allele-specific reverse primers Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Integrated DNA Technologies oligos
    Oligos, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 99/100, based on 1472 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/oligos/product/Integrated DNA Technologies
    Average 99 stars, based on 1472 article reviews
    Price from $9.99 to $1999.99
    oligos - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    97
    New England Biolabs ddei
    Ectopic Expression of ASGP-R1 in U87 Cells Increases Efficacy of GN3-SMN-ASO In Vitro (A) Major and minor transmembrane domain (TM)-containing ASGP-R isoform cDNAs were cloned into retroviral and lentiviral expression vectors as indicated. The lentiviral vector carries the selection marker puromycin. ASGP-R H2c has a short 18-amino acid deletion in the intracellular domain of the receptor. (B) Western blot confirms expression of major and minor ASGP-R isoforms in U87 glioblastoma cells. The expression level of H1a is approximately 6-fold higher than the endogenous expression level in HepG2 cells, normalized to tubulin. H2a and H2b isoforms are detectable when expressed alone, but are stabilized in the presence of H1a. (C) Micrographs of U87 cells expressing ASGPR-H1a and H2b alone, and in combination. Cells were stained for ASGP-R1 (red), ASGP-R2 (green), and DAPI (blue). Arrowheads indicate non-uniform distribution of ASGP-R H2b, consistent with ER localization. Scale bar, 50 μm. (D) The SMN ASOs used in this study bind to intron 7 of SMN2 and promote exon 7 inclusion. Full-length SMN2 mRNA was quantified by radioactive <t>RT-PCR;</t> the product was digested with <t>DdeI</t> to separate SMN1 from SMN2 products. (E) U87 cells expressing major and minor ASGP-R isoforms alone or in combination were incubated with 300 nM unconjugated (SMN-MOE) or GalNAc-conjugated SMN-MOE ASOs (GN3-SMN-MOE) for 5 days by free uptake. Representative radiograph shows full-length SMN2 (top band) and SMN2 Δ exon 7 (bottom band). (F) Quantification of full-length SMN2 in ASO-treated U87 cells. The differences among the means in the SMN group (p = 0.0055) and the GN3-SMN group (p
    Ddei, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 439 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ddei/product/New England Biolabs
    Average 97 stars, based on 439 article reviews
    Price from $9.99 to $1999.99
    ddei - by Bioz Stars, 2020-08
    97/100 stars
      Buy from Supplier

    93
    Eurofins allele specific reverse primer
    Ectopic Expression of ASGP-R1 in U87 Cells Increases Efficacy of GN3-SMN-ASO In Vitro (A) Major and minor transmembrane domain (TM)-containing ASGP-R isoform cDNAs were cloned into retroviral and lentiviral expression vectors as indicated. The lentiviral vector carries the selection marker puromycin. ASGP-R H2c has a short 18-amino acid deletion in the intracellular domain of the receptor. (B) Western blot confirms expression of major and minor ASGP-R isoforms in U87 glioblastoma cells. The expression level of H1a is approximately 6-fold higher than the endogenous expression level in HepG2 cells, normalized to tubulin. H2a and H2b isoforms are detectable when expressed alone, but are stabilized in the presence of H1a. (C) Micrographs of U87 cells expressing ASGPR-H1a and H2b alone, and in combination. Cells were stained for ASGP-R1 (red), ASGP-R2 (green), and DAPI (blue). Arrowheads indicate non-uniform distribution of ASGP-R H2b, consistent with ER localization. Scale bar, 50 μm. (D) The SMN ASOs used in this study bind to intron 7 of SMN2 and promote exon 7 inclusion. Full-length SMN2 mRNA was quantified by radioactive <t>RT-PCR;</t> the product was digested with <t>DdeI</t> to separate SMN1 from SMN2 products. (E) U87 cells expressing major and minor ASGP-R isoforms alone or in combination were incubated with 300 nM unconjugated (SMN-MOE) or GalNAc-conjugated SMN-MOE ASOs (GN3-SMN-MOE) for 5 days by free uptake. Representative radiograph shows full-length SMN2 (top band) and SMN2 Δ exon 7 (bottom band). (F) Quantification of full-length SMN2 in ASO-treated U87 cells. The differences among the means in the SMN group (p = 0.0055) and the GN3-SMN group (p
    Allele Specific Reverse Primer, supplied by Eurofins, used in various techniques. Bioz Stars score: 93/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/allele specific reverse primer/product/Eurofins
    Average 93 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    allele specific reverse primer - by Bioz Stars, 2020-08
    93/100 stars
      Buy from Supplier

    91
    TIB MOLBIOL allele specific reverse primers
    Ectopic Expression of ASGP-R1 in U87 Cells Increases Efficacy of GN3-SMN-ASO In Vitro (A) Major and minor transmembrane domain (TM)-containing ASGP-R isoform cDNAs were cloned into retroviral and lentiviral expression vectors as indicated. The lentiviral vector carries the selection marker puromycin. ASGP-R H2c has a short 18-amino acid deletion in the intracellular domain of the receptor. (B) Western blot confirms expression of major and minor ASGP-R isoforms in U87 glioblastoma cells. The expression level of H1a is approximately 6-fold higher than the endogenous expression level in HepG2 cells, normalized to tubulin. H2a and H2b isoforms are detectable when expressed alone, but are stabilized in the presence of H1a. (C) Micrographs of U87 cells expressing ASGPR-H1a and H2b alone, and in combination. Cells were stained for ASGP-R1 (red), ASGP-R2 (green), and DAPI (blue). Arrowheads indicate non-uniform distribution of ASGP-R H2b, consistent with ER localization. Scale bar, 50 μm. (D) The SMN ASOs used in this study bind to intron 7 of SMN2 and promote exon 7 inclusion. Full-length SMN2 mRNA was quantified by radioactive <t>RT-PCR;</t> the product was digested with <t>DdeI</t> to separate SMN1 from SMN2 products. (E) U87 cells expressing major and minor ASGP-R isoforms alone or in combination were incubated with 300 nM unconjugated (SMN-MOE) or GalNAc-conjugated SMN-MOE ASOs (GN3-SMN-MOE) for 5 days by free uptake. Representative radiograph shows full-length SMN2 (top band) and SMN2 Δ exon 7 (bottom band). (F) Quantification of full-length SMN2 in ASO-treated U87 cells. The differences among the means in the SMN group (p = 0.0055) and the GN3-SMN group (p
    Allele Specific Reverse Primers, supplied by TIB MOLBIOL, used in various techniques. Bioz Stars score: 91/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/allele specific reverse primers/product/TIB MOLBIOL
    Average 91 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    allele specific reverse primers - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

    99
    Thermo Fisher sirna oligos
    Reduction of eRNA expression results in reduced expression of nearby mRNAs a , Q-PCR analysis of Mmp9 eRNA, and Mmp9 , NCoA5 and Cx3cr1 mRNAs for wildtype and Rev-Erb DKO thioglycollate-elicited macrophages transfected with Ctrl or Mmp9 eRNA <t>siRNA</t> (N WT = 4, and N DKO = 4). b , Q-PCR analysis of Cx3cr1 eRNA, and Cx3cr1, Csrnp1 and Mmp9 mRNAs for wildtype and Rev-Erb DKO bone marrow-derived macrophages transfected with siRNA targeting Cx3cr1 eRNA (N WT = 6, and N DKO = 5). c , Q-PCR analysis of Mmp9 eRNA and Mmp9 and Cx3cr1 mRNAs in thioglycollate-elicited macrophages transfected with the indicated antisense <t>oligonucleotides</t> (ASO, n = 3-7 per condition). d , Q-PCR analysis of Cx3cr1 eRNA and Cx3cr1, Mmp9 and Csrnpl mRNAs in BMDMs transfected with the indicated antisense oligonucleotides (ASO, n = 3-7 per condition). Data in a-d represent mean + s.d., with expression normalized to 36B4 in all cases. For a-b, statistical significance was determined by two tails Student’s t-test; for c-d, one-way ANOVA with Tukey HSD test. P value, * P
    Sirna Oligos, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1998 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sirna oligos/product/Thermo Fisher
    Average 99 stars, based on 1998 article reviews
    Price from $9.99 to $1999.99
    sirna oligos - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    89
    Thermo Fisher relitm reverse sequence specific oligonucleotide kits
    Reduction of eRNA expression results in reduced expression of nearby mRNAs a , Q-PCR analysis of Mmp9 eRNA, and Mmp9 , NCoA5 and Cx3cr1 mRNAs for wildtype and Rev-Erb DKO thioglycollate-elicited macrophages transfected with Ctrl or Mmp9 eRNA <t>siRNA</t> (N WT = 4, and N DKO = 4). b , Q-PCR analysis of Cx3cr1 eRNA, and Cx3cr1, Csrnp1 and Mmp9 mRNAs for wildtype and Rev-Erb DKO bone marrow-derived macrophages transfected with siRNA targeting Cx3cr1 eRNA (N WT = 6, and N DKO = 5). c , Q-PCR analysis of Mmp9 eRNA and Mmp9 and Cx3cr1 mRNAs in thioglycollate-elicited macrophages transfected with the indicated antisense <t>oligonucleotides</t> (ASO, n = 3-7 per condition). d , Q-PCR analysis of Cx3cr1 eRNA and Cx3cr1, Mmp9 and Csrnpl mRNAs in BMDMs transfected with the indicated antisense oligonucleotides (ASO, n = 3-7 per condition). Data in a-d represent mean + s.d., with expression normalized to 36B4 in all cases. For a-b, statistical significance was determined by two tails Student’s t-test; for c-d, one-way ANOVA with Tukey HSD test. P value, * P
    Relitm Reverse Sequence Specific Oligonucleotide Kits, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 89/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/relitm reverse sequence specific oligonucleotide kits/product/Thermo Fisher
    Average 89 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    relitm reverse sequence specific oligonucleotide kits - by Bioz Stars, 2020-08
    89/100 stars
      Buy from Supplier

    99
    Thermo Fisher allele specific taqman mgb probes
    Reduction of eRNA expression results in reduced expression of nearby mRNAs a , Q-PCR analysis of Mmp9 eRNA, and Mmp9 , NCoA5 and Cx3cr1 mRNAs for wildtype and Rev-Erb DKO thioglycollate-elicited macrophages transfected with Ctrl or Mmp9 eRNA <t>siRNA</t> (N WT = 4, and N DKO = 4). b , Q-PCR analysis of Cx3cr1 eRNA, and Cx3cr1, Csrnp1 and Mmp9 mRNAs for wildtype and Rev-Erb DKO bone marrow-derived macrophages transfected with siRNA targeting Cx3cr1 eRNA (N WT = 6, and N DKO = 5). c , Q-PCR analysis of Mmp9 eRNA and Mmp9 and Cx3cr1 mRNAs in thioglycollate-elicited macrophages transfected with the indicated antisense <t>oligonucleotides</t> (ASO, n = 3-7 per condition). d , Q-PCR analysis of Cx3cr1 eRNA and Cx3cr1, Mmp9 and Csrnpl mRNAs in BMDMs transfected with the indicated antisense oligonucleotides (ASO, n = 3-7 per condition). Data in a-d represent mean + s.d., with expression normalized to 36B4 in all cases. For a-b, statistical significance was determined by two tails Student’s t-test; for c-d, one-way ANOVA with Tukey HSD test. P value, * P
    Allele Specific Taqman Mgb Probes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 97 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/allele specific taqman mgb probes/product/Thermo Fisher
    Average 99 stars, based on 97 article reviews
    Price from $9.99 to $1999.99
    allele specific taqman mgb probes - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    85
    Thermo Fisher real time reverse sequence specific oligonucleotide sso
    Reduction of eRNA expression results in reduced expression of nearby mRNAs a , Q-PCR analysis of Mmp9 eRNA, and Mmp9 , NCoA5 and Cx3cr1 mRNAs for wildtype and Rev-Erb DKO thioglycollate-elicited macrophages transfected with Ctrl or Mmp9 eRNA <t>siRNA</t> (N WT = 4, and N DKO = 4). b , Q-PCR analysis of Cx3cr1 eRNA, and Cx3cr1, Csrnp1 and Mmp9 mRNAs for wildtype and Rev-Erb DKO bone marrow-derived macrophages transfected with siRNA targeting Cx3cr1 eRNA (N WT = 6, and N DKO = 5). c , Q-PCR analysis of Mmp9 eRNA and Mmp9 and Cx3cr1 mRNAs in thioglycollate-elicited macrophages transfected with the indicated antisense <t>oligonucleotides</t> (ASO, n = 3-7 per condition). d , Q-PCR analysis of Cx3cr1 eRNA and Cx3cr1, Mmp9 and Csrnpl mRNAs in BMDMs transfected with the indicated antisense oligonucleotides (ASO, n = 3-7 per condition). Data in a-d represent mean + s.d., with expression normalized to 36B4 in all cases. For a-b, statistical significance was determined by two tails Student’s t-test; for c-d, one-way ANOVA with Tukey HSD test. P value, * P
    Real Time Reverse Sequence Specific Oligonucleotide Sso, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/real time reverse sequence specific oligonucleotide sso/product/Thermo Fisher
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    real time reverse sequence specific oligonucleotide sso - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    99
    Thermo Fisher allele specific rt pcr
    Reduction of eRNA expression results in reduced expression of nearby mRNAs a , Q-PCR analysis of Mmp9 eRNA, and Mmp9 , NCoA5 and Cx3cr1 mRNAs for wildtype and Rev-Erb DKO thioglycollate-elicited macrophages transfected with Ctrl or Mmp9 eRNA <t>siRNA</t> (N WT = 4, and N DKO = 4). b , Q-PCR analysis of Cx3cr1 eRNA, and Cx3cr1, Csrnp1 and Mmp9 mRNAs for wildtype and Rev-Erb DKO bone marrow-derived macrophages transfected with siRNA targeting Cx3cr1 eRNA (N WT = 6, and N DKO = 5). c , Q-PCR analysis of Mmp9 eRNA and Mmp9 and Cx3cr1 mRNAs in thioglycollate-elicited macrophages transfected with the indicated antisense <t>oligonucleotides</t> (ASO, n = 3-7 per condition). d , Q-PCR analysis of Cx3cr1 eRNA and Cx3cr1, Mmp9 and Csrnpl mRNAs in BMDMs transfected with the indicated antisense oligonucleotides (ASO, n = 3-7 per condition). Data in a-d represent mean + s.d., with expression normalized to 36B4 in all cases. For a-b, statistical significance was determined by two tails Student’s t-test; for c-d, one-way ANOVA with Tukey HSD test. P value, * P
    Allele Specific Rt Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/allele specific rt pcr/product/Thermo Fisher
    Average 99 stars, based on 49 article reviews
    Price from $9.99 to $1999.99
    allele specific rt pcr - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher oligo dt
    In vivo activity of a novel ligand-conjugated antisense (LICA) oligonucleotide. We treated TR; HSA LR bi-transgenic mice with saline or a novel LICA oligonucleotide, ASO 992948, targeting ACTA1 -CUG exp transcripts and measured DsRed and GFP fluorescence by serial in vivo spectroscopy. <t>LICA-oligo</t> doses were 2.5, 8.3, or 25 mg/kg twice weekly for 4 weeks (8 total doses) by subcutaneous injection ( N = 2 each group). However, due to low DsRed/GFP fluorescence measurements in gastrocnemius muscles, mice receiving the 2.5 mg/kg and 8.3 mg/kg doses received four additional injections over the next 2 weeks, for a total of 12 doses. a Serial DsRed/GFP measurements in gastrocnemius and lumbar paraspinal muscles through Day 42. The final dose in the saline (black circles) and 25 mg/kg groups (blue diamonds) was Day 24, while the final dose in the 2.5 (yellow triangles) and 8.3 mg/kg (gray triangles) groups was Day 37. Error bars indicate mean ± s.e.m. b Quantitation of ACTA1 -CUG exp transcript levels (copies per microliter cDNA) by droplet digital PCR in muscles collected at Day 42. Error bars indicate mean ± s.e.m. **** P
    Oligo Dt, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 25491 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/oligo dt/product/Thermo Fisher
    Average 99 stars, based on 25491 article reviews
    Price from $9.99 to $1999.99
    oligo dt - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    93
    LGC Biosearch common reverse primer
    In vivo activity of a novel ligand-conjugated antisense (LICA) oligonucleotide. We treated TR; HSA LR bi-transgenic mice with saline or a novel LICA oligonucleotide, ASO 992948, targeting ACTA1 -CUG exp transcripts and measured DsRed and GFP fluorescence by serial in vivo spectroscopy. <t>LICA-oligo</t> doses were 2.5, 8.3, or 25 mg/kg twice weekly for 4 weeks (8 total doses) by subcutaneous injection ( N = 2 each group). However, due to low DsRed/GFP fluorescence measurements in gastrocnemius muscles, mice receiving the 2.5 mg/kg and 8.3 mg/kg doses received four additional injections over the next 2 weeks, for a total of 12 doses. a Serial DsRed/GFP measurements in gastrocnemius and lumbar paraspinal muscles through Day 42. The final dose in the saline (black circles) and 25 mg/kg groups (blue diamonds) was Day 24, while the final dose in the 2.5 (yellow triangles) and 8.3 mg/kg (gray triangles) groups was Day 37. Error bars indicate mean ± s.e.m. b Quantitation of ACTA1 -CUG exp transcript levels (copies per microliter cDNA) by droplet digital PCR in muscles collected at Day 42. Error bars indicate mean ± s.e.m. **** P
    Common Reverse Primer, supplied by LGC Biosearch, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/common reverse primer/product/LGC Biosearch
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    common reverse primer - by Bioz Stars, 2020-08
    93/100 stars
      Buy from Supplier

    96
    Thermo Fisher gapdh
    Effect of antisense knockdown of TLR2, TLR3, and TLR4 on LPS- and poly(I-C)-induced expression of <t>TNF-α</t> in mouse BV-2 microglial cells. Cells received 0.5 µM of either ASO or ScO against TLR2 ( A ) and TLR4 ( B ). After 48 h of incubation, cells were stimulated with 1 µg/ml LPS for 6 h followed by analysis of TNF-α by RT-PCR. Cells received 0.5 µM of either ASO or ScO against TLR2 ( C ) and TLR3 ( D ). After 48 h of incubation, cells were stimulated with 100 µg/ml poly(I-C) for 6 h followed by analysis of TNF-α by RT-PCR. The relative expression ( bottom panels ) of TLRs or TNF-α <t>(TLR/GAPDH</t> or TNF-α/GAPDH) was measured after scanning the bands with a Fluor Chem 8800 Imaging System (Alpha Innotech Corporation). Results represent mean ± SD of three independent experiments. a , p
    Gapdh, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 14534 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gapdh/product/Thermo Fisher
    Average 96 stars, based on 14534 article reviews
    Price from $9.99 to $1999.99
    gapdh - by Bioz Stars, 2020-08
    96/100 stars
      Buy from Supplier

    99
    TaKaRa sybr premix ex taq
    Effect of antisense knockdown of TLR2, TLR3, and TLR4 on LPS- and poly(I-C)-induced expression of <t>TNF-α</t> in mouse BV-2 microglial cells. Cells received 0.5 µM of either ASO or ScO against TLR2 ( A ) and TLR4 ( B ). After 48 h of incubation, cells were stimulated with 1 µg/ml LPS for 6 h followed by analysis of TNF-α by RT-PCR. Cells received 0.5 µM of either ASO or ScO against TLR2 ( C ) and TLR3 ( D ). After 48 h of incubation, cells were stimulated with 100 µg/ml poly(I-C) for 6 h followed by analysis of TNF-α by RT-PCR. The relative expression ( bottom panels ) of TLRs or TNF-α <t>(TLR/GAPDH</t> or TNF-α/GAPDH) was measured after scanning the bands with a Fluor Chem 8800 Imaging System (Alpha Innotech Corporation). Results represent mean ± SD of three independent experiments. a , p
    Sybr Premix Ex Taq, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 46419 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sybr premix ex taq/product/TaKaRa
    Average 99 stars, based on 46419 article reviews
    Price from $9.99 to $1999.99
    sybr premix ex taq - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    99
    Promega gotaq dna polymerase
    Effect of antisense knockdown of TLR2, TLR3, and TLR4 on LPS- and poly(I-C)-induced expression of <t>TNF-α</t> in mouse BV-2 microglial cells. Cells received 0.5 µM of either ASO or ScO against TLR2 ( A ) and TLR4 ( B ). After 48 h of incubation, cells were stimulated with 1 µg/ml LPS for 6 h followed by analysis of TNF-α by RT-PCR. Cells received 0.5 µM of either ASO or ScO against TLR2 ( C ) and TLR3 ( D ). After 48 h of incubation, cells were stimulated with 100 µg/ml poly(I-C) for 6 h followed by analysis of TNF-α by RT-PCR. The relative expression ( bottom panels ) of TLRs or TNF-α <t>(TLR/GAPDH</t> or TNF-α/GAPDH) was measured after scanning the bands with a Fluor Chem 8800 Imaging System (Alpha Innotech Corporation). Results represent mean ± SD of three independent experiments. a , p
    Gotaq Dna Polymerase, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 12825 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gotaq dna polymerase/product/Promega
    Average 99 stars, based on 12825 article reviews
    Price from $9.99 to $1999.99
    gotaq dna polymerase - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    99
    Qiagen quantitect reverse transcription kit
    Effect of antisense knockdown of TLR2, TLR3, and TLR4 on LPS- and poly(I-C)-induced expression of <t>TNF-α</t> in mouse BV-2 microglial cells. Cells received 0.5 µM of either ASO or ScO against TLR2 ( A ) and TLR4 ( B ). After 48 h of incubation, cells were stimulated with 1 µg/ml LPS for 6 h followed by analysis of TNF-α by RT-PCR. Cells received 0.5 µM of either ASO or ScO against TLR2 ( C ) and TLR3 ( D ). After 48 h of incubation, cells were stimulated with 100 µg/ml poly(I-C) for 6 h followed by analysis of TNF-α by RT-PCR. The relative expression ( bottom panels ) of TLRs or TNF-α <t>(TLR/GAPDH</t> or TNF-α/GAPDH) was measured after scanning the bands with a Fluor Chem 8800 Imaging System (Alpha Innotech Corporation). Results represent mean ± SD of three independent experiments. a , p
    Quantitect Reverse Transcription Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 44197 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/quantitect reverse transcription kit/product/Qiagen
    Average 99 stars, based on 44197 article reviews
    Price from $9.99 to $1999.99
    quantitect reverse transcription kit - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher taqman microrna reverse transcription kit
    Effect of antisense knockdown of TLR2, TLR3, and TLR4 on LPS- and poly(I-C)-induced expression of <t>TNF-α</t> in mouse BV-2 microglial cells. Cells received 0.5 µM of either ASO or ScO against TLR2 ( A ) and TLR4 ( B ). After 48 h of incubation, cells were stimulated with 1 µg/ml LPS for 6 h followed by analysis of TNF-α by RT-PCR. Cells received 0.5 µM of either ASO or ScO against TLR2 ( C ) and TLR3 ( D ). After 48 h of incubation, cells were stimulated with 100 µg/ml poly(I-C) for 6 h followed by analysis of TNF-α by RT-PCR. The relative expression ( bottom panels ) of TLRs or TNF-α <t>(TLR/GAPDH</t> or TNF-α/GAPDH) was measured after scanning the bands with a Fluor Chem 8800 Imaging System (Alpha Innotech Corporation). Results represent mean ± SD of three independent experiments. a , p
    Taqman Microrna Reverse Transcription Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 26114 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taqman microrna reverse transcription kit/product/Thermo Fisher
    Average 99 stars, based on 26114 article reviews
    Price from $9.99 to $1999.99
    taqman microrna reverse transcription kit - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher high capacity cdna reverse transcription kit
    Knockdown of LESR2 reduces cell growth, cell cycle progression, and migration of LECs in vitro . (a, b) Cell growth profiles and cell growth rates of neonatal LECs over 48h after ASOKD (a) or CRISPRi-KD (b) of LESR2 using IncuCyte. Sample’s confluences were normalized to T 0 . Growth rates were calculated as the slope of linear regression and normalized to control ASO/sgRNA. (c) Representative flow cytometry plots of neonatal LECs after 24h LESR2-ASOKD. Cells were firstly gated with live/dead Zombie staining (upper plots). Resulting living cells were further gated for non-proliferating stages subG0 and G0, and proliferating stages G1, S, G2, and M, using propidium iodide (IP) and Ki-67 (lower plots). (d) Quantification of the cell cycle progression analysis of neonatal LECs after 24h LESR2-ASOKD. Bars represent percentages of gated living cells in subG0, G0, G1, S, G2, and M. Statistical analysis was performed on G0 populations. (e) Representative images of the wound closure assay (9h) in neonatal LECs after LESR2-ASOKD. Confluence mask is shown for all time points. Before scratch, cells were incubated for 2h with 2 μ g/mL Mitomycin C (proliferation inhibitor) at 37°C. Scale bar represents 200 μ m. (f) Quantification of the wound closure assay (up to 9h) of neonatal LECs after LESR2-ASOKD. Percentages were determined for each time point using TScratch 98 . (g) Schematic representation of 3’ RACE results depicting the three LESR2 transcripts expressed in LECs: LESR2-1 (approx. 1,100bp), LESR2-2 (approx. 1,200bp), LESR2-3 (approx. 600bp). <t>RNA-Seq</t> signal was visualized through the Zenbu genome browser 106 . LESR2 transcript sequences are listed in Supplementary Table 7. (h) Comparison of qPCR levels of GAPDH (polyA+), H2BK (polyA-), LESR2-1, LESR2-2, LESR2-3 after <t>cDNA</t> synthesis with either oligodT or random hexamers primers in neonatal LECs derived from 3 donors. (i) Expression of LESR2-1, LESR2-2, and LESR2-3 relative to housekeeping gene GAPDH in neonatal LECs derived from 3 donors. (j) Quantification of the cell cycle progression analysis of pCDH-empty vector (pCDH-EV) and pCDH-LESR2 infected neonatal LECs after 24h LESR2-ASOKD. Statistical analysis was performed on G0 populations. (k) Quantification of the wound closure assay (up to 9h) of pCDH-EV and pCDH-LESR2 infected neonatal LECs after LESR2-ASOKD. Data are displayed as mean + SD (n = 10 in a, f, and k; n = 5 in b; n = 3 in h, i, and j; n = 2 in d). Percentages represent LESR2 knockdown efficiencies after the experiments. **P
    High Capacity Cdna Reverse Transcription Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 117891 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/high capacity cdna reverse transcription kit/product/Thermo Fisher
    Average 99 stars, based on 117891 article reviews
    Price from $9.99 to $1999.99
    high capacity cdna reverse transcription kit - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher rnase h
    RNase H-mediated degradation of pre-formed ASO:RNA duplexes and in vitro -synthesized RNAs targeted by ASOs. (A) Schematic of the experimental setup for panels B and C. Applicable for some ASOs: Y, 8-oxo-dG residue; +, LNA sugar base. (B, C) Cleavage of pre-formed ASO:target RNA duplexes by RNase H. (B) Five femtomoles of 33 P-labeled substrate was treated with <t>RNase</t> H for the indicated times. The reaction products were collected, denatured by heating at 95°C for 2 min and analyzed by PAGE in native 15% gels. Arrows at right point to the substrate (S) and major cleavage product(s) (P). Results from one of three independent reproducible experiments are shown. (C) Kinetics of RNase H cleavage of different ASO:RNA duplexes. The amounts of radioactivity remaining in the uncleaved substrate were quantified using a Typhoon Trio instrument. Quantifications were performed for each gel. The obtained values were normalized to the radioactivity present in the substrate before adding RNase H (set to 100%). Each point corresponds to the average of three independent experiments. Error bars indicate the standard deviation. (D) Cleavage of FR3131 RNA by RNase H in the presence of different ASOs. The RNA and ASOs were mixed and incubated at 37°C for 10 min; then, RNase H was added to the reaction mixture. RNA samples were collected at the indicated time points and analyzed by electrophoresis in native 0.8% TAE agarose gels. The results from one of three independent reproducible experiments are shown. S: substrate; P1 and P2: cleavage products.
    Rnase H, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9985 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase h/product/Thermo Fisher
    Average 99 stars, based on 9985 article reviews
    Price from $9.99 to $1999.99
    rnase h - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    98
    Millipore p irs1
    Effects of miR-494 on the insulin signaling pathway. The mimic form of miR-494 was overexpressed in cells. Cells were starved for 4 h before treatment with 0, 10, 100 nM of insulin for 20 min, and the levels of p-Akt (Ser473 and Thr308), p-AS160, p-p70S6K, and p-GSK-3α/β were measured in C 2 C 12 myoblasts (A) and CHO IR/IRS-1 cells (D). Bars show densitometric quantitation of phosphorylations of Akt, GSK-3α/β, AS160 and p70S6K in C 2 C 12 myoblasts (B) and CHO IR/IRS-1 cells (E). (C) The level of <t>p-IRS1</t> (Tyr608) was measured in insulin-treated C 2 C 12 myoblasts. GAPDH and actin were used as loading controls. Levels of phospho-proteins between miR-494 transfected cells and control were compared at each concentration of insulin. The values are expressed as the means ± SEM. *P
    P Irs1, supplied by Millipore, used in various techniques. Bioz Stars score: 98/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p irs1/product/Millipore
    Average 98 stars, based on 9 article reviews
    Price from $9.99 to $1999.99
    p irs1 - by Bioz Stars, 2020-08
    98/100 stars
      Buy from Supplier

    96
    Millipore tubastatin a
    Effect of genetic HDAC6 knockdown and increased acetylation levels of α-tubulin by HDAC6 inhibition. a RT-PCR validation of knockdown by an antisense oligonucleotide (ASO) against HDAC6 in MNs. b Quantification of the RT-PCR results of the gel shown in a illustrating the HDAC6 knockdown by the ASO. c Knockdown of HDAC6 using an ASO in patient-derived MNs increased axonal transport based on tracking mitochondrial movement (total number, moving number, and ratio between total and moving mitochondria). Scrambled ASO was used as a negative control (NTC ASO). n = 20, n = 19, n = 18, n = 10, n = 11, n = 10 for R521R, R521R + NTC ASO, R521R + HDAC6 ASO, R521H, R521H + NTC ASO, R521H + HDAC6 ASO, respectively; ANOVA with post-hoc Tukey’s test, * P value of 0.05. d Western blot from MNs (R521H mutant line 2/2 and isogenic control R521R), with and without treatment with ACY-738 or <t>Tubastatin</t> A at day 31 of differentiation. The blot was probed with antibodies directed to acetylated α-tubulin, and GAPDH. e Schematic representation of our results indicating that HDAC6 inhibition rescues axonal transport defects in FUS-iPSC-derived motor neurons through increasing acetylation levels of α-tubulin
    Tubastatin A, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 128 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tubastatin a/product/Millipore
    Average 96 stars, based on 128 article reviews
    Price from $9.99 to $1999.99
    tubastatin a - by Bioz Stars, 2020-08
    96/100 stars
      Buy from Supplier

    95
    Thermo Fisher rpad protocol
    ( A ) EBV circBARTs are expressed in EBV-positive tumor samples. RNase R-treated (+) or RNase R-untreated (−) RNAs from 6 EBV-positive and 11 EBV-negative (red font) pathologically diagnosed PTLD, NPC tumor xenoexplants C17 and C15, and an EBV-positive AIDS-associated lymphoma were tested by DP1 RT-PCR for circBART-BSJ1 and circBART-BSJ2 ( Top ). All six EBV-positive PTLDs were strongly positive, and three of the EBV-negative PTLDs were weakly positive for circBART RNAs. RT-PCR for LMP2 ( Middle ) and GAPDH ( Bottom ) mRNAs are shown. RNase R treatment enriched circBART junctions and depleted linear LMP2 and GAPDH products. ( B ) Confirmation of cyclization for EBV circBARTs by RNase H treatment. In vitro RNase H assays using annealed ASOs targeting BSJ1 and BSJ2 showed depletion of junctional sequences after RNase H treatment as monitored by DP1 PCR for Akata, sLCL, and Raji cell RNAs, but not in B95-8 <t>RNA.</t> GAPDH mRNA amplification was not affected by RNase H treatment. ( C ) <t>RPAD</t> analysis of EBV circBART. RNase R treatment followed by RPAD diminished linear 18S ribosomal RNA transcripts but increased circBART transcripts. Relative RNA was determined by normalizing the qPCR Ct values RPAD+ RNA to untreated control RNA (RPAD−). The data represent the means ± SD from four replicates.
    Rpad Protocol, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rpad protocol/product/Thermo Fisher
    Average 95 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    rpad protocol - by Bioz Stars, 2020-08
    95/100 stars
      Buy from Supplier

    Image Search Results


    Ectopic Expression of ASGP-R1 in U87 Cells Increases Efficacy of GN3-SMN-ASO In Vitro (A) Major and minor transmembrane domain (TM)-containing ASGP-R isoform cDNAs were cloned into retroviral and lentiviral expression vectors as indicated. The lentiviral vector carries the selection marker puromycin. ASGP-R H2c has a short 18-amino acid deletion in the intracellular domain of the receptor. (B) Western blot confirms expression of major and minor ASGP-R isoforms in U87 glioblastoma cells. The expression level of H1a is approximately 6-fold higher than the endogenous expression level in HepG2 cells, normalized to tubulin. H2a and H2b isoforms are detectable when expressed alone, but are stabilized in the presence of H1a. (C) Micrographs of U87 cells expressing ASGPR-H1a and H2b alone, and in combination. Cells were stained for ASGP-R1 (red), ASGP-R2 (green), and DAPI (blue). Arrowheads indicate non-uniform distribution of ASGP-R H2b, consistent with ER localization. Scale bar, 50 μm. (D) The SMN ASOs used in this study bind to intron 7 of SMN2 and promote exon 7 inclusion. Full-length SMN2 mRNA was quantified by radioactive RT-PCR; the product was digested with DdeI to separate SMN1 from SMN2 products. (E) U87 cells expressing major and minor ASGP-R isoforms alone or in combination were incubated with 300 nM unconjugated (SMN-MOE) or GalNAc-conjugated SMN-MOE ASOs (GN3-SMN-MOE) for 5 days by free uptake. Representative radiograph shows full-length SMN2 (top band) and SMN2 Δ exon 7 (bottom band). (F) Quantification of full-length SMN2 in ASO-treated U87 cells. The differences among the means in the SMN group (p = 0.0055) and the GN3-SMN group (p

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Delivery of GalNAc-Conjugated Splice-Switching ASOs to Non-hepatic Cells through Ectopic Expression of Asialoglycoprotein Receptor

    doi: 10.1016/j.omtn.2019.02.024

    Figure Lengend Snippet: Ectopic Expression of ASGP-R1 in U87 Cells Increases Efficacy of GN3-SMN-ASO In Vitro (A) Major and minor transmembrane domain (TM)-containing ASGP-R isoform cDNAs were cloned into retroviral and lentiviral expression vectors as indicated. The lentiviral vector carries the selection marker puromycin. ASGP-R H2c has a short 18-amino acid deletion in the intracellular domain of the receptor. (B) Western blot confirms expression of major and minor ASGP-R isoforms in U87 glioblastoma cells. The expression level of H1a is approximately 6-fold higher than the endogenous expression level in HepG2 cells, normalized to tubulin. H2a and H2b isoforms are detectable when expressed alone, but are stabilized in the presence of H1a. (C) Micrographs of U87 cells expressing ASGPR-H1a and H2b alone, and in combination. Cells were stained for ASGP-R1 (red), ASGP-R2 (green), and DAPI (blue). Arrowheads indicate non-uniform distribution of ASGP-R H2b, consistent with ER localization. Scale bar, 50 μm. (D) The SMN ASOs used in this study bind to intron 7 of SMN2 and promote exon 7 inclusion. Full-length SMN2 mRNA was quantified by radioactive RT-PCR; the product was digested with DdeI to separate SMN1 from SMN2 products. (E) U87 cells expressing major and minor ASGP-R isoforms alone or in combination were incubated with 300 nM unconjugated (SMN-MOE) or GalNAc-conjugated SMN-MOE ASOs (GN3-SMN-MOE) for 5 days by free uptake. Representative radiograph shows full-length SMN2 (top band) and SMN2 Δ exon 7 (bottom band). (F) Quantification of full-length SMN2 in ASO-treated U87 cells. The differences among the means in the SMN group (p = 0.0055) and the GN3-SMN group (p

    Article Snippet: [α-32P]-dCTP radiolabeled PCR products were digested with DdeI (NEB, Ipswich, MA, USA) for 2 h at 37°C and separated on a 5% native polyacrylamide gel (Bio-Rad, Hercules, CA, USA), analyzed on a Typhoon 9410 phosphorimager (GE Healthcare), and quantified using Multi Gauge v2.3 (Fujifilm, Tokyo, Japan).

    Techniques: Expressing, Allele-specific Oligonucleotide, In Vitro, Clone Assay, Plasmid Preparation, Selection, Marker, Western Blot, Staining, Reverse Transcription Polymerase Chain Reaction, Incubation

    Reduction of eRNA expression results in reduced expression of nearby mRNAs a , Q-PCR analysis of Mmp9 eRNA, and Mmp9 , NCoA5 and Cx3cr1 mRNAs for wildtype and Rev-Erb DKO thioglycollate-elicited macrophages transfected with Ctrl or Mmp9 eRNA siRNA (N WT = 4, and N DKO = 4). b , Q-PCR analysis of Cx3cr1 eRNA, and Cx3cr1, Csrnp1 and Mmp9 mRNAs for wildtype and Rev-Erb DKO bone marrow-derived macrophages transfected with siRNA targeting Cx3cr1 eRNA (N WT = 6, and N DKO = 5). c , Q-PCR analysis of Mmp9 eRNA and Mmp9 and Cx3cr1 mRNAs in thioglycollate-elicited macrophages transfected with the indicated antisense oligonucleotides (ASO, n = 3-7 per condition). d , Q-PCR analysis of Cx3cr1 eRNA and Cx3cr1, Mmp9 and Csrnpl mRNAs in BMDMs transfected with the indicated antisense oligonucleotides (ASO, n = 3-7 per condition). Data in a-d represent mean + s.d., with expression normalized to 36B4 in all cases. For a-b, statistical significance was determined by two tails Student’s t-test; for c-d, one-way ANOVA with Tukey HSD test. P value, * P

    Journal: Nature

    Article Title: Rev-Erbs repress macrophage gene expression by inhibiting enhancer-directed transcription

    doi: 10.1038/nature12209

    Figure Lengend Snippet: Reduction of eRNA expression results in reduced expression of nearby mRNAs a , Q-PCR analysis of Mmp9 eRNA, and Mmp9 , NCoA5 and Cx3cr1 mRNAs for wildtype and Rev-Erb DKO thioglycollate-elicited macrophages transfected with Ctrl or Mmp9 eRNA siRNA (N WT = 4, and N DKO = 4). b , Q-PCR analysis of Cx3cr1 eRNA, and Cx3cr1, Csrnp1 and Mmp9 mRNAs for wildtype and Rev-Erb DKO bone marrow-derived macrophages transfected with siRNA targeting Cx3cr1 eRNA (N WT = 6, and N DKO = 5). c , Q-PCR analysis of Mmp9 eRNA and Mmp9 and Cx3cr1 mRNAs in thioglycollate-elicited macrophages transfected with the indicated antisense oligonucleotides (ASO, n = 3-7 per condition). d , Q-PCR analysis of Cx3cr1 eRNA and Cx3cr1, Mmp9 and Csrnpl mRNAs in BMDMs transfected with the indicated antisense oligonucleotides (ASO, n = 3-7 per condition). Data in a-d represent mean + s.d., with expression normalized to 36B4 in all cases. For a-b, statistical significance was determined by two tails Student’s t-test; for c-d, one-way ANOVA with Tukey HSD test. P value, * P

    Article Snippet: The following siRNA oligos were used for in vitro studies, and the underlined siRNA were used for in vivo experiments. siGENOME Non-targeting siRNA pool #2 (Thermo Science, D-001810): UAAGGCUAUGAAGAGAUAC , AUGUAUUGGCCUGUAUUAG, AUGAACGUGAAUUGCUCAA , UGGUUUACAUGUCGACUAA.

    Techniques: Expressing, Polymerase Chain Reaction, Transfection, Derivative Assay, Allele-specific Oligonucleotide

    miR-221 regulates CD44 protein expression post transcriptionally CD44 protein was measured in SNU-449 or Sk-Hep-1 cells transfected with 50 or 100 nM of anti-miR-221 or scrambled control (SC) oligo for 48 (A) or 96 h (B). CD44 mRNA was measured by qPCR in SNU-449 or Sk-Hep-1 cells transfected with 50 or 100 nM of anti-miR-221 or scrambled control (SC-ASO) oligo for 48 (C) or 96 h (D). (E) Huh7 cells were transfected with 100 nM of miR-221 mimic or negative control (NC) oligo for 72 h. CD44 expression was determined by immunoblotting.

    Journal: Biochemical and biophysical research communications

    Article Title: miR-221 REGULATES CD44 IN HEPATOCELLULAR CARCINOMA THROUGH THE PI3K-AKT-mTOR PATHWAY

    doi: 10.1016/j.bbrc.2017.04.121

    Figure Lengend Snippet: miR-221 regulates CD44 protein expression post transcriptionally CD44 protein was measured in SNU-449 or Sk-Hep-1 cells transfected with 50 or 100 nM of anti-miR-221 or scrambled control (SC) oligo for 48 (A) or 96 h (B). CD44 mRNA was measured by qPCR in SNU-449 or Sk-Hep-1 cells transfected with 50 or 100 nM of anti-miR-221 or scrambled control (SC-ASO) oligo for 48 (C) or 96 h (D). (E) Huh7 cells were transfected with 100 nM of miR-221 mimic or negative control (NC) oligo for 72 h. CD44 expression was determined by immunoblotting.

    Article Snippet: Anti-miR-221, anti-miR-708-5p and scrambled control oligo were synthesized from ThermoFisher (Lafayette, CO).

    Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Allele-specific Oligonucleotide, Negative Control

    miR-708-5p targets CD44 in HCC, anti-miR-708-5p rescues anti-miR-221 inhibition of CD44 (A) The differential expression of miRNAs in SNU-449 cells treated with anti-miR-221 or control oligo are presented as a volcano plot. Data were generated by profiling over 900 miRNAs by qPCR. Only those miRNAs that were expressed as defined in the Methods section are presented. The x-axis represents the log 2 fold change in the anti-miR-221 compared to control treated cells. The y-axis is the P values from the anti-miR-221 to control oligo comparison. The lines represent the fold change = 1.5 and P= 0.05 cutoffs. (B) The ability of miR-708-5p to target CD44 is shown using luciferase reporter assay in HEK293T cells transfected with miR-708-5p along with a vector containing the 3′ UTR of CD44 downstream of the luciferase gene. (C) SNU-449 cells were exposed to (left to right on immunoblot) media, media plus lipofectamine, 100 nM control oligo, 50 nM each of anti-miR-221/control oligo, 50 nM each of anti-miR-221/anti-miR-708-5p or 50 nM each of anti-miR-708-5p/control oligo. Total protein was resolved by gel electrophoresis and the immunoblot was probed for CD44 and GAPDH.

    Journal: Biochemical and biophysical research communications

    Article Title: miR-221 REGULATES CD44 IN HEPATOCELLULAR CARCINOMA THROUGH THE PI3K-AKT-mTOR PATHWAY

    doi: 10.1016/j.bbrc.2017.04.121

    Figure Lengend Snippet: miR-708-5p targets CD44 in HCC, anti-miR-708-5p rescues anti-miR-221 inhibition of CD44 (A) The differential expression of miRNAs in SNU-449 cells treated with anti-miR-221 or control oligo are presented as a volcano plot. Data were generated by profiling over 900 miRNAs by qPCR. Only those miRNAs that were expressed as defined in the Methods section are presented. The x-axis represents the log 2 fold change in the anti-miR-221 compared to control treated cells. The y-axis is the P values from the anti-miR-221 to control oligo comparison. The lines represent the fold change = 1.5 and P= 0.05 cutoffs. (B) The ability of miR-708-5p to target CD44 is shown using luciferase reporter assay in HEK293T cells transfected with miR-708-5p along with a vector containing the 3′ UTR of CD44 downstream of the luciferase gene. (C) SNU-449 cells were exposed to (left to right on immunoblot) media, media plus lipofectamine, 100 nM control oligo, 50 nM each of anti-miR-221/control oligo, 50 nM each of anti-miR-221/anti-miR-708-5p or 50 nM each of anti-miR-708-5p/control oligo. Total protein was resolved by gel electrophoresis and the immunoblot was probed for CD44 and GAPDH.

    Article Snippet: Anti-miR-221, anti-miR-708-5p and scrambled control oligo were synthesized from ThermoFisher (Lafayette, CO).

    Techniques: Inhibition, Expressing, Generated, Real-time Polymerase Chain Reaction, Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Nucleic Acid Electrophoresis

    miR-221 regulates CD44 expression through the mTOR pathway (A) SNU-449 cells were transfected with 50 or 100 nM of anti-miR-221 or control oligo for 48 and 96 h. Phosphorylated 4EBP-1 and other proteins were measured by immunoblotting. (B) SNU-423 and SNU-449 cells were treated with 2.5 or 5 μM of the ATP-competitive inhibitor of mTOR PP242 for 48 h. (C) The mRNA expression of CD44 and xCT was measured by qPCR in SNU-423 and SNU-449 cells treated with 2.5 or 5 μM of PP242 or vehicle control for 48 h.

    Journal: Biochemical and biophysical research communications

    Article Title: miR-221 REGULATES CD44 IN HEPATOCELLULAR CARCINOMA THROUGH THE PI3K-AKT-mTOR PATHWAY

    doi: 10.1016/j.bbrc.2017.04.121

    Figure Lengend Snippet: miR-221 regulates CD44 expression through the mTOR pathway (A) SNU-449 cells were transfected with 50 or 100 nM of anti-miR-221 or control oligo for 48 and 96 h. Phosphorylated 4EBP-1 and other proteins were measured by immunoblotting. (B) SNU-423 and SNU-449 cells were treated with 2.5 or 5 μM of the ATP-competitive inhibitor of mTOR PP242 for 48 h. (C) The mRNA expression of CD44 and xCT was measured by qPCR in SNU-423 and SNU-449 cells treated with 2.5 or 5 μM of PP242 or vehicle control for 48 h.

    Article Snippet: Anti-miR-221, anti-miR-708-5p and scrambled control oligo were synthesized from ThermoFisher (Lafayette, CO).

    Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction

    miR-495 inhibited cell migration and invasion in BGC-823 and HGC-27 cell lines and regulated EMT factors. Pre-miR-495, cells were transfected with miR-495 precursor to overexpress miR-495. miR-495 ASO, cells transfected with miR-495 antisense oligo (ASO) to inhibit miR-495. Pre-ASO control, the corresponding control group of pre-miR-495 or miR-495 ASO. qRT-PCR, Western blot and Transwell assays were performed at 48 hours after transfection. ( A ) miR-495 was successfully promoted or inhibited by cell transfection in BGC-823 and HGC-27 cells. ( B ) miR-495 inhibited, and its ASO promoted, cell migration in BGC-823 and HGC-27 cells. ( C ) miR-495 inhibited, and its ASO promoted, cell invasion in BGC-823 and HGC-27 cells. ( D ) miR-495 upregulated CDH1 and downregulated VIM and ACTA2 mRNA levels in BGC-823 cells. ( E ) miR-495 upregulated CDH1 and downregulated VIM and ACTA2 protein levels in BGC-823 cells. GAPDH mRNA and protein were used as internal references for qRT-PCR and Western blot, respectively. * p

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: MicroRNA-495 Inhibits Gastric Cancer Cell Migration and Invasion Possibly via Targeting High Mobility Group AT-Hook 2 (HMGA2)

    doi: 10.12659/MSM.898740

    Figure Lengend Snippet: miR-495 inhibited cell migration and invasion in BGC-823 and HGC-27 cell lines and regulated EMT factors. Pre-miR-495, cells were transfected with miR-495 precursor to overexpress miR-495. miR-495 ASO, cells transfected with miR-495 antisense oligo (ASO) to inhibit miR-495. Pre-ASO control, the corresponding control group of pre-miR-495 or miR-495 ASO. qRT-PCR, Western blot and Transwell assays were performed at 48 hours after transfection. ( A ) miR-495 was successfully promoted or inhibited by cell transfection in BGC-823 and HGC-27 cells. ( B ) miR-495 inhibited, and its ASO promoted, cell migration in BGC-823 and HGC-27 cells. ( C ) miR-495 inhibited, and its ASO promoted, cell invasion in BGC-823 and HGC-27 cells. ( D ) miR-495 upregulated CDH1 and downregulated VIM and ACTA2 mRNA levels in BGC-823 cells. ( E ) miR-495 upregulated CDH1 and downregulated VIM and ACTA2 protein levels in BGC-823 cells. GAPDH mRNA and protein were used as internal references for qRT-PCR and Western blot, respectively. * p

    Article Snippet: The miR-495-specific precursor miRNA (pre-miR495), antisense oligo (ASO) and the corresponding controls were synthesized by Ambion (Carlsbad, CA, USA) to alter the miR-495 levels.

    Techniques: Migration, Transfection, Allele-specific Oligonucleotide, Quantitative RT-PCR, Western Blot

    miR-495 directly binds to and inhibits HMGA2 . pre-miR-495, cells were transfected with miR-495 precursor to overexpress miR-495. miR-495 ASO, cells transfected with miR-495 antisense oligo (ASO) to inhibit miR-495. Pre-ASO control, the corresponding control group of pre-miR-495 or miR-495 ASO. wt, wildtype. mut, mutant type. ( A ) A schematic diagram of HMGA2 3′UTR-wt, and HMGA2 3′UTR-mut and their predicted interaction with miR-495. The mutated bases are underlined. ( B ) Dual-luciferase reporter assay results of altered HMGA2 3′UTR activity by miR-495 in cell lines BGC-823 and HGC-27. ( C ) qRT-PCR showing relative level of HMGA2 mRNA in transfected BGC-823 cells. ( D ) Relative HMGA2 protein level in transfected BGC-823 cells based on the Western blot result, as shown in ( E ). GAPDH mRNA and protein were used as internal references for qRT-PCR and Western blot, respectively. NS – not significant. * p

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: MicroRNA-495 Inhibits Gastric Cancer Cell Migration and Invasion Possibly via Targeting High Mobility Group AT-Hook 2 (HMGA2)

    doi: 10.12659/MSM.898740

    Figure Lengend Snippet: miR-495 directly binds to and inhibits HMGA2 . pre-miR-495, cells were transfected with miR-495 precursor to overexpress miR-495. miR-495 ASO, cells transfected with miR-495 antisense oligo (ASO) to inhibit miR-495. Pre-ASO control, the corresponding control group of pre-miR-495 or miR-495 ASO. wt, wildtype. mut, mutant type. ( A ) A schematic diagram of HMGA2 3′UTR-wt, and HMGA2 3′UTR-mut and their predicted interaction with miR-495. The mutated bases are underlined. ( B ) Dual-luciferase reporter assay results of altered HMGA2 3′UTR activity by miR-495 in cell lines BGC-823 and HGC-27. ( C ) qRT-PCR showing relative level of HMGA2 mRNA in transfected BGC-823 cells. ( D ) Relative HMGA2 protein level in transfected BGC-823 cells based on the Western blot result, as shown in ( E ). GAPDH mRNA and protein were used as internal references for qRT-PCR and Western blot, respectively. NS – not significant. * p

    Article Snippet: The miR-495-specific precursor miRNA (pre-miR495), antisense oligo (ASO) and the corresponding controls were synthesized by Ambion (Carlsbad, CA, USA) to alter the miR-495 levels.

    Techniques: Transfection, Allele-specific Oligonucleotide, Mutagenesis, Luciferase, Reporter Assay, Activity Assay, Quantitative RT-PCR, Western Blot

    In vivo activity of a novel ligand-conjugated antisense (LICA) oligonucleotide. We treated TR; HSA LR bi-transgenic mice with saline or a novel LICA oligonucleotide, ASO 992948, targeting ACTA1 -CUG exp transcripts and measured DsRed and GFP fluorescence by serial in vivo spectroscopy. LICA-oligo doses were 2.5, 8.3, or 25 mg/kg twice weekly for 4 weeks (8 total doses) by subcutaneous injection ( N = 2 each group). However, due to low DsRed/GFP fluorescence measurements in gastrocnemius muscles, mice receiving the 2.5 mg/kg and 8.3 mg/kg doses received four additional injections over the next 2 weeks, for a total of 12 doses. a Serial DsRed/GFP measurements in gastrocnemius and lumbar paraspinal muscles through Day 42. The final dose in the saline (black circles) and 25 mg/kg groups (blue diamonds) was Day 24, while the final dose in the 2.5 (yellow triangles) and 8.3 mg/kg (gray triangles) groups was Day 37. Error bars indicate mean ± s.e.m. b Quantitation of ACTA1 -CUG exp transcript levels (copies per microliter cDNA) by droplet digital PCR in muscles collected at Day 42. Error bars indicate mean ± s.e.m. **** P

    Journal: Nature Communications

    Article Title: Non-invasive monitoring of alternative splicing outcomes to identify candidate therapies for myotonic dystrophy type 1

    doi: 10.1038/s41467-018-07517-y

    Figure Lengend Snippet: In vivo activity of a novel ligand-conjugated antisense (LICA) oligonucleotide. We treated TR; HSA LR bi-transgenic mice with saline or a novel LICA oligonucleotide, ASO 992948, targeting ACTA1 -CUG exp transcripts and measured DsRed and GFP fluorescence by serial in vivo spectroscopy. LICA-oligo doses were 2.5, 8.3, or 25 mg/kg twice weekly for 4 weeks (8 total doses) by subcutaneous injection ( N = 2 each group). However, due to low DsRed/GFP fluorescence measurements in gastrocnemius muscles, mice receiving the 2.5 mg/kg and 8.3 mg/kg doses received four additional injections over the next 2 weeks, for a total of 12 doses. a Serial DsRed/GFP measurements in gastrocnemius and lumbar paraspinal muscles through Day 42. The final dose in the saline (black circles) and 25 mg/kg groups (blue diamonds) was Day 24, while the final dose in the 2.5 (yellow triangles) and 8.3 mg/kg (gray triangles) groups was Day 37. Error bars indicate mean ± s.e.m. b Quantitation of ACTA1 -CUG exp transcript levels (copies per microliter cDNA) by droplet digital PCR in muscles collected at Day 42. Error bars indicate mean ± s.e.m. **** P

    Article Snippet: We made cDNA using Superscript II reverse transcriptase (Life Technologies) and oligo dT, and performed PCR using Amplitaq Gold (Life Technologies) and gene-specific primers.

    Techniques: In Vivo, Activity Assay, Transgenic Assay, Mouse Assay, Allele-specific Oligonucleotide, Fluorescence, Spectroscopy, Injection, Quantitation Assay, Digital PCR

    In vivo comparison of LICA and the unconjugated parent ASO. We treated TR; HSA LR mice with LICA oligo 992948 (LICA) or ASO 445236 (ASO), which is the unconjugated parent of LICA oligo 992948 that targets the identical ACTA1 . ASOs were administered by subcutaneous injection of 12.5 mg/kg twice weekly for 4 weeks ( N = 3 each) (eight total doses). Treatment with saline ( N = 1) served as a control. a DsRed/GFP quantitative fluorescence in gastrocnemius (left) and lumbar paraspinal muscles (right) by serial in vivo spectroscopy in mice treated with saline (black circles), ASO (yellow triangles), or LICA (blue diamonds). Error bars indicate mean ± s.e.m. b Due to the higher baseline DsRed/GFP values in the gastrocnemius of mice randomized to receive the unconjugated ASO, we normalized each measurement of quantitative fluorescence in the gastrocnemius (left) and lumbar paraspinal muscles (right) to the Day 0 value, so that the Day 0 value for each muscle = 1. Error bars indicate mean ± s.e.m. c ddPCR quantitation of ACTA1 transcripts (copies per microliter cDNA) in muscles collected at imaging Day 28. **** P

    Journal: Nature Communications

    Article Title: Non-invasive monitoring of alternative splicing outcomes to identify candidate therapies for myotonic dystrophy type 1

    doi: 10.1038/s41467-018-07517-y

    Figure Lengend Snippet: In vivo comparison of LICA and the unconjugated parent ASO. We treated TR; HSA LR mice with LICA oligo 992948 (LICA) or ASO 445236 (ASO), which is the unconjugated parent of LICA oligo 992948 that targets the identical ACTA1 . ASOs were administered by subcutaneous injection of 12.5 mg/kg twice weekly for 4 weeks ( N = 3 each) (eight total doses). Treatment with saline ( N = 1) served as a control. a DsRed/GFP quantitative fluorescence in gastrocnemius (left) and lumbar paraspinal muscles (right) by serial in vivo spectroscopy in mice treated with saline (black circles), ASO (yellow triangles), or LICA (blue diamonds). Error bars indicate mean ± s.e.m. b Due to the higher baseline DsRed/GFP values in the gastrocnemius of mice randomized to receive the unconjugated ASO, we normalized each measurement of quantitative fluorescence in the gastrocnemius (left) and lumbar paraspinal muscles (right) to the Day 0 value, so that the Day 0 value for each muscle = 1. Error bars indicate mean ± s.e.m. c ddPCR quantitation of ACTA1 transcripts (copies per microliter cDNA) in muscles collected at imaging Day 28. **** P

    Article Snippet: We made cDNA using Superscript II reverse transcriptase (Life Technologies) and oligo dT, and performed PCR using Amplitaq Gold (Life Technologies) and gene-specific primers.

    Techniques: In Vivo, Allele-specific Oligonucleotide, Mouse Assay, Injection, Fluorescence, Spectroscopy, Quantitation Assay, Imaging

    Effect of antisense knockdown of TLR2, TLR3, and TLR4 on LPS- and poly(I-C)-induced expression of TNF-α in mouse BV-2 microglial cells. Cells received 0.5 µM of either ASO or ScO against TLR2 ( A ) and TLR4 ( B ). After 48 h of incubation, cells were stimulated with 1 µg/ml LPS for 6 h followed by analysis of TNF-α by RT-PCR. Cells received 0.5 µM of either ASO or ScO against TLR2 ( C ) and TLR3 ( D ). After 48 h of incubation, cells were stimulated with 100 µg/ml poly(I-C) for 6 h followed by analysis of TNF-α by RT-PCR. The relative expression ( bottom panels ) of TLRs or TNF-α (TLR/GAPDH or TNF-α/GAPDH) was measured after scanning the bands with a Fluor Chem 8800 Imaging System (Alpha Innotech Corporation). Results represent mean ± SD of three independent experiments. a , p

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Fibrillar Amyloid-? Peptides Activate Microglia via TLR2: Implications for Alzheimer's Disease 1

    doi:

    Figure Lengend Snippet: Effect of antisense knockdown of TLR2, TLR3, and TLR4 on LPS- and poly(I-C)-induced expression of TNF-α in mouse BV-2 microglial cells. Cells received 0.5 µM of either ASO or ScO against TLR2 ( A ) and TLR4 ( B ). After 48 h of incubation, cells were stimulated with 1 µg/ml LPS for 6 h followed by analysis of TNF-α by RT-PCR. Cells received 0.5 µM of either ASO or ScO against TLR2 ( C ) and TLR3 ( D ). After 48 h of incubation, cells were stimulated with 100 µg/ml poly(I-C) for 6 h followed by analysis of TNF-α by RT-PCR. The relative expression ( bottom panels ) of TLRs or TNF-α (TLR/GAPDH or TNF-α/GAPDH) was measured after scanning the bands with a Fluor Chem 8800 Imaging System (Alpha Innotech Corporation). Results represent mean ± SD of three independent experiments. a , p

    Article Snippet: All primers and FAM-labeled probes for mouse TNF-α, iNOS, IL-6, IL-1β, and GAPDH were obtained from Applied Biosystems.

    Techniques: Expressing, Allele-specific Oligonucleotide, Incubation, Reverse Transcription Polymerase Chain Reaction, Imaging

    Knockdown of LESR2 reduces cell growth, cell cycle progression, and migration of LECs in vitro . (a, b) Cell growth profiles and cell growth rates of neonatal LECs over 48h after ASOKD (a) or CRISPRi-KD (b) of LESR2 using IncuCyte. Sample’s confluences were normalized to T 0 . Growth rates were calculated as the slope of linear regression and normalized to control ASO/sgRNA. (c) Representative flow cytometry plots of neonatal LECs after 24h LESR2-ASOKD. Cells were firstly gated with live/dead Zombie staining (upper plots). Resulting living cells were further gated for non-proliferating stages subG0 and G0, and proliferating stages G1, S, G2, and M, using propidium iodide (IP) and Ki-67 (lower plots). (d) Quantification of the cell cycle progression analysis of neonatal LECs after 24h LESR2-ASOKD. Bars represent percentages of gated living cells in subG0, G0, G1, S, G2, and M. Statistical analysis was performed on G0 populations. (e) Representative images of the wound closure assay (9h) in neonatal LECs after LESR2-ASOKD. Confluence mask is shown for all time points. Before scratch, cells were incubated for 2h with 2 μ g/mL Mitomycin C (proliferation inhibitor) at 37°C. Scale bar represents 200 μ m. (f) Quantification of the wound closure assay (up to 9h) of neonatal LECs after LESR2-ASOKD. Percentages were determined for each time point using TScratch 98 . (g) Schematic representation of 3’ RACE results depicting the three LESR2 transcripts expressed in LECs: LESR2-1 (approx. 1,100bp), LESR2-2 (approx. 1,200bp), LESR2-3 (approx. 600bp). RNA-Seq signal was visualized through the Zenbu genome browser 106 . LESR2 transcript sequences are listed in Supplementary Table 7. (h) Comparison of qPCR levels of GAPDH (polyA+), H2BK (polyA-), LESR2-1, LESR2-2, LESR2-3 after cDNA synthesis with either oligodT or random hexamers primers in neonatal LECs derived from 3 donors. (i) Expression of LESR2-1, LESR2-2, and LESR2-3 relative to housekeeping gene GAPDH in neonatal LECs derived from 3 donors. (j) Quantification of the cell cycle progression analysis of pCDH-empty vector (pCDH-EV) and pCDH-LESR2 infected neonatal LECs after 24h LESR2-ASOKD. Statistical analysis was performed on G0 populations. (k) Quantification of the wound closure assay (up to 9h) of pCDH-EV and pCDH-LESR2 infected neonatal LECs after LESR2-ASOKD. Data are displayed as mean + SD (n = 10 in a, f, and k; n = 5 in b; n = 3 in h, i, and j; n = 2 in d). Percentages represent LESR2 knockdown efficiencies after the experiments. **P

    Journal: bioRxiv

    Article Title: LESR2 is a lymphatic endothelial-specific lncRNA that governs cell proliferation and migration through KLF4 and SEMA3C

    doi: 10.1101/2020.05.25.114546

    Figure Lengend Snippet: Knockdown of LESR2 reduces cell growth, cell cycle progression, and migration of LECs in vitro . (a, b) Cell growth profiles and cell growth rates of neonatal LECs over 48h after ASOKD (a) or CRISPRi-KD (b) of LESR2 using IncuCyte. Sample’s confluences were normalized to T 0 . Growth rates were calculated as the slope of linear regression and normalized to control ASO/sgRNA. (c) Representative flow cytometry plots of neonatal LECs after 24h LESR2-ASOKD. Cells were firstly gated with live/dead Zombie staining (upper plots). Resulting living cells were further gated for non-proliferating stages subG0 and G0, and proliferating stages G1, S, G2, and M, using propidium iodide (IP) and Ki-67 (lower plots). (d) Quantification of the cell cycle progression analysis of neonatal LECs after 24h LESR2-ASOKD. Bars represent percentages of gated living cells in subG0, G0, G1, S, G2, and M. Statistical analysis was performed on G0 populations. (e) Representative images of the wound closure assay (9h) in neonatal LECs after LESR2-ASOKD. Confluence mask is shown for all time points. Before scratch, cells were incubated for 2h with 2 μ g/mL Mitomycin C (proliferation inhibitor) at 37°C. Scale bar represents 200 μ m. (f) Quantification of the wound closure assay (up to 9h) of neonatal LECs after LESR2-ASOKD. Percentages were determined for each time point using TScratch 98 . (g) Schematic representation of 3’ RACE results depicting the three LESR2 transcripts expressed in LECs: LESR2-1 (approx. 1,100bp), LESR2-2 (approx. 1,200bp), LESR2-3 (approx. 600bp). RNA-Seq signal was visualized through the Zenbu genome browser 106 . LESR2 transcript sequences are listed in Supplementary Table 7. (h) Comparison of qPCR levels of GAPDH (polyA+), H2BK (polyA-), LESR2-1, LESR2-2, LESR2-3 after cDNA synthesis with either oligodT or random hexamers primers in neonatal LECs derived from 3 donors. (i) Expression of LESR2-1, LESR2-2, and LESR2-3 relative to housekeeping gene GAPDH in neonatal LECs derived from 3 donors. (j) Quantification of the cell cycle progression analysis of pCDH-empty vector (pCDH-EV) and pCDH-LESR2 infected neonatal LECs after 24h LESR2-ASOKD. Statistical analysis was performed on G0 populations. (k) Quantification of the wound closure assay (up to 9h) of pCDH-EV and pCDH-LESR2 infected neonatal LECs after LESR2-ASOKD. Data are displayed as mean + SD (n = 10 in a, f, and k; n = 5 in b; n = 3 in h, i, and j; n = 2 in d). Percentages represent LESR2 knockdown efficiencies after the experiments. **P

    Article Snippet: Equal amounts of total RNA were reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), according to the manufacturer’s instructions.

    Techniques: Migration, In Vitro, Allele-specific Oligonucleotide, Flow Cytometry, Staining, Wound Closure Assay, Incubation, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Derivative Assay, Expressing, Plasmid Preparation, Infection

    RNase H-mediated degradation of pre-formed ASO:RNA duplexes and in vitro -synthesized RNAs targeted by ASOs. (A) Schematic of the experimental setup for panels B and C. Applicable for some ASOs: Y, 8-oxo-dG residue; +, LNA sugar base. (B, C) Cleavage of pre-formed ASO:target RNA duplexes by RNase H. (B) Five femtomoles of 33 P-labeled substrate was treated with RNase H for the indicated times. The reaction products were collected, denatured by heating at 95°C for 2 min and analyzed by PAGE in native 15% gels. Arrows at right point to the substrate (S) and major cleavage product(s) (P). Results from one of three independent reproducible experiments are shown. (C) Kinetics of RNase H cleavage of different ASO:RNA duplexes. The amounts of radioactivity remaining in the uncleaved substrate were quantified using a Typhoon Trio instrument. Quantifications were performed for each gel. The obtained values were normalized to the radioactivity present in the substrate before adding RNase H (set to 100%). Each point corresponds to the average of three independent experiments. Error bars indicate the standard deviation. (D) Cleavage of FR3131 RNA by RNase H in the presence of different ASOs. The RNA and ASOs were mixed and incubated at 37°C for 10 min; then, RNase H was added to the reaction mixture. RNA samples were collected at the indicated time points and analyzed by electrophoresis in native 0.8% TAE agarose gels. The results from one of three independent reproducible experiments are shown. S: substrate; P1 and P2: cleavage products.

    Journal: PLoS ONE

    Article Title: RNA Interference-Guided Targeting of Hepatitis C Virus Replication with Antisense Locked Nucleic Acid-Based Oligonucleotides Containing 8-oxo-dG Modifications

    doi: 10.1371/journal.pone.0128686

    Figure Lengend Snippet: RNase H-mediated degradation of pre-formed ASO:RNA duplexes and in vitro -synthesized RNAs targeted by ASOs. (A) Schematic of the experimental setup for panels B and C. Applicable for some ASOs: Y, 8-oxo-dG residue; +, LNA sugar base. (B, C) Cleavage of pre-formed ASO:target RNA duplexes by RNase H. (B) Five femtomoles of 33 P-labeled substrate was treated with RNase H for the indicated times. The reaction products were collected, denatured by heating at 95°C for 2 min and analyzed by PAGE in native 15% gels. Arrows at right point to the substrate (S) and major cleavage product(s) (P). Results from one of three independent reproducible experiments are shown. (C) Kinetics of RNase H cleavage of different ASO:RNA duplexes. The amounts of radioactivity remaining in the uncleaved substrate were quantified using a Typhoon Trio instrument. Quantifications were performed for each gel. The obtained values were normalized to the radioactivity present in the substrate before adding RNase H (set to 100%). Each point corresponds to the average of three independent experiments. Error bars indicate the standard deviation. (D) Cleavage of FR3131 RNA by RNase H in the presence of different ASOs. The RNA and ASOs were mixed and incubated at 37°C for 10 min; then, RNase H was added to the reaction mixture. RNA samples were collected at the indicated time points and analyzed by electrophoresis in native 0.8% TAE agarose gels. The results from one of three independent reproducible experiments are shown. S: substrate; P1 and P2: cleavage products.

    Article Snippet: This target RNA was pre-incubated with D4676, DM4676, LD4676 or LDM4676 for 10 min at 37°C; next, RNase H was added to the reaction mixture.

    Techniques: Allele-specific Oligonucleotide, In Vitro, Synthesized, Labeling, Polyacrylamide Gel Electrophoresis, Radioactivity, Standard Deviation, Incubation, Electrophoresis

    Effects of miR-494 on the insulin signaling pathway. The mimic form of miR-494 was overexpressed in cells. Cells were starved for 4 h before treatment with 0, 10, 100 nM of insulin for 20 min, and the levels of p-Akt (Ser473 and Thr308), p-AS160, p-p70S6K, and p-GSK-3α/β were measured in C 2 C 12 myoblasts (A) and CHO IR/IRS-1 cells (D). Bars show densitometric quantitation of phosphorylations of Akt, GSK-3α/β, AS160 and p70S6K in C 2 C 12 myoblasts (B) and CHO IR/IRS-1 cells (E). (C) The level of p-IRS1 (Tyr608) was measured in insulin-treated C 2 C 12 myoblasts. GAPDH and actin were used as loading controls. Levels of phospho-proteins between miR-494 transfected cells and control were compared at each concentration of insulin. The values are expressed as the means ± SEM. *P

    Journal: PLoS ONE

    Article Title: MicroRNA-494, Upregulated by Tumor Necrosis Factor-?, Desensitizes Insulin Effect in C2C12 Muscle Cells

    doi: 10.1371/journal.pone.0083471

    Figure Lengend Snippet: Effects of miR-494 on the insulin signaling pathway. The mimic form of miR-494 was overexpressed in cells. Cells were starved for 4 h before treatment with 0, 10, 100 nM of insulin for 20 min, and the levels of p-Akt (Ser473 and Thr308), p-AS160, p-p70S6K, and p-GSK-3α/β were measured in C 2 C 12 myoblasts (A) and CHO IR/IRS-1 cells (D). Bars show densitometric quantitation of phosphorylations of Akt, GSK-3α/β, AS160 and p70S6K in C 2 C 12 myoblasts (B) and CHO IR/IRS-1 cells (E). (C) The level of p-IRS1 (Tyr608) was measured in insulin-treated C 2 C 12 myoblasts. GAPDH and actin were used as loading controls. Levels of phospho-proteins between miR-494 transfected cells and control were compared at each concentration of insulin. The values are expressed as the means ± SEM. *P

    Article Snippet: Antibody for p-IRS1 (Y612) was from Millipore (Billerica, MA, USA).

    Techniques: Quantitation Assay, Transfection, Concentration Assay

    Regulation of putative target genes by miR-494. (A) The secondary structure of mouse premature(pre)-miR-494 (miRbase, Accession MI0003532). (B) Alignment of the seed matches in conserved sites of Stxbp5, Igf1r, Rab35, Irs1 genes at 3’ untranslated region (3’ UTR) and seed types of each target gene. Entrez accession numbers and binding position at 3’ UTR are addressed for the representative transcript of each gene. Complementary sequences binding to miR-494 are shown in bold letters, with the m8 and A1 extensions highlighted in bold italic letters. Gene abbreviations: Stxbp5 , syntaxin binding protein 5; Igf1r , insulin-like growth factor 1 receptor; Irs1, insulin receptor substrate 1 (C) RT-PCR analysis for predicted target genes in miR-494 mimic- or miR-494 ASO-transfected C 2 C 12 cells. Gapdh gene was used as a loading control. (E) Immunoblot analysis of molecules related to the insulin signaling pathway. Cells were transfected with miR-494 mimic and subjected to immunoblot analysis. Antibodies for PTEN, p-PTP1B (S50) and IRS-1 were used. GAPDH was used as a loading control. The values were expressed as the means ± SEM and compared between miR-494 mimic transfected cells and control. *P

    Journal: PLoS ONE

    Article Title: MicroRNA-494, Upregulated by Tumor Necrosis Factor-?, Desensitizes Insulin Effect in C2C12 Muscle Cells

    doi: 10.1371/journal.pone.0083471

    Figure Lengend Snippet: Regulation of putative target genes by miR-494. (A) The secondary structure of mouse premature(pre)-miR-494 (miRbase, Accession MI0003532). (B) Alignment of the seed matches in conserved sites of Stxbp5, Igf1r, Rab35, Irs1 genes at 3’ untranslated region (3’ UTR) and seed types of each target gene. Entrez accession numbers and binding position at 3’ UTR are addressed for the representative transcript of each gene. Complementary sequences binding to miR-494 are shown in bold letters, with the m8 and A1 extensions highlighted in bold italic letters. Gene abbreviations: Stxbp5 , syntaxin binding protein 5; Igf1r , insulin-like growth factor 1 receptor; Irs1, insulin receptor substrate 1 (C) RT-PCR analysis for predicted target genes in miR-494 mimic- or miR-494 ASO-transfected C 2 C 12 cells. Gapdh gene was used as a loading control. (E) Immunoblot analysis of molecules related to the insulin signaling pathway. Cells were transfected with miR-494 mimic and subjected to immunoblot analysis. Antibodies for PTEN, p-PTP1B (S50) and IRS-1 were used. GAPDH was used as a loading control. The values were expressed as the means ± SEM and compared between miR-494 mimic transfected cells and control. *P

    Article Snippet: Antibody for p-IRS1 (Y612) was from Millipore (Billerica, MA, USA).

    Techniques: Binding Assay, Reverse Transcription Polymerase Chain Reaction, Allele-specific Oligonucleotide, Transfection

    Effect of genetic HDAC6 knockdown and increased acetylation levels of α-tubulin by HDAC6 inhibition. a RT-PCR validation of knockdown by an antisense oligonucleotide (ASO) against HDAC6 in MNs. b Quantification of the RT-PCR results of the gel shown in a illustrating the HDAC6 knockdown by the ASO. c Knockdown of HDAC6 using an ASO in patient-derived MNs increased axonal transport based on tracking mitochondrial movement (total number, moving number, and ratio between total and moving mitochondria). Scrambled ASO was used as a negative control (NTC ASO). n = 20, n = 19, n = 18, n = 10, n = 11, n = 10 for R521R, R521R + NTC ASO, R521R + HDAC6 ASO, R521H, R521H + NTC ASO, R521H + HDAC6 ASO, respectively; ANOVA with post-hoc Tukey’s test, * P value of 0.05. d Western blot from MNs (R521H mutant line 2/2 and isogenic control R521R), with and without treatment with ACY-738 or Tubastatin A at day 31 of differentiation. The blot was probed with antibodies directed to acetylated α-tubulin, and GAPDH. e Schematic representation of our results indicating that HDAC6 inhibition rescues axonal transport defects in FUS-iPSC-derived motor neurons through increasing acetylation levels of α-tubulin

    Journal: Nature Communications

    Article Title: HDAC6 inhibition reverses axonal transport defects in motor neurons derived from FUS-ALS patients

    doi: 10.1038/s41467-017-00911-y

    Figure Lengend Snippet: Effect of genetic HDAC6 knockdown and increased acetylation levels of α-tubulin by HDAC6 inhibition. a RT-PCR validation of knockdown by an antisense oligonucleotide (ASO) against HDAC6 in MNs. b Quantification of the RT-PCR results of the gel shown in a illustrating the HDAC6 knockdown by the ASO. c Knockdown of HDAC6 using an ASO in patient-derived MNs increased axonal transport based on tracking mitochondrial movement (total number, moving number, and ratio between total and moving mitochondria). Scrambled ASO was used as a negative control (NTC ASO). n = 20, n = 19, n = 18, n = 10, n = 11, n = 10 for R521R, R521R + NTC ASO, R521R + HDAC6 ASO, R521H, R521H + NTC ASO, R521H + HDAC6 ASO, respectively; ANOVA with post-hoc Tukey’s test, * P value of 0.05. d Western blot from MNs (R521H mutant line 2/2 and isogenic control R521R), with and without treatment with ACY-738 or Tubastatin A at day 31 of differentiation. The blot was probed with antibodies directed to acetylated α-tubulin, and GAPDH. e Schematic representation of our results indicating that HDAC6 inhibition rescues axonal transport defects in FUS-iPSC-derived motor neurons through increasing acetylation levels of α-tubulin

    Article Snippet: For rescue experiments, motor neurons were treated overnight with either 1 µM Tubastatin A (Sigma), 1 µM ACY-738 (Acetylon Pharmaceuticals Inc., Boston, USA) or an equivalent amount of DMSO.

    Techniques: Inhibition, Reverse Transcription Polymerase Chain Reaction, Allele-specific Oligonucleotide, Derivative Assay, Negative Control, Western Blot, Mutagenesis

    ( A ) EBV circBARTs are expressed in EBV-positive tumor samples. RNase R-treated (+) or RNase R-untreated (−) RNAs from 6 EBV-positive and 11 EBV-negative (red font) pathologically diagnosed PTLD, NPC tumor xenoexplants C17 and C15, and an EBV-positive AIDS-associated lymphoma were tested by DP1 RT-PCR for circBART-BSJ1 and circBART-BSJ2 ( Top ). All six EBV-positive PTLDs were strongly positive, and three of the EBV-negative PTLDs were weakly positive for circBART RNAs. RT-PCR for LMP2 ( Middle ) and GAPDH ( Bottom ) mRNAs are shown. RNase R treatment enriched circBART junctions and depleted linear LMP2 and GAPDH products. ( B ) Confirmation of cyclization for EBV circBARTs by RNase H treatment. In vitro RNase H assays using annealed ASOs targeting BSJ1 and BSJ2 showed depletion of junctional sequences after RNase H treatment as monitored by DP1 PCR for Akata, sLCL, and Raji cell RNAs, but not in B95-8 RNA. GAPDH mRNA amplification was not affected by RNase H treatment. ( C ) RPAD analysis of EBV circBART. RNase R treatment followed by RPAD diminished linear 18S ribosomal RNA transcripts but increased circBART transcripts. Relative RNA was determined by normalizing the qPCR Ct values RPAD+ RNA to untreated control RNA (RPAD−). The data represent the means ± SD from four replicates.

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

    Article Title: Circular DNA tumor viruses make circular RNAs

    doi: 10.1073/pnas.1811728115

    Figure Lengend Snippet: ( A ) EBV circBARTs are expressed in EBV-positive tumor samples. RNase R-treated (+) or RNase R-untreated (−) RNAs from 6 EBV-positive and 11 EBV-negative (red font) pathologically diagnosed PTLD, NPC tumor xenoexplants C17 and C15, and an EBV-positive AIDS-associated lymphoma were tested by DP1 RT-PCR for circBART-BSJ1 and circBART-BSJ2 ( Top ). All six EBV-positive PTLDs were strongly positive, and three of the EBV-negative PTLDs were weakly positive for circBART RNAs. RT-PCR for LMP2 ( Middle ) and GAPDH ( Bottom ) mRNAs are shown. RNase R treatment enriched circBART junctions and depleted linear LMP2 and GAPDH products. ( B ) Confirmation of cyclization for EBV circBARTs by RNase H treatment. In vitro RNase H assays using annealed ASOs targeting BSJ1 and BSJ2 showed depletion of junctional sequences after RNase H treatment as monitored by DP1 PCR for Akata, sLCL, and Raji cell RNAs, but not in B95-8 RNA. GAPDH mRNA amplification was not affected by RNase H treatment. ( C ) RPAD analysis of EBV circBART. RNase R treatment followed by RPAD diminished linear 18S ribosomal RNA transcripts but increased circBART transcripts. Relative RNA was determined by normalizing the qPCR Ct values RPAD+ RNA to untreated control RNA (RPAD−). The data represent the means ± SD from four replicates.

    Article Snippet: This was followed by polyadenylation (E-PAP, AM1350; Thermo Fisher) with a subsequent poly(A)+ RNA depletion using Poly(A)Purist MAG Kit (AM1922; Thermo Fisher) (RPAD protocol) as described by Panda et al. ( ).

    Techniques: Reverse Transcription Polymerase Chain Reaction, In Vitro, Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction

    Identification and validation of KSHV circRNAs. ( A ) Identification of KSHV RNase R-resistant RNAs. Comparison of deposited BCBL1 poly(A) + RNA sequences (SRX2323239, BCBL1 polyA+seq ) and RNase R-treated RNAs from BCBL1 cells with and without NaB/TPA revealed KSHV RNase R-resistant RNAs with potential BSJs from KSHV circRNAs. Two expanded views ( Bottom ), spanning the PAN/K7.3 and the vIRF4 regions, have BSJs identified by CIRI2 alignment to the deposited BCBL1 HQ404500 genome ( Table 2 ). For circvIRF4 (nucleotides 85,600 to 88,400), backsplicing from a cryptic donor site in exon 2 to a cryptic acceptor site in exon 1 (red arrow) generates a single 632-bp RNA plasmid. Predicted MaxEnt splice sites with entropy scores are shown, as in Fig. 1 A . For circPAN/K7.3 (nucleotides 28,200 to 29,300), multiple cyclized RNAs from both sense and antisense orientations were identified by BSJ analysis ( Table 2 ). Shown here are predicted circRNA sequences in K7.3 sense (green arrows) and PAN sense (dark blue arrows) orientations from untreated BCBL1 cells. A divergent PCR primer pair (DP5) was designed to detect the most common circRNAs from this locus. ( B ) The circvIRF4 and circPAN/K7.3 are detected in KSHV-positive PELs. RNAs were extracted from DMSO or NaB/TPA-treated KSHV-positive BC1, BCBL1, and BCP1 and KSHV-negative BJAB cell lines and tested with DP3 and DP5 divergent primer RT-PCR (see A for primer location). Nuclease-resistant circvIRF4 was present in all untreated KSHV-positive cell lines but markedly diminished after NaB/TPA induction. In contrast, circPAN/K7.3 products were detected from all KSHV-positive cell lines and markedly increased after NaB/TPA treatment. The circPAN/K7.3 banding patterns varied between cell lines and with virus induction. Viral interleukin-6 (vIL6) and cellular GAPDH mRNA RT-PCR amplification is shown for comparison. ( C ) KSHV circRNAs in KS patient tissues. LANA mRNAs were detected in six out of seven KS tissues. The circvIRF4 BSJ was found in three KS samples (KS4, KS6, and KS8) which also showed higher levels of LANA mRNA. Various RNase R-resistant circPAN/K7.3 isoforms (∼250 bp to 700 bp) were detected in KS4, KS6, and KS9. BJAB and BC-1 RNAs were used as virus negative and positive controls, respectively. ( D ) Confirmation of cyclization for circvIRF4. In vitro RNase H assays ( Left ) using annealed ASO showed depletion of the circvIRF4 junctional sequences after RNase H treatment for BC1 and BCBL RNAs, but not in KSHV-negative BJAB RNAs. RPAD analysis ( Right ) revealed that ribosomal 18S RNA is selectively diminished compared with circvIRF4 RNA. Data represent the mean ± SD from three replicates.

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

    Article Title: Circular DNA tumor viruses make circular RNAs

    doi: 10.1073/pnas.1811728115

    Figure Lengend Snippet: Identification and validation of KSHV circRNAs. ( A ) Identification of KSHV RNase R-resistant RNAs. Comparison of deposited BCBL1 poly(A) + RNA sequences (SRX2323239, BCBL1 polyA+seq ) and RNase R-treated RNAs from BCBL1 cells with and without NaB/TPA revealed KSHV RNase R-resistant RNAs with potential BSJs from KSHV circRNAs. Two expanded views ( Bottom ), spanning the PAN/K7.3 and the vIRF4 regions, have BSJs identified by CIRI2 alignment to the deposited BCBL1 HQ404500 genome ( Table 2 ). For circvIRF4 (nucleotides 85,600 to 88,400), backsplicing from a cryptic donor site in exon 2 to a cryptic acceptor site in exon 1 (red arrow) generates a single 632-bp RNA plasmid. Predicted MaxEnt splice sites with entropy scores are shown, as in Fig. 1 A . For circPAN/K7.3 (nucleotides 28,200 to 29,300), multiple cyclized RNAs from both sense and antisense orientations were identified by BSJ analysis ( Table 2 ). Shown here are predicted circRNA sequences in K7.3 sense (green arrows) and PAN sense (dark blue arrows) orientations from untreated BCBL1 cells. A divergent PCR primer pair (DP5) was designed to detect the most common circRNAs from this locus. ( B ) The circvIRF4 and circPAN/K7.3 are detected in KSHV-positive PELs. RNAs were extracted from DMSO or NaB/TPA-treated KSHV-positive BC1, BCBL1, and BCP1 and KSHV-negative BJAB cell lines and tested with DP3 and DP5 divergent primer RT-PCR (see A for primer location). Nuclease-resistant circvIRF4 was present in all untreated KSHV-positive cell lines but markedly diminished after NaB/TPA induction. In contrast, circPAN/K7.3 products were detected from all KSHV-positive cell lines and markedly increased after NaB/TPA treatment. The circPAN/K7.3 banding patterns varied between cell lines and with virus induction. Viral interleukin-6 (vIL6) and cellular GAPDH mRNA RT-PCR amplification is shown for comparison. ( C ) KSHV circRNAs in KS patient tissues. LANA mRNAs were detected in six out of seven KS tissues. The circvIRF4 BSJ was found in three KS samples (KS4, KS6, and KS8) which also showed higher levels of LANA mRNA. Various RNase R-resistant circPAN/K7.3 isoforms (∼250 bp to 700 bp) were detected in KS4, KS6, and KS9. BJAB and BC-1 RNAs were used as virus negative and positive controls, respectively. ( D ) Confirmation of cyclization for circvIRF4. In vitro RNase H assays ( Left ) using annealed ASO showed depletion of the circvIRF4 junctional sequences after RNase H treatment for BC1 and BCBL RNAs, but not in KSHV-negative BJAB RNAs. RPAD analysis ( Right ) revealed that ribosomal 18S RNA is selectively diminished compared with circvIRF4 RNA. Data represent the mean ± SD from three replicates.

    Article Snippet: This was followed by polyadenylation (E-PAP, AM1350; Thermo Fisher) with a subsequent poly(A)+ RNA depletion using Poly(A)Purist MAG Kit (AM1922; Thermo Fisher) (RPAD protocol) as described by Panda et al. ( ).

    Techniques: Plasmid Preparation, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Amplification, In Vitro, Allele-specific Oligonucleotide