Structured Review

Applichem dntps
Dntps, supplied by Applichem, used in various techniques. Bioz Stars score: 96/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 96 stars, based on 6 article reviews
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dntps - by Bioz Stars, 2020-04
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Related Articles

Conditioned Place Preference:

Article Title: Alcohol enhances the psychostimulant and conditioning effects of mephedrone in adolescent mice; postulation of unique roles of D3 receptors and BDNF in place preference acquisition) Alcohol enhances the psychostimulant and conditioning effects of mephedrone in adolescent mice; postulation of unique roles of D3 receptors and BDNF in place preference acquisition
Article Snippet: Furthermore, Drd3 mRNA levels from CPP experiments using the antagonists SB‐277011A and ANA‐12 were also determined. .. Briefly, complementary DNA was synthesized in a total volume of 20 μL by mixing 2 µg of total RNA, 125 ng of random hexamers (Roche), in the presence of 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol, 20 U RNasin (Invitrogen), 0.5 mM dNTPs (AppliChem), 200 U M‐MLV reverse transcriptase (Invitrogen) and 50 mM Tris–HCl buffer, pH 8.3.

Clone Assay:

Article Title: Amplified Host Defense by Toll-Like Receptor-Mediated Downregulation of the Glucocorticoid-Induced Leucine Zipper (GILZ) in Macrophages
Article Snippet: For TNF, IL6, CXCL8, GILZ , and ACTB , one 25 μL reaction mix contained 2.5 U Taq polymerase, 500 nM sense and antisense primers, 60–100 nM probe, 200 μM dNTPs, 3–4 mM MgCl2 , 2.5 μL 10x Taq buffer, 3 μL Template, and molecular biology grade water (Applichem, #A7398). .. Standard curves were generated by serial dilution of the PCR product cloned into pGEMTeasy (Promega, #A1360).

Article Title: Toll-Like Receptor 2 Release by Macrophages: An Anti-inflammatory Program Induced by Glucocorticoids and Lipopolysaccharide
Article Snippet: For ACTB, CXCL10, IL10, TLR1, TLR2, TLR4, TLR6 , and TNF , one 25 μL reaction mix contained 2.5 U Taq polymerase, 500 nM sense and antisense primers, 60-100 nM probe, 200 μM dNTPs, 3-4 mM MgCl2 , 2.5 μL 10x Taq buffer, 3 μL Template, and molecular biology grade water (Applichem, #A7398). .. Standard curves were generated by using a dilution series of the PCR product cloned into pGEMTeasy (Promega, #A1360) ( , , ).

Article Title: Amplified Host Defense by Toll-Like Receptor-Mediated Downregulation of the Glucocorticoid-Induced Leucine Zipper (GILZ) in Macrophages
Article Snippet: For TNF, IL6, CXCL8, GILZ , and ACTB , one 25 μL reaction mix contained 2.5 U Taq polymerase, 500 nM sense and antisense primers, 60–100 nM probe, 200 μM dNTPs, 3–4 mM MgCl2 , 2.5 μL 10x Taq buffer, 3 μL Template, and molecular biology grade water (Applichem, #A7398). .. Standard curves were generated by serial dilution of the PCR product cloned into pGEMTeasy (Promega, #A1360).

Transfection:

Article Title: Networking of differentially expressed genes in human cancer cells resistant to methotrexate
Article Snippet: For determining gene-node mRNA levels upon siRNA treatment, cells were treated as described above and total RNA was prepared 48 hours after transfection using the same reagent. .. In either case, complementary DNA was synthesized in a total volume of 20 μl by mixing 500 ng of total RNA and 125 ng of random hexamers (Roche, Mannheim, Germany) in the presence of 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol, 20 units of RNasin (Promega, Madison, WI, USA), 0.5 mM dNTPs (AppliChem, Darmstadt, Germany), 200 units of M-MLV reverse transcriptase (Invitrogen) and 50 mM Tris-HCl buffer, pH 8.3.

Article Title: Regulation of Sp1 by cell cycle related proteins
Article Snippet: 1 μg of expression vectors for E2F, DP1, p21, CyclinD1, Stat3, p53, SKP2, NFκB, p21, CDK4, BRCA2 and Rb was transfected using Fugene 6. .. Total RNA was extracted from HeLa cells using the Ultraspec™ RNA reagent (Biotecx) in accordance with the manufacturer's instructions. cDNA was synthesized in a total volume of 20 μl from RNA samples by mixing 1 μg of total RNA, 125 ng of random hexamers (Roche Molecular Biochemicals), in the presence of 75 mM KCl, 3 mM MgCl2 ,10 mM dithiothreitol, 20 units of RNAsin (Promega), 0.5 mM dNTPs (AppliChem), 200 units of M-MLV reverse transcriptase (Invitrogen) and 50 mM Tris-HCl buffer, pH 8.3.

Amplification:

Article Title: Overexpression of S100A4 in human cancer cell lines resistant to methotrexate
Article Snippet: Complementary DNA was synthesized in a total volume of 20 μl by mixing 500 ng of total RNA, 125 ng of random hexamers (Roche), in the presence of 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol, 20 units of RNasin (Promega), 0.5 mM dNTPs (AppliChem), 200 units of M-MLV reverse transcriptase (Invitrogen) and 50 mM Tris-HCl buffer, pH 8.3. .. The cDNA product was used for subsequent Real-time PCR amplification using an ABI Prism 7000 Sequence Detection System (Applied Biosystems) with 25 ng of the cDNA mixture and the assays-on-demand from Applied Biosystems Hs00243202_m1 for S100A4 and Hs00356991_m1 for APRT.

Article Title: Networking of differentially expressed genes in human cancer cells resistant to methotrexate
Article Snippet: In either case, complementary DNA was synthesized in a total volume of 20 μl by mixing 500 ng of total RNA and 125 ng of random hexamers (Roche, Mannheim, Germany) in the presence of 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol, 20 units of RNasin (Promega, Madison, WI, USA), 0.5 mM dNTPs (AppliChem, Darmstadt, Germany), 200 units of M-MLV reverse transcriptase (Invitrogen) and 50 mM Tris-HCl buffer, pH 8.3. .. The cDNA product was used for subsequent real-time PCR amplification using an ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) with 25 ng of the cDNA mixture, the assays-on-demand from Applied Biosystems Hs00758822_s1 for DHFR and Hs00356991_m1 for Adenine Phosphoribosyltransferase (APRT) or the following primers: DKK1 , 5'-AGTACTGCGCTAGTCCCACC-3' and 5'-CTGGAATACCCATCCAAGGTGC-3'; UGT1A , 5'-TAAGTGGCTACCCCAAAACG-3' and 5'-CTCCAGCTCCCTTAGTCTCC-3'; EEF1A1 , 5'-CGTCATTGGACACGTAGATTCGGG-3' and 5'-GGAGCCCTTTCCCATCTCAGC-3'.

Article Title: Regulation of Sp1 by cell cycle related proteins
Article Snippet: Total RNA was extracted from HeLa cells using the Ultraspec™ RNA reagent (Biotecx) in accordance with the manufacturer's instructions. cDNA was synthesized in a total volume of 20 μl from RNA samples by mixing 1 μg of total RNA, 125 ng of random hexamers (Roche Molecular Biochemicals), in the presence of 75 mM KCl, 3 mM MgCl2 ,10 mM dithiothreitol, 20 units of RNAsin (Promega), 0.5 mM dNTPs (AppliChem), 200 units of M-MLV reverse transcriptase (Invitrogen) and 50 mM Tris-HCl buffer, pH 8.3. .. The cDNA product was used for subsequent amplification by Real Time-PCR.

Article Title: Role of Caveolin 1, E-Cadherin, Enolase 2 and PKCalpha on resistance to methotrexate in human HT29 colon cancer cells
Article Snippet: Complementary DNA was synthesized in a total volume of 20 μl from RNA samples by mixing 500 ng of total RNA, 125 ng of random hexamers (Roche), in the presence of 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol, 20 units of RNasin (Promega), 0.5 mM dNTPs (AppliChem), 200 units of M-MLV reverse transcriptase (Invitrogen) and 50 mM Tris-HCl buffer, pH 8.3. .. The reaction mixture was incubated at 37°C for 60 min and the cDNA product was used for subsequent Real-time PCR amplification using SYBR Green.

Article Title: Alcohol enhances the psychostimulant and conditioning effects of mephedrone in adolescent mice; postulation of unique roles of D3 receptors and BDNF in place preference acquisition) Alcohol enhances the psychostimulant and conditioning effects of mephedrone in adolescent mice; postulation of unique roles of D3 receptors and BDNF in place preference acquisition
Article Snippet: Briefly, complementary DNA was synthesized in a total volume of 20 μL by mixing 2 µg of total RNA, 125 ng of random hexamers (Roche), in the presence of 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol, 20 U RNasin (Invitrogen), 0.5 mM dNTPs (AppliChem), 200 U M‐MLV reverse transcriptase (Invitrogen) and 50 mM Tris–HCl buffer, pH 8.3. .. The cDNA product was used for subsequent real‐time PCR amplification using an ABI Prism 7000 Sequence Detection System (Applied Biosystems) with 25 ng of the cDNA mixture and the assays‐on‐demand from Applied Biosystems Mm00432887_m1 for Drd3 , Mm01350708_m1 for Muted , Mm00444512_m1 for Syt10 , Mm00777068_m1 for Nfu1 , Mm04208715_m1 for Arpc5 , Mm00513979_m1 for Gpx6 , Mm04230607_s1 for BDNF and Mm00607939_s1 for Actb as an endogenous control.

Synthesized:

Article Title: Overexpression of S100A4 in human cancer cell lines resistant to methotrexate
Article Snippet: .. Complementary DNA was synthesized in a total volume of 20 μl by mixing 500 ng of total RNA, 125 ng of random hexamers (Roche), in the presence of 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol, 20 units of RNasin (Promega), 0.5 mM dNTPs (AppliChem), 200 units of M-MLV reverse transcriptase (Invitrogen) and 50 mM Tris-HCl buffer, pH 8.3. ..

Article Title: Networking of differentially expressed genes in human cancer cells resistant to methotrexate
Article Snippet: .. In either case, complementary DNA was synthesized in a total volume of 20 μl by mixing 500 ng of total RNA and 125 ng of random hexamers (Roche, Mannheim, Germany) in the presence of 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol, 20 units of RNasin (Promega, Madison, WI, USA), 0.5 mM dNTPs (AppliChem, Darmstadt, Germany), 200 units of M-MLV reverse transcriptase (Invitrogen) and 50 mM Tris-HCl buffer, pH 8.3. ..

Article Title: Regulation of Sp1 by cell cycle related proteins
Article Snippet: .. Total RNA was extracted from HeLa cells using the Ultraspec™ RNA reagent (Biotecx) in accordance with the manufacturer's instructions. cDNA was synthesized in a total volume of 20 μl from RNA samples by mixing 1 μg of total RNA, 125 ng of random hexamers (Roche Molecular Biochemicals), in the presence of 75 mM KCl, 3 mM MgCl2 ,10 mM dithiothreitol, 20 units of RNAsin (Promega), 0.5 mM dNTPs (AppliChem), 200 units of M-MLV reverse transcriptase (Invitrogen) and 50 mM Tris-HCl buffer, pH 8.3. ..

Article Title: Role of Caveolin 1, E-Cadherin, Enolase 2 and PKCalpha on resistance to methotrexate in human HT29 colon cancer cells
Article Snippet: .. Complementary DNA was synthesized in a total volume of 20 μl from RNA samples by mixing 500 ng of total RNA, 125 ng of random hexamers (Roche), in the presence of 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol, 20 units of RNasin (Promega), 0.5 mM dNTPs (AppliChem), 200 units of M-MLV reverse transcriptase (Invitrogen) and 50 mM Tris-HCl buffer, pH 8.3. .. The reaction mixture was incubated at 37°C for 60 min and the cDNA product was used for subsequent Real-time PCR amplification using SYBR Green.

Article Title: Alcohol enhances the psychostimulant and conditioning effects of mephedrone in adolescent mice; postulation of unique roles of D3 receptors and BDNF in place preference acquisition) Alcohol enhances the psychostimulant and conditioning effects of mephedrone in adolescent mice; postulation of unique roles of D3 receptors and BDNF in place preference acquisition
Article Snippet: .. Briefly, complementary DNA was synthesized in a total volume of 20 μL by mixing 2 µg of total RNA, 125 ng of random hexamers (Roche), in the presence of 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol, 20 U RNasin (Invitrogen), 0.5 mM dNTPs (AppliChem), 200 U M‐MLV reverse transcriptase (Invitrogen) and 50 mM Tris–HCl buffer, pH 8.3. ..

Isolation:

Article Title: Amplified Host Defense by Toll-Like Receptor-Mediated Downregulation of the Glucocorticoid-Induced Leucine Zipper (GILZ) in Macrophages
Article Snippet: Paragraph title: RNA Isolation, Reverse Transcription, and Quantitative RT-PCR ... For TNF, IL6, CXCL8, GILZ , and ACTB , one 25 μL reaction mix contained 2.5 U Taq polymerase, 500 nM sense and antisense primers, 60–100 nM probe, 200 μM dNTPs, 3–4 mM MgCl2 , 2.5 μL 10x Taq buffer, 3 μL Template, and molecular biology grade water (Applichem, #A7398).

Article Title: Toll-Like Receptor 2 Release by Macrophages: An Anti-inflammatory Program Induced by Glucocorticoids and Lipopolysaccharide
Article Snippet: Paragraph title: RNA Isolation, Reverse Transcription, and Quantitative RT-PCR ... For ACTB, CXCL10, IL10, TLR1, TLR2, TLR4, TLR6 , and TNF , one 25 μL reaction mix contained 2.5 U Taq polymerase, 500 nM sense and antisense primers, 60-100 nM probe, 200 μM dNTPs, 3-4 mM MgCl2 , 2.5 μL 10x Taq buffer, 3 μL Template, and molecular biology grade water (Applichem, #A7398).

Generated:

Article Title: Amplified Host Defense by Toll-Like Receptor-Mediated Downregulation of the Glucocorticoid-Induced Leucine Zipper (GILZ) in Macrophages
Article Snippet: For TNF, IL6, CXCL8, GILZ , and ACTB , one 25 μL reaction mix contained 2.5 U Taq polymerase, 500 nM sense and antisense primers, 60–100 nM probe, 200 μM dNTPs, 3–4 mM MgCl2 , 2.5 μL 10x Taq buffer, 3 μL Template, and molecular biology grade water (Applichem, #A7398). .. Standard curves were generated by serial dilution of the PCR product cloned into pGEMTeasy (Promega, #A1360).

Article Title: Toll-Like Receptor 2 Release by Macrophages: An Anti-inflammatory Program Induced by Glucocorticoids and Lipopolysaccharide
Article Snippet: For ACTB, CXCL10, IL10, TLR1, TLR2, TLR4, TLR6 , and TNF , one 25 μL reaction mix contained 2.5 U Taq polymerase, 500 nM sense and antisense primers, 60-100 nM probe, 200 μM dNTPs, 3-4 mM MgCl2 , 2.5 μL 10x Taq buffer, 3 μL Template, and molecular biology grade water (Applichem, #A7398). .. Standard curves were generated by using a dilution series of the PCR product cloned into pGEMTeasy (Promega, #A1360) ( , , ).

Article Title: Amplified Host Defense by Toll-Like Receptor-Mediated Downregulation of the Glucocorticoid-Induced Leucine Zipper (GILZ) in Macrophages
Article Snippet: For TNF, IL6, CXCL8, GILZ , and ACTB , one 25 μL reaction mix contained 2.5 U Taq polymerase, 500 nM sense and antisense primers, 60–100 nM probe, 200 μM dNTPs, 3–4 mM MgCl2 , 2.5 μL 10x Taq buffer, 3 μL Template, and molecular biology grade water (Applichem, #A7398). .. Standard curves were generated by serial dilution of the PCR product cloned into pGEMTeasy (Promega, #A1360).

Quantitative RT-PCR:

Article Title: Amplified Host Defense by Toll-Like Receptor-Mediated Downregulation of the Glucocorticoid-Induced Leucine Zipper (GILZ) in Macrophages
Article Snippet: Paragraph title: RNA Isolation, Reverse Transcription, and Quantitative RT-PCR ... For TNF, IL6, CXCL8, GILZ , and ACTB , one 25 μL reaction mix contained 2.5 U Taq polymerase, 500 nM sense and antisense primers, 60–100 nM probe, 200 μM dNTPs, 3–4 mM MgCl2 , 2.5 μL 10x Taq buffer, 3 μL Template, and molecular biology grade water (Applichem, #A7398).

Article Title: Networking of differentially expressed genes in human cancer cells resistant to methotrexate
Article Snippet: Paragraph title: Real-time RT-PCR ... In either case, complementary DNA was synthesized in a total volume of 20 μl by mixing 500 ng of total RNA and 125 ng of random hexamers (Roche, Mannheim, Germany) in the presence of 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol, 20 units of RNasin (Promega, Madison, WI, USA), 0.5 mM dNTPs (AppliChem, Darmstadt, Germany), 200 units of M-MLV reverse transcriptase (Invitrogen) and 50 mM Tris-HCl buffer, pH 8.3.

Article Title: Toll-Like Receptor 2 Release by Macrophages: An Anti-inflammatory Program Induced by Glucocorticoids and Lipopolysaccharide
Article Snippet: Paragraph title: RNA Isolation, Reverse Transcription, and Quantitative RT-PCR ... For ACTB, CXCL10, IL10, TLR1, TLR2, TLR4, TLR6 , and TNF , one 25 μL reaction mix contained 2.5 U Taq polymerase, 500 nM sense and antisense primers, 60-100 nM probe, 200 μM dNTPs, 3-4 mM MgCl2 , 2.5 μL 10x Taq buffer, 3 μL Template, and molecular biology grade water (Applichem, #A7398).

SYBR Green Assay:

Article Title: Role of Caveolin 1, E-Cadherin, Enolase 2 and PKCalpha on resistance to methotrexate in human HT29 colon cancer cells
Article Snippet: Complementary DNA was synthesized in a total volume of 20 μl from RNA samples by mixing 500 ng of total RNA, 125 ng of random hexamers (Roche), in the presence of 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol, 20 units of RNasin (Promega), 0.5 mM dNTPs (AppliChem), 200 units of M-MLV reverse transcriptase (Invitrogen) and 50 mM Tris-HCl buffer, pH 8.3. .. The reaction mixture was incubated at 37°C for 60 min and the cDNA product was used for subsequent Real-time PCR amplification using SYBR Green.

Article Title: The Redox State of Cytochrome C Modulates Resistance to Methotrexate in Human MCF7 Breast Cancer Cells
Article Snippet: Complementary DNA (cDNA) was synthetized in a total volume of 20 µl by mixing 1 µg of total RNA, 125 ng of random hexamers (Roche, Mannheim, Germany), in the presence of 75 mM KCl, 3 mM MgCl2 , 50 mM Tris-HCl buffer, pH 8.3, 10 mM dithiothreitol (Invitrogen, Life Technologies, Madrid, Spain), 20 units of RNAsin (Promega, Madrid, Spain), 0.5 mM dNTPs (Applichem, Ecogen, Barcelona, Spain) and 200 units of MLV-reverse transcriptase (Invitrogen, Life Technologies, Madrid, Spain). .. Gene expression levels were quantified by SYBR Green RT-Real Time PCR reaction in a final volume of 20 µl with specific forward and reverse primers, using the StepOnePlus™ detection system (Applied Biosystems, Life Technologies, Madrid, Spain).

Polymerase Chain Reaction:

Article Title: Overexpression of S100A4 in human cancer cell lines resistant to methotrexate
Article Snippet: Paragraph title: RT-Real-Time PCR ... Complementary DNA was synthesized in a total volume of 20 μl by mixing 500 ng of total RNA, 125 ng of random hexamers (Roche), in the presence of 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol, 20 units of RNasin (Promega), 0.5 mM dNTPs (AppliChem), 200 units of M-MLV reverse transcriptase (Invitrogen) and 50 mM Tris-HCl buffer, pH 8.3.

Article Title: Amplified Host Defense by Toll-Like Receptor-Mediated Downregulation of the Glucocorticoid-Induced Leucine Zipper (GILZ) in Macrophages
Article Snippet: For TNF, IL6, CXCL8, GILZ , and ACTB , one 25 μL reaction mix contained 2.5 U Taq polymerase, 500 nM sense and antisense primers, 60–100 nM probe, 200 μM dNTPs, 3–4 mM MgCl2 , 2.5 μL 10x Taq buffer, 3 μL Template, and molecular biology grade water (Applichem, #A7398). .. Standard curves were generated by serial dilution of the PCR product cloned into pGEMTeasy (Promega, #A1360).

Article Title: Toll-Like Receptor 2 Release by Macrophages: An Anti-inflammatory Program Induced by Glucocorticoids and Lipopolysaccharide
Article Snippet: For ACTB, CXCL10, IL10, TLR1, TLR2, TLR4, TLR6 , and TNF , one 25 μL reaction mix contained 2.5 U Taq polymerase, 500 nM sense and antisense primers, 60-100 nM probe, 200 μM dNTPs, 3-4 mM MgCl2 , 2.5 μL 10x Taq buffer, 3 μL Template, and molecular biology grade water (Applichem, #A7398). .. Standard curves were generated by using a dilution series of the PCR product cloned into pGEMTeasy (Promega, #A1360) ( , , ).

Article Title: Amplified Host Defense by Toll-Like Receptor-Mediated Downregulation of the Glucocorticoid-Induced Leucine Zipper (GILZ) in Macrophages
Article Snippet: For TNF, IL6, CXCL8, GILZ , and ACTB , one 25 μL reaction mix contained 2.5 U Taq polymerase, 500 nM sense and antisense primers, 60–100 nM probe, 200 μM dNTPs, 3–4 mM MgCl2 , 2.5 μL 10x Taq buffer, 3 μL Template, and molecular biology grade water (Applichem, #A7398). .. Standard curves were generated by serial dilution of the PCR product cloned into pGEMTeasy (Promega, #A1360).

Article Title: Role of Caveolin 1, E-Cadherin, Enolase 2 and PKCalpha on resistance to methotrexate in human HT29 colon cancer cells
Article Snippet: Paragraph title: RT-Real-Time PCR ... Complementary DNA was synthesized in a total volume of 20 μl from RNA samples by mixing 500 ng of total RNA, 125 ng of random hexamers (Roche), in the presence of 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol, 20 units of RNasin (Promega), 0.5 mM dNTPs (AppliChem), 200 units of M-MLV reverse transcriptase (Invitrogen) and 50 mM Tris-HCl buffer, pH 8.3.

Article Title: The Redox State of Cytochrome C Modulates Resistance to Methotrexate in Human MCF7 Breast Cancer Cells
Article Snippet: Paragraph title: RT-Real-Time PCR ... Complementary DNA (cDNA) was synthetized in a total volume of 20 µl by mixing 1 µg of total RNA, 125 ng of random hexamers (Roche, Mannheim, Germany), in the presence of 75 mM KCl, 3 mM MgCl2 , 50 mM Tris-HCl buffer, pH 8.3, 10 mM dithiothreitol (Invitrogen, Life Technologies, Madrid, Spain), 20 units of RNAsin (Promega, Madrid, Spain), 0.5 mM dNTPs (Applichem, Ecogen, Barcelona, Spain) and 200 units of MLV-reverse transcriptase (Invitrogen, Life Technologies, Madrid, Spain).

Incubation:

Article Title: Role of Caveolin 1, E-Cadherin, Enolase 2 and PKCalpha on resistance to methotrexate in human HT29 colon cancer cells
Article Snippet: Complementary DNA was synthesized in a total volume of 20 μl from RNA samples by mixing 500 ng of total RNA, 125 ng of random hexamers (Roche), in the presence of 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol, 20 units of RNasin (Promega), 0.5 mM dNTPs (AppliChem), 200 units of M-MLV reverse transcriptase (Invitrogen) and 50 mM Tris-HCl buffer, pH 8.3. .. The reaction mixture was incubated at 37°C for 60 min and the cDNA product was used for subsequent Real-time PCR amplification using SYBR Green.

Expressing:

Article Title: Regulation of Sp1 by cell cycle related proteins
Article Snippet: 1 μg of expression vectors for E2F, DP1, p21, CyclinD1, Stat3, p53, SKP2, NFκB, p21, CDK4, BRCA2 and Rb was transfected using Fugene 6. .. Total RNA was extracted from HeLa cells using the Ultraspec™ RNA reagent (Biotecx) in accordance with the manufacturer's instructions. cDNA was synthesized in a total volume of 20 μl from RNA samples by mixing 1 μg of total RNA, 125 ng of random hexamers (Roche Molecular Biochemicals), in the presence of 75 mM KCl, 3 mM MgCl2 ,10 mM dithiothreitol, 20 units of RNAsin (Promega), 0.5 mM dNTPs (AppliChem), 200 units of M-MLV reverse transcriptase (Invitrogen) and 50 mM Tris-HCl buffer, pH 8.3.

Article Title: The Redox State of Cytochrome C Modulates Resistance to Methotrexate in Human MCF7 Breast Cancer Cells
Article Snippet: Complementary DNA (cDNA) was synthetized in a total volume of 20 µl by mixing 1 µg of total RNA, 125 ng of random hexamers (Roche, Mannheim, Germany), in the presence of 75 mM KCl, 3 mM MgCl2 , 50 mM Tris-HCl buffer, pH 8.3, 10 mM dithiothreitol (Invitrogen, Life Technologies, Madrid, Spain), 20 units of RNAsin (Promega, Madrid, Spain), 0.5 mM dNTPs (Applichem, Ecogen, Barcelona, Spain) and 200 units of MLV-reverse transcriptase (Invitrogen, Life Technologies, Madrid, Spain). .. Gene expression levels were quantified by SYBR Green RT-Real Time PCR reaction in a final volume of 20 µl with specific forward and reverse primers, using the StepOnePlus™ detection system (Applied Biosystems, Life Technologies, Madrid, Spain).

Article Title: Alcohol enhances the psychostimulant and conditioning effects of mephedrone in adolescent mice; postulation of unique roles of D3 receptors and BDNF in place preference acquisition) Alcohol enhances the psychostimulant and conditioning effects of mephedrone in adolescent mice; postulation of unique roles of D3 receptors and BDNF in place preference acquisition
Article Snippet: Briefly, complementary DNA was synthesized in a total volume of 20 μL by mixing 2 µg of total RNA, 125 ng of random hexamers (Roche), in the presence of 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol, 20 U RNasin (Invitrogen), 0.5 mM dNTPs (AppliChem), 200 U M‐MLV reverse transcriptase (Invitrogen) and 50 mM Tris–HCl buffer, pH 8.3. .. Fold‐changes in gene expression were calculated using the standard δδCt method (Livak and Schmittgen, ).

Serial Dilution:

Article Title: Amplified Host Defense by Toll-Like Receptor-Mediated Downregulation of the Glucocorticoid-Induced Leucine Zipper (GILZ) in Macrophages
Article Snippet: For TNF, IL6, CXCL8, GILZ , and ACTB , one 25 μL reaction mix contained 2.5 U Taq polymerase, 500 nM sense and antisense primers, 60–100 nM probe, 200 μM dNTPs, 3–4 mM MgCl2 , 2.5 μL 10x Taq buffer, 3 μL Template, and molecular biology grade water (Applichem, #A7398). .. Standard curves were generated by serial dilution of the PCR product cloned into pGEMTeasy (Promega, #A1360).

Article Title: Amplified Host Defense by Toll-Like Receptor-Mediated Downregulation of the Glucocorticoid-Induced Leucine Zipper (GILZ) in Macrophages
Article Snippet: For TNF, IL6, CXCL8, GILZ , and ACTB , one 25 μL reaction mix contained 2.5 U Taq polymerase, 500 nM sense and antisense primers, 60–100 nM probe, 200 μM dNTPs, 3–4 mM MgCl2 , 2.5 μL 10x Taq buffer, 3 μL Template, and molecular biology grade water (Applichem, #A7398). .. Standard curves were generated by serial dilution of the PCR product cloned into pGEMTeasy (Promega, #A1360).

Sequencing:

Article Title: Overexpression of S100A4 in human cancer cell lines resistant to methotrexate
Article Snippet: Complementary DNA was synthesized in a total volume of 20 μl by mixing 500 ng of total RNA, 125 ng of random hexamers (Roche), in the presence of 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol, 20 units of RNasin (Promega), 0.5 mM dNTPs (AppliChem), 200 units of M-MLV reverse transcriptase (Invitrogen) and 50 mM Tris-HCl buffer, pH 8.3. .. The cDNA product was used for subsequent Real-time PCR amplification using an ABI Prism 7000 Sequence Detection System (Applied Biosystems) with 25 ng of the cDNA mixture and the assays-on-demand from Applied Biosystems Hs00243202_m1 for S100A4 and Hs00356991_m1 for APRT.

Article Title: Networking of differentially expressed genes in human cancer cells resistant to methotrexate
Article Snippet: In either case, complementary DNA was synthesized in a total volume of 20 μl by mixing 500 ng of total RNA and 125 ng of random hexamers (Roche, Mannheim, Germany) in the presence of 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol, 20 units of RNasin (Promega, Madison, WI, USA), 0.5 mM dNTPs (AppliChem, Darmstadt, Germany), 200 units of M-MLV reverse transcriptase (Invitrogen) and 50 mM Tris-HCl buffer, pH 8.3. .. The cDNA product was used for subsequent real-time PCR amplification using an ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) with 25 ng of the cDNA mixture, the assays-on-demand from Applied Biosystems Hs00758822_s1 for DHFR and Hs00356991_m1 for Adenine Phosphoribosyltransferase (APRT) or the following primers: DKK1 , 5'-AGTACTGCGCTAGTCCCACC-3' and 5'-CTGGAATACCCATCCAAGGTGC-3'; UGT1A , 5'-TAAGTGGCTACCCCAAAACG-3' and 5'-CTCCAGCTCCCTTAGTCTCC-3'; EEF1A1 , 5'-CGTCATTGGACACGTAGATTCGGG-3' and 5'-GGAGCCCTTTCCCATCTCAGC-3'.

Article Title: Regulation of Sp1 by cell cycle related proteins
Article Snippet: Total RNA was extracted from HeLa cells using the Ultraspec™ RNA reagent (Biotecx) in accordance with the manufacturer's instructions. cDNA was synthesized in a total volume of 20 μl from RNA samples by mixing 1 μg of total RNA, 125 ng of random hexamers (Roche Molecular Biochemicals), in the presence of 75 mM KCl, 3 mM MgCl2 ,10 mM dithiothreitol, 20 units of RNAsin (Promega), 0.5 mM dNTPs (AppliChem), 200 units of M-MLV reverse transcriptase (Invitrogen) and 50 mM Tris-HCl buffer, pH 8.3. .. Sp1 mRNA levels were determined in an ABI Prism 7000 Sequence Detection System (Applied Biosystems) using 3 μl of the cDNA mixture and the Assays-on-demand Hs00412720_m1 for Sp1 and Hs99999901_s1 for 18S RNA (both from Applied Biosystems).

Article Title: Alcohol enhances the psychostimulant and conditioning effects of mephedrone in adolescent mice; postulation of unique roles of D3 receptors and BDNF in place preference acquisition) Alcohol enhances the psychostimulant and conditioning effects of mephedrone in adolescent mice; postulation of unique roles of D3 receptors and BDNF in place preference acquisition
Article Snippet: Briefly, complementary DNA was synthesized in a total volume of 20 μL by mixing 2 µg of total RNA, 125 ng of random hexamers (Roche), in the presence of 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol, 20 U RNasin (Invitrogen), 0.5 mM dNTPs (AppliChem), 200 U M‐MLV reverse transcriptase (Invitrogen) and 50 mM Tris–HCl buffer, pH 8.3. .. The cDNA product was used for subsequent real‐time PCR amplification using an ABI Prism 7000 Sequence Detection System (Applied Biosystems) with 25 ng of the cDNA mixture and the assays‐on‐demand from Applied Biosystems Mm00432887_m1 for Drd3 , Mm01350708_m1 for Muted , Mm00444512_m1 for Syt10 , Mm00777068_m1 for Nfu1 , Mm04208715_m1 for Arpc5 , Mm00513979_m1 for Gpx6 , Mm04230607_s1 for BDNF and Mm00607939_s1 for Actb as an endogenous control.

Real-time Polymerase Chain Reaction:

Article Title: Overexpression of S100A4 in human cancer cell lines resistant to methotrexate
Article Snippet: Complementary DNA was synthesized in a total volume of 20 μl by mixing 500 ng of total RNA, 125 ng of random hexamers (Roche), in the presence of 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol, 20 units of RNasin (Promega), 0.5 mM dNTPs (AppliChem), 200 units of M-MLV reverse transcriptase (Invitrogen) and 50 mM Tris-HCl buffer, pH 8.3. .. The cDNA product was used for subsequent Real-time PCR amplification using an ABI Prism 7000 Sequence Detection System (Applied Biosystems) with 25 ng of the cDNA mixture and the assays-on-demand from Applied Biosystems Hs00243202_m1 for S100A4 and Hs00356991_m1 for APRT.

Article Title: Amplified Host Defense by Toll-Like Receptor-Mediated Downregulation of the Glucocorticoid-Induced Leucine Zipper (GILZ) in Macrophages
Article Snippet: The CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad) was used for real-time RT-PCR. .. For TNF, IL6, CXCL8, GILZ , and ACTB , one 25 μL reaction mix contained 2.5 U Taq polymerase, 500 nM sense and antisense primers, 60–100 nM probe, 200 μM dNTPs, 3–4 mM MgCl2 , 2.5 μL 10x Taq buffer, 3 μL Template, and molecular biology grade water (Applichem, #A7398).

Article Title: Networking of differentially expressed genes in human cancer cells resistant to methotrexate
Article Snippet: In either case, complementary DNA was synthesized in a total volume of 20 μl by mixing 500 ng of total RNA and 125 ng of random hexamers (Roche, Mannheim, Germany) in the presence of 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol, 20 units of RNasin (Promega, Madison, WI, USA), 0.5 mM dNTPs (AppliChem, Darmstadt, Germany), 200 units of M-MLV reverse transcriptase (Invitrogen) and 50 mM Tris-HCl buffer, pH 8.3. .. The cDNA product was used for subsequent real-time PCR amplification using an ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) with 25 ng of the cDNA mixture, the assays-on-demand from Applied Biosystems Hs00758822_s1 for DHFR and Hs00356991_m1 for Adenine Phosphoribosyltransferase (APRT) or the following primers: DKK1 , 5'-AGTACTGCGCTAGTCCCACC-3' and 5'-CTGGAATACCCATCCAAGGTGC-3'; UGT1A , 5'-TAAGTGGCTACCCCAAAACG-3' and 5'-CTCCAGCTCCCTTAGTCTCC-3'; EEF1A1 , 5'-CGTCATTGGACACGTAGATTCGGG-3' and 5'-GGAGCCCTTTCCCATCTCAGC-3'.

Article Title: Toll-Like Receptor 2 Release by Macrophages: An Anti-inflammatory Program Induced by Glucocorticoids and Lipopolysaccharide
Article Snippet: The CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad) was used for real-time RT-PCR. .. For ACTB, CXCL10, IL10, TLR1, TLR2, TLR4, TLR6 , and TNF , one 25 μL reaction mix contained 2.5 U Taq polymerase, 500 nM sense and antisense primers, 60-100 nM probe, 200 μM dNTPs, 3-4 mM MgCl2 , 2.5 μL 10x Taq buffer, 3 μL Template, and molecular biology grade water (Applichem, #A7398).

Article Title: Amplified Host Defense by Toll-Like Receptor-Mediated Downregulation of the Glucocorticoid-Induced Leucine Zipper (GILZ) in Macrophages
Article Snippet: The CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad) was used for real-time RT-PCR. .. For TNF, IL6, CXCL8, GILZ , and ACTB , one 25 μL reaction mix contained 2.5 U Taq polymerase, 500 nM sense and antisense primers, 60–100 nM probe, 200 μM dNTPs, 3–4 mM MgCl2 , 2.5 μL 10x Taq buffer, 3 μL Template, and molecular biology grade water (Applichem, #A7398).

Article Title: Regulation of Sp1 by cell cycle related proteins
Article Snippet: Total RNA was extracted from HeLa cells using the Ultraspec™ RNA reagent (Biotecx) in accordance with the manufacturer's instructions. cDNA was synthesized in a total volume of 20 μl from RNA samples by mixing 1 μg of total RNA, 125 ng of random hexamers (Roche Molecular Biochemicals), in the presence of 75 mM KCl, 3 mM MgCl2 ,10 mM dithiothreitol, 20 units of RNAsin (Promega), 0.5 mM dNTPs (AppliChem), 200 units of M-MLV reverse transcriptase (Invitrogen) and 50 mM Tris-HCl buffer, pH 8.3. .. The cDNA product was used for subsequent amplification by Real Time-PCR.

Article Title: Role of Caveolin 1, E-Cadherin, Enolase 2 and PKCalpha on resistance to methotrexate in human HT29 colon cancer cells
Article Snippet: Complementary DNA was synthesized in a total volume of 20 μl from RNA samples by mixing 500 ng of total RNA, 125 ng of random hexamers (Roche), in the presence of 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol, 20 units of RNasin (Promega), 0.5 mM dNTPs (AppliChem), 200 units of M-MLV reverse transcriptase (Invitrogen) and 50 mM Tris-HCl buffer, pH 8.3. .. The reaction mixture was incubated at 37°C for 60 min and the cDNA product was used for subsequent Real-time PCR amplification using SYBR Green.

Article Title: Alcohol enhances the psychostimulant and conditioning effects of mephedrone in adolescent mice; postulation of unique roles of D3 receptors and BDNF in place preference acquisition) Alcohol enhances the psychostimulant and conditioning effects of mephedrone in adolescent mice; postulation of unique roles of D3 receptors and BDNF in place preference acquisition
Article Snippet: Paragraph title: Real‐time PCR (qPCR) ... Briefly, complementary DNA was synthesized in a total volume of 20 μL by mixing 2 µg of total RNA, 125 ng of random hexamers (Roche), in the presence of 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol, 20 U RNasin (Invitrogen), 0.5 mM dNTPs (AppliChem), 200 U M‐MLV reverse transcriptase (Invitrogen) and 50 mM Tris–HCl buffer, pH 8.3.

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