oligo dt primer  (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 oligo dt primer
    Expression of FasR, FADD, and caspase 8 in PBL and various catfish LCL. Total <t>RNA</t> was isolated from T (G14D), cytotoxic T (TS 32.17), B (1G8, 3B11), macrophage (42TA), and fibroblast (CCO) cell lines as well as PBLs from two fish and reverse transcribed using an <t>oligo[dT]</t> primer. The resulting cDNA was used in PCR along with gene-specific primers. Actin was included as a positive control. The presence or absence of FasL cross-reactive proteins as determined previously by Western blotting is indicated: + 37 denotes the 37,000- M r membrane form of FasL, whereas + 15 denotes the 15,000- M r soluble form. ND denotes not detected
    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/oligo dt primer/product/Thermo Fisher
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    Images

    1) Product Images from "Identification and characterization of a FasL-like protein and cDNAs encoding the channel catfish death-inducing signaling complex"

    Article Title: Identification and characterization of a FasL-like protein and cDNAs encoding the channel catfish death-inducing signaling complex

    Journal: Immunogenetics

    doi: 10.1007/s00251-004-0701-2

    Expression of FasR, FADD, and caspase 8 in PBL and various catfish LCL. Total RNA was isolated from T (G14D), cytotoxic T (TS 32.17), B (1G8, 3B11), macrophage (42TA), and fibroblast (CCO) cell lines as well as PBLs from two fish and reverse transcribed using an oligo[dT] primer. The resulting cDNA was used in PCR along with gene-specific primers. Actin was included as a positive control. The presence or absence of FasL cross-reactive proteins as determined previously by Western blotting is indicated: + 37 denotes the 37,000- M r membrane form of FasL, whereas + 15 denotes the 15,000- M r soluble form. ND denotes not detected
    Figure Legend Snippet: Expression of FasR, FADD, and caspase 8 in PBL and various catfish LCL. Total RNA was isolated from T (G14D), cytotoxic T (TS 32.17), B (1G8, 3B11), macrophage (42TA), and fibroblast (CCO) cell lines as well as PBLs from two fish and reverse transcribed using an oligo[dT] primer. The resulting cDNA was used in PCR along with gene-specific primers. Actin was included as a positive control. The presence or absence of FasL cross-reactive proteins as determined previously by Western blotting is indicated: + 37 denotes the 37,000- M r membrane form of FasL, whereas + 15 denotes the 15,000- M r soluble form. ND denotes not detected

    Techniques Used: Expressing, Isolation, Fluorescence In Situ Hybridization, Polymerase Chain Reaction, Positive Control, Western Blot

    2) Product Images from "Activation of Multiple Apoptotic Pathways in Human Nasopharyngeal Carcinoma Cells by the Prenylated Isoflavone, Osajin"

    Article Title: Activation of Multiple Apoptotic Pathways in Human Nasopharyngeal Carcinoma Cells by the Prenylated Isoflavone, Osajin

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018308

    Effect of osajin on the expression of Fas/CD95, FasL/CD95L, GRP78/BiP, CHOP/GADD153 and Bcl-2 family proteins. TW04 cells were treated with various concentrations of osajin for 24 h. ( A ) RNA was isolated from cells treated with 5 µM or 7.5 µM osajin. Two µg of RNA was reversely transcribed into cDNA using oligo (dT) primers. RT-PCR analysis was performed using primers specific for Fas, FasL, GRP78, CHOP, Bax and Bcl-2 genes and also the internal control gene, β-actin. ( B ) Cell lysates were prepared for SDS-PAGE followed by Western blot for Fas, FasL, GRP78, CHOP, Bax and Bcl-2, with β-actin as a loading control. ( C ) to ( E ) Results are presented as the relative densities of protein bands normalized to β-actin. The data shown are the means ± SE of three individual experiments (* P
    Figure Legend Snippet: Effect of osajin on the expression of Fas/CD95, FasL/CD95L, GRP78/BiP, CHOP/GADD153 and Bcl-2 family proteins. TW04 cells were treated with various concentrations of osajin for 24 h. ( A ) RNA was isolated from cells treated with 5 µM or 7.5 µM osajin. Two µg of RNA was reversely transcribed into cDNA using oligo (dT) primers. RT-PCR analysis was performed using primers specific for Fas, FasL, GRP78, CHOP, Bax and Bcl-2 genes and also the internal control gene, β-actin. ( B ) Cell lysates were prepared for SDS-PAGE followed by Western blot for Fas, FasL, GRP78, CHOP, Bax and Bcl-2, with β-actin as a loading control. ( C ) to ( E ) Results are presented as the relative densities of protein bands normalized to β-actin. The data shown are the means ± SE of three individual experiments (* P

    Techniques Used: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, SDS Page, Western Blot

    3) Product Images from "Genome-Wide Profiling of Cap-Independent Translation Enhancing Elements in the Human Genome"

    Article Title: Genome-Wide Profiling of Cap-Independent Translation Enhancing Elements in the Human Genome

    Journal: Nature methods

    doi: 10.1038/nmeth.2522

    In vitro selection of RNA elements that mediate cap-independent (CI) translation ( a ) A library of human genomic DNA fragments was inserted into a DNA cassette containing all of the sequence information necessary for mRNA display. For each selection round, the dsDNA pool was in vitro transcribed into ssRNA, conjugated to a DNA-puromycin linker, and translated in vitro . Uncapped mRNA sequences that initiate translation of an intact ORF become covalently linked to a His-6 protein affinity tag encoded in the RNA message. Functional molecules are recovered, reverse transcribed, and amplified by PCR to generate the DNA for the next selection cycle. ( b ) RNA-protein fusion molecules are generated via the natural peptidyl transferase activity of the ribosome. ( c ) Selection progress was monitored by measuring the fraction of S 35 -labeled fusion molecules recovered from the oligo-dT and Ni-NTA affinity columns.
    Figure Legend Snippet: In vitro selection of RNA elements that mediate cap-independent (CI) translation ( a ) A library of human genomic DNA fragments was inserted into a DNA cassette containing all of the sequence information necessary for mRNA display. For each selection round, the dsDNA pool was in vitro transcribed into ssRNA, conjugated to a DNA-puromycin linker, and translated in vitro . Uncapped mRNA sequences that initiate translation of an intact ORF become covalently linked to a His-6 protein affinity tag encoded in the RNA message. Functional molecules are recovered, reverse transcribed, and amplified by PCR to generate the DNA for the next selection cycle. ( b ) RNA-protein fusion molecules are generated via the natural peptidyl transferase activity of the ribosome. ( c ) Selection progress was monitored by measuring the fraction of S 35 -labeled fusion molecules recovered from the oligo-dT and Ni-NTA affinity columns.

    Techniques Used: In Vitro, Selection, Sequencing, Functional Assay, Amplification, Polymerase Chain Reaction, Generated, Activity Assay, Labeling

    4) Product Images from "Enrichment of processed pseudogene transcripts in L1-ribonucleoprotein particles"

    Article Title: Enrichment of processed pseudogene transcripts in L1-ribonucleoprotein particles

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddt225

    qRT-PCR to determine the relative amount of other RNAs compared with L1 RNA in L1 RNPs. ( A ) Total RNA was isolated from RNPs after transfecting FL-O1F by Trizol extraction and treated with DNAse. cDNA was synthesized using an oligo-dT (12 Ts) primer and
    Figure Legend Snippet: qRT-PCR to determine the relative amount of other RNAs compared with L1 RNA in L1 RNPs. ( A ) Total RNA was isolated from RNPs after transfecting FL-O1F by Trizol extraction and treated with DNAse. cDNA was synthesized using an oligo-dT (12 Ts) primer and

    Techniques Used: Quantitative RT-PCR, Isolation, Synthesized

    Enrichment of RPLP1, GAPDH, β-actin and L1 transcript in RNPs compared with total lysate. ( A ) For construct FL-O1F, equal amounts of RNA purified from total lysate, unbound lysate and purified RNPs were subjected to cDNA synthesis using an oligo-dT
    Figure Legend Snippet: Enrichment of RPLP1, GAPDH, β-actin and L1 transcript in RNPs compared with total lysate. ( A ) For construct FL-O1F, equal amounts of RNA purified from total lysate, unbound lysate and purified RNPs were subjected to cDNA synthesis using an oligo-dT

    Techniques Used: Construct, Purification

    5) Product Images from "MyoD inhibits Fstl1 and Utrn expression by inducing transcription of miR-206"

    Article Title: MyoD inhibits Fstl1 and Utrn expression by inducing transcription of miR-206

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200603039

    Fstl1 and Utrn mRNA contain miR-206 target sites. RT-PCR analysis of Fstl1 and Utrn using a miR-206 oligo to prime first-strand synthesis. After collection of RNA from M+M MDER cells either maintained in growth medium (0 h −β) or cultured in DM with β-estradiol (48 h +β), reverse transcription was performed in which either a DNA oligo corresponding to miR-206, a random hexamer oligo mixture (positive control), or no primer (negative control) was used. PCR was subsequently performed with primers specific for Fstl1, Utrn, Timm17b, and desmin, as indicated. Timm17b and Desmin are not predicted targets of miR-206 and are used to demonstrate the specificity of miR-206 oligo priming.
    Figure Legend Snippet: Fstl1 and Utrn mRNA contain miR-206 target sites. RT-PCR analysis of Fstl1 and Utrn using a miR-206 oligo to prime first-strand synthesis. After collection of RNA from M+M MDER cells either maintained in growth medium (0 h −β) or cultured in DM with β-estradiol (48 h +β), reverse transcription was performed in which either a DNA oligo corresponding to miR-206, a random hexamer oligo mixture (positive control), or no primer (negative control) was used. PCR was subsequently performed with primers specific for Fstl1, Utrn, Timm17b, and desmin, as indicated. Timm17b and Desmin are not predicted targets of miR-206 and are used to demonstrate the specificity of miR-206 oligo priming.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Cell Culture, Random Hexamer Labeling, Positive Control, Negative Control, Polymerase Chain Reaction

    6) Product Images from "TcTASV: A Novel Protein Family in Trypanosoma cruzi Identified from a Subtractive Trypomastigote cDNA Library"

    Article Title: TcTASV: A Novel Protein Family in Trypanosoma cruzi Identified from a Subtractive Trypomastigote cDNA Library

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0000841

    The TcT-E element (TcT-E elem ) is present in multiple copies in the T. cruzi genome and is associated with different coding regions. (A) Identification of an enriched 280-bp element in the TcT-E library. In silico screening of the TcT-E library using the FL-160-2 3′ UTR as bait depicted a large number of clones displaying homology with nucleotides 372–472. After analyzing a multiple sequence alignment of the identified TcT-E clones, a 280-bp consensus sequence with 3′ and 5′ polypyrimidine tracts (bold) and a variable number of TAA repeats (bold underlined) was obtained, and defined as TcT-Eelement (TcT-E elem ). (B) Analysis of TcT-E elem copy number. T. cruzi genomic DNA (CL-Brener strain) was digested with restriction enzymes having no internal site within the TcT-E elem , electrophoresed on TAE-agarose gel and transferred by standard procedures. A probe specific for the TcT-E elem was synthesized and labelled by PCR with 32 P. (C) The mRNA of the CDSs located upstream of the TcT-E are preferentially expressed in trypomastigotes. Northern blots probed with the complete ORF Tcruzi_1863-4-1211-93 ( http://TriTrypDB.org ). (D) The TcT-E elem is present 30–70 bp downstream of a stop codon in many coding sequences in T.cruzi . The consensus sequence of TcT-E elem was used as bait to search the T. cruzi database; WGSs with more than 80% identity to TcT-E elem and longer than 1000 bp were retrieved and analyzed. The schematic map of the relative position of TcT-E elem in relation to coding sequences in T. cruzi genome is shown. (E) Mature mRNA transcripts contain both the TcT-E elem and different CDSs. Total RNA from trypomastigotes (T) and epimastigotes (E) was purified and treated with RQ1 DNase. First strand cDNA was synthesized by RT using an oligo dT primer. PCR was performed using a 5′ primer specific for the T. cruzi miniexon (ME) and a 3′ antisense primer corresponding to the 3′ region of the TcT-E elem . Alternatively, PCR was performed with primers corresponding to the 5′ and 3′ conserved regions of most CDSs (CDS-L and CDS-R) or with a 5′primer specific for the CDS and a 3′ primer specific for the TcT-E elem . The relative position of the primers is indicated in Fig. 2D . PCR- and RT- denote the negative controls for each reaction.
    Figure Legend Snippet: The TcT-E element (TcT-E elem ) is present in multiple copies in the T. cruzi genome and is associated with different coding regions. (A) Identification of an enriched 280-bp element in the TcT-E library. In silico screening of the TcT-E library using the FL-160-2 3′ UTR as bait depicted a large number of clones displaying homology with nucleotides 372–472. After analyzing a multiple sequence alignment of the identified TcT-E clones, a 280-bp consensus sequence with 3′ and 5′ polypyrimidine tracts (bold) and a variable number of TAA repeats (bold underlined) was obtained, and defined as TcT-Eelement (TcT-E elem ). (B) Analysis of TcT-E elem copy number. T. cruzi genomic DNA (CL-Brener strain) was digested with restriction enzymes having no internal site within the TcT-E elem , electrophoresed on TAE-agarose gel and transferred by standard procedures. A probe specific for the TcT-E elem was synthesized and labelled by PCR with 32 P. (C) The mRNA of the CDSs located upstream of the TcT-E are preferentially expressed in trypomastigotes. Northern blots probed with the complete ORF Tcruzi_1863-4-1211-93 ( http://TriTrypDB.org ). (D) The TcT-E elem is present 30–70 bp downstream of a stop codon in many coding sequences in T.cruzi . The consensus sequence of TcT-E elem was used as bait to search the T. cruzi database; WGSs with more than 80% identity to TcT-E elem and longer than 1000 bp were retrieved and analyzed. The schematic map of the relative position of TcT-E elem in relation to coding sequences in T. cruzi genome is shown. (E) Mature mRNA transcripts contain both the TcT-E elem and different CDSs. Total RNA from trypomastigotes (T) and epimastigotes (E) was purified and treated with RQ1 DNase. First strand cDNA was synthesized by RT using an oligo dT primer. PCR was performed using a 5′ primer specific for the T. cruzi miniexon (ME) and a 3′ antisense primer corresponding to the 3′ region of the TcT-E elem . Alternatively, PCR was performed with primers corresponding to the 5′ and 3′ conserved regions of most CDSs (CDS-L and CDS-R) or with a 5′primer specific for the CDS and a 3′ primer specific for the TcT-E elem . The relative position of the primers is indicated in Fig. 2D . PCR- and RT- denote the negative controls for each reaction.

    Techniques Used: In Silico, Clone Assay, Sequencing, Agarose Gel Electrophoresis, Synthesized, Polymerase Chain Reaction, Northern Blot, Purification

    7) Product Images from "The Epstein-Barr Virus-Encoded MicroRNA MiR-BART9 Promotes Tumor Metastasis by Targeting E-Cadherin in Nasopharyngeal Carcinoma"

    Article Title: The Epstein-Barr Virus-Encoded MicroRNA MiR-BART9 Promotes Tumor Metastasis by Targeting E-Cadherin in Nasopharyngeal Carcinoma

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003974

    E-cadherin plays a pivotal role in miR-BART9-mediated migration and invasion in NPC cells. (A) Protein level of E-cadherin was increased after introducing pcDNA6/His-CDH1, which contains CDH1 open reading frame without 3′-UTR. Transwell migration assay (B) and Matrigel invasion assay (C) for miR-BART9- or LacZ-expressing BM1 cells with or without ectopic expression of E-cadherin. (D) HK1-EBV cells were transfected with 10 nM siRNA negative control (si-Neg) or CDH1 siRNA (si-CDH1). Expression of E-cadherin was examined by Western blotting. GAPDH was used as a loading control. (E) Transwell migration assay (Upper) and Matrigel invasion assay (Middle) of HK1-EBV cells treated with an LNA-modified miR-BART9 antisense oligo (anti-BART9), scramble control (anti-Ctrl), or anti-BART9 plus E-cadherin siRNA (si-CHD1). Images of cells adhered to the lower surface of the filter insert from a representative experiment are shown. The numbers of migratory or invasive cells were quantified using image J and are expressed as the fold change relative to the appropriate cell line (bar graphs). The data are expressed as the means ± SEM from three independent experiments and two-tailed Student's t-tests were performed (*, P
    Figure Legend Snippet: E-cadherin plays a pivotal role in miR-BART9-mediated migration and invasion in NPC cells. (A) Protein level of E-cadherin was increased after introducing pcDNA6/His-CDH1, which contains CDH1 open reading frame without 3′-UTR. Transwell migration assay (B) and Matrigel invasion assay (C) for miR-BART9- or LacZ-expressing BM1 cells with or without ectopic expression of E-cadherin. (D) HK1-EBV cells were transfected with 10 nM siRNA negative control (si-Neg) or CDH1 siRNA (si-CDH1). Expression of E-cadherin was examined by Western blotting. GAPDH was used as a loading control. (E) Transwell migration assay (Upper) and Matrigel invasion assay (Middle) of HK1-EBV cells treated with an LNA-modified miR-BART9 antisense oligo (anti-BART9), scramble control (anti-Ctrl), or anti-BART9 plus E-cadherin siRNA (si-CHD1). Images of cells adhered to the lower surface of the filter insert from a representative experiment are shown. The numbers of migratory or invasive cells were quantified using image J and are expressed as the fold change relative to the appropriate cell line (bar graphs). The data are expressed as the means ± SEM from three independent experiments and two-tailed Student's t-tests were performed (*, P

    Techniques Used: Migration, Transwell Migration Assay, Invasion Assay, Expressing, Transfection, Negative Control, Western Blot, Modification, Two Tailed Test

    Depletion of endogenous miR-BART9 suppresses the migration and invasiveness of EBV-positive NPC cells. (A) LNA-modified anti-BART9 efficiently decreases the level of mature miR-BART9 in EBV-positive HK1-EBV and C666-1 cells. HK1-EBV and C666-1 cells were treated with a 12.5 nM concentration of an LNA-modified miR-BART9 antisense oligo (anti-BART9) or a scramble control (anti-Ctrl) for 48 hr. The expression level of miR-BART9 was determined via qPCR. (B) HK1-EBV cells were treated with anti-BART9 or anti-Ctrl for 24 hr and the plated for colony formation assays. Colony formation activity was determined via crystal violet staining after 11 days in culture. (C, D) Transwell migration assay (C) and Matrigel invasion assay (D) for HK1-EBV and C666-1 cells. Cells were treated with a 12.5 nM concentration of an LNA-modified miR-BART9 antisense oligo (anti-BART9) or a scramble control (anti-Ctrl) for 48 hr before the migration or invasion assay. Images of cells adhered to the lower surface of the filter insert from a representative experiment are shown. (Left panel). The numbers of migratory or invasive cells were quantified using image J and expressed as the fold change relative to the appropriate cell line (bar graphs). (E) Expression levels of LMP1, LMP2A and EBNA1 in HK1-EBV and C666-1 cells treated with a 12.5 nM concentration of an LNA-modified miR-BART9 antisense oligo (anti-BART9) or a scramble control (anti-Ctrl). Total RNA was collected 48 hr after transfection and mRNA levels were determined via qPCR. The data were normalized to cellular EEF1A1 levels and expressed as the fold change relative to the appropriate cell line. The bar graphs in (B), (C), (D), (E) show means ± SEM from three independent experiments and two-tailed Student's t-tests were performed (*, P
    Figure Legend Snippet: Depletion of endogenous miR-BART9 suppresses the migration and invasiveness of EBV-positive NPC cells. (A) LNA-modified anti-BART9 efficiently decreases the level of mature miR-BART9 in EBV-positive HK1-EBV and C666-1 cells. HK1-EBV and C666-1 cells were treated with a 12.5 nM concentration of an LNA-modified miR-BART9 antisense oligo (anti-BART9) or a scramble control (anti-Ctrl) for 48 hr. The expression level of miR-BART9 was determined via qPCR. (B) HK1-EBV cells were treated with anti-BART9 or anti-Ctrl for 24 hr and the plated for colony formation assays. Colony formation activity was determined via crystal violet staining after 11 days in culture. (C, D) Transwell migration assay (C) and Matrigel invasion assay (D) for HK1-EBV and C666-1 cells. Cells were treated with a 12.5 nM concentration of an LNA-modified miR-BART9 antisense oligo (anti-BART9) or a scramble control (anti-Ctrl) for 48 hr before the migration or invasion assay. Images of cells adhered to the lower surface of the filter insert from a representative experiment are shown. (Left panel). The numbers of migratory or invasive cells were quantified using image J and expressed as the fold change relative to the appropriate cell line (bar graphs). (E) Expression levels of LMP1, LMP2A and EBNA1 in HK1-EBV and C666-1 cells treated with a 12.5 nM concentration of an LNA-modified miR-BART9 antisense oligo (anti-BART9) or a scramble control (anti-Ctrl). Total RNA was collected 48 hr after transfection and mRNA levels were determined via qPCR. The data were normalized to cellular EEF1A1 levels and expressed as the fold change relative to the appropriate cell line. The bar graphs in (B), (C), (D), (E) show means ± SEM from three independent experiments and two-tailed Student's t-tests were performed (*, P

    Techniques Used: Migration, Modification, Concentration Assay, Expressing, Real-time Polymerase Chain Reaction, Activity Assay, Staining, Transwell Migration Assay, Invasion Assay, Transfection, Two Tailed Test

    miR-BART9 promotes the migration and invasion of EBV-negative NPC cells. Transwell migration assay (A) and Matrigel invasion assay (B) for miR-BART9- or LacZ-expressing EBV-negative NPC cells. BM1, TW04 and HK1 cells were infected with the miR-BART9 (BART9) or control (LacZ) vector. Cells expressing miR-BART9 or LacZ were directly compared in terms of migratory activity or invasion activity in the same assay (Left panel). In a separate assay, cells expressing miR-BART9 were treated with a 12.5 nM concentration of an LNA-modified miR-BART9 antisense oligo (anti-BART9) or a scramble control (anti-Ctrl) for 48 hr before the migration or invasion assay (middle panel). Images of cells adhered to the lower surface of the filter insert from a representative experiment are shown. The numbers of migratory or invasive cells were quantified using image J and expressed as the fold change relative to the appropriate cell line (bar graphs). The data are expressed as the means ± SEM from three independent experiments and two-tailed Student's t-tests were performed (*, P
    Figure Legend Snippet: miR-BART9 promotes the migration and invasion of EBV-negative NPC cells. Transwell migration assay (A) and Matrigel invasion assay (B) for miR-BART9- or LacZ-expressing EBV-negative NPC cells. BM1, TW04 and HK1 cells were infected with the miR-BART9 (BART9) or control (LacZ) vector. Cells expressing miR-BART9 or LacZ were directly compared in terms of migratory activity or invasion activity in the same assay (Left panel). In a separate assay, cells expressing miR-BART9 were treated with a 12.5 nM concentration of an LNA-modified miR-BART9 antisense oligo (anti-BART9) or a scramble control (anti-Ctrl) for 48 hr before the migration or invasion assay (middle panel). Images of cells adhered to the lower surface of the filter insert from a representative experiment are shown. The numbers of migratory or invasive cells were quantified using image J and expressed as the fold change relative to the appropriate cell line (bar graphs). The data are expressed as the means ± SEM from three independent experiments and two-tailed Student's t-tests were performed (*, P

    Techniques Used: Migration, Transwell Migration Assay, Invasion Assay, Expressing, Infection, Plasmid Preparation, Activity Assay, Concentration Assay, Modification, Two Tailed Test

    8) Product Images from "Combinatorial Action of miRNAs Regulates Transcriptional and Post-Transcriptional Gene Silencing following invivo PNS Injury"

    Article Title: Combinatorial Action of miRNAs Regulates Transcriptional and Post-Transcriptional Gene Silencing following invivo PNS Injury

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039674

    miRNAs induce post-transcriptional gene silencing through association with Ago-2 in functional complexes. (A). Protein expression of Ago-1, Ago-2 and Dicer in sciatic nerves before and after nerve injury to confirm the expression of the miRNA processing machinery proteins. Actin was used as a loading control. (B) Cytoplasmic lysates isolated from control sciatic nerves and injured distal segments (10 nerves each) were immunoprecipitated with Ago-2 antibody (Cell Signaling, USA) or IgG control. A portion (1/3 rd ) of the sample was used for each of the analysis and the experiment was repeated twice. Input, No-Antibody (No AB) and Ago-2 immunoprecipitated protein was analyzed by western blotting with Ago-2 antibody, which shows enrichment of Ago-2 in the 24 hour post-injury samples. (C). For RNA-IPs (RIPs), mRNAs that were co-immunoprecipitated with Ago-2 in vivo, were reverse transcribed using oligo-dT primer and genes of interest were PCR amplified using gene-specific primers (Krox-20 = 1274 bp, C-Jun = 689 bp, Nanog = 753 bp, QKI-6 = 1345 bp, Sox-2 = 958 bp and ID-2 = 588 bp). (D). microRNAs that were co-immunoprecipitated with Ago-2 in complex with their targeted mRNAs, were reverse-transcribed with microRNA specific RT-primers using Multiscribe RT kit. Ago-2 associated microRNAs were detected by real time qPCR with miRNA specific Taqman probe/primer sets. Data were normalized to input and an internal control. Fold difference (2 -ΔΔCT ) in the association of individual microRNAs with Ago-2 protein between injured and control nerves was plotted as log 2 median ratio and error is expressed as standard deviation.
    Figure Legend Snippet: miRNAs induce post-transcriptional gene silencing through association with Ago-2 in functional complexes. (A). Protein expression of Ago-1, Ago-2 and Dicer in sciatic nerves before and after nerve injury to confirm the expression of the miRNA processing machinery proteins. Actin was used as a loading control. (B) Cytoplasmic lysates isolated from control sciatic nerves and injured distal segments (10 nerves each) were immunoprecipitated with Ago-2 antibody (Cell Signaling, USA) or IgG control. A portion (1/3 rd ) of the sample was used for each of the analysis and the experiment was repeated twice. Input, No-Antibody (No AB) and Ago-2 immunoprecipitated protein was analyzed by western blotting with Ago-2 antibody, which shows enrichment of Ago-2 in the 24 hour post-injury samples. (C). For RNA-IPs (RIPs), mRNAs that were co-immunoprecipitated with Ago-2 in vivo, were reverse transcribed using oligo-dT primer and genes of interest were PCR amplified using gene-specific primers (Krox-20 = 1274 bp, C-Jun = 689 bp, Nanog = 753 bp, QKI-6 = 1345 bp, Sox-2 = 958 bp and ID-2 = 588 bp). (D). microRNAs that were co-immunoprecipitated with Ago-2 in complex with their targeted mRNAs, were reverse-transcribed with microRNA specific RT-primers using Multiscribe RT kit. Ago-2 associated microRNAs were detected by real time qPCR with miRNA specific Taqman probe/primer sets. Data were normalized to input and an internal control. Fold difference (2 -ΔΔCT ) in the association of individual microRNAs with Ago-2 protein between injured and control nerves was plotted as log 2 median ratio and error is expressed as standard deviation.

    Techniques Used: Functional Assay, Expressing, Isolation, Immunoprecipitation, Western Blot, In Vivo, Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction, Standard Deviation

    9) Product Images from "Induction of the cyclic nucleotide phosphodiesterase PDE4B is essential for LPS-activated TNF-? responses"

    Article Title: Induction of the cyclic nucleotide phosphodiesterase PDE4B is essential for LPS-activated TNF-? responses

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

    doi: 10.1073/pnas.122041599

    LPS effects on PDE4B mRNA steady state. ( A and B ) THP-1 cells were incubated in the absence or presence of 1 μg/ml LPS and/or 100 μM of dibutyryl cAMP (dbcAMP) for 3 h. Extraction of total RNA and Northern blot analysis were performed as described in Materials and Methods . Twenty-five micrograms of total RNA were loaded onto each lane. Numbers on the left are sizes of DNA molecular weight markers in kb. ( C ) Circulating leukocytes from wild-type and PDE4B −/− mice were cultured in the absence or presence of 100 ng/ml of LPS for 3 h. Extraction of total RNA from leukocytes (pooled from four animals per group), RT-PCR using primers corresponding to mouse PDE4B cDNA sequence, and Southern blot analysis were performed as described in Materials and Methods . Results derived from cDNAs prepared with random primers ( Left ) and oligo dT primers ( Right ) are reported. Amplification of a GAPDH fragment was included to monitor the amount of RNA in each sample.
    Figure Legend Snippet: LPS effects on PDE4B mRNA steady state. ( A and B ) THP-1 cells were incubated in the absence or presence of 1 μg/ml LPS and/or 100 μM of dibutyryl cAMP (dbcAMP) for 3 h. Extraction of total RNA and Northern blot analysis were performed as described in Materials and Methods . Twenty-five micrograms of total RNA were loaded onto each lane. Numbers on the left are sizes of DNA molecular weight markers in kb. ( C ) Circulating leukocytes from wild-type and PDE4B −/− mice were cultured in the absence or presence of 100 ng/ml of LPS for 3 h. Extraction of total RNA from leukocytes (pooled from four animals per group), RT-PCR using primers corresponding to mouse PDE4B cDNA sequence, and Southern blot analysis were performed as described in Materials and Methods . Results derived from cDNAs prepared with random primers ( Left ) and oligo dT primers ( Right ) are reported. Amplification of a GAPDH fragment was included to monitor the amount of RNA in each sample.

    Techniques Used: Incubation, Northern Blot, Molecular Weight, Mouse Assay, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Sequencing, Southern Blot, Derivative Assay, Amplification

    10) Product Images from "Characterization and Transposon Mutagenesis of the Maize (Zea mays) Pho1 Gene Family"

    Article Title: Characterization and Transposon Mutagenesis of the Maize (Zea mays) Pho1 Gene Family

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0161882

    Accumulation of Pho1;2 sense and antisense transcripts under contrasting phosphate conditions. (A) Schematic of Pho1;2 genes indicating the position of primers (solid triangles; see Table 1 ). (B) Amplification of fragments corresponding to the mature sense-RNAs of ZmPho1;2a , ZmPho1;2b and SbPho1;2 from oligo-dT primed cDNA prepared from roots and shoot of 10-day-old seedlings, fertilized with Hoagland solution adjusted to 1mM (+P) or 0mM (-P) inorganic phosphate. Primer pairs used for amplification indicated by letter codes on the right-hand side of panel. (C) Amplification of fragments corresponding to putative anti-sense transcripts encoded by ZmPho1;2a , ZmPho1;2b from cDNA as Panel A. Primary PCR (left) and nested PCR performed from 1:100,000 dilution of primary reaction (right). Amplification of ZmUBQ and SbUBQ fragments and amplification from genomic DNA template (G) were used as controls. Primer pairs indicated as in (B).
    Figure Legend Snippet: Accumulation of Pho1;2 sense and antisense transcripts under contrasting phosphate conditions. (A) Schematic of Pho1;2 genes indicating the position of primers (solid triangles; see Table 1 ). (B) Amplification of fragments corresponding to the mature sense-RNAs of ZmPho1;2a , ZmPho1;2b and SbPho1;2 from oligo-dT primed cDNA prepared from roots and shoot of 10-day-old seedlings, fertilized with Hoagland solution adjusted to 1mM (+P) or 0mM (-P) inorganic phosphate. Primer pairs used for amplification indicated by letter codes on the right-hand side of panel. (C) Amplification of fragments corresponding to putative anti-sense transcripts encoded by ZmPho1;2a , ZmPho1;2b from cDNA as Panel A. Primary PCR (left) and nested PCR performed from 1:100,000 dilution of primary reaction (right). Amplification of ZmUBQ and SbUBQ fragments and amplification from genomic DNA template (G) were used as controls. Primer pairs indicated as in (B).

    Techniques Used: Amplification, Polymerase Chain Reaction, Nested PCR

    11) Product Images from "Identification of a consensus element recognized and cleaved by IRE1?"

    Article Title: Identification of a consensus element recognized and cleaved by IRE1?

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq452

    Scheme of the screening approach used to identify cleavage targets of IRE1α. ( A ) Schematic representation of the screening procedure. Cellular RNAs were subjected to in vitro cleavage by the cytoplasmic domain of human IRE1α, or by GFP as a mock treatment. Then the RNAs were reverse transcribed with oligo-dT primers and treated with RNase H. The resulting RNA fragments, corresponding to the 5′ sides of the cleaved RNAs (represented by the gray bar in the figure), were purified and used as probes in the exon microarray analysis. ( B ) Efficient cleavage and production of a probe fragment in the case of the XBP1 mRNA. Left panel: total RNA (5 µg) was incubated with IRE1α or GFP and analyzed by northern blotting with a probe specific for the 5′ fragment of the XBP1 mRNA. Right panel: the RNA samples, as described for the left panel, were reverse transcribed and treated with RNaseH before blotting.
    Figure Legend Snippet: Scheme of the screening approach used to identify cleavage targets of IRE1α. ( A ) Schematic representation of the screening procedure. Cellular RNAs were subjected to in vitro cleavage by the cytoplasmic domain of human IRE1α, or by GFP as a mock treatment. Then the RNAs were reverse transcribed with oligo-dT primers and treated with RNase H. The resulting RNA fragments, corresponding to the 5′ sides of the cleaved RNAs (represented by the gray bar in the figure), were purified and used as probes in the exon microarray analysis. ( B ) Efficient cleavage and production of a probe fragment in the case of the XBP1 mRNA. Left panel: total RNA (5 µg) was incubated with IRE1α or GFP and analyzed by northern blotting with a probe specific for the 5′ fragment of the XBP1 mRNA. Right panel: the RNA samples, as described for the left panel, were reverse transcribed and treated with RNaseH before blotting.

    Techniques Used: In Vitro, Purification, Microarray, Incubation, Northern Blot

    12) Product Images from "A noncoding RNA containing a SINE-B1 motif associates with meiotic metaphase chromatin and has an indispensable function during spermatogenesis"

    Article Title: A noncoding RNA containing a SINE-B1 motif associates with meiotic metaphase chromatin and has an indispensable function during spermatogenesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0179585

    RT-PCR and ISH analyses of R53 RNA. ( A) Schematic representation of the R53 gene locus. The location of Dig-labeled RNA and Dig-oligo DNA probes used for ISH are indicated with arrows, and the region of B1F motif in the R53 sequence is indicated by the box. (B) The cDNA sequence of R53 transcript (587 bp) was detected in adult mouse tissues by RT-PCR using R53A (R53F and R53R) as primers (30 cycles) and is shown together with G3pdh products (22 cycles) as standards. Single-stranded cDNA was prepared from adult wild-type liver (Lv), spleen (Sp), kidney (Kd), brain (Br) and testis (Ts) tissues, in addition to Mvh homozygote (-/-) testis tissue. (C) ISH images of adult testis sections probed with Dig-antisense R53 RNA (a, b, d) and Dig-sense R53 RNA (c). (d) High-magnification view of the outlined frame in (b). The scale bars in (a-c) and (d) indicate 100 μm and 20 μm, respectively. Additionally, dotted-line arrows represent the position of R53-S1 primer pair used in Fig 4 and the positions of oligo DNA probes (S2, AS2 and pB1D) used in Fig 4 and S3 Fig are also indicated in the schema.
    Figure Legend Snippet: RT-PCR and ISH analyses of R53 RNA. ( A) Schematic representation of the R53 gene locus. The location of Dig-labeled RNA and Dig-oligo DNA probes used for ISH are indicated with arrows, and the region of B1F motif in the R53 sequence is indicated by the box. (B) The cDNA sequence of R53 transcript (587 bp) was detected in adult mouse tissues by RT-PCR using R53A (R53F and R53R) as primers (30 cycles) and is shown together with G3pdh products (22 cycles) as standards. Single-stranded cDNA was prepared from adult wild-type liver (Lv), spleen (Sp), kidney (Kd), brain (Br) and testis (Ts) tissues, in addition to Mvh homozygote (-/-) testis tissue. (C) ISH images of adult testis sections probed with Dig-antisense R53 RNA (a, b, d) and Dig-sense R53 RNA (c). (d) High-magnification view of the outlined frame in (b). The scale bars in (a-c) and (d) indicate 100 μm and 20 μm, respectively. Additionally, dotted-line arrows represent the position of R53-S1 primer pair used in Fig 4 and the positions of oligo DNA probes (S2, AS2 and pB1D) used in Fig 4 and S3 Fig are also indicated in the schema.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, In Situ Hybridization, Labeling, Sequencing

    QRT-PCR analyses of the subcellular localization of the RNAs in the testicular cells. Testicular cells were prepared from the testes of mice 22 days after birth; this stage approximately corresponds to the time of the highest level of R53 expression ( Fig 5B ). (A) Gel electrophoreses of PCR products using primer pairs for SCP3 (19 cycles), BC1 (22 cycles), Xist (35 cycles), β-actin (18 cycles), R53 RNA (35 cycles, R53-S2 for the primer detecting a R53-B1F-containing sequence downstream of that of R53-S1) and genotyping of the Jmjd1C locus (genome, 35 cycles) in the cytoplasmic extract (Cyt), nuclear soluble (Nuc) and chromatin-bound (Chr) fractions are shown. The genotyping PCR (qJ1C used as the primer) to detect a sequence in the third intron of wild-type Jmjd1C gene was performed to demonstrate that there was almost no contamination of the genomic DNA in any fraction. All the primer pairs used are listed in S1 Table . (B) Reverse-transcription was performed using total RNA prepared from the chromatin-bound fraction and oligo-dT primer with (RT+) or without reverse transcriptase (RT-). As in (A), PCR was performed using 2 sets of primers for R53 RNA (35 cycles) to testify that the PCR products were derived from RT-dependent cDNA. The PCR specificity for R53 transcript was confirmed by sequencing analyses of the PCR products. (C) The amounts of each transcript in the three subcellular fractions were quantitatively analyzed. The values indicated are the relative levels of each transcript against total values of the three subcellular fractions (set as 100%). The error bars indicate the SEM (n = 6).
    Figure Legend Snippet: QRT-PCR analyses of the subcellular localization of the RNAs in the testicular cells. Testicular cells were prepared from the testes of mice 22 days after birth; this stage approximately corresponds to the time of the highest level of R53 expression ( Fig 5B ). (A) Gel electrophoreses of PCR products using primer pairs for SCP3 (19 cycles), BC1 (22 cycles), Xist (35 cycles), β-actin (18 cycles), R53 RNA (35 cycles, R53-S2 for the primer detecting a R53-B1F-containing sequence downstream of that of R53-S1) and genotyping of the Jmjd1C locus (genome, 35 cycles) in the cytoplasmic extract (Cyt), nuclear soluble (Nuc) and chromatin-bound (Chr) fractions are shown. The genotyping PCR (qJ1C used as the primer) to detect a sequence in the third intron of wild-type Jmjd1C gene was performed to demonstrate that there was almost no contamination of the genomic DNA in any fraction. All the primer pairs used are listed in S1 Table . (B) Reverse-transcription was performed using total RNA prepared from the chromatin-bound fraction and oligo-dT primer with (RT+) or without reverse transcriptase (RT-). As in (A), PCR was performed using 2 sets of primers for R53 RNA (35 cycles) to testify that the PCR products were derived from RT-dependent cDNA. The PCR specificity for R53 transcript was confirmed by sequencing analyses of the PCR products. (C) The amounts of each transcript in the three subcellular fractions were quantitatively analyzed. The values indicated are the relative levels of each transcript against total values of the three subcellular fractions (set as 100%). The error bars indicate the SEM (n = 6).

    Techniques Used: Quantitative RT-PCR, Mouse Assay, Expressing, Polymerase Chain Reaction, Sequencing, Derivative Assay

    13) Product Images from "Intracerebral delivery of small interfering RNAs (siRNAs) using adenoviral vector protects mice against lethal peripheral rabies challenge"

    Article Title: Intracerebral delivery of small interfering RNAs (siRNAs) using adenoviral vector protects mice against lethal peripheral rabies challenge

    Journal: Virus Research

    doi: 10.1016/j.virusres.2011.08.004

    Relative levels of RABV-N and L transcripts in different adenovirus-treated and RABV-PV-11-infected BHK-21 cells quantified by real-time PCR. BHK-21 cells were first treated with different adenoviruses expressing siRNAs and then infected with RABV-PV-11 strain at a MOI of 0.01. At 24 h post-infection, the total RNA was isolated from infected cells and transcribed into cDNA using oligo-dT primer. The RABV-N and L gene transcripts in cDNAs were quantified using gene-specific primers in real-time PCR. GAPDH gene transcript was used as reference for normalization. Data presented are from six independent experiments and n-fold changes in target mRNA transcript levels were determined. *One-way ANOVA statistical analysis on log 10 -transformed data, followed by comparison with control using Dunnett's method.
    Figure Legend Snippet: Relative levels of RABV-N and L transcripts in different adenovirus-treated and RABV-PV-11-infected BHK-21 cells quantified by real-time PCR. BHK-21 cells were first treated with different adenoviruses expressing siRNAs and then infected with RABV-PV-11 strain at a MOI of 0.01. At 24 h post-infection, the total RNA was isolated from infected cells and transcribed into cDNA using oligo-dT primer. The RABV-N and L gene transcripts in cDNAs were quantified using gene-specific primers in real-time PCR. GAPDH gene transcript was used as reference for normalization. Data presented are from six independent experiments and n-fold changes in target mRNA transcript levels were determined. *One-way ANOVA statistical analysis on log 10 -transformed data, followed by comparison with control using Dunnett's method.

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

    Related Articles

    Transfection:

    Article Title: A redox mechanism underlying nucleolar stress sensing by nucleophosmin
    Article Snippet: .. Plasmids DNA and siRNA oligo were transfected into cells with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. ..

    Article Title: Glucocorticoid impairs cell-cell communication by autophagy-mediated degradation of connexin 43 in osteocytes
    Article Snippet: .. Transfection MLO-Y4 cells (1×105 /well) were plated in type-I collagen coated six-well culture plates, and then transfected with various concentrations (12.5nM, 25nM, 50nM and 100nM) of Atg5 siRNA oligo cocktail (Stealth siRNAs: MSS247019, MSS247020, MSS247021, from Thermo Fisher® , USA) or negative control using Lipofectamine® 3000 Transfection Reagent (Invitrogen® , USA) following the manufacturer's protocol. .. Confocal microscopy MLO-Y4 cells cultured on glass coverslips and calvarial primary osteocytes ex vivo cultured in 96-wells plates were fixed for 15mins and and 2hrs at RT with 4% PFA respectively.

    Article Title: miR-411 is up-regulated in FSHD myoblasts and suppresses myogenic factors
    Article Snippet: .. Cell Transfection Cells were transfected with 30 mM Ambion pre-miR-411 precursor oligos (Life Technologies) using Lipofectamine 2000 (Life Technologies) following the manufacture protocol. .. Briefly, 4×103 /cm2 C2 C12 cells were plated in growth medium one day before the transfection.

    Article Title: Downregulation of cytoplasmic DNases is implicated in cytoplasmic DNA accumulation and SASP in senescent cells
    Article Snippet: .. RNAi was performed by the transfection of siRNA oligos using the Lipofectamine™ RNAiMAX transfection reagent (Thermo Fisher Scientific), according to the manufacturer’s instructions. ..

    Amplification:

    Article Title: iFISH is a publically available resource enabling versatile DNA FISH to study genome architecture
    Article Snippet: .. We then amplified the oligos in each well by real-time PCR using the SYBR Select Master Mix (Thermo Fisher Scientific, cat. no. 4472913). .. We designed PCR primers that would anneal to the F and R adapters specific for each oligo in the pool, and incorporate the C adapter and T7 promoter sequence, on the 5’ side of the F and R adapters, respectively.

    Synthesized:

    Article Title: Human RAD9 checkpoint control/proapoptotic protein can activate transcription of p21
    Article Snippet: .. Complementary oligos containing the p53-binding site were synthesized commercially (Invitrogen) and annealed in buffer (100 mM NaCl/5 mM Tris·Cl, pH 7.5/10 mM MgCl2 /0.02 mM EDTA/1 mM DTT). .. These probes were end-labeled with [γ-32 P]ATP by using T4 polynucleotide kinase (New England Biolabs).

    Magnetic Beads:

    Article Title: Transposable elements become active and mobile in the genomes of aging mammalian somatic tissues
    Article Snippet: .. Polyadenylated mRNA was prepared using two consecutive purifications with oligo-dT magnetic beads, following the mRNA Direct Dynabeads Kit protocol provided by the manufacturer (Invitrogen). .. The eluted mRNA was quantified using the Qubit 2.0 RNA HS Assay Kit (Invitrogen).

    Negative Control:

    Article Title: Glucocorticoid impairs cell-cell communication by autophagy-mediated degradation of connexin 43 in osteocytes
    Article Snippet: .. Transfection MLO-Y4 cells (1×105 /well) were plated in type-I collagen coated six-well culture plates, and then transfected with various concentrations (12.5nM, 25nM, 50nM and 100nM) of Atg5 siRNA oligo cocktail (Stealth siRNAs: MSS247019, MSS247020, MSS247021, from Thermo Fisher® , USA) or negative control using Lipofectamine® 3000 Transfection Reagent (Invitrogen® , USA) following the manufacturer's protocol. .. Confocal microscopy MLO-Y4 cells cultured on glass coverslips and calvarial primary osteocytes ex vivo cultured in 96-wells plates were fixed for 15mins and and 2hrs at RT with 4% PFA respectively.

    Real-time Polymerase Chain Reaction:

    Article Title: iFISH is a publically available resource enabling versatile DNA FISH to study genome architecture
    Article Snippet: .. We then amplified the oligos in each well by real-time PCR using the SYBR Select Master Mix (Thermo Fisher Scientific, cat. no. 4472913). .. We designed PCR primers that would anneal to the F and R adapters specific for each oligo in the pool, and incorporate the C adapter and T7 promoter sequence, on the 5’ side of the F and R adapters, respectively.

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    Thermo Fisher oligos
    iFISH implementation. a Scheme of iFISH4U. Pre-designed genome-wide databases of <t>oligos</t> (left) are used as input by the iFISH4U web interface (center) to select oligos within one or more user-specified genomic regions, based on the indicated features. Features 1–3 are used while designing single probes, whereas all the four features are used to design multiple probes on the same chromosome. The black dashed boxes indicate examples of probes within the same region of interest, with the same number of oligos (vertical bars), but suboptimal size (1), homogeneity (2), or centrality (3), whereas the orange box represents the probe of choice having optimal size, homogeneity and centrality. b Cumulative distribution of the distances between consecutive oligos in the human 40-mers database. c Median standard deviation (s.d.) of the distance between consecutive oligos, inside non-overlapping genomic windows of the indicated size, in the 40-mers database and OligoMiner (OM) hg19 databases. OMB, OM ‘Balance’. OMC, OM ‘Coverage’. OMS, OM ‘Stringent’. d Percentage of non-overlapping genomic windows of the indicated size, containing at least 96 oligos, in the 40-mers database and OM hg19 databases. e Scheme of oligos in iFISH probes. Each probe consists of n oligos differing in the T sequence. f Location of the 330 iFISH probes targeting all the human autosomes and chrX. Red dots, individually tested probes (see Fig 2a, b ). g Scheme of the pipeline used to produce iFISH probes. (1) Up to 12,000 oligos, corresponding to a maximum of 125 probes each containing 96 oligos, are synthesized on an array and then pooled together. (2) The oligo-pool is dispensed into n 96-well plates, depending on the total number of probes ( p ) and colors per probe ( c ). (3) In each well, the oligos corresponding to the same probe are selectively amplified using a probe-specific <t>PCR</t> primer pair that incorporates the T7 promoter sequence (T7) and color adapter sequence (C), and (4) successfully amplified probes are purified and linearly amplified by in vitro transcription (IVT). (5) Purified IVT products are reverse transcribed (RT), (6) RNA is hydrolyzed, and finally (7) single-stranded DNA (ssDNA) is purified to obtain ready-to-use probes
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    iFISH implementation. a Scheme of iFISH4U. Pre-designed genome-wide databases of oligos (left) are used as input by the iFISH4U web interface (center) to select oligos within one or more user-specified genomic regions, based on the indicated features. Features 1–3 are used while designing single probes, whereas all the four features are used to design multiple probes on the same chromosome. The black dashed boxes indicate examples of probes within the same region of interest, with the same number of oligos (vertical bars), but suboptimal size (1), homogeneity (2), or centrality (3), whereas the orange box represents the probe of choice having optimal size, homogeneity and centrality. b Cumulative distribution of the distances between consecutive oligos in the human 40-mers database. c Median standard deviation (s.d.) of the distance between consecutive oligos, inside non-overlapping genomic windows of the indicated size, in the 40-mers database and OligoMiner (OM) hg19 databases. OMB, OM ‘Balance’. OMC, OM ‘Coverage’. OMS, OM ‘Stringent’. d Percentage of non-overlapping genomic windows of the indicated size, containing at least 96 oligos, in the 40-mers database and OM hg19 databases. e Scheme of oligos in iFISH probes. Each probe consists of n oligos differing in the T sequence. f Location of the 330 iFISH probes targeting all the human autosomes and chrX. Red dots, individually tested probes (see Fig 2a, b ). g Scheme of the pipeline used to produce iFISH probes. (1) Up to 12,000 oligos, corresponding to a maximum of 125 probes each containing 96 oligos, are synthesized on an array and then pooled together. (2) The oligo-pool is dispensed into n 96-well plates, depending on the total number of probes ( p ) and colors per probe ( c ). (3) In each well, the oligos corresponding to the same probe are selectively amplified using a probe-specific PCR primer pair that incorporates the T7 promoter sequence (T7) and color adapter sequence (C), and (4) successfully amplified probes are purified and linearly amplified by in vitro transcription (IVT). (5) Purified IVT products are reverse transcribed (RT), (6) RNA is hydrolyzed, and finally (7) single-stranded DNA (ssDNA) is purified to obtain ready-to-use probes

    Journal: Nature Communications

    Article Title: iFISH is a publically available resource enabling versatile DNA FISH to study genome architecture

    doi: 10.1038/s41467-019-09616-w

    Figure Lengend Snippet: iFISH implementation. a Scheme of iFISH4U. Pre-designed genome-wide databases of oligos (left) are used as input by the iFISH4U web interface (center) to select oligos within one or more user-specified genomic regions, based on the indicated features. Features 1–3 are used while designing single probes, whereas all the four features are used to design multiple probes on the same chromosome. The black dashed boxes indicate examples of probes within the same region of interest, with the same number of oligos (vertical bars), but suboptimal size (1), homogeneity (2), or centrality (3), whereas the orange box represents the probe of choice having optimal size, homogeneity and centrality. b Cumulative distribution of the distances between consecutive oligos in the human 40-mers database. c Median standard deviation (s.d.) of the distance between consecutive oligos, inside non-overlapping genomic windows of the indicated size, in the 40-mers database and OligoMiner (OM) hg19 databases. OMB, OM ‘Balance’. OMC, OM ‘Coverage’. OMS, OM ‘Stringent’. d Percentage of non-overlapping genomic windows of the indicated size, containing at least 96 oligos, in the 40-mers database and OM hg19 databases. e Scheme of oligos in iFISH probes. Each probe consists of n oligos differing in the T sequence. f Location of the 330 iFISH probes targeting all the human autosomes and chrX. Red dots, individually tested probes (see Fig 2a, b ). g Scheme of the pipeline used to produce iFISH probes. (1) Up to 12,000 oligos, corresponding to a maximum of 125 probes each containing 96 oligos, are synthesized on an array and then pooled together. (2) The oligo-pool is dispensed into n 96-well plates, depending on the total number of probes ( p ) and colors per probe ( c ). (3) In each well, the oligos corresponding to the same probe are selectively amplified using a probe-specific PCR primer pair that incorporates the T7 promoter sequence (T7) and color adapter sequence (C), and (4) successfully amplified probes are purified and linearly amplified by in vitro transcription (IVT). (5) Purified IVT products are reverse transcribed (RT), (6) RNA is hydrolyzed, and finally (7) single-stranded DNA (ssDNA) is purified to obtain ready-to-use probes

    Article Snippet: We then amplified the oligos in each well by real-time PCR using the SYBR Select Master Mix (Thermo Fisher Scientific, cat. no. 4472913).

    Techniques: Genome Wide, Standard Deviation, Sequencing, Synthesized, Amplification, Polymerase Chain Reaction, Purification, In Vitro

    Induction of SASP by activating the cytoplasmic DNA sensing pathway. a Senescent TIG-3 cells induced by oncogenic Ras expression (lane 2–4) were transfected with previously validated siRNA oligos indicated at the top of the panel for twice at 2 day intervals. These cells were then subjected to western blotting using antibodies shown right ( a ), RT-qPCR analysis of SASP factor gene expression ( b ), analysis of intracellular ROS levels ( c ) or immunofluorescence staining for markers of DNA damage (γ-H2AX [red], phosphor-Ser/Thr ATM/ATR (pST/Q) substrate [green] and 40,6-diamidino-2-phenylindole [blue]) on day 4 ( d ). The representative data from three independent experiments are shown. Tubulin was used as a loading control ( a ). For all graphs, error bars indicate mean ± standard deviation (s.d.) of triplicate measurements. (* P

    Journal: Nature Communications

    Article Title: Downregulation of cytoplasmic DNases is implicated in cytoplasmic DNA accumulation and SASP in senescent cells

    doi: 10.1038/s41467-018-03555-8

    Figure Lengend Snippet: Induction of SASP by activating the cytoplasmic DNA sensing pathway. a Senescent TIG-3 cells induced by oncogenic Ras expression (lane 2–4) were transfected with previously validated siRNA oligos indicated at the top of the panel for twice at 2 day intervals. These cells were then subjected to western blotting using antibodies shown right ( a ), RT-qPCR analysis of SASP factor gene expression ( b ), analysis of intracellular ROS levels ( c ) or immunofluorescence staining for markers of DNA damage (γ-H2AX [red], phosphor-Ser/Thr ATM/ATR (pST/Q) substrate [green] and 40,6-diamidino-2-phenylindole [blue]) on day 4 ( d ). The representative data from three independent experiments are shown. Tubulin was used as a loading control ( a ). For all graphs, error bars indicate mean ± standard deviation (s.d.) of triplicate measurements. (* P

    Article Snippet: RNAi was performed by the transfection of siRNA oligos using the Lipofectamine™ RNAiMAX transfection reagent (Thermo Fisher Scientific), according to the manufacturer’s instructions.

    Techniques: Expressing, Transfection, Western Blot, Quantitative RT-PCR, Immunofluorescence, Staining, Standard Deviation

    The knockdown of cytoplasmic DNases activates the IFN-β pathway. a – c Pre-senescent TIG-3 cells were subjected to transfection with indicated siRNA oligos twice (at 2 day intervals). These cells were then subjected to western blotting using antibodies shown right ( a ), isolation of cytoplasmic fraction followed by qPCR analysis of chromosomal DNA ( b ) or qPCR analysis of SASP factor gene expression ( c ). Tubulin was used as a loading control ( a ). The representative data from three independent experiments are shown. For all graphs, error bars indicate mean ± standard deviation (s.d.) of triplicate measurements. (** P

    Journal: Nature Communications

    Article Title: Downregulation of cytoplasmic DNases is implicated in cytoplasmic DNA accumulation and SASP in senescent cells

    doi: 10.1038/s41467-018-03555-8

    Figure Lengend Snippet: The knockdown of cytoplasmic DNases activates the IFN-β pathway. a – c Pre-senescent TIG-3 cells were subjected to transfection with indicated siRNA oligos twice (at 2 day intervals). These cells were then subjected to western blotting using antibodies shown right ( a ), isolation of cytoplasmic fraction followed by qPCR analysis of chromosomal DNA ( b ) or qPCR analysis of SASP factor gene expression ( c ). Tubulin was used as a loading control ( a ). The representative data from three independent experiments are shown. For all graphs, error bars indicate mean ± standard deviation (s.d.) of triplicate measurements. (** P

    Article Snippet: RNAi was performed by the transfection of siRNA oligos using the Lipofectamine™ RNAiMAX transfection reagent (Thermo Fisher Scientific), according to the manufacturer’s instructions.

    Techniques: Transfection, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Expressing, Standard Deviation

    Cx43 is degraded by autophagy following Dex treatment ( A ) Inhibition of lysosome activity by CLQ blocked Dex-induced Cx43 degradation. Total cellular proteins (TCPs) from MLO-Y4 cells co-stimulated with 10 −6 M Dex and 50μM CLQ for 24hrs were immunoblotted with antibody against Cx43. β-actin served as loading and normalization control. ( B ) The protein levels of Cx43 were quantified and expressed as a ratio against β-actin. ( C ) Attenuation of autophagy by Atg5 silencing blocks Dex-induced Cx43 degradation. TCPs form Atg5 siRNA transfected MLO-Y4 cells with/without Dex treatment for 24hrs were immunoblotted with antibodies against Cx43, Atg5 and LC3-I/II. β-actin served as loading and normalization control. The protein levels Cx43 ( D ) and Atg5 ( E ), were quantified and expressed as a ratio against β-actin. ( F ) The protein levels of lipidated LC3-II were expressed as a ratio against LC3-I. All data presented are representative of at least three independent experiments. *p

    Journal: Oncotarget

    Article Title: Glucocorticoid impairs cell-cell communication by autophagy-mediated degradation of connexin 43 in osteocytes

    doi: 10.18632/oncotarget.9034

    Figure Lengend Snippet: Cx43 is degraded by autophagy following Dex treatment ( A ) Inhibition of lysosome activity by CLQ blocked Dex-induced Cx43 degradation. Total cellular proteins (TCPs) from MLO-Y4 cells co-stimulated with 10 −6 M Dex and 50μM CLQ for 24hrs were immunoblotted with antibody against Cx43. β-actin served as loading and normalization control. ( B ) The protein levels of Cx43 were quantified and expressed as a ratio against β-actin. ( C ) Attenuation of autophagy by Atg5 silencing blocks Dex-induced Cx43 degradation. TCPs form Atg5 siRNA transfected MLO-Y4 cells with/without Dex treatment for 24hrs were immunoblotted with antibodies against Cx43, Atg5 and LC3-I/II. β-actin served as loading and normalization control. The protein levels Cx43 ( D ) and Atg5 ( E ), were quantified and expressed as a ratio against β-actin. ( F ) The protein levels of lipidated LC3-II were expressed as a ratio against LC3-I. All data presented are representative of at least three independent experiments. *p

    Article Snippet: Transfection MLO-Y4 cells (1×105 /well) were plated in type-I collagen coated six-well culture plates, and then transfected with various concentrations (12.5nM, 25nM, 50nM and 100nM) of Atg5 siRNA oligo cocktail (Stealth siRNAs: MSS247019, MSS247020, MSS247021, from Thermo Fisher® , USA) or negative control using Lipofectamine® 3000 Transfection Reagent (Invitrogen® , USA) following the manufacturer's protocol.

    Techniques: Inhibition, Activity Assay, Transfection

    RacGAP1 is required for pseudopod extension and invasion. (A) A2780 cells were transfected with control or RacGAP1-specific SMARTpool oligonucleotides, seeded onto CDMs, and stimulated with cRGDfV as indicated. Images were captured every 10 min using a 20× objective lens. Representative images are shown. Bar, 50 µm. (B) Pseudopod length ( n > 400/condition) was measured for all moving cells within the 20th frame. (C) A2780 cells were transfected as in A and seeded into inverted invasion assays after 16 h in the presence or absence of FN and cRGDfV as indicated. The yellow line indicates the level of invasion under control conditions. (D) A2780 cells stably expressing GFP or FLAG-RacGAP1 WT were transfected with control or RacGAP RNAi oligo #6, treated as in C, and seeded into inverted invasion assays in the presence of cRGDfV and FN. (E) MDA-MB-231 cells were transfected as in A and seeded into inverted invasion assays in the presence of FN. (F) H1299 cells stably expressing mutant p53 (273H) or control vector (VEC) were transfected as in A and seeded into inverted invasion assays in the presence of FN. Data represent means ± SEM from at least three independent experiments. *, P

    Journal: The Journal of Cell Biology

    Article Title: RCP-driven ?5?1 recycling suppresses Rac and promotes RhoA activity via the RacGAP1-IQGAP1 complex

    doi: 10.1083/jcb.201302041

    Figure Lengend Snippet: RacGAP1 is required for pseudopod extension and invasion. (A) A2780 cells were transfected with control or RacGAP1-specific SMARTpool oligonucleotides, seeded onto CDMs, and stimulated with cRGDfV as indicated. Images were captured every 10 min using a 20× objective lens. Representative images are shown. Bar, 50 µm. (B) Pseudopod length ( n > 400/condition) was measured for all moving cells within the 20th frame. (C) A2780 cells were transfected as in A and seeded into inverted invasion assays after 16 h in the presence or absence of FN and cRGDfV as indicated. The yellow line indicates the level of invasion under control conditions. (D) A2780 cells stably expressing GFP or FLAG-RacGAP1 WT were transfected with control or RacGAP RNAi oligo #6, treated as in C, and seeded into inverted invasion assays in the presence of cRGDfV and FN. (E) MDA-MB-231 cells were transfected as in A and seeded into inverted invasion assays in the presence of FN. (F) H1299 cells stably expressing mutant p53 (273H) or control vector (VEC) were transfected as in A and seeded into inverted invasion assays in the presence of FN. Data represent means ± SEM from at least three independent experiments. *, P

    Article Snippet: Plasmids and reagents RNAi oligonucleotides (oligo) were purchased from Thermo Fisher Scientific as follows: ON-TARGETplus nontargeting siRNA (single oligo or pool as appropriate); IQGAP1 #1 (5′-GAACGUGGCUUAUGAGUAC-3′); IQGAP1 #2 (J-004694-08); RacGAP1 (SMARTpool, oligo 6, 5′-GCGAAGUGCUCUGGAUGUU-3′; and oligo 8, 5′-GAAGUCACAUCUGCCUGUU-3′); Rac1 (SMARTpool or Rac1 #1, 5′-CGGCACCACUGUCCCAACA-3′); RhoA (SMARTpool or RhoA #1, 5′-AUGGAAAGCAGGUAGAGUU-3′); and RCP (J-015968-10). shRNA vectors for PKB/Akt isoforms were prepared using mU6Pro and the following sequences: Akt1 #1, 5′-GCTACTTCCTCCTCAAGAA-3′; Akt1 #2, 5′-CGAGTTTGAGTACCTGAAG-3′; Akt2 #1, 5′-CGTGGTGAATACATCAAGA-3′; and Akt2# 2, 5′-TCTGTCATCAAAGAAGGCT-3′.

    Techniques: Transfection, Stable Transfection, Expressing, Multiple Displacement Amplification, Mutagenesis, Plasmid Preparation

    Integrin trafficking suppresses Rac activity and activates RhoA through the RacGAP1–IQGAP1 complex. (A) A2780 cells were subjected to control or RacGAP1 oligo #6 RNAi and allowed to recover for 24 h. Cells were then transfected with Raichu-Rac or Raichu-RhoA as indicated and seeded onto CDM. FLIM was performed, and FRET efficiency at the cell front was calculated as in Fig. 6 (A–C ; n ≥ 15/condition). (B) A2780 cells were subjected to control or IQGAP1 oligo #1 RNAi and allowed to recover for 24 h. Cells were then transfected with Raichu-Rac or Raichu-RhoA as indicated and seeded onto the CDM. FLIM was performed, and FRET efficiency at the cell front was calculated as in Fig. 6 (A–C ; n ≥ 8/condition). (C) A2780 cells stably expressing RacGAP1 WT , RacGAP1 249A , or RacGAP1 249D were transfected with Raichu-Rac and seeded onto CDMs. FLIM was performed, and FRET efficiency at the cell front was calculated as in Fig. 6 (A–C) . Representative images are shown ( n ≥ 8/condition). (D) A2780 cells stably expressing RacGAP1 WT , RacGAP1 249A , or RacGAP1 249D were transfected with Raichu-RhoA and seeded onto CDMs. FLIM was performed, and FRET efficiency at the cell front was calculated as in Fig. 6 (A–C) . Representative images are shown ( n ≥ 4/condition). Zoomed insets correspond to areas indicated by dotted ROIs. Yellow lines represent the baseline activity as determined by an inactive mutant of the probe. Data represent means ± SEM from at least three independent experiments. *, P

    Journal: The Journal of Cell Biology

    Article Title: RCP-driven ?5?1 recycling suppresses Rac and promotes RhoA activity via the RacGAP1-IQGAP1 complex

    doi: 10.1083/jcb.201302041

    Figure Lengend Snippet: Integrin trafficking suppresses Rac activity and activates RhoA through the RacGAP1–IQGAP1 complex. (A) A2780 cells were subjected to control or RacGAP1 oligo #6 RNAi and allowed to recover for 24 h. Cells were then transfected with Raichu-Rac or Raichu-RhoA as indicated and seeded onto CDM. FLIM was performed, and FRET efficiency at the cell front was calculated as in Fig. 6 (A–C ; n ≥ 15/condition). (B) A2780 cells were subjected to control or IQGAP1 oligo #1 RNAi and allowed to recover for 24 h. Cells were then transfected with Raichu-Rac or Raichu-RhoA as indicated and seeded onto the CDM. FLIM was performed, and FRET efficiency at the cell front was calculated as in Fig. 6 (A–C ; n ≥ 8/condition). (C) A2780 cells stably expressing RacGAP1 WT , RacGAP1 249A , or RacGAP1 249D were transfected with Raichu-Rac and seeded onto CDMs. FLIM was performed, and FRET efficiency at the cell front was calculated as in Fig. 6 (A–C) . Representative images are shown ( n ≥ 8/condition). (D) A2780 cells stably expressing RacGAP1 WT , RacGAP1 249A , or RacGAP1 249D were transfected with Raichu-RhoA and seeded onto CDMs. FLIM was performed, and FRET efficiency at the cell front was calculated as in Fig. 6 (A–C) . Representative images are shown ( n ≥ 4/condition). Zoomed insets correspond to areas indicated by dotted ROIs. Yellow lines represent the baseline activity as determined by an inactive mutant of the probe. Data represent means ± SEM from at least three independent experiments. *, P

    Article Snippet: Plasmids and reagents RNAi oligonucleotides (oligo) were purchased from Thermo Fisher Scientific as follows: ON-TARGETplus nontargeting siRNA (single oligo or pool as appropriate); IQGAP1 #1 (5′-GAACGUGGCUUAUGAGUAC-3′); IQGAP1 #2 (J-004694-08); RacGAP1 (SMARTpool, oligo 6, 5′-GCGAAGUGCUCUGGAUGUU-3′; and oligo 8, 5′-GAAGUCACAUCUGCCUGUU-3′); Rac1 (SMARTpool or Rac1 #1, 5′-CGGCACCACUGUCCCAACA-3′); RhoA (SMARTpool or RhoA #1, 5′-AUGGAAAGCAGGUAGAGUU-3′); and RCP (J-015968-10). shRNA vectors for PKB/Akt isoforms were prepared using mU6Pro and the following sequences: Akt1 #1, 5′-GCTACTTCCTCCTCAAGAA-3′; Akt1 #2, 5′-CGAGTTTGAGTACCTGAAG-3′; Akt2 #1, 5′-CGTGGTGAATACATCAAGA-3′; and Akt2# 2, 5′-TCTGTCATCAAAGAAGGCT-3′.

    Techniques: Activity Assay, Transfection, Stable Transfection, Expressing, Mutagenesis