the genetic relationship of sequences from each time point. For participant Pan18, the plasma samples were collected ∼1 year and 6 months before initiation of antiretroviral therapy and 14 days following the analytical treatment interruption. Peripheral blood samples were collected at baseline, 2 h after the first dose of panobinostat (TP1), 32 days after the first dose of panobinostat (TP2) and 38 days after the final panobinostat dose. Intestinal lamina propria mononuclear cells were collected at baseline (1 week before the first panobinostat dose) and during week 4 of the panobinostat trial. For participant Vor16, peripheral blood samples were collected at baseline, 7 days after the first dose of vorinostat (TP1), 14 days after the first dose of vorinostat (TP2) and 7 days after the final vorinostat dose. ( b ) Average pairwise distance of cell-associated DNA (Pan n =12, Vor n =12) before and DNA (Pan n =12, Vor n =14) and cell-associated RNA (Pan n = 8, Vor n =3) during vorinostat and panobinostat administration, as well as the plasma HIV-1 RNA following an ATI for the panobinostat trial ( n =7). Each data point represents the group mean±s.e.m. The Wilcoxon signed rank test was used to generate the P values. * P ≤0.05.

    Techniques Used: Infection

    A significant proportion of cell-associated HIV-1 RNA is defective. The percentage of defective virus detected in CD4 + T cells collected from HIV-infected participants on ART before, during and following administration of panobinostat (baseline DNA n =14, RNA n =7; on HDACi DNA n =15, RNA n =9; post HDACi DNA n =14, RNA n =6) or vorinostat (baseline DNA n =14, RNA n =5; on HDACi DNA n =15, RNA n =6; post HDACi DNA n =14, RNA n =3). Each data point represents the group mean, and the error bars represent the s.e.m. * P ≤0.05, ** P ≤0.01. The Wilcoxon signed rank test was used to generate the P values.
    Figure Legend Snippet: A significant proportion of cell-associated HIV-1 RNA is defective. The percentage of defective virus detected in CD4 + T cells collected from HIV-infected participants on ART before, during and following administration of panobinostat (baseline DNA n =14, RNA n =7; on HDACi DNA n =15, RNA n =9; post HDACi DNA n =14, RNA n =6) or vorinostat (baseline DNA n =14, RNA n =5; on HDACi DNA n =15, RNA n =6; post HDACi DNA n =14, RNA n =3). Each data point represents the group mean, and the error bars represent the s.e.m. * P ≤0.05, ** P ≤0.01. The Wilcoxon signed rank test was used to generate the P values.

    Techniques Used: Infection

    57) 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

    58) Product Images from "Picoeukaryotic Diversity And Activity in the Northwestern Pacific Ocean Based on rDNA and rRNA High-Throughput Sequencing"

    Article Title: Picoeukaryotic Diversity And Activity in the Northwestern Pacific Ocean Based on rDNA and rRNA High-Throughput Sequencing

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.03259

    Cluster diagram of the Bray-Curtis dissimilarities based on the log-transformed relative OTU abundance for each sample. The separated DNA (A) and RNA (B) community groups (with asterisks at the node) exist with significant compositional differences (DNA: r 2 = 0.31718, P = 0.001; RNA: r 2 = 0.27023, P = 0.001) as determined by PERMANOVA. The percent dissimilarity among samples is shown along the horizontal axes.
    Figure Legend Snippet: Cluster diagram of the Bray-Curtis dissimilarities based on the log-transformed relative OTU abundance for each sample. The separated DNA (A) and RNA (B) community groups (with asterisks at the node) exist with significant compositional differences (DNA: r 2 = 0.31718, P = 0.001; RNA: r 2 = 0.27023, P = 0.001) as determined by PERMANOVA. The percent dissimilarity among samples is shown along the horizontal axes.

    Techniques Used: Transformation Assay

    Number of OTUs unveiled by DNA and RNA surveys. The pie chart indicated the OTU numbers mainly at class level; the inner ring represented these classes belong to the phylum; the outer ring indicated the number of OTUs at super-group level.
    Figure Legend Snippet: Number of OTUs unveiled by DNA and RNA surveys. The pie chart indicated the OTU numbers mainly at class level; the inner ring represented these classes belong to the phylum; the outer ring indicated the number of OTUs at super-group level.

    Techniques Used:

    Number of OTUs of higher taxonomic groups (sequences proportion over 1% of the total DNA or RNA abundance) based on the DNA-derived (A) and RNA-derived (B) approaches for each sample. Others refer to groups with relatively lower abundance (sequences proportion
    Figure Legend Snippet: Number of OTUs of higher taxonomic groups (sequences proportion over 1% of the total DNA or RNA abundance) based on the DNA-derived (A) and RNA-derived (B) approaches for each sample. Others refer to groups with relatively lower abundance (sequences proportion

    Techniques Used: Derivative Assay

    Relative abundance of higher taxonomic groups (sequences proportion over 1% of the total DNA or RNA abundance) based on the DNA-derived (A) and RNA-derived (B) approaches for each sample. Others refer to groups with relatively lower abundance (sequences proportion
    Figure Legend Snippet: Relative abundance of higher taxonomic groups (sequences proportion over 1% of the total DNA or RNA abundance) based on the DNA-derived (A) and RNA-derived (B) approaches for each sample. Others refer to groups with relatively lower abundance (sequences proportion

    Techniques Used: Derivative Assay

    (A) Scatter diagram of PC 1 and PC 2 derived from the relative abundance of higher taxonomic groups in the DNA and RNA databases detected in each sample, blue color indicated the sequences was derived from the DNA survey and red color indicated the sequences was derived from the RNA survey. Ma, Mamiellophyceae; Sp, Spirotrichea; Pe, Pelagophyceae; Ba, Bacillariophyta; Dict, Dictyochophyceae; Prym, Prymnesiophyceae. Boxplot representing the RNA: DNA ratios for Mamiellophyceae (B) , Pelagophyceae (C) , and Spirotrichea (D) in all and individual samples.
    Figure Legend Snippet: (A) Scatter diagram of PC 1 and PC 2 derived from the relative abundance of higher taxonomic groups in the DNA and RNA databases detected in each sample, blue color indicated the sequences was derived from the DNA survey and red color indicated the sequences was derived from the RNA survey. Ma, Mamiellophyceae; Sp, Spirotrichea; Pe, Pelagophyceae; Ba, Bacillariophyta; Dict, Dictyochophyceae; Prym, Prymnesiophyceae. Boxplot representing the RNA: DNA ratios for Mamiellophyceae (B) , Pelagophyceae (C) , and Spirotrichea (D) in all and individual samples.

    Techniques Used: Derivative Assay

    59) Product Images from "Genome editing of oncogenes with ZFNs and TALENs: caveats in nuclease design"

    Article Title: Genome editing of oncogenes with ZFNs and TALENs: caveats in nuclease design

    Journal: Cancer Cell International

    doi: 10.1186/s12935-018-0666-0

    TALEN mediated editing in SiHa and CaSki cell lines. a , b SiHa control, c , d TALEN treated SiHa cells showed single spots (green; arrows) indicating the presence of 53bp1, e , f CaSki control, g , h TALEN treated CaSki cell showed multiple spots (green; arrows) indicating the presence of DNA double strand breaks. Magnification ×600
    Figure Legend Snippet: TALEN mediated editing in SiHa and CaSki cell lines. a , b SiHa control, c , d TALEN treated SiHa cells showed single spots (green; arrows) indicating the presence of 53bp1, e , f CaSki control, g , h TALEN treated CaSki cell showed multiple spots (green; arrows) indicating the presence of DNA double strand breaks. Magnification ×600

    Techniques Used:

    60) Product Images from "Intratumoural Heterogeneity Underlies Distinct Therapy Responses and Treatment Resistance in Glioblastoma"

    Article Title: Intratumoural Heterogeneity Underlies Distinct Therapy Responses and Treatment Resistance in Glioblastoma

    Journal: Cancers

    doi: 10.3390/cancers11020190

    Comprehensive genomic analyses of single-cell clones. ( A ) Genomic proportion of copy number alteration. For clonal samples B–F, > 84% of their genome is between copy number 3 and 4 (ranging from 84.82% to 85.9%, shown as dark grey bars) as compared to ≤3% for the other samples. This global whole genome duplication typically affected both alleles, preserving heterozygosity in these samples. ( B ) Number of somatic structural variants identified per sample with the type indicated by colours. ( C ) Hierarchical clustering of somatic substitution variant allele frequencies across tumour and clonal samples. The absence of a variant in the normal control cortex sample is demonstrated by a variant allele frequency of zero (dark blue). ( D ) Hierarchical clustering of Log2-normalised and gene-scaled RNA seq gene expression of genes with most variable expression as identified as contributing to the first principle component. Positive values indicate a sample with the highest expression and negative values with the lowest expression. ( E ) Hierarchical clustering of 1000 most variable β-values from DNA methylation sequencing data. Β-value of 1 indicates completely methylated and 0 unmethylated CpGs.
    Figure Legend Snippet: Comprehensive genomic analyses of single-cell clones. ( A ) Genomic proportion of copy number alteration. For clonal samples B–F, > 84% of their genome is between copy number 3 and 4 (ranging from 84.82% to 85.9%, shown as dark grey bars) as compared to ≤3% for the other samples. This global whole genome duplication typically affected both alleles, preserving heterozygosity in these samples. ( B ) Number of somatic structural variants identified per sample with the type indicated by colours. ( C ) Hierarchical clustering of somatic substitution variant allele frequencies across tumour and clonal samples. The absence of a variant in the normal control cortex sample is demonstrated by a variant allele frequency of zero (dark blue). ( D ) Hierarchical clustering of Log2-normalised and gene-scaled RNA seq gene expression of genes with most variable expression as identified as contributing to the first principle component. Positive values indicate a sample with the highest expression and negative values with the lowest expression. ( E ) Hierarchical clustering of 1000 most variable β-values from DNA methylation sequencing data. Β-value of 1 indicates completely methylated and 0 unmethylated CpGs.

    Techniques Used: Clone Assay, Preserving, Variant Assay, RNA Sequencing Assay, Expressing, DNA Methylation Assay, Sequencing, Methylation

    61) Product Images from "A C9ORF72 BAC mouse model recapitulates key epigenetic perturbations of ALS/FTD"

    Article Title: A C9ORF72 BAC mouse model recapitulates key epigenetic perturbations of ALS/FTD

    Journal: Molecular Neurodegeneration

    doi: 10.1186/s13024-017-0185-9

    DNA methylation is acquired independently of RNA-DNA hybrid formation at the C9ORF72 locus. Relative quantification of all three C9ORF72 transcript variants in iPSC-derived motor neurons stably expressing a C9ORF72-specifc shRNA (shC9) or a scrambled CTL (shCTL) ( a ). DNA-RNA immunoprecipitation at the C9ORF72 promoter of shC9 and shCTL motor neurons, relative quantification was measured using two sets of primers, designed upstream ( b ) and downstream ( c ) of the repeat expansion, RNase H treatment was performed prior to pull-down as a negative control. DNA methylation levels at the C9ORF72 promoter were assessed using bisulfite pyrosequencing across 16 CpG dinucleotides; positions relative to the transcription start site are indicated on the x-axis ( d ). Fragile X patient-derived iPSC-neurons (FXS) were used as a negative control. All experiments were performed in duplicates ( N = 2 from a single biological sample for each iPSC line examined). Significance is indicated by p
    Figure Legend Snippet: DNA methylation is acquired independently of RNA-DNA hybrid formation at the C9ORF72 locus. Relative quantification of all three C9ORF72 transcript variants in iPSC-derived motor neurons stably expressing a C9ORF72-specifc shRNA (shC9) or a scrambled CTL (shCTL) ( a ). DNA-RNA immunoprecipitation at the C9ORF72 promoter of shC9 and shCTL motor neurons, relative quantification was measured using two sets of primers, designed upstream ( b ) and downstream ( c ) of the repeat expansion, RNase H treatment was performed prior to pull-down as a negative control. DNA methylation levels at the C9ORF72 promoter were assessed using bisulfite pyrosequencing across 16 CpG dinucleotides; positions relative to the transcription start site are indicated on the x-axis ( d ). Fragile X patient-derived iPSC-neurons (FXS) were used as a negative control. All experiments were performed in duplicates ( N = 2 from a single biological sample for each iPSC line examined). Significance is indicated by p

    Techniques Used: DNA Methylation Assay, Derivative Assay, Stable Transfection, Expressing, shRNA, CTL Assay, Immunoprecipitation, Negative Control

    62) 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 LCT13 L1ASP in CRC. (A) Top: Schematic diagram of the LCT13 genomic locus on human chromosome 7 (chr7:93,204,042–93,540,485; center) with indicated the positions of the CALCR, TFPI-2 , and GNGT1 genes and of the 2 intact intergenic LINE1s (L1) present in this region. Middle: enlargement of the LINE-1 (L1PA2: chr7:93,213,393–93,221,079, with an SVA_D spanning the interval 93,214,544–93,216,214) from which LCT13 originates with the regions (black bars) tested by bisulfite or hMeDIP and ChIP. Bottom: enlargement of the LCT13 spliced transcript with indicated its exon structure [LINE-1 5′UTR fragment in light gray (chr7:93,220,882–93,221,083) and, in dark gray, the 2 GNGT1 exons (93,536,051–93,536,154 and 93,540,102–93,540,235), part of the LCT13 transcript]. Also indicated are the positions of the Taqman assay used for LCT13 expression studies located at the splice junction (black bar) and of the primers used for 5′RACE (arrows). All coordinates are from hg19 annotations; scale is in kilobase pairs (kb). (B) Bar charts showing the expression of LCT13 measured by real time RT-PCR and expressed relatively to the geometric mean of 3 reference genes in matched normal (dark gray, N) and tumor (light gray, T) tissues from 6 colorectal cancer patients (left panel) and 6 cell lines (right panel). NC: normal colon, commercially sourced total RNA from 7 healthy donors pooled together. (C) Bar charts of the methylation levels measured by bisulfite sequencing in the tissues of the 6 patients and cell lines presented in B.
    Figure Legend Snippet: Relationship between methylation and expression of LCT13 L1ASP in CRC. (A) Top: Schematic diagram of the LCT13 genomic locus on human chromosome 7 (chr7:93,204,042–93,540,485; center) with indicated the positions of the CALCR, TFPI-2 , and GNGT1 genes and of the 2 intact intergenic LINE1s (L1) present in this region. Middle: enlargement of the LINE-1 (L1PA2: chr7:93,213,393–93,221,079, with an SVA_D spanning the interval 93,214,544–93,216,214) from which LCT13 originates with the regions (black bars) tested by bisulfite or hMeDIP and ChIP. Bottom: enlargement of the LCT13 spliced transcript with indicated its exon structure [LINE-1 5′UTR fragment in light gray (chr7:93,220,882–93,221,083) and, in dark gray, the 2 GNGT1 exons (93,536,051–93,536,154 and 93,540,102–93,540,235), part of the LCT13 transcript]. Also indicated are the positions of the Taqman assay used for LCT13 expression studies located at the splice junction (black bar) and of the primers used for 5′RACE (arrows). All coordinates are from hg19 annotations; scale is in kilobase pairs (kb). (B) Bar charts showing the expression of LCT13 measured by real time RT-PCR and expressed relatively to the geometric mean of 3 reference genes in matched normal (dark gray, N) and tumor (light gray, T) tissues from 6 colorectal cancer patients (left panel) and 6 cell lines (right panel). NC: normal colon, commercially sourced total RNA from 7 healthy donors pooled together. (C) Bar charts of the methylation levels measured by bisulfite sequencing in the tissues of the 6 patients and cell lines presented in B.

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

    63) Product Images from "Optimizing exosomal RNA isolation for RNA-Seq analyses of archival sera specimens"

    Article Title: Optimizing exosomal RNA isolation for RNA-Seq analyses of archival sera specimens

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0196913

    Comparison of sera exosomal RNA using four different RNA extraction methods. (A) Total RNA yield from ultracentrifugation (UC) and ExoQuick (EQ) treated samples using the RNeasy Mini Kit combined with TRIzol LS, the RNeasy Mini Kit alone, conventional RNA precipitation, and AllPrep DNA/RNA Mini Kit. (B) Demonstration of RNA quality measured by OD 260 /OD 280 in EQ and UC treated samples. Data are shown as the mean ± SD from six independent patient samples. *** P
    Figure Legend Snippet: Comparison of sera exosomal RNA using four different RNA extraction methods. (A) Total RNA yield from ultracentrifugation (UC) and ExoQuick (EQ) treated samples using the RNeasy Mini Kit combined with TRIzol LS, the RNeasy Mini Kit alone, conventional RNA precipitation, and AllPrep DNA/RNA Mini Kit. (B) Demonstration of RNA quality measured by OD 260 /OD 280 in EQ and UC treated samples. Data are shown as the mean ± SD from six independent patient samples. *** P

    Techniques Used: RNA Extraction

    Minimal sera volume for exosome detection and RNA extraction. (A) Representative immunoblots for CD63 comparing exosome enrichment using ultracentrifugation (UC) and ExoQuick (EQ) among samples of varying volumes. (B) Scatter dot plots depict total exosomal RNA recovered per sample volume using EQ. (C) Scatter dot plots show RNA quality measured by OD 260 /OD 280 per sample volume. Data are shown as the mean ± standard deviation (SD) from six independent patient samples. P -values were calculated using one-way ANOVA.
    Figure Legend Snippet: Minimal sera volume for exosome detection and RNA extraction. (A) Representative immunoblots for CD63 comparing exosome enrichment using ultracentrifugation (UC) and ExoQuick (EQ) among samples of varying volumes. (B) Scatter dot plots depict total exosomal RNA recovered per sample volume using EQ. (C) Scatter dot plots show RNA quality measured by OD 260 /OD 280 per sample volume. Data are shown as the mean ± standard deviation (SD) from six independent patient samples. P -values were calculated using one-way ANOVA.

    Techniques Used: RNA Extraction, Western Blot, Standard Deviation

    Exosomal RNAs are stable over two decades. Dot plots of total RNA yield and OD 260 /OD 280 from 105 EQ-treated archival patient samples using the RNeasy Mini Kit combined with TRIzol LS. Storage time for each sample is indicated on the x-axis. The Spearman’s rank correlations for RNA yield versus storing time and OD 260 /OD 280 versus storage time are 0.185 ( P = 0.06) and 0.04 ( P = 0.70), respectively.
    Figure Legend Snippet: Exosomal RNAs are stable over two decades. Dot plots of total RNA yield and OD 260 /OD 280 from 105 EQ-treated archival patient samples using the RNeasy Mini Kit combined with TRIzol LS. Storage time for each sample is indicated on the x-axis. The Spearman’s rank correlations for RNA yield versus storing time and OD 260 /OD 280 versus storage time are 0.185 ( P = 0.06) and 0.04 ( P = 0.70), respectively.

    Techniques Used:

    Flow chart for evaluation of exosomal RNAs from cell-free sera as biomarkers for human diseases. Graphic summary of the workflow including time allotment for preparation for cell-free serum (steps ①-③), comparison of methods for exosome enrichment (step ④), validation by transmission electron microscopy (TEM) and immunoblotting for CD63 or other exosomal markers (step ⑤), RNA extraction (step ⑥), and preparation of RNA-Seq libraries (step ⑦). 10–100 nanograms RNA can be used for library preparation with the NEBNext Ultra Directional RNA Library Prep Kit. Step ② is an optional centrifugation step that can be included to ensure the most efficient removal of trace amounts of cell debris and shedding microvesicles. A validation step can be performed with RT-qPCR for specific candidate RNA following RNA-Seq analysis (step ⑧).
    Figure Legend Snippet: Flow chart for evaluation of exosomal RNAs from cell-free sera as biomarkers for human diseases. Graphic summary of the workflow including time allotment for preparation for cell-free serum (steps ①-③), comparison of methods for exosome enrichment (step ④), validation by transmission electron microscopy (TEM) and immunoblotting for CD63 or other exosomal markers (step ⑤), RNA extraction (step ⑥), and preparation of RNA-Seq libraries (step ⑦). 10–100 nanograms RNA can be used for library preparation with the NEBNext Ultra Directional RNA Library Prep Kit. Step ② is an optional centrifugation step that can be included to ensure the most efficient removal of trace amounts of cell debris and shedding microvesicles. A validation step can be performed with RT-qPCR for specific candidate RNA following RNA-Seq analysis (step ⑧).

    Techniques Used: Flow Cytometry, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, RNA Extraction, RNA Sequencing Assay, Centrifugation, Quantitative RT-PCR

    Exosomal RNA-Seq libraries from archival sera specimens. (A) Bioanalyzer results for 5 independent specimens demonstrate inserted size of exosomal RNAs. (B) Number of mapped reads generated from RNA-Seq libraries for 5 independent specimens with 20 ng exosomal RNAs. RNA-Seq library concentrations were calculated using three different methods: Nanodrop, BioAnalyzer (BioA), and RT-qPCR (qPCR). The Spearman’s rank correlations for Nanodrop versus reads, BioAnalyzer versus reads, and qPCR versus reads are 0.7 ( P = 0.23), 0.6 ( P = 0.35), and 0.7 ( P = 0.23), respectively. (C) Relative abundance of biotypes detected for 5 exosomal RNA-Seq libraries from healthy female subjects. PC = protein coding, PP = processed pseudogene, lincRNA = long intergenic non-coding RNA, UP = unprocessed pseudogene, snRNA = small nuclear RNA, miRNA = microRNA, TEC = to be experimentally confirmed, snoRNA = small nucleolar RNA, TUP = transcribed unprocessed pseudogene.
    Figure Legend Snippet: Exosomal RNA-Seq libraries from archival sera specimens. (A) Bioanalyzer results for 5 independent specimens demonstrate inserted size of exosomal RNAs. (B) Number of mapped reads generated from RNA-Seq libraries for 5 independent specimens with 20 ng exosomal RNAs. RNA-Seq library concentrations were calculated using three different methods: Nanodrop, BioAnalyzer (BioA), and RT-qPCR (qPCR). The Spearman’s rank correlations for Nanodrop versus reads, BioAnalyzer versus reads, and qPCR versus reads are 0.7 ( P = 0.23), 0.6 ( P = 0.35), and 0.7 ( P = 0.23), respectively. (C) Relative abundance of biotypes detected for 5 exosomal RNA-Seq libraries from healthy female subjects. PC = protein coding, PP = processed pseudogene, lincRNA = long intergenic non-coding RNA, UP = unprocessed pseudogene, snRNA = small nuclear RNA, miRNA = microRNA, TEC = to be experimentally confirmed, snoRNA = small nucleolar RNA, TUP = transcribed unprocessed pseudogene.

    Techniques Used: RNA Sequencing Assay, Generated, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

    64) Product Images from "A comparative analysis of RNA sequencing methods with ribosome RNA depletion for degraded and low-input total RNA from formalin-fixed and paraffin-embedded samples"

    Article Title: A comparative analysis of RNA sequencing methods with ribosome RNA depletion for degraded and low-input total RNA from formalin-fixed and paraffin-embedded samples

    Journal: BMC Genomics

    doi: 10.1186/s12864-019-6166-3

    Genome alignment profiles of four RNA-seq kits with paired FFPE and FF samples. For FF RNA from GM 12878 cell line, all the four kits got similar alignment profiles while the input RNA of TaKaRa kit was 10 ng and it of the others was 100 ng. For FFPE RNA from GM 12878 cell line, the library with TaKaRa kit produced more exon profiles with 10 ng total RNA input
    Figure Legend Snippet: Genome alignment profiles of four RNA-seq kits with paired FFPE and FF samples. For FF RNA from GM 12878 cell line, all the four kits got similar alignment profiles while the input RNA of TaKaRa kit was 10 ng and it of the others was 100 ng. For FFPE RNA from GM 12878 cell line, the library with TaKaRa kit produced more exon profiles with 10 ng total RNA input

    Techniques Used: RNA Sequencing Assay, Formalin-fixed Paraffin-Embedded, Produced

    Comparison of transcripts quantification in FFPE and FF samples across four kits. High concordance in transcript quantifications were got between FF and FFPE samples using any kit. For either FFPE or FF RNA from GM 12878, the Pearson R between TaKaRa kit and the other three kits were lower and higher similarity was got among KAPA, Vazyme and QIAGEN kits
    Figure Legend Snippet: Comparison of transcripts quantification in FFPE and FF samples across four kits. High concordance in transcript quantifications were got between FF and FFPE samples using any kit. For either FFPE or FF RNA from GM 12878, the Pearson R between TaKaRa kit and the other three kits were lower and higher similarity was got among KAPA, Vazyme and QIAGEN kits

    Techniques Used: Formalin-fixed Paraffin-Embedded

    The distribution of transcripts of four RNA-seq kits with paired FFPE and FF samples. For FF RNA from GM 12878 cell line, more low-expressed transcripts were detected in the library of TaKaRa with only 10 ng total RNA input. For FFPE RNA from GM 12878 cell line, similar transcripts were detected while the input RNA of TaKaRa kit was 10 ng and it of the others was 100 ng
    Figure Legend Snippet: The distribution of transcripts of four RNA-seq kits with paired FFPE and FF samples. For FF RNA from GM 12878 cell line, more low-expressed transcripts were detected in the library of TaKaRa with only 10 ng total RNA input. For FFPE RNA from GM 12878 cell line, similar transcripts were detected while the input RNA of TaKaRa kit was 10 ng and it of the others was 100 ng

    Techniques Used: RNA Sequencing Assay, Formalin-fixed Paraffin-Embedded

    65) Product Images from "Molecular homology and difference between spontaneous canine mammary cancer and human breast cancer"

    Article Title: Molecular homology and difference between spontaneous canine mammary cancer and human breast cancer

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-14-0392

    Coding sequence mutations are frequent in canine simple carcinomas; chromatin-modification genes are downregulated in canine complex carcinomas A , the fractions (the Y-axis) of somatic base substitution types of simple carcinomas (IDs indicated by the X-axis) detected by WES. The total number of significantly mutated genes in each tumor is also shown, and tumor 5 has many DNA repair genes mutated. B , synonymous (green dots) and non-synonymous substitutions (yellow dots), and a nonsense mutation (red star) uncovered in the USH2A gene in tumor 5. C , the base substitution (compared to the dog reference genome) rates of the three sample types in coding regions with 30-300X RNA-seq read coverage. The p-values were calculated by t-tests. D , the heatmap of 751 genes differentially expressed at FDR ≤ 0.2 between simple and complex carcinomas (red: upregulation; green: downregulation). The right panel illustrates the enriched functions of each gene cluster indicated, and the 35 chromatin modifiers downregulated in complex carcinomas are specified below.
    Figure Legend Snippet: Coding sequence mutations are frequent in canine simple carcinomas; chromatin-modification genes are downregulated in canine complex carcinomas A , the fractions (the Y-axis) of somatic base substitution types of simple carcinomas (IDs indicated by the X-axis) detected by WES. The total number of significantly mutated genes in each tumor is also shown, and tumor 5 has many DNA repair genes mutated. B , synonymous (green dots) and non-synonymous substitutions (yellow dots), and a nonsense mutation (red star) uncovered in the USH2A gene in tumor 5. C , the base substitution (compared to the dog reference genome) rates of the three sample types in coding regions with 30-300X RNA-seq read coverage. The p-values were calculated by t-tests. D , the heatmap of 751 genes differentially expressed at FDR ≤ 0.2 between simple and complex carcinomas (red: upregulation; green: downregulation). The right panel illustrates the enriched functions of each gene cluster indicated, and the 35 chromatin modifiers downregulated in complex carcinomas are specified below.

    Techniques Used: Sequencing, Modification, Mutagenesis, RNA Sequencing Assay

    66) 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

    67) Product Images from "Long Noncoding RNA MEG3 Is an Epigenetic Determinant of Oncogenic Signaling in Functional Pancreatic Neuroendocrine Tumor Cells"

    Article Title: Long Noncoding RNA MEG3 Is an Epigenetic Determinant of Oncogenic Signaling in Functional Pancreatic Neuroendocrine Tumor Cells

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00278-17

    ChIRP-Seq reveals m-Meg3 enrichment at multiple m-c-Met loci. (A) Representative agarose gel image showing the specificity of the m-Meg3 ChIRP probes. RNA was isolated after m-Meg3 ChIRP from the V3 (vector) and the M5 (stable MIN6-4N cells stably expressing the m-Meg3-3 isoform which lacks exon 4) cell lines. The RNA was then used for RT-PCR. Input corresponds to the RT-PCR product using RNA isolated before m-Meg3 ChIRP-Seq. Odd and even correspond to RT-PCR using RNA after m-Meg3 ChIRP with probes located at odd and even locations on the m-Meg3 RNA. The gel image represents products of the RT-PCR performed with primers 1F/1R (flanking exon 7 and exon 8) that recognize all m-Meg3 isoforms. gapdh served as the negative control. cDNAs from three replicates of V3 and M5 ChIRP-Seq were pooled due to low yields and sequenced. (B) m-Meg3 ChIRP-PCR in a stable cell line expressing full-length m-Meg3. RNA was isolated after m-Meg3 ChIRP from the 9V (vector) and the 14M (stable MIN6-4N cells with the m-Meg3-1 isoform, which encompasses all 10 exons) cell lines. The RNA was then used for RT-PCR. Input corresponds to RT-PCR from RNA isolated before m-Meg3 ChIRP. RT-PCR was performed with primers 1F/1R (flanking exons 7 and 8) that recognize all m-Meg3 isoforms and further confirmed with the ex3F/ex4R primer pair (flanking exons 3 and 4), specific for the m-Meg3-1 isoform. gapdh served as the negative control. (C) m-Meg3 enrichment patterns at discrete m-c-Met genomic regions in different m-Meg3 stable cell lines. DNA was isolated after m-Meg3 ChIRP from two different m-Meg-3 stable MIN6-4N cell lines and their respective vector controls. The DNA was then subjected to whole-genome amplification (WGA) and subsequent purification. The purified WGA DNA was then used to set up qPCRs in duplicate with primers specific for m-c-Met genomic regions identified by m-Meg3 ChIRP-Seq, namely, the m-c-Met upstream region, the m-c-Met exon 18 region, the m-c-Met exon 20 region, and also the previously identified kb +63 enhancer. The qPCR data are represented as percent input of DNA.
    Figure Legend Snippet: ChIRP-Seq reveals m-Meg3 enrichment at multiple m-c-Met loci. (A) Representative agarose gel image showing the specificity of the m-Meg3 ChIRP probes. RNA was isolated after m-Meg3 ChIRP from the V3 (vector) and the M5 (stable MIN6-4N cells stably expressing the m-Meg3-3 isoform which lacks exon 4) cell lines. The RNA was then used for RT-PCR. Input corresponds to the RT-PCR product using RNA isolated before m-Meg3 ChIRP-Seq. Odd and even correspond to RT-PCR using RNA after m-Meg3 ChIRP with probes located at odd and even locations on the m-Meg3 RNA. The gel image represents products of the RT-PCR performed with primers 1F/1R (flanking exon 7 and exon 8) that recognize all m-Meg3 isoforms. gapdh served as the negative control. cDNAs from three replicates of V3 and M5 ChIRP-Seq were pooled due to low yields and sequenced. (B) m-Meg3 ChIRP-PCR in a stable cell line expressing full-length m-Meg3. RNA was isolated after m-Meg3 ChIRP from the 9V (vector) and the 14M (stable MIN6-4N cells with the m-Meg3-1 isoform, which encompasses all 10 exons) cell lines. The RNA was then used for RT-PCR. Input corresponds to RT-PCR from RNA isolated before m-Meg3 ChIRP. RT-PCR was performed with primers 1F/1R (flanking exons 7 and 8) that recognize all m-Meg3 isoforms and further confirmed with the ex3F/ex4R primer pair (flanking exons 3 and 4), specific for the m-Meg3-1 isoform. gapdh served as the negative control. (C) m-Meg3 enrichment patterns at discrete m-c-Met genomic regions in different m-Meg3 stable cell lines. DNA was isolated after m-Meg3 ChIRP from two different m-Meg-3 stable MIN6-4N cell lines and their respective vector controls. The DNA was then subjected to whole-genome amplification (WGA) and subsequent purification. The purified WGA DNA was then used to set up qPCRs in duplicate with primers specific for m-c-Met genomic regions identified by m-Meg3 ChIRP-Seq, namely, the m-c-Met upstream region, the m-c-Met exon 18 region, the m-c-Met exon 20 region, and also the previously identified kb +63 enhancer. The qPCR data are represented as percent input of DNA.

    Techniques Used: Agarose Gel Electrophoresis, Isolation, Plasmid Preparation, Stable Transfection, Expressing, Reverse Transcription Polymerase Chain Reaction, Negative Control, Polymerase Chain Reaction, Whole Genome Amplification, Purification, Real-time Polymerase Chain Reaction

    m-Meg3 TFO-9 regulates the m-c-Met transcript. (A) Schematic of full-length m-Meg3-1 exon structure showing the TFO-9 coordinates. Full-length m-Meg3-1 is depicted, with exons 1 to 10 numbered (top) and the sequence length in base pairs (bottom). TFO-9, a GA- and GT-rich 16-mer sequence, is shown mapping to the C-terminal portion of exon 10 in m-Meg3-1. TFO-9, spanning bp 1822 to 1838, was predicted by Triplexator to form triplexes with double-stranded DNA. (B) Effect of TFO-9 on the expression of m-Meg3 and m-c-Met transcripts. RNA was isolated at 48 h and 96 h posttransfection from MIN6-4N cells transiently transfected with TFO RNA oligonucleotides. Purified RNA converted to cDNA was subjected to qPCR analyses with primers specific for m-Meg3-1 or m-c-Met. The data represent an average from three independent experiments and multiple technical replicates (mean ± SD) *, P ≤ 0.05.
    Figure Legend Snippet: m-Meg3 TFO-9 regulates the m-c-Met transcript. (A) Schematic of full-length m-Meg3-1 exon structure showing the TFO-9 coordinates. Full-length m-Meg3-1 is depicted, with exons 1 to 10 numbered (top) and the sequence length in base pairs (bottom). TFO-9, a GA- and GT-rich 16-mer sequence, is shown mapping to the C-terminal portion of exon 10 in m-Meg3-1. TFO-9, spanning bp 1822 to 1838, was predicted by Triplexator to form triplexes with double-stranded DNA. (B) Effect of TFO-9 on the expression of m-Meg3 and m-c-Met transcripts. RNA was isolated at 48 h and 96 h posttransfection from MIN6-4N cells transiently transfected with TFO RNA oligonucleotides. Purified RNA converted to cDNA was subjected to qPCR analyses with primers specific for m-Meg3-1 or m-c-Met. The data represent an average from three independent experiments and multiple technical replicates (mean ± SD) *, P ≤ 0.05.

    Techniques Used: Sequencing, Expressing, Isolation, Transfection, Purification, Real-time Polymerase Chain Reaction

    m-Meg3 associates with PRC2 components in mouse insulinoma cell line models. (A and B) Association of m-Meg3 RNA with EZH2 and H3K27me3. RNA-ChIP in Men1 cells (Meg3 proficient) was performed using the antibodies directed toward EZH2 (A) and H3K27me3 (B). qPCR was performed on cDNA obtained from RNA isolated after RNA-ChIP. qPCR data were calculated as percent RNA input and are shown as the average from two independent biological replicates and multiple technical replicates (mean ± SD). IgG served as the negative control for the RNA-ChIP assay. (C and D) EZH2 and H3K27me3 enrichment at the m-c-Met regions identified by m-Meg3 ChIRP-Seq. ChIP assay was performed in Men1 cells using the indicated antibodies. DNA isolated after ChIP was used for qPCR, with primers specific for the m-c-Met upstream region, the +63-kb enhancer, the m-c-Met exon 18 region, and the m-c-Met exon 20 region. EZH2 (C) and H3K27me3 (D) enrichment was calculated as percent chromatin DNA input and constitutes the average from three independent biological replicates and multiple technical replicates (mean ± SD). (E and F) Enhancer-signature histone modifications at the m-c-Met loci identified by m-Meg3 ChIRP-Seq. MIN6-4N cells were subjected to ChIP assays and qPCR analyses to detect the enrichment of H3K27Ac (E) and H3K4me1 (F) at the m-c-Met upstream region, the kb +63 enhancer, the m-c-Met exon 18 region, and the m-c-Met exon 20 region. The data represent an average from two independent biological replicates and multiple technical replicates (mean ± SD).
    Figure Legend Snippet: m-Meg3 associates with PRC2 components in mouse insulinoma cell line models. (A and B) Association of m-Meg3 RNA with EZH2 and H3K27me3. RNA-ChIP in Men1 cells (Meg3 proficient) was performed using the antibodies directed toward EZH2 (A) and H3K27me3 (B). qPCR was performed on cDNA obtained from RNA isolated after RNA-ChIP. qPCR data were calculated as percent RNA input and are shown as the average from two independent biological replicates and multiple technical replicates (mean ± SD). IgG served as the negative control for the RNA-ChIP assay. (C and D) EZH2 and H3K27me3 enrichment at the m-c-Met regions identified by m-Meg3 ChIRP-Seq. ChIP assay was performed in Men1 cells using the indicated antibodies. DNA isolated after ChIP was used for qPCR, with primers specific for the m-c-Met upstream region, the +63-kb enhancer, the m-c-Met exon 18 region, and the m-c-Met exon 20 region. EZH2 (C) and H3K27me3 (D) enrichment was calculated as percent chromatin DNA input and constitutes the average from three independent biological replicates and multiple technical replicates (mean ± SD). (E and F) Enhancer-signature histone modifications at the m-c-Met loci identified by m-Meg3 ChIRP-Seq. MIN6-4N cells were subjected to ChIP assays and qPCR analyses to detect the enrichment of H3K27Ac (E) and H3K4me1 (F) at the m-c-Met upstream region, the kb +63 enhancer, the m-c-Met exon 18 region, and the m-c-Met exon 20 region. The data represent an average from two independent biological replicates and multiple technical replicates (mean ± SD).

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Isolation, Negative Control

    68) Product Images from "Distinct high resolution genome profiles of early onset and late onset colorectal cancer integrated with gene expression data identify candidate susceptibility loci"

    Article Title: Distinct high resolution genome profiles of early onset and late onset colorectal cancer integrated with gene expression data identify candidate susceptibility loci

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-9-100

    Study design comparing the tumor genomes of early onset and late onset colorectal cancer patients . Tumor samples from early onset and late onset CRC patients were analyzed with aCGH (DNA/genome level) and the data were integrated with the mRNA expression (RNA/transcriptome level) of the same samples. About 10 000 genes show corresponding DNA and RNA level. Exclusion of genes located outside chromosomal regions with statistically significant difference between the two patient groups resulted in 107 genes, summarized in Additional file 6 . Further, exclusion of genes whose mRNA expression levels were not statistically significant between patients groups resulted in a short list of seven potential predisposing genes, as summarized in Table 3.
    Figure Legend Snippet: Study design comparing the tumor genomes of early onset and late onset colorectal cancer patients . Tumor samples from early onset and late onset CRC patients were analyzed with aCGH (DNA/genome level) and the data were integrated with the mRNA expression (RNA/transcriptome level) of the same samples. About 10 000 genes show corresponding DNA and RNA level. Exclusion of genes located outside chromosomal regions with statistically significant difference between the two patient groups resulted in 107 genes, summarized in Additional file 6 . Further, exclusion of genes whose mRNA expression levels were not statistically significant between patients groups resulted in a short list of seven potential predisposing genes, as summarized in Table 3.

    Techniques Used: Expressing

    69) Product Images from "Delivery of the 7-dehydrocholesterol reductase gene to the central nervous system using adeno-associated virus vector in a mouse model of Smith-Lemli-Opitz Syndrome"

    Article Title: Delivery of the 7-dehydrocholesterol reductase gene to the central nervous system using adeno-associated virus vector in a mouse model of Smith-Lemli-Opitz Syndrome

    Journal: Molecular Genetics and Metabolism Reports

    doi: 10.1016/j.ymgmr.2015.07.006

    Expression of mRNA in brain. RNA extracts from brains of AAV-DHCR7 treated (lanes 1–11) and saline treated (lanes 12–17) mice were tested for h DHCR7 mRNA (A) and mouse GAPDH mRNA (B). Non-reverse transcriptase treated samples showed the absence of h DHCR7 or GAPDH specific PCR product confirming that the amplification products result from mRNA derived cDNA rather than from the presence of contaminating DNA (data not shown).
    Figure Legend Snippet: Expression of mRNA in brain. RNA extracts from brains of AAV-DHCR7 treated (lanes 1–11) and saline treated (lanes 12–17) mice were tested for h DHCR7 mRNA (A) and mouse GAPDH mRNA (B). Non-reverse transcriptase treated samples showed the absence of h DHCR7 or GAPDH specific PCR product confirming that the amplification products result from mRNA derived cDNA rather than from the presence of contaminating DNA (data not shown).

    Techniques Used: Expressing, Mouse Assay, Polymerase Chain Reaction, Amplification, Derivative Assay

    70) 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

    71) Product Images from "Functional Characteristics of the Flying Squirrel's Cecal Microbiota under a Leaf-Based Diet, Based on Multiple Meta-Omic Profiling"

    Article Title: Functional Characteristics of the Flying Squirrel's Cecal Microbiota under a Leaf-Based Diet, Based on Multiple Meta-Omic Profiling

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2017.02622

    Abundance distributions of COGs in metagenomes (DNA-level) and metatranscriptomes (RNA-level) of cecal microbiota from two flying squirrels (FS1 and FS2). Only abundant COGs that constituted > 0.5% in either library are shown.
    Figure Legend Snippet: Abundance distributions of COGs in metagenomes (DNA-level) and metatranscriptomes (RNA-level) of cecal microbiota from two flying squirrels (FS1 and FS2). Only abundant COGs that constituted > 0.5% in either library are shown.

    Techniques Used:

    Glycoside hydrolases (GHs) detected in metagenomes (DNA-level) and metatranscriptomes (RNA-level) of cecal microbiota from two flying squirrels (FS1 and FS2). Only abundant GHs that constituted > 0.05% in either library are shown.
    Figure Legend Snippet: Glycoside hydrolases (GHs) detected in metagenomes (DNA-level) and metatranscriptomes (RNA-level) of cecal microbiota from two flying squirrels (FS1 and FS2). Only abundant GHs that constituted > 0.05% in either library are shown.

    Techniques Used:

    Genus-level taxonomic compositions of metagenomes (DNA-level) and metatranscriptomes (RNA-level) of cecal microbiota from two flying squirrels (FS1 and FS2). Top 30 abundant genera that constituted > 0.5% in either library are shown, with their phyla in parentheses.
    Figure Legend Snippet: Genus-level taxonomic compositions of metagenomes (DNA-level) and metatranscriptomes (RNA-level) of cecal microbiota from two flying squirrels (FS1 and FS2). Top 30 abundant genera that constituted > 0.5% in either library are shown, with their phyla in parentheses.

    Techniques Used:

    Abundance distributions of KOs in metagenomes (DNA-level) and metatranscriptomes (RNA-level) of cecal microbiota from two flying squirrels (FS1 and FS2). Only abundant KOs that constituted > 0.5% in either library are shown.
    Figure Legend Snippet: Abundance distributions of KOs in metagenomes (DNA-level) and metatranscriptomes (RNA-level) of cecal microbiota from two flying squirrels (FS1 and FS2). Only abundant KOs that constituted > 0.5% in either library are shown.

    Techniques Used:

    72) Product Images from "A Next-Generation Sequencing-Based Molecular Approach to Characterize a Tick Vector in Lyme Disease"

    Article Title: A Next-Generation Sequencing-Based Molecular Approach to Characterize a Tick Vector in Lyme Disease

    Journal: OMICS : a Journal of Integrative Biology

    doi: 10.1089/omi.2018.0089

    Workflow for analysis of next-generation sequencing data. Workflow employed for analysis of (A) whole-genome and (B) RNA-seq sequencing data. Genomic DNA and RNA sequences for Ixodida (taxonomy ID: txid6935) and bacterial genomes were downloaded from
    Figure Legend Snippet: Workflow for analysis of next-generation sequencing data. Workflow employed for analysis of (A) whole-genome and (B) RNA-seq sequencing data. Genomic DNA and RNA sequences for Ixodida (taxonomy ID: txid6935) and bacterial genomes were downloaded from

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

    73) Product Images from "Optimizing exosomal RNA isolation for RNA-Seq analyses of archival sera specimens"

    Article Title: Optimizing exosomal RNA isolation for RNA-Seq analyses of archival sera specimens

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0196913

    Comparison of sera exosomal RNA using four different RNA extraction methods. (A) Total RNA yield from ultracentrifugation (UC) and ExoQuick (EQ) treated samples using the RNeasy Mini Kit combined with TRIzol LS, the RNeasy Mini Kit alone, conventional RNA precipitation, and AllPrep DNA/RNA Mini Kit. (B) Demonstration of RNA quality measured by OD 260 /OD 280 in EQ and UC treated samples. Data are shown as the mean ± SD from six independent patient samples. *** P
    Figure Legend Snippet: Comparison of sera exosomal RNA using four different RNA extraction methods. (A) Total RNA yield from ultracentrifugation (UC) and ExoQuick (EQ) treated samples using the RNeasy Mini Kit combined with TRIzol LS, the RNeasy Mini Kit alone, conventional RNA precipitation, and AllPrep DNA/RNA Mini Kit. (B) Demonstration of RNA quality measured by OD 260 /OD 280 in EQ and UC treated samples. Data are shown as the mean ± SD from six independent patient samples. *** P

    Techniques Used: RNA Extraction

    74) Product Images from "Optimizing exosomal RNA isolation for RNA-Seq analyses of archival sera specimens"

    Article Title: Optimizing exosomal RNA isolation for RNA-Seq analyses of archival sera specimens

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0196913

    Comparison of sera exosomal RNA using four different RNA extraction methods. (A) Total RNA yield from ultracentrifugation (UC) and ExoQuick (EQ) treated samples using the RNeasy Mini Kit combined with TRIzol LS, the RNeasy Mini Kit alone, conventional RNA precipitation, and AllPrep DNA/RNA Mini Kit. (B) Demonstration of RNA quality measured by OD 260 /OD 280 in EQ and UC treated samples. Data are shown as the mean ± SD from six independent patient samples. *** P
    Figure Legend Snippet: Comparison of sera exosomal RNA using four different RNA extraction methods. (A) Total RNA yield from ultracentrifugation (UC) and ExoQuick (EQ) treated samples using the RNeasy Mini Kit combined with TRIzol LS, the RNeasy Mini Kit alone, conventional RNA precipitation, and AllPrep DNA/RNA Mini Kit. (B) Demonstration of RNA quality measured by OD 260 /OD 280 in EQ and UC treated samples. Data are shown as the mean ± SD from six independent patient samples. *** P

    Techniques Used: RNA Extraction

    75) Product Images from "Picoeukaryotic Diversity And Activity in the Northwestern Pacific Ocean Based on rDNA and rRNA High-Throughput Sequencing"

    Article Title: Picoeukaryotic Diversity And Activity in the Northwestern Pacific Ocean Based on rDNA and rRNA High-Throughput Sequencing

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.03259

    Cluster diagram of the Bray-Curtis dissimilarities based on the log-transformed relative OTU abundance for each sample. The separated DNA (A) and RNA (B) community groups (with asterisks at the node) exist with significant compositional differences (DNA: r 2 = 0.31718, P = 0.001; RNA: r 2 = 0.27023, P = 0.001) as determined by PERMANOVA. The percent dissimilarity among samples is shown along the horizontal axes.
    Figure Legend Snippet: Cluster diagram of the Bray-Curtis dissimilarities based on the log-transformed relative OTU abundance for each sample. The separated DNA (A) and RNA (B) community groups (with asterisks at the node) exist with significant compositional differences (DNA: r 2 = 0.31718, P = 0.001; RNA: r 2 = 0.27023, P = 0.001) as determined by PERMANOVA. The percent dissimilarity among samples is shown along the horizontal axes.

    Techniques Used: Transformation Assay

    Number of OTUs unveiled by DNA and RNA surveys. The pie chart indicated the OTU numbers mainly at class level; the inner ring represented these classes belong to the phylum; the outer ring indicated the number of OTUs at super-group level.
    Figure Legend Snippet: Number of OTUs unveiled by DNA and RNA surveys. The pie chart indicated the OTU numbers mainly at class level; the inner ring represented these classes belong to the phylum; the outer ring indicated the number of OTUs at super-group level.

    Techniques Used:

    Number of OTUs of higher taxonomic groups (sequences proportion over 1% of the total DNA or RNA abundance) based on the DNA-derived (A) and RNA-derived (B) approaches for each sample. Others refer to groups with relatively lower abundance (sequences proportion
    Figure Legend Snippet: Number of OTUs of higher taxonomic groups (sequences proportion over 1% of the total DNA or RNA abundance) based on the DNA-derived (A) and RNA-derived (B) approaches for each sample. Others refer to groups with relatively lower abundance (sequences proportion

    Techniques Used: Derivative Assay

    Relative abundance of higher taxonomic groups (sequences proportion over 1% of the total DNA or RNA abundance) based on the DNA-derived (A) and RNA-derived (B) approaches for each sample. Others refer to groups with relatively lower abundance (sequences proportion
    Figure Legend Snippet: Relative abundance of higher taxonomic groups (sequences proportion over 1% of the total DNA or RNA abundance) based on the DNA-derived (A) and RNA-derived (B) approaches for each sample. Others refer to groups with relatively lower abundance (sequences proportion

    Techniques Used: Derivative Assay

    (A) Scatter diagram of PC 1 and PC 2 derived from the relative abundance of higher taxonomic groups in the DNA and RNA databases detected in each sample, blue color indicated the sequences was derived from the DNA survey and red color indicated the sequences was derived from the RNA survey. Ma, Mamiellophyceae; Sp, Spirotrichea; Pe, Pelagophyceae; Ba, Bacillariophyta; Dict, Dictyochophyceae; Prym, Prymnesiophyceae. Boxplot representing the RNA: DNA ratios for Mamiellophyceae (B) , Pelagophyceae (C) , and Spirotrichea (D) in all and individual samples.
    Figure Legend Snippet: (A) Scatter diagram of PC 1 and PC 2 derived from the relative abundance of higher taxonomic groups in the DNA and RNA databases detected in each sample, blue color indicated the sequences was derived from the DNA survey and red color indicated the sequences was derived from the RNA survey. Ma, Mamiellophyceae; Sp, Spirotrichea; Pe, Pelagophyceae; Ba, Bacillariophyta; Dict, Dictyochophyceae; Prym, Prymnesiophyceae. Boxplot representing the RNA: DNA ratios for Mamiellophyceae (B) , Pelagophyceae (C) , and Spirotrichea (D) in all and individual samples.

    Techniques Used: Derivative Assay

    Related Articles

    Synthesized:

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    Cytometry:

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    Quantitative RT-PCR:

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

    Article Title: Decreased expression of the immediate early protein, ICP4, by deletion of the tegument protein VP22 of equine herpesvirus type 1
    Article Snippet: After 1 hr adsorption, cells were washed three times with MEM and incubated at 37°C in a 5% CO2 atmosphere in 1 ml/well of MEM, and this time point was defined as 0 hrpi. .. Total RNA and DNA of infected cells were extracted using AllPrep DNA/RNA Mini Kit (QIAGEN K.K., Hilden, Germany) at 0 and 1 hrpi. cDNA was synthesized from 0.5 µ g of total RNA using ReverTra Ace (TOYOBO, Osaka, Japan).

    Real-time Polymerase Chain Reaction:

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    Article Title: Clinical and Mucosal Immune Correlates of HIV-1 Semen Levels in Antiretroviral-Naive Men
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    Article Title: Decreased expression of the immediate early protein, ICP4, by deletion of the tegument protein VP22 of equine herpesvirus type 1
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    Incubation:

    Article Title: Decreased expression of the immediate early protein, ICP4, by deletion of the tegument protein VP22 of equine herpesvirus type 1
    Article Snippet: After 1 hr adsorption, cells were washed three times with MEM and incubated at 37°C in a 5% CO2 atmosphere in 1 ml/well of MEM, and this time point was defined as 0 hrpi. .. Total RNA and DNA of infected cells were extracted using AllPrep DNA/RNA Mini Kit (QIAGEN K.K., Hilden, Germany) at 0 and 1 hrpi. cDNA was synthesized from 0.5 µ g of total RNA using ReverTra Ace (TOYOBO, Osaka, Japan).

    Expressing:

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

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    Article Title: Loss of spermatogonia and wide-spread DNA methylation defects in newborn male mice deficient in DNMT3L
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    Infection:

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    Polymerase Chain Reaction:

    Article Title: Increased HIV-1 transcriptional activity and infectious burden in peripheral blood and gut-associated CD4+ T cells expressing CD30
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    Article Title: Decreased expression of the immediate early protein, ICP4, by deletion of the tegument protein VP22 of equine herpesvirus type 1
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    Recombinant:

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    Nucleic Acid Purification:

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    Methylation:

    Article Title: Loss of spermatogonia and wide-spread DNA methylation defects in newborn male mice deficient in DNMT3L
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    Mutagenesis:

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    Isolation:

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    Article Title: FIV establishes a latent infection in feline peripheral blood CD4+ T lymphocytes in vivo during the asymptomatic phase of infection
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    Article Snippet: .. DNA/RNA extraction and APOE genotyping Genomic DNA was isolated from frozen PMB using the AllPrep DNA/RNA Mini Kit (Qiagen). .. Nucleic acid concentrations were measured by NanoPhotometer (Implen), and samples were stored at –20°C prior to use.

    Article Title: Characterization of the fecal and mucosa-associated microbiota in dogs with colorectal epithelial tumors
    Article Snippet: .. Isolation of DNA and RNA from mucosal samples and cDNA synthesis Using the AllPrep DNA/RNA Mini Kit (Qiagen), RNA and DNA were isolated from ~ 8 mg of mucosal tissue that had been preserved in Allprotect Tissue Reagent (Qiagen). .. The manufacturer’s instructions were followed except for extended homogenization and additional enzymatic lysis steps as reported in [ ].

    Article Title: Loss of spermatogonia and wide-spread DNA methylation defects in newborn male mice deficient in DNMT3L
    Article Snippet: Paragraph title: Isolation of Dnmt3L mutant germ cells and DNA Methylation Analysis ... RNA and DNA were simultaneously extracted using the AllPrep DNA/RNA Mini kit according to the manufacturer's protocol (Qiagen); the RNA was used in qRT-PCR analyses as described above, while DNA methylation analyses were carried out on the DNA.

    Purification:

    Article Title: Characterization of the fecal and mucosa-associated microbiota in dogs with colorectal epithelial tumors
    Article Snippet: Isolation of DNA and RNA from mucosal samples and cDNA synthesis Using the AllPrep DNA/RNA Mini Kit (Qiagen), RNA and DNA were isolated from ~ 8 mg of mucosal tissue that had been preserved in Allprotect Tissue Reagent (Qiagen). .. For optimal RNA purification, on column DNAse treatment was included as described in the DNA/RNA Mini Kit protocol.

    Article Title: Simultaneous purification of DNA and RNA from microbiota in a single colonic mucosal biopsy
    Article Snippet: .. A further optimised tissue homogenisation and lysis protocol comprising mechanical bead beating, lysis buffer replacement and enzymatic treatment, in combination with the AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) resulted in efficient and simultaneous purification of microbial and human RNA and DNA from a single mucosal colonic tissue sample. .. The present work provides a unique possibility to study RNA and DNA from the same mucosal biopsy sample, making a direct comparison between metabolically active microbes and total microbial DNA.

    Article Title: Clinical and Mucosal Immune Correlates of HIV-1 Semen Levels in Antiretroviral-Naive Men
    Article Snippet: .. Bacterial Load Quantification For bacterial load quantification, 500 μL of thawed SP was lysed using a combination of chemical and mechanical methods and purified using AllPrep DNA/RNA Mini Kit (QIAGEN). ..

    Article Title: Simultaneous purification of DNA and RNA from microbiota in a single colonic mucosal biopsy
    Article Snippet: .. Total RNA and DNA were purified from colonic mucosal tissue using the AllPrep DNA/RNA Mini Kit (Qiagen). .. Manufacturer’s instructions were followed with the exception of the lysis steps, where three tissue lysis protocols (Protocol 1, 2 and 3) were performed and evaluated (Fig. ; Additional file ).

    Sequencing:

    Article Title: Loss of spermatogonia and wide-spread DNA methylation defects in newborn male mice deficient in DNMT3L
    Article Snippet: RNA and DNA were simultaneously extracted using the AllPrep DNA/RNA Mini kit according to the manufacturer's protocol (Qiagen); the RNA was used in qRT-PCR analyses as described above, while DNA methylation analyses were carried out on the DNA. .. For a given sequence, primers were designed to flank the restriction sites of interest and real-time PCR was performed on the different digested templates using the QuantiTect ™ SYBR® Green PCR kit (Qiagen) according to the manufacturer's suggested conditions for use of the Mx3000P PCR machine (Stratagene).

    Mouse Assay:

    Article Title: Loss of spermatogonia and wide-spread DNA methylation defects in newborn male mice deficient in DNMT3L
    Article Snippet: Isolation of Dnmt3L mutant germ cells and DNA Methylation Analysis Dnmt3L +/- females were crossed with GOF18/deltaPE-Oct-4/GFP males to obtain [Dnmt3L +/- , GFP+ ] mice. .. RNA and DNA were simultaneously extracted using the AllPrep DNA/RNA Mini kit according to the manufacturer's protocol (Qiagen); the RNA was used in qRT-PCR analyses as described above, while DNA methylation analyses were carried out on the DNA.

    SYBR Green Assay:

    Article Title: Loss of spermatogonia and wide-spread DNA methylation defects in newborn male mice deficient in DNMT3L
    Article Snippet: RNA and DNA were simultaneously extracted using the AllPrep DNA/RNA Mini kit according to the manufacturer's protocol (Qiagen); the RNA was used in qRT-PCR analyses as described above, while DNA methylation analyses were carried out on the DNA. .. For a given sequence, primers were designed to flank the restriction sites of interest and real-time PCR was performed on the different digested templates using the QuantiTect ™ SYBR® Green PCR kit (Qiagen) according to the manufacturer's suggested conditions for use of the Mx3000P PCR machine (Stratagene).

    Homogenization:

    Article Title: Characterization of the fecal and mucosa-associated microbiota in dogs with colorectal epithelial tumors
    Article Snippet: Isolation of DNA and RNA from mucosal samples and cDNA synthesis Using the AllPrep DNA/RNA Mini Kit (Qiagen), RNA and DNA were isolated from ~ 8 mg of mucosal tissue that had been preserved in Allprotect Tissue Reagent (Qiagen). .. The manufacturer’s instructions were followed except for extended homogenization and additional enzymatic lysis steps as reported in [ ].

    Article Title: Simultaneous purification of DNA and RNA from microbiota in a single colonic mucosal biopsy
    Article Snippet: .. A further optimised tissue homogenisation and lysis protocol comprising mechanical bead beating, lysis buffer replacement and enzymatic treatment, in combination with the AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) resulted in efficient and simultaneous purification of microbial and human RNA and DNA from a single mucosal colonic tissue sample. .. The present work provides a unique possibility to study RNA and DNA from the same mucosal biopsy sample, making a direct comparison between metabolically active microbes and total microbial DNA.

    DNA Methylation Assay:

    Article Title: Loss of spermatogonia and wide-spread DNA methylation defects in newborn male mice deficient in DNMT3L
    Article Snippet: .. RNA and DNA were simultaneously extracted using the AllPrep DNA/RNA Mini kit according to the manufacturer's protocol (Qiagen); the RNA was used in qRT-PCR analyses as described above, while DNA methylation analyses were carried out on the DNA. .. Quantitative analysis of DNA methylation using real-time PCR, or qAMP, was conducted to analyze the DNA methylation status of a number of sequences as described [ ].

    Concentration Assay:

    Article Title: Association between TLR-9 polymorphisms and colon cancer susceptibility in Saudi Arabian female patients
    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. .. The isolated RNA concentration, purity, and quality were determined using an Agilent 2100 Bioanalyzer system and Agilent Small RNA analysis kit (Agilent Technologies, Waldbronn, Germany) according to the manufacturer’s instructions.

    DNA Purification:

    Article Title: Increased HIV-1 transcriptional activity and infectious burden in peripheral blood and gut-associated CD4+ T cells expressing CD30
    Article Snippet: .. Quantification of cell-associated HIV-1 RNA and DNA Purification of HIV-1 DNA and RNA from sorted cell populations was achieved using a Qiagen AllPrep DNA/RNA mini Kit, and following the manufacturer’s standard protocol, with an additional DNAse treatment (QIAgen). .. Given the small number of CD30+ cells that could be obtained from flow sorting, these cell fractions were spiked into uninfected carrier PBMC to maximize HIV-1 DNA and RNA recovery and normalize extraction efficiency between CD30+ and CD30- CD4+ T cell populations.

    Lysis:

    Article Title: Characterization of the fecal and mucosa-associated microbiota in dogs with colorectal epithelial tumors
    Article Snippet: Isolation of DNA and RNA from mucosal samples and cDNA synthesis Using the AllPrep DNA/RNA Mini Kit (Qiagen), RNA and DNA were isolated from ~ 8 mg of mucosal tissue that had been preserved in Allprotect Tissue Reagent (Qiagen). .. The manufacturer’s instructions were followed except for extended homogenization and additional enzymatic lysis steps as reported in [ ].

    Article Title: Simultaneous purification of DNA and RNA from microbiota in a single colonic mucosal biopsy
    Article Snippet: .. A further optimised tissue homogenisation and lysis protocol comprising mechanical bead beating, lysis buffer replacement and enzymatic treatment, in combination with the AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) resulted in efficient and simultaneous purification of microbial and human RNA and DNA from a single mucosal colonic tissue sample. .. The present work provides a unique possibility to study RNA and DNA from the same mucosal biopsy sample, making a direct comparison between metabolically active microbes and total microbial DNA.

    Article Title: Simultaneous purification of DNA and RNA from microbiota in a single colonic mucosal biopsy
    Article Snippet: Total RNA and DNA were purified from colonic mucosal tissue using the AllPrep DNA/RNA Mini Kit (Qiagen). .. Manufacturer’s instructions were followed with the exception of the lysis steps, where three tissue lysis protocols (Protocol 1, 2 and 3) were performed and evaluated (Fig. ; Additional file ).

    Article Title: Simultaneous purification of DNA and RNA from microbiota in a single colonic mucosal biopsy
    Article Snippet: .. The finding of bead beating and enzymatic lysis in combination with the AllPrep DNA/RNA Mini Kit being superior in breaking the ‘hard-to-lyse’ cell walls of the Firmicutes phylum (Fig. ) resulted in development of protocol 3 in an attempt to use enzymatic lysis and bead beating and still preserve microbial and human RNA. .. Protocol 3 is a combination of protocol 2 and a modified version of the enzymatic lysis procedure for stool samples published by Franzosa and colleagues [ ].

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    Qiagen allprep dna rna mini kit
    TLR-9 mRNA expression in colon cancer cells and colon cancer tissues. Notes: Total <t>RNA</t> 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 <t>DNA;</t> GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
    Allprep Dna Rna Mini Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    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

    Article Snippet: A further optimised tissue homogenisation and lysis protocol comprising mechanical bead beating, lysis buffer replacement and enzymatic treatment, in combination with the AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) resulted in efficient and simultaneous purification of microbial and human RNA and DNA from a single mucosal colonic tissue sample.

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