Diagnostic Assay:Article Title: Loss-of-Function Mutations in the WNT Co-receptor LRP6 Cause Autosomal-Dominant Oligodontia
Article Snippet: Informed consent for WES as a part of the diagnostic process (approved by the medical ethical committee of UMC Utrecht) was obtained for all subjects included in this study. .. In brief, with a Covaris S2 sonicator, we sheared 1 ug of purified gDNA (QIAGEN) into 100–500 bp fragments that we then blunt ended, 5′ phosphorylated, and A-tailed by using KapaBiosystems reagents.
Amplification:Article Title: Loss-of-Function Mutations in the WNT Co-receptor LRP6 Cause Autosomal-Dominant Oligodontia
Article Snippet: In brief, with a Covaris S2 sonicator, we sheared 1 ug of purified gDNA (QIAGEN) into 100–500 bp fragments that we then blunt ended, 5′ phosphorylated, and A-tailed by using KapaBiosystems reagents. .. Next, we ligated adaptors containing the Illumina barcode sequences to each sample and amplified for seven PCR cycles.
Article Title: Age related changes in microglial phenotype vary between CNS regions: Grey versus white matter differences
Article Snippet: Total RNA was extracted from brain tissue using RNeasy mini kits (Qiagen, Crawley, UK) and treated with DNAse I to remove any contaminating gDNA (Qiagen). cDNA was synthesised using reverse transcription reagents from Applied Biosystems (Warrington, UK). .. SYBR green super mix (BioRad, Hemel Hempstead, UK) was used to detect amplification of primer products.
Article Title: Expression levels of hnRNP K and p21WAF1/CIP1 are associated with resistance to radiochemotherapy independent of p53 pathway activation in rectal adenocarcinoma
Article Snippet: In brief, genomic DNA was quantified and target enrichment was processed with the GeneRead DNAseq Panel PCR V2 kit (Qiagen GmbH) applying 40 ng genomic DNA quantified with a Qubit® dsDNA HS kit and a Qubit® 2.0 Fluorometer (Life Technologies; Thermo Fisher Scientific, Inc.). .. Amplification of adapter-ligated DNA was conducted using NEXTflex primers (Bioo Scientific) and the HiFi PCR Master mix (GeneRead DNA I Amp kit; Qiagen GmbH) with an initial activation step of 98°C (2 min), five cycles of denaturation at 98°C (20 sec), annealing at 60°C (30 sec) and extension at 72°C (30 sec), followed by a final extension at 72°C (10 min).
SYBR Green Assay:Article Title: Age related changes in microglial phenotype vary between CNS regions: Grey versus white matter differences
Article Snippet: Total RNA was extracted from brain tissue using RNeasy mini kits (Qiagen, Crawley, UK) and treated with DNAse I to remove any contaminating gDNA (Qiagen). cDNA was synthesised using reverse transcription reagents from Applied Biosystems (Warrington, UK). .. SYBR green super mix (BioRad, Hemel Hempstead, UK) was used to detect amplification of primer products.
Random Hexamer Labeling:Article Title: Calcium transport in bovine rumen epithelium as affected by luminal Ca concentrations and Ca sources
Article Snippet: Isolation of RNA and reverse transcription The kit for isolation of RNA contained special spin columns to eliminate gDNA (Qiagen, Hilden, Germany). .. Concentration and quality of RNA were determined by UV absorbance and 200 ng were reverse transcribed using random hexamer primers and TaqMan-Reverse Transcription Reagents (Applied Biosystems, Darmstadt, Germany).
Modification:Article Title: Integration of Human Immunodeficiency Virus Type 1 in Untreated Infection Occurs Preferentially within Genes
Article Snippet: Genomic DNA was isolated from PBMCs with a QIAamp DNA blood minikit (QIAGEN); genomic DNA was isolated from brains, lymph nodes, and spleens with a genomic DNA purification kit (Gentra Systems) according to the manufacturer's protocol. .. Our procedure to identify integration sites is a modification of that previously described ( ).
Hybridization:Article Title: Virulence differences in Toxoplasma mediated by amplification of a family of polymorphic pseudokinases
Article Snippet: Freshly egressed parasites grown in HFF cells were harvested as described above, and gDNA was purified using the DNAeasy kit (Qiagen) with the addition of RNase Cocktail (Ambion). gDNA was sheared by nebulization (Invitrogen) for 3 min at ∼ 40 psi, precipitated, washed in 70% ethanol, and resuspended in water. .. Hybridization intensities were processed and normalized with the software package R ( ) using the following R library functions: bg.correct = rma and normalize = quantiles.
Ligation:Article Title: Expression levels of hnRNP K and p21WAF1/CIP1 are associated with resistance to radiochemotherapy independent of p53 pathway activation in rectal adenocarcinoma
Article Snippet: In brief, genomic DNA was quantified and target enrichment was processed with the GeneRead DNAseq Panel PCR V2 kit (Qiagen GmbH) applying 40 ng genomic DNA quantified with a Qubit® dsDNA HS kit and a Qubit® 2.0 Fluorometer (Life Technologies; Thermo Fisher Scientific, Inc.). .. End repair, A-addition and ligation to NEXTflex-96 DNA barcodes (Bioo Scientific, Austin, TX, USA) were performed using the GeneRead DNA Library I Core kit (Qiagen GmbH).
Article Title: Integration of Human Immunodeficiency Virus Type 1 in Untreated Infection Occurs Preferentially within Genes
Article Snippet: Genomic DNA was isolated from PBMCs with a QIAamp DNA blood minikit (QIAGEN); genomic DNA was isolated from brains, lymph nodes, and spleens with a genomic DNA purification kit (Gentra Systems) according to the manufacturer's protocol. .. Genomic DNA preparations were digested with PstI or combinations of SpeI, XbaI, and NheI (SpeI, XbaI, and NheI have compatible ends for ligation).
Cell Culture:Article Title: Concerted copy number variation balances ribosomal DNA dosage in human and mouse genomes
Article Snippet: Paragraph title: Cell Culture and BPA Treatment. ... Cells were harvested 24 h after the treatment with BPA, and gDNA was isolated using DNeasy Blood and Tissue kit following the method described by the manufacturer (Qiagen). gDNA samples were treated with RNase A, and concentration was estimated with NanoDrop (Thermo Scientific).
Generated:Article Title: Long-term Correction of Very Long-chain Acyl-CoA Dehydrogenase Deficiency in Mice Using AAV9 Gene Therapy
Article Snippet: All animal procedures were approved by University of Massachusetts Medical School Institutional Animal Care and Use Committee as well as University of Florida Institutional Animal Care and Use Committee in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care International specifications. rAAV vectors . rAAV9/2 pseudotyped vectors were generated to express human VLCAD under the transcriptional control of the cytomegalovirus enhancer/chicken β-actin promoter. rAAV vectors were produced, purified and tittered as previously described (UMMS Gene Therapy Center, Worcester, MA). .. Animals were sacrificed at indicated time points and tissues were harvested in a manner to avoid cross-contamination, snap frozen in liquid nitrogen and stored at −80 °C. gDNA was extracted using a DNeasy blood and tissue kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. gDNA concentrations were determined using an Eppendorf Biophotometer (Eppendorf, Hamburg, Germany). rAAV genome copies in the gDNA from heart, liver, tibialis anterior, and brown fat were quantified by quantitative-PCR with an ABI 7900 HT sequence detection system (Applied Biosystems, Carlsbad, CA) according to the manufacturer's instructions and results were analyzed using SDS 2.3 software.
Polymerase Chain Reaction:Article Title: Loss-of-Function Mutations in the WNT Co-receptor LRP6 Cause Autosomal-Dominant Oligodontia
Article Snippet: In brief, with a Covaris S2 sonicator, we sheared 1 ug of purified gDNA (QIAGEN) into 100–500 bp fragments that we then blunt ended, 5′ phosphorylated, and A-tailed by using KapaBiosystems reagents. .. Next, we ligated adaptors containing the Illumina barcode sequences to each sample and amplified for seven PCR cycles.
Article Title: Expression levels of hnRNP K and p21WAF1/CIP1 are associated with resistance to radiochemotherapy independent of p53 pathway activation in rectal adenocarcinoma
Article Snippet: .. In brief, genomic DNA was quantified and target enrichment was processed with the GeneRead DNAseq Panel PCR V2 kit (Qiagen GmbH) applying 40 ng genomic DNA quantified with a Qubit® dsDNA HS kit and a Qubit® 2.0 Fluorometer (Life Technologies; Thermo Fisher Scientific, Inc.). .. An initial activation step (95°C; 15 min) was followed by 25 cycles of denaturing at 95°C (15 sec) and annealing at 60°C (4 min), followed by extension at 72°C for 10 min. All purification and size selection steps were performed using Agencourt AMPure XP magnetic beads (Beckman Coulter, Inc., Brea, CA, USA).
Sonication:Article Title: Epigenetic Enhancer Marks and Transcription Factor Binding Influence Vκ Gene Rearrangement in Pre-B Cells and Pro-B Cells
Article Snippet: .. gDNA and cDNA library preparation for Igκ repertoire deep sequencing Sorted cells were split into two fractions: one for genomic DNA (gDNA) extraction (DNeasy, Qiagen) and the other for RNA extraction (RNeasy Plus, Qiagen). gDNA libraries were prepared as recently described with several modifications ( ) (Figure ). gDNA was sonicated to a range of 500–1,000 bp using a Bioruptor sonicator (Diagenode). ..
Software:Article Title: Long-term Correction of Very Long-chain Acyl-CoA Dehydrogenase Deficiency in Mice Using AAV9 Gene Therapy
Article Snippet: .. Animals were sacrificed at indicated time points and tissues were harvested in a manner to avoid cross-contamination, snap frozen in liquid nitrogen and stored at −80 °C. gDNA was extracted using a DNeasy blood and tissue kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. gDNA concentrations were determined using an Eppendorf Biophotometer (Eppendorf, Hamburg, Germany). rAAV genome copies in the gDNA from heart, liver, tibialis anterior, and brown fat were quantified by quantitative-PCR with an ABI 7900 HT sequence detection system (Applied Biosystems, Carlsbad, CA) according to the manufacturer's instructions and results were analyzed using SDS 2.3 software. .. Briefly, primers and probe were designed to amplify SV40 poly-A tail of the rAAV9-CBA-VLCAD vector.
Article Title: Virulence differences in Toxoplasma mediated by amplification of a family of polymorphic pseudokinases
Article Snippet: Freshly egressed parasites grown in HFF cells were harvested as described above, and gDNA was purified using the DNAeasy kit (Qiagen) with the addition of RNase Cocktail (Ambion). gDNA was sheared by nebulization (Invitrogen) for 3 min at ∼ 40 psi, precipitated, washed in 70% ethanol, and resuspended in water. .. Hybridization intensities were processed and normalized with the software package R ( ) using the following R library functions: bg.correct = rma and normalize = quantiles.
DNA Extraction:Article Title: Designing a SCAR molecular marker for monitoring Trichoderma cf. harzianum in experimental communities * in experimental communities * #
Article Snippet: .. Destructive samplings were carried out 0, 3, 7, and 10 d after inoculation with T . cf. harzianum BpT10a and the gDNA was extracted with the PowerSoil™ DNA extraction kit (MoBio Laboratories Inc.). gDNA was quantified using the Take3™ Epoch MicroVolume Plate reader (Biotek) and 1 ng was used as the template for the qPCR reactions, which were performed under the same conditions above mentioned. .. The fungal gDNA-mix standard curve was used to calculate the copy number of the SCAR marker in each community.
Article Title: Genomic Comparison of Campylobacter spp. and Their Potential for Zoonotic Transmission between Birds, Primates, and Livestock
Article Snippet: Paragraph title: DNA extraction, library preparation, and next-generation sequencing. ... Briefly, bacteria were lysed with an enzyme preparation, vortexed, and processed according to the manufacturer's recommendations to obtain purified gDNA (Qiagen).
Article Title: Long-term Correction of Very Long-chain Acyl-CoA Dehydrogenase Deficiency in Mice Using AAV9 Gene Therapy
Article Snippet: Genomic DNA extraction and quantitative-PCR . .. Animals were sacrificed at indicated time points and tissues were harvested in a manner to avoid cross-contamination, snap frozen in liquid nitrogen and stored at −80 °C. gDNA was extracted using a DNeasy blood and tissue kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. gDNA concentrations were determined using an Eppendorf Biophotometer (Eppendorf, Hamburg, Germany). rAAV genome copies in the gDNA from heart, liver, tibialis anterior, and brown fat were quantified by quantitative-PCR with an ABI 7900 HT sequence detection system (Applied Biosystems, Carlsbad, CA) according to the manufacturer's instructions and results were analyzed using SDS 2.3 software.
Magnetic Beads:Article Title: Expression levels of hnRNP K and p21WAF1/CIP1 are associated with resistance to radiochemotherapy independent of p53 pathway activation in rectal adenocarcinoma
Article Snippet: In brief, genomic DNA was quantified and target enrichment was processed with the GeneRead DNAseq Panel PCR V2 kit (Qiagen GmbH) applying 40 ng genomic DNA quantified with a Qubit® dsDNA HS kit and a Qubit® 2.0 Fluorometer (Life Technologies; Thermo Fisher Scientific, Inc.). .. An initial activation step (95°C; 15 min) was followed by 25 cycles of denaturing at 95°C (15 sec) and annealing at 60°C (4 min), followed by extension at 72°C for 10 min. All purification and size selection steps were performed using Agencourt AMPure XP magnetic beads (Beckman Coulter, Inc., Brea, CA, USA).
Mutagenesis:Article Title: Expression levels of hnRNP K and p21WAF1/CIP1 are associated with resistance to radiochemotherapy independent of p53 pathway activation in rectal adenocarcinoma
Article Snippet: Next-generation sequencing In addition to KRAS mutation analysis by Sanger sequencing, samples with low tumor cell content were analyzed by more sensitive next-generation sequencing with the use of a Custom GeneRead DNASeq Panel (Qiagen GmbH, Hilden, Germany) consisting of 189 amplicons for mutation analysis of 19 cancer-related genes, (NRAS, H3F3A, RET, KRAS, AKT1, TP53, ERBB2, H3F3B, GNA11, ALK, GNAS, CTNNB1, PIK3CA, PDGFRA, KIT, EGFR, MET, BRAF, GNAQ ), according to the manufacturer’s protocol. .. In brief, genomic DNA was quantified and target enrichment was processed with the GeneRead DNAseq Panel PCR V2 kit (Qiagen GmbH) applying 40 ng genomic DNA quantified with a Qubit® dsDNA HS kit and a Qubit® 2.0 Fluorometer (Life Technologies; Thermo Fisher Scientific, Inc.).
Isolation:Article Title: Calcium transport in bovine rumen epithelium as affected by luminal Ca concentrations and Ca sources
Article Snippet: .. Isolation of RNA and reverse transcription The kit for isolation of RNA contained special spin columns to eliminate gDNA (Qiagen, Hilden, Germany). ..
Article Title: Genomic Comparison of Campylobacter spp. and Their Potential for Zoonotic Transmission between Birds, Primates, and Livestock
Article Snippet: DNA was extracted using whole-genome isolation kits (Qiagen, Valencia, CA, USA) with previously published modifications ( ). .. Briefly, bacteria were lysed with an enzyme preparation, vortexed, and processed according to the manufacturer's recommendations to obtain purified gDNA (Qiagen).
Article Title: Integration of Human Immunodeficiency Virus Type 1 in Untreated Infection Occurs Preferentially within Genes
Article Snippet: .. Genomic DNA was isolated from PBMCs with a QIAamp DNA blood minikit (QIAGEN); genomic DNA was isolated from brains, lymph nodes, and spleens with a genomic DNA purification kit (Gentra Systems) according to the manufacturer's protocol. .. Our procedure to identify integration sites is a modification of that previously described ( ).
Article Title: Concerted copy number variation balances ribosomal DNA dosage in human and mouse genomes
Article Snippet: .. Cells were harvested 24 h after the treatment with BPA, and gDNA was isolated using DNeasy Blood and Tissue kit following the method described by the manufacturer (Qiagen). gDNA samples were treated with RNase A, and concentration was estimated with NanoDrop (Thermo Scientific). ..
Article Title: Developmental Expression of CYP2B6: A Comprehensive Analysis of mRNA Expression, Protein Content and Bupropion Hydroxylase Activity and the Impact of Genetic Variation
Article Snippet: .. gDNA was isolated from ∼25 mg of tissue using a DNeasy Tissue Kit (Qiagen) or an Illustra Tissue and Cells Genomic Prep Kit (GE Healthcare). gDNA was assessed by agarose gel electrophoresis for quality; concentration was measured using a NanoDrop 1000 spectrophotometer (Thermo-Scientific, Waltham, MA). gDNA samples were diluted to 15 ng/ µ l. Genotyping was carried out on an Applied Biosystems 7900 HT Real-Time PCR System using predesigned Life Technologies TaqMan assays for CYP2B6 rs34223104 (−82T > C); rs3745274 (516G > T, Q172H), rs28399499 (983T > C, I328T), and rs3211371 (1459C > T R487C). .. Reactions were scaled to 8 µ l and contained 4 µ l KAPA PROBE FAST Universal 2X qPCR Master Mix (KAPA Biosystems, Woburn, MA), and 0.2 or 0.4 µ l TaqMan assay mix, respectively, depending on whether it was a 20× or 40× mix, and 12–18 ng gDNA.
Multiplex Assay:Article Title: Epigenetic Enhancer Marks and Transcription Factor Binding Influence Vκ Gene Rearrangement in Pre-B Cells and Pro-B Cells
Article Snippet: gDNA and cDNA library preparation for Igκ repertoire deep sequencing Sorted cells were split into two fractions: one for genomic DNA (gDNA) extraction (DNeasy, Qiagen) and the other for RNA extraction (RNeasy Plus, Qiagen). gDNA libraries were prepared as recently described with several modifications ( ) (Figure ). gDNA was sonicated to a range of 500–1,000 bp using a Bioruptor sonicator (Diagenode). .. Library barcoding was performed using NEBNext Multiplex Oligos for Illumina (E7600S).
Labeling:Article Title: Virulence differences in Toxoplasma mediated by amplification of a family of polymorphic pseudokinases
Article Snippet: Freshly egressed parasites grown in HFF cells were harvested as described above, and gDNA was purified using the DNAeasy kit (Qiagen) with the addition of RNase Cocktail (Ambion). gDNA was sheared by nebulization (Invitrogen) for 3 min at ∼ 40 psi, precipitated, washed in 70% ethanol, and resuspended in water. .. The BioPrime Labeling System (Invitrogen) was used to label 600 ng gDNA per reaction for 2 h at 37 °C.
Purification:Article Title: Loss-of-Function Mutations in the WNT Co-receptor LRP6 Cause Autosomal-Dominant Oligodontia
Article Snippet: .. In brief, with a Covaris S2 sonicator, we sheared 1 ug of purified gDNA (QIAGEN) into 100–500 bp fragments that we then blunt ended, 5′ phosphorylated, and A-tailed by using KapaBiosystems reagents. .. Next, we ligated adaptors containing the Illumina barcode sequences to each sample and amplified for seven PCR cycles.
Article Title: Expression levels of hnRNP K and p21WAF1/CIP1 are associated with resistance to radiochemotherapy independent of p53 pathway activation in rectal adenocarcinoma
Article Snippet: In brief, genomic DNA was quantified and target enrichment was processed with the GeneRead DNAseq Panel PCR V2 kit (Qiagen GmbH) applying 40 ng genomic DNA quantified with a Qubit® dsDNA HS kit and a Qubit® 2.0 Fluorometer (Life Technologies; Thermo Fisher Scientific, Inc.). .. An initial activation step (95°C; 15 min) was followed by 25 cycles of denaturing at 95°C (15 sec) and annealing at 60°C (4 min), followed by extension at 72°C for 10 min. All purification and size selection steps were performed using Agencourt AMPure XP magnetic beads (Beckman Coulter, Inc., Brea, CA, USA).
Article Title: Genomic Comparison of Campylobacter spp. and Their Potential for Zoonotic Transmission between Birds, Primates, and Livestock
Article Snippet: .. Briefly, bacteria were lysed with an enzyme preparation, vortexed, and processed according to the manufacturer's recommendations to obtain purified gDNA (Qiagen). .. Genomic DNA purity and integrity were assessed on the Agilent 2200 TapeStation with the genomic DNA ScreenTape (Agilent Technologies, Santa Clara, CA, USA) as previously described ( , , ).
Article Title: Long-term Correction of Very Long-chain Acyl-CoA Dehydrogenase Deficiency in Mice Using AAV9 Gene Therapy
Article Snippet: All animal procedures were approved by University of Massachusetts Medical School Institutional Animal Care and Use Committee as well as University of Florida Institutional Animal Care and Use Committee in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care International specifications. rAAV vectors . rAAV9/2 pseudotyped vectors were generated to express human VLCAD under the transcriptional control of the cytomegalovirus enhancer/chicken β-actin promoter. rAAV vectors were produced, purified and tittered as previously described (UMMS Gene Therapy Center, Worcester, MA). .. Animals were sacrificed at indicated time points and tissues were harvested in a manner to avoid cross-contamination, snap frozen in liquid nitrogen and stored at −80 °C. gDNA was extracted using a DNeasy blood and tissue kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. gDNA concentrations were determined using an Eppendorf Biophotometer (Eppendorf, Hamburg, Germany). rAAV genome copies in the gDNA from heart, liver, tibialis anterior, and brown fat were quantified by quantitative-PCR with an ABI 7900 HT sequence detection system (Applied Biosystems, Carlsbad, CA) according to the manufacturer's instructions and results were analyzed using SDS 2.3 software.
Article Title: Virulence differences in Toxoplasma mediated by amplification of a family of polymorphic pseudokinases
Article Snippet: .. Freshly egressed parasites grown in HFF cells were harvested as described above, and gDNA was purified using the DNAeasy kit (Qiagen) with the addition of RNase Cocktail (Ambion). gDNA was sheared by nebulization (Invitrogen) for 3 min at ∼ 40 psi, precipitated, washed in 70% ethanol, and resuspended in water. .. The BioPrime Labeling System (Invitrogen) was used to label 600 ng gDNA per reaction for 2 h at 37 °C.
Sequencing:Article Title: Epigenetic Enhancer Marks and Transcription Factor Binding Influence Vκ Gene Rearrangement in Pre-B Cells and Pro-B Cells
Article Snippet: .. gDNA and cDNA library preparation for Igκ repertoire deep sequencing Sorted cells were split into two fractions: one for genomic DNA (gDNA) extraction (DNeasy, Qiagen) and the other for RNA extraction (RNeasy Plus, Qiagen). gDNA libraries were prepared as recently described with several modifications ( ) (Figure ). gDNA was sonicated to a range of 500–1,000 bp using a Bioruptor sonicator (Diagenode). ..
Article Title: Loss-of-Function Mutations in the WNT Co-receptor LRP6 Cause Autosomal-Dominant Oligodontia
Article Snippet: Informed consent for Sanger sequencing analysis was obtained for all subjects included in this study. .. In brief, with a Covaris S2 sonicator, we sheared 1 ug of purified gDNA (QIAGEN) into 100–500 bp fragments that we then blunt ended, 5′ phosphorylated, and A-tailed by using KapaBiosystems reagents.
Article Title: Expression levels of hnRNP K and p21WAF1/CIP1 are associated with resistance to radiochemotherapy independent of p53 pathway activation in rectal adenocarcinoma
Article Snippet: Next-generation sequencing In addition to KRAS mutation analysis by Sanger sequencing, samples with low tumor cell content were analyzed by more sensitive next-generation sequencing with the use of a Custom GeneRead DNASeq Panel (Qiagen GmbH, Hilden, Germany) consisting of 189 amplicons for mutation analysis of 19 cancer-related genes, (NRAS, H3F3A, RET, KRAS, AKT1, TP53, ERBB2, H3F3B, GNA11, ALK, GNAS, CTNNB1, PIK3CA, PDGFRA, KIT, EGFR, MET, BRAF, GNAQ ), according to the manufacturer’s protocol. .. In brief, genomic DNA was quantified and target enrichment was processed with the GeneRead DNAseq Panel PCR V2 kit (Qiagen GmbH) applying 40 ng genomic DNA quantified with a Qubit® dsDNA HS kit and a Qubit® 2.0 Fluorometer (Life Technologies; Thermo Fisher Scientific, Inc.).
Article Title: Long-term Correction of Very Long-chain Acyl-CoA Dehydrogenase Deficiency in Mice Using AAV9 Gene Therapy
Article Snippet: .. Animals were sacrificed at indicated time points and tissues were harvested in a manner to avoid cross-contamination, snap frozen in liquid nitrogen and stored at −80 °C. gDNA was extracted using a DNeasy blood and tissue kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. gDNA concentrations were determined using an Eppendorf Biophotometer (Eppendorf, Hamburg, Germany). rAAV genome copies in the gDNA from heart, liver, tibialis anterior, and brown fat were quantified by quantitative-PCR with an ABI 7900 HT sequence detection system (Applied Biosystems, Carlsbad, CA) according to the manufacturer's instructions and results were analyzed using SDS 2.3 software. .. Briefly, primers and probe were designed to amplify SV40 poly-A tail of the rAAV9-CBA-VLCAD vector.
cDNA Library Assay:Article Title: Epigenetic Enhancer Marks and Transcription Factor Binding Influence Vκ Gene Rearrangement in Pre-B Cells and Pro-B Cells
Article Snippet: .. gDNA and cDNA library preparation for Igκ repertoire deep sequencing Sorted cells were split into two fractions: one for genomic DNA (gDNA) extraction (DNeasy, Qiagen) and the other for RNA extraction (RNeasy Plus, Qiagen). gDNA libraries were prepared as recently described with several modifications ( ) (Figure ). gDNA was sonicated to a range of 500–1,000 bp using a Bioruptor sonicator (Diagenode). ..
Mouse Assay:Article Title: Long-term Correction of Very Long-chain Acyl-CoA Dehydrogenase Deficiency in Mice Using AAV9 Gene Therapy
Article Snippet: For cold fast challenge, after the 18-hour fast, mice were singly housed in minimal bedding cages and placed in a 4 °C cold room for 150 minutes or until rectal temperatures were below 20 °C. .. Animals were sacrificed at indicated time points and tissues were harvested in a manner to avoid cross-contamination, snap frozen in liquid nitrogen and stored at −80 °C. gDNA was extracted using a DNeasy blood and tissue kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. gDNA concentrations were determined using an Eppendorf Biophotometer (Eppendorf, Hamburg, Germany). rAAV genome copies in the gDNA from heart, liver, tibialis anterior, and brown fat were quantified by quantitative-PCR with an ABI 7900 HT sequence detection system (Applied Biosystems, Carlsbad, CA) according to the manufacturer's instructions and results were analyzed using SDS 2.3 software.
RNA Extraction:Article Title: Epigenetic Enhancer Marks and Transcription Factor Binding Influence Vκ Gene Rearrangement in Pre-B Cells and Pro-B Cells
Article Snippet: .. gDNA and cDNA library preparation for Igκ repertoire deep sequencing Sorted cells were split into two fractions: one for genomic DNA (gDNA) extraction (DNeasy, Qiagen) and the other for RNA extraction (RNeasy Plus, Qiagen). gDNA libraries were prepared as recently described with several modifications ( ) (Figure ). gDNA was sonicated to a range of 500–1,000 bp using a Bioruptor sonicator (Diagenode). ..
Plasmid Preparation:Article Title: Long-term Correction of Very Long-chain Acyl-CoA Dehydrogenase Deficiency in Mice Using AAV9 Gene Therapy
Article Snippet: Animals were sacrificed at indicated time points and tissues were harvested in a manner to avoid cross-contamination, snap frozen in liquid nitrogen and stored at −80 °C. gDNA was extracted using a DNeasy blood and tissue kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. gDNA concentrations were determined using an Eppendorf Biophotometer (Eppendorf, Hamburg, Germany). rAAV genome copies in the gDNA from heart, liver, tibialis anterior, and brown fat were quantified by quantitative-PCR with an ABI 7900 HT sequence detection system (Applied Biosystems, Carlsbad, CA) according to the manufacturer's instructions and results were analyzed using SDS 2.3 software. .. Briefly, primers and probe were designed to amplify SV40 poly-A tail of the rAAV9-CBA-VLCAD vector.
TaqMan Assay:Article Title: Developmental Expression of CYP2B6: A Comprehensive Analysis of mRNA Expression, Protein Content and Bupropion Hydroxylase Activity and the Impact of Genetic Variation
Article Snippet: gDNA was isolated from ∼25 mg of tissue using a DNeasy Tissue Kit (Qiagen) or an Illustra Tissue and Cells Genomic Prep Kit (GE Healthcare). gDNA was assessed by agarose gel electrophoresis for quality; concentration was measured using a NanoDrop 1000 spectrophotometer (Thermo-Scientific, Waltham, MA). gDNA samples were diluted to 15 ng/ µ l. Genotyping was carried out on an Applied Biosystems 7900 HT Real-Time PCR System using predesigned Life Technologies TaqMan assays for CYP2B6 rs34223104 (−82T > C); rs3745274 (516G > T, Q172H), rs28399499 (983T > C, I328T), and rs3211371 (1459C > T R487C). .. Reactions were scaled to 8 µ l and contained 4 µ l KAPA PROBE FAST Universal 2X qPCR Master Mix (KAPA Biosystems, Woburn, MA), and 0.2 or 0.4 µ l TaqMan assay mix, respectively, depending on whether it was a 20× or 40× mix, and 12–18 ng gDNA.
Real-time Polymerase Chain Reaction:Article Title: Age related changes in microglial phenotype vary between CNS regions: Grey versus white matter differences
Article Snippet: Paragraph title: Quantitative PCR ... Total RNA was extracted from brain tissue using RNeasy mini kits (Qiagen, Crawley, UK) and treated with DNAse I to remove any contaminating gDNA (Qiagen). cDNA was synthesised using reverse transcription reagents from Applied Biosystems (Warrington, UK).
Article Title: Designing a SCAR molecular marker for monitoring Trichoderma cf. harzianum in experimental communities * in experimental communities * #
Article Snippet: .. Destructive samplings were carried out 0, 3, 7, and 10 d after inoculation with T . cf. harzianum BpT10a and the gDNA was extracted with the PowerSoil™ DNA extraction kit (MoBio Laboratories Inc.). gDNA was quantified using the Take3™ Epoch MicroVolume Plate reader (Biotek) and 1 ng was used as the template for the qPCR reactions, which were performed under the same conditions above mentioned. .. The fungal gDNA-mix standard curve was used to calculate the copy number of the SCAR marker in each community.
Article Title: Long-term Correction of Very Long-chain Acyl-CoA Dehydrogenase Deficiency in Mice Using AAV9 Gene Therapy
Article Snippet: .. Animals were sacrificed at indicated time points and tissues were harvested in a manner to avoid cross-contamination, snap frozen in liquid nitrogen and stored at −80 °C. gDNA was extracted using a DNeasy blood and tissue kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. gDNA concentrations were determined using an Eppendorf Biophotometer (Eppendorf, Hamburg, Germany). rAAV genome copies in the gDNA from heart, liver, tibialis anterior, and brown fat were quantified by quantitative-PCR with an ABI 7900 HT sequence detection system (Applied Biosystems, Carlsbad, CA) according to the manufacturer's instructions and results were analyzed using SDS 2.3 software. .. Briefly, primers and probe were designed to amplify SV40 poly-A tail of the rAAV9-CBA-VLCAD vector.
Article Title: Developmental Expression of CYP2B6: A Comprehensive Analysis of mRNA Expression, Protein Content and Bupropion Hydroxylase Activity and the Impact of Genetic Variation
Article Snippet: .. gDNA was isolated from ∼25 mg of tissue using a DNeasy Tissue Kit (Qiagen) or an Illustra Tissue and Cells Genomic Prep Kit (GE Healthcare). gDNA was assessed by agarose gel electrophoresis for quality; concentration was measured using a NanoDrop 1000 spectrophotometer (Thermo-Scientific, Waltham, MA). gDNA samples were diluted to 15 ng/ µ l. Genotyping was carried out on an Applied Biosystems 7900 HT Real-Time PCR System using predesigned Life Technologies TaqMan assays for CYP2B6 rs34223104 (−82T > C); rs3745274 (516G > T, Q172H), rs28399499 (983T > C, I328T), and rs3211371 (1459C > T R487C). .. Reactions were scaled to 8 µ l and contained 4 µ l KAPA PROBE FAST Universal 2X qPCR Master Mix (KAPA Biosystems, Woburn, MA), and 0.2 or 0.4 µ l TaqMan assay mix, respectively, depending on whether it was a 20× or 40× mix, and 12–18 ng gDNA.
Functional Assay:Article Title: Virulence differences in Toxoplasma mediated by amplification of a family of polymorphic pseudokinases
Article Snippet: Freshly egressed parasites grown in HFF cells were harvested as described above, and gDNA was purified using the DNAeasy kit (Qiagen) with the addition of RNase Cocktail (Ambion). gDNA was sheared by nebulization (Invitrogen) for 3 min at ∼ 40 psi, precipitated, washed in 70% ethanol, and resuspended in water. .. A total of 5 μg labeled DNA was hybridized to Affymetrix T. gondii GeneChips ( ) performed at the Functional Genomics Core Facility at Montana State University ( ).
Selection:Article Title: Expression levels of hnRNP K and p21WAF1/CIP1 are associated with resistance to radiochemotherapy independent of p53 pathway activation in rectal adenocarcinoma
Article Snippet: In brief, genomic DNA was quantified and target enrichment was processed with the GeneRead DNAseq Panel PCR V2 kit (Qiagen GmbH) applying 40 ng genomic DNA quantified with a Qubit® dsDNA HS kit and a Qubit® 2.0 Fluorometer (Life Technologies; Thermo Fisher Scientific, Inc.). .. An initial activation step (95°C; 15 min) was followed by 25 cycles of denaturing at 95°C (15 sec) and annealing at 60°C (4 min), followed by extension at 72°C for 10 min. All purification and size selection steps were performed using Agencourt AMPure XP magnetic beads (Beckman Coulter, Inc., Brea, CA, USA).
Agarose Gel Electrophoresis:Article Title: Developmental Expression of CYP2B6: A Comprehensive Analysis of mRNA Expression, Protein Content and Bupropion Hydroxylase Activity and the Impact of Genetic Variation
Article Snippet: .. gDNA was isolated from ∼25 mg of tissue using a DNeasy Tissue Kit (Qiagen) or an Illustra Tissue and Cells Genomic Prep Kit (GE Healthcare). gDNA was assessed by agarose gel electrophoresis for quality; concentration was measured using a NanoDrop 1000 spectrophotometer (Thermo-Scientific, Waltham, MA). gDNA samples were diluted to 15 ng/ µ l. Genotyping was carried out on an Applied Biosystems 7900 HT Real-Time PCR System using predesigned Life Technologies TaqMan assays for CYP2B6 rs34223104 (−82T > C); rs3745274 (516G > T, Q172H), rs28399499 (983T > C, I328T), and rs3211371 (1459C > T R487C). .. Reactions were scaled to 8 µ l and contained 4 µ l KAPA PROBE FAST Universal 2X qPCR Master Mix (KAPA Biosystems, Woburn, MA), and 0.2 or 0.4 µ l TaqMan assay mix, respectively, depending on whether it was a 20× or 40× mix, and 12–18 ng gDNA.
Size-exclusion Chromatography:Article Title: Expression levels of hnRNP K and p21WAF1/CIP1 are associated with resistance to radiochemotherapy independent of p53 pathway activation in rectal adenocarcinoma
Article Snippet: In brief, genomic DNA was quantified and target enrichment was processed with the GeneRead DNAseq Panel PCR V2 kit (Qiagen GmbH) applying 40 ng genomic DNA quantified with a Qubit® dsDNA HS kit and a Qubit® 2.0 Fluorometer (Life Technologies; Thermo Fisher Scientific, Inc.). .. An initial activation step (95°C; 15 min) was followed by 25 cycles of denaturing at 95°C (15 sec) and annealing at 60°C (4 min), followed by extension at 72°C for 10 min. All purification and size selection steps were performed using Agencourt AMPure XP magnetic beads (Beckman Coulter, Inc., Brea, CA, USA).
Next-Generation Sequencing:Article Title: Expression levels of hnRNP K and p21WAF1/CIP1 are associated with resistance to radiochemotherapy independent of p53 pathway activation in rectal adenocarcinoma
Article Snippet: Paragraph title: Next-generation sequencing ... In brief, genomic DNA was quantified and target enrichment was processed with the GeneRead DNAseq Panel PCR V2 kit (Qiagen GmbH) applying 40 ng genomic DNA quantified with a Qubit® dsDNA HS kit and a Qubit® 2.0 Fluorometer (Life Technologies; Thermo Fisher Scientific, Inc.).
Article Title: Genomic Comparison of Campylobacter spp. and Their Potential for Zoonotic Transmission between Birds, Primates, and Livestock
Article Snippet: Paragraph title: DNA extraction, library preparation, and next-generation sequencing. ... Briefly, bacteria were lysed with an enzyme preparation, vortexed, and processed according to the manufacturer's recommendations to obtain purified gDNA (Qiagen).
Incubation:Article Title: Designing a SCAR molecular marker for monitoring Trichoderma cf. harzianum in experimental communities * in experimental communities * #
Article Snippet: Each community was inoculated with 2×104 spores of each fungal isolate and incubated at 25 °C; after 3 d they were inoculated with 2×104 spores of T . cf. harzianum BpT10a. .. Destructive samplings were carried out 0, 3, 7, and 10 d after inoculation with T . cf. harzianum BpT10a and the gDNA was extracted with the PowerSoil™ DNA extraction kit (MoBio Laboratories Inc.). gDNA was quantified using the Take3™ Epoch MicroVolume Plate reader (Biotek) and 1 ng was used as the template for the qPCR reactions, which were performed under the same conditions above mentioned.
Spectrophotometry:Article Title: Developmental Expression of CYP2B6: A Comprehensive Analysis of mRNA Expression, Protein Content and Bupropion Hydroxylase Activity and the Impact of Genetic Variation
Article Snippet: .. gDNA was isolated from ∼25 mg of tissue using a DNeasy Tissue Kit (Qiagen) or an Illustra Tissue and Cells Genomic Prep Kit (GE Healthcare). gDNA was assessed by agarose gel electrophoresis for quality; concentration was measured using a NanoDrop 1000 spectrophotometer (Thermo-Scientific, Waltham, MA). gDNA samples were diluted to 15 ng/ µ l. Genotyping was carried out on an Applied Biosystems 7900 HT Real-Time PCR System using predesigned Life Technologies TaqMan assays for CYP2B6 rs34223104 (−82T > C); rs3745274 (516G > T, Q172H), rs28399499 (983T > C, I328T), and rs3211371 (1459C > T R487C). .. Reactions were scaled to 8 µ l and contained 4 µ l KAPA PROBE FAST Universal 2X qPCR Master Mix (KAPA Biosystems, Woburn, MA), and 0.2 or 0.4 µ l TaqMan assay mix, respectively, depending on whether it was a 20× or 40× mix, and 12–18 ng gDNA.
Activation Assay:Article Title: Expression levels of hnRNP K and p21WAF1/CIP1 are associated with resistance to radiochemotherapy independent of p53 pathway activation in rectal adenocarcinoma
Article Snippet: In brief, genomic DNA was quantified and target enrichment was processed with the GeneRead DNAseq Panel PCR V2 kit (Qiagen GmbH) applying 40 ng genomic DNA quantified with a Qubit® dsDNA HS kit and a Qubit® 2.0 Fluorometer (Life Technologies; Thermo Fisher Scientific, Inc.). .. An initial activation step (95°C; 15 min) was followed by 25 cycles of denaturing at 95°C (15 sec) and annealing at 60°C (4 min), followed by extension at 72°C for 10 min. All purification and size selection steps were performed using Agencourt AMPure XP magnetic beads (Beckman Coulter, Inc., Brea, CA, USA).
Produced:Article Title: Long-term Correction of Very Long-chain Acyl-CoA Dehydrogenase Deficiency in Mice Using AAV9 Gene Therapy
Article Snippet: All animal procedures were approved by University of Massachusetts Medical School Institutional Animal Care and Use Committee as well as University of Florida Institutional Animal Care and Use Committee in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care International specifications. rAAV vectors . rAAV9/2 pseudotyped vectors were generated to express human VLCAD under the transcriptional control of the cytomegalovirus enhancer/chicken β-actin promoter. rAAV vectors were produced, purified and tittered as previously described (UMMS Gene Therapy Center, Worcester, MA). .. Animals were sacrificed at indicated time points and tissues were harvested in a manner to avoid cross-contamination, snap frozen in liquid nitrogen and stored at −80 °C. gDNA was extracted using a DNeasy blood and tissue kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. gDNA concentrations were determined using an Eppendorf Biophotometer (Eppendorf, Hamburg, Germany). rAAV genome copies in the gDNA from heart, liver, tibialis anterior, and brown fat were quantified by quantitative-PCR with an ABI 7900 HT sequence detection system (Applied Biosystems, Carlsbad, CA) according to the manufacturer's instructions and results were analyzed using SDS 2.3 software.
Concentration Assay:Article Title: Calcium transport in bovine rumen epithelium as affected by luminal Ca concentrations and Ca sources
Article Snippet: Isolation of RNA and reverse transcription The kit for isolation of RNA contained special spin columns to eliminate gDNA (Qiagen, Hilden, Germany). .. Concentration and quality of RNA were determined by UV absorbance and 200 ng were reverse transcribed using random hexamer primers and TaqMan-Reverse Transcription Reagents (Applied Biosystems, Darmstadt, Germany).
Article Title: Concerted copy number variation balances ribosomal DNA dosage in human and mouse genomes
Article Snippet: .. Cells were harvested 24 h after the treatment with BPA, and gDNA was isolated using DNeasy Blood and Tissue kit following the method described by the manufacturer (Qiagen). gDNA samples were treated with RNase A, and concentration was estimated with NanoDrop (Thermo Scientific). ..
Article Title: Developmental Expression of CYP2B6: A Comprehensive Analysis of mRNA Expression, Protein Content and Bupropion Hydroxylase Activity and the Impact of Genetic Variation
Article Snippet: .. gDNA was isolated from ∼25 mg of tissue using a DNeasy Tissue Kit (Qiagen) or an Illustra Tissue and Cells Genomic Prep Kit (GE Healthcare). gDNA was assessed by agarose gel electrophoresis for quality; concentration was measured using a NanoDrop 1000 spectrophotometer (Thermo-Scientific, Waltham, MA). gDNA samples were diluted to 15 ng/ µ l. Genotyping was carried out on an Applied Biosystems 7900 HT Real-Time PCR System using predesigned Life Technologies TaqMan assays for CYP2B6 rs34223104 (−82T > C); rs3745274 (516G > T, Q172H), rs28399499 (983T > C, I328T), and rs3211371 (1459C > T R487C). .. Reactions were scaled to 8 µ l and contained 4 µ l KAPA PROBE FAST Universal 2X qPCR Master Mix (KAPA Biosystems, Woburn, MA), and 0.2 or 0.4 µ l TaqMan assay mix, respectively, depending on whether it was a 20× or 40× mix, and 12–18 ng gDNA.
DNA Purification:Article Title: Integration of Human Immunodeficiency Virus Type 1 in Untreated Infection Occurs Preferentially within Genes
Article Snippet: .. Genomic DNA was isolated from PBMCs with a QIAamp DNA blood minikit (QIAGEN); genomic DNA was isolated from brains, lymph nodes, and spleens with a genomic DNA purification kit (Gentra Systems) according to the manufacturer's protocol. .. Our procedure to identify integration sites is a modification of that previously described ( ).
Marker:Article Title: Designing a SCAR molecular marker for monitoring Trichoderma cf. harzianum in experimental communities * in experimental communities * #
Article Snippet: Destructive samplings were carried out 0, 3, 7, and 10 d after inoculation with T . cf. harzianum BpT10a and the gDNA was extracted with the PowerSoil™ DNA extraction kit (MoBio Laboratories Inc.). gDNA was quantified using the Take3™ Epoch MicroVolume Plate reader (Biotek) and 1 ng was used as the template for the qPCR reactions, which were performed under the same conditions above mentioned. .. The fungal gDNA-mix standard curve was used to calculate the copy number of the SCAR marker in each community.
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![Gene expression and hypothalamic Pomc <t>DNA</t> methylation changes in offspring at weaning. a mRNA expression levels of Pomc , Agrp , Npy , and b Ob-Rb in the <t>ARC.</t> c Relative mRNA levels of Mc4r and Npy1r in the PVN analyzed by qRT-PCR in the 3-week-old offspring of LF- or HF-fed dams (Student’s t -test, n = 8). d Map of the Pro-opiomelanocortin ( Pomc ) gene promoter and enhancer region including functional regulatory elements and CpG dinucleotides (red lines). e Methylation analyzes of hypothalamic Pomc promoter (− 150 bp to transcription start site [TSS]) (Student’s t -test, D-LF, n = 6; D-HF, n = 7) and f , g of neuronal Pomc enhancer region 1 and 2 in the offspring of LF- or HF-fed mothers at 3 weeks of age (Student’s t -test, n = 8). Data are shown as mean ± SEM. * p](https://storage.googleapis.com/bioz_article_images/PMC6113193/41366_2018_94_Fig2_HTML.jpg)
