oligo-dt primers Search Results


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  • 99
    Thermo Fisher oligodt oligonucleotides
    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 <t>oligodT</t> 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.
    Oligodt Oligonucleotides, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen oligodt
    Characteristics of dsRNA-induced primary and secondary siRNAs. ( A and B ) Strand bias of siRNA. Blots described in Figure 2 D and E were hybridized with two adjacent 50-nt strand-specific oligonucleotide probes located in the centre of the ND169 dsRNA fragment (A) or a single <t>oligo</t> in the polylinker sequence (B) (upper blot: antisense-orientated probe, lower blot: sense-orientated probe). Arrowheads indicate small amounts of ∼23-nt ND169 siRNAs from both strands. ( C ) Properties of 5′- and 3′-ends of dsRNA-induced siRNA were analysed by treatment with CIP, Terminator (Ter) and periodate followed by β-elimination (P/β). Treatment of total <t>RNA</t> with CIP alkaline phosphatase, removing all 5′ phosphates, resulted in a ∼0.5-nt slower migration of siRNA in comparison to untreated samples. This was found for polylinker-specific ∼23-nt siRNA (upper blot) and ND169 -specific ∼22-nt siRNA (middle blot). Treatment of total RNA with Terminator 5′-monophosphate-specific exonuclease (Ter) degraded both classes of siRNA. Periodate treatment and subsequent β-elimination (P/β) resulted in ∼1.5-nt faster migration of both classes of siRNAs as it was also observed for the 3′-unmodified control oligo. The second P/β-lane (right) represents the latter one with increased contrast. A 5′-monophosphorylated (grey arrowhead) and a 5′-unphosphorylated (black arrowhead) 22-nt RNA oligonucleotide, both lacking a 3′ modification, were added to each reaction as a control (lower blot). The lower panels show hybridization to glutamine tRNA as a loading control.
    Oligodt, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 255 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Price from $9.99 to $1999.99
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    99
    Promega oligodt 15
    Characteristics of dsRNA-induced primary and secondary siRNAs. ( A and B ) Strand bias of siRNA. Blots described in Figure 2 D and E were hybridized with two adjacent 50-nt strand-specific oligonucleotide probes located in the centre of the ND169 dsRNA fragment (A) or a single <t>oligo</t> in the polylinker sequence (B) (upper blot: antisense-orientated probe, lower blot: sense-orientated probe). Arrowheads indicate small amounts of ∼23-nt ND169 siRNAs from both strands. ( C ) Properties of 5′- and 3′-ends of dsRNA-induced siRNA were analysed by treatment with CIP, Terminator (Ter) and periodate followed by β-elimination (P/β). Treatment of total <t>RNA</t> with CIP alkaline phosphatase, removing all 5′ phosphates, resulted in a ∼0.5-nt slower migration of siRNA in comparison to untreated samples. This was found for polylinker-specific ∼23-nt siRNA (upper blot) and ND169 -specific ∼22-nt siRNA (middle blot). Treatment of total RNA with Terminator 5′-monophosphate-specific exonuclease (Ter) degraded both classes of siRNA. Periodate treatment and subsequent β-elimination (P/β) resulted in ∼1.5-nt faster migration of both classes of siRNAs as it was also observed for the 3′-unmodified control oligo. The second P/β-lane (right) represents the latter one with increased contrast. A 5′-monophosphorylated (grey arrowhead) and a 5′-unphosphorylated (black arrowhead) 22-nt RNA oligonucleotide, both lacking a 3′ modification, were added to each reaction as a control (lower blot). The lower panels show hybridization to glutamine tRNA as a loading control.
    Oligodt 15, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Price from $9.99 to $1999.99
    oligodt 15 - by Bioz Stars, 2020-05
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    92
    Promega oligo dt
    Characteristics of dsRNA-induced primary and secondary siRNAs. ( A and B ) Strand bias of siRNA. Blots described in Figure 2 D and E were hybridized with two adjacent 50-nt strand-specific oligonucleotide probes located in the centre of the ND169 dsRNA fragment (A) or a single <t>oligo</t> in the polylinker sequence (B) (upper blot: antisense-orientated probe, lower blot: sense-orientated probe). Arrowheads indicate small amounts of ∼23-nt ND169 siRNAs from both strands. ( C ) Properties of 5′- and 3′-ends of dsRNA-induced siRNA were analysed by treatment with CIP, Terminator (Ter) and periodate followed by β-elimination (P/β). Treatment of total <t>RNA</t> with CIP alkaline phosphatase, removing all 5′ phosphates, resulted in a ∼0.5-nt slower migration of siRNA in comparison to untreated samples. This was found for polylinker-specific ∼23-nt siRNA (upper blot) and ND169 -specific ∼22-nt siRNA (middle blot). Treatment of total RNA with Terminator 5′-monophosphate-specific exonuclease (Ter) degraded both classes of siRNA. Periodate treatment and subsequent β-elimination (P/β) resulted in ∼1.5-nt faster migration of both classes of siRNAs as it was also observed for the 3′-unmodified control oligo. The second P/β-lane (right) represents the latter one with increased contrast. A 5′-monophosphorylated (grey arrowhead) and a 5′-unphosphorylated (black arrowhead) 22-nt RNA oligonucleotide, both lacking a 3′ modification, were added to each reaction as a control (lower blot). The lower panels show hybridization to glutamine tRNA as a loading control.
    Oligo Dt, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 158 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 158 article reviews
    Price from $9.99 to $1999.99
    oligo dt - by Bioz Stars, 2020-05
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    Image Search Results


    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

    Characteristics of dsRNA-induced primary and secondary siRNAs. ( A and B ) Strand bias of siRNA. Blots described in Figure 2 D and E were hybridized with two adjacent 50-nt strand-specific oligonucleotide probes located in the centre of the ND169 dsRNA fragment (A) or a single oligo in the polylinker sequence (B) (upper blot: antisense-orientated probe, lower blot: sense-orientated probe). Arrowheads indicate small amounts of ∼23-nt ND169 siRNAs from both strands. ( C ) Properties of 5′- and 3′-ends of dsRNA-induced siRNA were analysed by treatment with CIP, Terminator (Ter) and periodate followed by β-elimination (P/β). Treatment of total RNA with CIP alkaline phosphatase, removing all 5′ phosphates, resulted in a ∼0.5-nt slower migration of siRNA in comparison to untreated samples. This was found for polylinker-specific ∼23-nt siRNA (upper blot) and ND169 -specific ∼22-nt siRNA (middle blot). Treatment of total RNA with Terminator 5′-monophosphate-specific exonuclease (Ter) degraded both classes of siRNA. Periodate treatment and subsequent β-elimination (P/β) resulted in ∼1.5-nt faster migration of both classes of siRNAs as it was also observed for the 3′-unmodified control oligo. The second P/β-lane (right) represents the latter one with increased contrast. A 5′-monophosphorylated (grey arrowhead) and a 5′-unphosphorylated (black arrowhead) 22-nt RNA oligonucleotide, both lacking a 3′ modification, were added to each reaction as a control (lower blot). The lower panels show hybridization to glutamine tRNA as a loading control.

    Journal: Nucleic Acids Research

    Article Title: Distinct RNA-dependent RNA polymerases are required for RNAi triggered by double-stranded RNA versus truncated transgenes in Paramecium tetraurelia

    doi: 10.1093/nar/gkq131

    Figure Lengend Snippet: Characteristics of dsRNA-induced primary and secondary siRNAs. ( A and B ) Strand bias of siRNA. Blots described in Figure 2 D and E were hybridized with two adjacent 50-nt strand-specific oligonucleotide probes located in the centre of the ND169 dsRNA fragment (A) or a single oligo in the polylinker sequence (B) (upper blot: antisense-orientated probe, lower blot: sense-orientated probe). Arrowheads indicate small amounts of ∼23-nt ND169 siRNAs from both strands. ( C ) Properties of 5′- and 3′-ends of dsRNA-induced siRNA were analysed by treatment with CIP, Terminator (Ter) and periodate followed by β-elimination (P/β). Treatment of total RNA with CIP alkaline phosphatase, removing all 5′ phosphates, resulted in a ∼0.5-nt slower migration of siRNA in comparison to untreated samples. This was found for polylinker-specific ∼23-nt siRNA (upper blot) and ND169 -specific ∼22-nt siRNA (middle blot). Treatment of total RNA with Terminator 5′-monophosphate-specific exonuclease (Ter) degraded both classes of siRNA. Periodate treatment and subsequent β-elimination (P/β) resulted in ∼1.5-nt faster migration of both classes of siRNAs as it was also observed for the 3′-unmodified control oligo. The second P/β-lane (right) represents the latter one with increased contrast. A 5′-monophosphorylated (grey arrowhead) and a 5′-unphosphorylated (black arrowhead) 22-nt RNA oligonucleotide, both lacking a 3′ modification, were added to each reaction as a control (lower blot). The lower panels show hybridization to glutamine tRNA as a loading control.

    Article Snippet: RNA (500 ng) was reverse transcribed using an oligo-dT primer. cDNA was amplified with the QuantiTectTM SYBR® Green PCR Kit (Qiagen, Hilden, Germany).

    Techniques: Sequencing, Migration, Modification, Hybridization

    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

    The MTNR1A gene is downregulated by a piRNA transcript. a , The 500 bp piRNA genomic cluster was ligated into a linearised XbaI pRL-CMV vector (Promega) at the 3′ end of the Renilla luciferase reporter gene; this plasmid is reported as pRL-PIWI. The histogram shows the renilla/luciferase expression ratio after transfection of HEK 293 cells with increasing concentrations of the pRL-PIWI plasmid and the chemically synthesised piRNA mimic (50 nM, 100 nM, 200 nM, 300 nM). b , The histogram shows the levels of MTNR1A mRNA in HEK 293 cells after transfection with increasing concentrations of the piRNA mimic (50 nM, 100 nM, 200 nM). As controls of transfection we used HEK 293 cells without piRNA mimic and HEK 293 cells trasfected with 200 nM of a dsRNA 30 bp oligo not related with the MTNR1A gene. The bar reported as control is a mean value of these two experiments. GAPDH mRNA was used as a control. The ratio of MTNR1A mRNA/ GAPDH mRNA was set to 1 in the control (no piRNA mimic transfection). Quantitation of expression levels was determined by RT-qPCR. c , Western blots of HEK 293 cells transfected with the piRNA mimic in increasing concentrations (50 nM, 100 nM, 200 nM) were probed with an anti-MTNR1A antibody. Beta-actin was used as a loading control. The histogram shows the ratio of MTNR1A/beta-actin. The ratio was normalised to 1 in the control transfection (as described above). The mean value of three quantitations is shown; error bars correspond to s.d.

    Journal: PLoS ONE

    Article Title: piR_015520 Belongs to Piwi-Associated RNAs Regulates Expression of the Human Melatonin Receptor 1A Gene

    doi: 10.1371/journal.pone.0022727

    Figure Lengend Snippet: The MTNR1A gene is downregulated by a piRNA transcript. a , The 500 bp piRNA genomic cluster was ligated into a linearised XbaI pRL-CMV vector (Promega) at the 3′ end of the Renilla luciferase reporter gene; this plasmid is reported as pRL-PIWI. The histogram shows the renilla/luciferase expression ratio after transfection of HEK 293 cells with increasing concentrations of the pRL-PIWI plasmid and the chemically synthesised piRNA mimic (50 nM, 100 nM, 200 nM, 300 nM). b , The histogram shows the levels of MTNR1A mRNA in HEK 293 cells after transfection with increasing concentrations of the piRNA mimic (50 nM, 100 nM, 200 nM). As controls of transfection we used HEK 293 cells without piRNA mimic and HEK 293 cells trasfected with 200 nM of a dsRNA 30 bp oligo not related with the MTNR1A gene. The bar reported as control is a mean value of these two experiments. GAPDH mRNA was used as a control. The ratio of MTNR1A mRNA/ GAPDH mRNA was set to 1 in the control (no piRNA mimic transfection). Quantitation of expression levels was determined by RT-qPCR. c , Western blots of HEK 293 cells transfected with the piRNA mimic in increasing concentrations (50 nM, 100 nM, 200 nM) were probed with an anti-MTNR1A antibody. Beta-actin was used as a loading control. The histogram shows the ratio of MTNR1A/beta-actin. The ratio was normalised to 1 in the control transfection (as described above). The mean value of three quantitations is shown; error bars correspond to s.d.

    Article Snippet: Reverse transcriptase was used to convert RNA (including precursor miRNA, mature miRNA, other small noncoding RNA, and mRNA) to cDNA using both oligo-dT and random primers. qPCR reactions were performed in triplicate using an oligo-dT primer with a universal tag sequence on the 5′ end, together with the piRNA-specific primer. qPCR reactions were prepared using the miScript SYBR Green PCR Kit (Qiagen) following the manufacturer's directions.

    Techniques: Plasmid Preparation, Luciferase, Expressing, Transfection, Quantitation Assay, Quantitative RT-PCR, Western Blot