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  • 99
    Millipore reverse transcription polymerase chain reaction rt pcr
    Reverse Transcription Polymerase Chain Reaction Rt Pcr, supplied by Millipore, 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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    Thermo Fisher one step reverse transcription polymerase chain reaction rt pcr
    Expression of <t>RCAS-MYC</t> and RCAS-β-catenin in Ntv-a mouse brain cultures. (A) RCAS vector carrying exogenous gene (human c-MYC cDNA) 3′ of the retroviral env gene. Although RCAS vectors replicate only in avian cells, the exogenous gene is expressed in both avian and mammalian cells as a spliced message from the constitutive retroviral promoter, LTR. (B) Western blot showing expression of human MYC protein (67 kDa) in a mouse brain culture infected with RCAS-MYC retrovirus. No expression was detected in cells infected with control virus carrying LACZ (9E10 antibody, 1:100). (C) <t>RT-PCR</t> analysis of brain cultures from three different Ntv-a mice (lanes 1–3) infected with RCAS-MYC or RCAS-β-catenin and two uninfected cultures (lanes 4 and 5). Total RNA was used as template in RT-PCR reactions with oligonucleotide primers specific for either human MYC or CTNNB1 coding sequences within the integrated RCAS proviruses. The MYC product (838 bp) or CTNNB1 (819 bp) product was present in all infected cultures, but in none of the uninfected cultures. As a positive control, we used RNA from virus producer cell lines, DF1-MYC or DF1-CTNNB1 (lane 6). Parallel PCRs showed no amplification from residual traces of genomic DNA in the RNA samples (lanes 1–6).
    One Step Reverse Transcription Polymerase Chain Reaction Rt Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega reverse transcription polymerase chain reaction rt pcr kit
    Expression of <t>RCAS-MYC</t> and RCAS-β-catenin in Ntv-a mouse brain cultures. (A) RCAS vector carrying exogenous gene (human c-MYC cDNA) 3′ of the retroviral env gene. Although RCAS vectors replicate only in avian cells, the exogenous gene is expressed in both avian and mammalian cells as a spliced message from the constitutive retroviral promoter, LTR. (B) Western blot showing expression of human MYC protein (67 kDa) in a mouse brain culture infected with RCAS-MYC retrovirus. No expression was detected in cells infected with control virus carrying LACZ (9E10 antibody, 1:100). (C) <t>RT-PCR</t> analysis of brain cultures from three different Ntv-a mice (lanes 1–3) infected with RCAS-MYC or RCAS-β-catenin and two uninfected cultures (lanes 4 and 5). Total RNA was used as template in RT-PCR reactions with oligonucleotide primers specific for either human MYC or CTNNB1 coding sequences within the integrated RCAS proviruses. The MYC product (838 bp) or CTNNB1 (819 bp) product was present in all infected cultures, but in none of the uninfected cultures. As a positive control, we used RNA from virus producer cell lines, DF1-MYC or DF1-CTNNB1 (lane 6). Parallel PCRs showed no amplification from residual traces of genomic DNA in the RNA samples (lanes 1–6).
    Reverse Transcription Polymerase Chain Reaction Rt Pcr Kit, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher reverse transcription polymerase chain reaction rt pcr kit
    Changes in SpSOS1 and <t>SpAHA1</t> mRNAs in S . portulacastrum exposed to salinity. Seedlings were treated with 400, 600 or 800 mM NaCl. The expression of SpSOS1 (A, B) and SpAHA1 (C, D) was analyzed in roots (A, C) and leaves (B, D) at different time intervals (0, 3, 6, 9, 12, 24 and 48 h) under salt treatment using real-time <t>PCR.</t> The expression value at the initial time (0 h) was set to 1, and the expression levels at the other time points under salinity treatment were calculated as the fold of the value at 0 h. Values are expressed as the means±SE (n = 3).
    Reverse Transcription Polymerase Chain Reaction Rt Pcr Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa reverse transcription polymerase chain reaction rt pcr kit
    Changes in BCL-2 family expression caused by rapamycin treatment and FAK down-regulation. REH-empty vector or REH-FAK shRNA cells were cultured with or without rapamycin (100 nM). After 10 h, <t>RNA</t> was extracted from these cells and mRNA expression was detected using quantitative real-time <t>PCR.</t> The expression of pro-apoptosis genes, such as BIK, PUMA, BMF, BAX, and MCL-1S, was higher in the combination treatment group than in the rapamycin-only group. The expression of anti-apoptosis genes, such as BCL-2, was decreased in the combination treatment group compared with the rapamycin-only group. The results represent the mean ± S.D. of three experiments performed in triplicate. * p
    Reverse Transcription Polymerase Chain Reaction Rt Pcr Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 119 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Labnet reverse transcription polymerase chain reaction rt pcr machine
    Changes in BCL-2 family expression caused by rapamycin treatment and FAK down-regulation. REH-empty vector or REH-FAK shRNA cells were cultured with or without rapamycin (100 nM). After 10 h, <t>RNA</t> was extracted from these cells and mRNA expression was detected using quantitative real-time <t>PCR.</t> The expression of pro-apoptosis genes, such as BIK, PUMA, BMF, BAX, and MCL-1S, was higher in the combination treatment group than in the rapamycin-only group. The expression of anti-apoptosis genes, such as BCL-2, was decreased in the combination treatment group compared with the rapamycin-only group. The results represent the mean ± S.D. of three experiments performed in triplicate. * p
    Reverse Transcription Polymerase Chain Reaction Rt Pcr Machine, supplied by Labnet, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa reverse transcription polymerase chain reaction rt pcr analysis
    <t>Pluripotency</t> of patient-derived iPSCs. (A) Embryoid bodies expressed differentiation markers specific to all three germ layers. To examine the differentiation potential of iPSCs, we evaluated their ability to form embryoid bodies by using floating culture assays. <t>RT-PCR</t> was used to examine the expression of differentiation markers in the embryoid bodies. AFP was used as an endodermal marker, MSX1 as a mesodermal marker, and MAP as an ectodermal marker, with β-actin used as an internal control. (B) Teratoma formation in G-OFiPSCs. Patient-derived iPSCs (G-OF1iPS clones 1 and 6, and G-OF2iPS clones 5 and 8) were injected at a concentration of 1 × 10 6 cells in 20 μl of PBS into 8–10-week-old male CB17 SCID mice. Tumors were collected 9–12 weeks after the injections. Histological examination demonstrated that tissues originating from all three embryonic germ layers were present, including cartilage (mesoderm), gut-like epithelium (endoderm), and neural tube-like structures (ectoderm).
    Reverse Transcription Polymerase Chain Reaction Rt Pcr Analysis, supplied by TaKaRa, used in various techniques. Bioz Stars score: 93/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche reverse transcription polymerase chain reaction rt pcr kit
    <t>Pluripotency</t> of patient-derived iPSCs. (A) Embryoid bodies expressed differentiation markers specific to all three germ layers. To examine the differentiation potential of iPSCs, we evaluated their ability to form embryoid bodies by using floating culture assays. <t>RT-PCR</t> was used to examine the expression of differentiation markers in the embryoid bodies. AFP was used as an endodermal marker, MSX1 as a mesodermal marker, and MAP as an ectodermal marker, with β-actin used as an internal control. (B) Teratoma formation in G-OFiPSCs. Patient-derived iPSCs (G-OF1iPS clones 1 and 6, and G-OF2iPS clones 5 and 8) were injected at a concentration of 1 × 10 6 cells in 20 μl of PBS into 8–10-week-old male CB17 SCID mice. Tumors were collected 9–12 weeks after the injections. Histological examination demonstrated that tissues originating from all three embryonic germ layers were present, including cartilage (mesoderm), gut-like epithelium (endoderm), and neural tube-like structures (ectoderm).
    Reverse Transcription Polymerase Chain Reaction Rt Pcr Kit, supplied by Roche, used in various techniques. Bioz Stars score: 95/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tiangen biotech co reverse transcription polymerase chain reaction rt pcr kits
    <t>Pluripotency</t> of patient-derived iPSCs. (A) Embryoid bodies expressed differentiation markers specific to all three germ layers. To examine the differentiation potential of iPSCs, we evaluated their ability to form embryoid bodies by using floating culture assays. <t>RT-PCR</t> was used to examine the expression of differentiation markers in the embryoid bodies. AFP was used as an endodermal marker, MSX1 as a mesodermal marker, and MAP as an ectodermal marker, with β-actin used as an internal control. (B) Teratoma formation in G-OFiPSCs. Patient-derived iPSCs (G-OF1iPS clones 1 and 6, and G-OF2iPS clones 5 and 8) were injected at a concentration of 1 × 10 6 cells in 20 μl of PBS into 8–10-week-old male CB17 SCID mice. Tumors were collected 9–12 weeks after the injections. Histological examination demonstrated that tissues originating from all three embryonic germ layers were present, including cartilage (mesoderm), gut-like epithelium (endoderm), and neural tube-like structures (ectoderm).
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    Thermo Fisher reverse transcription polymerase chain reaction rt pcr primers
    <t>Pluripotency</t> of patient-derived iPSCs. (A) Embryoid bodies expressed differentiation markers specific to all three germ layers. To examine the differentiation potential of iPSCs, we evaluated their ability to form embryoid bodies by using floating culture assays. <t>RT-PCR</t> was used to examine the expression of differentiation markers in the embryoid bodies. AFP was used as an endodermal marker, MSX1 as a mesodermal marker, and MAP as an ectodermal marker, with β-actin used as an internal control. (B) Teratoma formation in G-OFiPSCs. Patient-derived iPSCs (G-OF1iPS clones 1 and 6, and G-OF2iPS clones 5 and 8) were injected at a concentration of 1 × 10 6 cells in 20 μl of PBS into 8–10-week-old male CB17 SCID mice. Tumors were collected 9–12 weeks after the injections. Histological examination demonstrated that tissues originating from all three embryonic germ layers were present, including cartilage (mesoderm), gut-like epithelium (endoderm), and neural tube-like structures (ectoderm).
    Reverse Transcription Polymerase Chain Reaction Rt Pcr Primers, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher reverse transcription polymerase chain reaction rt pcr system
    <t>Pluripotency</t> of patient-derived iPSCs. (A) Embryoid bodies expressed differentiation markers specific to all three germ layers. To examine the differentiation potential of iPSCs, we evaluated their ability to form embryoid bodies by using floating culture assays. <t>RT-PCR</t> was used to examine the expression of differentiation markers in the embryoid bodies. AFP was used as an endodermal marker, MSX1 as a mesodermal marker, and MAP as an ectodermal marker, with β-actin used as an internal control. (B) Teratoma formation in G-OFiPSCs. Patient-derived iPSCs (G-OF1iPS clones 1 and 6, and G-OF2iPS clones 5 and 8) were injected at a concentration of 1 × 10 6 cells in 20 μl of PBS into 8–10-week-old male CB17 SCID mice. Tumors were collected 9–12 weeks after the injections. Histological examination demonstrated that tissues originating from all three embryonic germ layers were present, including cartilage (mesoderm), gut-like epithelium (endoderm), and neural tube-like structures (ectoderm).
    Reverse Transcription Polymerase Chain Reaction Rt Pcr System, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher reverse transcription polymerase chain reaction rt pcr semi quantitative rt pcr
    <t>Pluripotency</t> of patient-derived iPSCs. (A) Embryoid bodies expressed differentiation markers specific to all three germ layers. To examine the differentiation potential of iPSCs, we evaluated their ability to form embryoid bodies by using floating culture assays. <t>RT-PCR</t> was used to examine the expression of differentiation markers in the embryoid bodies. AFP was used as an endodermal marker, MSX1 as a mesodermal marker, and MAP as an ectodermal marker, with β-actin used as an internal control. (B) Teratoma formation in G-OFiPSCs. Patient-derived iPSCs (G-OF1iPS clones 1 and 6, and G-OF2iPS clones 5 and 8) were injected at a concentration of 1 × 10 6 cells in 20 μl of PBS into 8–10-week-old male CB17 SCID mice. Tumors were collected 9–12 weeks after the injections. Histological examination demonstrated that tissues originating from all three embryonic germ layers were present, including cartilage (mesoderm), gut-like epithelium (endoderm), and neural tube-like structures (ectoderm).
    Reverse Transcription Polymerase Chain Reaction Rt Pcr Semi Quantitative Rt Pcr, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Toyobo reverse transcription polymerase chain reaction rt pcr kit
    <t>Pluripotency</t> of patient-derived iPSCs. (A) Embryoid bodies expressed differentiation markers specific to all three germ layers. To examine the differentiation potential of iPSCs, we evaluated their ability to form embryoid bodies by using floating culture assays. <t>RT-PCR</t> was used to examine the expression of differentiation markers in the embryoid bodies. AFP was used as an endodermal marker, MSX1 as a mesodermal marker, and MAP as an ectodermal marker, with β-actin used as an internal control. (B) Teratoma formation in G-OFiPSCs. Patient-derived iPSCs (G-OF1iPS clones 1 and 6, and G-OF2iPS clones 5 and 8) were injected at a concentration of 1 × 10 6 cells in 20 μl of PBS into 8–10-week-old male CB17 SCID mice. Tumors were collected 9–12 weeks after the injections. Histological examination demonstrated that tissues originating from all three embryonic germ layers were present, including cartilage (mesoderm), gut-like epithelium (endoderm), and neural tube-like structures (ectoderm).
    Reverse Transcription Polymerase Chain Reaction Rt Pcr Kit, supplied by Toyobo, used in various techniques. Bioz Stars score: 95/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    iNtRON Biotechnology reverse transcription polymerase chain reaction rt pcr kits
    <t>Pluripotency</t> of patient-derived iPSCs. (A) Embryoid bodies expressed differentiation markers specific to all three germ layers. To examine the differentiation potential of iPSCs, we evaluated their ability to form embryoid bodies by using floating culture assays. <t>RT-PCR</t> was used to examine the expression of differentiation markers in the embryoid bodies. AFP was used as an endodermal marker, MSX1 as a mesodermal marker, and MAP as an ectodermal marker, with β-actin used as an internal control. (B) Teratoma formation in G-OFiPSCs. Patient-derived iPSCs (G-OF1iPS clones 1 and 6, and G-OF2iPS clones 5 and 8) were injected at a concentration of 1 × 10 6 cells in 20 μl of PBS into 8–10-week-old male CB17 SCID mice. Tumors were collected 9–12 weeks after the injections. Histological examination demonstrated that tissues originating from all three embryonic germ layers were present, including cartilage (mesoderm), gut-like epithelium (endoderm), and neural tube-like structures (ectoderm).
    Reverse Transcription Polymerase Chain Reaction Rt Pcr Kits, supplied by iNtRON Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega reverse transcription polymerase chain reaction rt pcr system
    <t>RT/PCR</t> amplification of LAS GP1 and TNF-α mRNAs in MDM. RNAs from MDM infected with LAS (lanes 1–6) or MOP (lanes 8–11) were extracted at 12 (lanes 1, 3, 5, 8, 10) or 24 (lanes 2, 4, 6, 9, 11) hours p. i. and used in RT-PCR with LAS GP1, GAPDH or TNF-α primers. <t>RNA</t> from mock-infected cells treated with LPS was used as a positive control for natural TNF-α mRNA expression ( lane 7 , 382-bp amplicon). 2,000 copies of exogenous synthesized DNA was included in PCR as TNF-α internal calibration standard, ICS ( lanes 5–8 , 482-bp amplicon). M, positions of DNA markers: ICS, TNF standard; TNF, wild type TNF-α cDNA. Note that wild type TNF-α cDNA and GAPDH cDNA amplicons have the same size, 382 bp.
    Reverse Transcription Polymerase Chain Reaction Rt Pcr System, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Eppendorf AG reverse transcription polymerase chain reaction rt pcr mastercycler
    <t>RT/PCR</t> amplification of LAS GP1 and TNF-α mRNAs in MDM. RNAs from MDM infected with LAS (lanes 1–6) or MOP (lanes 8–11) were extracted at 12 (lanes 1, 3, 5, 8, 10) or 24 (lanes 2, 4, 6, 9, 11) hours p. i. and used in RT-PCR with LAS GP1, GAPDH or TNF-α primers. <t>RNA</t> from mock-infected cells treated with LPS was used as a positive control for natural TNF-α mRNA expression ( lane 7 , 382-bp amplicon). 2,000 copies of exogenous synthesized DNA was included in PCR as TNF-α internal calibration standard, ICS ( lanes 5–8 , 482-bp amplicon). M, positions of DNA markers: ICS, TNF standard; TNF, wild type TNF-α cDNA. Note that wild type TNF-α cDNA and GAPDH cDNA amplicons have the same size, 382 bp.
    Reverse Transcription Polymerase Chain Reaction Rt Pcr Mastercycler, supplied by Eppendorf AG, used in various techniques. Bioz Stars score: 95/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa reverse transcription polymerase chain reaction rt pcr protocol
    ID proteins inhibit E-cadherin expression in breast cells. Notes: ( A ) Western blot analysis of MCF10A cells with stable expression of <t>ID1,</t> ID2, or ID3. ( B ) Real-time <t>PCR</t> analysis of E-cadherin mRNA abundance in cell lines in ( A ). ( C ) Western blot analysis of E-cadherin protein abundance in cell lines in ( A ). ( D ) Assessment of E-cadherin promoter activity, using luciferase reporter assays, in HEK293T cells. The 1.38 kb DNA sequence containing E-cadherin promoter region was cloned upstream of the luciferase gene in a reporter construct (top image). The relative luciferase activities of luciferase reporters with E-cadherin promoter were determined in HEK293T cells, which were cotransfected with the ID1, ID2, ID3, and control vectors. For ( B ) and ( D , bottom image), data represent mean values, with error bars indicating SEM. ** P
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    Thermo Fisher reverse transcription polymerase chain reaction rt pcr trizol
    ID proteins inhibit E-cadherin expression in breast cells. Notes: ( A ) Western blot analysis of MCF10A cells with stable expression of <t>ID1,</t> ID2, or ID3. ( B ) Real-time <t>PCR</t> analysis of E-cadherin mRNA abundance in cell lines in ( A ). ( C ) Western blot analysis of E-cadherin protein abundance in cell lines in ( A ). ( D ) Assessment of E-cadherin promoter activity, using luciferase reporter assays, in HEK293T cells. The 1.38 kb DNA sequence containing E-cadherin promoter region was cloned upstream of the luciferase gene in a reporter construct (top image). The relative luciferase activities of luciferase reporters with E-cadherin promoter were determined in HEK293T cells, which were cotransfected with the ID1, ID2, ID3, and control vectors. For ( B ) and ( D , bottom image), data represent mean values, with error bars indicating SEM. ** P
    Reverse Transcription Polymerase Chain Reaction Rt Pcr Trizol, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    iNtRON Biotechnology conventional reverse transcription polymerase chain reaction rt pcr
    ID proteins inhibit E-cadherin expression in breast cells. Notes: ( A ) Western blot analysis of MCF10A cells with stable expression of <t>ID1,</t> ID2, or ID3. ( B ) Real-time <t>PCR</t> analysis of E-cadherin mRNA abundance in cell lines in ( A ). ( C ) Western blot analysis of E-cadherin protein abundance in cell lines in ( A ). ( D ) Assessment of E-cadherin promoter activity, using luciferase reporter assays, in HEK293T cells. The 1.38 kb DNA sequence containing E-cadherin promoter region was cloned upstream of the luciferase gene in a reporter construct (top image). The relative luciferase activities of luciferase reporters with E-cadherin promoter were determined in HEK293T cells, which were cotransfected with the ID1, ID2, ID3, and control vectors. For ( B ) and ( D , bottom image), data represent mean values, with error bars indicating SEM. ** P
    Conventional Reverse Transcription Polymerase Chain Reaction Rt Pcr, supplied by iNtRON Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa reverse transcription polymerase chain reaction amplification rt pcr kit
    ID proteins inhibit E-cadherin expression in breast cells. Notes: ( A ) Western blot analysis of MCF10A cells with stable expression of <t>ID1,</t> ID2, or ID3. ( B ) Real-time <t>PCR</t> analysis of E-cadherin mRNA abundance in cell lines in ( A ). ( C ) Western blot analysis of E-cadherin protein abundance in cell lines in ( A ). ( D ) Assessment of E-cadherin promoter activity, using luciferase reporter assays, in HEK293T cells. The 1.38 kb DNA sequence containing E-cadherin promoter region was cloned upstream of the luciferase gene in a reporter construct (top image). The relative luciferase activities of luciferase reporters with E-cadherin promoter were determined in HEK293T cells, which were cotransfected with the ID1, ID2, ID3, and control vectors. For ( B ) and ( D , bottom image), data represent mean values, with error bars indicating SEM. ** P
    Reverse Transcription Polymerase Chain Reaction Amplification Rt Pcr Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa reverse transcription polymerase chain reaction rt pcr reagents
    ID proteins inhibit E-cadherin expression in breast cells. Notes: ( A ) Western blot analysis of MCF10A cells with stable expression of <t>ID1,</t> ID2, or ID3. ( B ) Real-time <t>PCR</t> analysis of E-cadherin mRNA abundance in cell lines in ( A ). ( C ) Western blot analysis of E-cadherin protein abundance in cell lines in ( A ). ( D ) Assessment of E-cadherin promoter activity, using luciferase reporter assays, in HEK293T cells. The 1.38 kb DNA sequence containing E-cadherin promoter region was cloned upstream of the luciferase gene in a reporter construct (top image). The relative luciferase activities of luciferase reporters with E-cadherin promoter were determined in HEK293T cells, which were cotransfected with the ID1, ID2, ID3, and control vectors. For ( B ) and ( D , bottom image), data represent mean values, with error bars indicating SEM. ** P
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    ID proteins inhibit E-cadherin expression in breast cells. Notes: ( A ) Western blot analysis of MCF10A cells with stable expression of <t>ID1,</t> ID2, or ID3. ( B ) Real-time <t>PCR</t> analysis of E-cadherin mRNA abundance in cell lines in ( A ). ( C ) Western blot analysis of E-cadherin protein abundance in cell lines in ( A ). ( D ) Assessment of E-cadherin promoter activity, using luciferase reporter assays, in HEK293T cells. The 1.38 kb DNA sequence containing E-cadherin promoter region was cloned upstream of the luciferase gene in a reporter construct (top image). The relative luciferase activities of luciferase reporters with E-cadherin promoter were determined in HEK293T cells, which were cotransfected with the ID1, ID2, ID3, and control vectors. For ( B ) and ( D , bottom image), data represent mean values, with error bars indicating SEM. ** P
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    ID proteins inhibit E-cadherin expression in breast cells. Notes: ( A ) Western blot analysis of MCF10A cells with stable expression of <t>ID1,</t> ID2, or ID3. ( B ) Real-time <t>PCR</t> analysis of E-cadherin mRNA abundance in cell lines in ( A ). ( C ) Western blot analysis of E-cadherin protein abundance in cell lines in ( A ). ( D ) Assessment of E-cadherin promoter activity, using luciferase reporter assays, in HEK293T cells. The 1.38 kb DNA sequence containing E-cadherin promoter region was cloned upstream of the luciferase gene in a reporter construct (top image). The relative luciferase activities of luciferase reporters with E-cadherin promoter were determined in HEK293T cells, which were cotransfected with the ID1, ID2, ID3, and control vectors. For ( B ) and ( D , bottom image), data represent mean values, with error bars indicating SEM. ** P
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    ID proteins inhibit E-cadherin expression in breast cells. Notes: ( A ) Western blot analysis of MCF10A cells with stable expression of <t>ID1,</t> ID2, or ID3. ( B ) Real-time <t>PCR</t> analysis of E-cadherin mRNA abundance in cell lines in ( A ). ( C ) Western blot analysis of E-cadherin protein abundance in cell lines in ( A ). ( D ) Assessment of E-cadherin promoter activity, using luciferase reporter assays, in HEK293T cells. The 1.38 kb DNA sequence containing E-cadherin promoter region was cloned upstream of the luciferase gene in a reporter construct (top image). The relative luciferase activities of luciferase reporters with E-cadherin promoter were determined in HEK293T cells, which were cotransfected with the ID1, ID2, ID3, and control vectors. For ( B ) and ( D , bottom image), data represent mean values, with error bars indicating SEM. ** P
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    ID proteins inhibit E-cadherin expression in breast cells. Notes: ( A ) Western blot analysis of MCF10A cells with stable expression of <t>ID1,</t> ID2, or ID3. ( B ) Real-time <t>PCR</t> analysis of E-cadherin mRNA abundance in cell lines in ( A ). ( C ) Western blot analysis of E-cadherin protein abundance in cell lines in ( A ). ( D ) Assessment of E-cadherin promoter activity, using luciferase reporter assays, in HEK293T cells. The 1.38 kb DNA sequence containing E-cadherin promoter region was cloned upstream of the luciferase gene in a reporter construct (top image). The relative luciferase activities of luciferase reporters with E-cadherin promoter were determined in HEK293T cells, which were cotransfected with the ID1, ID2, ID3, and control vectors. For ( B ) and ( D , bottom image), data represent mean values, with error bars indicating SEM. ** P
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    Image Search Results


    Expression of RCAS-MYC and RCAS-β-catenin in Ntv-a mouse brain cultures. (A) RCAS vector carrying exogenous gene (human c-MYC cDNA) 3′ of the retroviral env gene. Although RCAS vectors replicate only in avian cells, the exogenous gene is expressed in both avian and mammalian cells as a spliced message from the constitutive retroviral promoter, LTR. (B) Western blot showing expression of human MYC protein (67 kDa) in a mouse brain culture infected with RCAS-MYC retrovirus. No expression was detected in cells infected with control virus carrying LACZ (9E10 antibody, 1:100). (C) RT-PCR analysis of brain cultures from three different Ntv-a mice (lanes 1–3) infected with RCAS-MYC or RCAS-β-catenin and two uninfected cultures (lanes 4 and 5). Total RNA was used as template in RT-PCR reactions with oligonucleotide primers specific for either human MYC or CTNNB1 coding sequences within the integrated RCAS proviruses. The MYC product (838 bp) or CTNNB1 (819 bp) product was present in all infected cultures, but in none of the uninfected cultures. As a positive control, we used RNA from virus producer cell lines, DF1-MYC or DF1-CTNNB1 (lane 6). Parallel PCRs showed no amplification from residual traces of genomic DNA in the RNA samples (lanes 1–6).

    Journal: Neoplasia (New York, N.Y.)

    Article Title: MYC Expression Promotes the Proliferation of Neural Progenitor Cells in Culture and In Vivo 1

    doi:

    Figure Lengend Snippet: Expression of RCAS-MYC and RCAS-β-catenin in Ntv-a mouse brain cultures. (A) RCAS vector carrying exogenous gene (human c-MYC cDNA) 3′ of the retroviral env gene. Although RCAS vectors replicate only in avian cells, the exogenous gene is expressed in both avian and mammalian cells as a spliced message from the constitutive retroviral promoter, LTR. (B) Western blot showing expression of human MYC protein (67 kDa) in a mouse brain culture infected with RCAS-MYC retrovirus. No expression was detected in cells infected with control virus carrying LACZ (9E10 antibody, 1:100). (C) RT-PCR analysis of brain cultures from three different Ntv-a mice (lanes 1–3) infected with RCAS-MYC or RCAS-β-catenin and two uninfected cultures (lanes 4 and 5). Total RNA was used as template in RT-PCR reactions with oligonucleotide primers specific for either human MYC or CTNNB1 coding sequences within the integrated RCAS proviruses. The MYC product (838 bp) or CTNNB1 (819 bp) product was present in all infected cultures, but in none of the uninfected cultures. As a positive control, we used RNA from virus producer cell lines, DF1-MYC or DF1-CTNNB1 (lane 6). Parallel PCRs showed no amplification from residual traces of genomic DNA in the RNA samples (lanes 1–6).

    Article Snippet: To detect human MYC and CTNNB1 mRNA in mouse cells infected with RCAS vectors, we used total RNA (100 ng) as template in one-step reverse transcription polymerase chain reaction (RT-PCR) (Gibco BRL Superscript) with oligonucleotide primers specific for human MYC and CTNNB1 coding sequences within the integrated RCAS provirus.

    Techniques: Expressing, Plasmid Preparation, Western Blot, Infection, Reverse Transcription Polymerase Chain Reaction, Mouse Assay, Positive Control, Amplification

    Changes in SpSOS1 and SpAHA1 mRNAs in S . portulacastrum exposed to salinity. Seedlings were treated with 400, 600 or 800 mM NaCl. The expression of SpSOS1 (A, B) and SpAHA1 (C, D) was analyzed in roots (A, C) and leaves (B, D) at different time intervals (0, 3, 6, 9, 12, 24 and 48 h) under salt treatment using real-time PCR. The expression value at the initial time (0 h) was set to 1, and the expression levels at the other time points under salinity treatment were calculated as the fold of the value at 0 h. Values are expressed as the means±SE (n = 3).

    Journal: PLoS ONE

    Article Title: SpAHA1 and SpSOS1 Coordinate in Transgenic Yeast to Improve Salt Tolerance

    doi: 10.1371/journal.pone.0137447

    Figure Lengend Snippet: Changes in SpSOS1 and SpAHA1 mRNAs in S . portulacastrum exposed to salinity. Seedlings were treated with 400, 600 or 800 mM NaCl. The expression of SpSOS1 (A, B) and SpAHA1 (C, D) was analyzed in roots (A, C) and leaves (B, D) at different time intervals (0, 3, 6, 9, 12, 24 and 48 h) under salt treatment using real-time PCR. The expression value at the initial time (0 h) was set to 1, and the expression levels at the other time points under salinity treatment were calculated as the fold of the value at 0 h. Values are expressed as the means±SE (n = 3).

    Article Snippet: Cloning of SpSOS1 and SpAHA1 Total RNA isolation and cDNA synthesis were performed using a reverse transcription polymerase chain reaction (RT-PCR) kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Changes in BCL-2 family expression caused by rapamycin treatment and FAK down-regulation. REH-empty vector or REH-FAK shRNA cells were cultured with or without rapamycin (100 nM). After 10 h, RNA was extracted from these cells and mRNA expression was detected using quantitative real-time PCR. The expression of pro-apoptosis genes, such as BIK, PUMA, BMF, BAX, and MCL-1S, was higher in the combination treatment group than in the rapamycin-only group. The expression of anti-apoptosis genes, such as BCL-2, was decreased in the combination treatment group compared with the rapamycin-only group. The results represent the mean ± S.D. of three experiments performed in triplicate. * p

    Journal: Journal of Hematology & Oncology

    Article Title: Synergism between the mTOR inhibitor rapamycin and FAK down-regulation in the treatment of acute lymphoblastic leukemia

    doi: 10.1186/s13045-016-0241-x

    Figure Lengend Snippet: Changes in BCL-2 family expression caused by rapamycin treatment and FAK down-regulation. REH-empty vector or REH-FAK shRNA cells were cultured with or without rapamycin (100 nM). After 10 h, RNA was extracted from these cells and mRNA expression was detected using quantitative real-time PCR. The expression of pro-apoptosis genes, such as BIK, PUMA, BMF, BAX, and MCL-1S, was higher in the combination treatment group than in the rapamycin-only group. The expression of anti-apoptosis genes, such as BCL-2, was decreased in the combination treatment group compared with the rapamycin-only group. The results represent the mean ± S.D. of three experiments performed in triplicate. * p

    Article Snippet: Total RNA was extracted using the TRIzol reagent (Invitrogen, USA) according to the manufacturer’s protocols. cDNA was prepared from 1 μg of total RNA using a reverse transcription-polymerase chain reaction (RT-PCR) kit (Takara, Japan) with oligodT according to the manufacturer’s instructions. cDNA samples were then analyzed via quantitative real-time PCR using SYBR Green (Takara, Japan) in an ABI Step One Real-Time PCR machine (Applied Biosystems, Foster City, CA), with 40 cycles of 95 °C for 15 s and 60 °C for 30 s. The efficiency of cDNA synthesis was estimated using hGAPDH as a house-keeping gene.

    Techniques: Expressing, Plasmid Preparation, shRNA, Cell Culture, Real-time Polymerase Chain Reaction

    Telomerase activity, as assessed by stretch polymerase chain reaction in the (A) pEGFP-C3-PinX1, (B) pEGFP-C3, (C) Lipofectamine 2000 only, (D) control and (E) PinX1-FAM-siRNA groups. ECV-304 cells showed weak positive telomerase activity, whereas Eca-109 exhibited strong positive activity. These findings indicated that pEGFP-C3-PinX1 successfully inhibited telomerase activity, whereas the PinX1-FAM-siRNA, Lipofectamine 2000 only and pEGFP-C3 treatments did not alter telomerase activity.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Biological significance of PinX1 telomerase inhibitor in esophageal carcinoma treatment

    doi: 10.3892/etm.2016.3561

    Figure Lengend Snippet: Telomerase activity, as assessed by stretch polymerase chain reaction in the (A) pEGFP-C3-PinX1, (B) pEGFP-C3, (C) Lipofectamine 2000 only, (D) control and (E) PinX1-FAM-siRNA groups. ECV-304 cells showed weak positive telomerase activity, whereas Eca-109 exhibited strong positive activity. These findings indicated that pEGFP-C3-PinX1 successfully inhibited telomerase activity, whereas the PinX1-FAM-siRNA, Lipofectamine 2000 only and pEGFP-C3 treatments did not alter telomerase activity.

    Article Snippet: Austrian newborn cow blood serum was purchased from Weijia Company (Guangzhou, China); Lipofectamine 2000 and all primers (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA); reverse transcription-polymerase chain reaction (RT-PCR) detection kit (Takara Biotechnology Co., Ltd., Dalian, China); Thiazolyl blue tetrazolium bromide for the MTT assay (Sigma-Aldrich St. Louis, MO, USA); telomerase activity detection kit (Toyobo, Co., Ltd., Osaka, Japan).

    Techniques: Activity Assay, Polymerase Chain Reaction

    Pluripotency of patient-derived iPSCs. (A) Embryoid bodies expressed differentiation markers specific to all three germ layers. To examine the differentiation potential of iPSCs, we evaluated their ability to form embryoid bodies by using floating culture assays. RT-PCR was used to examine the expression of differentiation markers in the embryoid bodies. AFP was used as an endodermal marker, MSX1 as a mesodermal marker, and MAP as an ectodermal marker, with β-actin used as an internal control. (B) Teratoma formation in G-OFiPSCs. Patient-derived iPSCs (G-OF1iPS clones 1 and 6, and G-OF2iPS clones 5 and 8) were injected at a concentration of 1 × 10 6 cells in 20 μl of PBS into 8–10-week-old male CB17 SCID mice. Tumors were collected 9–12 weeks after the injections. Histological examination demonstrated that tissues originating from all three embryonic germ layers were present, including cartilage (mesoderm), gut-like epithelium (endoderm), and neural tube-like structures (ectoderm).

    Journal: PLoS ONE

    Article Title: Gorlin syndrome-derived induced pluripotent stem cells are hypersensitive to hedgehog-mediated osteogenic induction

    doi: 10.1371/journal.pone.0186879

    Figure Lengend Snippet: Pluripotency of patient-derived iPSCs. (A) Embryoid bodies expressed differentiation markers specific to all three germ layers. To examine the differentiation potential of iPSCs, we evaluated their ability to form embryoid bodies by using floating culture assays. RT-PCR was used to examine the expression of differentiation markers in the embryoid bodies. AFP was used as an endodermal marker, MSX1 as a mesodermal marker, and MAP as an ectodermal marker, with β-actin used as an internal control. (B) Teratoma formation in G-OFiPSCs. Patient-derived iPSCs (G-OF1iPS clones 1 and 6, and G-OF2iPS clones 5 and 8) were injected at a concentration of 1 × 10 6 cells in 20 μl of PBS into 8–10-week-old male CB17 SCID mice. Tumors were collected 9–12 weeks after the injections. Histological examination demonstrated that tissues originating from all three embryonic germ layers were present, including cartilage (mesoderm), gut-like epithelium (endoderm), and neural tube-like structures (ectoderm).

    Article Snippet: The stemness and pluripotency of the iPSCs were analyzed using reverse transcription polymerase chain reaction (RT-PCR) analysis with ExTaq DNA polymerase (Takara Biotechnology, Shiga, Japan).

    Techniques: Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Marker, Clone Assay, Injection, Concentration Assay, Mouse Assay

    Hh-dependent induction of osteogenic marker ALP in patient-derived iPSCs. (A) Control iPSCs (KD iPSCs, 201B7iPS cells, and Nips B2 iPSCs) and patient-derived iPSCs (G-OF1iPS1, and G-OF2iPS5) were cultured in OBM, OBM with FIT (bFGF, IGF-1, and TGF-β1), or OBM with SAG. FIT- and SAG-induced ALP activity significantly increased in patient-derived iPSCs compared with control iPSCs. (B) A sample of each supernatant was transferred to tubes for measurement of ALP activity as the hydrolysis of ρ-nitrophenyl phosphate using a LabAssay ALP kit and protein concentration using a Protein assay rapid kit. G-OF1iPS1 was showed a high ALP activity compared with 201B7,especially cultured in OBM with SAG. (C) KDiPSCs and G-OF1iPS1 cells were cultured in OBM (control), or OBM supplemented with 100 ng/ml BMP2/7 for 24 h. qRT-PCR analysis of RUNX2 was conducted using Premix Ex Taq reagent according to the manufacturer’s instructions. RUNX2 levels were normalized to those of GAPDH. A significant difference was observed in G-OF1iPS1. Especially, BMP2/7-induced RUNX2 expression promoted an approximate 2-fold increase in the number of G-OFiPSCs compared with KDiPSCs. Values represent the mean ± SD. Bonferroni correction for multiple comparisons was applied, * p

    Journal: PLoS ONE

    Article Title: Gorlin syndrome-derived induced pluripotent stem cells are hypersensitive to hedgehog-mediated osteogenic induction

    doi: 10.1371/journal.pone.0186879

    Figure Lengend Snippet: Hh-dependent induction of osteogenic marker ALP in patient-derived iPSCs. (A) Control iPSCs (KD iPSCs, 201B7iPS cells, and Nips B2 iPSCs) and patient-derived iPSCs (G-OF1iPS1, and G-OF2iPS5) were cultured in OBM, OBM with FIT (bFGF, IGF-1, and TGF-β1), or OBM with SAG. FIT- and SAG-induced ALP activity significantly increased in patient-derived iPSCs compared with control iPSCs. (B) A sample of each supernatant was transferred to tubes for measurement of ALP activity as the hydrolysis of ρ-nitrophenyl phosphate using a LabAssay ALP kit and protein concentration using a Protein assay rapid kit. G-OF1iPS1 was showed a high ALP activity compared with 201B7,especially cultured in OBM with SAG. (C) KDiPSCs and G-OF1iPS1 cells were cultured in OBM (control), or OBM supplemented with 100 ng/ml BMP2/7 for 24 h. qRT-PCR analysis of RUNX2 was conducted using Premix Ex Taq reagent according to the manufacturer’s instructions. RUNX2 levels were normalized to those of GAPDH. A significant difference was observed in G-OF1iPS1. Especially, BMP2/7-induced RUNX2 expression promoted an approximate 2-fold increase in the number of G-OFiPSCs compared with KDiPSCs. Values represent the mean ± SD. Bonferroni correction for multiple comparisons was applied, * p

    Article Snippet: The stemness and pluripotency of the iPSCs were analyzed using reverse transcription polymerase chain reaction (RT-PCR) analysis with ExTaq DNA polymerase (Takara Biotechnology, Shiga, Japan).

    Techniques: Marker, ALP Assay, Derivative Assay, Cell Culture, Activity Assay, Protein Concentration, Quantitative RT-PCR, Expressing

    Distinct expression patterns of Hh-associated genes in control and patient-derived iPSCs under osteogenic conditions. We investigated two controls (KDiPS and 201B7) and four Gorlin iPS cases derived from different patients. Because we used single RT 2 Profiler PCR Array plate for each case, we did not performed statistical analysis. (A) Osteogeic induction promoted characteristic changes in the expression levels of the following genes associated with the canonical Hh pathway: GLI1 , GLI2 , GLI3 , and SHH . Compared with the basal conditions, GLI1 and GLI2 expression levels decreased in both controls (KDiPSCs and 201B7) and G-OFiPSCs grown in OBM, whereas GLI3 expression increased only in G-OFiPSCs grown in OBM. Basal expression levels of SHH was significantly lower in G-OFiPSCs compared with controls (KDiPSCs and 201B7). However, OBM enhanced SHH expression levels in G-OFiPSCs but not in control cells. (B) The basal expression levels of many Wnt genes were lower in patient-derived iPSCs than in control iPSCs. With the exception of WNT2 , all of the Wnt genes evaluated were upregulated in G-OFiPSCs grown in OBM and downregulated in control cells (KDiPSCs and 201B7) grown in OBM. (C) Changes in BMP2 , BMP6 , and RUNX2 expression levels were greater in patient-derived iPSCs than in controls (KD iPSCs and 201B7). The ΔΔC t value 10 days after OBM treatment (Post) was determined, and the fold-change relative to pre-induction (Pre) KDiPSCs was calculated for each gene.

    Journal: PLoS ONE

    Article Title: Gorlin syndrome-derived induced pluripotent stem cells are hypersensitive to hedgehog-mediated osteogenic induction

    doi: 10.1371/journal.pone.0186879

    Figure Lengend Snippet: Distinct expression patterns of Hh-associated genes in control and patient-derived iPSCs under osteogenic conditions. We investigated two controls (KDiPS and 201B7) and four Gorlin iPS cases derived from different patients. Because we used single RT 2 Profiler PCR Array plate for each case, we did not performed statistical analysis. (A) Osteogeic induction promoted characteristic changes in the expression levels of the following genes associated with the canonical Hh pathway: GLI1 , GLI2 , GLI3 , and SHH . Compared with the basal conditions, GLI1 and GLI2 expression levels decreased in both controls (KDiPSCs and 201B7) and G-OFiPSCs grown in OBM, whereas GLI3 expression increased only in G-OFiPSCs grown in OBM. Basal expression levels of SHH was significantly lower in G-OFiPSCs compared with controls (KDiPSCs and 201B7). However, OBM enhanced SHH expression levels in G-OFiPSCs but not in control cells. (B) The basal expression levels of many Wnt genes were lower in patient-derived iPSCs than in control iPSCs. With the exception of WNT2 , all of the Wnt genes evaluated were upregulated in G-OFiPSCs grown in OBM and downregulated in control cells (KDiPSCs and 201B7) grown in OBM. (C) Changes in BMP2 , BMP6 , and RUNX2 expression levels were greater in patient-derived iPSCs than in controls (KD iPSCs and 201B7). The ΔΔC t value 10 days after OBM treatment (Post) was determined, and the fold-change relative to pre-induction (Pre) KDiPSCs was calculated for each gene.

    Article Snippet: The stemness and pluripotency of the iPSCs were analyzed using reverse transcription polymerase chain reaction (RT-PCR) analysis with ExTaq DNA polymerase (Takara Biotechnology, Shiga, Japan).

    Techniques: Expressing, Derivative Assay, Polymerase Chain Reaction

    Generation of iPSCs from Gorlin syndrome fibroblasts (G-OFs). (A) We collected fibroblasts from surgically resected unaffected oral skin areas. Typical spindle shape fibroblasts were obtained (a). Fibroblasts (1 × 10 5 /well) were transfected with the Sendai virus vector SeVdp(KOSM)302L. The resulting iPSC colonies were selected 21–25 days after transfection and several clones were subsequently expanded (b). Stable clones (two clones of Patient 1, four clones of Patient 2, two clones of Patient 3, and six clones of Patient 4) exhibited the characteristic morphology of human iPSCs. Representative iPSCs (from passage number 6) are shown (c) (scale bar: 400 μm). (B) We confirmed the infection and silencing of SeVdp using RT-PCR. Cells expressed SeVdp mRNA one day after infection (TF). SeVdp was not detected in any G-OFs (F) or G-OFiPSCs. (C) RT-PCR analysis of embryonic stem cell marker genes in patient-derived iPSCs (G-OF1iPS clones 1 and 6; G-OF2iPS clones 3, 5, 8, and 10; G-OF3iPS clones 25 and 26; and G-OF4iPS clones 3, 8, 12, 15, 18, and 23) and control iPSCs (KDiPSCs). β-actin was used as a loading control. The target genes evaluated included OCT3/4 , SOX2 , NANOG , REX1 , c-MYC , and KLF4 .

    Journal: PLoS ONE

    Article Title: Gorlin syndrome-derived induced pluripotent stem cells are hypersensitive to hedgehog-mediated osteogenic induction

    doi: 10.1371/journal.pone.0186879

    Figure Lengend Snippet: Generation of iPSCs from Gorlin syndrome fibroblasts (G-OFs). (A) We collected fibroblasts from surgically resected unaffected oral skin areas. Typical spindle shape fibroblasts were obtained (a). Fibroblasts (1 × 10 5 /well) were transfected with the Sendai virus vector SeVdp(KOSM)302L. The resulting iPSC colonies were selected 21–25 days after transfection and several clones were subsequently expanded (b). Stable clones (two clones of Patient 1, four clones of Patient 2, two clones of Patient 3, and six clones of Patient 4) exhibited the characteristic morphology of human iPSCs. Representative iPSCs (from passage number 6) are shown (c) (scale bar: 400 μm). (B) We confirmed the infection and silencing of SeVdp using RT-PCR. Cells expressed SeVdp mRNA one day after infection (TF). SeVdp was not detected in any G-OFs (F) or G-OFiPSCs. (C) RT-PCR analysis of embryonic stem cell marker genes in patient-derived iPSCs (G-OF1iPS clones 1 and 6; G-OF2iPS clones 3, 5, 8, and 10; G-OF3iPS clones 25 and 26; and G-OF4iPS clones 3, 8, 12, 15, 18, and 23) and control iPSCs (KDiPSCs). β-actin was used as a loading control. The target genes evaluated included OCT3/4 , SOX2 , NANOG , REX1 , c-MYC , and KLF4 .

    Article Snippet: The stemness and pluripotency of the iPSCs were analyzed using reverse transcription polymerase chain reaction (RT-PCR) analysis with ExTaq DNA polymerase (Takara Biotechnology, Shiga, Japan).

    Techniques: Transfection, Plasmid Preparation, Clone Assay, Infection, Reverse Transcription Polymerase Chain Reaction, Marker, Derivative Assay

    RT/PCR amplification of LAS GP1 and TNF-α mRNAs in MDM. RNAs from MDM infected with LAS (lanes 1–6) or MOP (lanes 8–11) were extracted at 12 (lanes 1, 3, 5, 8, 10) or 24 (lanes 2, 4, 6, 9, 11) hours p. i. and used in RT-PCR with LAS GP1, GAPDH or TNF-α primers. RNA from mock-infected cells treated with LPS was used as a positive control for natural TNF-α mRNA expression ( lane 7 , 382-bp amplicon). 2,000 copies of exogenous synthesized DNA was included in PCR as TNF-α internal calibration standard, ICS ( lanes 5–8 , 482-bp amplicon). M, positions of DNA markers: ICS, TNF standard; TNF, wild type TNF-α cDNA. Note that wild type TNF-α cDNA and GAPDH cDNA amplicons have the same size, 382 bp.

    Journal: Journal of medical virology

    Article Title: Lassa and Mopeia Virus Replication in Human Monocytes/Macrophages and in Endothelial Cells: Different Effects on IL-8 and TNF-? Gene Expression

    doi:

    Figure Lengend Snippet: RT/PCR amplification of LAS GP1 and TNF-α mRNAs in MDM. RNAs from MDM infected with LAS (lanes 1–6) or MOP (lanes 8–11) were extracted at 12 (lanes 1, 3, 5, 8, 10) or 24 (lanes 2, 4, 6, 9, 11) hours p. i. and used in RT-PCR with LAS GP1, GAPDH or TNF-α primers. RNA from mock-infected cells treated with LPS was used as a positive control for natural TNF-α mRNA expression ( lane 7 , 382-bp amplicon). 2,000 copies of exogenous synthesized DNA was included in PCR as TNF-α internal calibration standard, ICS ( lanes 5–8 , 482-bp amplicon). M, positions of DNA markers: ICS, TNF standard; TNF, wild type TNF-α cDNA. Note that wild type TNF-α cDNA and GAPDH cDNA amplicons have the same size, 382 bp.

    Article Snippet: For detection of Lassa virus-specific RNA in infected cells the Access reverse transcription-polymerase chain reaction (RT-PCR) System (Promega, Madison, WI) was used as described previously [ ] with the Lassa GP1 primer set (5′ACCGGGG ATCCTA GGCATTT, nt 5–24, forward; 5′-ATATAATGATGACTGTTGTTC TTTGTGCA, nt 311–339, reverse).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Infection, Positive Control, Expressing, Synthesized, Polymerase Chain Reaction

    Quantitation of TNF-α mRNA expression in human LPS-stimulated and Lassa-infected MDM by competitive PCR. A. Gel-based analysis of TNF-α mRNA expression in LPS-MDM infected with Lassa virus. MDM grown in T25 flasks were stimulated with LPS, infected with Lassa virus (MOI = 5) and incubated for 12 and 24 hours. At 12 and 24 hours p. i. RNA was extracted, converted into cDNA and equivalent amounts of cDNA (based on GAPDH) were amplified. Quantitative PCR was performed with biotin-labeled TNF-α primers using different concentrations of cytokine template (1, 1:10, and 1:100 dilutions) and a constant amount (2,000 copies) of ICS (BioSource). 5 ul-aliquots were analyzed on 2% agarose. M, positions of DNA markers, 500 and 250 bp. B. Quantitation of PCR products. 25 ul from each PCR reaction was denatured under alkaline pH and serial dilutions (1:40–1:320) of TNF-α and ICS amplicons were made in hybridization buffer in ICS- or TNF-α-specific oligonucleotide capture wells. After hybridization the captured amplicons were detected by streptavidin conjugates. Numbers of TNF-α mRNA copies were calculated as follows: Total TNF-α Optical Density (OD)/Total ICS OD × 2 × Input copy number of ICS × cDNA dilution.

    Journal: Journal of medical virology

    Article Title: Lassa and Mopeia Virus Replication in Human Monocytes/Macrophages and in Endothelial Cells: Different Effects on IL-8 and TNF-? Gene Expression

    doi:

    Figure Lengend Snippet: Quantitation of TNF-α mRNA expression in human LPS-stimulated and Lassa-infected MDM by competitive PCR. A. Gel-based analysis of TNF-α mRNA expression in LPS-MDM infected with Lassa virus. MDM grown in T25 flasks were stimulated with LPS, infected with Lassa virus (MOI = 5) and incubated for 12 and 24 hours. At 12 and 24 hours p. i. RNA was extracted, converted into cDNA and equivalent amounts of cDNA (based on GAPDH) were amplified. Quantitative PCR was performed with biotin-labeled TNF-α primers using different concentrations of cytokine template (1, 1:10, and 1:100 dilutions) and a constant amount (2,000 copies) of ICS (BioSource). 5 ul-aliquots were analyzed on 2% agarose. M, positions of DNA markers, 500 and 250 bp. B. Quantitation of PCR products. 25 ul from each PCR reaction was denatured under alkaline pH and serial dilutions (1:40–1:320) of TNF-α and ICS amplicons were made in hybridization buffer in ICS- or TNF-α-specific oligonucleotide capture wells. After hybridization the captured amplicons were detected by streptavidin conjugates. Numbers of TNF-α mRNA copies were calculated as follows: Total TNF-α Optical Density (OD)/Total ICS OD × 2 × Input copy number of ICS × cDNA dilution.

    Article Snippet: For detection of Lassa virus-specific RNA in infected cells the Access reverse transcription-polymerase chain reaction (RT-PCR) System (Promega, Madison, WI) was used as described previously [ ] with the Lassa GP1 primer set (5′ACCGGGG ATCCTA GGCATTT, nt 5–24, forward; 5′-ATATAATGATGACTGTTGTTC TTTGTGCA, nt 311–339, reverse).

    Techniques: Quantitation Assay, Expressing, Infection, Polymerase Chain Reaction, Incubation, Amplification, Real-time Polymerase Chain Reaction, Labeling, Hybridization

    ID proteins inhibit E-cadherin expression in breast cells. Notes: ( A ) Western blot analysis of MCF10A cells with stable expression of ID1, ID2, or ID3. ( B ) Real-time PCR analysis of E-cadherin mRNA abundance in cell lines in ( A ). ( C ) Western blot analysis of E-cadherin protein abundance in cell lines in ( A ). ( D ) Assessment of E-cadherin promoter activity, using luciferase reporter assays, in HEK293T cells. The 1.38 kb DNA sequence containing E-cadherin promoter region was cloned upstream of the luciferase gene in a reporter construct (top image). The relative luciferase activities of luciferase reporters with E-cadherin promoter were determined in HEK293T cells, which were cotransfected with the ID1, ID2, ID3, and control vectors. For ( B ) and ( D , bottom image), data represent mean values, with error bars indicating SEM. ** P

    Journal: OncoTargets and therapy

    Article Title: ID2 predicts poor prognosis in breast cancer, especially in triple-negative breast cancer, and inhibits E-cadherin expression

    doi: 10.2147/OTT.S64759

    Figure Lengend Snippet: ID proteins inhibit E-cadherin expression in breast cells. Notes: ( A ) Western blot analysis of MCF10A cells with stable expression of ID1, ID2, or ID3. ( B ) Real-time PCR analysis of E-cadherin mRNA abundance in cell lines in ( A ). ( C ) Western blot analysis of E-cadherin protein abundance in cell lines in ( A ). ( D ) Assessment of E-cadherin promoter activity, using luciferase reporter assays, in HEK293T cells. The 1.38 kb DNA sequence containing E-cadherin promoter region was cloned upstream of the luciferase gene in a reporter construct (top image). The relative luciferase activities of luciferase reporters with E-cadherin promoter were determined in HEK293T cells, which were cotransfected with the ID1, ID2, ID3, and control vectors. For ( B ) and ( D , bottom image), data represent mean values, with error bars indicating SEM. ** P

    Article Snippet: Plasmid constructs The human full-length coding sequences of ID1, ID2, and ID3 were cloned using a standard reverse-transcription polymerase chain reaction (RT-PCR) protocol (Takara Bio Company, Dalian, Japan).

    Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Activity Assay, Luciferase, Sequencing, Clone Assay, Construct