DNA from glioma cells was extracted using the commercial DNA Sample Preparation Kit (Roche, Basel, Switzerland). Mutation analysis was performed by multi parallel sequencing (NGS) using the hybrid-capture-based target enrichment. A custom KAPA HyperChoice MAX Library (Roche) for enrichment of the coding and 30 bp upstream and downstream overlaps of selected panel of genes (TP53, EGFR, IDH1, IDH2, PTEN, PIK3CA) was used. Paired-end cluster generation and sequencing was performed by NGS system Illumina MiniSeq. Sequencing data analysis were performed by NextGENe software (Softgenetics) and Varsome Clinical Platform. MGMT methylation analysis was performed on DNA in FFPE using DNA Sample Preparation Kit (Roche). Bisulfite conversion of isolated DNA was performed using the EZ DNA Methylation-Gold Kit (Zymo Research). Detection and quantification of the hypermethylation status of the O (6)-methylguanine-DNA methyltransferase (MGMT) promoter was performed by the methylation-specific real-time PCR method using the CE-IVD marked geneMAP MGMT Methylation Analysis Kit (GenmarkSalgik).
Dna sample preparation kit
Manufactured by Roche
Sourced in United States
The DNA Sample Preparation Kit is a laboratory equipment designed for the extraction and purification of DNA from various biological samples. The kit contains the necessary reagents and components to perform the DNA isolation process in a standardized and efficient manner. It is intended for use in molecular biology applications that require high-quality DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Ez dna methylation gold kit, by Zymo Research (2 mentions)
Nextgene software, by SoftGenetics (2 mentions)
Cobas z 480, by Roche (1 mentions)
Cobas kras mutation test, by Roche (1 mentions)
Kapa hyperchoice max library, by Roche (1 mentions)
Ampure xp bead, by Illumina (1 mentions)
Pcr buffer, by Qiagen (1 mentions)
Hotstartaq, by Qiagen (1 mentions)
Ion reporter software, by Thermo Fisher Scientific (1 mentions)
Ion 530 chip, by Thermo Fisher Scientific (1 mentions)
Ion ampliseq library kit 2, by Thermo Fisher Scientific (1 mentions)
As lmd, by Leica (1 mentions)
Cobas 4800 braf v600 mutation test, by Roche (1 mentions)
Agilent feature extraction software version 10.7.3.1, by Agilent Technologies (1 mentions)
Qiamp dna blood mini kit, by Qiagen (1 mentions)
Kapa library preparation hyper plus kit, by Roche (1 mentions)
V2 kits, by Illumina (1 mentions)
Seqcap ez choice library, by Roche (1 mentions)
10 protocols using dna sample preparation kit
1
Multilevel Genomic Profiling of Glioma Samples
2
BRAF V600E Mutation Detection
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DNA was extracted from the same surgical paraffin-embedded tissue blocks using DNA Sample Preparation Kit (Roche USA) following the manufacturer’s instructions. Exon 15 mutation of the BRAF gene (V600E) was assessed using a Cobus 4800 BRAF V600 Mutation Test Kit (Roche, USA) and a Cobas z480 following the manufacturer’s instructions at the Molecular Pathology Laboratory of Chuiyangliu Hospital.
3
KRAS and BRAF Mutation Detection
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KRAS and BRAF mutational status was performed in the Pathology Department of Fundacion Jimenez Diaz Hospital. For this, DNA was extracted with a DNA Sample Preparation Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. KRAS mutational status of each sample was determined by Real-Time PCR with a cobas®KRAS Mutation Test (Roche Diagnostics). BRAF V600E mutation testing was performed by cobas®BRAF mutation test IVD (Roche Diagnostics).
4
FFPE DNA Sequencing Workflow
5
Methylation Analysis of Glioma Samples
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Bisulfite conversion of template DNA was performed using the EZ DNA methylation Gold Kit according to the manufacturer’s instructions (Zymo Research, Irvine, CA, USA) immediately after extracting DNA from glioma tissue samples using the DNA Sample Preparation Kit (Roche, Pleasanton, CA, USA). MGMT methylation was then detected using MethyLight real-time methylation-specific PCR. To verify DNA integrity and the quality of the bisulfite conversion and the PCR reaction, the methylation of CDH1 (E-cadherin) and Alu-M5 was analyzed in parallel with that of MGMT, and a commercial methylated and bisulfite-converted DNA standard (Zymo Research) was analyzed as a control alongside all DNA extracts from tissue samples.
Each 10 µl PCR reaction mixture for detection of MGMT, E-cadherin, and Alu-M5 promoter methylation contained 1x PCR buffer (Qiagen, Hiden, Germany), 1 mM MgCl2, 0.2 mM dNTPs, 0.5 U HotStarTaq (Qiagen, Hiden, Germany), and the relevant primers and probe (Table 1 ). The PCR program involved denaturation at 95 °C for 15 min then 40 cycles of 95 °C for 30 s, 65 °C for 50 s, and 72 °C for 60 s.
Each 10 µl PCR reaction mixture for detection of MGMT, E-cadherin, and Alu-M5 promoter methylation contained 1x PCR buffer (Qiagen, Hiden, Germany), 1 mM MgCl2, 0.2 mM dNTPs, 0.5 U HotStarTaq (Qiagen, Hiden, Germany), and the relevant primers and probe (
6
Targeted Tumor DNA Sequencing Protocol
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Tumor DNA was extracted from all samples at the central lab after wet macrodissection according to the DNA Sample Preparation Kit (Roche). A total of 20 ng of DNA was used to build the Oncomine Focus DNA Assay panel libraries (Thermo Fisher Scientific, Inc.), using the Ion AmpliSeq Library kit 2.0 (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. A total of 30 uniquely barcoded library samples were pooled for sequencing per run on an Ion 530 chip (Thermo Fisher Scientific, Inc.) for an expected mean read depth of 300×.
BAM files derived from processed raw data were generated with the Ion Reporter Software (v. 5.10.5.0; Thermo Fisher Scientific, Inc.) and analyzed for single-nucleotide variants, indels [variant allele frequency (VAF) > 10%], and CNVs [for sample with a median absolute pairwise difference (MAPD) ≤ 0.5] by the Oncomine Focus w2.4 - DNA - Single Sample (v. 5.10) pipeline. Finally, a custom filter chain was applied to report only likely somatic mutations with a VAF ≥ 0.1 and a minor allele frequency or global allele frequency in ExAC or 5,000 exomes databases ≤1.0E-6. Mutations must also be nonsynonymous and occur in exonic or splice-site regions. MET, EGFR, and KRAS amplification were defined by the presence of CNV ≥ 4.
BAM files derived from processed raw data were generated with the Ion Reporter Software (v. 5.10.5.0; Thermo Fisher Scientific, Inc.) and analyzed for single-nucleotide variants, indels [variant allele frequency (VAF) > 10%], and CNVs [for sample with a median absolute pairwise difference (MAPD) ≤ 0.5] by the Oncomine Focus w2.4 - DNA - Single Sample (v. 5.10) pipeline. Finally, a custom filter chain was applied to report only likely somatic mutations with a VAF ≥ 0.1 and a minor allele frequency or global allele frequency in ExAC or 5,000 exomes databases ≤1.0E-6. Mutations must also be nonsynonymous and occur in exonic or splice-site regions. MET, EGFR, and KRAS amplification were defined by the presence of CNV ≥ 4.
7
BRAF V600E Mutation Detection
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Tissue samples were obtained by laser capture microdissection using the system Leica AS LMD (Leica Microsystems, Wetzlar, Germany) according to the manufacturer's instructions. Representative 4-μm-thick paraffin sections containing the PMC stained with hematoxylin were deposited in specific frame slides for laser microdissection so that in each case, only tumor tissue could be selected for subsequent DNA extraction as previously reported 37 (Fig. 1). DNA was extracted using the DNA Sample Preparation Kit (Roche Diagnostics, San Cugat del Vallés, Spain) and amplified by polymerase chain reaction. Samples were screened for somatic BRAF V600E gene mutations using real-time polymerase chain reaction (Cobas 4800 BRAF V600 Mutation Test; Roche Diagnostics), with appropriate positive and negative controls.
8
DNA Extraction and Array Profiling for Hematological Cancers
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DNA was extracted from fresh biopsy material or cytogenetic fixed-cell suspension by QIAmp DNA Blood Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s recommendation. DNA of patient 10 was isolated from paraffin sections using DNA Sample Preparation Kit (Roche, Basel, Switzerland). The reference DNA was used from two pools (male and female) from normal individuals and run as a same-sex control. The control DNA for patient 10 was isolated from paraffin sections of normal lymph node. For SNP/aCGH analysis, CytoSure Haematological Cancer and SNP Array (8x60k) (Oxford Gene Technology [OGT], Yarnton, UK) was used. Total genomic DNA of 600 ng was processed in accordance with the manufacturer’s protocol. Each patient and reference DNA was labeled with Cy3 and Cy5 dyes, respectively. Purification of labeled products, hybridization, and postwash of the array were carried out according to OGT’s recommendation. Array slides were scanned with Agilent G2565CA scanner, and images were quantified using Agilent Feature Extraction Software (version 10.7.3.1) (Agilent, Santa Clara, CA, USA).
9
Renal Tumor Diagnosis and Management
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This paper reports on MA in three men and one woman, aged between 19 and 65 (mean age 47.5); patient data are provided in Table 3 . The diagnosis was confirmed and subsequently reviewed following WHO-recommended criteria [4 ]. In all cases, MA presented as a single, asymptomatic mass discovered incidentally during imaging procedures; CT scan confirmed the presence of a solitary, space-occupying, solid renal tumor. A nephrectomy was performed in all patients.
Immunohistochemical staining was performed on H&E- stained sections cut from paraffin blocks to assess expression of WT1, vimentin, racemase, CK7, CD10 and RCC. Testing for the BRAF gene mutation V600 was carried out by real-time PCR (Cobas® 4800) using the DNA Sample Preparation Kit and the BRAF Mutation Test (Roche), which detects the BRAF gene mutations V600E, V600K and V600D in formalin-fixed, paraffin-embedded tissue.
Immunohistochemical staining was performed on H&E- stained sections cut from paraffin blocks to assess expression of WT1, vimentin, racemase, CK7, CD10 and RCC. Testing for the BRAF gene mutation V600 was carried out by real-time PCR (Cobas® 4800) using the DNA Sample Preparation Kit and the BRAF Mutation Test (Roche), which detects the BRAF gene mutations V600E, V600K and V600D in formalin-fixed, paraffin-embedded tissue.
10
Targeted NGS Profiling of Common Oncogenes
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DNA was extracted from formalin-fixed, paraffin embedded tumour tissue after deparaffinization in xylene and rehydration in ethanol using the commercial DNA Sample Preparation Kit (Roche, Basel, Switzerland) according to the manufacturer's protocol. Mutation analysis was performed by Massively Parallel Sequencing (NGS). Indexed Illumina NGS library was constructed from 100 ng tumour cell line DNA using KAPA Library Preparation Hyper Plus Kit (Kapa Biosystems). Hybrid selection was performed with a custom SeqCap EZ Choice Library (Roche NimbleGen). The library was designed using the genome build hg19 NCBI Build 37.1/GRCh37 (input genomic regions: KRAS NM_004985.4 -full coding regions; NRAS NM_002524.4 -full coding regions; BRAF NM_004333.4 -exons 11 a 15; PTEN NM_000314.4 -full coding regions). Paired-end cluster generation and sequencing were performed according to standard protocols from Illumina, using v2 kits. Sequencing data analysis and variant annotation was performed using NextGENe software (Softgenetics) with minimum 5% variant allele frequency filtering.
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