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
    Price:
    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 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    oligo dt primers - by Bioz Stars, 2021-05
    86/100 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.

    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.

    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.

    Synthesized:

    Article Title: Deficiency of fibroblast growth factor 21 (FGF21) promotes hepatocellular carcinoma (HCC) in mice on a long term obesogenic diet
    Article Snippet: Total RNA was isolated using the RNeasy Lipid Tissue Kit (Qiagen); DNA digestion step was included to prevent contamination of genomic DNA. .. Complementary DNA was synthesized from 0.5 μg of RNA using a mixture of oligo (dT) and random hexamer primers with Quantiscript Reverse Transcriptase (QuantiTect Reverse Transcription Kit; Qiagen). .. Quantitative polymerase chain reaction was performed using a 7800HT thermal cycler (Applied Biosystems, Foster City, CA) and SYBR green master mix (Applied Biosystems).

    Article Title: TPX2 silencing mediated by joint action of microvesicles and ultrasonic radiation inhibits the migration and invasion of SKOV3 cells
    Article Snippet: Total RNA was extracted using TRIzol (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. .. The concentration of extracted RNA was read using a UV spectrophotometer (Thermo Fisher Scientific, Inc.). cDNA was synthesized using: A reverse transcriptase, a poly (A) polymerase and tagged oligo (dT) primers in a 20 µl reaction volume for RT-qPCR (miScript; Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's protocol. .. Each RT-qPCR contained 2 µl of cDNA, 400 nM of each sense and antisense primer, and 2× SYBR-Green PCR Master Mix (Takara Bio, Inc., Otsu, Japan).

    Article Title: The liver-specific microRNA miR-122 controls systemic iron homeostasis in mice
    Article Snippet: Independent of the method of qPCR analysis, relative miRNA expression levels were calculated using qBase ( ) and determined relative to the appropriate reference gene(s). .. cDNA for the 3′–rapid amplification of cDNA ends (3′-RACE) reaction was synthesized as described above, except that reverse transcription was primed using an oligo(dT)-adapter primer (Qiagen). .. 3′-RACE was carried out in a final volume of 50 μl containing 1 pmol of gene-specific primers (Supplemental Table 3), 20 ng cDNA, 25 mM dNTP mix, and 2.5 U Thermo-Start Taq polymerase (Thermo-Fisher).

    Random Hexamer Labeling:

    Article Title: Deficiency of fibroblast growth factor 21 (FGF21) promotes hepatocellular carcinoma (HCC) in mice on a long term obesogenic diet
    Article Snippet: Total RNA was isolated using the RNeasy Lipid Tissue Kit (Qiagen); DNA digestion step was included to prevent contamination of genomic DNA. .. Complementary DNA was synthesized from 0.5 μg of RNA using a mixture of oligo (dT) and random hexamer primers with Quantiscript Reverse Transcriptase (QuantiTect Reverse Transcription Kit; Qiagen). .. Quantitative polymerase chain reaction was performed using a 7800HT thermal cycler (Applied Biosystems, Foster City, CA) and SYBR green master mix (Applied Biosystems).

    Quantitative RT-PCR:

    Article Title: Expansion of functional personalized cells with specific transgene combinations
    Article Snippet: For expression analysis, total RNA was extracted from harvested cell pellets using the RNeasy Mini Kit (Qiagen) in combination with QIAschredder (Qiagen). .. 5 µg of total RNA were reversely transcribed using the Ready-To-Go First Strand Beads (GE Healthcare) and OligodT primers. qRT-PCR was performed with Qiagen SYBR Green RT-PCR Mix on Light Cycler 480 (Roche). ..

    Article Title: TPX2 silencing mediated by joint action of microvesicles and ultrasonic radiation inhibits the migration and invasion of SKOV3 cells
    Article Snippet: Total RNA was extracted using TRIzol (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. .. The concentration of extracted RNA was read using a UV spectrophotometer (Thermo Fisher Scientific, Inc.). cDNA was synthesized using: A reverse transcriptase, a poly (A) polymerase and tagged oligo (dT) primers in a 20 µl reaction volume for RT-qPCR (miScript; Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's protocol. .. Each RT-qPCR contained 2 µl of cDNA, 400 nM of each sense and antisense primer, and 2× SYBR-Green PCR Master Mix (Takara Bio, Inc., Otsu, Japan).

    SYBR Green Assay:

    Article Title: Expansion of functional personalized cells with specific transgene combinations
    Article Snippet: For expression analysis, total RNA was extracted from harvested cell pellets using the RNeasy Mini Kit (Qiagen) in combination with QIAschredder (Qiagen). .. 5 µg of total RNA were reversely transcribed using the Ready-To-Go First Strand Beads (GE Healthcare) and OligodT primers. qRT-PCR was performed with Qiagen SYBR Green RT-PCR Mix on Light Cycler 480 (Roche). ..

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Expansion of functional personalized cells with specific transgene combinations
    Article Snippet: For expression analysis, total RNA was extracted from harvested cell pellets using the RNeasy Mini Kit (Qiagen) in combination with QIAschredder (Qiagen). .. 5 µg of total RNA were reversely transcribed using the Ready-To-Go First Strand Beads (GE Healthcare) and OligodT primers. qRT-PCR was performed with Qiagen SYBR Green RT-PCR Mix on Light Cycler 480 (Roche). ..

    Concentration Assay:

    Article Title: TPX2 silencing mediated by joint action of microvesicles and ultrasonic radiation inhibits the migration and invasion of SKOV3 cells
    Article Snippet: Total RNA was extracted using TRIzol (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. .. The concentration of extracted RNA was read using a UV spectrophotometer (Thermo Fisher Scientific, Inc.). cDNA was synthesized using: A reverse transcriptase, a poly (A) polymerase and tagged oligo (dT) primers in a 20 µl reaction volume for RT-qPCR (miScript; Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's protocol. .. Each RT-qPCR contained 2 µl of cDNA, 400 nM of each sense and antisense primer, and 2× SYBR-Green PCR Master Mix (Takara Bio, Inc., Otsu, Japan).

    Spectrophotometry:

    Article Title: TPX2 silencing mediated by joint action of microvesicles and ultrasonic radiation inhibits the migration and invasion of SKOV3 cells
    Article Snippet: Total RNA was extracted using TRIzol (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols. .. The concentration of extracted RNA was read using a UV spectrophotometer (Thermo Fisher Scientific, Inc.). cDNA was synthesized using: A reverse transcriptase, a poly (A) polymerase and tagged oligo (dT) primers in a 20 µl reaction volume for RT-qPCR (miScript; Qiagen, Inc., Valencia, CA, USA) according to the manufacturer's protocol. .. Each RT-qPCR contained 2 µl of cDNA, 400 nM of each sense and antisense primer, and 2× SYBR-Green PCR Master Mix (Takara Bio, Inc., Otsu, Japan).

    Amplification:

    Article Title: The liver-specific microRNA miR-122 controls systemic iron homeostasis in mice
    Article Snippet: Independent of the method of qPCR analysis, relative miRNA expression levels were calculated using qBase ( ) and determined relative to the appropriate reference gene(s). .. cDNA for the 3′–rapid amplification of cDNA ends (3′-RACE) reaction was synthesized as described above, except that reverse transcription was primed using an oligo(dT)-adapter primer (Qiagen). .. 3′-RACE was carried out in a final volume of 50 μl containing 1 pmol of gene-specific primers (Supplemental Table 3), 20 ng cDNA, 25 mM dNTP mix, and 2.5 U Thermo-Start Taq polymerase (Thermo-Fisher).

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    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: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/oligo dt primer/product/Qiagen
    Average 86 stars, based on 1 article reviews
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    oligo dt primer - by Bioz Stars, 2021-05
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    Qiagen oligo dt primers
    KLF2 and SMAD7 genes were upregulated by stimulation with SCF or NGF . A: HMC-1 (V560G c-Kit) cells were either incubated with serum (serum) or in serum free medium for 17 h, then treated without (-) or with (+) imatinib (5 μM) for 4 h prior to stimulation without (-) or with SCF (100 ng/ml) or NGF (100 ng/ml) for 30 (30) and 120 min (120). Total RNA was isolated and, after reverse transcription with <t>Oligo</t> dT-primers, cDNA was analyzed by semi-quantitative <t>PCR</t> for expression of EGR1, KLF2, SMAD7 and actin (ACTB). Water (H 2 O) and RNA (RNA) serve as negative controls. bp: base pairs of the DNA marker. B: Expression of KLF2 was quantified using TaqMan-PCR as in figure 3. Relative expression levels compared to GUSB were normalized to gene expression in serum-starved HMC-1(V560G c-Kit) cells. Average values from six independent PCR reactions + SEM are shown.
    Oligo Dt Primers, supplied by Qiagen, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/oligo dt primers/product/Qiagen
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    oligo dt primers - by Bioz Stars, 2021-05
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    86
    Qiagen epor sirna oligo
    Effects of <t>EpoR</t> knockdown in vitro A. EpoR mRNA expression. Quantitative RT-PCR of EpoR in melanoma cells with control scrambled or EpoR <t>shRNA.</t> * indicates p
    Epor Sirna Oligo, supplied by Qiagen, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    Qiagen oligo dt
    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 <t>mRNA</t> 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 <t>oligo</t> 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.
    Oligo Dt, supplied by Qiagen, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/oligo dt/product/Qiagen
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    oligo dt - by Bioz Stars, 2021-05
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    Image Search Results


    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

    Effects of EpoR knockdown in vitro A. EpoR mRNA expression. Quantitative RT-PCR of EpoR in melanoma cells with control scrambled or EpoR shRNA. * indicates p

    Journal: Oncogene

    Article Title: Erythropoietin Receptor Contributes to Melanoma Cell Survival in vivo

    doi: 10.1038/onc.2011.366

    Figure Lengend Snippet: Effects of EpoR knockdown in vitro A. EpoR mRNA expression. Quantitative RT-PCR of EpoR in melanoma cells with control scrambled or EpoR shRNA. * indicates p

    Article Snippet: EpoR siRNA oligo (Hs EpoR 5 Cat No: S102780351) was purchased from Qiagen.

    Techniques: In Vitro, Expressing, Quantitative RT-PCR, shRNA

    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