oligo dt primers  (Qiagen)

 
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    Name:
    Oligo dT Primers
    Description:
    For cDNA synthesis using oligo dT primers in TurboCapture strips or plates Kit contents 100 l of oligo dT primers 0 4 g ul for cDNA synthesis with TurboCapture mRNA Kits
    Catalog Number:
    79237
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    27.7
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    Oligo dT Primers
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    Structured Review

    Qiagen oligo dt primers
    Oligo dT Primers
    For cDNA synthesis using oligo dT primers in TurboCapture strips or plates Kit contents 100 l of oligo dT primers 0 4 g ul for cDNA synthesis with TurboCapture mRNA Kits
    https://www.bioz.com/result/oligo dt primers/product/Qiagen
    Average 99 stars, based on 176 article reviews
    Price from $9.99 to $1999.99
    oligo dt primers - by Bioz Stars, 2020-11
    99/100 stars

    Images

    1) Product Images from "Identification of novel target genes of nerve growth factor (NGF) in human mastocytoma cell line (HMC-1 (V560G c-Kit)) by transcriptome analysis"

    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

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-12-196

    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.
    Figure Legend 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.

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

    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: Annexin A5 suppresses cyclooxygenase-2 expression by downregulating the protein kinase C-ζ–nuclear factor-κB signaling pathway in prostate cancer cells
    Article Snippet: .. Total RNA (0.5 μg) was reverse-transcribed at 37 °C for 1 h in 20 μL total volume containing 5× RT buffer, 10 mM dNTPs, 40 U RNase inhibitor, 200 U Moloney murine leukemia virus reverse transcriptase, and 100 pmol oligo-dT primer. qPCR was performed using the Rotor-Gene SYBR1 PCR Kit, as recommended by the manufacturer, and analyzed by using QIAGEN Rotor-Gene Q Series software. .. Each reaction contained 10 μL of 2×SYBR1 Green PCR Master Mix, 1 μM oligonucleotide primers, and 2 μL of cDNA in a final volume of 20 μL.

    Article Title: piR_015520 Belongs to Piwi-Associated RNAs Regulates Expression of the Human Melatonin Receptor 1A Gene
    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. .. The second assay used for expression profiling was performed with the Custom TaqMan® Small RNA Assays kit (Applied Biosystems).

    Amplification:

    Article Title: Distinct RNA-dependent RNA polymerases are required for RNAi triggered by double-stranded RNA versus truncated transgenes in Paramecium tetraurelia
    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). .. Data from surface antigen expression were set in relation to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which was found to be constantly expressed during vegetative growth and RDR silencing.

    Article Title: Transcriptional Control of an Essential Ribozyme in Drosophila Reveals an Ancient Evolutionary Divide in Animals
    Article Snippet: .. Reverse transcription and PCR cDNAs were prepared using an oligo dT primer (for mRNAs) or gene specific primers (for RPRs) by reverse transcription using an Omniscript RT kit (Qiagen). cDNAs were amplified with Taq DNA polymerase (NEB) using the recommended conditions and the following primer pairs: Forward DmelRPR, 5′-AGTCAGTTGCAAACTAGCATC-3′ and Reverse Dmel RPR, 5′- AGTCAGTCACAGATTAGTCTGAATTG-3′ ; Forward GFP, 5′-TAAGATATCATGGTGAGCAAGGG-3′ and Reverse GFP, 5′- ACCTCTAGATTACTTGTACAGCTCGTCC-3′ ; Forward Oda, 5′-GTCCTTCGGTAGAGCGACAT-3′ and Reverse Oda, 5′- GCACCATCTCGACTTCGTCT-3′ . .. Northern blot analysis D. melanogaster and D. virilis RPRs were detected using full-length anti-sense RNA probes labeled with [α-32 P]-ATP in an in vitro transcription reaction.

    Polymerase Chain Reaction:

    Article Title: Annexin A5 suppresses cyclooxygenase-2 expression by downregulating the protein kinase C-ζ–nuclear factor-κB signaling pathway in prostate cancer cells
    Article Snippet: .. Total RNA (0.5 μg) was reverse-transcribed at 37 °C for 1 h in 20 μL total volume containing 5× RT buffer, 10 mM dNTPs, 40 U RNase inhibitor, 200 U Moloney murine leukemia virus reverse transcriptase, and 100 pmol oligo-dT primer. qPCR was performed using the Rotor-Gene SYBR1 PCR Kit, as recommended by the manufacturer, and analyzed by using QIAGEN Rotor-Gene Q Series software. .. Each reaction contained 10 μL of 2×SYBR1 Green PCR Master Mix, 1 μM oligonucleotide primers, and 2 μL of cDNA in a final volume of 20 μL.

    Article Title: Defective structural RNA processing in relapsing-remitting multiple sclerosis
    Article Snippet: .. Complementary DNA (cDNA) was reverse transcribed from total RNA using the SuperScript III First-Strand Synthesis Kit (Life Technologies) using oligo-dT or random hexamer primers and purified using the Qiagen QiaQuick PCR purification kit. .. Real-time qPCR (Bio-Rad iCycleriQ Real Time PCR System) was performed in duplicate using SYBR green in 15 μL reaction volumes.

    Article Title: Distinct RNA-dependent RNA polymerases are required for RNAi triggered by double-stranded RNA versus truncated transgenes in Paramecium tetraurelia
    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). .. Data from surface antigen expression were set in relation to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which was found to be constantly expressed during vegetative growth and RDR silencing.

    Article Title: Transcriptional Control of an Essential Ribozyme in Drosophila Reveals an Ancient Evolutionary Divide in Animals
    Article Snippet: .. Reverse transcription and PCR cDNAs were prepared using an oligo dT primer (for mRNAs) or gene specific primers (for RPRs) by reverse transcription using an Omniscript RT kit (Qiagen). cDNAs were amplified with Taq DNA polymerase (NEB) using the recommended conditions and the following primer pairs: Forward DmelRPR, 5′-AGTCAGTTGCAAACTAGCATC-3′ and Reverse Dmel RPR, 5′- AGTCAGTCACAGATTAGTCTGAATTG-3′ ; Forward GFP, 5′-TAAGATATCATGGTGAGCAAGGG-3′ and Reverse GFP, 5′- ACCTCTAGATTACTTGTACAGCTCGTCC-3′ ; Forward Oda, 5′-GTCCTTCGGTAGAGCGACAT-3′ and Reverse Oda, 5′- GCACCATCTCGACTTCGTCT-3′ . .. Northern blot analysis D. melanogaster and D. virilis RPRs were detected using full-length anti-sense RNA probes labeled with [α-32 P]-ATP in an in vitro transcription reaction.

    Article Title: piR_015520 Belongs to Piwi-Associated RNAs Regulates Expression of the Human Melatonin Receptor 1A Gene
    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. .. The second assay used for expression profiling was performed with the Custom TaqMan® Small RNA Assays kit (Applied Biosystems).

    Article Title: Determinants of RNA Quality from FFPE Samples
    Article Snippet: .. Two-step RT-PCR was performed using the QuantiTect Reverse Transcription Kit (QIAGEN GmbH, Hilden, Germany) for cDNA synthesis either according to manufacturer's recommendations, or using oligo-dT primer in place of the supplied primer mix, followed by PCR using the QuantiTect SYBR Green PCR Kit (QIAGEN GmbH, Hilden, Germany). .. Thermal cycling conditions following the 15 minutes at 94°C activation step were: 15 seconds at 94°C, 30 seconds at 50°C, 30 seconds at 72°C for 40 cycles.

    Quantitative RT-PCR:

    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
    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. ..

    Purification:

    Article Title: Defective structural RNA processing in relapsing-remitting multiple sclerosis
    Article Snippet: .. Complementary DNA (cDNA) was reverse transcribed from total RNA using the SuperScript III First-Strand Synthesis Kit (Life Technologies) using oligo-dT or random hexamer primers and purified using the Qiagen QiaQuick PCR purification kit. .. Real-time qPCR (Bio-Rad iCycleriQ Real Time PCR System) was performed in duplicate using SYBR green in 15 μL reaction volumes.

    SYBR Green Assay:

    Article Title: Distinct RNA-dependent RNA polymerases are required for RNAi triggered by double-stranded RNA versus truncated transgenes in Paramecium tetraurelia
    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). .. Data from surface antigen expression were set in relation to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which was found to be constantly expressed during vegetative growth and RDR silencing.

    Article Title: piR_015520 Belongs to Piwi-Associated RNAs Regulates Expression of the Human Melatonin Receptor 1A Gene
    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. .. The second assay used for expression profiling was performed with the Custom TaqMan® Small RNA Assays kit (Applied Biosystems).

    Article Title: Determinants of RNA Quality from FFPE Samples
    Article Snippet: .. Two-step RT-PCR was performed using the QuantiTect Reverse Transcription Kit (QIAGEN GmbH, Hilden, Germany) for cDNA synthesis either according to manufacturer's recommendations, or using oligo-dT primer in place of the supplied primer mix, followed by PCR using the QuantiTect SYBR Green PCR Kit (QIAGEN GmbH, Hilden, Germany). .. Thermal cycling conditions following the 15 minutes at 94°C activation step were: 15 seconds at 94°C, 30 seconds at 50°C, 30 seconds at 72°C for 40 cycles.

    Reverse Transcription Polymerase Chain Reaction:

    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
    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. ..

    Article Title: Determinants of RNA Quality from FFPE Samples
    Article Snippet: .. Two-step RT-PCR was performed using the QuantiTect Reverse Transcription Kit (QIAGEN GmbH, Hilden, Germany) for cDNA synthesis either according to manufacturer's recommendations, or using oligo-dT primer in place of the supplied primer mix, followed by PCR using the QuantiTect SYBR Green PCR Kit (QIAGEN GmbH, Hilden, Germany). .. Thermal cycling conditions following the 15 minutes at 94°C activation step were: 15 seconds at 94°C, 30 seconds at 50°C, 30 seconds at 72°C for 40 cycles.

    Random Hexamer Labeling:

    Article Title: Defective structural RNA processing in relapsing-remitting multiple sclerosis
    Article Snippet: .. Complementary DNA (cDNA) was reverse transcribed from total RNA using the SuperScript III First-Strand Synthesis Kit (Life Technologies) using oligo-dT or random hexamer primers and purified using the Qiagen QiaQuick PCR purification kit. .. Real-time qPCR (Bio-Rad iCycleriQ Real Time PCR System) was performed in duplicate using SYBR green in 15 μL reaction volumes.

    Sequencing:

    Article Title: piR_015520 Belongs to Piwi-Associated RNAs Regulates Expression of the Human Melatonin Receptor 1A Gene
    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. .. The second assay used for expression profiling was performed with the Custom TaqMan® Small RNA Assays kit (Applied Biosystems).

    Software:

    Article Title: Annexin A5 suppresses cyclooxygenase-2 expression by downregulating the protein kinase C-ζ–nuclear factor-κB signaling pathway in prostate cancer cells
    Article Snippet: .. Total RNA (0.5 μg) was reverse-transcribed at 37 °C for 1 h in 20 μL total volume containing 5× RT buffer, 10 mM dNTPs, 40 U RNase inhibitor, 200 U Moloney murine leukemia virus reverse transcriptase, and 100 pmol oligo-dT primer. qPCR was performed using the Rotor-Gene SYBR1 PCR Kit, as recommended by the manufacturer, and analyzed by using QIAGEN Rotor-Gene Q Series software. .. Each reaction contained 10 μL of 2×SYBR1 Green PCR Master Mix, 1 μM oligonucleotide primers, and 2 μL of cDNA in a final volume of 20 μL.

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  • 93
    Qiagen oligo
    SC35 acts on exon 10 of tau pre-mRNA to promote tau exon 10 inclusion. ( A ) Tau pre-mRNA could be immunoprecipitated by SC35. pCI/SI9–SI10 was co-transfected with pCEP4/SC35-HA into HEK-293T cells. SC35 was immunoprecipitated with anti-HA antibody. Co-immunoprecipitated pre-mRNA of tau with SC35 was determined by RT–PCR with random primer or <t>oligo-dT</t> for generating cDNA and with two sets of primers specific to introns 9 and 10 as indicated for amplifying the cDNA derived from tau pre-mRNA. The RT–PCR product was separated by agarose electrophoresis and quantitated by densitometry and presented in B from two separated experiments. ( C ) Schematic of SC35 deletion mutants. ( D ) Tau pre-mRNA was immunoprecipitated by deletion mutants of SC35 differentially. Different deletion mutants of SC35 showed in panel C tagged with HA were overexpressed in pCI/SI9–SI10 transfected HEK-293T cells. <t>RNA-IP</t> was carried out with anti-HA antibody and co-immunoprecipitated pre-mRNA of tau was measured by RT–PCR as in panel A. Total pre-mRNA of tau, Input, was also measured by RT–PCR with same primers. The immunoprecipitated deletion mutations of SC35 were examined by western blot using anti-HA antibody (lower panel). ( E ) Deletion mutations of SC35 promoted tau exon 10 inclusion differentially. pCI/SI9–SI10 was co-transfected with different deletion mutants of SC35 into HEK-293T. Total RNA was extracted and subjected to RT–PCR for measurement of tau exon 10 splicing after 36 h transfection.
    Oligo, supplied by Qiagen, used in various techniques. Bioz Stars score: 93/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/oligo/product/Qiagen
    Average 93 stars, based on 105 article reviews
    Price from $9.99 to $1999.99
    oligo - by Bioz Stars, 2020-11
    93/100 stars
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    99
    Qiagen oligo dt primer
    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 Primer, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 290 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/oligo dt primer/product/Qiagen
    Average 99 stars, based on 290 article reviews
    Price from $9.99 to $1999.99
    oligo dt primer - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    91
    Qiagen oligo dt 18
    TDP-43 acts on the (UG)n elements of tau mRNA 3΄-UTR. ( A ) Schematic of (UG)n elements in tau mRNA 3΄-UTR. Two sets of primers specific to (UG)n elements, as indicated for amplifying the cDNA derived from tau mRNA 3΄-UTR. ( B and C ) TDP-43 acts on two (UG)n elements in tau mRNA 3΄-UTR. pEGFP/tau 3΄-UTR was co-transfected with pCI/TDP-43·HA into HEK-293FT cells. TDP-43 was immunoprecipitated with anti-HA. Co-immunoprecipitated 3΄-UTR of tau mRNA covering (UG)n element 1 (Site 1) or (UG)n element 2 (Site 2) with TDP-43 was reverse-transcribed to cDNA with random primer or <t>oligo-dT</t> 18 and amplified by PCR with two sets of primers. The RT-PCR products from RNA-IP or cell lysate (Input) were separated by agarose electrophoresis (B) and quantitated by densitometry (C). ( D ) Physiological TDP-43 acts on tau mRNA 3΄-UTR. TDP-43 was immunoprecipitated from SH-SY5Y cells with anti-TDP-43 (H-8). Co-immunoprecipitated 3΄-UTR of tau mRNA with TDP-43 was reverse-transcribed to cDNA with oligo-dT 18 and amplified by PCR with two sets of primers. The RT-PCR products from RNA-IP or cell lysate (Input) were separated by agarose electrophoresis. The data are presented as mean ± SD ( n = 3). * P
    Oligo Dt 18, supplied by Qiagen, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/oligo dt 18/product/Qiagen
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    oligo dt 18 - by Bioz Stars, 2020-11
    91/100 stars
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    Image Search Results


    SC35 acts on exon 10 of tau pre-mRNA to promote tau exon 10 inclusion. ( A ) Tau pre-mRNA could be immunoprecipitated by SC35. pCI/SI9–SI10 was co-transfected with pCEP4/SC35-HA into HEK-293T cells. SC35 was immunoprecipitated with anti-HA antibody. Co-immunoprecipitated pre-mRNA of tau with SC35 was determined by RT–PCR with random primer or oligo-dT for generating cDNA and with two sets of primers specific to introns 9 and 10 as indicated for amplifying the cDNA derived from tau pre-mRNA. The RT–PCR product was separated by agarose electrophoresis and quantitated by densitometry and presented in B from two separated experiments. ( C ) Schematic of SC35 deletion mutants. ( D ) Tau pre-mRNA was immunoprecipitated by deletion mutants of SC35 differentially. Different deletion mutants of SC35 showed in panel C tagged with HA were overexpressed in pCI/SI9–SI10 transfected HEK-293T cells. RNA-IP was carried out with anti-HA antibody and co-immunoprecipitated pre-mRNA of tau was measured by RT–PCR as in panel A. Total pre-mRNA of tau, Input, was also measured by RT–PCR with same primers. The immunoprecipitated deletion mutations of SC35 were examined by western blot using anti-HA antibody (lower panel). ( E ) Deletion mutations of SC35 promoted tau exon 10 inclusion differentially. pCI/SI9–SI10 was co-transfected with different deletion mutants of SC35 into HEK-293T. Total RNA was extracted and subjected to RT–PCR for measurement of tau exon 10 splicing after 36 h transfection.

    Journal: Nucleic Acids Research

    Article Title: Regulation of the alternative splicing of tau exon 10 by SC35 and Dyrk1A

    doi: 10.1093/nar/gkr195

    Figure Lengend Snippet: SC35 acts on exon 10 of tau pre-mRNA to promote tau exon 10 inclusion. ( A ) Tau pre-mRNA could be immunoprecipitated by SC35. pCI/SI9–SI10 was co-transfected with pCEP4/SC35-HA into HEK-293T cells. SC35 was immunoprecipitated with anti-HA antibody. Co-immunoprecipitated pre-mRNA of tau with SC35 was determined by RT–PCR with random primer or oligo-dT for generating cDNA and with two sets of primers specific to introns 9 and 10 as indicated for amplifying the cDNA derived from tau pre-mRNA. The RT–PCR product was separated by agarose electrophoresis and quantitated by densitometry and presented in B from two separated experiments. ( C ) Schematic of SC35 deletion mutants. ( D ) Tau pre-mRNA was immunoprecipitated by deletion mutants of SC35 differentially. Different deletion mutants of SC35 showed in panel C tagged with HA were overexpressed in pCI/SI9–SI10 transfected HEK-293T cells. RNA-IP was carried out with anti-HA antibody and co-immunoprecipitated pre-mRNA of tau was measured by RT–PCR as in panel A. Total pre-mRNA of tau, Input, was also measured by RT–PCR with same primers. The immunoprecipitated deletion mutations of SC35 were examined by western blot using anti-HA antibody (lower panel). ( E ) Deletion mutations of SC35 promoted tau exon 10 inclusion differentially. pCI/SI9–SI10 was co-transfected with different deletion mutants of SC35 into HEK-293T. Total RNA was extracted and subjected to RT–PCR for measurement of tau exon 10 splicing after 36 h transfection.

    Article Snippet: Six hundred nanograms of total RNA was used for first-strand cDNA synthesis with oligo (dT)18 by using an Omniscript reverse transcription kit (Qiagen GmbH).

    Techniques: Immunoprecipitation, Transfection, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Electrophoresis, 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

    TDP-43 acts on the (UG)n elements of tau mRNA 3΄-UTR. ( A ) Schematic of (UG)n elements in tau mRNA 3΄-UTR. Two sets of primers specific to (UG)n elements, as indicated for amplifying the cDNA derived from tau mRNA 3΄-UTR. ( B and C ) TDP-43 acts on two (UG)n elements in tau mRNA 3΄-UTR. pEGFP/tau 3΄-UTR was co-transfected with pCI/TDP-43·HA into HEK-293FT cells. TDP-43 was immunoprecipitated with anti-HA. Co-immunoprecipitated 3΄-UTR of tau mRNA covering (UG)n element 1 (Site 1) or (UG)n element 2 (Site 2) with TDP-43 was reverse-transcribed to cDNA with random primer or oligo-dT 18 and amplified by PCR with two sets of primers. The RT-PCR products from RNA-IP or cell lysate (Input) were separated by agarose electrophoresis (B) and quantitated by densitometry (C). ( D ) Physiological TDP-43 acts on tau mRNA 3΄-UTR. TDP-43 was immunoprecipitated from SH-SY5Y cells with anti-TDP-43 (H-8). Co-immunoprecipitated 3΄-UTR of tau mRNA with TDP-43 was reverse-transcribed to cDNA with oligo-dT 18 and amplified by PCR with two sets of primers. The RT-PCR products from RNA-IP or cell lysate (Input) were separated by agarose electrophoresis. The data are presented as mean ± SD ( n = 3). * P

    Journal: Nucleic Acids Research

    Article Title: TDP-43 suppresses tau expression via promoting its mRNA instability

    doi: 10.1093/nar/gkx175

    Figure Lengend Snippet: TDP-43 acts on the (UG)n elements of tau mRNA 3΄-UTR. ( A ) Schematic of (UG)n elements in tau mRNA 3΄-UTR. Two sets of primers specific to (UG)n elements, as indicated for amplifying the cDNA derived from tau mRNA 3΄-UTR. ( B and C ) TDP-43 acts on two (UG)n elements in tau mRNA 3΄-UTR. pEGFP/tau 3΄-UTR was co-transfected with pCI/TDP-43·HA into HEK-293FT cells. TDP-43 was immunoprecipitated with anti-HA. Co-immunoprecipitated 3΄-UTR of tau mRNA covering (UG)n element 1 (Site 1) or (UG)n element 2 (Site 2) with TDP-43 was reverse-transcribed to cDNA with random primer or oligo-dT 18 and amplified by PCR with two sets of primers. The RT-PCR products from RNA-IP or cell lysate (Input) were separated by agarose electrophoresis (B) and quantitated by densitometry (C). ( D ) Physiological TDP-43 acts on tau mRNA 3΄-UTR. TDP-43 was immunoprecipitated from SH-SY5Y cells with anti-TDP-43 (H-8). Co-immunoprecipitated 3΄-UTR of tau mRNA with TDP-43 was reverse-transcribed to cDNA with oligo-dT 18 and amplified by PCR with two sets of primers. The RT-PCR products from RNA-IP or cell lysate (Input) were separated by agarose electrophoresis. The data are presented as mean ± SD ( n = 3). * P

    Article Snippet: One microgram of total RNA was used for first-strand cDNA synthesis with oligo-(dT)18 by using the Omniscript reverse transcription kit (Qiagen).

    Techniques: Derivative Assay, Transfection, Immunoprecipitation, Amplification, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Electrophoresis