poly ra oligo dt assays  (Thermo Fisher)


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    Name:
    Oligo dT Primer 50 µM
    Description:
    These are the same Ambion primers that are currently included in the RETROscript Kit SKU AM1710 They are provided at a stock concentration of 50 µM and are functionally tested using the RETROscript Kit
    Catalog Number:
    am5730g
    Price:
    None
    Applications:
    PCR & Real-Time PCR|RT-PCR|Reverse Transcription|Two-Step RT-PCR
    Category:
    Oligos Primers Probes Nucleotides
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    Structured Review

    Thermo Fisher poly ra oligo dt assays
    These are the same Ambion primers that are currently included in the RETROscript Kit SKU AM1710 They are provided at a stock concentration of 50 µM and are functionally tested using the RETROscript Kit
    https://www.bioz.com/result/poly ra oligo dt assays/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    poly ra oligo dt assays - by Bioz Stars, 2020-05
    99/100 stars

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    Related Articles

    Clonogenic Assay:

    Article Title: Berberine Radiosensitizes Human Esophageal Cancer Cells by Downregulating Homologous Recombination Repair Protein RAD51
    Article Snippet: .. RAD51 depletion by RNA interference RAD51 siRNA duplex 1, 2 and 3 and the negative oligo were obtained from GenePharma (Shanghai, China). siRNA duplexes or negative oligo (200 nM) were transfected into KYSE30 and KYSE450 cells using lipofectamine2000 (Invitrogen) for 24 h. RAD51 protein levels were determined by western blotting analysis and the levels of radiosensitivity were determined by clonogenic assay described above. .. Sequences for small interfering RNA for RAD51 RNAi are as follows: siRAD51-1: CGAUGUGAAGAAAUUGGAATT siRAD51-2: GACUGGAUCUAUCACAGAATT siRAD51-3: GCAGUGAUGUCCUGGAUAATT

    Transfection:

    Article Title: A Novel Gonadotropin-Releasing Hormone 1 (Gnrh1) Enhancer-Derived Noncoding RNA Regulates Gnrh1 Gene Expression in GnRH Neuronal Cell Models
    Article Snippet: .. Negative siRNA control targeting luciferase mRNA and custom-designed siRNA duplex oligos targeting GnRH-E1 RNA (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA) and Lipofectamine RNAiMax transfection reagent (Thermo Fisher Scientific, Carlsbad, CA) were prepared according to the manufacturer’s recommendations, in Gibco OptiMEM (Thermo Fisher Scientific, Carlsbad, CA) at a concentration of 10 μM. siRNA was transfected into GT1-7 cells at a concentration of 900 pmol per well. .. Data Analysis All transient transfections, RT-PCR and quantitative RT-PCR were independently repeated three times or more.

    Article Title: Nodal Signaling via an Autocrine Pathway Promotes Proliferation of Mouse Spermatogonial Stem/Progenitor Cells through Smad2/3 and Oct-4 Activation
    Article Snippet: .. We found that Nodal siRNAs could be efficiently transfected into C18-4 cells using Lipofectamine™ 2000 as shown by a high uptake of the BLOCK-iT™ fluorescent oligo, whose fluorescent signal correlates with the delivery of active siRNAs (Invitrogen). .. Nodal siRNAs could knock down the expression of Nodal mRNA and mature Nodal protein in C18-4 cells.

    Article Title: Cooperative Regulation of NSCLC Angiogenic Potential by Macrophage Migration Inhibitory Factor and its Homolog, D-Dopachrome Tautomerase 1
    Article Snippet: .. Cells were transfected with 50 nM annealed siRNA oligos using Oligofectamine reagent (Invitrogen) following the manufacturer’s protocol. .. The targeted base sequence for human MIF was: 5’-CCTTCTGGTGGGGAGAAAT-3’ (Dharmacon, Lafayette, CO).

    Article Title: Berberine Radiosensitizes Human Esophageal Cancer Cells by Downregulating Homologous Recombination Repair Protein RAD51
    Article Snippet: .. RAD51 depletion by RNA interference RAD51 siRNA duplex 1, 2 and 3 and the negative oligo were obtained from GenePharma (Shanghai, China). siRNA duplexes or negative oligo (200 nM) were transfected into KYSE30 and KYSE450 cells using lipofectamine2000 (Invitrogen) for 24 h. RAD51 protein levels were determined by western blotting analysis and the levels of radiosensitivity were determined by clonogenic assay described above. .. Sequences for small interfering RNA for RAD51 RNAi are as follows: siRAD51-1: CGAUGUGAAGAAAUUGGAATT siRAD51-2: GACUGGAUCUAUCACAGAATT siRAD51-3: GCAGUGAUGUCCUGGAUAATT

    Article Title: Down-regulation of TERE1/UBIAD1 activated Ras-MAPK signalling and induced cell proliferation
    Article Snippet: .. SiRNA oligos were synthesized by Invitrogen as follows: Negative control siRNA, 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) 5′-ACGUGACACGUUCGGAGAATT-3′(antisense); TERE1-291 siRNA, 5′-GUAAUUUGGUCAACACUUATT-3′(sense) 5′-UAAGUGUUGACCAAAUUACCG3-′(antisense); TERE1-22 siRNA, 5′-CAAGGGCAUUGACCACAAATT-3′ (sense) 5′-UUUGUGGUCAAUGCCCUUGGA-3′ (antisense) For each transfection, oligomer–Lipofectamine™ 2000 complexes were prepared by mixing siRNA oligos with Lipofectamine™ 2000 and added to each well containing cells and medium. ..

    Article Title: DISTINCT ROLES FOR N-CADHERIN LINKED C-SRC AND FYN KINASES IN LENS DEVELOPMENT
    Article Snippet: .. As an indicator of transfected cells in the immunolocalization studies lens cultures were co-transfected with BLOCK-iT fluorescent oligo (Invitrogen, Camarillo, CA). .. Lenses were isolated from E10 chicken embryos (B & E Eggs, York Springs, PA) and microdissected as previously described ( ) to yield four distinct regions of differentiation; central anterior epithelium (EC), equatorial epithelium (EQ), cortical fiber cells (FP), and central fiber zone (FC), as modeled in .

    Luciferase:

    Article Title: A Novel Gonadotropin-Releasing Hormone 1 (Gnrh1) Enhancer-Derived Noncoding RNA Regulates Gnrh1 Gene Expression in GnRH Neuronal Cell Models
    Article Snippet: .. Negative siRNA control targeting luciferase mRNA and custom-designed siRNA duplex oligos targeting GnRH-E1 RNA (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA) and Lipofectamine RNAiMax transfection reagent (Thermo Fisher Scientific, Carlsbad, CA) were prepared according to the manufacturer’s recommendations, in Gibco OptiMEM (Thermo Fisher Scientific, Carlsbad, CA) at a concentration of 10 μM. siRNA was transfected into GT1-7 cells at a concentration of 900 pmol per well. .. Data Analysis All transient transfections, RT-PCR and quantitative RT-PCR were independently repeated three times or more.

    Article Title: Respiratory Syncytial Virus Utilizes a tRNA Fragment to Suppress Antiviral Responses Through a Novel Targeting Mechanism
    Article Snippet: .. To investigate the effect of RSV-induced tRF5-GluCTC on APOER2 mRNA expression A549 cells were cotransfected with Pp -WT sensor plasmids (firefly plasmids), pRL-CMV plasmids expressing Rr (renilla luciferase), and anti-tRF5-GluCTC oligo or its control (anti-ctrl), using Lipofetamine 2000 according to the manufacturer's instruction (Invitrogen, Grand Island, NY). ..

    Synthesized:

    Article Title: Down-regulation of TERE1/UBIAD1 activated Ras-MAPK signalling and induced cell proliferation
    Article Snippet: .. SiRNA oligos were synthesized by Invitrogen as follows: Negative control siRNA, 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) 5′-ACGUGACACGUUCGGAGAATT-3′(antisense); TERE1-291 siRNA, 5′-GUAAUUUGGUCAACACUUATT-3′(sense) 5′-UAAGUGUUGACCAAAUUACCG3-′(antisense); TERE1-22 siRNA, 5′-CAAGGGCAUUGACCACAAATT-3′ (sense) 5′-UUUGUGGUCAAUGCCCUUGGA-3′ (antisense) For each transfection, oligomer–Lipofectamine™ 2000 complexes were prepared by mixing siRNA oligos with Lipofectamine™ 2000 and added to each well containing cells and medium. ..

    Ligation:

    Article Title: Structural bias in T4 RNA ligase-mediated 3?-adapter ligation
    Article Snippet: .. To assess the frequency of nucleotides at each randomized position in the oligo pool, the random RNA oligos were subjected to Ion Torrent sequencing without undergoing adapter ligation ( ). ..

    Negative Control:

    Article Title: Down-regulation of TERE1/UBIAD1 activated Ras-MAPK signalling and induced cell proliferation
    Article Snippet: .. SiRNA oligos were synthesized by Invitrogen as follows: Negative control siRNA, 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense) 5′-ACGUGACACGUUCGGAGAATT-3′(antisense); TERE1-291 siRNA, 5′-GUAAUUUGGUCAACACUUATT-3′(sense) 5′-UAAGUGUUGACCAAAUUACCG3-′(antisense); TERE1-22 siRNA, 5′-CAAGGGCAUUGACCACAAATT-3′ (sense) 5′-UUUGUGGUCAAUGCCCUUGGA-3′ (antisense) For each transfection, oligomer–Lipofectamine™ 2000 complexes were prepared by mixing siRNA oligos with Lipofectamine™ 2000 and added to each well containing cells and medium. ..

    Blocking Assay:

    Article Title: Nodal Signaling via an Autocrine Pathway Promotes Proliferation of Mouse Spermatogonial Stem/Progenitor Cells through Smad2/3 and Oct-4 Activation
    Article Snippet: .. We found that Nodal siRNAs could be efficiently transfected into C18-4 cells using Lipofectamine™ 2000 as shown by a high uptake of the BLOCK-iT™ fluorescent oligo, whose fluorescent signal correlates with the delivery of active siRNAs (Invitrogen). .. Nodal siRNAs could knock down the expression of Nodal mRNA and mature Nodal protein in C18-4 cells.

    Article Title: DISTINCT ROLES FOR N-CADHERIN LINKED C-SRC AND FYN KINASES IN LENS DEVELOPMENT
    Article Snippet: .. As an indicator of transfected cells in the immunolocalization studies lens cultures were co-transfected with BLOCK-iT fluorescent oligo (Invitrogen, Camarillo, CA). .. Lenses were isolated from E10 chicken embryos (B & E Eggs, York Springs, PA) and microdissected as previously described ( ) to yield four distinct regions of differentiation; central anterior epithelium (EC), equatorial epithelium (EQ), cortical fiber cells (FP), and central fiber zone (FC), as modeled in .

    Concentration Assay:

    Article Title: A Novel Gonadotropin-Releasing Hormone 1 (Gnrh1) Enhancer-Derived Noncoding RNA Regulates Gnrh1 Gene Expression in GnRH Neuronal Cell Models
    Article Snippet: .. Negative siRNA control targeting luciferase mRNA and custom-designed siRNA duplex oligos targeting GnRH-E1 RNA (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA) and Lipofectamine RNAiMax transfection reagent (Thermo Fisher Scientific, Carlsbad, CA) were prepared according to the manufacturer’s recommendations, in Gibco OptiMEM (Thermo Fisher Scientific, Carlsbad, CA) at a concentration of 10 μM. siRNA was transfected into GT1-7 cells at a concentration of 900 pmol per well. .. Data Analysis All transient transfections, RT-PCR and quantitative RT-PCR were independently repeated three times or more.

    Expressing:

    Article Title: Respiratory Syncytial Virus Utilizes a tRNA Fragment to Suppress Antiviral Responses Through a Novel Targeting Mechanism
    Article Snippet: .. To investigate the effect of RSV-induced tRF5-GluCTC on APOER2 mRNA expression A549 cells were cotransfected with Pp -WT sensor plasmids (firefly plasmids), pRL-CMV plasmids expressing Rr (renilla luciferase), and anti-tRF5-GluCTC oligo or its control (anti-ctrl), using Lipofetamine 2000 according to the manufacturer's instruction (Invitrogen, Grand Island, NY). ..

    Sequencing:

    Article Title: Structural bias in T4 RNA ligase-mediated 3?-adapter ligation
    Article Snippet: .. To assess the frequency of nucleotides at each randomized position in the oligo pool, the random RNA oligos were subjected to Ion Torrent sequencing without undergoing adapter ligation ( ). ..

    Western Blot:

    Article Title: Berberine Radiosensitizes Human Esophageal Cancer Cells by Downregulating Homologous Recombination Repair Protein RAD51
    Article Snippet: .. RAD51 depletion by RNA interference RAD51 siRNA duplex 1, 2 and 3 and the negative oligo were obtained from GenePharma (Shanghai, China). siRNA duplexes or negative oligo (200 nM) were transfected into KYSE30 and KYSE450 cells using lipofectamine2000 (Invitrogen) for 24 h. RAD51 protein levels were determined by western blotting analysis and the levels of radiosensitivity were determined by clonogenic assay described above. .. Sequences for small interfering RNA for RAD51 RNAi are as follows: siRAD51-1: CGAUGUGAAGAAAUUGGAATT siRAD51-2: GACUGGAUCUAUCACAGAATT siRAD51-3: GCAGUGAUGUCCUGGAUAATT

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  • 99
    Thermo Fisher biotinylated rna oligos
    hnRNPM and ESRP1 show common motif enrichment downstream from discordantly regulated exons and compete for shared binding sites. ( A ) K-mer enrichment analysis showing the top three enriched 6-mers in introns flanking hnRNPM and ESRP1 coregulated cassette exons. Two identical GU-rich motifs (GUGUGU and GUGGUG, black box) were enriched downstream from cassette exons in ESRP1-enhanced and hnRNPM-repressed events. The RBFOX motif (UGCAUG, underlined) was enriched downstream from ESRP1-repressed and hnRNPM-enhanced events. ( B , C ) <t>RNA</t> motif map analysis of GU-rich motifs in introns flanking hnRNPM-ESRP1 coregulated exons with respect to ESRP1 regulation ( B ) and hnRNPM regulation ( C ) reveals enrichment of GU-rich motifs in downstream introns of ESRP1-enhanced and hnRNPM-repressed cassette exon splicing events. Inclusion events (green). Skipping events (red). Control events (black). ( D ) Genome browser plot of RNA sequencing data sets showing hnRNPM knockdown promotes APLP2 exon 7 inclusion and ESRP1 depletion promotes skipping. Black bars indicate constitutive exons. Green bar indicates variable exon 7. Yellow bars indicate location of two clusters of GU-rich motifs within 250 nucleotides (nt) downstream from APLP2 exon 7. ( E ) Immunoblot of HA-tagged ESRP1 overexpression in MDA-MB-231 cells and endogenous hnRNPM. ( F ) ESRP1-HA overexpression in MDA-MB-231 cells results in increased APLP2 exon 7 inclusion. (*) Indicates nonspecific band. ( G ) ( Upper panel) The two GU-rich regions identified within 250 nt downstream from APLP2 exon 7 were used to design RNA probes GU1 and GU2 containing stretches of GU nucleotides underlined and in red with mutant probes GU1-mut and GU2-mut with mutated sequences colored in blue. ( Lower panel) RNA pull-down analysis using RNA probes blotting for endogenous hnRNPM and overexpressed ESRP1-HA in MDA-MB-231 cells shows that hnRNPM and ESRP1 bind common GU-rich sequences. ( H ) RNA pull-down experiments using a static amount of the <t>biotinylated</t> GU2 RNA probe and cell lysate assaying for hnRNPM in the MDA-MB-231 cell line, which does not express ESRP1, and the same line with ESRP1-HA overexpression. A total of 2.5% input was provided as a loading control for both samples. Overexpression of ESRP1 leads to less hnRNPM binding, suggesting that ESRP1 competes for the same GU2 binding site. (Error bars = S.E.M, n = 3, [***] P -value
    Biotinylated Rna Oligos, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotinylated rna oligos/product/Thermo Fisher
    Average 99 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    biotinylated rna oligos - by Bioz Stars, 2020-05
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher rna
    Inhibition effect of LPS on LCFA absorption in IPEC-J2 cells was related to m 6 A methylation. IPEC-J2 cells were treated with 10 μg/ml LPS for various times, and cells without LPS stimulation were used as controls. ( A ) The composition of fatty acids in whole lysate measured by GC/MS. ( B ) m 6 A dot blot to measure the m 6 A levels with purified <t>mRNA</t> from total <t>RNA.</t> Methylene blue staining was used for loading control. The right panel shows the relative levels quantified by densitometry and normalized to control. The data are expressed as the mean ± SEM. Statistically significant difference relative to the control (0 h) ( n = 3, biological replicates). * p
    Rna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3653 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna/product/Thermo Fisher
    Average 99 stars, based on 3653 article reviews
    Price from $9.99 to $1999.99
    rna - by Bioz Stars, 2020-05
    99/100 stars
      Buy from Supplier

    Image Search Results


    hnRNPM and ESRP1 show common motif enrichment downstream from discordantly regulated exons and compete for shared binding sites. ( A ) K-mer enrichment analysis showing the top three enriched 6-mers in introns flanking hnRNPM and ESRP1 coregulated cassette exons. Two identical GU-rich motifs (GUGUGU and GUGGUG, black box) were enriched downstream from cassette exons in ESRP1-enhanced and hnRNPM-repressed events. The RBFOX motif (UGCAUG, underlined) was enriched downstream from ESRP1-repressed and hnRNPM-enhanced events. ( B , C ) RNA motif map analysis of GU-rich motifs in introns flanking hnRNPM-ESRP1 coregulated exons with respect to ESRP1 regulation ( B ) and hnRNPM regulation ( C ) reveals enrichment of GU-rich motifs in downstream introns of ESRP1-enhanced and hnRNPM-repressed cassette exon splicing events. Inclusion events (green). Skipping events (red). Control events (black). ( D ) Genome browser plot of RNA sequencing data sets showing hnRNPM knockdown promotes APLP2 exon 7 inclusion and ESRP1 depletion promotes skipping. Black bars indicate constitutive exons. Green bar indicates variable exon 7. Yellow bars indicate location of two clusters of GU-rich motifs within 250 nucleotides (nt) downstream from APLP2 exon 7. ( E ) Immunoblot of HA-tagged ESRP1 overexpression in MDA-MB-231 cells and endogenous hnRNPM. ( F ) ESRP1-HA overexpression in MDA-MB-231 cells results in increased APLP2 exon 7 inclusion. (*) Indicates nonspecific band. ( G ) ( Upper panel) The two GU-rich regions identified within 250 nt downstream from APLP2 exon 7 were used to design RNA probes GU1 and GU2 containing stretches of GU nucleotides underlined and in red with mutant probes GU1-mut and GU2-mut with mutated sequences colored in blue. ( Lower panel) RNA pull-down analysis using RNA probes blotting for endogenous hnRNPM and overexpressed ESRP1-HA in MDA-MB-231 cells shows that hnRNPM and ESRP1 bind common GU-rich sequences. ( H ) RNA pull-down experiments using a static amount of the biotinylated GU2 RNA probe and cell lysate assaying for hnRNPM in the MDA-MB-231 cell line, which does not express ESRP1, and the same line with ESRP1-HA overexpression. A total of 2.5% input was provided as a loading control for both samples. Overexpression of ESRP1 leads to less hnRNPM binding, suggesting that ESRP1 competes for the same GU2 binding site. (Error bars = S.E.M, n = 3, [***] P -value

    Journal: RNA

    Article Title: Coregulation of alternative splicing by hnRNPM and ESRP1 during EMT

    doi: 10.1261/rna.066712.118

    Figure Lengend Snippet: hnRNPM and ESRP1 show common motif enrichment downstream from discordantly regulated exons and compete for shared binding sites. ( A ) K-mer enrichment analysis showing the top three enriched 6-mers in introns flanking hnRNPM and ESRP1 coregulated cassette exons. Two identical GU-rich motifs (GUGUGU and GUGGUG, black box) were enriched downstream from cassette exons in ESRP1-enhanced and hnRNPM-repressed events. The RBFOX motif (UGCAUG, underlined) was enriched downstream from ESRP1-repressed and hnRNPM-enhanced events. ( B , C ) RNA motif map analysis of GU-rich motifs in introns flanking hnRNPM-ESRP1 coregulated exons with respect to ESRP1 regulation ( B ) and hnRNPM regulation ( C ) reveals enrichment of GU-rich motifs in downstream introns of ESRP1-enhanced and hnRNPM-repressed cassette exon splicing events. Inclusion events (green). Skipping events (red). Control events (black). ( D ) Genome browser plot of RNA sequencing data sets showing hnRNPM knockdown promotes APLP2 exon 7 inclusion and ESRP1 depletion promotes skipping. Black bars indicate constitutive exons. Green bar indicates variable exon 7. Yellow bars indicate location of two clusters of GU-rich motifs within 250 nucleotides (nt) downstream from APLP2 exon 7. ( E ) Immunoblot of HA-tagged ESRP1 overexpression in MDA-MB-231 cells and endogenous hnRNPM. ( F ) ESRP1-HA overexpression in MDA-MB-231 cells results in increased APLP2 exon 7 inclusion. (*) Indicates nonspecific band. ( G ) ( Upper panel) The two GU-rich regions identified within 250 nt downstream from APLP2 exon 7 were used to design RNA probes GU1 and GU2 containing stretches of GU nucleotides underlined and in red with mutant probes GU1-mut and GU2-mut with mutated sequences colored in blue. ( Lower panel) RNA pull-down analysis using RNA probes blotting for endogenous hnRNPM and overexpressed ESRP1-HA in MDA-MB-231 cells shows that hnRNPM and ESRP1 bind common GU-rich sequences. ( H ) RNA pull-down experiments using a static amount of the biotinylated GU2 RNA probe and cell lysate assaying for hnRNPM in the MDA-MB-231 cell line, which does not express ESRP1, and the same line with ESRP1-HA overexpression. A total of 2.5% input was provided as a loading control for both samples. Overexpression of ESRP1 leads to less hnRNPM binding, suggesting that ESRP1 competes for the same GU2 binding site. (Error bars = S.E.M, n = 3, [***] P -value

    Article Snippet: Biotinylated RNA oligos (10 µL at 40 µM) were immobilized on 50 µL of streptavidin beads (50% slurry; Thermo Fisher) in a total volume of 400 µL 1× binding buffer (20 mM Tris, 200 mM NaCl, 6 mM EDTA, 5 mM sodium fluoride, 5 mM β-glycerophosphate, 2 mg/mL aprotinin, pH 7.5) for 2 h at 4°C in a rotating shaker.

    Techniques: Binding Assay, RNA Sequencing Assay, Over Expression, Multiple Displacement Amplification, Mutagenesis

    Hematopoietic stem cells receive microRNA loaded extracellular vesicles released by Cy5-RNA positive telocytes. (A) Experimental set-up : Cardiac telocytes are transfected with lipofectamine/Cy5-RNA complexes and release extracellular vesicles loaded with fluorescent-RNA into culture supernatant; hematopoietic stem cells incubated with supernatant receive the Cy5-RNA vesicles and become fluorescent; in control experiments, telocytes are not transfected with oligoRNA in absence of lipofectamine, the released vesicles does not have Cy5-RNA and the corresponding hematopoietic stem cells are negative for Cy5 fluorescence. (B) TC transfection with Cy5-RNA : Rat or mouse telocytes are positive for fluorescent Cy5 after 3–5 hrs incubation with a mixture of Cy5-RNA oligo and Lipofectamine RNAiMAX (red filled areas). Controls, non-fluorescent telocytes, represent cells incubated with oligoRNA in absence of lipids (dot plots). (C) Cy5-RNA positive hematopoietic stem/progenitor cells : Hematopoietic progenitors (LSK) are fluorescent after incubation with supernatant derived for Cy5-RNA transfected telocytes (sample), but not in case of supernatant derived from telocytes negative for Cy5 (control). LSK cells Lineage/7AAD negative, Sca1 positive and c-Kit positive. N = 4, P = 0.03 (D) Representative FACS plots of data presented in (C) . Right plot: Hematopoietic stem cells (HSC) and their multipotent progeny (MPP), defined as cells LSK Flt3 negative, are positive for Cy5 when cultured in presence of medium conditioned by Cy5-RNA transfected telocytes. Left plot: in control experiments, HSC/MPP have been incubated with conditioned supernatant derived from non-fluorescent telocytes.

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Telocytes transfer extracellular vesicles loaded with microRNAs to stem cells

    doi: 10.1111/jcmm.12529

    Figure Lengend Snippet: Hematopoietic stem cells receive microRNA loaded extracellular vesicles released by Cy5-RNA positive telocytes. (A) Experimental set-up : Cardiac telocytes are transfected with lipofectamine/Cy5-RNA complexes and release extracellular vesicles loaded with fluorescent-RNA into culture supernatant; hematopoietic stem cells incubated with supernatant receive the Cy5-RNA vesicles and become fluorescent; in control experiments, telocytes are not transfected with oligoRNA in absence of lipofectamine, the released vesicles does not have Cy5-RNA and the corresponding hematopoietic stem cells are negative for Cy5 fluorescence. (B) TC transfection with Cy5-RNA : Rat or mouse telocytes are positive for fluorescent Cy5 after 3–5 hrs incubation with a mixture of Cy5-RNA oligo and Lipofectamine RNAiMAX (red filled areas). Controls, non-fluorescent telocytes, represent cells incubated with oligoRNA in absence of lipids (dot plots). (C) Cy5-RNA positive hematopoietic stem/progenitor cells : Hematopoietic progenitors (LSK) are fluorescent after incubation with supernatant derived for Cy5-RNA transfected telocytes (sample), but not in case of supernatant derived from telocytes negative for Cy5 (control). LSK cells Lineage/7AAD negative, Sca1 positive and c-Kit positive. N = 4, P = 0.03 (D) Representative FACS plots of data presented in (C) . Right plot: Hematopoietic stem cells (HSC) and their multipotent progeny (MPP), defined as cells LSK Flt3 negative, are positive for Cy5 when cultured in presence of medium conditioned by Cy5-RNA transfected telocytes. Left plot: in control experiments, HSC/MPP have been incubated with conditioned supernatant derived from non-fluorescent telocytes.

    Article Snippet: The EV donor cells were transfected with a mixture of RNA oligo and lipofectamine RNAiMAX (# 13778-075; Thermo Fisher Scientific, Waltham, MA, USA) in culture medium supplemented with 2% FBS.

    Techniques: Transfection, Incubation, Fluorescence, Derivative Assay, FACS, Cell Culture

    Inhibition effect of LPS on LCFA absorption in IPEC-J2 cells was related to m 6 A methylation. IPEC-J2 cells were treated with 10 μg/ml LPS for various times, and cells without LPS stimulation were used as controls. ( A ) The composition of fatty acids in whole lysate measured by GC/MS. ( B ) m 6 A dot blot to measure the m 6 A levels with purified mRNA from total RNA. Methylene blue staining was used for loading control. The right panel shows the relative levels quantified by densitometry and normalized to control. The data are expressed as the mean ± SEM. Statistically significant difference relative to the control (0 h) ( n = 3, biological replicates). * p

    Journal: The Journal of Immunology Author Choice

    Article Title: Mettl3 Deficiency Sustains Long-Chain Fatty Acid Absorption through Suppressing Traf6-Dependent Inflammation Response

    doi: 10.4049/jimmunol.1801151

    Figure Lengend Snippet: Inhibition effect of LPS on LCFA absorption in IPEC-J2 cells was related to m 6 A methylation. IPEC-J2 cells were treated with 10 μg/ml LPS for various times, and cells without LPS stimulation were used as controls. ( A ) The composition of fatty acids in whole lysate measured by GC/MS. ( B ) m 6 A dot blot to measure the m 6 A levels with purified mRNA from total RNA. Methylene blue staining was used for loading control. The right panel shows the relative levels quantified by densitometry and normalized to control. The data are expressed as the mean ± SEM. Statistically significant difference relative to the control (0 h) ( n = 3, biological replicates). * p

    Article Snippet: mRNA was purified from total RNA using Dynabeads Oligo(dT)25 (Thermo Fisher Scientific).

    Techniques: Inhibition, Methylation, Gas Chromatography-Mass Spectrometry, Dot Blot, Purification, Staining

    Quantitative Proteomics of ATF4 Translation (A) Schematic of SILAC. MEF cells were cultured in “light” or “heavy” media for 5 passages before treating “heavy” cells with amino acid starvation for 2 hr. Atf4 mRNA and associated proteins were purified by a biotinylated probe followed by mass spectrometry. (B) Relative ratio of Atf4 mRNA-associated 60S and 40S ribosomal proteins before and after amino acid starvation. Two biological replicates are shown. (C) A scatter plot shows Atf4 mRNA-associated proteins before and after amino acid starvation. The original peptide score (log 2 ) and starvation-induced fold changes are shown in the x-axis and the y-axis, respectively. RNA-binding proteins (RBPs) are highlighted with ALKBH5 indicated. (D) Validation of Atf4 mRNA-associated ALKBH5 by immunoblotting using the same sample as (D). (E) Schematic of zero distance cross linking methodology. 4-Thiouridine (s 4 U)-labelled RNAs were crosslinked to directly associated proteins using 365 nm UV. Gapdh or Atf4 mRNAs were purified by biotinylated probes followed by immunoblotting. .

    Journal: Molecular cell

    Article Title: N6-Methyladenosine Guides mRNA Alternative Translation during Integrated Stress Response

    doi: 10.1016/j.molcel.2018.01.019

    Figure Lengend Snippet: Quantitative Proteomics of ATF4 Translation (A) Schematic of SILAC. MEF cells were cultured in “light” or “heavy” media for 5 passages before treating “heavy” cells with amino acid starvation for 2 hr. Atf4 mRNA and associated proteins were purified by a biotinylated probe followed by mass spectrometry. (B) Relative ratio of Atf4 mRNA-associated 60S and 40S ribosomal proteins before and after amino acid starvation. Two biological replicates are shown. (C) A scatter plot shows Atf4 mRNA-associated proteins before and after amino acid starvation. The original peptide score (log 2 ) and starvation-induced fold changes are shown in the x-axis and the y-axis, respectively. RNA-binding proteins (RBPs) are highlighted with ALKBH5 indicated. (D) Validation of Atf4 mRNA-associated ALKBH5 by immunoblotting using the same sample as (D). (E) Schematic of zero distance cross linking methodology. 4-Thiouridine (s 4 U)-labelled RNAs were crosslinked to directly associated proteins using 365 nm UV. Gapdh or Atf4 mRNAs were purified by biotinylated probes followed by immunoblotting. .

    Article Snippet: mRNA was purified from total RNA using Dynabeads® Oligo(dT)25 (Thermo Fisher).

    Techniques: Cell Culture, Purification, Mass Spectrometry, RNA Binding Assay