rna sequence  (Thermo Fisher)


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    Structured Review

    Thermo Fisher rna sequence
    Slot blot hybridization analysis ( A and B ) to detect enrichment of intracellular <t>RNA-protein</t> complexes composed of FR-α mRNA cis- element bound to <t>hnRNP-E1</t> following exposure of HeLa-IU 1 - HF cells to increasing physiologically relevant concentrations
    Rna Sequence, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna sequence/product/Thermo Fisher
    Average 90 stars, based on 18 article reviews
    Price from $9.99 to $1999.99
    rna sequence - by Bioz Stars, 2020-09
    90/100 stars

    Images

    1) Product Images from "Incrimination of Heterogeneous Nuclear Ribonucleoprotein E1 (hnRNP-E1) as a Candidate Sensor of Physiological Folate Deficiency *"

    Article Title: Incrimination of Heterogeneous Nuclear Ribonucleoprotein E1 (hnRNP-E1) as a Candidate Sensor of Physiological Folate Deficiency *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.230938

    Slot blot hybridization analysis ( A and B ) to detect enrichment of intracellular RNA-protein complexes composed of FR-α mRNA cis- element bound to hnRNP-E1 following exposure of HeLa-IU 1 - HF cells to increasing physiologically relevant concentrations
    Figure Legend Snippet: Slot blot hybridization analysis ( A and B ) to detect enrichment of intracellular RNA-protein complexes composed of FR-α mRNA cis- element bound to hnRNP-E1 following exposure of HeLa-IU 1 - HF cells to increasing physiologically relevant concentrations

    Techniques Used: Dot Blot, Hybridization

    Effect of RNA interference using siRNA against hnRNP-E1 mRNA on the rates of biosynthesis of newly synthesized hnRNP-E1 in HeLa-IU 1 - HF cells with simultaneous evaluation of the biosynthetic rate of newly synthesized FR under basal conditions and after
    Figure Legend Snippet: Effect of RNA interference using siRNA against hnRNP-E1 mRNA on the rates of biosynthesis of newly synthesized hnRNP-E1 in HeLa-IU 1 - HF cells with simultaneous evaluation of the biosynthetic rate of newly synthesized FR under basal conditions and after

    Techniques Used: Synthesized

    Comparison of the Influence of dl - and l -Homocysteine on RNA-Protein Interactions Involving 18-Base FR-α mRNA cis-Element (and Various Other mRNAs) and Purified hnRNP-E1
    Figure Legend Snippet: Comparison of the Influence of dl - and l -Homocysteine on RNA-Protein Interactions Involving 18-Base FR-α mRNA cis-Element (and Various Other mRNAs) and Purified hnRNP-E1

    Techniques Used: Purification

    Related Articles

    Clone Assay:

    Article Title: The C-Terminal Domain of CENP-C Displays Multiple and Critical Functions for Mammalian Centromere Formation
    Article Snippet: .. Anti CENP-C antibodies: the DNA sequence encoding amino-terminal half of human CENP-C (amino acids 23–410) was cloned into pFastBac HTb vector (Invitrogen). .. The histidine-tagged recombinant protein was expressed in SF9 cells using Bac-to-Bac baculovirus expression system (Invitrogen) and affinity purified by nickel column chromatography.

    Article Title: A natural antisense transcript regulates Zeb2/Sip1 gene expression during Snail1-induced epithelial–mesenchymal transition
    Article Snippet: .. The DNA sequence containing the IRES element was cloned by amplifying the sequences +525/+2377, +1118/+2377, +1719/+2377, and +2356/+2998 (from the main transcription start site) using high-fidelity Pfx Platinium polymerase (Invitrogen). .. Oligonucleotides include EcoRI and NcoI sites for facilitating cloning of the fragment in plasmids pRF or pRFL (pRF without promotor), kindly provided by Dr. Raúl Méndez, (Centre de Regulació Genòmica, Barcelona, Spain).

    Amplification:

    Article Title: First Report of Oryctes rhinoceros nudivirus (Coleoptera: Scarabaeidae) Causing Severe Disease in Allomyrina dichotoma in Korea
    Article Snippet: .. The viral DNA fragment amplified by AdV-F1/R1, -F2/R2, and -F3/R3 was isolated from agarose gel, and DNA sequences were determined with an Applied Biosystems 3730xl DNA Analyzer (MacroGen, Seoul, Korea). ..

    Article Title: Formulation in DDA-MPLA-TDB Liposome Enhances the Immunogenicity and Protective Efficacy of a DNA Vaccine against Mycobacterium tuberculosis Infection
    Article Snippet: .. Construction of Recombinant Eukaryotic Expression Plasmid pCMFO The DNA sequence of CMFO was consisted of the tandem-fusion genes encoding the signal peptide of human tissue plasminogen activator (SPtPA ) and CMFO, which was amplified by polymerase chain reaction (PCR) with primers (F: 5′-CCCAAGCTTGCCACCATGGATGCAATGAAGA-3′, R: 5′-CGGGATCCTTATCGCGGCATCCTGCGCCAGACGAAC-3′) from commercially synthesized plasmid pDC316-CMFO (Life Invitrogen of Shanghai, Shanghai, China). .. For amplification, the Phusion high-fidelity DNA polymerase (Thermo Fisher Scientific, Inc., MA, USA) was used.

    Article Title: High-Level Vancomycin-Resistant Staphylococcus aureus Isolates Associated with a Polymicrobial Biofilm ▿
    Article Snippet: .. DNA sequences of the PCR products were determined using dRhodamine dye terminator cycle sequencing (Applied Biosystems) and the same primers as those used for PCR amplification. .. DNA sequences of the forward and reverse strands were determined from independently amplified PCR fragments.

    Agarose Gel Electrophoresis:

    Article Title: First Report of Oryctes rhinoceros nudivirus (Coleoptera: Scarabaeidae) Causing Severe Disease in Allomyrina dichotoma in Korea
    Article Snippet: .. The viral DNA fragment amplified by AdV-F1/R1, -F2/R2, and -F3/R3 was isolated from agarose gel, and DNA sequences were determined with an Applied Biosystems 3730xl DNA Analyzer (MacroGen, Seoul, Korea). ..

    Synthesized:

    Article Title: Sp100 colocalizes with HPV replication foci and restricts the productive stage of the infectious cycle
    Article Snippet: .. N-terminal, EE epitope-tagged versions of each Sp100 isoforms were generated by restriction digestion of pEYFP-Sp100A, pEYFP-Sp100B, pEYFP-Sp100C, pEYFP-Sp100HMG, pEYFP-Sp100B-W655Q with NheI and XhoI to remove the N-terminal EYFP tag and it was replaced with a synthesized DNA sequence containing a T7/SP6 promoter, β-actin mRNA leader sequence, and an EE epitope tag (GeneART; Hamburg, Germany). ..

    Article Title: Formulation in DDA-MPLA-TDB Liposome Enhances the Immunogenicity and Protective Efficacy of a DNA Vaccine against Mycobacterium tuberculosis Infection
    Article Snippet: .. Construction of Recombinant Eukaryotic Expression Plasmid pCMFO The DNA sequence of CMFO was consisted of the tandem-fusion genes encoding the signal peptide of human tissue plasminogen activator (SPtPA ) and CMFO, which was amplified by polymerase chain reaction (PCR) with primers (F: 5′-CCCAAGCTTGCCACCATGGATGCAATGAAGA-3′, R: 5′-CGGGATCCTTATCGCGGCATCCTGCGCCAGACGAAC-3′) from commercially synthesized plasmid pDC316-CMFO (Life Invitrogen of Shanghai, Shanghai, China). .. For amplification, the Phusion high-fidelity DNA polymerase (Thermo Fisher Scientific, Inc., MA, USA) was used.

    Isolation:

    Article Title: First Report of Oryctes rhinoceros nudivirus (Coleoptera: Scarabaeidae) Causing Severe Disease in Allomyrina dichotoma in Korea
    Article Snippet: .. The viral DNA fragment amplified by AdV-F1/R1, -F2/R2, and -F3/R3 was isolated from agarose gel, and DNA sequences were determined with an Applied Biosystems 3730xl DNA Analyzer (MacroGen, Seoul, Korea). ..

    Chromosome Walking:

    Article Title: The Unique Morgue Ubiquitination Protein Is Conserved in a Diverse but Restricted Set of Invertebrates
    Article Snippet: .. The complete DNA sequence of the 2.2-kb EST was obtained via a primer-walking approach using primers derived from the pCMV Sport 6.1 plasmid vector (Invitrogen Corp.) and the insert. .. Total RNA was extracted from approximately 30 D. pulex adult water fleas obtained from the Carolina Biological Supply Company (Burlington, NC) using the RNeasy Fibrous Tissue Mini Kit (QIAGEN Inc.).

    Sequencing:

    Article Title: Sp100 colocalizes with HPV replication foci and restricts the productive stage of the infectious cycle
    Article Snippet: .. N-terminal, EE epitope-tagged versions of each Sp100 isoforms were generated by restriction digestion of pEYFP-Sp100A, pEYFP-Sp100B, pEYFP-Sp100C, pEYFP-Sp100HMG, pEYFP-Sp100B-W655Q with NheI and XhoI to remove the N-terminal EYFP tag and it was replaced with a synthesized DNA sequence containing a T7/SP6 promoter, β-actin mRNA leader sequence, and an EE epitope tag (GeneART; Hamburg, Germany). ..

    Article Title: The C-Terminal Domain of CENP-C Displays Multiple and Critical Functions for Mammalian Centromere Formation
    Article Snippet: .. Anti CENP-C antibodies: the DNA sequence encoding amino-terminal half of human CENP-C (amino acids 23–410) was cloned into pFastBac HTb vector (Invitrogen). .. The histidine-tagged recombinant protein was expressed in SF9 cells using Bac-to-Bac baculovirus expression system (Invitrogen) and affinity purified by nickel column chromatography.

    Article Title: The hr1 and Fusion Peptide Regions of the Subgroup B Avian Sarcoma and Leukosis Virus Envelope Glycoprotein Influence Low pH-Dependent Membrane Fusion
    Article Snippet: .. The DNA sequences of the envB genes were determined using Big Dye sequencing (Applied Biosystems, Foster City, CA). .. PCR amplification was also used to screen individual single cell clones for the Δ 152–164 mutation using primers (5′-cagaactacaactgctagg-3′) and (5′-cggtttcgaggagttagagg-3′) which generates either a 209 bp (wild-type) or a 170 bp (Δ 152–164 mutant) product.

    Article Title: Formulation in DDA-MPLA-TDB Liposome Enhances the Immunogenicity and Protective Efficacy of a DNA Vaccine against Mycobacterium tuberculosis Infection
    Article Snippet: .. Construction of Recombinant Eukaryotic Expression Plasmid pCMFO The DNA sequence of CMFO was consisted of the tandem-fusion genes encoding the signal peptide of human tissue plasminogen activator (SPtPA ) and CMFO, which was amplified by polymerase chain reaction (PCR) with primers (F: 5′-CCCAAGCTTGCCACCATGGATGCAATGAAGA-3′, R: 5′-CGGGATCCTTATCGCGGCATCCTGCGCCAGACGAAC-3′) from commercially synthesized plasmid pDC316-CMFO (Life Invitrogen of Shanghai, Shanghai, China). .. For amplification, the Phusion high-fidelity DNA polymerase (Thermo Fisher Scientific, Inc., MA, USA) was used.

    Article Title: The Unique Morgue Ubiquitination Protein Is Conserved in a Diverse but Restricted Set of Invertebrates
    Article Snippet: .. The complete DNA sequence of the 2.2-kb EST was obtained via a primer-walking approach using primers derived from the pCMV Sport 6.1 plasmid vector (Invitrogen Corp.) and the insert. .. Total RNA was extracted from approximately 30 D. pulex adult water fleas obtained from the Carolina Biological Supply Company (Burlington, NC) using the RNeasy Fibrous Tissue Mini Kit (QIAGEN Inc.).

    Article Title: A natural antisense transcript regulates Zeb2/Sip1 gene expression during Snail1-induced epithelial–mesenchymal transition
    Article Snippet: .. The DNA sequence containing the IRES element was cloned by amplifying the sequences +525/+2377, +1118/+2377, +1719/+2377, and +2356/+2998 (from the main transcription start site) using high-fidelity Pfx Platinium polymerase (Invitrogen). .. Oligonucleotides include EcoRI and NcoI sites for facilitating cloning of the fragment in plasmids pRF or pRFL (pRF without promotor), kindly provided by Dr. Raúl Méndez, (Centre de Regulació Genòmica, Barcelona, Spain).

    Article Title: High-Level Vancomycin-Resistant Staphylococcus aureus Isolates Associated with a Polymicrobial Biofilm ▿
    Article Snippet: .. DNA sequences of the PCR products were determined using dRhodamine dye terminator cycle sequencing (Applied Biosystems) and the same primers as those used for PCR amplification. .. DNA sequences of the forward and reverse strands were determined from independently amplified PCR fragments.

    Generated:

    Article Title: Sp100 colocalizes with HPV replication foci and restricts the productive stage of the infectious cycle
    Article Snippet: .. N-terminal, EE epitope-tagged versions of each Sp100 isoforms were generated by restriction digestion of pEYFP-Sp100A, pEYFP-Sp100B, pEYFP-Sp100C, pEYFP-Sp100HMG, pEYFP-Sp100B-W655Q with NheI and XhoI to remove the N-terminal EYFP tag and it was replaced with a synthesized DNA sequence containing a T7/SP6 promoter, β-actin mRNA leader sequence, and an EE epitope tag (GeneART; Hamburg, Germany). ..

    Plasmid Preparation:

    Article Title: The C-Terminal Domain of CENP-C Displays Multiple and Critical Functions for Mammalian Centromere Formation
    Article Snippet: .. Anti CENP-C antibodies: the DNA sequence encoding amino-terminal half of human CENP-C (amino acids 23–410) was cloned into pFastBac HTb vector (Invitrogen). .. The histidine-tagged recombinant protein was expressed in SF9 cells using Bac-to-Bac baculovirus expression system (Invitrogen) and affinity purified by nickel column chromatography.

    Article Title: Formulation in DDA-MPLA-TDB Liposome Enhances the Immunogenicity and Protective Efficacy of a DNA Vaccine against Mycobacterium tuberculosis Infection
    Article Snippet: .. Construction of Recombinant Eukaryotic Expression Plasmid pCMFO The DNA sequence of CMFO was consisted of the tandem-fusion genes encoding the signal peptide of human tissue plasminogen activator (SPtPA ) and CMFO, which was amplified by polymerase chain reaction (PCR) with primers (F: 5′-CCCAAGCTTGCCACCATGGATGCAATGAAGA-3′, R: 5′-CGGGATCCTTATCGCGGCATCCTGCGCCAGACGAAC-3′) from commercially synthesized plasmid pDC316-CMFO (Life Invitrogen of Shanghai, Shanghai, China). .. For amplification, the Phusion high-fidelity DNA polymerase (Thermo Fisher Scientific, Inc., MA, USA) was used.

    Article Title: The Unique Morgue Ubiquitination Protein Is Conserved in a Diverse but Restricted Set of Invertebrates
    Article Snippet: .. The complete DNA sequence of the 2.2-kb EST was obtained via a primer-walking approach using primers derived from the pCMV Sport 6.1 plasmid vector (Invitrogen Corp.) and the insert. .. Total RNA was extracted from approximately 30 D. pulex adult water fleas obtained from the Carolina Biological Supply Company (Burlington, NC) using the RNeasy Fibrous Tissue Mini Kit (QIAGEN Inc.).

    Expressing:

    Article Title: Formulation in DDA-MPLA-TDB Liposome Enhances the Immunogenicity and Protective Efficacy of a DNA Vaccine against Mycobacterium tuberculosis Infection
    Article Snippet: .. Construction of Recombinant Eukaryotic Expression Plasmid pCMFO The DNA sequence of CMFO was consisted of the tandem-fusion genes encoding the signal peptide of human tissue plasminogen activator (SPtPA ) and CMFO, which was amplified by polymerase chain reaction (PCR) with primers (F: 5′-CCCAAGCTTGCCACCATGGATGCAATGAAGA-3′, R: 5′-CGGGATCCTTATCGCGGCATCCTGCGCCAGACGAAC-3′) from commercially synthesized plasmid pDC316-CMFO (Life Invitrogen of Shanghai, Shanghai, China). .. For amplification, the Phusion high-fidelity DNA polymerase (Thermo Fisher Scientific, Inc., MA, USA) was used.

    Polymerase Chain Reaction:

    Article Title: Formulation in DDA-MPLA-TDB Liposome Enhances the Immunogenicity and Protective Efficacy of a DNA Vaccine against Mycobacterium tuberculosis Infection
    Article Snippet: .. Construction of Recombinant Eukaryotic Expression Plasmid pCMFO The DNA sequence of CMFO was consisted of the tandem-fusion genes encoding the signal peptide of human tissue plasminogen activator (SPtPA ) and CMFO, which was amplified by polymerase chain reaction (PCR) with primers (F: 5′-CCCAAGCTTGCCACCATGGATGCAATGAAGA-3′, R: 5′-CGGGATCCTTATCGCGGCATCCTGCGCCAGACGAAC-3′) from commercially synthesized plasmid pDC316-CMFO (Life Invitrogen of Shanghai, Shanghai, China). .. For amplification, the Phusion high-fidelity DNA polymerase (Thermo Fisher Scientific, Inc., MA, USA) was used.

    Article Title: High-Level Vancomycin-Resistant Staphylococcus aureus Isolates Associated with a Polymicrobial Biofilm ▿
    Article Snippet: .. DNA sequences of the PCR products were determined using dRhodamine dye terminator cycle sequencing (Applied Biosystems) and the same primers as those used for PCR amplification. .. DNA sequences of the forward and reverse strands were determined from independently amplified PCR fragments.

    Recombinant:

    Article Title: Formulation in DDA-MPLA-TDB Liposome Enhances the Immunogenicity and Protective Efficacy of a DNA Vaccine against Mycobacterium tuberculosis Infection
    Article Snippet: .. Construction of Recombinant Eukaryotic Expression Plasmid pCMFO The DNA sequence of CMFO was consisted of the tandem-fusion genes encoding the signal peptide of human tissue plasminogen activator (SPtPA ) and CMFO, which was amplified by polymerase chain reaction (PCR) with primers (F: 5′-CCCAAGCTTGCCACCATGGATGCAATGAAGA-3′, R: 5′-CGGGATCCTTATCGCGGCATCCTGCGCCAGACGAAC-3′) from commercially synthesized plasmid pDC316-CMFO (Life Invitrogen of Shanghai, Shanghai, China). .. For amplification, the Phusion high-fidelity DNA polymerase (Thermo Fisher Scientific, Inc., MA, USA) was used.

    Derivative Assay:

    Article Title: The Unique Morgue Ubiquitination Protein Is Conserved in a Diverse but Restricted Set of Invertebrates
    Article Snippet: .. The complete DNA sequence of the 2.2-kb EST was obtained via a primer-walking approach using primers derived from the pCMV Sport 6.1 plasmid vector (Invitrogen Corp.) and the insert. .. Total RNA was extracted from approximately 30 D. pulex adult water fleas obtained from the Carolina Biological Supply Company (Burlington, NC) using the RNeasy Fibrous Tissue Mini Kit (QIAGEN Inc.).

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    Thermo Fisher whole drg rna sequencing l4 drg
    Spinal Cord Injury (SCI) induces an acute transcriptional response in the dorsal root ganglion <t>(DRG)</t> that differs from sciatic nerve injury (SNI). A ) Schematic of the experimental design for L4 DRG RNA sequencing after SNI (n=3) and SCI (n=4). B) Proportional Venn diagrams for differentially expressed (DE) genes upregulated (red) or downregulated (blue) after SNI and SCI (p-adj
    Whole Drg Rna Sequencing L4 Drg, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/whole drg rna sequencing l4 drg/product/Thermo Fisher
    Average 84 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    whole drg rna sequencing l4 drg - by Bioz Stars, 2020-09
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    92
    Thermo Fisher sequencing total rna
    Main flow diagram of <t>transcriptome</t> sequencing. Abbreviations: QC, quality control; KEGG, Kyoto Encyclopedia of Genes and Genomes; lncRNA, long noncoding <t>RNA;</t> GO, gene ontology.
    Sequencing Total Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 853 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sequencing total rna/product/Thermo Fisher
    Average 92 stars, based on 853 article reviews
    Price from $9.99 to $1999.99
    sequencing total rna - by Bioz Stars, 2020-09
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    89
    Thermo Fisher short hairpin rna shrna sequences
    Silencing of LRRK2 expression rescues ArfGAP1-induced neurite shortening. (A) Validation of short hairpin <t>RNA</t> <t>(shRNA)-mediated</t> silencing of endogenous LRRK2 expression in rat primary cortical neuronal cultures. Lentiviral vectors expressing non-silencing shRNA (LV-sh-control) or LRRK2-specific shRNA (LV-sh-LRRK2) were used to infect primary neurons at increasing viral doses ranging from 1.66 to 25 ng of P24 antigen per ml of media. Western blot analysis of neuronal extracts reveals the viral dose-dependent knockdown of LRRK2 with LRRK2-specific shRNAs but not control shRNA using two independent rabbit LRRK2 antibodies (MJFF-2/c41-2 or JH5514). β-tubulin indicates equivalent protein loading. (B) Silencing of endogenous LRRK2 expression in GFP-labeled cortical neurons with lentiviral-shRNA vectors revealed by confocal fluorescence microscopy with a rabbit anti-LRRK2 antibody (JH5514). (C) Primary cortical neurons were infected with lentiviral vectors expressing shRNAs (non-silencing control or LRRK2-specific) at DIV 2, subsequently transfected with ArfGAP1-YFP and DsRed constructs at a 10∶1 molar ratio at DIV 3, and fixed at DIV 6. Fluorescent microscopic images reveal the co-labeling of cortical neurons with ArfGAP1-YFP and DsRed. DsRed images were pseudo-colored with ICA for neurite length measurements. Neuronal soma ( arrows ) and axonal processes ( arrowheads ) are indicated. Scale bars: 400 µm. (D) Analysis of DsRed-positive axonal processes reveals a robust shortening of axons induced by ArfGAP1 expression and increased axon length induced by silencing of LRRK2 with LV-sh-LRRK2 vector, compared to DsRed alone (control). Silencing of LRRK2 with LV-sh-LRRK2 produces a partial rescue of ArfGAP1-induced axon shortening compared to LV-sh-control. Bars represent mean (± SEM) length of axons expressed as a percent of DsRed alone (control/LV-sh-control) from > 85 DsRed-positive neurons from two independent experiments/cultures. ** P
    Short Hairpin Rna Shrna Sequences, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/short hairpin rna shrna sequences/product/Thermo Fisher
    Average 89 stars, based on 25 article reviews
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    Image Search Results


    Spinal Cord Injury (SCI) induces an acute transcriptional response in the dorsal root ganglion (DRG) that differs from sciatic nerve injury (SNI). A ) Schematic of the experimental design for L4 DRG RNA sequencing after SNI (n=3) and SCI (n=4). B) Proportional Venn diagrams for differentially expressed (DE) genes upregulated (red) or downregulated (blue) after SNI and SCI (p-adj

    Journal: bioRxiv

    Article Title: Ascending dorsal column sensory neurons respond to spinal cord injury and downregulate genes related to lipid metabolism

    doi: 10.1101/2020.05.07.083584

    Figure Lengend Snippet: Spinal Cord Injury (SCI) induces an acute transcriptional response in the dorsal root ganglion (DRG) that differs from sciatic nerve injury (SNI). A ) Schematic of the experimental design for L4 DRG RNA sequencing after SNI (n=3) and SCI (n=4). B) Proportional Venn diagrams for differentially expressed (DE) genes upregulated (red) or downregulated (blue) after SNI and SCI (p-adj

    Article Snippet: Whole DRG RNA sequencing L4 DRG were dissected and homogenized in 300μl of lysis buffer on ice and RNA purified using the PureLink RNA Mini kit (Thermo Fisher), which was submitted to the Genome Technology Access Center (GTAC) at Washington University for library preparation and sequencing.

    Techniques: RNA Sequencing Assay

    Spinal Cord Injury (SCI) does not induce a pro-growth state in dorsal column (DC) neurons in vitro . A ) Schematic of the experimental design and peripheral and central projects of major sensory neuron subtypes of L4 dorsal root ganglion (DRG) neurons. B ) Representative images of L4 dorsal root ganglion (DRG) neurons from Thy1YFP16 mice labeled with NF200 (blue) and TrkA (red) antibodies. C ) Quantification of B indicating percent overlap of YFP, NF200, and TrkA labeling in Thy1YFP16 mice (n=4). D ) Representative images of L4 DRG neurons labeled with ATF3 and Islet1 antibodies in Thy1YFP16 mice in naive or 3 days after sciatic nerve injury (SNI) or SCI. E ) Quantification of D indicating percentage of ATF3 positive, Islet-1 labeled neuronal nuclei in all neurons, as well as YFP negative and YFP positive neurons, for each condition (n=3/group; 2-way ANOVA). White arrowheads point to ATF3 positive neuronal nuclei. F ) Representative images of cultured cells from L4 DRG labeled for all neurons (TUJ1) and YFP positive neurons in naïve or 3 days after SNI or SCI. G ) Quantification of F indicating the percentage of neurons extending neurites and the average radial length of neurites, in YFP negative and YFP positive neurons, for each condition (n=4/group; 2-way ANOVA). Yellow arrows point to the cell body of YFP positive neurons. *p

    Journal: bioRxiv

    Article Title: Ascending dorsal column sensory neurons respond to spinal cord injury and downregulate genes related to lipid metabolism

    doi: 10.1101/2020.05.07.083584

    Figure Lengend Snippet: Spinal Cord Injury (SCI) does not induce a pro-growth state in dorsal column (DC) neurons in vitro . A ) Schematic of the experimental design and peripheral and central projects of major sensory neuron subtypes of L4 dorsal root ganglion (DRG) neurons. B ) Representative images of L4 dorsal root ganglion (DRG) neurons from Thy1YFP16 mice labeled with NF200 (blue) and TrkA (red) antibodies. C ) Quantification of B indicating percent overlap of YFP, NF200, and TrkA labeling in Thy1YFP16 mice (n=4). D ) Representative images of L4 DRG neurons labeled with ATF3 and Islet1 antibodies in Thy1YFP16 mice in naive or 3 days after sciatic nerve injury (SNI) or SCI. E ) Quantification of D indicating percentage of ATF3 positive, Islet-1 labeled neuronal nuclei in all neurons, as well as YFP negative and YFP positive neurons, for each condition (n=3/group; 2-way ANOVA). White arrowheads point to ATF3 positive neuronal nuclei. F ) Representative images of cultured cells from L4 DRG labeled for all neurons (TUJ1) and YFP positive neurons in naïve or 3 days after SNI or SCI. G ) Quantification of F indicating the percentage of neurons extending neurites and the average radial length of neurites, in YFP negative and YFP positive neurons, for each condition (n=4/group; 2-way ANOVA). Yellow arrows point to the cell body of YFP positive neurons. *p

    Article Snippet: Whole DRG RNA sequencing L4 DRG were dissected and homogenized in 300μl of lysis buffer on ice and RNA purified using the PureLink RNA Mini kit (Thermo Fisher), which was submitted to the Genome Technology Access Center (GTAC) at Washington University for library preparation and sequencing.

    Techniques: In Vitro, Mouse Assay, Labeling, Cell Culture

    Main flow diagram of transcriptome sequencing. Abbreviations: QC, quality control; KEGG, Kyoto Encyclopedia of Genes and Genomes; lncRNA, long noncoding RNA; GO, gene ontology.

    Journal: International Journal of Nanomedicine

    Article Title: Impairments of spatial learning and memory following intrahippocampal injection in rats of 3-mercaptopropionic acid-modified CdTe quantum dots and molecular mechanisms

    doi: 10.2147/IJN.S104985

    Figure Lengend Snippet: Main flow diagram of transcriptome sequencing. Abbreviations: QC, quality control; KEGG, Kyoto Encyclopedia of Genes and Genomes; lncRNA, long noncoding RNA; GO, gene ontology.

    Article Snippet: Transcriptome-library preparation and sequencing Total RNA was obtained from rats exposed to 1,600 μg/mL 2.2 nm or 3.5 nm MPA-modified CdTe QDs using Trizol (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol.

    Techniques: Flow Cytometry, Sequencing

    Mitochondrial stress activates the ATF4 pathway in vitro and in vivo independent of the UPR mt . (A) mRNA expression analysis of the ATF4 pathway and UPR mt genes in HeLa cells 6 and 24 h after exposure to paraquat (PQ) at a final concentration of 400 µM. (B and C) Enrichment score plots from GSEA using RNA sequencing data of cells treated with (B) the TRAP1 inhibitor GTPP and (C) the LONP1 inhibitor CDDO ( GSE75247 ). List of mitochondrial stress genes (mt-stress genes; Table S5) was used as the gene set of interest. FDR q-val, false discovery rate adjusted p-value; NES, normalized enrichment score. (D and E) mRNA expression analysis of the ATF4 pathway and UPR mt genes in HeLa cells after (D) knockdown of LONP1 using two different shRNAs and (E) 6 and 24 h of exposure to mdivi-1 at a final concentration of 50 µM. (F and G) Enrichment score plots from GSEA using microarray data (F) from hearts of mice deficient in ClpP ( GSE40207 ) and (G) from brains of mice deficient in Htra2 ( GSE13035 ). NOM p-val, nominal p-value. (H and I) Barcode plot representing the enrichment in the core mt-stress genes in (H) mitochondrial myopathies versus nonmitochondrial myopathies ( GSE43698 ) and (I) myopathies caused by mtDNA deletions ( GSE1462 ). Enrichment was analyzed using the fold-change relative to control. p-value was calculated using the ROAST test, which confirmed that gene expression changes induced by mitochondrial stress significantly correlate with gene expression changes caused by mitochondrial myopathies. Black vertical lines represent the genes, and the red and blue rectangles the cutoff for the up-regulated and down-regulated genes, respectively. The black horizontal line indicates neutral enrichment, whereas the red line shows the enrichment of up-regulated genes. All experiments were independently performed at least two times, using triplicates for each condition; data are presented as mean ± SEM of a representative experiment; *, P

    Journal: The Journal of Cell Biology

    Article Title: Multi-omics analysis identifies ATF4 as a key regulator of the mitochondrial stress response in mammals

    doi: 10.1083/jcb.201702058

    Figure Lengend Snippet: Mitochondrial stress activates the ATF4 pathway in vitro and in vivo independent of the UPR mt . (A) mRNA expression analysis of the ATF4 pathway and UPR mt genes in HeLa cells 6 and 24 h after exposure to paraquat (PQ) at a final concentration of 400 µM. (B and C) Enrichment score plots from GSEA using RNA sequencing data of cells treated with (B) the TRAP1 inhibitor GTPP and (C) the LONP1 inhibitor CDDO ( GSE75247 ). List of mitochondrial stress genes (mt-stress genes; Table S5) was used as the gene set of interest. FDR q-val, false discovery rate adjusted p-value; NES, normalized enrichment score. (D and E) mRNA expression analysis of the ATF4 pathway and UPR mt genes in HeLa cells after (D) knockdown of LONP1 using two different shRNAs and (E) 6 and 24 h of exposure to mdivi-1 at a final concentration of 50 µM. (F and G) Enrichment score plots from GSEA using microarray data (F) from hearts of mice deficient in ClpP ( GSE40207 ) and (G) from brains of mice deficient in Htra2 ( GSE13035 ). NOM p-val, nominal p-value. (H and I) Barcode plot representing the enrichment in the core mt-stress genes in (H) mitochondrial myopathies versus nonmitochondrial myopathies ( GSE43698 ) and (I) myopathies caused by mtDNA deletions ( GSE1462 ). Enrichment was analyzed using the fold-change relative to control. p-value was calculated using the ROAST test, which confirmed that gene expression changes induced by mitochondrial stress significantly correlate with gene expression changes caused by mitochondrial myopathies. Black vertical lines represent the genes, and the red and blue rectangles the cutoff for the up-regulated and down-regulated genes, respectively. The black horizontal line indicates neutral enrichment, whereas the red line shows the enrichment of up-regulated genes. All experiments were independently performed at least two times, using triplicates for each condition; data are presented as mean ± SEM of a representative experiment; *, P

    Article Snippet: RNA sequencing library preparation and analysis Total RNA from HeLa cells was extracted using TRIzol (Thermo Fisher Scientific) and purified by column using RNeasy Mini kit (QIAGEN).

    Techniques: In Vitro, In Vivo, Expressing, Concentration Assay, RNA Sequencing Assay, Microarray, Mouse Assay

    RNA sequencing and proteomic analysis upon mitochondrial stress. (A) Schematic representation of the omics experiments. HeLa cells were treated with compounds for 24 h, and the transcriptome, proteome, and metabolome were analyzed. Control cells were treated with DMSO. (B) Pie charts representing the total number of genes and proteins identified in all conditions by RNA sequencing and TMT quantitative proteomic analysis. Number of mitochondrial genes and proteins are represented in both charts, as well as the percentage relative to the total. (C) Heatmap analysis of the transcriptome (top) and proteome (bottom). Hierarchical clustering of samples (columns) and genes/proteins (rows) was based on Pearson’s correlation coefficient to measure the distance and the mean to cluster the samples. (D) Multidimensional scaling (MDS) using the distances between transcript expression profiles (top) and principal-component analysis (PCA) of the proteomic profiles (bottom) show a similar clustering between the duplicates and treatments. BCV, biological coefficient of variation; Comp, principal component. (E) Bar plots representing the number of differentially expressed (DE) genes (top) and proteins (bottom) in each condition (FDR 5%). Percentage of DE mitochondrial genes and proteins is shown, observing a greater impact on the mitochondrial proteome than on the mitochondrial transcriptome. Acti, actinonin; Dox, doxycycline; MB, MitoBloCK-6.

    Journal: The Journal of Cell Biology

    Article Title: Multi-omics analysis identifies ATF4 as a key regulator of the mitochondrial stress response in mammals

    doi: 10.1083/jcb.201702058

    Figure Lengend Snippet: RNA sequencing and proteomic analysis upon mitochondrial stress. (A) Schematic representation of the omics experiments. HeLa cells were treated with compounds for 24 h, and the transcriptome, proteome, and metabolome were analyzed. Control cells were treated with DMSO. (B) Pie charts representing the total number of genes and proteins identified in all conditions by RNA sequencing and TMT quantitative proteomic analysis. Number of mitochondrial genes and proteins are represented in both charts, as well as the percentage relative to the total. (C) Heatmap analysis of the transcriptome (top) and proteome (bottom). Hierarchical clustering of samples (columns) and genes/proteins (rows) was based on Pearson’s correlation coefficient to measure the distance and the mean to cluster the samples. (D) Multidimensional scaling (MDS) using the distances between transcript expression profiles (top) and principal-component analysis (PCA) of the proteomic profiles (bottom) show a similar clustering between the duplicates and treatments. BCV, biological coefficient of variation; Comp, principal component. (E) Bar plots representing the number of differentially expressed (DE) genes (top) and proteins (bottom) in each condition (FDR 5%). Percentage of DE mitochondrial genes and proteins is shown, observing a greater impact on the mitochondrial proteome than on the mitochondrial transcriptome. Acti, actinonin; Dox, doxycycline; MB, MitoBloCK-6.

    Article Snippet: RNA sequencing library preparation and analysis Total RNA from HeLa cells was extracted using TRIzol (Thermo Fisher Scientific) and purified by column using RNeasy Mini kit (QIAGEN).

    Techniques: RNA Sequencing Assay, Expressing

    Silencing of LRRK2 expression rescues ArfGAP1-induced neurite shortening. (A) Validation of short hairpin RNA (shRNA)-mediated silencing of endogenous LRRK2 expression in rat primary cortical neuronal cultures. Lentiviral vectors expressing non-silencing shRNA (LV-sh-control) or LRRK2-specific shRNA (LV-sh-LRRK2) were used to infect primary neurons at increasing viral doses ranging from 1.66 to 25 ng of P24 antigen per ml of media. Western blot analysis of neuronal extracts reveals the viral dose-dependent knockdown of LRRK2 with LRRK2-specific shRNAs but not control shRNA using two independent rabbit LRRK2 antibodies (MJFF-2/c41-2 or JH5514). β-tubulin indicates equivalent protein loading. (B) Silencing of endogenous LRRK2 expression in GFP-labeled cortical neurons with lentiviral-shRNA vectors revealed by confocal fluorescence microscopy with a rabbit anti-LRRK2 antibody (JH5514). (C) Primary cortical neurons were infected with lentiviral vectors expressing shRNAs (non-silencing control or LRRK2-specific) at DIV 2, subsequently transfected with ArfGAP1-YFP and DsRed constructs at a 10∶1 molar ratio at DIV 3, and fixed at DIV 6. Fluorescent microscopic images reveal the co-labeling of cortical neurons with ArfGAP1-YFP and DsRed. DsRed images were pseudo-colored with ICA for neurite length measurements. Neuronal soma ( arrows ) and axonal processes ( arrowheads ) are indicated. Scale bars: 400 µm. (D) Analysis of DsRed-positive axonal processes reveals a robust shortening of axons induced by ArfGAP1 expression and increased axon length induced by silencing of LRRK2 with LV-sh-LRRK2 vector, compared to DsRed alone (control). Silencing of LRRK2 with LV-sh-LRRK2 produces a partial rescue of ArfGAP1-induced axon shortening compared to LV-sh-control. Bars represent mean (± SEM) length of axons expressed as a percent of DsRed alone (control/LV-sh-control) from > 85 DsRed-positive neurons from two independent experiments/cultures. ** P

    Journal: PLoS Genetics

    Article Title: GTPase Activity and Neuronal Toxicity of Parkinson's Disease-Associated LRRK2 Is Regulated by ArfGAP1

    doi: 10.1371/journal.pgen.1002526

    Figure Lengend Snippet: Silencing of LRRK2 expression rescues ArfGAP1-induced neurite shortening. (A) Validation of short hairpin RNA (shRNA)-mediated silencing of endogenous LRRK2 expression in rat primary cortical neuronal cultures. Lentiviral vectors expressing non-silencing shRNA (LV-sh-control) or LRRK2-specific shRNA (LV-sh-LRRK2) were used to infect primary neurons at increasing viral doses ranging from 1.66 to 25 ng of P24 antigen per ml of media. Western blot analysis of neuronal extracts reveals the viral dose-dependent knockdown of LRRK2 with LRRK2-specific shRNAs but not control shRNA using two independent rabbit LRRK2 antibodies (MJFF-2/c41-2 or JH5514). β-tubulin indicates equivalent protein loading. (B) Silencing of endogenous LRRK2 expression in GFP-labeled cortical neurons with lentiviral-shRNA vectors revealed by confocal fluorescence microscopy with a rabbit anti-LRRK2 antibody (JH5514). (C) Primary cortical neurons were infected with lentiviral vectors expressing shRNAs (non-silencing control or LRRK2-specific) at DIV 2, subsequently transfected with ArfGAP1-YFP and DsRed constructs at a 10∶1 molar ratio at DIV 3, and fixed at DIV 6. Fluorescent microscopic images reveal the co-labeling of cortical neurons with ArfGAP1-YFP and DsRed. DsRed images were pseudo-colored with ICA for neurite length measurements. Neuronal soma ( arrows ) and axonal processes ( arrowheads ) are indicated. Scale bars: 400 µm. (D) Analysis of DsRed-positive axonal processes reveals a robust shortening of axons induced by ArfGAP1 expression and increased axon length induced by silencing of LRRK2 with LV-sh-LRRK2 vector, compared to DsRed alone (control). Silencing of LRRK2 with LV-sh-LRRK2 produces a partial rescue of ArfGAP1-induced axon shortening compared to LV-sh-control. Bars represent mean (± SEM) length of axons expressed as a percent of DsRed alone (control/LV-sh-control) from > 85 DsRed-positive neurons from two independent experiments/cultures. ** P

    Article Snippet: Short hairpin RNA (shRNA) sequences in lentiviral plasmid pLKO.1 targeting rat ArfGAP1 (sh-ArfGAP1 #1, TRCN0000047321; sh-ArfGAP1 #2, TRCN0000100750) or rat LRRK2 (sh-LRRK2, TRC0000021461) were obtained from Thermo Fisher Scientific (Open Biosystems, Huntsville, AL, USA).

    Techniques: Expressing, shRNA, Western Blot, Labeling, Fluorescence, Microscopy, Infection, Transfection, Construct, Plasmid Preparation

    ArfGAP1 is expressed in the mammalian brain. (A) Western blot analysis of ArfGAP1 protein levels in mouse brain, with highest levels detected in cerebral cortex ( Ctx ) and cerebellum ( Cb ), moderate levels in striatum ( Str ) and ventral midbrain ( VMB ), and lowest levels in olfactory bulb ( Olf ) and spinal cord ( SC ). LRRK2 is detected at highest levels in striatum, cortex and cerebellum. β-tubulin indicates equivalent protein loading. (B) Subcellular fractionation of mouse whole brain tissue. ArfGAP1 is broadly detected with particular enrichment in membrane-associated fractions including the heavy (P2) and synaptosomal (LP1) membrane fractions, and soluble cytosolic (S1 and S3) and synaptosomal cytosolic (LS1) fractions. LRRK2 (MJFF-2/c41-2) is also broadly detected with enrichment in membrane-associated fractions, including heavy (P2) and light (P3) membrane, synaptosomal (LP1) and synaptic vesicle (LP2) fractions, in addition to soluble cytosolic (S1 and S2) and synaptosomal cytosolic (LS1) fractions. ArfGAP1 and LRRK2 co-associate in the S1, P2, S2, LP1 and LS1 fractions. The distribution of marker proteins demonstrates the enrichment of mitochondria/heavy membranes (TIM23; P2 and LP1), endoplasmic reticulum (PDI; P2, P3, LP1 and LP2), Golgi (Giantin; P2 and P3), synaptosomes/synaptic vesicles (synaptophysin 1; P2, P3, LP1 and LP2) and synaptosomal/synaptic vesicle cytosolic (α-synuclein; LS1 and LS2). (C) Confocal fluorescence microscopy reveals the localization of endogenous ArfGAP1 to the Golgi complex and cytoplasm of MAP2-positive cortical neurons and tyrosine hydroxylase (TH)-positive dopaminergic neurons in rat primary cortical and midbrain cultures, respectively. (D) Validation of short hairpin RNA (shRNA)-mediated silencing of endogenous ArfGAP1 expression in rat primary cortical neuronal cultures. Lentiviral vectors expressing non-silencing shRNA (LV-sh-control) or ArfGAP1-specific shRNAs (LV-sh-ArfGAP1 #1 and LV-sh-ArfGAP1 #2) were used to infect primary neurons at increasing viral doses ranging from 3.3 to 50 ng of P24 antigen per ml of media. Western blot analysis of neuronal extracts reveals the viral dose-dependent knockdown of ArfGAP1 with ArfGAP1-specific shRNAs but not control shRNA. β-tubulin indicates equivalent protein loading. (E) Silencing of endogenous ArfGAP1 expression in GFP-labeled cortical neurons with lentiviral-shRNA vectors revealed by confocal fluorescence microscopy with a rabbit anti-ArfGAP1 antibody. Molecular mass markers are indicated in kilodalton (kDa). Scale bars: 10 µm.

    Journal: PLoS Genetics

    Article Title: GTPase Activity and Neuronal Toxicity of Parkinson's Disease-Associated LRRK2 Is Regulated by ArfGAP1

    doi: 10.1371/journal.pgen.1002526

    Figure Lengend Snippet: ArfGAP1 is expressed in the mammalian brain. (A) Western blot analysis of ArfGAP1 protein levels in mouse brain, with highest levels detected in cerebral cortex ( Ctx ) and cerebellum ( Cb ), moderate levels in striatum ( Str ) and ventral midbrain ( VMB ), and lowest levels in olfactory bulb ( Olf ) and spinal cord ( SC ). LRRK2 is detected at highest levels in striatum, cortex and cerebellum. β-tubulin indicates equivalent protein loading. (B) Subcellular fractionation of mouse whole brain tissue. ArfGAP1 is broadly detected with particular enrichment in membrane-associated fractions including the heavy (P2) and synaptosomal (LP1) membrane fractions, and soluble cytosolic (S1 and S3) and synaptosomal cytosolic (LS1) fractions. LRRK2 (MJFF-2/c41-2) is also broadly detected with enrichment in membrane-associated fractions, including heavy (P2) and light (P3) membrane, synaptosomal (LP1) and synaptic vesicle (LP2) fractions, in addition to soluble cytosolic (S1 and S2) and synaptosomal cytosolic (LS1) fractions. ArfGAP1 and LRRK2 co-associate in the S1, P2, S2, LP1 and LS1 fractions. The distribution of marker proteins demonstrates the enrichment of mitochondria/heavy membranes (TIM23; P2 and LP1), endoplasmic reticulum (PDI; P2, P3, LP1 and LP2), Golgi (Giantin; P2 and P3), synaptosomes/synaptic vesicles (synaptophysin 1; P2, P3, LP1 and LP2) and synaptosomal/synaptic vesicle cytosolic (α-synuclein; LS1 and LS2). (C) Confocal fluorescence microscopy reveals the localization of endogenous ArfGAP1 to the Golgi complex and cytoplasm of MAP2-positive cortical neurons and tyrosine hydroxylase (TH)-positive dopaminergic neurons in rat primary cortical and midbrain cultures, respectively. (D) Validation of short hairpin RNA (shRNA)-mediated silencing of endogenous ArfGAP1 expression in rat primary cortical neuronal cultures. Lentiviral vectors expressing non-silencing shRNA (LV-sh-control) or ArfGAP1-specific shRNAs (LV-sh-ArfGAP1 #1 and LV-sh-ArfGAP1 #2) were used to infect primary neurons at increasing viral doses ranging from 3.3 to 50 ng of P24 antigen per ml of media. Western blot analysis of neuronal extracts reveals the viral dose-dependent knockdown of ArfGAP1 with ArfGAP1-specific shRNAs but not control shRNA. β-tubulin indicates equivalent protein loading. (E) Silencing of endogenous ArfGAP1 expression in GFP-labeled cortical neurons with lentiviral-shRNA vectors revealed by confocal fluorescence microscopy with a rabbit anti-ArfGAP1 antibody. Molecular mass markers are indicated in kilodalton (kDa). Scale bars: 10 µm.

    Article Snippet: Short hairpin RNA (shRNA) sequences in lentiviral plasmid pLKO.1 targeting rat ArfGAP1 (sh-ArfGAP1 #1, TRCN0000047321; sh-ArfGAP1 #2, TRCN0000100750) or rat LRRK2 (sh-LRRK2, TRC0000021461) were obtained from Thermo Fisher Scientific (Open Biosystems, Huntsville, AL, USA).

    Techniques: Western Blot, Fractionation, Marker, Fluorescence, Microscopy, shRNA, Expressing, Labeling