Structured Review

Thermo Fisher oligo
Oligo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1988 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/oligo/product/Thermo Fisher
Average 99 stars, based on 1988 article reviews
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oligo - by Bioz Stars, 2020-04
99/100 stars

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Related Articles

Clone Assay:

Article Title: The Drosophila Small Conductance Calcium-Activated Potassium Channel Negatively Regulates Nociception
Article Snippet: For the cloning of SK-M and SK-V rescue constructs, RT-PCR was performed on total RNA extracts (TRIzol, Life Technologies) from Canton-S third instar larvae. .. The SuperScript III reverse transcriptase enzyme (Life Technologies) and Oligo(dT)12–18 primer (Life Technologies) were used to direct the synthesis of first strand cDNA.

Amplification:

Article Title: The Drosophila Small Conductance Calcium-Activated Potassium Channel Negatively Regulates Nociception
Article Snippet: The SuperScript III reverse transcriptase enzyme (Life Technologies) and Oligo(dT)12–18 primer (Life Technologies) were used to direct the synthesis of first strand cDNA. .. For SK-M and SK-V cDNA amplification, PCR was performed using the reverse primer 5ʹ-TCAGCTAGAATGTGGAAACAGCAT-3ʹ, and either the forward primer 5ʹ-ATGTCAATTCA GAAGCTTAACGAC-3ʹ to target the SK-M isoform or the forward primer 5ʹ-ATGTCGCCGGCCTTCTGC-3ʹ to target the SK-V isoform.

Article Title: Gene structures and processing of Arabidopsis thaliana HYL1-dependent pri-miRNAs
Article Snippet: Quantitative real-time PCR profiling of pri-miRNAs Reverse transcriptase reactions were performed using (i) Oligo(dT)12–18 Primer (Invitrogen) or (ii) Oligo(dT)18 Primer (Fermentas, Vilnius, Lithuania) and SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturers instructions. .. The efficiency of cDNA synthesis was assessed by real-time PCR amplification of an UBQ10 (At4g05320 ) cDNA fragment ( , ).

Synthesized:

Article Title: Optimizing Laser Capture Microdissection Protocol for Isolating Zone-Specific Cell Populations from Mandibular Condylar Cartilage
Article Snippet: Standard curves were generated for each primer pair with serial dilutions of cDNA synthesized using RNA isolated from a mixture of MCC and FCC specimens. .. The isolated genetic material was used as a template to synthesize the first-strand cDNA using Superscript III Reverse Transcriptase (Invitrogen, CA, USA) and oligo (dT)12–18 (Invitrogen, CA, USA) according to the manufacturer instructions.

Article Title: Propionate and butyrate induce gene expression of monocarboxylate transporter 4 and cluster of differentiation 147 in cultured rumen epithelial cells derived from preweaning dairy calves
Article Snippet: .. RNA concentration and absorbance ratio were measured by spectrophotometer. cDNA in a total volume of 20 µL was synthesized from 1 µg of RNA with 2.5 µM of Oligo (dT)20 primer at 50 °C for 60 min using Thermo Script reverse transcriptase (15 U/µL, cat. #: 12236-014, Thermo Fisher Scientific) in the presence of 40 units RNaseOUT and subsequently incubated with 1 µL of RNase H (2 U/µL) for 20 min at 37 °C in accordance with the manufacturer′s instructions. .. Real-time PCR analysis with Brilliant Ⅱ SYBR Green QPCR Master Mix (Agilent, CA) was performed in duplicate using Mx4000 (Agilent).

Article Title: An Arabidopsis thaliana Plasma Membrane Proton Pump Is Essential for Pollen Development
Article Snippet: .. RNA was isolated from plants heterozygous for either the aha3-1 or the aha3-5 allele using the QIAGEN RNeasy mini kit (Valencia, CA). cDNA was synthesized using a modified oligo(dT) primer (5′-24T + A/C/G/T) and SuperScript II reverse transcriptase (Invitrogen, San Diego). .. Nested primers specific to AHA3 cDNA and to the T-DNA border sequence (primer JL202) were used to amplify PCR products corresponding to the mutant aha3 transcripts.

Article Title: Targeting TM4SF5 with anti-TM4SF5 monoclonal antibody suppresses the growth and motility of human pancreatic cancer cells
Article Snippet: Then, 2 µg of total RNA was reverse-transcribed in the first-strand synthesis buffer containing 6 µg/ml oligo(dT) primer, 50 U M-MLV reverse transcriptase, 2 mM dNTP, 10 mM DTT, and 40 U RNaseOUT™ recombinant ribonuclease inhibitor (Invitrogen; Thermo Fisher Scientific, Inc.). .. The reaction was carried out at 37°C for 50 min and heat inactivated at 70°C for 15 min. One microliter of the synthesized cDNA solution was subjected to a semi-quantitative PCR of 25 (for GAPDH) or 30 (for TM4SF5) cycles consisting of denaturation for 40 sec at 95°C, annealing for 40 sec at 58°C, and extension for 40 sec at 72°C.

Quantitative RT-PCR:

Article Title: Optimizing Laser Capture Microdissection Protocol for Isolating Zone-Specific Cell Populations from Mandibular Condylar Cartilage
Article Snippet: The isolated genetic material was used as a template to synthesize the first-strand cDNA using Superscript III Reverse Transcriptase (Invitrogen, CA, USA) and oligo (dT)12–18 (Invitrogen, CA, USA) according to the manufacturer instructions. .. The qRT-PCR was performed using a Step One Plus RT-PCR system (Applied Biosystems, CA, USA) and Power SYBR® Green PCR master mix (Applied Biosystems, Warrington, UK).

Article Title: LINT, a Novel dL(3)mbt-Containing Complex, Represses Malignant Brain Tumour Signature Genes
Article Snippet: Paragraph title: qRT–PCR ... 1.5 µg of RNA was applied to RT by incubation with 0.5 µg of Oligo(T)17 primer and 100 U of M-MLV reverse transcriptase (Invitrogen). cDNA was analyzed by qPCR, which was performed using Absolute SybrGreen Mix (Thermo Fisher) and the Mx3000P real-time detection system (Agilent).

Article Title: MCAM knockdown impairs PPARγ expression and 3T3-L1 fibroblasts differentiation to adipocytes
Article Snippet: Paragraph title: RNA extraction and RT-qPCR analysis ... 2 μg of RNA and 0.5 μg of Oligo (dT)15 were used for reverse transcription with RevertAid Reverse Transcriptase (Thermo Fisher Scientific). mRNA relative expression was determined by quantitative real-time PCR using a Bio-Rad MyIQ2 thermal cycler.

Article Title: Assessing a species thermal tolerance through a multiparameter approach: the case study of the deep-sea hydrothermal vent shrimp Rimicaris exoculata
Article Snippet: Paragraph title: Quantification of hsp70 expression using real-time quantitative RT-PCR ... The RNA (0.5 μg) was treated to remove DNA contamination by using the Turbo-DNAse kit (Ambion), and then reversely transcribed to cDNA with the oligo(dT)18 primer and Superscript II Reverse Transcriptase kit (Invitrogen) according to the manufacturer’s instructions.

Real-time Polymerase Chain Reaction:

Article Title: LINT, a Novel dL(3)mbt-Containing Complex, Represses Malignant Brain Tumour Signature Genes
Article Snippet: .. 1.5 µg of RNA was applied to RT by incubation with 0.5 µg of Oligo(T)17 primer and 100 U of M-MLV reverse transcriptase (Invitrogen). cDNA was analyzed by qPCR, which was performed using Absolute SybrGreen Mix (Thermo Fisher) and the Mx3000P real-time detection system (Agilent). ..

Article Title: Propionate and butyrate induce gene expression of monocarboxylate transporter 4 and cluster of differentiation 147 in cultured rumen epithelial cells derived from preweaning dairy calves
Article Snippet: RNA concentration and absorbance ratio were measured by spectrophotometer. cDNA in a total volume of 20 µL was synthesized from 1 µg of RNA with 2.5 µM of Oligo (dT)20 primer at 50 °C for 60 min using Thermo Script reverse transcriptase (15 U/µL, cat. #: 12236-014, Thermo Fisher Scientific) in the presence of 40 units RNaseOUT and subsequently incubated with 1 µL of RNase H (2 U/µL) for 20 min at 37 °C in accordance with the manufacturer′s instructions. .. Real-time PCR analysis with Brilliant ⠡ SYBR Green QPCR Master Mix (Agilent, CA) was performed in duplicate using Mx4000 (Agilent).

Article Title: MCAM knockdown impairs PPARγ expression and 3T3-L1 fibroblasts differentiation to adipocytes
Article Snippet: .. 2 μg of RNA and 0.5 μg of Oligo (dT)15 were used for reverse transcription with RevertAid Reverse Transcriptase (Thermo Fisher Scientific). mRNA relative expression was determined by quantitative real-time PCR using a Bio-Rad MyIQ2 thermal cycler. .. Each PCR reaction was performed using Platinum Taq Polymerase (Invitrogen) in a final volume of 25 μL, containing 5 μL of a 1:10 dilution of first-strand cDNA and 5 pmol of each primer.

Article Title: Fluorescent Reporter Zebrafish Line for Estrogenic Compound Screening Generated Using a CRISPR/Cas9-Mediated Knock-in System.
Article Snippet: An increasing number of compounds in our diet and environment are being identified as estrogenic, causing serious and detrimental effects on human, animal, and ecosystem health. .. An increasing number of compounds in our diet and environment are being identified as estrogenic, causing serious and detrimental effects on human, animal, and ecosystem health.

Article Title: Assessing a species thermal tolerance through a multiparameter approach: the case study of the deep-sea hydrothermal vent shrimp Rimicaris exoculata
Article Snippet: The RNA (0.5 μg) was treated to remove DNA contamination by using the Turbo-DNAse kit (Ambion), and then reversely transcribed to cDNA with the oligo(dT)18 primer and Superscript II Reverse Transcriptase kit (Invitrogen) according to the manufacturer’s instructions. .. The expression of hsp70 gene was assessed by qPCR with specific primers (see ).

Article Title: Gene structures and processing of Arabidopsis thaliana HYL1-dependent pri-miRNAs
Article Snippet: .. Quantitative real-time PCR profiling of pri-miRNAs Reverse transcriptase reactions were performed using (i) Oligo(dT)12–18 Primer (Invitrogen) or (ii) Oligo(dT)18 Primer (Fermentas, Vilnius, Lithuania) and SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturers instructions. .. The efficiency of cDNA synthesis was assessed by real-time PCR amplification of an UBQ10 (At4g05320 ) cDNA fragment ( , ).

Incubation:

Article Title: LINT, a Novel dL(3)mbt-Containing Complex, Represses Malignant Brain Tumour Signature Genes
Article Snippet: .. 1.5 µg of RNA was applied to RT by incubation with 0.5 µg of Oligo(T)17 primer and 100 U of M-MLV reverse transcriptase (Invitrogen). cDNA was analyzed by qPCR, which was performed using Absolute SybrGreen Mix (Thermo Fisher) and the Mx3000P real-time detection system (Agilent). ..

Article Title: Propionate and butyrate induce gene expression of monocarboxylate transporter 4 and cluster of differentiation 147 in cultured rumen epithelial cells derived from preweaning dairy calves
Article Snippet: .. RNA concentration and absorbance ratio were measured by spectrophotometer. cDNA in a total volume of 20 µL was synthesized from 1 µg of RNA with 2.5 µM of Oligo (dT)20 primer at 50 °C for 60 min using Thermo Script reverse transcriptase (15 U/µL, cat. #: 12236-014, Thermo Fisher Scientific) in the presence of 40 units RNaseOUT and subsequently incubated with 1 µL of RNase H (2 U/µL) for 20 min at 37 °C in accordance with the manufacturer′s instructions. .. Real-time PCR analysis with Brilliant Ⅱ SYBR Green QPCR Master Mix (Agilent, CA) was performed in duplicate using Mx4000 (Agilent).

Infection:

Article Title: Cooperativity between the 3’ untranslated region microRNA binding sites is critical for the virulence of eastern equine encephalitis virus
Article Snippet: Identification of EEEV escape mutants RNA was isolated as described from in vitro cultured RAW cells or BMDCs at 48 hr post infection or EEEV-infected (1x103 pfu bilaterally in footpad) and brain (D5), serum (24 hpi), and CVLN (D5) samples were harvested and placed in Tri-Reagent for RNA analysis. .. RT was performed using 50 μM Oligo(dT) (Thermo Fisher) as previously described [ ] with a 48°C extension temperature. cDNA was diluted with H2 O and 10ul was used in a GoTaq PCR reaction (GoTaq Green Mastermix, Fisher Scientific) with the following conditions: 95°C 2 min, (95°C 45s, 60°C 30s, 73°C 60s) x 40 cycles, 73°C 7 min.

Expressing:

Article Title: Propionate and butyrate induce gene expression of monocarboxylate transporter 4 and cluster of differentiation 147 in cultured rumen epithelial cells derived from preweaning dairy calves
Article Snippet: Paragraph title: Quantification of Gene Expression Levels in the Ruminal Tissues and Cultured Cells ... RNA concentration and absorbance ratio were measured by spectrophotometer. cDNA in a total volume of 20 µL was synthesized from 1 µg of RNA with 2.5 µM of Oligo (dT)20 primer at 50 °C for 60 min using Thermo Script reverse transcriptase (15 U/µL, cat. #: 12236-014, Thermo Fisher Scientific) in the presence of 40 units RNaseOUT and subsequently incubated with 1 µL of RNase H (2 U/µL) for 20 min at 37 °C in accordance with the manufacturer′s instructions.

Article Title: MCAM knockdown impairs PPARγ expression and 3T3-L1 fibroblasts differentiation to adipocytes
Article Snippet: .. 2 μg of RNA and 0.5 μg of Oligo (dT)15 were used for reverse transcription with RevertAid Reverse Transcriptase (Thermo Fisher Scientific). mRNA relative expression was determined by quantitative real-time PCR using a Bio-Rad MyIQ2 thermal cycler. .. Each PCR reaction was performed using Platinum Taq Polymerase (Invitrogen) in a final volume of 25 μL, containing 5 μL of a 1:10 dilution of first-strand cDNA and 5 pmol of each primer.

Article Title: Fluorescent Reporter Zebrafish Line for Estrogenic Compound Screening Generated Using a CRISPR/Cas9-Mediated Knock-in System.
Article Snippet: An increasing number of compounds in our diet and environment are being identified as estrogenic, causing serious and detrimental effects on human, animal, and ecosystem health. .. An increasing number of compounds in our diet and environment are being identified as estrogenic, causing serious and detrimental effects on human, animal, and ecosystem health.

Article Title: Assessing a species thermal tolerance through a multiparameter approach: the case study of the deep-sea hydrothermal vent shrimp Rimicaris exoculata
Article Snippet: Paragraph title: Quantification of hsp70 expression using real-time quantitative RT-PCR ... The RNA (0.5 μg) was treated to remove DNA contamination by using the Turbo-DNAse kit (Ambion), and then reversely transcribed to cDNA with the oligo(dT)18 primer and Superscript II Reverse Transcriptase kit (Invitrogen) according to the manufacturer’s instructions.

Modification:

Article Title: An Arabidopsis thaliana Plasma Membrane Proton Pump Is Essential for Pollen Development
Article Snippet: .. RNA was isolated from plants heterozygous for either the aha3-1 or the aha3-5 allele using the QIAGEN RNeasy mini kit (Valencia, CA). cDNA was synthesized using a modified oligo(dT) primer (5′-24T + A/C/G/T) and SuperScript II reverse transcriptase (Invitrogen, San Diego). .. Nested primers specific to AHA3 cDNA and to the T-DNA border sequence (primer JL202) were used to amplify PCR products corresponding to the mutant aha3 transcripts.

Activated Clotting Time Assay:

Article Title: Propionate and butyrate induce gene expression of monocarboxylate transporter 4 and cluster of differentiation 147 in cultured rumen epithelial cells derived from preweaning dairy calves
Article Snippet: RNA concentration and absorbance ratio were measured by spectrophotometer. cDNA in a total volume of 20 µL was synthesized from 1 µg of RNA with 2.5 µM of Oligo (dT)20 primer at 50 °C for 60 min using Thermo Script reverse transcriptase (15 U/µL, cat. #: 12236-014, Thermo Fisher Scientific) in the presence of 40 units RNaseOUT and subsequently incubated with 1 µL of RNase H (2 U/µL) for 20 min at 37 °C in accordance with the manufacturer′s instructions. .. The following primers were used at the final concentration of 0.25 µM for real-time PCR: MCT1 ( SLC16A1 , , 148 bp) forward (exon2), 5′-TTA ATG CCA CCA CCA GTG AA-3′; reverse (exon3), 5′-AAG CCA CTG CCT GAC AAG AT-3′; MCT4 ( SLC16A3 , , 85 bp) forward (exon4), 5′-TCC AGC TCT ACC TCA CCA CA-3′; reverse (exon5), 5′-TAG CGG TTG AGC ATG ATG AG-3′; CD147 ( BSG , , 63 bp) forward (bridging exon2-3 junction), 5′-CGG AGT ATG AGG TGG ACT CAG AA-3′, reverse (exon3), 5′-GCT CCG GAA GGA AGA TGC A-3′; GAPDH ( , 120 bp) forward (exon3), 5′-AGT TCA ACG GCA CAG TCA AG-3′; reverse (exon4), 5′-CAT ACT CAG CAC CAG CAT CA-3′.

Cell Culture:

Article Title: Propionate and butyrate induce gene expression of monocarboxylate transporter 4 and cluster of differentiation 147 in cultured rumen epithelial cells derived from preweaning dairy calves
Article Snippet: Paragraph title: Quantification of Gene Expression Levels in the Ruminal Tissues and Cultured Cells ... RNA concentration and absorbance ratio were measured by spectrophotometer. cDNA in a total volume of 20 µL was synthesized from 1 µg of RNA with 2.5 µM of Oligo (dT)20 primer at 50 °C for 60 min using Thermo Script reverse transcriptase (15 U/µL, cat. #: 12236-014, Thermo Fisher Scientific) in the presence of 40 units RNaseOUT and subsequently incubated with 1 µL of RNase H (2 U/µL) for 20 min at 37 °C in accordance with the manufacturer′s instructions.

Article Title: Cooperativity between the 3’ untranslated region microRNA binding sites is critical for the virulence of eastern equine encephalitis virus
Article Snippet: Identification of EEEV escape mutants RNA was isolated as described from in vitro cultured RAW cells or BMDCs at 48 hr post infection or EEEV-infected (1x103 pfu bilaterally in footpad) and brain (D5), serum (24 hpi), and CVLN (D5) samples were harvested and placed in Tri-Reagent for RNA analysis. .. RT was performed using 50 μM Oligo(dT) (Thermo Fisher) as previously described [ ] with a 48°C extension temperature. cDNA was diluted with H2 O and 10ul was used in a GoTaq PCR reaction (GoTaq Green Mastermix, Fisher Scientific) with the following conditions: 95°C 2 min, (95°C 45s, 60°C 30s, 73°C 60s) x 40 cycles, 73°C 7 min.

Generated:

Article Title: Optimizing Laser Capture Microdissection Protocol for Isolating Zone-Specific Cell Populations from Mandibular Condylar Cartilage
Article Snippet: Standard curves were generated for each primer pair with serial dilutions of cDNA synthesized using RNA isolated from a mixture of MCC and FCC specimens. .. The isolated genetic material was used as a template to synthesize the first-strand cDNA using Superscript III Reverse Transcriptase (Invitrogen, CA, USA) and oligo (dT)12–18 (Invitrogen, CA, USA) according to the manufacturer instructions.

Polymerase Chain Reaction:

Article Title: Optimizing Laser Capture Microdissection Protocol for Isolating Zone-Specific Cell Populations from Mandibular Condylar Cartilage
Article Snippet: The isolated genetic material was used as a template to synthesize the first-strand cDNA using Superscript III Reverse Transcriptase (Invitrogen, CA, USA) and oligo (dT)12–18 (Invitrogen, CA, USA) according to the manufacturer instructions. .. The qRT-PCR was performed using a Step One Plus RT-PCR system (Applied Biosystems, CA, USA) and Power SYBR® Green PCR master mix (Applied Biosystems, Warrington, UK).

Article Title: The Drosophila Small Conductance Calcium-Activated Potassium Channel Negatively Regulates Nociception
Article Snippet: The SuperScript III reverse transcriptase enzyme (Life Technologies) and Oligo(dT)12–18 primer (Life Technologies) were used to direct the synthesis of first strand cDNA. .. For SK-M and SK-V cDNA amplification, PCR was performed using the reverse primer 5ʹ-TCAGCTAGAATGTGGAAACAGCAT-3ʹ, and either the forward primer 5ʹ-ATGTCAATTCA GAAGCTTAACGAC-3ʹ to target the SK-M isoform or the forward primer 5ʹ-ATGTCGCCGGCCTTCTGC-3ʹ to target the SK-V isoform.

Article Title: MCAM knockdown impairs PPARγ expression and 3T3-L1 fibroblasts differentiation to adipocytes
Article Snippet: 2 μg of RNA and 0.5 μg of Oligo (dT)15 were used for reverse transcription with RevertAid Reverse Transcriptase (Thermo Fisher Scientific). mRNA relative expression was determined by quantitative real-time PCR using a Bio-Rad MyIQ2 thermal cycler. .. Each PCR reaction was performed using Platinum Taq Polymerase (Invitrogen) in a final volume of 25 μL, containing 5 μL of a 1:10 dilution of first-strand cDNA and 5 pmol of each primer.

Article Title: An Arabidopsis thaliana Plasma Membrane Proton Pump Is Essential for Pollen Development
Article Snippet: RNA was isolated from plants heterozygous for either the aha3-1 or the aha3-5 allele using the QIAGEN RNeasy mini kit (Valencia, CA). cDNA was synthesized using a modified oligo(dT) primer (5′-24T + A/C/G/T) and SuperScript II reverse transcriptase (Invitrogen, San Diego). .. Nested primers specific to AHA3 cDNA and to the T-DNA border sequence (primer JL202) were used to amplify PCR products corresponding to the mutant aha3 transcripts.

Article Title: Targeting TM4SF5 with anti-TM4SF5 monoclonal antibody suppresses the growth and motility of human pancreatic cancer cells
Article Snippet: Then, 2 µg of total RNA was reverse-transcribed in the first-strand synthesis buffer containing 6 µg/ml oligo(dT) primer, 50 U M-MLV reverse transcriptase, 2 mM dNTP, 10 mM DTT, and 40 U RNaseOUT™ recombinant ribonuclease inhibitor (Invitrogen; Thermo Fisher Scientific, Inc.). .. The reaction was carried out at 37°C for 50 min and heat inactivated at 70°C for 15 min. One microliter of the synthesized cDNA solution was subjected to a semi-quantitative PCR of 25 (for GAPDH) or 30 (for TM4SF5) cycles consisting of denaturation for 40 sec at 95°C, annealing for 40 sec at 58°C, and extension for 40 sec at 72°C.

Article Title: Cooperativity between the 3’ untranslated region microRNA binding sites is critical for the virulence of eastern equine encephalitis virus
Article Snippet: .. RT was performed using 50 μM Oligo(dT) (Thermo Fisher) as previously described [ ] with a 48°C extension temperature. cDNA was diluted with H2 O and 10ul was used in a GoTaq PCR reaction (GoTaq Green Mastermix, Fisher Scientific) with the following conditions: 95°C 2 min, (95°C 45s, 60°C 30s, 73°C 60s) x 40 cycles, 73°C 7 min. ..

DNA Sequencing:

Article Title: An Arabidopsis thaliana Plasma Membrane Proton Pump Is Essential for Pollen Development
Article Snippet: RNA was isolated from plants heterozygous for either the aha3-1 or the aha3-5 allele using the QIAGEN RNeasy mini kit (Valencia, CA). cDNA was synthesized using a modified oligo(dT) primer (5′-24T + A/C/G/T) and SuperScript II reverse transcriptase (Invitrogen, San Diego). .. DNA sequencing of PCR products confirmed the location of the junction between AHA3 cDNA and T-DNA.

Sequencing:

Article Title: The Drosophila Small Conductance Calcium-Activated Potassium Channel Negatively Regulates Nociception
Article Snippet: The SuperScript III reverse transcriptase enzyme (Life Technologies) and Oligo(dT)12–18 primer (Life Technologies) were used to direct the synthesis of first strand cDNA. .. The complete sequence for the newly cloned SK-V isoform was deposited in GenBank (accession number MH001552).

Article Title: An Arabidopsis thaliana Plasma Membrane Proton Pump Is Essential for Pollen Development
Article Snippet: RNA was isolated from plants heterozygous for either the aha3-1 or the aha3-5 allele using the QIAGEN RNeasy mini kit (Valencia, CA). cDNA was synthesized using a modified oligo(dT) primer (5′-24T + A/C/G/T) and SuperScript II reverse transcriptase (Invitrogen, San Diego). .. Nested primers specific to AHA3 cDNA and to the T-DNA border sequence (primer JL202) were used to amplify PCR products corresponding to the mutant aha3 transcripts.

Article Title: Cooperativity between the 3’ untranslated region microRNA binding sites is critical for the virulence of eastern equine encephalitis virus
Article Snippet: RT was performed using 50 μM Oligo(dT) (Thermo Fisher) as previously described [ ] with a 48°C extension temperature. cDNA was diluted with H2 O and 10ul was used in a GoTaq PCR reaction (GoTaq Green Mastermix, Fisher Scientific) with the following conditions: 95°C 2 min, (95°C 45s, 60°C 30s, 73°C 60s) x 40 cycles, 73°C 7 min. .. Sequencing was performed by the University of Pittsburgh HSCRF Genomics Research Core and analyzed using CLC Genomics Workbench (Qiagen).

Recombinant:

Article Title: Targeting TM4SF5 with anti-TM4SF5 monoclonal antibody suppresses the growth and motility of human pancreatic cancer cells
Article Snippet: .. Then, 2 µg of total RNA was reverse-transcribed in the first-strand synthesis buffer containing 6 µg/ml oligo(dT) primer, 50 U M-MLV reverse transcriptase, 2 mM dNTP, 10 mM DTT, and 40 U RNaseOUT™ recombinant ribonuclease inhibitor (Invitrogen; Thermo Fisher Scientific, Inc.). .. The reaction was carried out at 37°C for 50 min and heat inactivated at 70°C for 15 min. One microliter of the synthesized cDNA solution was subjected to a semi-quantitative PCR of 25 (for GAPDH) or 30 (for TM4SF5) cycles consisting of denaturation for 40 sec at 95°C, annealing for 40 sec at 58°C, and extension for 40 sec at 72°C.

Imaging:

Article Title: Fluorescent Reporter Zebrafish Line for Estrogenic Compound Screening Generated Using a CRISPR/Cas9-Mediated Knock-in System.
Article Snippet: An increasing number of compounds in our diet and environment are being identified as estrogenic, causing serious and detrimental effects on human, animal, and ecosystem health. .. An increasing number of compounds in our diet and environment are being identified as estrogenic, causing serious and detrimental effects on human, animal, and ecosystem health.

Fluorescence:

Article Title: Fluorescent Reporter Zebrafish Line for Estrogenic Compound Screening Generated Using a CRISPR/Cas9-Mediated Knock-in System.
Article Snippet: An increasing number of compounds in our diet and environment are being identified as estrogenic, causing serious and detrimental effects on human, animal, and ecosystem health. .. An increasing number of compounds in our diet and environment are being identified as estrogenic, causing serious and detrimental effects on human, animal, and ecosystem health.

Magnetic Beads:

Article Title: A novel synthesis and detection method for cap-associated adenosine modifications in mouse mRNA
Article Snippet: .. The poly(A) RNA was purified twice using oligo(dT) cellulose (Fluka) followed by oligo(dT) magnetic beads (Invitrogen). .. All samples were quantified and assayed for poly(A) purity using an Agilent Bioanalyser.

Mutagenesis:

Article Title: An Arabidopsis thaliana Plasma Membrane Proton Pump Is Essential for Pollen Development
Article Snippet: Paragraph title: Mutant transcript identification: ... RNA was isolated from plants heterozygous for either the aha3-1 or the aha3-5 allele using the QIAGEN RNeasy mini kit (Valencia, CA). cDNA was synthesized using a modified oligo(dT) primer (5′-24T + A/C/G/T) and SuperScript II reverse transcriptase (Invitrogen, San Diego).

Isolation:

Article Title: Optimizing Laser Capture Microdissection Protocol for Isolating Zone-Specific Cell Populations from Mandibular Condylar Cartilage
Article Snippet: .. The isolated genetic material was used as a template to synthesize the first-strand cDNA using Superscript III Reverse Transcriptase (Invitrogen, CA, USA) and oligo (dT)12–18 (Invitrogen, CA, USA) according to the manufacturer instructions. .. The qRT-PCR was performed using a Step One Plus RT-PCR system (Applied Biosystems, CA, USA) and Power SYBR® Green PCR master mix (Applied Biosystems, Warrington, UK).

Article Title: LINT, a Novel dL(3)mbt-Containing Complex, Represses Malignant Brain Tumour Signature Genes
Article Snippet: qRT–PCR Total RNA from Kc cells or 3rd instar larvae was isolated using the peqGOLD total RNA kit (Peqlab). .. 1.5 µg of RNA was applied to RT by incubation with 0.5 µg of Oligo(T)17 primer and 100 U of M-MLV reverse transcriptase (Invitrogen). cDNA was analyzed by qPCR, which was performed using Absolute SybrGreen Mix (Thermo Fisher) and the Mx3000P real-time detection system (Agilent).

Article Title: Propionate and butyrate induce gene expression of monocarboxylate transporter 4 and cluster of differentiation 147 in cultured rumen epithelial cells derived from preweaning dairy calves
Article Snippet: Total RNA was then extracted from the cells using the RNAqueous-Micro Total RNA Isolation Kit (Thermo Fisher Scientific) followed by 1 µL of DNase I treatment for 20 min at 37 °C. .. RNA concentration and absorbance ratio were measured by spectrophotometer. cDNA in a total volume of 20 µL was synthesized from 1 µg of RNA with 2.5 µM of Oligo (dT)20 primer at 50 °C for 60 min using Thermo Script reverse transcriptase (15 U/µL, cat. #: 12236-014, Thermo Fisher Scientific) in the presence of 40 units RNaseOUT and subsequently incubated with 1 µL of RNase H (2 U/µL) for 20 min at 37 °C in accordance with the manufacturer′s instructions.

Article Title: An Arabidopsis thaliana Plasma Membrane Proton Pump Is Essential for Pollen Development
Article Snippet: .. RNA was isolated from plants heterozygous for either the aha3-1 or the aha3-5 allele using the QIAGEN RNeasy mini kit (Valencia, CA). cDNA was synthesized using a modified oligo(dT) primer (5′-24T + A/C/G/T) and SuperScript II reverse transcriptase (Invitrogen, San Diego). .. Nested primers specific to AHA3 cDNA and to the T-DNA border sequence (primer JL202) were used to amplify PCR products corresponding to the mutant aha3 transcripts.

Article Title: Targeting TM4SF5 with anti-TM4SF5 monoclonal antibody suppresses the growth and motility of human pancreatic cancer cells
Article Snippet: Reverse transcription (RT) PCR Total RNA was isolated with the TRI Reagent® according to the manufacturer's instructions (MRC). .. Then, 2 µg of total RNA was reverse-transcribed in the first-strand synthesis buffer containing 6 µg/ml oligo(dT) primer, 50 U M-MLV reverse transcriptase, 2 mM dNTP, 10 mM DTT, and 40 U RNaseOUT™ recombinant ribonuclease inhibitor (Invitrogen; Thermo Fisher Scientific, Inc.).

Article Title: Cooperativity between the 3’ untranslated region microRNA binding sites is critical for the virulence of eastern equine encephalitis virus
Article Snippet: Identification of EEEV escape mutants RNA was isolated as described from in vitro cultured RAW cells or BMDCs at 48 hr post infection or EEEV-infected (1x103 pfu bilaterally in footpad) and brain (D5), serum (24 hpi), and CVLN (D5) samples were harvested and placed in Tri-Reagent for RNA analysis. .. RT was performed using 50 μM Oligo(dT) (Thermo Fisher) as previously described [ ] with a 48°C extension temperature. cDNA was diluted with H2 O and 10ul was used in a GoTaq PCR reaction (GoTaq Green Mastermix, Fisher Scientific) with the following conditions: 95°C 2 min, (95°C 45s, 60°C 30s, 73°C 60s) x 40 cycles, 73°C 7 min.

Size-exclusion Chromatography:

Article Title: Targeting TM4SF5 with anti-TM4SF5 monoclonal antibody suppresses the growth and motility of human pancreatic cancer cells
Article Snippet: Then, 2 µg of total RNA was reverse-transcribed in the first-strand synthesis buffer containing 6 µg/ml oligo(dT) primer, 50 U M-MLV reverse transcriptase, 2 mM dNTP, 10 mM DTT, and 40 U RNaseOUT™ recombinant ribonuclease inhibitor (Invitrogen; Thermo Fisher Scientific, Inc.). .. The reaction was carried out at 37°C for 50 min and heat inactivated at 70°C for 15 min. One microliter of the synthesized cDNA solution was subjected to a semi-quantitative PCR of 25 (for GAPDH) or 30 (for TM4SF5) cycles consisting of denaturation for 40 sec at 95°C, annealing for 40 sec at 58°C, and extension for 40 sec at 72°C.

Purification:

Article Title: Optimizing Laser Capture Microdissection Protocol for Isolating Zone-Specific Cell Populations from Mandibular Condylar Cartilage
Article Snippet: Total RNA was isolated and purified in accordance with the manufacturer's instructions (RNeasy Fibrous Tissue Mini Kit, Qiagen, Germany). .. The isolated genetic material was used as a template to synthesize the first-strand cDNA using Superscript III Reverse Transcriptase (Invitrogen, CA, USA) and oligo (dT)12–18 (Invitrogen, CA, USA) according to the manufacturer instructions.

Article Title: MCAM knockdown impairs PPARγ expression and 3T3-L1 fibroblasts differentiation to adipocytes
Article Snippet: Total RNA purification from 3T3-L1 fibroblasts was performed with TRI Reagent (Molecular Research Center) according to the manufacturer’s instructions, pellets were dissolved in nuclease-free water and RNA concentration was determined with NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). .. 2 μg of RNA and 0.5 μg of Oligo (dT)15 were used for reverse transcription with RevertAid Reverse Transcriptase (Thermo Fisher Scientific). mRNA relative expression was determined by quantitative real-time PCR using a Bio-Rad MyIQ2 thermal cycler.

Article Title: A novel synthesis and detection method for cap-associated adenosine modifications in mouse mRNA
Article Snippet: .. The poly(A) RNA was purified twice using oligo(dT) cellulose (Fluka) followed by oligo(dT) magnetic beads (Invitrogen). .. All samples were quantified and assayed for poly(A) purity using an Agilent Bioanalyser.

Reverse Transcription Polymerase Chain Reaction:

Article Title: Optimizing Laser Capture Microdissection Protocol for Isolating Zone-Specific Cell Populations from Mandibular Condylar Cartilage
Article Snippet: The isolated genetic material was used as a template to synthesize the first-strand cDNA using Superscript III Reverse Transcriptase (Invitrogen, CA, USA) and oligo (dT)12–18 (Invitrogen, CA, USA) according to the manufacturer instructions. .. The qRT-PCR was performed using a Step One Plus RT-PCR system (Applied Biosystems, CA, USA) and Power SYBR® Green PCR master mix (Applied Biosystems, Warrington, UK).

Article Title: The Drosophila Small Conductance Calcium-Activated Potassium Channel Negatively Regulates Nociception
Article Snippet: For the cloning of SK-M and SK-V rescue constructs, RT-PCR was performed on total RNA extracts (TRIzol, Life Technologies) from Canton-S third instar larvae. .. The SuperScript III reverse transcriptase enzyme (Life Technologies) and Oligo(dT)12–18 primer (Life Technologies) were used to direct the synthesis of first strand cDNA.

Article Title: Targeting TM4SF5 with anti-TM4SF5 monoclonal antibody suppresses the growth and motility of human pancreatic cancer cells
Article Snippet: Paragraph title: Reverse transcription (RT) PCR ... Then, 2 µg of total RNA was reverse-transcribed in the first-strand synthesis buffer containing 6 µg/ml oligo(dT) primer, 50 U M-MLV reverse transcriptase, 2 mM dNTP, 10 mM DTT, and 40 U RNaseOUT™ recombinant ribonuclease inhibitor (Invitrogen; Thermo Fisher Scientific, Inc.).

Construct:

Article Title: The Drosophila Small Conductance Calcium-Activated Potassium Channel Negatively Regulates Nociception
Article Snippet: For the cloning of SK-M and SK-V rescue constructs, RT-PCR was performed on total RNA extracts (TRIzol, Life Technologies) from Canton-S third instar larvae. .. The SuperScript III reverse transcriptase enzyme (Life Technologies) and Oligo(dT)12–18 primer (Life Technologies) were used to direct the synthesis of first strand cDNA.

Chloramphenicol Acetyltransferase Assay:

Article Title: Propionate and butyrate induce gene expression of monocarboxylate transporter 4 and cluster of differentiation 147 in cultured rumen epithelial cells derived from preweaning dairy calves
Article Snippet: RNA concentration and absorbance ratio were measured by spectrophotometer. cDNA in a total volume of 20 µL was synthesized from 1 µg of RNA with 2.5 µM of Oligo (dT)20 primer at 50 °C for 60 min using Thermo Script reverse transcriptase (15 U/µL, cat. #: 12236-014, Thermo Fisher Scientific) in the presence of 40 units RNaseOUT and subsequently incubated with 1 µL of RNase H (2 U/µL) for 20 min at 37 °C in accordance with the manufacturer′s instructions. .. The following primers were used at the final concentration of 0.25 µM for real-time PCR: MCT1 ( SLC16A1 , , 148 bp) forward (exon2), 5′-TTA ATG CCA CCA CCA GTG AA-3′; reverse (exon3), 5′-AAG CCA CTG CCT GAC AAG AT-3′; MCT4 ( SLC16A3 , , 85 bp) forward (exon4), 5′-TCC AGC TCT ACC TCA CCA CA-3′; reverse (exon5), 5′-TAG CGG TTG AGC ATG ATG AG-3′; CD147 ( BSG , , 63 bp) forward (bridging exon2-3 junction), 5′-CGG AGT ATG AGG TGG ACT CAG AA-3′, reverse (exon3), 5′-GCT CCG GAA GGA AGA TGC A-3′; GAPDH ( , 120 bp) forward (exon3), 5′-AGT TCA ACG GCA CAG TCA AG-3′; reverse (exon4), 5′-CAT ACT CAG CAC CAG CAT CA-3′.

Plasmid Preparation:

Article Title: The Drosophila Small Conductance Calcium-Activated Potassium Channel Negatively Regulates Nociception
Article Snippet: The SuperScript III reverse transcriptase enzyme (Life Technologies) and Oligo(dT)12–18 primer (Life Technologies) were used to direct the synthesis of first strand cDNA. .. The SK-M and SK-V PCR products were subsequently cloned into the TOPO-XL vector (Life Technologies) and fully sequenced.

Software:

Article Title: MCAM knockdown impairs PPARγ expression and 3T3-L1 fibroblasts differentiation to adipocytes
Article Snippet: 2 μg of RNA and 0.5 μg of Oligo (dT)15 were used for reverse transcription with RevertAid Reverse Transcriptase (Thermo Fisher Scientific). mRNA relative expression was determined by quantitative real-time PCR using a Bio-Rad MyIQ2 thermal cycler. .. MCAM and Perilipin 1 ( Plin1 ) primers were designed using Primer3 v0.4.0 software [ ], Rplp0 , Peroxisome proliferator activated receptor gamma isoform 2 ( PPARγ2 ), and Adiponectin ( Adipoq ) primer sequences were obtained from published reports [ – ].

SYBR Green Assay:

Article Title: Optimizing Laser Capture Microdissection Protocol for Isolating Zone-Specific Cell Populations from Mandibular Condylar Cartilage
Article Snippet: The isolated genetic material was used as a template to synthesize the first-strand cDNA using Superscript III Reverse Transcriptase (Invitrogen, CA, USA) and oligo (dT)12–18 (Invitrogen, CA, USA) according to the manufacturer instructions. .. The qRT-PCR was performed using a Step One Plus RT-PCR system (Applied Biosystems, CA, USA) and Power SYBR® Green PCR master mix (Applied Biosystems, Warrington, UK).

Article Title: Propionate and butyrate induce gene expression of monocarboxylate transporter 4 and cluster of differentiation 147 in cultured rumen epithelial cells derived from preweaning dairy calves
Article Snippet: RNA concentration and absorbance ratio were measured by spectrophotometer. cDNA in a total volume of 20 µL was synthesized from 1 µg of RNA with 2.5 µM of Oligo (dT)20 primer at 50 °C for 60 min using Thermo Script reverse transcriptase (15 U/µL, cat. #: 12236-014, Thermo Fisher Scientific) in the presence of 40 units RNaseOUT and subsequently incubated with 1 µL of RNase H (2 U/µL) for 20 min at 37 °C in accordance with the manufacturer′s instructions. .. Real-time PCR analysis with Brilliant ⠡ SYBR Green QPCR Master Mix (Agilent, CA) was performed in duplicate using Mx4000 (Agilent).

Article Title: Assessing a species thermal tolerance through a multiparameter approach: the case study of the deep-sea hydrothermal vent shrimp Rimicaris exoculata
Article Snippet: The RNA (0.5 μg) was treated to remove DNA contamination by using the Turbo-DNAse kit (Ambion), and then reversely transcribed to cDNA with the oligo(dT)18 primer and Superscript II Reverse Transcriptase kit (Invitrogen) according to the manufacturer’s instructions. .. All reactions were performed on the LightCycler® 480 II Real-Time PCR Detection System (Roche, France), using Sybr Green I Master (Roche, France).

Article Title: Gene structures and processing of Arabidopsis thaliana HYL1-dependent pri-miRNAs
Article Snippet: Quantitative real-time PCR profiling of pri-miRNAs Reverse transcriptase reactions were performed using (i) Oligo(dT)12–18 Primer (Invitrogen) or (ii) Oligo(dT)18 Primer (Fermentas, Vilnius, Lithuania) and SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturers instructions. .. Real-time PCRs were performed using a 7900HT Fast Real-Time PCR System (Applied Biosystems Applera, Darmstadt, Germany) and SYBR Green to monitor dsDNA synthesis.

RNA Extraction:

Article Title: MCAM knockdown impairs PPARγ expression and 3T3-L1 fibroblasts differentiation to adipocytes
Article Snippet: Paragraph title: RNA extraction and RT-qPCR analysis ... 2 μg of RNA and 0.5 μg of Oligo (dT)15 were used for reverse transcription with RevertAid Reverse Transcriptase (Thermo Fisher Scientific). mRNA relative expression was determined by quantitative real-time PCR using a Bio-Rad MyIQ2 thermal cycler.

Article Title: Fluorescent Reporter Zebrafish Line for Estrogenic Compound Screening Generated Using a CRISPR/Cas9-Mediated Knock-in System.
Article Snippet: An increasing number of compounds in our diet and environment are being identified as estrogenic, causing serious and detrimental effects on human, animal, and ecosystem health. .. An increasing number of compounds in our diet and environment are being identified as estrogenic, causing serious and detrimental effects on human, animal, and ecosystem health.

Selection:

Article Title: Fluorescent Reporter Zebrafish Line for Estrogenic Compound Screening Generated Using a CRISPR/Cas9-Mediated Knock-in System.
Article Snippet: An increasing number of compounds in our diet and environment are being identified as estrogenic, causing serious and detrimental effects on human, animal, and ecosystem health. .. An increasing number of compounds in our diet and environment are being identified as estrogenic, causing serious and detrimental effects on human, animal, and ecosystem health.

In Vitro:

Article Title: Cooperativity between the 3’ untranslated region microRNA binding sites is critical for the virulence of eastern equine encephalitis virus
Article Snippet: Identification of EEEV escape mutants RNA was isolated as described from in vitro cultured RAW cells or BMDCs at 48 hr post infection or EEEV-infected (1x103 pfu bilaterally in footpad) and brain (D5), serum (24 hpi), and CVLN (D5) samples were harvested and placed in Tri-Reagent for RNA analysis. .. RT was performed using 50 μM Oligo(dT) (Thermo Fisher) as previously described [ ] with a 48°C extension temperature. cDNA was diluted with H2 O and 10ul was used in a GoTaq PCR reaction (GoTaq Green Mastermix, Fisher Scientific) with the following conditions: 95°C 2 min, (95°C 45s, 60°C 30s, 73°C 60s) x 40 cycles, 73°C 7 min.

Spectrophotometry:

Article Title: Propionate and butyrate induce gene expression of monocarboxylate transporter 4 and cluster of differentiation 147 in cultured rumen epithelial cells derived from preweaning dairy calves
Article Snippet: .. RNA concentration and absorbance ratio were measured by spectrophotometer. cDNA in a total volume of 20 µL was synthesized from 1 µg of RNA with 2.5 µM of Oligo (dT)20 primer at 50 °C for 60 min using Thermo Script reverse transcriptase (15 U/µL, cat. #: 12236-014, Thermo Fisher Scientific) in the presence of 40 units RNaseOUT and subsequently incubated with 1 µL of RNase H (2 U/µL) for 20 min at 37 °C in accordance with the manufacturer′s instructions. .. Real-time PCR analysis with Brilliant Ⅱ SYBR Green QPCR Master Mix (Agilent, CA) was performed in duplicate using Mx4000 (Agilent).

Article Title: MCAM knockdown impairs PPARγ expression and 3T3-L1 fibroblasts differentiation to adipocytes
Article Snippet: Total RNA purification from 3T3-L1 fibroblasts was performed with TRI Reagent (Molecular Research Center) according to the manufacturer’s instructions, pellets were dissolved in nuclease-free water and RNA concentration was determined with NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). .. 2 μg of RNA and 0.5 μg of Oligo (dT)15 were used for reverse transcription with RevertAid Reverse Transcriptase (Thermo Fisher Scientific). mRNA relative expression was determined by quantitative real-time PCR using a Bio-Rad MyIQ2 thermal cycler.

Article Title: Fluorescent Reporter Zebrafish Line for Estrogenic Compound Screening Generated Using a CRISPR/Cas9-Mediated Knock-in System.
Article Snippet: An increasing number of compounds in our diet and environment are being identified as estrogenic, causing serious and detrimental effects on human, animal, and ecosystem health. .. An increasing number of compounds in our diet and environment are being identified as estrogenic, causing serious and detrimental effects on human, animal, and ecosystem health.

Concentration Assay:

Article Title: Propionate and butyrate induce gene expression of monocarboxylate transporter 4 and cluster of differentiation 147 in cultured rumen epithelial cells derived from preweaning dairy calves
Article Snippet: .. RNA concentration and absorbance ratio were measured by spectrophotometer. cDNA in a total volume of 20 µL was synthesized from 1 µg of RNA with 2.5 µM of Oligo (dT)20 primer at 50 °C for 60 min using Thermo Script reverse transcriptase (15 U/µL, cat. #: 12236-014, Thermo Fisher Scientific) in the presence of 40 units RNaseOUT and subsequently incubated with 1 µL of RNase H (2 U/µL) for 20 min at 37 °C in accordance with the manufacturer′s instructions. .. Real-time PCR analysis with Brilliant Ⅱ SYBR Green QPCR Master Mix (Agilent, CA) was performed in duplicate using Mx4000 (Agilent).

Article Title: MCAM knockdown impairs PPARγ expression and 3T3-L1 fibroblasts differentiation to adipocytes
Article Snippet: Total RNA purification from 3T3-L1 fibroblasts was performed with TRI Reagent (Molecular Research Center) according to the manufacturer’s instructions, pellets were dissolved in nuclease-free water and RNA concentration was determined with NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). .. 2 μg of RNA and 0.5 μg of Oligo (dT)15 were used for reverse transcription with RevertAid Reverse Transcriptase (Thermo Fisher Scientific). mRNA relative expression was determined by quantitative real-time PCR using a Bio-Rad MyIQ2 thermal cycler.

CTG Assay:

Article Title: Propionate and butyrate induce gene expression of monocarboxylate transporter 4 and cluster of differentiation 147 in cultured rumen epithelial cells derived from preweaning dairy calves
Article Snippet: RNA concentration and absorbance ratio were measured by spectrophotometer. cDNA in a total volume of 20 µL was synthesized from 1 µg of RNA with 2.5 µM of Oligo (dT)20 primer at 50 °C for 60 min using Thermo Script reverse transcriptase (15 U/µL, cat. #: 12236-014, Thermo Fisher Scientific) in the presence of 40 units RNaseOUT and subsequently incubated with 1 µL of RNase H (2 U/µL) for 20 min at 37 °C in accordance with the manufacturer′s instructions. .. The following primers were used at the final concentration of 0.25 µM for real-time PCR: MCT1 ( SLC16A1 , , 148 bp) forward (exon2), 5′-TTA ATG CCA CCA CCA GTG AA-3′; reverse (exon3), 5′-AAG CCA CTG CCT GAC AAG AT-3′; MCT4 ( SLC16A3 , , 85 bp) forward (exon4), 5′-TCC AGC TCT ACC TCA CCA CA-3′; reverse (exon5), 5′-TAG CGG TTG AGC ATG ATG AG-3′; CD147 ( BSG , , 63 bp) forward (bridging exon2-3 junction), 5′-CGG AGT ATG AGG TGG ACT CAG AA-3′, reverse (exon3), 5′-GCT CCG GAA GGA AGA TGC A-3′; GAPDH ( , 120 bp) forward (exon3), 5′-AGT TCA ACG GCA CAG TCA AG-3′; reverse (exon4), 5′-CAT ACT CAG CAC CAG CAT CA-3′.

Standard Deviation:

Article Title: LINT, a Novel dL(3)mbt-Containing Complex, Represses Malignant Brain Tumour Signature Genes
Article Snippet: 1.5 µg of RNA was applied to RT by incubation with 0.5 µg of Oligo(T)17 primer and 100 U of M-MLV reverse transcriptase (Invitrogen). cDNA was analyzed by qPCR, which was performed using Absolute SybrGreen Mix (Thermo Fisher) and the Mx3000P real-time detection system (Agilent). .. Standard deviation was calculated from triplicates, error bars are indicated accordingly.

Lysis:

Article Title: Propionate and butyrate induce gene expression of monocarboxylate transporter 4 and cluster of differentiation 147 in cultured rumen epithelial cells derived from preweaning dairy calves
Article Snippet: Cultured rumen epithelial cells were lysed with Lysis solution 24 h following treatment. .. RNA concentration and absorbance ratio were measured by spectrophotometer. cDNA in a total volume of 20 µL was synthesized from 1 µg of RNA with 2.5 µM of Oligo (dT)20 primer at 50 °C for 60 min using Thermo Script reverse transcriptase (15 U/µL, cat. #: 12236-014, Thermo Fisher Scientific) in the presence of 40 units RNaseOUT and subsequently incubated with 1 µL of RNase H (2 U/µL) for 20 min at 37 °C in accordance with the manufacturer′s instructions.

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  • 93
    Thermo Fisher guide oligo
    Most telomeres in T . brucei WT cells have a 3′ overhang that ends in 5′ TTAGGG 3′. ( a ) A diagram showing the principle of the adaptor ligation assay. Top, a natural chromosome end with a 3′ overhang (left) is ligated with an adaptor (right). The light bar of the adaptor represents the unique <t>oligo</t> and is radiolabeled at its 5′ end. The darker bar of the adaptor represents the common region of all guide oligos. Only when the adaptor matches the telomere end perfectly can the adaptor be ligated to the chromosome end and eventually migrate with the long telomere fragment in agarose gel electrophoresis. Bottom, the sequence of the unique oligo and seven different guide oligos are shown. The T . brucei genomic <t>DNA</t> from BF ( b ) and PF ( c ) WT cells (either treated with or without Exo T) was ligated to radioisotope-labeled different adaptors (TG1–6 and TGNS, as indicated on top of each lane), digested with AluI and MboI, and separated by agarose gel electrophoresis. The ethidium bromide-stained gel is shown on the left and the image of the gel exposed to a phosphorimager (exposed) is shown on the right. 1 kb DNA ladder (ThermoFisher) was used as a molecular weight marker. ( d ) Quantification of the adaptor ligation results in both BF and PF WT cells. Average values are calculated from five (BF) or three (PF) independent experiments. Error bars represent standard deviation.
    Guide Oligo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/guide oligo/product/Thermo Fisher
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    guide oligo - by Bioz Stars, 2020-04
    93/100 stars
      Buy from Supplier

    99
    Thermo Fisher sirna oligos
    DDR1 affects IR expression (a) IR protein and mRNA expression in DDR1-depleted cells. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected with either a pool of four scramble or four DDR1 <t>siRNA</t> <t>oligos.</t> After 48h, cells were lysed and analyzed by SDS-PAGE and immunoblotted with the indicated primary antibodies. β-actin was used as control for protein loading. Blot is representative of three independent experiments. The histograms represent the mean±SEM of densitometric analysis after normalization against β-actin. In the same transfected cell lines, IR mRNA levels were evaluated by qRT-PCR analysis and values were normalized using human β-actin as housekeeping control gene. In parallel, DDR1 mRNA was evaluated by qRT-PCR to confirm DDR1 depletion (graphs on the right). (b) IR protein and mRNA expression after DDR1 overexpression. Breast cancer cells were transiently transfected with either constitutive empty (pCMV6-EV) or human DDR1 (pCMV6-DDR1) expressing vectors. After 48h, cells were lysed, analyzed by SDS-PAGE and immunoblotted with the indicated primary antibodies. β-actin was used as control for protein loading. The top panels show a representative blot of three independent experiments. The histograms represent the mean±SEM of densitometric analysis after normalization over β-actin. In the same transfected cells, IR mRNA levels were evaluated by qRT-PCR analysis and values were normalized using human β-actin as housekeeping control gene. The overexpression of DDR1 was confirmed measuring DDR1 mRNA by qRT-PCR (graphs on the right). (a-b) *0.01
    Sirna Oligos, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 284 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sirna oligos/product/Thermo Fisher
    Average 99 stars, based on 284 article reviews
    Price from $9.99 to $1999.99
    sirna oligos - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    86
    Thermo Fisher sirna oligos targeting rock1
    Loss of either <t>ROCK1</t> or ROCK2 expression results in impaired cell migration. Confluent HaCaT-NSC (A,B), HaCaT-ROCK1-KD (C,D) and HaCaT-ROCK2-KD (E,F) monolayers were scratched with a pipette tip. Phase contrast images were taken immediately and 6 hours after wounding and representative phase-contrast images are shown (A–F). Relative wound closure was calculated for HaCaT-ROCK1-KD and HaCaT-ROCK2-KD cells (G) as well as SCC12f keratinocytes transiently transfected with <t>siRNA</t> directed against ROCK1 and ROCK2 (H). The means and standard errors of 3 separate experiments are shown and statistical significance calculated using Mann-Whitney test (*** p
    Sirna Oligos Targeting Rock1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sirna oligos targeting rock1/product/Thermo Fisher
    Average 86 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    sirna oligos targeting rock1 - by Bioz Stars, 2020-04
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    Image Search Results


    Most telomeres in T . brucei WT cells have a 3′ overhang that ends in 5′ TTAGGG 3′. ( a ) A diagram showing the principle of the adaptor ligation assay. Top, a natural chromosome end with a 3′ overhang (left) is ligated with an adaptor (right). The light bar of the adaptor represents the unique oligo and is radiolabeled at its 5′ end. The darker bar of the adaptor represents the common region of all guide oligos. Only when the adaptor matches the telomere end perfectly can the adaptor be ligated to the chromosome end and eventually migrate with the long telomere fragment in agarose gel electrophoresis. Bottom, the sequence of the unique oligo and seven different guide oligos are shown. The T . brucei genomic DNA from BF ( b ) and PF ( c ) WT cells (either treated with or without Exo T) was ligated to radioisotope-labeled different adaptors (TG1–6 and TGNS, as indicated on top of each lane), digested with AluI and MboI, and separated by agarose gel electrophoresis. The ethidium bromide-stained gel is shown on the left and the image of the gel exposed to a phosphorimager (exposed) is shown on the right. 1 kb DNA ladder (ThermoFisher) was used as a molecular weight marker. ( d ) Quantification of the adaptor ligation results in both BF and PF WT cells. Average values are calculated from five (BF) or three (PF) independent experiments. Error bars represent standard deviation.

    Journal: Scientific Reports

    Article Title: Telomerase activity is required for the telomere G-overhang structure in Trypanosoma brucei

    doi: 10.1038/s41598-017-16182-y

    Figure Lengend Snippet: Most telomeres in T . brucei WT cells have a 3′ overhang that ends in 5′ TTAGGG 3′. ( a ) A diagram showing the principle of the adaptor ligation assay. Top, a natural chromosome end with a 3′ overhang (left) is ligated with an adaptor (right). The light bar of the adaptor represents the unique oligo and is radiolabeled at its 5′ end. The darker bar of the adaptor represents the common region of all guide oligos. Only when the adaptor matches the telomere end perfectly can the adaptor be ligated to the chromosome end and eventually migrate with the long telomere fragment in agarose gel electrophoresis. Bottom, the sequence of the unique oligo and seven different guide oligos are shown. The T . brucei genomic DNA from BF ( b ) and PF ( c ) WT cells (either treated with or without Exo T) was ligated to radioisotope-labeled different adaptors (TG1–6 and TGNS, as indicated on top of each lane), digested with AluI and MboI, and separated by agarose gel electrophoresis. The ethidium bromide-stained gel is shown on the left and the image of the gel exposed to a phosphorimager (exposed) is shown on the right. 1 kb DNA ladder (ThermoFisher) was used as a molecular weight marker. ( d ) Quantification of the adaptor ligation results in both BF and PF WT cells. Average values are calculated from five (BF) or three (PF) independent experiments. Error bars represent standard deviation.

    Article Snippet: Briefly, 100 ng genomic DNA was ligated to each guide oligo (10 pmole) using T4 DNA ligase (ThermoFisher).

    Techniques: Ligation, Agarose Gel Electrophoresis, Sequencing, Labeling, Staining, Molecular Weight, Marker, Standard Deviation

    Telomeres in T . brucei cells prefer to have a 5′ end with a sequence of 5′ CCTAAC 3′. ( a ) A diagram showing the principle of the Telomere Oligo Ligation-mediated PCR (TOLP) assay. Seven different telomere guide oligos are separately ligated to intact genomic DNA. Only when the oligo is aligned immediately next to the 5′ end of the telomere can the oligo be ligated to the chromosome DNA. Subsequently the ligation products are amplified by PCR using a forward primer with the sequence of (TTAGGG) 3 and a backward primer with the sequence identical to the common regions of all guide oligos. The PCR products were then separated by agarose gel electrophoresis followed by Southern analysis using a telomeric probe. ( b ) TOLP was performed using genomic DNA isolated from WT BF cells that was treated with or without T7 exonuclease. The ethidium bromide-stained gel is shown on the top, and the Southern hybridization result using a telomeric probe is shown on the bottom. ( c ) Quantification of the TOLP signals from different guide oligos is shown. Average is calculated from three independent experiments. Error bars represent standard deviation.

    Journal: Scientific Reports

    Article Title: Telomerase activity is required for the telomere G-overhang structure in Trypanosoma brucei

    doi: 10.1038/s41598-017-16182-y

    Figure Lengend Snippet: Telomeres in T . brucei cells prefer to have a 5′ end with a sequence of 5′ CCTAAC 3′. ( a ) A diagram showing the principle of the Telomere Oligo Ligation-mediated PCR (TOLP) assay. Seven different telomere guide oligos are separately ligated to intact genomic DNA. Only when the oligo is aligned immediately next to the 5′ end of the telomere can the oligo be ligated to the chromosome DNA. Subsequently the ligation products are amplified by PCR using a forward primer with the sequence of (TTAGGG) 3 and a backward primer with the sequence identical to the common regions of all guide oligos. The PCR products were then separated by agarose gel electrophoresis followed by Southern analysis using a telomeric probe. ( b ) TOLP was performed using genomic DNA isolated from WT BF cells that was treated with or without T7 exonuclease. The ethidium bromide-stained gel is shown on the top, and the Southern hybridization result using a telomeric probe is shown on the bottom. ( c ) Quantification of the TOLP signals from different guide oligos is shown. Average is calculated from three independent experiments. Error bars represent standard deviation.

    Article Snippet: Briefly, 100 ng genomic DNA was ligated to each guide oligo (10 pmole) using T4 DNA ligase (ThermoFisher).

    Techniques: Sequencing, Ligation, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Isolation, Staining, Hybridization, Standard Deviation

    DDR1 affects IR expression (a) IR protein and mRNA expression in DDR1-depleted cells. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected with either a pool of four scramble or four DDR1 siRNA oligos. After 48h, cells were lysed and analyzed by SDS-PAGE and immunoblotted with the indicated primary antibodies. β-actin was used as control for protein loading. Blot is representative of three independent experiments. The histograms represent the mean±SEM of densitometric analysis after normalization against β-actin. In the same transfected cell lines, IR mRNA levels were evaluated by qRT-PCR analysis and values were normalized using human β-actin as housekeeping control gene. In parallel, DDR1 mRNA was evaluated by qRT-PCR to confirm DDR1 depletion (graphs on the right). (b) IR protein and mRNA expression after DDR1 overexpression. Breast cancer cells were transiently transfected with either constitutive empty (pCMV6-EV) or human DDR1 (pCMV6-DDR1) expressing vectors. After 48h, cells were lysed, analyzed by SDS-PAGE and immunoblotted with the indicated primary antibodies. β-actin was used as control for protein loading. The top panels show a representative blot of three independent experiments. The histograms represent the mean±SEM of densitometric analysis after normalization over β-actin. In the same transfected cells, IR mRNA levels were evaluated by qRT-PCR analysis and values were normalized using human β-actin as housekeeping control gene. The overexpression of DDR1 was confirmed measuring DDR1 mRNA by qRT-PCR (graphs on the right). (a-b) *0.01

    Journal: Oncotarget

    Article Title: Discoidin domain receptor 1 modulates insulin receptor signaling and biological responses in breast cancer cells

    doi: 10.18632/oncotarget.18020

    Figure Lengend Snippet: DDR1 affects IR expression (a) IR protein and mRNA expression in DDR1-depleted cells. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected with either a pool of four scramble or four DDR1 siRNA oligos. After 48h, cells were lysed and analyzed by SDS-PAGE and immunoblotted with the indicated primary antibodies. β-actin was used as control for protein loading. Blot is representative of three independent experiments. The histograms represent the mean±SEM of densitometric analysis after normalization against β-actin. In the same transfected cell lines, IR mRNA levels were evaluated by qRT-PCR analysis and values were normalized using human β-actin as housekeeping control gene. In parallel, DDR1 mRNA was evaluated by qRT-PCR to confirm DDR1 depletion (graphs on the right). (b) IR protein and mRNA expression after DDR1 overexpression. Breast cancer cells were transiently transfected with either constitutive empty (pCMV6-EV) or human DDR1 (pCMV6-DDR1) expressing vectors. After 48h, cells were lysed, analyzed by SDS-PAGE and immunoblotted with the indicated primary antibodies. β-actin was used as control for protein loading. The top panels show a representative blot of three independent experiments. The histograms represent the mean±SEM of densitometric analysis after normalization over β-actin. In the same transfected cells, IR mRNA levels were evaluated by qRT-PCR analysis and values were normalized using human β-actin as housekeeping control gene. The overexpression of DDR1 was confirmed measuring DDR1 mRNA by qRT-PCR (graphs on the right). (a-b) *0.01

    Article Snippet: The specific silencer Select Pre-designed pool of four siRNA oligos for DDR1 (Human DDR1 siGENOME SMARTpool Cat M-003111–04) and the negative control, consisting of a pool of four scramble siRNAs were from Thermo Fisher Scientific Dharmacon (NYSE:TMO).

    Techniques: Expressing, Multiple Displacement Amplification, Transfection, SDS Page, Quantitative RT-PCR, Over Expression

    DDR1 affects IR – dependent transcription factors (a) Protein levels of IR-dependent transcription factors after DDR1 depletion. MCF-7 breast cancer cells were transiently transfected with either a pool of four scramble or four DDR1 specific siRNA oligos. After 48h, protein expression of IR, and of p53, HMGA, and Sp1 transcription factors was evaluated by western blotting. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization against β-actin. (b) Protein levels of IR-related transcription factors after DDR1 overexpression. MCF-7 cells were transiently transfected with either either pCMV6-EV or pCMV6-DDR1 encoding vectors. After 48h, protein expression of IR, p53, HMGA, and Sp1 was evaluated by western blotting. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization against β-actin. (c) mRNA expression of IR-related transcription factors after DDR1 depletion. MCF-7 cells transfected as in (a) were evaluated by qRT-PCR analysis for IR, p53, HMGA, and Sp1 mRNA levels. Values were normalized using human β-actin as housekeeping control gene. DDR1 mRNA levels were also evaluated as control for DDR1 depletion efficiency. Values represent the mean±SEM of three independent experiments. (d) mRNA expression of IR-related transcription factors after DDR1 overexpression. In cells transfected as in (b) , IR, p53, HMGA, and Sp1 mRNA levels were evaluated by qRT-PCR analysis and values were normalized using human β-actin as housekeeping control gene. DDR1 mRNA levels were also evaluated as silencing control. Values represent the mean±SEM of three independent experiments. (a-d) NS, p > 0.05; *0.01

    Journal: Oncotarget

    Article Title: Discoidin domain receptor 1 modulates insulin receptor signaling and biological responses in breast cancer cells

    doi: 10.18632/oncotarget.18020

    Figure Lengend Snippet: DDR1 affects IR – dependent transcription factors (a) Protein levels of IR-dependent transcription factors after DDR1 depletion. MCF-7 breast cancer cells were transiently transfected with either a pool of four scramble or four DDR1 specific siRNA oligos. After 48h, protein expression of IR, and of p53, HMGA, and Sp1 transcription factors was evaluated by western blotting. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization against β-actin. (b) Protein levels of IR-related transcription factors after DDR1 overexpression. MCF-7 cells were transiently transfected with either either pCMV6-EV or pCMV6-DDR1 encoding vectors. After 48h, protein expression of IR, p53, HMGA, and Sp1 was evaluated by western blotting. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization against β-actin. (c) mRNA expression of IR-related transcription factors after DDR1 depletion. MCF-7 cells transfected as in (a) were evaluated by qRT-PCR analysis for IR, p53, HMGA, and Sp1 mRNA levels. Values were normalized using human β-actin as housekeeping control gene. DDR1 mRNA levels were also evaluated as control for DDR1 depletion efficiency. Values represent the mean±SEM of three independent experiments. (d) mRNA expression of IR-related transcription factors after DDR1 overexpression. In cells transfected as in (b) , IR, p53, HMGA, and Sp1 mRNA levels were evaluated by qRT-PCR analysis and values were normalized using human β-actin as housekeeping control gene. DDR1 mRNA levels were also evaluated as silencing control. Values represent the mean±SEM of three independent experiments. (a-d) NS, p > 0.05; *0.01

    Article Snippet: The specific silencer Select Pre-designed pool of four siRNA oligos for DDR1 (Human DDR1 siGENOME SMARTpool Cat M-003111–04) and the negative control, consisting of a pool of four scramble siRNAs were from Thermo Fisher Scientific Dharmacon (NYSE:TMO).

    Techniques: Transfection, Expressing, Western Blot, Over Expression, Quantitative RT-PCR

    DDR1 stabilizes IR both at the protein and mRNA level in MCF-7 cells (a) DDR1 depletion affects IR proteasomal degradation. MCF-7 cells were transiently transfected with either a pool of four scramble or four DDR1-specific siRNA oligos. After 48h, cells were treated with 1μM of the proteasome inhibitor MG132 for 6h. (b) DDR1 depletion does not affect IR lysosomal degradation. MCF-7 cells were transiently transfected as in (a) . After 48 h, cells were treated with 10μM of the cloroquine (CHL) for 2, 6, and 24h. (c) Cycloheximide treatment in DDR1 overexpressing cells. MCF-7 cells were transiently transfected with either pCMV6-EV or pCMV6-DDR1 encoding vectors. After 24 h, cells were treated with 100 μM of cycloheximide (CHX) for 8, 16, and 24h. (d) Actinomycin D treatment in DDR1 overexpressing cells. MCF-7 cells were transiently transfected as in (c) . After 24h, cells were treated with 2.5μg/mL of actinomycin D (ACT D) for 2, 4, 6, and 16h. IR mRNA levels were evaluated by qRT-PCR analysis and values were normalized using human β-actin as housekeeping control gene. DDR1 overexpression was confirmed by western blot analysis as shown on the right of the histogram. (a-c) Samples were analyzed by western blotting for DDR1 and IR expression. β-actin antibody was used as control. A representative blot of three independent experiments is shown. The histogram represents the mean±SEM of densitometric analysis after normalization against β-actin. (a-d) NS, p > 0.05; *0.01

    Journal: Oncotarget

    Article Title: Discoidin domain receptor 1 modulates insulin receptor signaling and biological responses in breast cancer cells

    doi: 10.18632/oncotarget.18020

    Figure Lengend Snippet: DDR1 stabilizes IR both at the protein and mRNA level in MCF-7 cells (a) DDR1 depletion affects IR proteasomal degradation. MCF-7 cells were transiently transfected with either a pool of four scramble or four DDR1-specific siRNA oligos. After 48h, cells were treated with 1μM of the proteasome inhibitor MG132 for 6h. (b) DDR1 depletion does not affect IR lysosomal degradation. MCF-7 cells were transiently transfected as in (a) . After 48 h, cells were treated with 10μM of the cloroquine (CHL) for 2, 6, and 24h. (c) Cycloheximide treatment in DDR1 overexpressing cells. MCF-7 cells were transiently transfected with either pCMV6-EV or pCMV6-DDR1 encoding vectors. After 24 h, cells were treated with 100 μM of cycloheximide (CHX) for 8, 16, and 24h. (d) Actinomycin D treatment in DDR1 overexpressing cells. MCF-7 cells were transiently transfected as in (c) . After 24h, cells were treated with 2.5μg/mL of actinomycin D (ACT D) for 2, 4, 6, and 16h. IR mRNA levels were evaluated by qRT-PCR analysis and values were normalized using human β-actin as housekeeping control gene. DDR1 overexpression was confirmed by western blot analysis as shown on the right of the histogram. (a-c) Samples were analyzed by western blotting for DDR1 and IR expression. β-actin antibody was used as control. A representative blot of three independent experiments is shown. The histogram represents the mean±SEM of densitometric analysis after normalization against β-actin. (a-d) NS, p > 0.05; *0.01

    Article Snippet: The specific silencer Select Pre-designed pool of four siRNA oligos for DDR1 (Human DDR1 siGENOME SMARTpool Cat M-003111–04) and the negative control, consisting of a pool of four scramble siRNAs were from Thermo Fisher Scientific Dharmacon (NYSE:TMO).

    Techniques: Transfection, Activated Clotting Time Assay, Quantitative RT-PCR, Over Expression, Western Blot, Expressing

    DDR1 depletion affects insulin and IGF-2 mediated biological effects in human cancer cells (a) Western blot before and after DDR1 depletion. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected with either a pool of four DDR1 siRNA oligos or scramble siRNA oligos. After 24h, cells were grown in medium containing 2.5% of CS-FCS for 24h. DDR1 depletion was confirmed for each cells line by western blot analysis as shown in the panel. (b) Cell proliferation. Cell viability was evaluated by MTT assay. Values are expressed as percentages of untreated scramble-transfected cells (basal) and represent the mean±SEM of three independent experiments in triplicate. (c) Cell cycle progression. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected as in (a) . After 24h, cells were grown in medium containing 0.1% of BSA for additional 24h. Cells were then incubated with or without insulin or IGF-2 at a dose of 10nM for additional 48h and analyzed for cell-cycle profiles. Cell populations positive for propidium iodine staining were evaluated by FACS analysis, and G0/G1 and G2/M phases were scored. The graph shows the percentage of cells in S and G2/M phases. Values are expressed as percent of basal (untreated scramble transfected cells) and are the mean±SEM of three independent experiments. (d) Cell invasion. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected as in (a) . After 24h, cells were grown in medium containing 0.1% of BSA for additional 24 h. Cells were then removed from plates with 0.01% trypsin and seeded on polycarbonate filters coated with 25μg/mL fibronectin. Cells were allowed to migrate for 6h (MCF-7 and MDA-MB-157) or 8h (BT-474 cells) in response to 10nM of insulin or IGF-2 added to the lower chamber. Values are mean±SEM of three independent experiments done in duplicate and are expressed as percent increase over untreated scramble cells (basal). (e) Colony formation. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected as in (a) , and seeded in soft-agar, as described in Materials and Methods. Cells were plated in triplicate and cultured in serum free medium containing 2.5% CS-FCS for 3 weeks. Colonies developed only from plated MCF-7 and MDA-MB-157 cells but not from BT-474. Colonies were stained with MTT and then photographed. The histogram represents the mean number of colonies shown in (e) . Error bars indicate SEM (n = 3 wells). (b-e) *0.01

    Journal: Oncotarget

    Article Title: Discoidin domain receptor 1 modulates insulin receptor signaling and biological responses in breast cancer cells

    doi: 10.18632/oncotarget.18020

    Figure Lengend Snippet: DDR1 depletion affects insulin and IGF-2 mediated biological effects in human cancer cells (a) Western blot before and after DDR1 depletion. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected with either a pool of four DDR1 siRNA oligos or scramble siRNA oligos. After 24h, cells were grown in medium containing 2.5% of CS-FCS for 24h. DDR1 depletion was confirmed for each cells line by western blot analysis as shown in the panel. (b) Cell proliferation. Cell viability was evaluated by MTT assay. Values are expressed as percentages of untreated scramble-transfected cells (basal) and represent the mean±SEM of three independent experiments in triplicate. (c) Cell cycle progression. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected as in (a) . After 24h, cells were grown in medium containing 0.1% of BSA for additional 24h. Cells were then incubated with or without insulin or IGF-2 at a dose of 10nM for additional 48h and analyzed for cell-cycle profiles. Cell populations positive for propidium iodine staining were evaluated by FACS analysis, and G0/G1 and G2/M phases were scored. The graph shows the percentage of cells in S and G2/M phases. Values are expressed as percent of basal (untreated scramble transfected cells) and are the mean±SEM of three independent experiments. (d) Cell invasion. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected as in (a) . After 24h, cells were grown in medium containing 0.1% of BSA for additional 24 h. Cells were then removed from plates with 0.01% trypsin and seeded on polycarbonate filters coated with 25μg/mL fibronectin. Cells were allowed to migrate for 6h (MCF-7 and MDA-MB-157) or 8h (BT-474 cells) in response to 10nM of insulin or IGF-2 added to the lower chamber. Values are mean±SEM of three independent experiments done in duplicate and are expressed as percent increase over untreated scramble cells (basal). (e) Colony formation. MCF-7, MDA-MB-157 and BT-474 breast cancer cells were transiently transfected as in (a) , and seeded in soft-agar, as described in Materials and Methods. Cells were plated in triplicate and cultured in serum free medium containing 2.5% CS-FCS for 3 weeks. Colonies developed only from plated MCF-7 and MDA-MB-157 cells but not from BT-474. Colonies were stained with MTT and then photographed. The histogram represents the mean number of colonies shown in (e) . Error bars indicate SEM (n = 3 wells). (b-e) *0.01

    Article Snippet: The specific silencer Select Pre-designed pool of four siRNA oligos for DDR1 (Human DDR1 siGENOME SMARTpool Cat M-003111–04) and the negative control, consisting of a pool of four scramble siRNAs were from Thermo Fisher Scientific Dharmacon (NYSE:TMO).

    Techniques: Western Blot, Multiple Displacement Amplification, Transfection, MTT Assay, Incubation, Staining, FACS, Cell Culture

    DDR1 depletion or overexpression affects insulin and IGF-2 downstream signaling in human breast cancer cells (a) Insulin and IGF-2 signaling after DDR1 depletion. MCF-7, BT-474 and MDA-MB-157 breast cancer cells were transiently transfected with a pool of four scramble or of four siRNA oligos against DDR1. After 48h, cells were grown in medium containing 2.5% of CS-FCS for 24h and then stimulated with or without 10nM of insulin or IGF-2 for 5 minutes. Cells were then lysed and analyzed by SDS-PAGE and immunoblotted with the indicated primary antibodies. β-actin was used as control for protein loading. The top panels show a representative of three experiments. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization of each phosphoprotein against β-actin. (b) Insulin and IGF-2 signaling after DDR1 overexpression. MCF-7, BT-474 and MDA-MB-157 breast cancer cells were transiently transfected with plasmids encoding either the human DDR1 cDNA (pCMV6-DDR1) or the corresponding empty vector (pCMV6-EV). After 48h, cells were grown in medium containing 2.5% of CS-FCS for 24h and then stimulated with or without 10nM of insulin or IGF-2 for 5 minutes. The activation of downstream signaling was assessed as in (a) . Blots are representative of three independent experiments. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization of each phosphoproteins against β-actin. (a-b) NS, p > 0.05; *0.01

    Journal: Oncotarget

    Article Title: Discoidin domain receptor 1 modulates insulin receptor signaling and biological responses in breast cancer cells

    doi: 10.18632/oncotarget.18020

    Figure Lengend Snippet: DDR1 depletion or overexpression affects insulin and IGF-2 downstream signaling in human breast cancer cells (a) Insulin and IGF-2 signaling after DDR1 depletion. MCF-7, BT-474 and MDA-MB-157 breast cancer cells were transiently transfected with a pool of four scramble or of four siRNA oligos against DDR1. After 48h, cells were grown in medium containing 2.5% of CS-FCS for 24h and then stimulated with or without 10nM of insulin or IGF-2 for 5 minutes. Cells were then lysed and analyzed by SDS-PAGE and immunoblotted with the indicated primary antibodies. β-actin was used as control for protein loading. The top panels show a representative of three experiments. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization of each phosphoprotein against β-actin. (b) Insulin and IGF-2 signaling after DDR1 overexpression. MCF-7, BT-474 and MDA-MB-157 breast cancer cells were transiently transfected with plasmids encoding either the human DDR1 cDNA (pCMV6-DDR1) or the corresponding empty vector (pCMV6-EV). After 48h, cells were grown in medium containing 2.5% of CS-FCS for 24h and then stimulated with or without 10nM of insulin or IGF-2 for 5 minutes. The activation of downstream signaling was assessed as in (a) . Blots are representative of three independent experiments. The histograms represent the mean±SEM of densitometric analysis of three independent experiments after normalization of each phosphoproteins against β-actin. (a-b) NS, p > 0.05; *0.01

    Article Snippet: The specific silencer Select Pre-designed pool of four siRNA oligos for DDR1 (Human DDR1 siGENOME SMARTpool Cat M-003111–04) and the negative control, consisting of a pool of four scramble siRNAs were from Thermo Fisher Scientific Dharmacon (NYSE:TMO).

    Techniques: Over Expression, Multiple Displacement Amplification, Transfection, SDS Page, Plasmid Preparation, Activation Assay

    Induction of SASP by activating the cytoplasmic DNA sensing pathway. a Senescent TIG-3 cells induced by oncogenic Ras expression (lane 2–4) were transfected with previously validated siRNA oligos indicated at the top of the panel for twice at 2 day intervals. These cells were then subjected to western blotting using antibodies shown right ( a ), RT-qPCR analysis of SASP factor gene expression ( b ), analysis of intracellular ROS levels ( c ) or immunofluorescence staining for markers of DNA damage (γ-H2AX [red], phosphor-Ser/Thr ATM/ATR (pST/Q) substrate [green] and 40,6-diamidino-2-phenylindole [blue]) on day 4 ( d ). The representative data from three independent experiments are shown. Tubulin was used as a loading control ( a ). For all graphs, error bars indicate mean ± standard deviation (s.d.) of triplicate measurements. (* P

    Journal: Nature Communications

    Article Title: Downregulation of cytoplasmic DNases is implicated in cytoplasmic DNA accumulation and SASP in senescent cells

    doi: 10.1038/s41467-018-03555-8

    Figure Lengend Snippet: Induction of SASP by activating the cytoplasmic DNA sensing pathway. a Senescent TIG-3 cells induced by oncogenic Ras expression (lane 2–4) were transfected with previously validated siRNA oligos indicated at the top of the panel for twice at 2 day intervals. These cells were then subjected to western blotting using antibodies shown right ( a ), RT-qPCR analysis of SASP factor gene expression ( b ), analysis of intracellular ROS levels ( c ) or immunofluorescence staining for markers of DNA damage (γ-H2AX [red], phosphor-Ser/Thr ATM/ATR (pST/Q) substrate [green] and 40,6-diamidino-2-phenylindole [blue]) on day 4 ( d ). The representative data from three independent experiments are shown. Tubulin was used as a loading control ( a ). For all graphs, error bars indicate mean ± standard deviation (s.d.) of triplicate measurements. (* P

    Article Snippet: RNAi was performed by the transfection of siRNA oligos using the Lipofectamine™ RNAiMAX transfection reagent (Thermo Fisher Scientific), according to the manufacturer’s instructions.

    Techniques: Expressing, Transfection, Western Blot, Quantitative RT-PCR, Immunofluorescence, Staining, Standard Deviation

    The knockdown of cytoplasmic DNases activates the IFN-β pathway. a – c Pre-senescent TIG-3 cells were subjected to transfection with indicated siRNA oligos twice (at 2 day intervals). These cells were then subjected to western blotting using antibodies shown right ( a ), isolation of cytoplasmic fraction followed by qPCR analysis of chromosomal DNA ( b ) or qPCR analysis of SASP factor gene expression ( c ). Tubulin was used as a loading control ( a ). The representative data from three independent experiments are shown. For all graphs, error bars indicate mean ± standard deviation (s.d.) of triplicate measurements. (** P

    Journal: Nature Communications

    Article Title: Downregulation of cytoplasmic DNases is implicated in cytoplasmic DNA accumulation and SASP in senescent cells

    doi: 10.1038/s41467-018-03555-8

    Figure Lengend Snippet: The knockdown of cytoplasmic DNases activates the IFN-β pathway. a – c Pre-senescent TIG-3 cells were subjected to transfection with indicated siRNA oligos twice (at 2 day intervals). These cells were then subjected to western blotting using antibodies shown right ( a ), isolation of cytoplasmic fraction followed by qPCR analysis of chromosomal DNA ( b ) or qPCR analysis of SASP factor gene expression ( c ). Tubulin was used as a loading control ( a ). The representative data from three independent experiments are shown. For all graphs, error bars indicate mean ± standard deviation (s.d.) of triplicate measurements. (** P

    Article Snippet: RNAi was performed by the transfection of siRNA oligos using the Lipofectamine™ RNAiMAX transfection reagent (Thermo Fisher Scientific), according to the manufacturer’s instructions.

    Techniques: Transfection, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Expressing, Standard Deviation

    Loss of either ROCK1 or ROCK2 expression results in impaired cell migration. Confluent HaCaT-NSC (A,B), HaCaT-ROCK1-KD (C,D) and HaCaT-ROCK2-KD (E,F) monolayers were scratched with a pipette tip. Phase contrast images were taken immediately and 6 hours after wounding and representative phase-contrast images are shown (A–F). Relative wound closure was calculated for HaCaT-ROCK1-KD and HaCaT-ROCK2-KD cells (G) as well as SCC12f keratinocytes transiently transfected with siRNA directed against ROCK1 and ROCK2 (H). The means and standard errors of 3 separate experiments are shown and statistical significance calculated using Mann-Whitney test (*** p

    Journal: PLoS ONE

    Article Title: Differential Regulation of Adhesion Complex Turnover by ROCK1 and ROCK2

    doi: 10.1371/journal.pone.0031423

    Figure Lengend Snippet: Loss of either ROCK1 or ROCK2 expression results in impaired cell migration. Confluent HaCaT-NSC (A,B), HaCaT-ROCK1-KD (C,D) and HaCaT-ROCK2-KD (E,F) monolayers were scratched with a pipette tip. Phase contrast images were taken immediately and 6 hours after wounding and representative phase-contrast images are shown (A–F). Relative wound closure was calculated for HaCaT-ROCK1-KD and HaCaT-ROCK2-KD cells (G) as well as SCC12f keratinocytes transiently transfected with siRNA directed against ROCK1 and ROCK2 (H). The means and standard errors of 3 separate experiments are shown and statistical significance calculated using Mann-Whitney test (*** p

    Article Snippet: In some experiments SCC12f were transiently transfected with siRNA oligos targeting ROCK1 (J-003536-06-0020, J-003536-07-0020, Thermo Scientific, USA), ROCK2 (S102223746, S102223753, QIAGEN, UK) or NSC (S103650325, QIAGEN, UK) as described elsewhere .

    Techniques: Expressing, Migration, Transferring, Transfection, MANN-WHITNEY

    Stable and transient knock-down of ROCK1 and ROCK2 expression in two different keratinocyte cell lines. A: HaCaT keratinocytes stably expressing control (NSC), ROCK1 (ROCK1 KD) or ROCK2 (ROCK2 KD) shRNA vectors or B: SCC12f keratinocytes transiently transfected with NSC, ROCK1 or ROCK2 RNAi oligos were lysed and immunoblotted with antibodies against ROCK1, ROCK2 or tubulin as a loading control.

    Journal: PLoS ONE

    Article Title: Differential Regulation of Adhesion Complex Turnover by ROCK1 and ROCK2

    doi: 10.1371/journal.pone.0031423

    Figure Lengend Snippet: Stable and transient knock-down of ROCK1 and ROCK2 expression in two different keratinocyte cell lines. A: HaCaT keratinocytes stably expressing control (NSC), ROCK1 (ROCK1 KD) or ROCK2 (ROCK2 KD) shRNA vectors or B: SCC12f keratinocytes transiently transfected with NSC, ROCK1 or ROCK2 RNAi oligos were lysed and immunoblotted with antibodies against ROCK1, ROCK2 or tubulin as a loading control.

    Article Snippet: In some experiments SCC12f were transiently transfected with siRNA oligos targeting ROCK1 (J-003536-06-0020, J-003536-07-0020, Thermo Scientific, USA), ROCK2 (S102223746, S102223753, QIAGEN, UK) or NSC (S103650325, QIAGEN, UK) as described elsewhere .

    Techniques: Expressing, Stable Transfection, shRNA, Transfection