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  • 99
    Integrated DNA Technologies dna oligonucleotides
    Method to generate a Random Deletion Library in HIV-1 A. Overview schematic of method to create a barcoded random deletion library: ( 1 ) Transposon cassettes harboring unique restriction sites are inserted into plasmids via in vitro transposition. ( 2 ) Transposons are excised to linearize the insertion library with a meganuclease. ( 3 ) Deletions are performed by chewback from both <t>DNA</t> termini by simultaneous treatment with enzyme blend. Mean deletion size is modulated by adjusting duration of chewback. ( 4 ) The chewed termini are end-repaired, dA-tailed, then joined by ligation to a T-tailed 60bp unique barcode cassette. B. Schematic of the “TN5MK” synthetic meganuclease transposon cassette used in library construction: TN5MK is composed of an antibiotic resistance gene, neomycin phosphotransferase I (npt), flanked by meganuclease restriction sites for I-SceI and I-CeuI and Tn5 mosaic ends (gray triangles) at the termini. The transposon cassette also contains a unique internal BamHI recognition site. C. The HIV-1 molecular clone pNL4-3, is a 14825 bp plasmid harboring the 9709 bp NL4-3 provirus (HIV-1 subtype B). NL4-3 is a chimera of two viruses (NY5 and LAV). D.Library insertion, excision, barcoding details: Circular DNA ( 1 ) is linearized by digestion with a meganuclease (I-SceI or I-CeuI), which cleaves at recognition sites encoded on the inserted transposon. This creates linear DNA with 4 base 3’ overhangs ( 2 ). Deletions are created by bidirectional chewback. Treatment with two exonucleases (T4 and RecJf) creates a population of truncated deletion mutants with ragged ends ( 3 ). Ragged DNA ends are blunted and then prepared for barcode cassette ligation by 5’ dephosphorylation and addition of a single 3’-dA ( 4 ). Deletion mutants are re-ligated in presence of a barcode cassette with single 3’-dT overhangs and 5’ phosphoryl groups to create barcoded circular DNAs with 2 nicks separated by 60 bp ( 5 ). E. Insertion libraries following I-SceI (S) or I-CeuI (C) digestion. Digestion of pNL43 insertion library shows excisions of the TN5MK transposon (1.4kb) and upward shift of the supercoiled library vs. the undigested library. Lanes: (M) 2 log DNA ladder, ( 1 ) undigested insertion library, ( 2 ) I-SceI digested insertion library, ( 3 ) I-CeuI digested insertion library. F. Location of TN5MK insertions for a subset of 7559 transposon integrations (3844 were unique). G.Determination of enzymatic chewback rate for deletion size: The chewback rate was determined by treating a 4 kb fragment of linear <t>dsDNA</t> with RecJf and T4 exonucleases in the presence of SSB and no dNTPs for increasing amounts of time, then halting enzymatic activity. Reactions were performed in triplicate. DNA concentrations were established by quantifying fluorescence of PicoGreen in a plate reader in comparison to a dsDNA standard of known concentration. H.Validation of Deletion Library: The pNL4-3 insertion library and pNL4-3 deletion library were either not digested (Ø) or cut with I-CeuI (C) and then subjected to binary treatment with RecBCD, which digests linear DNA to completion. Lanes 1-4 are the pNL43 insertion library and Lanes 5-8 are the pNL43 deletion library. I.pNL4-31 is composed of 23,851 tagged mutants with a range of deletion sizes. The right-skewed (i.e. right-tailed) histogram of deletion sizes in pNL4-31, with bins of 100 bp (shown in blue) is well-fit by a Gamma distribution (green, broken-line). Inset: Number of deletions detected within each region of the HIV genome. J.Deletion Depth Profile over the full HIV-1 genome. Calculation of the deletion depth profile of the pNL4-31 genome indicates that each base is covered by hundreds to thousands of deletion mutants. Two regions where deletions are not tolerated in the plasmid backbone are ori, the origin of replication and bla, -lactamase, the resistance marker
    Dna Oligonucleotides, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 99/100, based on 2965 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs nebnext multiplex oligos for illumina
    The newly developed mTSS-Capture method combined with paired-end sequencing maps genome-wide nucleosome distribution in primary patient samples and identifies bona fide nucleosome characteristics, concordant with other human nucleosome mapping studies A . Work-flow of the mTSS-seq method. Following MNase digestion using a titration of MNase, populations of mononucleosomally protected DNA and subnucleosomal fragments are isolated, and prepared as libraries for <t>Illumina</t> sequencing. Solution-based sequence capture is performed using biotinylated <t>oligos,</t> enabling the enrichment of fragments within 2kb of each transcription start site in the human genome. Paired-end 50bp sequencing was then performed on each index. B . Alignment of the mTSS-seq midpoints to the human genome using the UCSC genome browser for LAC patient #4137 Normal tissue is shown for chr11, hg19 ( http://genome.ucsc.edu ). Zooming in twice at 100X allows for further visualization of the sequence capture oligos surrounding the TSS in a 500kb and a 5kb region showing the ATM locus. C . Averaged, normalized reads per million (y-axis) from mTSS-seq plotted as fragments (gray) and midpoints (black), centered on and surrounding 2kb of the TSS for ~22,000 open reading frames in hg19 (x-axis). DNase I-hypersensitivity (GSM736580; green) and RNA polymerase II from ChIP-seq (GSM935299; blue) data from A549 cells are shown. (D) LAC patient 4137 Normal nucleosomal midpoints (blue track) were plotted in the UCSC genome browser against the published human lymphocyte nucleosome distribution maps by Gaffney et. al. (green track) for the ZNF451 and CCDC97 loci. Sequence capture oligos and corresponding RefSeq gene models are shown for each locus. Correlations are shown for ZNF451 and CCDC87, respectively.
    Nebnext Multiplex Oligos For Illumina, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1655 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher oligo
    RT-PCR confirmation of antisense transcription. a) A graphic representation of um02794 transcription. The grey line represents the genomic sequence (middle), the blue arrow represents predicted gene structure, and the red (top) and green (bottom) arrows represent sense and anti-sense ESTs respectively. The range of the genome coordinates was included. b) Detecting antisense transcripts corresponding to um02794 via strand specific RT-PCR. In lanes 2 to 5 first strand synthesis was carried out on <t>RNA</t> of CM grown haploid cells. In lanes 6 to 9 first strand synthesis was carried out on RNA of MN grown haploid cells. First synthesis reactions of lanes 2 and 6 were prepared using sense strand specific primers; lanes 3 and 7 anti sense specific primers; lane 4 and 8 <t>oligo</t> dT and lanes 5 and 9 DEPC-treated water. Lane 10 used genomic DNA from U. maydis strain 521 and lane 11 used water a PCR template. Lane 1 and 12: Full Ranger DNA ladder.
    Oligo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 38400 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Promega oligo
    RT-PCR confirmation of antisense transcription. a) A graphic representation of um02794 transcription. The grey line represents the genomic sequence (middle), the blue arrow represents predicted gene structure, and the red (top) and green (bottom) arrows represent sense and anti-sense ESTs respectively. The range of the genome coordinates was included. b) Detecting antisense transcripts corresponding to um02794 via strand specific RT-PCR. In lanes 2 to 5 first strand synthesis was carried out on <t>RNA</t> of CM grown haploid cells. In lanes 6 to 9 first strand synthesis was carried out on RNA of MN grown haploid cells. First synthesis reactions of lanes 2 and 6 were prepared using sense strand specific primers; lanes 3 and 7 anti sense specific primers; lane 4 and 8 <t>oligo</t> dT and lanes 5 and 9 DEPC-treated water. Lane 10 used genomic DNA from U. maydis strain 521 and lane 11 used water a PCR template. Lane 1 and 12: Full Ranger DNA ladder.
    Oligo, supplied by Promega, used in various techniques. Bioz Stars score: 94/100, based on 9214 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega oligo dt primer
    USP21 modulates Hh signaling and Gli1 transcriptional activity. (A) NIH3T3 cells were treated with USP21-targeting siRNA oligonucleotides (#10, #11, #12) or a non-targeting control (NT3), serum-starved and then incubated with SAG (100 nM) for 4 h before mRNA extraction. USP21 and Gli1 mRNA levels were measured by performing quantitative reverse-transcription PCR and normalised to <t>RNA</t> levels of Pol2 ( n =3 independent experiments, mean±s.d.). x -axis labels given in bottom panel also apply to the top panel. (B) HEK293T cells were treated with USP21-targeting siRNA oligonucleotides (#5, #6) or non-targeting control <t>oligo</t> (NT3), or were treated with transfection reagent alone (Mock). 24 h before lysis, cells were transfected with Gli-responsive firefly luciferase and Renilla luciferase reporters (pGLB3B-12Gli-Luc and pRL-Renilla-TK), as well as with a Gli1 expression construct (HA–Gli1). Efficiency of the knockdown was assessed by western blotting ( n =2, bars show the range). (C) HEK293T cells were transfected with Gli-responsive firefly luciferase, Renilla luciferase reporters, HA–Gli1 and the indicated amounts of GFP, GFP–USP21 or GFP–USP21CS plasmids. Protein expression was assessed by western blotting (IB, immunoblot) for GFP. (GFP, n =5; USP21 and USP21CS, n =6; mean±s.d.; one-way ANOVA and Dunnett's test, ** P
    Oligo Dt Primer, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 4100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Thermo Fisher oligo d
    USP21 modulates Hh signaling and Gli1 transcriptional activity. (A) NIH3T3 cells were treated with USP21-targeting siRNA oligonucleotides (#10, #11, #12) or a non-targeting control (NT3), serum-starved and then incubated with SAG (100 nM) for 4 h before mRNA extraction. USP21 and Gli1 mRNA levels were measured by performing quantitative reverse-transcription PCR and normalised to <t>RNA</t> levels of Pol2 ( n =3 independent experiments, mean±s.d.). x -axis labels given in bottom panel also apply to the top panel. (B) HEK293T cells were treated with USP21-targeting siRNA oligonucleotides (#5, #6) or non-targeting control <t>oligo</t> (NT3), or were treated with transfection reagent alone (Mock). 24 h before lysis, cells were transfected with Gli-responsive firefly luciferase and Renilla luciferase reporters (pGLB3B-12Gli-Luc and pRL-Renilla-TK), as well as with a Gli1 expression construct (HA–Gli1). Efficiency of the knockdown was assessed by western blotting ( n =2, bars show the range). (C) HEK293T cells were transfected with Gli-responsive firefly luciferase, Renilla luciferase reporters, HA–Gli1 and the indicated amounts of GFP, GFP–USP21 or GFP–USP21CS plasmids. Protein expression was assessed by western blotting (IB, immunoblot) for GFP. (GFP, n =5; USP21 and USP21CS, n =6; mean±s.d.; one-way ANOVA and Dunnett's test, ** P
    Oligo D, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 2342 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs nebnext multiplex oligos
    USP21 modulates Hh signaling and Gli1 transcriptional activity. (A) NIH3T3 cells were treated with USP21-targeting siRNA oligonucleotides (#10, #11, #12) or a non-targeting control (NT3), serum-starved and then incubated with SAG (100 nM) for 4 h before mRNA extraction. USP21 and Gli1 mRNA levels were measured by performing quantitative reverse-transcription PCR and normalised to <t>RNA</t> levels of Pol2 ( n =3 independent experiments, mean±s.d.). x -axis labels given in bottom panel also apply to the top panel. (B) HEK293T cells were treated with USP21-targeting siRNA oligonucleotides (#5, #6) or non-targeting control <t>oligo</t> (NT3), or were treated with transfection reagent alone (Mock). 24 h before lysis, cells were transfected with Gli-responsive firefly luciferase and Renilla luciferase reporters (pGLB3B-12Gli-Luc and pRL-Renilla-TK), as well as with a Gli1 expression construct (HA–Gli1). Efficiency of the knockdown was assessed by western blotting ( n =2, bars show the range). (C) HEK293T cells were transfected with Gli-responsive firefly luciferase, Renilla luciferase reporters, HA–Gli1 and the indicated amounts of GFP, GFP–USP21 or GFP–USP21CS plasmids. Protein expression was assessed by western blotting (IB, immunoblot) for GFP. (GFP, n =5; USP21 and USP21CS, n =6; mean±s.d.; one-way ANOVA and Dunnett's test, ** P
    Nebnext Multiplex Oligos, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4661 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega oligo dt primers
    p120 308 is a null allele. (A) p120 308 deletes the entire p120 coding region, but does not affect other genes. Schematic as in Fig. 1 A. Genomic DNA from single wild-type or homozygous mutant flies was PCR amplified using primer pairs from the indicated regions between LD05623 and CG17486 . (B) The p120 mutants are mRNA nulls. <t>cDNA</t> generated from <t>oligo-dT-primed</t> total RNA from p120 mutants and wild-type was amplified with primers spanning the p120 third intron. An unrelated gene, CG2905 , is a control. A DNA control confirmed we were examining mRNA.
    Oligo Dt Primers, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 4572 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs oligos
    Toeprint analyses of translation initiation complexes on <t>atpA</t> and atpA – gfp RNAs. In vitro transcribed atpA – atpA ( left panels ) and atpA – gfp ( right panels ) RNAs (20 nM) were extended from [γ- 32 P] ATP-labeled <t>oligos</t> that anneal 120-nt downstream of the initiator AUGs, either alone or in the presence of tRNA Met , 30S subunit or both. Sequencing reactions were run alongside. The treatments are indicated at the top of each panel. The positions of the toeprints respective to the initiator AUG (+1) are indicated with lines on the side. a Toeprint reactions using 100-nM 30S subunit, b Inset showing the toeprint area of experiments similar to ( a ) with 200-nM 30S subunit. The figure shows representative autoradiographs obtained from three independent experiments
    Oligos, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1801 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore oligo
    Toeprint analyses of translation initiation complexes on <t>atpA</t> and atpA – gfp RNAs. In vitro transcribed atpA – atpA ( left panels ) and atpA – gfp ( right panels ) RNAs (20 nM) were extended from [γ- 32 P] ATP-labeled <t>oligos</t> that anneal 120-nt downstream of the initiator AUGs, either alone or in the presence of tRNA Met , 30S subunit or both. Sequencing reactions were run alongside. The treatments are indicated at the top of each panel. The positions of the toeprints respective to the initiator AUG (+1) are indicated with lines on the side. a Toeprint reactions using 100-nM 30S subunit, b Inset showing the toeprint area of experiments similar to ( a ) with 200-nM 30S subunit. The figure shows representative autoradiographs obtained from three independent experiments
    Oligo, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1764 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen oligo dt primers
    KLF2 and SMAD7 genes were upregulated by stimulation with SCF or NGF . A: HMC-1 (V560G c-Kit) cells were either incubated with serum (serum) or in serum free medium for 17 h, then treated without (-) or with (+) imatinib (5 μM) for 4 h prior to stimulation without (-) or with SCF (100 ng/ml) or NGF (100 ng/ml) for 30 (30) and 120 min (120). Total RNA was isolated and, after reverse transcription with <t>Oligo</t> dT-primers, cDNA was analyzed by semi-quantitative <t>PCR</t> for expression of EGR1, KLF2, SMAD7 and actin (ACTB). Water (H 2 O) and RNA (RNA) serve as negative controls. bp: base pairs of the DNA marker. B: Expression of KLF2 was quantified using TaqMan-PCR as in figure 3. Relative expression levels compared to GUSB were normalized to gene expression in serum-starved HMC-1(V560G c-Kit) cells. Average values from six independent PCR reactions + SEM are shown.
    Oligo Dt Primers, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1677 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Agilent technologies whole human genome oligo microarray
    TOP1MT has a wide expression range in the NCI-60 panel. ( A ) TOP1MT mRNA expression intensity across the cell line panel from the NCI (NCI-60), ranked from the highest TOP1MT expresser (leukemia RPMI-8226, top) to the lowest (lung NCI-H226, bottom). Tissues of origin are designated by colored circles (see legend below). Names of the individual cell lines are reported. Expression values were obtained with Agilent Whole Human Genome <t>Oligo</t> <t>Microarray</t> chips, and quality filtering and normalization were performed using GeneSpring GX 10.0 software (Agilent, Santa Clara, CA, USA). A value of one corresponds to a 2-fold higher intensity than the 75th percentile of expression for all probes on the entire array. Values are on a log 2 scale. ( B ) Normalized TOP1MT mRNA expressions sorted by level (highest to lowest) across the NCI-60. Individual cell lines (filled dots) are colored according to tissue of origin and arranged by the intensity values in panel A. Inset: frequency distribution plot showing the number of cell lines from the NCI-60 panel by TOP1MT normalized intensity. x -axis, normalized TOP1MT intensity; y -axis, frequency of cell lines for any given TOP1MT intensity in the x -axis.
    Whole Human Genome Oligo Microarray, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 1382 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Integrated DNA Technologies rna oligonucleotides
    Multi-trigger systems can be composed in which each <t>RNA/DNA</t> hybrid contains a responsive DNA structural element. ( A ) A system comprising a 3-input AND gate and a NOT gate can be constructed by pairing sH ^CTGF.20/8 (activated by the connective tissue growth factor (CTGF) derived trigger) with aH ∨ KRAS (repressed by the Kirsten rat sarcoma proto-oncogene (KRAS) mRNA derived trigger). Co-incubation of the two hybrids results in no interaction. Both hybrids and the CTGF trigger are required for dsRNA release, while the presence of the KRAS trigger will inhibit strand exchange. ( B ) The multi-trigger system was assessed by 10% acrylamide non-denaturing PAGE. The fraction of DsiRNA released is indicated in the gel depicted, in the presence of indicated trigger combinations following 30 min incubation at 37 °C. The sH and aH hybrid were present at equimolar concentration, while the triggers were added at a 2-fold or 3-fold excess, as indicated. In samples when both triggers are present, they were added to premixed hybrids sequentially (KRAS followed by CTGF). The antisense hybrid and DsiRNA control in were assembled using a 5′-AlexaFluor546 labeled antisense RNA strand for the purpose of visualization and quantification.
    Rna Oligonucleotides, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 99/100, based on 655 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Method to generate a Random Deletion Library in HIV-1 A. Overview schematic of method to create a barcoded random deletion library: ( 1 ) Transposon cassettes harboring unique restriction sites are inserted into plasmids via in vitro transposition. ( 2 ) Transposons are excised to linearize the insertion library with a meganuclease. ( 3 ) Deletions are performed by chewback from both DNA termini by simultaneous treatment with enzyme blend. Mean deletion size is modulated by adjusting duration of chewback. ( 4 ) The chewed termini are end-repaired, dA-tailed, then joined by ligation to a T-tailed 60bp unique barcode cassette. B. Schematic of the “TN5MK” synthetic meganuclease transposon cassette used in library construction: TN5MK is composed of an antibiotic resistance gene, neomycin phosphotransferase I (npt), flanked by meganuclease restriction sites for I-SceI and I-CeuI and Tn5 mosaic ends (gray triangles) at the termini. The transposon cassette also contains a unique internal BamHI recognition site. C. The HIV-1 molecular clone pNL4-3, is a 14825 bp plasmid harboring the 9709 bp NL4-3 provirus (HIV-1 subtype B). NL4-3 is a chimera of two viruses (NY5 and LAV). D.Library insertion, excision, barcoding details: Circular DNA ( 1 ) is linearized by digestion with a meganuclease (I-SceI or I-CeuI), which cleaves at recognition sites encoded on the inserted transposon. This creates linear DNA with 4 base 3’ overhangs ( 2 ). Deletions are created by bidirectional chewback. Treatment with two exonucleases (T4 and RecJf) creates a population of truncated deletion mutants with ragged ends ( 3 ). Ragged DNA ends are blunted and then prepared for barcode cassette ligation by 5’ dephosphorylation and addition of a single 3’-dA ( 4 ). Deletion mutants are re-ligated in presence of a barcode cassette with single 3’-dT overhangs and 5’ phosphoryl groups to create barcoded circular DNAs with 2 nicks separated by 60 bp ( 5 ). E. Insertion libraries following I-SceI (S) or I-CeuI (C) digestion. Digestion of pNL43 insertion library shows excisions of the TN5MK transposon (1.4kb) and upward shift of the supercoiled library vs. the undigested library. Lanes: (M) 2 log DNA ladder, ( 1 ) undigested insertion library, ( 2 ) I-SceI digested insertion library, ( 3 ) I-CeuI digested insertion library. F. Location of TN5MK insertions for a subset of 7559 transposon integrations (3844 were unique). G.Determination of enzymatic chewback rate for deletion size: The chewback rate was determined by treating a 4 kb fragment of linear dsDNA with RecJf and T4 exonucleases in the presence of SSB and no dNTPs for increasing amounts of time, then halting enzymatic activity. Reactions were performed in triplicate. DNA concentrations were established by quantifying fluorescence of PicoGreen in a plate reader in comparison to a dsDNA standard of known concentration. H.Validation of Deletion Library: The pNL4-3 insertion library and pNL4-3 deletion library were either not digested (Ø) or cut with I-CeuI (C) and then subjected to binary treatment with RecBCD, which digests linear DNA to completion. Lanes 1-4 are the pNL43 insertion library and Lanes 5-8 are the pNL43 deletion library. I.pNL4-31 is composed of 23,851 tagged mutants with a range of deletion sizes. The right-skewed (i.e. right-tailed) histogram of deletion sizes in pNL4-31, with bins of 100 bp (shown in blue) is well-fit by a Gamma distribution (green, broken-line). Inset: Number of deletions detected within each region of the HIV genome. J.Deletion Depth Profile over the full HIV-1 genome. Calculation of the deletion depth profile of the pNL4-31 genome indicates that each base is covered by hundreds to thousands of deletion mutants. Two regions where deletions are not tolerated in the plasmid backbone are ori, the origin of replication and bla, -lactamase, the resistance marker

    Journal: bioRxiv

    Article Title: RanDeL-seq: A high-throughput method to map viral cis- and trans-acting elements

    doi: 10.1101/2020.07.01.183574

    Figure Lengend Snippet: Method to generate a Random Deletion Library in HIV-1 A. Overview schematic of method to create a barcoded random deletion library: ( 1 ) Transposon cassettes harboring unique restriction sites are inserted into plasmids via in vitro transposition. ( 2 ) Transposons are excised to linearize the insertion library with a meganuclease. ( 3 ) Deletions are performed by chewback from both DNA termini by simultaneous treatment with enzyme blend. Mean deletion size is modulated by adjusting duration of chewback. ( 4 ) The chewed termini are end-repaired, dA-tailed, then joined by ligation to a T-tailed 60bp unique barcode cassette. B. Schematic of the “TN5MK” synthetic meganuclease transposon cassette used in library construction: TN5MK is composed of an antibiotic resistance gene, neomycin phosphotransferase I (npt), flanked by meganuclease restriction sites for I-SceI and I-CeuI and Tn5 mosaic ends (gray triangles) at the termini. The transposon cassette also contains a unique internal BamHI recognition site. C. The HIV-1 molecular clone pNL4-3, is a 14825 bp plasmid harboring the 9709 bp NL4-3 provirus (HIV-1 subtype B). NL4-3 is a chimera of two viruses (NY5 and LAV). D.Library insertion, excision, barcoding details: Circular DNA ( 1 ) is linearized by digestion with a meganuclease (I-SceI or I-CeuI), which cleaves at recognition sites encoded on the inserted transposon. This creates linear DNA with 4 base 3’ overhangs ( 2 ). Deletions are created by bidirectional chewback. Treatment with two exonucleases (T4 and RecJf) creates a population of truncated deletion mutants with ragged ends ( 3 ). Ragged DNA ends are blunted and then prepared for barcode cassette ligation by 5’ dephosphorylation and addition of a single 3’-dA ( 4 ). Deletion mutants are re-ligated in presence of a barcode cassette with single 3’-dT overhangs and 5’ phosphoryl groups to create barcoded circular DNAs with 2 nicks separated by 60 bp ( 5 ). E. Insertion libraries following I-SceI (S) or I-CeuI (C) digestion. Digestion of pNL43 insertion library shows excisions of the TN5MK transposon (1.4kb) and upward shift of the supercoiled library vs. the undigested library. Lanes: (M) 2 log DNA ladder, ( 1 ) undigested insertion library, ( 2 ) I-SceI digested insertion library, ( 3 ) I-CeuI digested insertion library. F. Location of TN5MK insertions for a subset of 7559 transposon integrations (3844 were unique). G.Determination of enzymatic chewback rate for deletion size: The chewback rate was determined by treating a 4 kb fragment of linear dsDNA with RecJf and T4 exonucleases in the presence of SSB and no dNTPs for increasing amounts of time, then halting enzymatic activity. Reactions were performed in triplicate. DNA concentrations were established by quantifying fluorescence of PicoGreen in a plate reader in comparison to a dsDNA standard of known concentration. H.Validation of Deletion Library: The pNL4-3 insertion library and pNL4-3 deletion library were either not digested (Ø) or cut with I-CeuI (C) and then subjected to binary treatment with RecBCD, which digests linear DNA to completion. Lanes 1-4 are the pNL43 insertion library and Lanes 5-8 are the pNL43 deletion library. I.pNL4-31 is composed of 23,851 tagged mutants with a range of deletion sizes. The right-skewed (i.e. right-tailed) histogram of deletion sizes in pNL4-31, with bins of 100 bp (shown in blue) is well-fit by a Gamma distribution (green, broken-line). Inset: Number of deletions detected within each region of the HIV genome. J.Deletion Depth Profile over the full HIV-1 genome. Calculation of the deletion depth profile of the pNL4-31 genome indicates that each base is covered by hundreds to thousands of deletion mutants. Two regions where deletions are not tolerated in the plasmid backbone are ori, the origin of replication and bla, -lactamase, the resistance marker

    Article Snippet: DNA oligonucleotides and synthetic dsDNA were obtained from Integrated DNA Technologies (Coralville, IA, USA).

    Techniques: In Vitro, Ligation, Plasmid Preparation, De-Phosphorylation Assay, Activity Assay, Fluorescence, Concentration Assay, Marker

    Enhancement of T4 DNA ligase activity by supplemental oligonucleotides. (a) Unsuccessful 4-bp duplex reactions could be salvaged by utilizing a supplementary oligonucleotide, designed to complement the first oligonucleotide-dsDNA duplex but is unphosphorylated to prevent ligation of itself. Two hour ligation of the 4-bp reaction at 16°C supplemented with 3.33 μM of the hexamer, shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (b) Ligation reaction of an octamer supplemented with a second octamer in which one is used for ligation and the other is used to extend the duplex. A two hour ligation at 16°C of serial concentrations of the octamer with 3.33 μM of the supplementary octamer shows significant ligation (■) compared to reactions without the supplemental octamer (◆). (c) Unsuccessful 3-bp duplex reactions could be salvaged by utilizing a supplementary hexamer that hybridized at all six positions. A two hour ligation of the 3-bp reaction at 16°C with 3.33 μM supplementary hexamer shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (d) Ligation using a hexamer pair at 4°C for 16 hours shows limited improvement (■) compared to the unsupplemented (◆) control.

    Journal: BMC Research Notes

    Article Title: Efficient assembly of very short oligonucleotides using T4 DNA Ligase

    doi: 10.1186/1756-0500-3-291

    Figure Lengend Snippet: Enhancement of T4 DNA ligase activity by supplemental oligonucleotides. (a) Unsuccessful 4-bp duplex reactions could be salvaged by utilizing a supplementary oligonucleotide, designed to complement the first oligonucleotide-dsDNA duplex but is unphosphorylated to prevent ligation of itself. Two hour ligation of the 4-bp reaction at 16°C supplemented with 3.33 μM of the hexamer, shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (b) Ligation reaction of an octamer supplemented with a second octamer in which one is used for ligation and the other is used to extend the duplex. A two hour ligation at 16°C of serial concentrations of the octamer with 3.33 μM of the supplementary octamer shows significant ligation (■) compared to reactions without the supplemental octamer (◆). (c) Unsuccessful 3-bp duplex reactions could be salvaged by utilizing a supplementary hexamer that hybridized at all six positions. A two hour ligation of the 3-bp reaction at 16°C with 3.33 μM supplementary hexamer shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (d) Ligation using a hexamer pair at 4°C for 16 hours shows limited improvement (■) compared to the unsupplemented (◆) control.

    Article Snippet: Preparation of immobilized dsDNA All oligos, including those 5'-biotinylated, 3'-FAM6, and 5'-phosphorylated were synthesized by Integrated DNA Technologies (IDT Inc., IA, USA).

    Techniques: Activity Assay, Ligation

    Evaluation of minimal oligonucleotide substrate requirements for T4 DNA ligase. (a) Schematic diagram of an immobilized DNA strand used in ligation assays and DNA construction. M-270 Dynabeads (Invitrogen) are attached through a streptavidin-biotin linkage to the 5' end of a double stranded DNA. The free end is designed with a variable 5' overhang, complementary to labeled oligonucleotides used in ligation. An additional BbsI restriction site and a forward primer site are included in the case of DNA construction. (b) Increasing concentrations of 5'-phosphorylated, 3'-fluorescently labeled oligonucleotide are ligated to 5 pmoles of immobilized dsDNA with a complementary overhang. Reactions were performed for one hour at 16°C and washed with TE to remove unligated substrate. Successful ligation kinetics are observed at the 5-bp duplex length (▲), but no significant ligation occurs at lengths of 4-bp (■) or 3-bp (◆).

    Journal: BMC Research Notes

    Article Title: Efficient assembly of very short oligonucleotides using T4 DNA Ligase

    doi: 10.1186/1756-0500-3-291

    Figure Lengend Snippet: Evaluation of minimal oligonucleotide substrate requirements for T4 DNA ligase. (a) Schematic diagram of an immobilized DNA strand used in ligation assays and DNA construction. M-270 Dynabeads (Invitrogen) are attached through a streptavidin-biotin linkage to the 5' end of a double stranded DNA. The free end is designed with a variable 5' overhang, complementary to labeled oligonucleotides used in ligation. An additional BbsI restriction site and a forward primer site are included in the case of DNA construction. (b) Increasing concentrations of 5'-phosphorylated, 3'-fluorescently labeled oligonucleotide are ligated to 5 pmoles of immobilized dsDNA with a complementary overhang. Reactions were performed for one hour at 16°C and washed with TE to remove unligated substrate. Successful ligation kinetics are observed at the 5-bp duplex length (▲), but no significant ligation occurs at lengths of 4-bp (■) or 3-bp (◆).

    Article Snippet: Preparation of immobilized dsDNA All oligos, including those 5'-biotinylated, 3'-FAM6, and 5'-phosphorylated were synthesized by Integrated DNA Technologies (IDT Inc., IA, USA).

    Techniques: Ligation, Labeling

    The newly developed mTSS-Capture method combined with paired-end sequencing maps genome-wide nucleosome distribution in primary patient samples and identifies bona fide nucleosome characteristics, concordant with other human nucleosome mapping studies A . Work-flow of the mTSS-seq method. Following MNase digestion using a titration of MNase, populations of mononucleosomally protected DNA and subnucleosomal fragments are isolated, and prepared as libraries for Illumina sequencing. Solution-based sequence capture is performed using biotinylated oligos, enabling the enrichment of fragments within 2kb of each transcription start site in the human genome. Paired-end 50bp sequencing was then performed on each index. B . Alignment of the mTSS-seq midpoints to the human genome using the UCSC genome browser for LAC patient #4137 Normal tissue is shown for chr11, hg19 ( http://genome.ucsc.edu ). Zooming in twice at 100X allows for further visualization of the sequence capture oligos surrounding the TSS in a 500kb and a 5kb region showing the ATM locus. C . Averaged, normalized reads per million (y-axis) from mTSS-seq plotted as fragments (gray) and midpoints (black), centered on and surrounding 2kb of the TSS for ~22,000 open reading frames in hg19 (x-axis). DNase I-hypersensitivity (GSM736580; green) and RNA polymerase II from ChIP-seq (GSM935299; blue) data from A549 cells are shown. (D) LAC patient 4137 Normal nucleosomal midpoints (blue track) were plotted in the UCSC genome browser against the published human lymphocyte nucleosome distribution maps by Gaffney et. al. (green track) for the ZNF451 and CCDC97 loci. Sequence capture oligos and corresponding RefSeq gene models are shown for each locus. Correlations are shown for ZNF451 and CCDC87, respectively.

    Journal: Oncotarget

    Article Title: Comprehensive nucleosome mapping of the human genome in cancer progression

    doi: 10.18632/oncotarget.6811

    Figure Lengend Snippet: The newly developed mTSS-Capture method combined with paired-end sequencing maps genome-wide nucleosome distribution in primary patient samples and identifies bona fide nucleosome characteristics, concordant with other human nucleosome mapping studies A . Work-flow of the mTSS-seq method. Following MNase digestion using a titration of MNase, populations of mononucleosomally protected DNA and subnucleosomal fragments are isolated, and prepared as libraries for Illumina sequencing. Solution-based sequence capture is performed using biotinylated oligos, enabling the enrichment of fragments within 2kb of each transcription start site in the human genome. Paired-end 50bp sequencing was then performed on each index. B . Alignment of the mTSS-seq midpoints to the human genome using the UCSC genome browser for LAC patient #4137 Normal tissue is shown for chr11, hg19 ( http://genome.ucsc.edu ). Zooming in twice at 100X allows for further visualization of the sequence capture oligos surrounding the TSS in a 500kb and a 5kb region showing the ATM locus. C . Averaged, normalized reads per million (y-axis) from mTSS-seq plotted as fragments (gray) and midpoints (black), centered on and surrounding 2kb of the TSS for ~22,000 open reading frames in hg19 (x-axis). DNase I-hypersensitivity (GSM736580; green) and RNA polymerase II from ChIP-seq (GSM935299; blue) data from A549 cells are shown. (D) LAC patient 4137 Normal nucleosomal midpoints (blue track) were plotted in the UCSC genome browser against the published human lymphocyte nucleosome distribution maps by Gaffney et. al. (green track) for the ZNF451 and CCDC97 loci. Sequence capture oligos and corresponding RefSeq gene models are shown for each locus. Correlations are shown for ZNF451 and CCDC87, respectively.

    Article Snippet: Universal and indexed sequences were added through 8 cycles of PCR, using NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1, NEB #E7335S/L).

    Techniques: Sequencing, Genome Wide, Titration, Isolation, Chromatin Immunoprecipitation

    RT-PCR confirmation of antisense transcription. a) A graphic representation of um02794 transcription. The grey line represents the genomic sequence (middle), the blue arrow represents predicted gene structure, and the red (top) and green (bottom) arrows represent sense and anti-sense ESTs respectively. The range of the genome coordinates was included. b) Detecting antisense transcripts corresponding to um02794 via strand specific RT-PCR. In lanes 2 to 5 first strand synthesis was carried out on RNA of CM grown haploid cells. In lanes 6 to 9 first strand synthesis was carried out on RNA of MN grown haploid cells. First synthesis reactions of lanes 2 and 6 were prepared using sense strand specific primers; lanes 3 and 7 anti sense specific primers; lane 4 and 8 oligo dT and lanes 5 and 9 DEPC-treated water. Lane 10 used genomic DNA from U. maydis strain 521 and lane 11 used water a PCR template. Lane 1 and 12: Full Ranger DNA ladder.

    Journal: BMC Genomics

    Article Title: Gene discovery and transcript analyses in the corn smut pathogen Ustilago maydis: expressed sequence tag and genome sequence comparison

    doi: 10.1186/1471-2164-8-334

    Figure Lengend Snippet: RT-PCR confirmation of antisense transcription. a) A graphic representation of um02794 transcription. The grey line represents the genomic sequence (middle), the blue arrow represents predicted gene structure, and the red (top) and green (bottom) arrows represent sense and anti-sense ESTs respectively. The range of the genome coordinates was included. b) Detecting antisense transcripts corresponding to um02794 via strand specific RT-PCR. In lanes 2 to 5 first strand synthesis was carried out on RNA of CM grown haploid cells. In lanes 6 to 9 first strand synthesis was carried out on RNA of MN grown haploid cells. First synthesis reactions of lanes 2 and 6 were prepared using sense strand specific primers; lanes 3 and 7 anti sense specific primers; lane 4 and 8 oligo dT and lanes 5 and 9 DEPC-treated water. Lane 10 used genomic DNA from U. maydis strain 521 and lane 11 used water a PCR template. Lane 1 and 12: Full Ranger DNA ladder.

    Article Snippet: Reverse transcription was carried out on 5 μg of DNaseI treated RNA using Superscript III (Invitrogen) and oligo (dT)12–18 primers (Invitrogen).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing, Polymerase Chain Reaction

    Measurements of influenza transcription. HIV-1-infected and -uninfected macrophages were infected with influenza A(H1N1)pdm09. After 24h, cells were lysed, RNA extracted and cDNA synthetized using oligo.dT and UNI-12 primers. Quantitative real time PCR was performed for the M1 gene. As a control, ribavirin-treated and -untreated influenza-infected macrophages were submitted to the same procedure described above (inset). * P

    Journal: PLoS ONE

    Article Title: HIV-1 and Its gp120 Inhibits the Influenza A(H1N1)pdm09 Life Cycle in an IFITM3-Dependent Fashion

    doi: 10.1371/journal.pone.0101056

    Figure Lengend Snippet: Measurements of influenza transcription. HIV-1-infected and -uninfected macrophages were infected with influenza A(H1N1)pdm09. After 24h, cells were lysed, RNA extracted and cDNA synthetized using oligo.dT and UNI-12 primers. Quantitative real time PCR was performed for the M1 gene. As a control, ribavirin-treated and -untreated influenza-infected macrophages were submitted to the same procedure described above (inset). * P

    Article Snippet: CDNA was synthetized with SuperScript III (Life Technologies) using oligo.dT (Life Technologies) or UNI-12 (5′-AGCRAAAGCAGG-3′) as the primers for first-strand synthesis, for 1 h at 45°C.

    Techniques: Infection, Real-time Polymerase Chain Reaction

    Ferroportin is expressed in hPASMCs and regulated by hepcidin. Confluent hPASMCs were ( A ) either mock-treated or treated with 1 µg/mL hepcidin for 2.5 h and total RNA extracted using RNeasy kit, cDNA synthesised using oligo-dT primers and RT-PCR performed using SYBR green with human Fpn (ferroportin) primers and β-actin as housekeeping gene. The values were further normalised as fold changes to the control untreated cells at time zero. N = 4 ( B ) mock-treated for 24 h, cells lysed and total protein extracted, quantified by Bradford assay and 40 µg of protein separated on 10% SDS-PAGE and transferred onto nitrocellulose membranes. Western blotting was performed using Rabbit anti-Fpn as primary and Goat anti-rabbit IgG conjugated with horse-radish peroxidase as secondary antibodies. Human intestinal lysates (Abcam) were used as positive control. N = 3 ( C ) either mock-treated or treated with 1 µg/mL hepcidin for 24 h, cells lysed and total protein extracted. Fpn expression was quantitated using an ELISA kit (BlueGene Biotech) and normalised to total protein estimated by Bradford reagent. N = 4 ( D ) Confocal images of hPASMCs grown with (top panels) normal media or (bottom panels) treated with 1 µg/mL hepcidin for 20–22 h and immuno-stained with rabbit anti-Fpn antibody and goat anti-rabbit IgG secondary antibody tagged with Alexa-568. The cells were further counterstained with DAPI and images captured using Leica LSM 510 confocal microscope. Scale bar = 10 µm; N = 5. Student’s t test was performed; **p

    Journal: Scientific Reports

    Article Title: The Hepcidin/Ferroportin axis modulates proliferation of pulmonary artery smooth muscle cells

    doi: 10.1038/s41598-018-31095-0

    Figure Lengend Snippet: Ferroportin is expressed in hPASMCs and regulated by hepcidin. Confluent hPASMCs were ( A ) either mock-treated or treated with 1 µg/mL hepcidin for 2.5 h and total RNA extracted using RNeasy kit, cDNA synthesised using oligo-dT primers and RT-PCR performed using SYBR green with human Fpn (ferroportin) primers and β-actin as housekeeping gene. The values were further normalised as fold changes to the control untreated cells at time zero. N = 4 ( B ) mock-treated for 24 h, cells lysed and total protein extracted, quantified by Bradford assay and 40 µg of protein separated on 10% SDS-PAGE and transferred onto nitrocellulose membranes. Western blotting was performed using Rabbit anti-Fpn as primary and Goat anti-rabbit IgG conjugated with horse-radish peroxidase as secondary antibodies. Human intestinal lysates (Abcam) were used as positive control. N = 3 ( C ) either mock-treated or treated with 1 µg/mL hepcidin for 24 h, cells lysed and total protein extracted. Fpn expression was quantitated using an ELISA kit (BlueGene Biotech) and normalised to total protein estimated by Bradford reagent. N = 4 ( D ) Confocal images of hPASMCs grown with (top panels) normal media or (bottom panels) treated with 1 µg/mL hepcidin for 20–22 h and immuno-stained with rabbit anti-Fpn antibody and goat anti-rabbit IgG secondary antibody tagged with Alexa-568. The cells were further counterstained with DAPI and images captured using Leica LSM 510 confocal microscope. Scale bar = 10 µm; N = 5. Student’s t test was performed; **p

    Article Snippet: 0.6–1 μg of total RNA was used for reverse transcription to produce cDNA using oligo-dT primers (Invitrogen), 10 mM dNTP mix, RNasin plus (Promega) and M-MLV Reverse transcriptase enzyme (Invitrogen) according to manufacturer recommendations.

    Techniques: Reverse Transcription Polymerase Chain Reaction, SYBR Green Assay, Bradford Assay, SDS Page, Western Blot, Positive Control, Expressing, Enzyme-linked Immunosorbent Assay, Staining, Microscopy

    IL-6 increased hepcidin expression and down-regulated ferroportin in hPASMCs. Confluent hPASMCs were either mock-treated or treated with 10 ng/mL IL-6 ( A ) for 2.5 h and total RNA extracted using RNeasy kit, cDNA synthesised using oligo-dT primers and RT-PCR performed using SYBR green with human hepcidin (Hamp-1) primers and β-actin as housekeeping gene. The values were further normalised as fold changes to the control untreated cells at time zero. N = 4 ( B ) for 24 h, media supernatant collected and hepcidin secretion was quantitated using an ELISA kit (R D Systems). N = 4 ( C ) for 24 h cells lysed and total protein extracted. Ferroportin expression was quantitated using an ELISA kit (BlueGene Biotech) and normalised to total protein estimated by Bradford reagent. N = 4 ( D ) Confocal images of hPASMCs grown with (top panels) normal media or (bottom panels) treated with 10 ng/mL IL-6 for 20–22 h and immuno-stained with rabbit anti-Fpn antibody and goat anti-rabbit IgG secondary antibody tagged with Alexa-568. The cells were further counterstained with DAPI and images captured using Leica LSM 510 confocal microscope. Scale bar = 10 µm, N = 5. Student’s t test was performed; **p

    Journal: Scientific Reports

    Article Title: The Hepcidin/Ferroportin axis modulates proliferation of pulmonary artery smooth muscle cells

    doi: 10.1038/s41598-018-31095-0

    Figure Lengend Snippet: IL-6 increased hepcidin expression and down-regulated ferroportin in hPASMCs. Confluent hPASMCs were either mock-treated or treated with 10 ng/mL IL-6 ( A ) for 2.5 h and total RNA extracted using RNeasy kit, cDNA synthesised using oligo-dT primers and RT-PCR performed using SYBR green with human hepcidin (Hamp-1) primers and β-actin as housekeeping gene. The values were further normalised as fold changes to the control untreated cells at time zero. N = 4 ( B ) for 24 h, media supernatant collected and hepcidin secretion was quantitated using an ELISA kit (R D Systems). N = 4 ( C ) for 24 h cells lysed and total protein extracted. Ferroportin expression was quantitated using an ELISA kit (BlueGene Biotech) and normalised to total protein estimated by Bradford reagent. N = 4 ( D ) Confocal images of hPASMCs grown with (top panels) normal media or (bottom panels) treated with 10 ng/mL IL-6 for 20–22 h and immuno-stained with rabbit anti-Fpn antibody and goat anti-rabbit IgG secondary antibody tagged with Alexa-568. The cells were further counterstained with DAPI and images captured using Leica LSM 510 confocal microscope. Scale bar = 10 µm, N = 5. Student’s t test was performed; **p

    Article Snippet: 0.6–1 μg of total RNA was used for reverse transcription to produce cDNA using oligo-dT primers (Invitrogen), 10 mM dNTP mix, RNasin plus (Promega) and M-MLV Reverse transcriptase enzyme (Invitrogen) according to manufacturer recommendations.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, Staining, Microscopy

    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

    Journal: Immunogenetics

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

    doi: 10.1007/s00251-004-0701-2

    Figure Lengend 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

    Article Snippet: Total RNA (2.5 μg) was reverse transcribed with an oligo(dT) primer and Superscript II RNase H-minus reverse transcriptase (Invitrogen) according to the manufacturer's instructions.

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

    BAX knockdown by siRNA mimics promotes tumor growth in vivo. siNC and siBAX oligos were injected into A431 cell xenografts every three days. ( A ) Loss of BAX promotes subcutaneous tumor growth in a mouse xenograft model. Tumor volumes (mm 3 ) were plotted according to days. Tumor volume statistical data represent the average of three independent experiments ± s.d., respectively; ( B ) the mice were sacrificed at the end of the experiment and images taken along with the dissected tumors from three representative mice are shown. White arrows indicate the siNC-treated xenografts, while black arrows indicate siBAX-treated xenografts; ( C ) the expression of BAX was measured in the dissected tumors by qRT-PCR. qRT-PCR statistical data represent the average of three independent experiments ± s.d. Expression folds are shown with respect to NC where normalized copy numbers were set to 1; ( D ) the protein expression of BAX was detected in xenografts after siBAX treatment by Western blot; ( E ) histopathology analysis (IHC staining) of BAX on tumor sections. The quantification was done by counting positively-stained cells from 20 randomly-chosen fields from a total of five sections per tumor. Magnification, 400×, Scale bars, 50 µm; ( F ) TUNEL assay of apoptosis on tumor sections after BAX depletion. The quantification was done by counting positively-stained signals from 20 randomly-chosen fields from a total of five sections per tumor. Magnification, 400×, Scale bars, 50 µm. ** p

    Journal: International Journal of Molecular Sciences

    Article Title: Loss of BAX by miR-365 Promotes Cutaneous Squamous Cell Carcinoma Progression by Suppressing Apoptosis

    doi: 10.3390/ijms18061157

    Figure Lengend Snippet: BAX knockdown by siRNA mimics promotes tumor growth in vivo. siNC and siBAX oligos were injected into A431 cell xenografts every three days. ( A ) Loss of BAX promotes subcutaneous tumor growth in a mouse xenograft model. Tumor volumes (mm 3 ) were plotted according to days. Tumor volume statistical data represent the average of three independent experiments ± s.d., respectively; ( B ) the mice were sacrificed at the end of the experiment and images taken along with the dissected tumors from three representative mice are shown. White arrows indicate the siNC-treated xenografts, while black arrows indicate siBAX-treated xenografts; ( C ) the expression of BAX was measured in the dissected tumors by qRT-PCR. qRT-PCR statistical data represent the average of three independent experiments ± s.d. Expression folds are shown with respect to NC where normalized copy numbers were set to 1; ( D ) the protein expression of BAX was detected in xenografts after siBAX treatment by Western blot; ( E ) histopathology analysis (IHC staining) of BAX on tumor sections. The quantification was done by counting positively-stained cells from 20 randomly-chosen fields from a total of five sections per tumor. Magnification, 400×, Scale bars, 50 µm; ( F ) TUNEL assay of apoptosis on tumor sections after BAX depletion. The quantification was done by counting positively-stained signals from 20 randomly-chosen fields from a total of five sections per tumor. Magnification, 400×, Scale bars, 50 µm. ** p

    Article Snippet: Oligos were prepared by pre-incubating 3 nM siRNA per mouse with Lipofectamine 2000 (Life Technologies) for 15 min; injections were made using a final volume of 50 µL in serum free DMEM (Life Technologies).

    Techniques: In Vivo, Injection, Mouse Assay, Expressing, Quantitative RT-PCR, Western Blot, Histopathology, Immunohistochemistry, Staining, TUNEL Assay

    DDR1 affects IR expression (a) IR protein and mRNA expression in DDR1-depleted cells. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected with either a pool of four scramble or four DDR1 siRNA oligos. After 48h, cells were lysed and analyzed by SDS-PAGE and immunoblotted with the indicated primary antibodies. β-actin was used as control for protein loading. Blot is representative of three independent experiments. The histograms represent the mean±SEM of densitometric analysis after normalization against β-actin. In the same transfected cell lines, IR mRNA levels were evaluated by qRT-PCR analysis and values were normalized using human β-actin as housekeeping control gene. In parallel, DDR1 mRNA was evaluated by qRT-PCR to confirm DDR1 depletion (graphs on the right). (b) IR protein and mRNA expression after DDR1 overexpression. Breast cancer cells were transiently transfected with either constitutive empty (pCMV6-EV) or human DDR1 (pCMV6-DDR1) expressing vectors. After 48h, cells were lysed, analyzed by SDS-PAGE and immunoblotted with the indicated primary antibodies. β-actin was used as control for protein loading. The top panels show a representative blot of three independent experiments. The histograms represent the mean±SEM of densitometric analysis after normalization over β-actin. In the same transfected cells, IR mRNA levels were evaluated by qRT-PCR analysis and values were normalized using human β-actin as housekeeping control gene. The overexpression of DDR1 was confirmed measuring DDR1 mRNA by qRT-PCR (graphs on the right). (a-b) *0.01

    Journal: Oncotarget

    Article Title: Discoidin domain receptor 1 modulates insulin receptor signaling and biological responses in breast cancer cells

    doi: 10.18632/oncotarget.18020

    Figure Lengend Snippet: DDR1 affects IR expression (a) IR protein and mRNA expression in DDR1-depleted cells. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected with either a pool of four scramble or four DDR1 siRNA oligos. After 48h, cells were lysed and analyzed by SDS-PAGE and immunoblotted with the indicated primary antibodies. β-actin was used as control for protein loading. Blot is representative of three independent experiments. The histograms represent the mean±SEM of densitometric analysis after normalization against β-actin. In the same transfected cell lines, IR mRNA levels were evaluated by qRT-PCR analysis and values were normalized using human β-actin as housekeeping control gene. In parallel, DDR1 mRNA was evaluated by qRT-PCR to confirm DDR1 depletion (graphs on the right). (b) IR protein and mRNA expression after DDR1 overexpression. Breast cancer cells were transiently transfected with either constitutive empty (pCMV6-EV) or human DDR1 (pCMV6-DDR1) expressing vectors. After 48h, cells were lysed, analyzed by SDS-PAGE and immunoblotted with the indicated primary antibodies. β-actin was used as control for protein loading. The top panels show a representative blot of three independent experiments. The histograms represent the mean±SEM of densitometric analysis after normalization over β-actin. In the same transfected cells, IR mRNA levels were evaluated by qRT-PCR analysis and values were normalized using human β-actin as housekeeping control gene. The overexpression of DDR1 was confirmed measuring DDR1 mRNA by qRT-PCR (graphs on the right). (a-b) *0.01

    Article Snippet: The specific silencer Select Pre-designed pool of four siRNA oligos for DDR1 (Human DDR1 siGENOME SMARTpool Cat M-003111–04) and the negative control, consisting of a pool of four scramble siRNAs were from Thermo Fisher Scientific Dharmacon (NYSE:TMO).

    Techniques: Expressing, Multiple Displacement Amplification, Transfection, SDS Page, Quantitative RT-PCR, Over Expression

    DDR1 affects IR – dependent transcription factors (a) Protein levels of IR-dependent transcription factors after DDR1 depletion. MCF-7 breast cancer cells were transiently transfected with either a pool of four scramble or four DDR1 specific siRNA oligos. After 48h, protein expression of IR, and of p53, HMGA, and Sp1 transcription factors was evaluated by western blotting. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization against β-actin. (b) Protein levels of IR-related transcription factors after DDR1 overexpression. MCF-7 cells were transiently transfected with either either pCMV6-EV or pCMV6-DDR1 encoding vectors. After 48h, protein expression of IR, p53, HMGA, and Sp1 was evaluated by western blotting. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization against β-actin. (c) mRNA expression of IR-related transcription factors after DDR1 depletion. MCF-7 cells transfected as in (a) were evaluated by qRT-PCR analysis for IR, p53, HMGA, and Sp1 mRNA levels. Values were normalized using human β-actin as housekeeping control gene. DDR1 mRNA levels were also evaluated as control for DDR1 depletion efficiency. Values represent the mean±SEM of three independent experiments. (d) mRNA expression of IR-related transcription factors after DDR1 overexpression. In cells transfected as in (b) , IR, p53, HMGA, and Sp1 mRNA levels were evaluated by qRT-PCR analysis and values were normalized using human β-actin as housekeeping control gene. DDR1 mRNA levels were also evaluated as silencing control. Values represent the mean±SEM of three independent experiments. (a-d) NS, p > 0.05; *0.01

    Journal: Oncotarget

    Article Title: Discoidin domain receptor 1 modulates insulin receptor signaling and biological responses in breast cancer cells

    doi: 10.18632/oncotarget.18020

    Figure Lengend Snippet: DDR1 affects IR – dependent transcription factors (a) Protein levels of IR-dependent transcription factors after DDR1 depletion. MCF-7 breast cancer cells were transiently transfected with either a pool of four scramble or four DDR1 specific siRNA oligos. After 48h, protein expression of IR, and of p53, HMGA, and Sp1 transcription factors was evaluated by western blotting. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization against β-actin. (b) Protein levels of IR-related transcription factors after DDR1 overexpression. MCF-7 cells were transiently transfected with either either pCMV6-EV or pCMV6-DDR1 encoding vectors. After 48h, protein expression of IR, p53, HMGA, and Sp1 was evaluated by western blotting. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization against β-actin. (c) mRNA expression of IR-related transcription factors after DDR1 depletion. MCF-7 cells transfected as in (a) were evaluated by qRT-PCR analysis for IR, p53, HMGA, and Sp1 mRNA levels. Values were normalized using human β-actin as housekeeping control gene. DDR1 mRNA levels were also evaluated as control for DDR1 depletion efficiency. Values represent the mean±SEM of three independent experiments. (d) mRNA expression of IR-related transcription factors after DDR1 overexpression. In cells transfected as in (b) , IR, p53, HMGA, and Sp1 mRNA levels were evaluated by qRT-PCR analysis and values were normalized using human β-actin as housekeeping control gene. DDR1 mRNA levels were also evaluated as silencing control. Values represent the mean±SEM of three independent experiments. (a-d) NS, p > 0.05; *0.01

    Article Snippet: The specific silencer Select Pre-designed pool of four siRNA oligos for DDR1 (Human DDR1 siGENOME SMARTpool Cat M-003111–04) and the negative control, consisting of a pool of four scramble siRNAs were from Thermo Fisher Scientific Dharmacon (NYSE:TMO).

    Techniques: Transfection, Expressing, Western Blot, Over Expression, Quantitative RT-PCR

    DDR1 stabilizes IR both at the protein and mRNA level in MCF-7 cells (a) DDR1 depletion affects IR proteasomal degradation. MCF-7 cells were transiently transfected with either a pool of four scramble or four DDR1-specific siRNA oligos. After 48h, cells were treated with 1μM of the proteasome inhibitor MG132 for 6h. (b) DDR1 depletion does not affect IR lysosomal degradation. MCF-7 cells were transiently transfected as in (a) . After 48 h, cells were treated with 10μM of the cloroquine (CHL) for 2, 6, and 24h. (c) Cycloheximide treatment in DDR1 overexpressing cells. MCF-7 cells were transiently transfected with either pCMV6-EV or pCMV6-DDR1 encoding vectors. After 24 h, cells were treated with 100 μM of cycloheximide (CHX) for 8, 16, and 24h. (d) Actinomycin D treatment in DDR1 overexpressing cells. MCF-7 cells were transiently transfected as in (c) . After 24h, cells were treated with 2.5μg/mL of actinomycin D (ACT D) for 2, 4, 6, and 16h. IR mRNA levels were evaluated by qRT-PCR analysis and values were normalized using human β-actin as housekeeping control gene. DDR1 overexpression was confirmed by western blot analysis as shown on the right of the histogram. (a-c) Samples were analyzed by western blotting for DDR1 and IR expression. β-actin antibody was used as control. A representative blot of three independent experiments is shown. The histogram represents the mean±SEM of densitometric analysis after normalization against β-actin. (a-d) NS, p > 0.05; *0.01

    Journal: Oncotarget

    Article Title: Discoidin domain receptor 1 modulates insulin receptor signaling and biological responses in breast cancer cells

    doi: 10.18632/oncotarget.18020

    Figure Lengend Snippet: DDR1 stabilizes IR both at the protein and mRNA level in MCF-7 cells (a) DDR1 depletion affects IR proteasomal degradation. MCF-7 cells were transiently transfected with either a pool of four scramble or four DDR1-specific siRNA oligos. After 48h, cells were treated with 1μM of the proteasome inhibitor MG132 for 6h. (b) DDR1 depletion does not affect IR lysosomal degradation. MCF-7 cells were transiently transfected as in (a) . After 48 h, cells were treated with 10μM of the cloroquine (CHL) for 2, 6, and 24h. (c) Cycloheximide treatment in DDR1 overexpressing cells. MCF-7 cells were transiently transfected with either pCMV6-EV or pCMV6-DDR1 encoding vectors. After 24 h, cells were treated with 100 μM of cycloheximide (CHX) for 8, 16, and 24h. (d) Actinomycin D treatment in DDR1 overexpressing cells. MCF-7 cells were transiently transfected as in (c) . After 24h, cells were treated with 2.5μg/mL of actinomycin D (ACT D) for 2, 4, 6, and 16h. IR mRNA levels were evaluated by qRT-PCR analysis and values were normalized using human β-actin as housekeeping control gene. DDR1 overexpression was confirmed by western blot analysis as shown on the right of the histogram. (a-c) Samples were analyzed by western blotting for DDR1 and IR expression. β-actin antibody was used as control. A representative blot of three independent experiments is shown. The histogram represents the mean±SEM of densitometric analysis after normalization against β-actin. (a-d) NS, p > 0.05; *0.01

    Article Snippet: The specific silencer Select Pre-designed pool of four siRNA oligos for DDR1 (Human DDR1 siGENOME SMARTpool Cat M-003111–04) and the negative control, consisting of a pool of four scramble siRNAs were from Thermo Fisher Scientific Dharmacon (NYSE:TMO).

    Techniques: Transfection, Activated Clotting Time Assay, Quantitative RT-PCR, Over Expression, Western Blot, Expressing

    DDR1 depletion affects insulin and IGF-2 mediated biological effects in human cancer cells (a) Western blot before and after DDR1 depletion. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected with either a pool of four DDR1 siRNA oligos or scramble siRNA oligos. After 24h, cells were grown in medium containing 2.5% of CS-FCS for 24h. DDR1 depletion was confirmed for each cells line by western blot analysis as shown in the panel. (b) Cell proliferation. Cell viability was evaluated by MTT assay. Values are expressed as percentages of untreated scramble-transfected cells (basal) and represent the mean±SEM of three independent experiments in triplicate. (c) Cell cycle progression. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected as in (a) . After 24h, cells were grown in medium containing 0.1% of BSA for additional 24h. Cells were then incubated with or without insulin or IGF-2 at a dose of 10nM for additional 48h and analyzed for cell-cycle profiles. Cell populations positive for propidium iodine staining were evaluated by FACS analysis, and G0/G1 and G2/M phases were scored. The graph shows the percentage of cells in S and G2/M phases. Values are expressed as percent of basal (untreated scramble transfected cells) and are the mean±SEM of three independent experiments. (d) Cell invasion. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected as in (a) . After 24h, cells were grown in medium containing 0.1% of BSA for additional 24 h. Cells were then removed from plates with 0.01% trypsin and seeded on polycarbonate filters coated with 25μg/mL fibronectin. Cells were allowed to migrate for 6h (MCF-7 and MDA-MB-157) or 8h (BT-474 cells) in response to 10nM of insulin or IGF-2 added to the lower chamber. Values are mean±SEM of three independent experiments done in duplicate and are expressed as percent increase over untreated scramble cells (basal). (e) Colony formation. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected as in (a) , and seeded in soft-agar, as described in Materials and Methods. Cells were plated in triplicate and cultured in serum free medium containing 2.5% CS-FCS for 3 weeks. Colonies developed only from plated MCF-7 and MDA-MB-157 cells but not from BT-474. Colonies were stained with MTT and then photographed. The histogram represents the mean number of colonies shown in (e) . Error bars indicate SEM (n = 3 wells). (b-e) *0.01

    Journal: Oncotarget

    Article Title: Discoidin domain receptor 1 modulates insulin receptor signaling and biological responses in breast cancer cells

    doi: 10.18632/oncotarget.18020

    Figure Lengend Snippet: DDR1 depletion affects insulin and IGF-2 mediated biological effects in human cancer cells (a) Western blot before and after DDR1 depletion. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected with either a pool of four DDR1 siRNA oligos or scramble siRNA oligos. After 24h, cells were grown in medium containing 2.5% of CS-FCS for 24h. DDR1 depletion was confirmed for each cells line by western blot analysis as shown in the panel. (b) Cell proliferation. Cell viability was evaluated by MTT assay. Values are expressed as percentages of untreated scramble-transfected cells (basal) and represent the mean±SEM of three independent experiments in triplicate. (c) Cell cycle progression. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected as in (a) . After 24h, cells were grown in medium containing 0.1% of BSA for additional 24h. Cells were then incubated with or without insulin or IGF-2 at a dose of 10nM for additional 48h and analyzed for cell-cycle profiles. Cell populations positive for propidium iodine staining were evaluated by FACS analysis, and G0/G1 and G2/M phases were scored. The graph shows the percentage of cells in S and G2/M phases. Values are expressed as percent of basal (untreated scramble transfected cells) and are the mean±SEM of three independent experiments. (d) Cell invasion. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected as in (a) . After 24h, cells were grown in medium containing 0.1% of BSA for additional 24 h. Cells were then removed from plates with 0.01% trypsin and seeded on polycarbonate filters coated with 25μg/mL fibronectin. Cells were allowed to migrate for 6h (MCF-7 and MDA-MB-157) or 8h (BT-474 cells) in response to 10nM of insulin or IGF-2 added to the lower chamber. Values are mean±SEM of three independent experiments done in duplicate and are expressed as percent increase over untreated scramble cells (basal). (e) Colony formation. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected as in (a) , and seeded in soft-agar, as described in Materials and Methods. Cells were plated in triplicate and cultured in serum free medium containing 2.5% CS-FCS for 3 weeks. Colonies developed only from plated MCF-7 and MDA-MB-157 cells but not from BT-474. Colonies were stained with MTT and then photographed. The histogram represents the mean number of colonies shown in (e) . Error bars indicate SEM (n = 3 wells). (b-e) *0.01

    Article Snippet: The specific silencer Select Pre-designed pool of four siRNA oligos for DDR1 (Human DDR1 siGENOME SMARTpool Cat M-003111–04) and the negative control, consisting of a pool of four scramble siRNAs were from Thermo Fisher Scientific Dharmacon (NYSE:TMO).

    Techniques: Western Blot, Multiple Displacement Amplification, Transfection, MTT Assay, Incubation, Staining, FACS, Cell Culture

    DDR1 depletion or overexpression affects insulin and IGF-2 downstream signaling in human breast cancer cells (a) Insulin and IGF-2 signaling after DDR1 depletion. MCF-7, BT-474 and MDA-MB-157 breast cancer cells were transiently transfected with a pool of four scramble or of four siRNA oligos against DDR1. After 48h, cells were grown in medium containing 2.5% of CS-FCS for 24h and then stimulated with or without 10nM of insulin or IGF-2 for 5 minutes. Cells were then lysed and analyzed by SDS-PAGE and immunoblotted with the indicated primary antibodies. β-actin was used as control for protein loading. The top panels show a representative of three experiments. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization of each phosphoprotein against β-actin. (b) Insulin and IGF-2 signaling after DDR1 overexpression. MCF-7, BT-474 and MDA-MB-157 breast cancer cells were transiently transfected with plasmids encoding either the human DDR1 cDNA (pCMV6-DDR1) or the corresponding empty vector (pCMV6-EV). After 48h, cells were grown in medium containing 2.5% of CS-FCS for 24h and then stimulated with or without 10nM of insulin or IGF-2 for 5 minutes. The activation of downstream signaling was assessed as in (a) . Blots are representative of three independent experiments. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization of each phosphoproteins against β-actin. (a-b) NS, p > 0.05; *0.01

    Journal: Oncotarget

    Article Title: Discoidin domain receptor 1 modulates insulin receptor signaling and biological responses in breast cancer cells

    doi: 10.18632/oncotarget.18020

    Figure Lengend Snippet: DDR1 depletion or overexpression affects insulin and IGF-2 downstream signaling in human breast cancer cells (a) Insulin and IGF-2 signaling after DDR1 depletion. MCF-7, BT-474 and MDA-MB-157 breast cancer cells were transiently transfected with a pool of four scramble or of four siRNA oligos against DDR1. After 48h, cells were grown in medium containing 2.5% of CS-FCS for 24h and then stimulated with or without 10nM of insulin or IGF-2 for 5 minutes. Cells were then lysed and analyzed by SDS-PAGE and immunoblotted with the indicated primary antibodies. β-actin was used as control for protein loading. The top panels show a representative of three experiments. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization of each phosphoprotein against β-actin. (b) Insulin and IGF-2 signaling after DDR1 overexpression. MCF-7, BT-474 and MDA-MB-157 breast cancer cells were transiently transfected with plasmids encoding either the human DDR1 cDNA (pCMV6-DDR1) or the corresponding empty vector (pCMV6-EV). After 48h, cells were grown in medium containing 2.5% of CS-FCS for 24h and then stimulated with or without 10nM of insulin or IGF-2 for 5 minutes. The activation of downstream signaling was assessed as in (a) . Blots are representative of three independent experiments. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization of each phosphoproteins against β-actin. (a-b) NS, p > 0.05; *0.01

    Article Snippet: The specific silencer Select Pre-designed pool of four siRNA oligos for DDR1 (Human DDR1 siGENOME SMARTpool Cat M-003111–04) and the negative control, consisting of a pool of four scramble siRNAs were from Thermo Fisher Scientific Dharmacon (NYSE:TMO).

    Techniques: Over Expression, Multiple Displacement Amplification, Transfection, SDS Page, Plasmid Preparation, Activation Assay

    RNA Pol II kinetics with a slower transcription elongation rate affects pA site selection. ( A ) Pol II ChIP across polo and polo-snap intergenic region. In the top panel, a schematic diagram is shown where polo and snap genes are depicted to scale (adapted from genome.ucsc.edu). ChIP was performed using α-Rpb3 antibody on wild-type ( w 1118 ) and slow Pol II ( RpII215 mutant, C4 mutation) adult flies chromatin. Numbers below each graph bar represent the position of real-time PCR primers. ( B ) RpII215 mutant generates similar levels of polo mRNA as compared with w 1118 . Graph represents RT–qPCR quantification of polo mRNAs in adult flies, relative to rp49 mRNA. ( C ) RpII215 mutant presents similar levels of Polo protein as w 1118 . Western blot from total protein extracts of w 1118 and RpII215 third instar larvae brains. MA294 Polo and DM1A α-tubulin antibodies were used. Quantification was made by densitometry (see Materials and methods) and Polo/α-tubulin ratio was set at 1 for w 1118 . ( D ) Representative gel of fractionated 3′RACE products of polo mRNA from w 1118 and RpII215 adult flies is shown. Arrows indicate the bands corresponding to polo pA1 and pA2; −RT is a control reaction without reverse transcriptase. In both panels, phased-anchored oligodT was used for reverse transcription, as depicted in the diagram. ( E ) RpII215 shows an increase in the ratio of total/pA2 mRNAs. Diagram shows primer positions for qPCR analysis. Levels of total polo mRNAs and pA2 mRNAs were measured by real-time PCR and their ratio (total/pA2) in adult flies is shown. ( F ) RpII215 shows an increase in proximal site usage in several genes. RT–qPCR quantification was performed as in ( E ). cDNA synthesis was performed with random primers (see Materials and methods). The ratio for w 1118 was set at 1. For all the panels, error bars show s.e.m. from at least three independent experiments.

    Journal: The EMBO Journal

    Article Title: RNA polymerase II kinetics in polo polyadenylation signal selection

    doi: 10.1038/emboj.2011.156

    Figure Lengend Snippet: RNA Pol II kinetics with a slower transcription elongation rate affects pA site selection. ( A ) Pol II ChIP across polo and polo-snap intergenic region. In the top panel, a schematic diagram is shown where polo and snap genes are depicted to scale (adapted from genome.ucsc.edu). ChIP was performed using α-Rpb3 antibody on wild-type ( w 1118 ) and slow Pol II ( RpII215 mutant, C4 mutation) adult flies chromatin. Numbers below each graph bar represent the position of real-time PCR primers. ( B ) RpII215 mutant generates similar levels of polo mRNA as compared with w 1118 . Graph represents RT–qPCR quantification of polo mRNAs in adult flies, relative to rp49 mRNA. ( C ) RpII215 mutant presents similar levels of Polo protein as w 1118 . Western blot from total protein extracts of w 1118 and RpII215 third instar larvae brains. MA294 Polo and DM1A α-tubulin antibodies were used. Quantification was made by densitometry (see Materials and methods) and Polo/α-tubulin ratio was set at 1 for w 1118 . ( D ) Representative gel of fractionated 3′RACE products of polo mRNA from w 1118 and RpII215 adult flies is shown. Arrows indicate the bands corresponding to polo pA1 and pA2; −RT is a control reaction without reverse transcriptase. In both panels, phased-anchored oligodT was used for reverse transcription, as depicted in the diagram. ( E ) RpII215 shows an increase in the ratio of total/pA2 mRNAs. Diagram shows primer positions for qPCR analysis. Levels of total polo mRNAs and pA2 mRNAs were measured by real-time PCR and their ratio (total/pA2) in adult flies is shown. ( F ) RpII215 shows an increase in proximal site usage in several genes. RT–qPCR quantification was performed as in ( E ). cDNA synthesis was performed with random primers (see Materials and methods). The ratio for w 1118 was set at 1. For all the panels, error bars show s.e.m. from at least three independent experiments.

    Article Snippet: cDNA synthesis was performed using either oligodT or random hexamers with Superscript III following the manufacturer's instructions (Invitrogen). cDNA was then quantified in an iQ5 Bio-Rad real time PCR machine with iQ™ Sybr® Green Supermix. rp49 was used for normalization in all assays.

    Techniques: Selection, Chromatin Immunoprecipitation, Mutagenesis, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot

    USP21 modulates Hh signaling and Gli1 transcriptional activity. (A) NIH3T3 cells were treated with USP21-targeting siRNA oligonucleotides (#10, #11, #12) or a non-targeting control (NT3), serum-starved and then incubated with SAG (100 nM) for 4 h before mRNA extraction. USP21 and Gli1 mRNA levels were measured by performing quantitative reverse-transcription PCR and normalised to RNA levels of Pol2 ( n =3 independent experiments, mean±s.d.). x -axis labels given in bottom panel also apply to the top panel. (B) HEK293T cells were treated with USP21-targeting siRNA oligonucleotides (#5, #6) or non-targeting control oligo (NT3), or were treated with transfection reagent alone (Mock). 24 h before lysis, cells were transfected with Gli-responsive firefly luciferase and Renilla luciferase reporters (pGLB3B-12Gli-Luc and pRL-Renilla-TK), as well as with a Gli1 expression construct (HA–Gli1). Efficiency of the knockdown was assessed by western blotting ( n =2, bars show the range). (C) HEK293T cells were transfected with Gli-responsive firefly luciferase, Renilla luciferase reporters, HA–Gli1 and the indicated amounts of GFP, GFP–USP21 or GFP–USP21CS plasmids. Protein expression was assessed by western blotting (IB, immunoblot) for GFP. (GFP, n =5; USP21 and USP21CS, n =6; mean±s.d.; one-way ANOVA and Dunnett's test, ** P

    Journal: Journal of Cell Science

    Article Title: The centrosomal deubiquitylase USP21 regulates Gli1 transcriptional activity and stability

    doi: 10.1242/jcs.188516

    Figure Lengend Snippet: USP21 modulates Hh signaling and Gli1 transcriptional activity. (A) NIH3T3 cells were treated with USP21-targeting siRNA oligonucleotides (#10, #11, #12) or a non-targeting control (NT3), serum-starved and then incubated with SAG (100 nM) for 4 h before mRNA extraction. USP21 and Gli1 mRNA levels were measured by performing quantitative reverse-transcription PCR and normalised to RNA levels of Pol2 ( n =3 independent experiments, mean±s.d.). x -axis labels given in bottom panel also apply to the top panel. (B) HEK293T cells were treated with USP21-targeting siRNA oligonucleotides (#5, #6) or non-targeting control oligo (NT3), or were treated with transfection reagent alone (Mock). 24 h before lysis, cells were transfected with Gli-responsive firefly luciferase and Renilla luciferase reporters (pGLB3B-12Gli-Luc and pRL-Renilla-TK), as well as with a Gli1 expression construct (HA–Gli1). Efficiency of the knockdown was assessed by western blotting ( n =2, bars show the range). (C) HEK293T cells were transfected with Gli-responsive firefly luciferase, Renilla luciferase reporters, HA–Gli1 and the indicated amounts of GFP, GFP–USP21 or GFP–USP21CS plasmids. Protein expression was assessed by western blotting (IB, immunoblot) for GFP. (GFP, n =5; USP21 and USP21CS, n =6; mean±s.d.; one-way ANOVA and Dunnett's test, ** P

    Article Snippet: Quantitative reverse-transcription PCR Cells were lysed, and mRNA was extracted using the RNAeasy mini kit (Qiagen). cDNA synthesis was performed using 1 µg RNA with RevertAid H-minus M-MuLV reverse transcriptase (Fermentas) using an oligo-dT primer (Promega).

    Techniques: Activity Assay, Incubation, Polymerase Chain Reaction, Transfection, Lysis, Luciferase, Expressing, Construct, Western Blot

    p120 308 is a null allele. (A) p120 308 deletes the entire p120 coding region, but does not affect other genes. Schematic as in Fig. 1 A. Genomic DNA from single wild-type or homozygous mutant flies was PCR amplified using primer pairs from the indicated regions between LD05623 and CG17486 . (B) The p120 mutants are mRNA nulls. cDNA generated from oligo-dT-primed total RNA from p120 mutants and wild-type was amplified with primers spanning the p120 third intron. An unrelated gene, CG2905 , is a control. A DNA control confirmed we were examining mRNA.

    Journal: The Journal of Cell Biology

    Article Title: Drosophila p120catenin plays a supporting role in cell adhesion but is not an essential adherens junction component

    doi: 10.1083/jcb.200211083

    Figure Lengend Snippet: p120 308 is a null allele. (A) p120 308 deletes the entire p120 coding region, but does not affect other genes. Schematic as in Fig. 1 A. Genomic DNA from single wild-type or homozygous mutant flies was PCR amplified using primer pairs from the indicated regions between LD05623 and CG17486 . (B) The p120 mutants are mRNA nulls. cDNA generated from oligo-dT-primed total RNA from p120 mutants and wild-type was amplified with primers spanning the p120 third intron. An unrelated gene, CG2905 , is a control. A DNA control confirmed we were examining mRNA.

    Article Snippet: For RT-PCR, RNA was isolated from 20 larvae using TRIzol reagent and the manufacturer's protocol (Invitrogen Life Technologies), cDNA was generated using oligo-dT primers (Promega Reverse Transcription System), and 5 of 100 μl used for PCR.

    Techniques: Mutagenesis, Polymerase Chain Reaction, Amplification, Generated

    Toeprint analyses of translation initiation complexes on atpA and atpA – gfp RNAs. In vitro transcribed atpA – atpA ( left panels ) and atpA – gfp ( right panels ) RNAs (20 nM) were extended from [γ- 32 P] ATP-labeled oligos that anneal 120-nt downstream of the initiator AUGs, either alone or in the presence of tRNA Met , 30S subunit or both. Sequencing reactions were run alongside. The treatments are indicated at the top of each panel. The positions of the toeprints respective to the initiator AUG (+1) are indicated with lines on the side. a Toeprint reactions using 100-nM 30S subunit, b Inset showing the toeprint area of experiments similar to ( a ) with 200-nM 30S subunit. The figure shows representative autoradiographs obtained from three independent experiments

    Journal: Molecular Biotechnology

    Article Title: Molecular Factors Affecting the Accumulation of Recombinant Proteins in the Chlamydomonas reinhardtii Chloroplast

    doi: 10.1007/s12033-010-9348-4

    Figure Lengend Snippet: Toeprint analyses of translation initiation complexes on atpA and atpA – gfp RNAs. In vitro transcribed atpA – atpA ( left panels ) and atpA – gfp ( right panels ) RNAs (20 nM) were extended from [γ- 32 P] ATP-labeled oligos that anneal 120-nt downstream of the initiator AUGs, either alone or in the presence of tRNA Met , 30S subunit or both. Sequencing reactions were run alongside. The treatments are indicated at the top of each panel. The positions of the toeprints respective to the initiator AUG (+1) are indicated with lines on the side. a Toeprint reactions using 100-nM 30S subunit, b Inset showing the toeprint area of experiments similar to ( a ) with 200-nM 30S subunit. The figure shows representative autoradiographs obtained from three independent experiments

    Article Snippet: Twenty picomoles of the following oligos: 5′-gcaataccgtcacctacttgg-3′ (for atpA ) and 5′-ccataagttgcgtcacc-3′ (for gfp ) were labeled with T4 polynucleotide kinase (20 U, New England Biolabs, Ipswich, MA) according to the manufacturer’s instructions.

    Techniques: In Vitro, Labeling, Sequencing

    KLF2 and SMAD7 genes were upregulated by stimulation with SCF or NGF . A: HMC-1 (V560G c-Kit) cells were either incubated with serum (serum) or in serum free medium for 17 h, then treated without (-) or with (+) imatinib (5 μM) for 4 h prior to stimulation without (-) or with SCF (100 ng/ml) or NGF (100 ng/ml) for 30 (30) and 120 min (120). Total RNA was isolated and, after reverse transcription with Oligo dT-primers, cDNA was analyzed by semi-quantitative PCR for expression of EGR1, KLF2, SMAD7 and actin (ACTB). Water (H 2 O) and RNA (RNA) serve as negative controls. bp: base pairs of the DNA marker. B: Expression of KLF2 was quantified using TaqMan-PCR as in figure 3. Relative expression levels compared to GUSB were normalized to gene expression in serum-starved HMC-1(V560G c-Kit) cells. Average values from six independent PCR reactions + SEM are shown.

    Journal: BMC Genomics

    Article Title: Identification of novel target genes of nerve growth factor (NGF) in human mastocytoma cell line (HMC-1 (V560G c-Kit)) by transcriptome analysis

    doi: 10.1186/1471-2164-12-196

    Figure Lengend Snippet: KLF2 and SMAD7 genes were upregulated by stimulation with SCF or NGF . A: HMC-1 (V560G c-Kit) cells were either incubated with serum (serum) or in serum free medium for 17 h, then treated without (-) or with (+) imatinib (5 μM) for 4 h prior to stimulation without (-) or with SCF (100 ng/ml) or NGF (100 ng/ml) for 30 (30) and 120 min (120). Total RNA was isolated and, after reverse transcription with Oligo dT-primers, cDNA was analyzed by semi-quantitative PCR for expression of EGR1, KLF2, SMAD7 and actin (ACTB). Water (H 2 O) and RNA (RNA) serve as negative controls. bp: base pairs of the DNA marker. B: Expression of KLF2 was quantified using TaqMan-PCR as in figure 3. Relative expression levels compared to GUSB were normalized to gene expression in serum-starved HMC-1(V560G c-Kit) cells. Average values from six independent PCR reactions + SEM are shown.

    Article Snippet: RT-PCR and qRT-PCR Reverse transcription was carried out using oligo dT primers and the Omniscript reverse transcriptase kit (Qiagen) following the instructions provided.

    Techniques: Incubation, Isolation, Real-time Polymerase Chain Reaction, Expressing, Marker, Polymerase Chain Reaction

    TOP1MT has a wide expression range in the NCI-60 panel. ( A ) TOP1MT mRNA expression intensity across the cell line panel from the NCI (NCI-60), ranked from the highest TOP1MT expresser (leukemia RPMI-8226, top) to the lowest (lung NCI-H226, bottom). Tissues of origin are designated by colored circles (see legend below). Names of the individual cell lines are reported. Expression values were obtained with Agilent Whole Human Genome Oligo Microarray chips, and quality filtering and normalization were performed using GeneSpring GX 10.0 software (Agilent, Santa Clara, CA, USA). A value of one corresponds to a 2-fold higher intensity than the 75th percentile of expression for all probes on the entire array. Values are on a log 2 scale. ( B ) Normalized TOP1MT mRNA expressions sorted by level (highest to lowest) across the NCI-60. Individual cell lines (filled dots) are colored according to tissue of origin and arranged by the intensity values in panel A. Inset: frequency distribution plot showing the number of cell lines from the NCI-60 panel by TOP1MT normalized intensity. x -axis, normalized TOP1MT intensity; y -axis, frequency of cell lines for any given TOP1MT intensity in the x -axis.

    Journal: Nucleic Acids Research

    Article Title: Coordinated regulation of mitochondrial topoisomerase IB with mitochondrial nuclear encoded genes and MYC

    doi: 10.1093/nar/gkr208

    Figure Lengend Snippet: TOP1MT has a wide expression range in the NCI-60 panel. ( A ) TOP1MT mRNA expression intensity across the cell line panel from the NCI (NCI-60), ranked from the highest TOP1MT expresser (leukemia RPMI-8226, top) to the lowest (lung NCI-H226, bottom). Tissues of origin are designated by colored circles (see legend below). Names of the individual cell lines are reported. Expression values were obtained with Agilent Whole Human Genome Oligo Microarray chips, and quality filtering and normalization were performed using GeneSpring GX 10.0 software (Agilent, Santa Clara, CA, USA). A value of one corresponds to a 2-fold higher intensity than the 75th percentile of expression for all probes on the entire array. Values are on a log 2 scale. ( B ) Normalized TOP1MT mRNA expressions sorted by level (highest to lowest) across the NCI-60. Individual cell lines (filled dots) are colored according to tissue of origin and arranged by the intensity values in panel A. Inset: frequency distribution plot showing the number of cell lines from the NCI-60 panel by TOP1MT normalized intensity. x -axis, normalized TOP1MT intensity; y -axis, frequency of cell lines for any given TOP1MT intensity in the x -axis.

    Article Snippet: All 132 labeled samples were hybridized to the Agilent Whole Human Genome Oligo Microarray (WHG; G4112F, design ID 014850, Agilent Technologies, Santa Clara, CA, USA), containing 41 000 probes (22 144 unique gene IDs).

    Techniques: Expressing, Microarray, Software

    Multi-trigger systems can be composed in which each RNA/DNA hybrid contains a responsive DNA structural element. ( A ) A system comprising a 3-input AND gate and a NOT gate can be constructed by pairing sH ^CTGF.20/8 (activated by the connective tissue growth factor (CTGF) derived trigger) with aH ∨ KRAS (repressed by the Kirsten rat sarcoma proto-oncogene (KRAS) mRNA derived trigger). Co-incubation of the two hybrids results in no interaction. Both hybrids and the CTGF trigger are required for dsRNA release, while the presence of the KRAS trigger will inhibit strand exchange. ( B ) The multi-trigger system was assessed by 10% acrylamide non-denaturing PAGE. The fraction of DsiRNA released is indicated in the gel depicted, in the presence of indicated trigger combinations following 30 min incubation at 37 °C. The sH and aH hybrid were present at equimolar concentration, while the triggers were added at a 2-fold or 3-fold excess, as indicated. In samples when both triggers are present, they were added to premixed hybrids sequentially (KRAS followed by CTGF). The antisense hybrid and DsiRNA control in were assembled using a 5′-AlexaFluor546 labeled antisense RNA strand for the purpose of visualization and quantification.

    Journal: Nanomaterials

    Article Title: A Suite of Therapeutically-Inspired Nucleic Acid Logic Systems for Conditional Generation of Single-Stranded and Double-Stranded Oligonucleotides

    doi: 10.3390/nano9040615

    Figure Lengend Snippet: Multi-trigger systems can be composed in which each RNA/DNA hybrid contains a responsive DNA structural element. ( A ) A system comprising a 3-input AND gate and a NOT gate can be constructed by pairing sH ^CTGF.20/8 (activated by the connective tissue growth factor (CTGF) derived trigger) with aH ∨ KRAS (repressed by the Kirsten rat sarcoma proto-oncogene (KRAS) mRNA derived trigger). Co-incubation of the two hybrids results in no interaction. Both hybrids and the CTGF trigger are required for dsRNA release, while the presence of the KRAS trigger will inhibit strand exchange. ( B ) The multi-trigger system was assessed by 10% acrylamide non-denaturing PAGE. The fraction of DsiRNA released is indicated in the gel depicted, in the presence of indicated trigger combinations following 30 min incubation at 37 °C. The sH and aH hybrid were present at equimolar concentration, while the triggers were added at a 2-fold or 3-fold excess, as indicated. In samples when both triggers are present, they were added to premixed hybrids sequentially (KRAS followed by CTGF). The antisense hybrid and DsiRNA control in were assembled using a 5′-AlexaFluor546 labeled antisense RNA strand for the purpose of visualization and quantification.

    Article Snippet: Oligonucleotide Synthesis and Purification The DNA and RNA oligonucleotides used to assemble the conditional RNA/DNA constructs, including those that were fluorescently labeled, were purchased from Integrated DNA Technologies (IDT, Coralville, IA, USA) and reconstituted in nuclease-free water (Quality Biological, Gaithersburg, MD, USA) for use.

    Techniques: Construct, Derivative Assay, Incubation, Polyacrylamide Gel Electrophoresis, Concentration Assay, Labeling

    An RNA/DNA cognate pair system was designed to undergo conditional strand exchange by hybridizing to neighboring sites on an RNA trigger. ( A ) “Traditional” RNA/DNA hybrid pairs act as an 2-input AND gate. Hybridization between the single stranded toeholds of a sense hybrid ( sH ) and antisense hybrid ( aH ) initiates a thermodynamically driven strand exchange that generates a dsRNA duplex and DNA waste byproduct. ( B ) The “adjacent targeting” RNA/DNA hybrid system functions as a 3-input AND gate, requiring a hybrid pair as well as a specific RNA trigger sequence. The hybrid pair’s respective toeholds bind to regions of the trigger that are immediately upstream and downstream from one another. Anchoring the cognate hybrids in close proximity leads to initiation of the thermodynamically favorable strand exchange reaction and dsRNA release. ( C ) Five different cognate pairs of adjacent targeting hybrids were analyzed by 12% acrylamide non-denaturing PAGE for their ability to release a DsiRNA product. Each sense hybrid and the DsiRNA control assembly contained a 3′ 6-carboxyfluorescein (6-FAM) labeled sense RNA strand for visualization. The pairs of constructs differ in the number of DNA nucleotides inserted between the single-strand toehold and the RNA/DNA hybrid duplex. These inserted nucleotides were complementary between cognate hybrids, resulting in either 0, +1, +2, +3 or +4 DNA bp that can seed the strand exchange (colored orange). The presence or absence of each component is indicated above each lane. The samples in the gel depicted were all incubated for 180 min at 37 °C. ( D ) Analysis of the fraction of dsRNA released by hybrid pairs in the presence and absence of the RNA trigger following 30, 90 or 180 min incubations at 37 °C. Error bars indicate standard deviation of three replicate experiments. Indication of statistical significance between samples is reported in the supporting information.

    Journal: Nanomaterials

    Article Title: A Suite of Therapeutically-Inspired Nucleic Acid Logic Systems for Conditional Generation of Single-Stranded and Double-Stranded Oligonucleotides

    doi: 10.3390/nano9040615

    Figure Lengend Snippet: An RNA/DNA cognate pair system was designed to undergo conditional strand exchange by hybridizing to neighboring sites on an RNA trigger. ( A ) “Traditional” RNA/DNA hybrid pairs act as an 2-input AND gate. Hybridization between the single stranded toeholds of a sense hybrid ( sH ) and antisense hybrid ( aH ) initiates a thermodynamically driven strand exchange that generates a dsRNA duplex and DNA waste byproduct. ( B ) The “adjacent targeting” RNA/DNA hybrid system functions as a 3-input AND gate, requiring a hybrid pair as well as a specific RNA trigger sequence. The hybrid pair’s respective toeholds bind to regions of the trigger that are immediately upstream and downstream from one another. Anchoring the cognate hybrids in close proximity leads to initiation of the thermodynamically favorable strand exchange reaction and dsRNA release. ( C ) Five different cognate pairs of adjacent targeting hybrids were analyzed by 12% acrylamide non-denaturing PAGE for their ability to release a DsiRNA product. Each sense hybrid and the DsiRNA control assembly contained a 3′ 6-carboxyfluorescein (6-FAM) labeled sense RNA strand for visualization. The pairs of constructs differ in the number of DNA nucleotides inserted between the single-strand toehold and the RNA/DNA hybrid duplex. These inserted nucleotides were complementary between cognate hybrids, resulting in either 0, +1, +2, +3 or +4 DNA bp that can seed the strand exchange (colored orange). The presence or absence of each component is indicated above each lane. The samples in the gel depicted were all incubated for 180 min at 37 °C. ( D ) Analysis of the fraction of dsRNA released by hybrid pairs in the presence and absence of the RNA trigger following 30, 90 or 180 min incubations at 37 °C. Error bars indicate standard deviation of three replicate experiments. Indication of statistical significance between samples is reported in the supporting information.

    Article Snippet: Oligonucleotide Synthesis and Purification The DNA and RNA oligonucleotides used to assemble the conditional RNA/DNA constructs, including those that were fluorescently labeled, were purchased from Integrated DNA Technologies (IDT, Coralville, IA, USA) and reconstituted in nuclease-free water (Quality Biological, Gaithersburg, MD, USA) for use.

    Techniques: Activated Clotting Time Assay, Hybridization, Sequencing, Polyacrylamide Gel Electrophoresis, Labeling, Construct, Incubation, Standard Deviation

    Effects of DNA structural alteration on the degree of trigger-inducible dsRNA release. ( A ) Four different sense hybrids that are responsive to the connective tissue growth factor (CTGF) trigger were designed, each having different features within the structured DNA hairpin. The hairpins differed in the size of their loop or the length of their stem. Two different cognate antisense hybrids were designed and differ in the length of their single-stranded toehold. Sequence regions are indicated by lowercase letters and different colors to convey sequence identity or sequence complementarity. ( B , D ) DsiRNA release in the presence and absence of trigger was assessed by 10% acrylamide non-denaturing PAGE for each sense hybrid paired with a cognate antisense hybrid exhibiting either ( B ) a 12 nt toehold ( aH ^CTGF-cgnt.12 ) or ( D ) a 16 nt toehold ( aH ^CTGF-cgnt.16 ). Each sense hybrid and the DsiRNA control contained a 3′ 6-carboxyfluorescein (6-FAM) labeled sense RNA strand for visualization and quantification. Gels in both ( B ) and ( D ) depict samples that were incubated for 30 min at 37 °C. ( C , E ) Analysis of the fraction of dsRNA released by the four sense hybrids paired with ( C ) aH ^CTGF-cgnt.12 or ( E ) aH ^CTGF-cgnt.16 , in the presence and absence of the RNA trigger following 30, 90, or 180 min incubations at 37 °C. Error bars indicate standard deviation of three replicate experiments. Indication of statistical significance between samples is reported in the supporting information.

    Journal: Nanomaterials

    Article Title: A Suite of Therapeutically-Inspired Nucleic Acid Logic Systems for Conditional Generation of Single-Stranded and Double-Stranded Oligonucleotides

    doi: 10.3390/nano9040615

    Figure Lengend Snippet: Effects of DNA structural alteration on the degree of trigger-inducible dsRNA release. ( A ) Four different sense hybrids that are responsive to the connective tissue growth factor (CTGF) trigger were designed, each having different features within the structured DNA hairpin. The hairpins differed in the size of their loop or the length of their stem. Two different cognate antisense hybrids were designed and differ in the length of their single-stranded toehold. Sequence regions are indicated by lowercase letters and different colors to convey sequence identity or sequence complementarity. ( B , D ) DsiRNA release in the presence and absence of trigger was assessed by 10% acrylamide non-denaturing PAGE for each sense hybrid paired with a cognate antisense hybrid exhibiting either ( B ) a 12 nt toehold ( aH ^CTGF-cgnt.12 ) or ( D ) a 16 nt toehold ( aH ^CTGF-cgnt.16 ). Each sense hybrid and the DsiRNA control contained a 3′ 6-carboxyfluorescein (6-FAM) labeled sense RNA strand for visualization and quantification. Gels in both ( B ) and ( D ) depict samples that were incubated for 30 min at 37 °C. ( C , E ) Analysis of the fraction of dsRNA released by the four sense hybrids paired with ( C ) aH ^CTGF-cgnt.12 or ( E ) aH ^CTGF-cgnt.16 , in the presence and absence of the RNA trigger following 30, 90, or 180 min incubations at 37 °C. Error bars indicate standard deviation of three replicate experiments. Indication of statistical significance between samples is reported in the supporting information.

    Article Snippet: Oligonucleotide Synthesis and Purification The DNA and RNA oligonucleotides used to assemble the conditional RNA/DNA constructs, including those that were fluorescently labeled, were purchased from Integrated DNA Technologies (IDT, Coralville, IA, USA) and reconstituted in nuclease-free water (Quality Biological, Gaithersburg, MD, USA) for use.

    Techniques: Sequencing, Polyacrylamide Gel Electrophoresis, Labeling, Incubation, Standard Deviation

    Incorporation of a structured responsive element can generate a trigger-inducible RNA/DNA hybrid system. ( A ) The inducible hybrid system functions as a three-input AND gate. The sense hybrid sH ^CTGF.12/8 contains a responsive DNA hairpin composed of a 12 bp stem and an 8 nt loop, and is flanked by an extended 5′ single strand that acts as a diagnostic toehold. Trigger hybridization to the diagnostic toehold progresses through the hairpin stem and unzips the hairpin (sequence regions colored blue). This liberates a previously sequestered toehold within sH ^CTGF.12/8 which can then hybridize with the complementary toehold of the cognate antisense hybrid, aH ^CTGF-cgnt.12 . Hybridization of these exchange toeholds (sequence regions colored orange) initiates strand exchange and releases a dsRNA product. ( B ) The function of this conditional system was assessed by 8% acrylamide non-denaturing PAGE and total staining with ethidium bromide. DsiRNA release is observed when the sense and antisense hybrids are co-incubated in the presence of trigger (red box). Formation of the expected waste product is observed by comparison to a control assembly of the s’ and a’ DNA strands with the trigger molecule. All samples were incubated for 30 min at 37 °C. ( C ) Förster resonance energy transfer (FRET) analysis was performed as another method to verify conditional dsRNA formation. sH ^CTGF.12/8 was assembled using a 3′ 6-carboxyfluorescein (6-FAM) (ex/em 495/520 nm) labeled sense RNA strand. aH ^CTGF-cgnt.12 was assembled using a 5′-AlexaFluor546 (ex/em 555/570 nm) labeled antisense RNA strand. The hybrids were mixed and incubated at 37 °C for one hour in the presence or absence of the RNA trigger. Fluorescence emission spectra were recorded at t = 0 and t = 60 min using excitation at 475 nm.

    Journal: Nanomaterials

    Article Title: A Suite of Therapeutically-Inspired Nucleic Acid Logic Systems for Conditional Generation of Single-Stranded and Double-Stranded Oligonucleotides

    doi: 10.3390/nano9040615

    Figure Lengend Snippet: Incorporation of a structured responsive element can generate a trigger-inducible RNA/DNA hybrid system. ( A ) The inducible hybrid system functions as a three-input AND gate. The sense hybrid sH ^CTGF.12/8 contains a responsive DNA hairpin composed of a 12 bp stem and an 8 nt loop, and is flanked by an extended 5′ single strand that acts as a diagnostic toehold. Trigger hybridization to the diagnostic toehold progresses through the hairpin stem and unzips the hairpin (sequence regions colored blue). This liberates a previously sequestered toehold within sH ^CTGF.12/8 which can then hybridize with the complementary toehold of the cognate antisense hybrid, aH ^CTGF-cgnt.12 . Hybridization of these exchange toeholds (sequence regions colored orange) initiates strand exchange and releases a dsRNA product. ( B ) The function of this conditional system was assessed by 8% acrylamide non-denaturing PAGE and total staining with ethidium bromide. DsiRNA release is observed when the sense and antisense hybrids are co-incubated in the presence of trigger (red box). Formation of the expected waste product is observed by comparison to a control assembly of the s’ and a’ DNA strands with the trigger molecule. All samples were incubated for 30 min at 37 °C. ( C ) Förster resonance energy transfer (FRET) analysis was performed as another method to verify conditional dsRNA formation. sH ^CTGF.12/8 was assembled using a 3′ 6-carboxyfluorescein (6-FAM) (ex/em 495/520 nm) labeled sense RNA strand. aH ^CTGF-cgnt.12 was assembled using a 5′-AlexaFluor546 (ex/em 555/570 nm) labeled antisense RNA strand. The hybrids were mixed and incubated at 37 °C for one hour in the presence or absence of the RNA trigger. Fluorescence emission spectra were recorded at t = 0 and t = 60 min using excitation at 475 nm.

    Article Snippet: Oligonucleotide Synthesis and Purification The DNA and RNA oligonucleotides used to assemble the conditional RNA/DNA constructs, including those that were fluorescently labeled, were purchased from Integrated DNA Technologies (IDT, Coralville, IA, USA) and reconstituted in nuclease-free water (Quality Biological, Gaithersburg, MD, USA) for use.

    Techniques: Diagnostic Assay, Hybridization, Sequencing, Polyacrylamide Gel Electrophoresis, Staining, Incubation, Förster Resonance Energy Transfer, Labeling, Fluorescence