DNA sequencing was performed on 256 patients with available tumor samples to detect genetic aberrations. Genomic DNA was extracted from frozen tumor tissue by a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) or from formalin‐fixed paraffin‐embedded (FFPE) tumor tissue by a GeneRead DNA FFPE Tissue Kit (Qiagen), based on the manufacturer's guidelines. Whole exome sequencing (WES) data from 44 patients and whole genome sequencing (WGS) data from 9 patients have been reported in our previous manuscript about extranodal DLBCL [20 (link)]. Two hundred and three patients with FFPE tumor tissue or frozen tumor tissue were analyzed by targeted sequencing of 55 lymphoma‐associated genes using the methods defined in the supporting information of our previous study [20 (link)]. Sequencing data have been deposited on https://www.biosino.org/node in project OEP001143 .
Generead dna ffpe tissue kit
Manufactured by Qiagen
Sourced in Germany
The GeneRead DNA FFPE Tissue Kit is a laboratory equipment product designed for the extraction and purification of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue samples. The kit provides a streamlined workflow for obtaining high-quality genomic DNA from challenging FFPE specimens.
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Lab products found in correlation
Qiaamp dna mini kit, by Qiagen (3 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Trizol and rneasy mini kit, by Qiagen (1 mentions)
Qsep100 system, by Bioptic (1 mentions)
Miseq reagent micro kit v2, by Illumina (1 mentions)
Dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Sureselectxt human all exon v6, by Agilent Technologies (1 mentions)
Qiaamp dna blood mini kit, by Qiagen (1 mentions)
Generead dna quantimize kit, by Qiagen (1 mentions)
Generead dna formalin fixed paraffin embedded tissue kit, by Qiagen (1 mentions)
Wizard genomic dna purification kit, by Promega (1 mentions)
Nanodrop 2000c spectrophotometer, by Thermo Fisher Scientific (1 mentions)
9 protocols using generead dna ffpe tissue kit
1
Genetic Profiling of Extranodal DLBCL
2
Genomic DNA and RNA Extraction from Tumor Tissues
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Genomic DNA (gDNA) was extracted from frozen tumor tissues using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), or from formalin-fixed paraffin-embedded (FFPE) tumor tissues using a GeneRead DNA FFPE Tissue Kit (Qiagen, Hilden, Germany), based on the manufacturer’s guidelines. The concentration of gDNA was measured using Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), and the gDNA was quantified by Qsep100 System (BIOptic, China).
Total RNA was extracted from frozen tumor tissues using Trizol and RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA quantity was assessed on Nanodrop, and the integrity of total RNA was estimated by RNA 6000 Nano Kit on Aligent 2100 Bioanalyzer.
Total RNA was extracted from frozen tumor tissues using Trizol and RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA quantity was assessed on Nanodrop, and the integrity of total RNA was estimated by RNA 6000 Nano Kit on Aligent 2100 Bioanalyzer.
3
Whole-Exome and Whole-Genome Sequencing of Lymphoma Samples
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Genomic DNA was extracted from frozen tumor tissue by a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany), or from formalin‐fixed paraffin‐embedded (FFPE) tumor tissue by a GeneRead DNA FFPE Tissue Kit (Qiagen), based on the manufacturer's guidelines. For 216 patients, whole‐exome sequencing (WES) was carried out on frozen tumor tissue (n = 132), or on FFPE tumor tissue quality controlled by agarose gel electrophoresis (n = 84). For 109 patients, whole‐genome sequencing (WGS) was performed on frozen tumor tissue. WES (n = 25, divided into five groups) and WGS (n = 17) were performed on 42 matched peripheral blood samples randomly selected to build a somatic mutation calling principle and to exclude germ‐line polymorphisms. Three hundred forty‐five patients with FFPE tumor tissue were analyzed by targeted sequencing of 55 lymphoma‐associated genes. Genome Analysis Toolkit (GATK, v3.7.0), Haplotype Caller, and GATK Unified Genotyper were applied to call single nucleotide variations (SNVs) and indels, which were mapped to the genome location using the UCSC Genome Browser (http://genome.ucsc.edu ). The Refseq database (Human Reference Genome version hg19) was used as the reference genome. The filtration of detected SNVs and indels was performed by homemade pipeline with the above software. Details for DNA sequencing are provided in the Supporting Information Methods.
4
FFPE Tissue DNA Extraction and NGS Analysis
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DNA extraction and sequential analysis were performed using our stocked FFPE biopsy specimens. Paraffin blocks of specimens were deparaffinized, microdissected, and subjected to DNA extraction using a Gene-Read DNA FFPE Tissue Kit (Qiagen, Hilden, Germany). Next-generation sequencing (NGS) libraries were prepared via two-step tailed PCR; each pool contained 36 pairs of primers targeting frequently mutated regions of TP53. Pair-stich read sequencing was performed using MiSeq (Illumina,San Diego, CA, USA) and MiSeq Reagent Micro Kit v2 (300 cycles) (Illumina). Sequence variations with variant allele frequencies and depths of more than 5% and 100×, respectively, were identified as candidate mutations. More detailed sequencing protocols are available in our previous reports22 (link),23 (link).
5
Whole Exome Sequencing of FFPE Tumors
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WES was implemented on the formalin-fixed paraffin-embedded tumor tissue and matched peripheral blood samples. GeneRead DNA FFPE Tissue Kit (QIAGEN, GER) was employed for FFPE section extraction, while Mag-Bind® Blood & Tissue DNA HDQ 96 kit (OMEGA) was utilized for blood sample extraction. The dsDNA HS Assay Kit (ThermoFisher Scientific, USA) was used for DNA quantification. Sequencing libraries were built by SureSelect XT Human All Exon V6 (Agilent), and sequencing procedures were utilized by the NextSeq 550AR platform with 150-bp paired-end reads. SOAPnuke58 (link) was implemented to cut adapters and remove low-quality raw reads. Clean reads were aligned against the human reference genome (hg19) with BWA (v0.7.12)59 (link), and duplicated reads were removed by Sambamba (v0.5.4)60 (link). Subsequently, generated BAM files were used for downstream analysis.
6
Molecular Detection of Echinococcus granulosus
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The DNA was extracted from 200 μl of serum by QIAamp DNA Blood Minikit (Qiagen, Germany) according to the manufacturer’s instructions. Five 10 μm sections were cut from each embedded cyst tissue sample, and excess paraffin was trimmed. The prepared sections were submitted to the DNA extraction procedure of GeneRead DNA FFPE Tissue Kit (Qiagen, Germany) according to manufacturer’s instructions. The obtained genomic DNA of cyst and serum samples was stored at -20°C until analysis.
The DNA of E. granulosus was detected by PCR amplification of two mitochondrial genes, cox1 and nad1, in patient sera and cysts. The 400 bp fragments of cox1 and 450 bp of nad1 genes were amplified by primers as described by Bowles [13 (link)] and Sharbatkhori [14 (link)], respectively. The final mixture of the PCR reaction contained 25 μl of Taq DNA Polymerase Master Mix (2X) (Amplicon III, Denmark, Cat no. 180301), 0.5 μM of each primer, 3–5 μl DNA. PCR was conducted under the conditions: 94°C for 5 min initial denaturation; 35 cycles of 94°C for 45 s, 55°C for 30 s, 72°C for 35 s; and a final extension at 72°C for 5 min. PCR products were visualized on 1.5% agarose gel. To validate accuracy of PCR results, DNA extracted from the laminated layer of a hydatid cyst and distilled water were used as positive and negative controls, respectively, and processed with the samples in each PCR set.
The DNA of E. granulosus was detected by PCR amplification of two mitochondrial genes, cox1 and nad1, in patient sera and cysts. The 400 bp fragments of cox1 and 450 bp of nad1 genes were amplified by primers as described by Bowles [13 (link)] and Sharbatkhori [14 (link)], respectively. The final mixture of the PCR reaction contained 25 μl of Taq DNA Polymerase Master Mix (2X) (Amplicon III, Denmark, Cat no. 180301), 0.5 μM of each primer, 3–5 μl DNA. PCR was conducted under the conditions: 94°C for 5 min initial denaturation; 35 cycles of 94°C for 45 s, 55°C for 30 s, 72°C for 35 s; and a final extension at 72°C for 5 min. PCR products were visualized on 1.5% agarose gel. To validate accuracy of PCR results, DNA extracted from the laminated layer of a hydatid cyst and distilled water were used as positive and negative controls, respectively, and processed with the samples in each PCR set.
7
FFPE DNA Extraction Optimization
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DNA was isolated from LCM caps using GeneRead DNA FFPE Tissue Kit (Qiagen, Valencia, CA) according to manufacturer’s protocol. However, the deparaffinization step was omitted starting with the proteinase K digestion step followed by incubation at 56°C for 16 hr. Quality and amplifiable DNA material was assessed with the GeneRead DNA QuantiMIZE Kit (Qiagen, Valencia, CA).
8
Comprehensive Genomic Profiling of DLBCL
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Genomic DNA was isolated from frozen tumor tissue using a Wizard® Genomic DNA Purification Kit (Promega, Wisconsin-Madison, USA) and from formalin-fixed paraffin-embedded (FFPE) tumor tissue using a GeneRead DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. For 128 patients, whole genome sequencing (WGS) was performed on frozen tumor tissue. For 153 patients, whole exome sequencing (WES) was performed on frozen tumor tissue (n = 125) and FFPE tumor tissue quality-controlled by agarose gel electrophoresis (n = 28). Targeted sequencing was performed on formalin-fixed paraffin-embedded tumor samples of 386 patients with genomic DNA extracted using GeneRead DNA formalin-fixed paraffin-embedded Tissue Kit (Qiagen, Hilden, Germany). A targeted sequencing panel was assessed using a custom Sure Select library (Agilent Technologies), including 55 genes related to the pathogenesis of DLBCL according to literature, or associated with significant alternations in gene expression, as revealed by RNA sequencing.
9
Macrodissection and DNA Extraction from FFPE Tissue
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One H&E stained slide along with 5–8 unstained sections (five microns in thickness) were mounted on slides. Areas of interest were circled on the H&E slide by a pathologist and corresponding areas from the unstained slides were manually macrodissected using a razor blade, to remove contaminating normal cells. The paraffin fragments were placed in a 1.5 ml microcentrifuge tube, deparaffinized with xylene, vortexed, and centrifuged at 14,000 rpm × 5 min. The tissue pellet was washed twice with 100% ethanol. The DNA was extracted from the macrodissected sections using the kit GeneRead DNA FFPE tissue kit (Cat # 180134, Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. The isolated DNA was quantified using a NanoDrop 2000c spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).
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