For WGSS, genomic DNA from frozen tumour sections or buffy coat from blood (germline reference) was extracted using the Gentra Pure Gene DNA Kit and standard techniques, as previously described. This DNA was whole‐genome amplified (WGA), using the Repli‐G kit (Qiagen), prior to use in the Haloplex amplicon kit (Agilent) for deep sequencing. DNA from additional tumours, endometriosis and benign lesions were isolated from formalin‐fixed, paraffin‐embedded (FFPE) tissues by coring, macrodissection from haematoxylin and eosin (H&E)‐stained sections, or laser‐capture microdissection (LCM) from stained sections, depending on their size and location (see supplementary material, Table S1). DNA from cored or macrodissected FFPE tissue was extracted using the QIAamp DNA FFPE kit (Qiagen). DNA from LCM FFPE tissues was extracted using the Picopure DNA Kit (Life Technologies). Samples with < 150 ng yield were subject to whole‐genome amplification, using the Ovation FFPE Whole Genome Amplification Kit (Nugen) and Ovation dsDNA module (Nugen), as marked in Table S1 (see supplementary material).
Qiaamp dna ffpe kit
Manufactured by Qiagen
Sourced in Germany, United States, United Kingdom, Spain
The QIAamp DNA FFPE Kit is a laboratory equipment designed for the purification of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue samples. The kit utilizes a silica-based membrane technology to efficiently extract and isolate DNA from FFPE samples, which can be used for various downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop spectrophotometer, by Thermo Fisher Scientific (8 mentions)
Qiaamp circulating nucleic acid kit, by Qiagen (6 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (6 mentions)
Qubit 2, by Thermo Fisher Scientific (4 mentions)
2100 bioanalyzer, by Agilent Technologies (4 mentions)
Qiaamp dna mini kit, by Qiagen (3 mentions)
Rneasy ffpe kit, by Qiagen (3 mentions)
Nanodrop 2000 uv spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Tapestation 2200, by Agilent Technologies (2 mentions)
Icycler iq real time pcr detection system, by Bio-Rad (2 mentions)
Iq supermix, by Bio-Rad (2 mentions)
Ez dna methylation goldtm kit, by Zymo Research (2 mentions)
Kapa library quantification kit, by Roche (2 mentions)
Hiseq 4000 platform, by Illumina (2 mentions)
Kapa hifi hotstart readymix, by Roche (2 mentions)
Membrane slide, by Zeiss (2 mentions)
Relaxgene blood dna system, by Tiangen Biotech (2 mentions)
Qubit br kit, by Thermo Fisher Scientific (2 mentions)
Ez dna methylation kit, by Zymo Research (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
84 protocols using qiaamp dna ffpe kit
1
Whole-Genome Sequencing of Tumor and Endometriosis Samples
2
Robust DNA Extraction from FFPE Samples
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Tumour DNA was extracted from sections of FFPE tumour blocks and extracted with QIAamp® DNA FFPE kit (Qiagen, Hilden, Germany) following the protocol provided by the manufacturer, with an extra proteinase K digestion step as previously described [22 (link)] or by using the Maxwell® FFPE Plus DNA Kit with a Maxwell® RSC (Promega, Wisconsin, USA). The extracted DNA was quantified using Qubit® Fluorometer Invitrogen (ThermoFisher, Waltham, Massachusetts, USA).
3
Methylation Profiling of FFPE Glioma Samples
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DNA from formalin-fixed paraffin embedded (FFPE) tumors from patients included in the retrospective cohort was isolated with the QIAamp® DNA FFPE kit (Qiagen, Hilden, Germany) according to the supplier’s instructions with the addition of an extra digestion step with proteinase K overnight. DNA concentration was measured with the Qubit Fluorometer (Life technologies™, Carlsbad, CA, USA). Between 500 and 1000 ng DNA was bisulfite-converted with the EZ DNA methylation kit (D5001, Zymo Research, Irvine, CA, USA) and the methylation levels of restored bisulfite-converted DNA was determined with the Infinium MethylationEPIC BeadChip (Illumina®, San Diego, CA, USA) according to the protocols provided by the supplier.
Methylation analysis and normalization was performed as previously described [21 (link)]. Briefly, methylation data were processed with the statistical software R (version 3.6.1) using the Minfi [22 (link)] and ChAMP [22 (link)–24 (link)] packages. IDH mutational status was acquired using a published DNA methylation-based classifier [9 (link)]. 1p19q codeletion status was acquired through copy number variations inferred from the array. Correlation between the T2-FLAIR mismatch sign and DNA-methylation profiles was evaluated by unsupervised hierarchical clustering of the 5000 most variable CpG sites including only patients with IDH-mut non-codel gliomas (N = 29).
Methylation analysis and normalization was performed as previously described [21 (link)]. Briefly, methylation data were processed with the statistical software R (version 3.6.1) using the Minfi [22 (link)] and ChAMP [22 (link)–24 (link)] packages. IDH mutational status was acquired using a published DNA methylation-based classifier [9 (link)]. 1p19q codeletion status was acquired through copy number variations inferred from the array. Correlation between the T2-FLAIR mismatch sign and DNA-methylation profiles was evaluated by unsupervised hierarchical clustering of the 5000 most variable CpG sites including only patients with IDH-mut non-codel gliomas (N = 29).
4
LOH Detection in FFPE Tumor Samples
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DNA from formalin-fixed paraffin-embedded (FFPE) tumor samples was extracted using the QIAAmp DNA FFPE kit (Qiagen). Tissue samples of the proband available for LOH analysis are detailed in Supplementary Table 1. LOH of selected nucleotide variant was searched by SNaPshot analysis as previously described (16 (link)). PCR and extension primers details are available upon request. Extension products were analyzed on Applied Biosystems 3730 along with GeneScan 120LIZ molecular marker using the Genemapper software. In addition, visualization of the variant peaks on Sanger sequence traces was done using the Mutation Quantifier tool (Surveyor program, Softgenetics).
5
DNA Extraction and NGS Library Preparation
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All resected specimens were subjected to a uniform preparation protocol for formalin-fixed, paraffin-embedded (FFPE) specimens. Tumour and corresponding nontumour samples were collected from each patient.
DNA was extracted using a QIAamp DNA FFPE Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The quantification of genomic DNA samples was assessed with a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc., Wilmington, DE, USA).
DNA was stored at − 20 °C for future use. At least 500 ng of DNA was required to perform the NGS library preparation. The library was prepared by using KAPA Biosystems library preparation reagents. The library had an average fragment size of 140–200 bp. Hybrid capture was performed by using NimbleGen Capture reagents.
DNA was extracted using a QIAamp DNA FFPE Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The quantification of genomic DNA samples was assessed with a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc., Wilmington, DE, USA).
DNA was stored at − 20 °C for future use. At least 500 ng of DNA was required to perform the NGS library preparation. The library was prepared by using KAPA Biosystems library preparation reagents. The library had an average fragment size of 140–200 bp. Hybrid capture was performed by using NimbleGen Capture reagents.
6
Nucleic Acid Extraction from FFPE, FNA, and Fresh Tissue
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TNA was isolated from FFPEs, FNAs, and fresh-frozen tissue using the QIAamp DNA FFPE Kit (Qiagen, Valencia, CA) without RNase treatment according to the manufacturer's instructions. A subset of these TNA eluates was further treated with DNase I, which was subsequently heat killed, to prepare pure RNA isolations. Individual cell lines were cultured under reported optimal conditions (ATCC, Manassas, VA) and collected at the time of passage. TNA from cell pellets was isolated using the RNeasy Plus Mini Kit (Qiagen) without the DNA eliminator column. Elution was performed in RNAse-free water provided with the kit.
7
CRC Tumor Sampling and DNA Extraction
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The cohort consisted of 20 CRC patients (12 fresh frozen and 8 FFPE tumor samples) with well-differentiated colorectal cancer who underwent surgery at Saint-Antoine hospital, Paris, France. Tumor samples were snap-frozen in liquid nitrogen or formalin-fixed and paraffin embedded and stored in the Biobank of this Hospital. DNA isolation was performed using the QIAamp DNA Mini Kit (Qiagen) or QIAamp DNA FFPE kit (Qiagen) for fresh frozen or FFPE samples respectively, according to the manufacturer's instructions. The use of clinical and pathologic records was in agreement with French laws and ethical guidelines related to the protection of the patient.
8
DNA Purification from FFPE Samples
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For DNA purification from formalin-fixed paraffin-embedded (FFPE) samples, the QIAampDNA FFPE Kit (Qiagen, Hilden, Germany) was employed, following the manufacturer’s instructions. Concentrations of the purified DNAs were determined using the Qubit ds DNA HS assay (Life Technologies Gaithersburg, USA). Samples with ≥2.5 ng DNA/mL were diluted to this concentration.
9
Comprehensive EGFR Mutation Analysis
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EGFR mutations in exons 18-21 were tested at diagnosis on tumor biopsy/cell blocks or on tumor specimens. EGFR rare/uncommon mutations are defined as alterations with the exception of common sensitizing exon 19 deletion, L858R point mutation and T790M mutation, accounting for about 15% to 20% of all EGFR mutations. Complex or compound mutations are usually defined as the presence of two or more different EGFR mutations in the same tumor sample. For analyses of tissue samples, tumor DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tumor slices using the QIAamp DNA FFPE kit (Qiagen, Hilden, Germany), and quantified with a Nanodrop spectrophotometer (NanoVue, GE Healthcare, Milwaukee, WI, USA); DNA sequencing was carried out with Sanger sequencing, pyrosequencing, polymerase chain reaction (PCR)-based methods (easy EGFR kit, Diatech Pharmacogenetics, Jesi, Italy; EGFR mutation analysis kit EntroGen, EntroGen, USA), and mass spectrometry-based methods (Myriapod lung status kit, Sequenom MassARRAY, Diatech Pharmacogenetics, Jesi, Italy) (17 (link)).
10
Targeted ERBB Mutation Profiling in HER2+ Breast Cancer
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DNA extraction was performed using a QiaAMP DNA FFPE Kit from Qiagen (Hilden,
Germany) as per manufacturer’s protocol and quantified using QuBit. We designed
an Agena MassARRAY panel to assay for 67 novel ERBB gene family
somatic mutations in 227 HER2+ breast cancer patients (Supplementary Table 1 ). Typically, 10 ng per assay was used for
mass spectrometry-based genotyping (Agena MassARRAY, San Diego, CA, USA), which
was applied as previously described.14 (link) Reactions where >15% of the resultant mass ran in the mutant site were
scored as positive.
Germany) as per manufacturer’s protocol and quantified using QuBit. We designed
an Agena MassARRAY panel to assay for 67 novel ERBB gene family
somatic mutations in 227 HER2+ breast cancer patients (
mass spectrometry-based genotyping (Agena MassARRAY, San Diego, CA, USA), which
was applied as previously described.14 (link) Reactions where >15% of the resultant mass ran in the mutant site were
scored as positive.
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