Maxwell 16 ffpe tissue lev dna purification kit

Manufactured by Promega

Sourced in United States, Germany

The Maxwell 16 FFPE Tissue LEV DNA Purification Kit is a laboratory equipment product designed for the extraction and purification of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue samples. The kit utilizes magnetic bead technology to automate the DNA extraction process.

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51 protocols using maxwell 16 ffpe tissue lev dna purification kit

FFPE DNA Extraction and Quantification

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Formalin-fixed paraffin-embedded (FFPE) tissue blocks were obtained from the Department of Pathology at Kawasaki Medical School Hospital. DNA was extracted from the tumor and normal tissue at the primary site (breast) and from sites of metastasis and/or local recurrence. Samples of pleural or pericardial effusion were collected using the collodion bag method to generate cell blocks. Four 10-μm sections were cut from each paraffin block. Maxwell 16 FFPE Tissue LEV DNA purification kits (#AS1130; Promega, Madison, WI, USA) were employed for DNA extraction. DNA was quantified using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and Qubit dsDNA BR assay kits (#Q32850; Thermo Fisher Scientific). DNA quality was assessed by calculating the QC score (https://www.qiagen.com/us/resources/download.aspx?id=aae35658-5ef2-44b2-bd02-fbe73fe7737c&lang=en). DNA amplification for library construction was performed by quantitative polymerase chain reaction (qPCR) using QIAseq DNA QuantiMIZE Assay Kits (#DNQC-100Y-R; Qiagen, Hilden, Germany) [15 (link)].

Akahane T., Kanomata N., Harada O., Yamashita T., Kurebayashi J., Tanimoto A, & Moriya T. (2020). Targeted next-generation sequencing assays using triplet samples of normal breast tissue, primary breast cancer, and recurrent/metastatic lesions. BMC Cancer, 20, 944.

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Extraction and Quantification of FFPE DNA

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Formalin-fixed paraffin-embedded (FFPE) tissue blocks were obtained from the Department of Pathology at Kawasaki Medical School Hospital. DNA was extracted from the tumor and normal tissue at the primary site (breast) and from sites of metastasis and/or local recurrence. Samples of pleural or pericardial effusion were collected using the collodion bag method to generate cell blocks. Four 10-mm sections were cut from each paraffin block. Maxwell 16 FFPE Tissue LEV DNA purification kits (#AS1130; Promega, Madison, WI, USA) were employed for DNA extraction. DNA was quantified using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and Qubit dsDNA BR assay kits (#Q32850; Thermo Fisher Scientific). DNA quality was assessed by calculating the QC score (https://www.qiagen.com/us/resources/download.aspx? id=aae35658-5ef2-44b2-bd02-fbe73fe7737c&lang=en). DNA amplification for library construction was performed by quantitative polymerase chain reaction (qPCR) using QIAseq DNA QuantiMIZE Assay Kits (#DNQC-100Y-R; Qiagen, Hilden, Germany) [15] .

Akahane T., Kanomata N., Harada O., Yamashita T., Kurebayashi J., Tanimoto A., & Moriya T. (2020). Targeted next-generation sequencing assays using triplet samples of normal breast tissue, primary breast cancer, and recurrent/metastatic lesions.

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Molecular Cytogenetics Analysis of POC

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Sample types submitted for analysis included fresh tissue, FFPE tissue, chorionic villi, cultured cells, amniotic fluid, and extracted DNA. DNA extraction from fresh and FFPE POC tissues was performed using Maxwell 16 Tissue and Maxwell 16 FFPE Tissue LEV DNA Purification kits, respectively (Promega, Madison, WI). For FFPE blocks and slides, the area of interest containing fetal tissue was determined via examination of hematoxylin and eosin (H&E)-stained sections. Following the tissue demarcation, microdissection, and DNA extraction, sample restoration (for FFPE tissues) was performed prior to SNP array analysis. For BAC array-based comparative genomic hybridization (arrayCGH), "sample restoration" was not a step in the sample preparation.

Sahoo T., Dzidic N., Strecker M.N., Commander S., Travis M.K., Doherty C., Tyson R.W., Mendoza A.E., Stephenson M., Dise C.A., Benito C.W., Ziadie M.S, & Hovanes K. (2017). Comprehensive genetic analysis of pregnancy loss by chromosomal microarrays: outcomes, benefits, and challenges. Genetics in medicine : official journal of the American College of Medical Genetics, 19(1).

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Genetic Evaluation of Von Hippel-Lindau Disease

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Patients suspected of being affected with Von Hippel–Lindau disease (VHL) were evaluated on an NCI IRB-approved protocol (NCT00001238). Family history, clinical phenotype, tumor characteristics, and mutational assessment were evaluated. Germline VHL mutation testing was performed using Clinical Laboratory Improvement Amendments (CLIA) certified laboratories.
For patients without clinical suspicion of germline VHL, tumor mutation analysis for VHL alterations was performed on an NCI IRB approved protocol (NCT000026884). For this analysis, DNA was extracted from fresh frozen tissue using a Promega Maxwell 16 Tissue DNA Purification Kit or from formalin-fixed, paraffin-embedded tissue using a Maxwell 16 FFPE Tissue LEV DNA Purification Kit (Madison, WI, USA). Quantification of extracted DNA was performed using a ThermoScientific Nanodrop 1000 (Wilmington, DE, USA). A Qiagen Taq PCR Core Kit (Germantown, MD, USA) was used to amplify all three VHL exons. Purified DNA products were sequenced bidirectionally using the Big Dye Terminator v.1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s specifications and run on an ABI 3130xl Genetic Analyzer (Applied Biosystems). Forward and reverse sequences were evaluated using Sequencher 4.10.01 (Ann Arbor, MI, USA).

Turkbey B., Lindenberg M.L., Adler S., Kurdziel K.A., McKinney Y.L., Weaver J., Vocke C.D., Anver M., Bratslavsky G., Eclarinal P., Kwarteng G., Lin F.I., Yaqub-Ogun N., Merino M.J., Linehan W.M., Choyke P.L, & Metwalli A.R. (2016). PET/CT imaging of renal cell carcinoma with 18F-VM4-037: a phase II pilot study. Abdominal radiology (New York), 41(1), 109-118.

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VHL Gene Mutation Analysis from FFPE

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Total DNA was extracted from 3 to 4 tissue cylinders (diameter 0.6 mm) punched from each FFPE block and processed following the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Germany) or the Maxwell® 16 FFPE Tissue LEV DNA Purification Kit (Promega corporation,USA).
The first 162 base pairs of VHL are rarely mutated and were excluded from sequence analysis [15 ]. The primers used for amplification were 5’-agagtccggcccggaggaact-3’ forward, 5’-gaccgtgctatcgtccctgc-3’ reverse for exon 1, 5’-accggtgtggctctttaaca-3’ forward and 5’-tcctgtacttaccacaacaacctt-3’ reverse for exon 2, and 5’-gagaccctagtctgtcactgag-3’ forward and 5’-tcatcagtaccatcaaaagctga-3’ reverse for exon 3. The forward and reverse DNA sequences overlap and cover the VHL sequence excluding the first 162 base pairs (Additional file 1). Sequencing was performed as described previously [23 (link)]. The sequences were aligned and compared to the NCBI sequence AF010238 using the informatics tool Sequencher (Sequencher® version 5.3 sequence analysis software, Gene Codes Corporation, Ann Arbor, MI USA, [37 ]). All VHL mutations were validated by a second independent PCR and sequence analysis.

Razafinjatovo C., Bihr S., Mischo A., Vogl U., Schmidinger M., Moch H, & Schraml P. (2016). Characterization of VHL missense mutations in sporadic clear cell renal cell carcinoma: hotspots, affected binding domains, functional impact on pVHL and therapeutic relevance. BMC Cancer, 16, 638.

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Extraction and Analysis of Nucleic Acids from FFPE Tissue

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Tumor tissue genomic DNA was extracted using the Maxwell^® 16 FFPE Tissue LEV DNA Purification Kit (Promega, Madison, WI) according to manufacturer’s specifications. DNA yields varied from 2–50μg per case.
RNA isolation was performed using the Roche High Pure FFPET RNA Isolation spin-column kit according to manufacturer’s instructions. RNA was then quantified using the NanoDrop 1000 (Thermo Scientific), and its quality was assessed with the Bioanalyzer 2100 RNA Nano kit for Eukaryotic Total RNA (Agilent Technologies).

Nfonsam V., Xu W., Koblinski J, & Jandova J. (2016). Gene Expression Analysis of Sporadic Early-Onset Rectal Adenocarcinoma. Gastrointestinal cancer : research & therapy, 1(1), 1005.

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Genetic Polymorphism Analysis in Liver Transplantation

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The recipients’ genetic material for IFNL3 polymorphism testing was obtained from fragments of the patients’ own liver removed during transplantation and stored as paraffin-embedded blocks. The donors’ genetic material was obtained through time-zero biopsies preceding reperfusion and was stored as paraffin-embedded blocks.
DNA was isolated using the Maxwell^®16 (Promega GmbH, Germany) instrument for nucleic acid and protein isolation. The Maxwell^® 16 FFPE Tissue LEV DNA Purification Kit (Promega GmbH, Germany) was used. Nucleotide sequences within rs12979860 were determined using allele-specific probes and the Custom TaqMan^® SNP Genotyping Assay (Applied Biosystems, A Thermo Fisher Scientific Brand). Possible single-nucleotide polymorphisms (SNPs) C > T withinrs12979860 included CC, CT, and TT. IFNL3 rs8099917 G > T genotyping was performed using the TaqMan^® Pre-Designed SNP Genotyping Assay (Applied Biosystems, A Thermo Fisher Scientific Brand). Possible SNP variants included TT, GT, and GG.
IFNL3 rs12979860 genotyping was performed for 88% of the recipients and 96% of the donors as well as for 120 recipient–donor pairs. In the case of the rs8099917 polymorphism, successful genotyping was performed for 84% of the recipients and 96% of the donors as well as 115 recipient–donor pairs.

Wieczorek-Godlewska R., Płoski R., Perkowska-Ptasińska A., Tronina O., Sadowska A., Pacholczyk M., Lisik W, & Durlik M. (2017). The impact of the recipient and donor interferon lambda-3 polymorphism on the course of HCV infection following liver transplantation. Clinical and Experimental Hepatology, 3(3), 152-158.

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Optimized FFPE DNA Extraction and Quantification

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For DNA preparation from FFPE samples, we used the Maxwell 16 FFPE Tissue LEV DNA Purification Kit (Promega, Madison, WI, USA, Cat#AS1130) according to the manufacturer’s instructions. Thereafter, DNA concentration was measured using a Qubit 3.0 Fluorometer dsDNA BR Assay Kit (Life Technologies, Grand Island, NY, USA, Cat#Q32850), and DNA quality was monitored using the QIAseq DNA QuantiMIZE Kit (QIAGEN, Reston, VA, USA, Cat#333414). The extracted DNA was diluted to 5–10 ng/μL as a template, and PCR was performed using the QIAseq DNA QuantiMIZE Kit (QIAGEN, Cat#333414).

Higa N., Akahane T., Yokoyama S., Makino R., Yonezawa H., Uchida H., Takajo T., Kirishima M., Hamada T., Noguchi N., Otsuji R., Kuga D., Nagasaka S., Yamahata H., Yamamoto J., Yoshimoto K., Tanimoto A, & Hanaya R. (2023). Favorable prognostic impact of phosphatase and tensin homolog alterations in wild-type isocitrate dehydrogenase and telomerase reverse transcriptase promoter glioblastoma. Neuro-Oncology Advances, 5(1), vdad078.

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Comprehensive SI-NET Sample Analysis

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Tissue and tumor samples as well as patient data used in this study were provided by the University Cancer Center Frankfurt (UCT). Written informed consent was obtained from all patients and the study was approved by the institutional Review Boards of the UCT and the Ethics Committee at the University Hospital Frankfurt (project-number: SGI-OW-01/2013). All methods were performed in accordance with the declaration of Helsinki. Only patients with available FFPE tissue of each the primary lesion, the metastasis and the matched normal tissue were included. The definite diagnosis of SI-NET was confirmed by two expert gastrointestinal pathologists. Percentage of tumor content was assessed based on hematoxylin and eosin stained sections. After macrodissection, DNA was extracted with the Maxwell 16 FFPE tissue LEV DNA purification kit (Promega, Madison, WI) from FFPE material according to manufacturer’s recommendations. DNA yield was quantified with Quantus Fluorometer (Promega).

Walter D., Harter P.N., Battke F., Winkelmann R., Schneider M., Holzer K., Koch C., Bojunga J., Zeuzem S., Hansmann M.L., Peveling-Oberhag J, & Waidmann O. (2018). Genetic heterogeneity of primary lesion and metastasis in small intestine neuroendocrine tumors. Scientific Reports, 8, 3811.

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DNA Extraction and Sequence Analysis

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About 30–40 mg of frozen tissue and the left over cells of FNA were used for genomic DNA extraction using the Maxwell^® 16 Tissue DNA Purification Kit (Promega Corporation, Madison, WI, USA). Genomic DNA was eluted in 300 µl of water. For FFPE tissues, one or two 5 µm sections were deparaffinized and digested with proteinase K overnight. DNA extraction was performed using the Maxwell^® 16 FFPE Tissue LEV DNA Purification Kit (Promega Corporation). DNA was finally diluted in 50–100 µl of water. DNA concentration was measured using an UV spectrophotometer (SmartSpec Plus Spectrophotometer; Bio-Rad Laboratories, Inc., Hercules, CA, USA), and 30 ng of genomic DNA was used for PCR in a final volume of 20 µl using a master mix (Kapa2G fast hs ready mix; Resnova S.r.l., Genzano di Roma, Italy).
Primers and PCR conditions were specially defined to the purpose of the present study (Table I). The amplified DNA was analyzed on 2% agarose gels and purified using a commercial kit (Jetquick purification kit; Celbio, Milan, Italy). Sequence analysis was performed with an automated system employing fluorescent dye terminators (ABI Prism 3110; PerkinElmer, Inc., Waltham, MA, USA). Hot spot positions of NRAS, HRAS, KRAS, BRAF, AKT, PIK3CA, PTEN, TP53, and TERT oncogenes have been sequenced in this study.

Romei C., Tacito A., Molinaro E., Piaggi P., Cappagli V., Pieruzzi L., Matrone A., Viola D., Agate L., Torregrossa L., Ugolini C., Basolo F., De Napoli L., Curcio M., Ciampi R., Materazzi G., Vitti P, & Elisei R. (2018). Clinical, pathological and genetic features of anaplastic and poorly differentiated thyroid cancer: A single institute experience. Oncology Letters, 15(6), 9174-9182.

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