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ffpe dna isolation

Hossein Ayatollahi, Mohammad Hadi Sadeghian, Mohammad Reza Keramati, Ali Ayatollahi, Arezoo Shajiei, Maryam Sheikhi, Samane Bakhshi
BACKGROUND: Nowadays, definitive diagnosis of numerous diseases is based on the genetic and molecular findings. Therefore, preparation of fundamental materials for these evaluations is necessary. Deoxyribonucleic acid (DNA) is the first material for the molecular pathology and genetic analysis, and better results need more pure DNA. Furthermore, higher concentration of achieved DNA causes better results and higher amplifying ability for subsequent steps. We aim to evaluate five DNA extraction methods to compare DNA intimacy including purity, concentration, and amplifying ability with each other...
July 2016: Nigerian Medical Journal: Journal of the Nigeria Medical Association
Hilmi Kodaz, Ebru Taştekin, Bülent Erdoğan, İlhan Hacıbekiroğlu, Hilmi Tozkır, Hakan Gürkan, Esma Türkmen, Bora Demirkan, Sernaz Uzunoğlu, İrfan Çiçin
BACKGROUND: Lung cancer is one of the most lethal cancers. It is mainly classified into 2 groups: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Extrapulmonary small cell carcinomas (EPSCC) are very rare. The Ras oncogene controls most of the cellular functions in the cell. Overall, 21.6% of human cancers contain a Kirsten Ras (KRAS) mutation. SCLC and EPSCC have several similar features but their clinical course is different. AIMS: We investigated the KRAS mutation status in SCLC and EPSCC...
July 2016: Balkan Medical Journal
Monica Molano, Sepehr N Tabrizi, Suzanne M Garland, Jennifer M Roberts, Dorothy A Machalek, Samuel Phillips, David Chandler, Richard J Hillman, Andrew E Grulich, Fengyi Jin, I Mary Poynten, David J Templeton, Alyssa M Cornall
Incidence and mortality rates of anal cancer are increasing globally. More than 90% of anal squamous cell carcinomas (ASCC) are associated with human papillomavirus (HPV). Studies on HPV-related anogenital lesions have shown that patterns of methylation of viral and cellular DNA targets could potentially be developed as disease biomarkers. Lesion-specific DNA isolated from formalin-fixed paraffin-embedded (FFPE) tissues from existing or prospective patient cohorts may constitute a valuable resource for methylation analysis...
2016: PloS One
Beata Biesaga, Anna Janecka, Anna Mucha-Małecka, Agnieszka Adamczyk, Sława Szostek, Dorota Słonina, Krzysztof Halaszka, Marcin Przewoźnik
The aim of the present study was to compare HPV16 detection by quantitative polymerase chain reaction (qPCR) in relation to the quantity and quality of DNA isolated from 21 formalin fixed and paraffin embedded (FFPE) head and neck cancer tissues by three commercially available kits: EX-WAX™ DNA Extraction Kit (M) (Merck Millipore, Darmstadt, Germany), QIAamp(®) DNA FFPE Tissue (Q) (Qiagen, Hilden, Germany) and ReliaPrep™ FFPE gDNA Miniprep System (P) (Promega, Madison, USA). Quantity of extracted DNA was assessed spectrophometrically and fluorometrically...
October 2016: Journal of Virological Methods
Annette M Lim, Nicholas C Wong, Ruth Pidsley, Elena Zotenko, June Corry, Alexander Dobrovic, Susan J Clark, Danny Rischin, Benjamin Solomon
BACKGROUND: DNA methylation profiling of heterogeneous head and neck squamous cell carcinoma (HNSCC) cohorts has been reported to predict patient outcome. We investigated if a prognostic DNA methylation profile could be found in tumour tissue from a single uniform subsite, the oral tongue. The methylation status of 109 comprehensively annotated oral tongue squamous cell carcinoma (OTSCC) formalin-fixed paraffin-embedded (FFPE) samples from a single institution were examined with the Illumina HumanMethylation450K (HM450K) array...
2016: Clinical Epigenetics
Ying Liu, Deepak P Edward
AIM: To compare the effect of the two fixatives on tissue morphology and utility to obtain good quality nucleic acids for molecular analysis from micro-dissected retinal samples. METHODS: Enucleated specimens from New Zealand white rabbits were fixed in formalin or PAXgene fixative according to standard protocols, and then processed and embedded in paraffin for sectioning. Sections were stained with hematoxylin and eosin to assess the structural integrity of retina...
July 13, 2016: Current Eye Research
Rajesh R Singh, Meenakshi Mehrotra, Hui Chen, Alaa A Almohammedsalim, Ayesagul Sahin, Alex Bosamra, Keyur P Patel, Mark J Routbort, Xinyan Lu, Abraham Ronald, Bal Mukund Mishra, Shumaila Virani, L Jeffrey Medeiros, Rajyalakshmi Luthra
Gene copy number aberrations (CNAs) represent a major class of cancer-related genomic alterations that drive solid tumors. Comprehensive and sensitive detection of CNAs is challenging because of often low quality and quantity of DNA isolated from the formalin-fixed, paraffin-embedded (FFPE) solid tumor samples. Here, in a clinical molecular diagnostic laboratory, we tested the utility and validated a molecular inversion probe-based (MIP) array to routinely screen for CNAs in solid tumors. Using low-input FFPE DNA, the array detects genome-wide CNAs with a special focus on 900 cancer-related genes...
September 2016: Journal of Molecular Diagnostics: JMD
Mark Laible, Kornelia Schlombs, Katharina Kaiser, Elke Veltrup, Stefanie Herlein, Sotiris Lakis, Robert Stöhr, Sebastian Eidt, Arndt Hartmann, Ralph M Wirtz, Ugur Sahin
BACKGROUND: MammaTyper is a novel CE-marked in vitro diagnostic RT-qPCR assay which assigns routinely processed breast cancer specimens into the molecular subtypes Luminal A-like, Luminal B-like (HER2 positive or negative), HER2 positive (non-luminal) and Triple negative (ductal) according to the mRNA expression of ERBB2, ESR1, PGR and MKI67 and the St Gallen consensus surrogate clinical definition. Until now and regarding formalin-fixed, paraffin-embedded material (FFPE), this has been a task mostly accomplished by immunohistochemistry (IHC)...
2016: BMC Cancer
Sepideh Zununi Vahed, Abolfazl Barzegari, Yalda Rahbar Saadat, Somayeh Mohammadi, Nasser Samadi
INTRODUCTION: microRNAs (miRNAs) are considered to be novel molecular biomakers that could be exploited in the diagnosis and treatment of different diseases. The present study aimed to develop an efficient miRNA isolation method from different clinical specimens. METHODS: Total RNAs were isolated by Trizol reagent followed by precipitation of the large RNAs with potassium acetate (KCH3COOH), polyethylene glycol (PEG) 4000 and 6000, and lithium chloride (LiCl). Then, small RNAs were enriched and recovered from the supernatants by applying a combination of LiCl and ethanol...
2016: BioImpacts: BI
B L Vaatstra, D J Tisdall, M Blackwood, R A Fairley
CASE HISTORY: A retrospective study was conducted to investigate 11 outbreaks of presumptive fatal adenovirus infection diagnosed through two New Zealand diagnostic laboratories during 2014 and 2015. Outbreaks occurred in 6-12-month-old Friesian or Friesian cross cattle during autumn, winter and spring. Individual outbreaks were short in duration, with mortality rates ranging from 3/250 to 20/600 (1.2 to 3.3%). CLINICAL AND PATHOLOGICAL FINDINGS: Clinical signs included severe diarrhoea, depression, recumbency, and death...
September 2016: New Zealand Veterinary Journal
Candice Perry, Joon-Yong Chung, Kris Ylaya, Chel Hun Choi, Amari Simpson, Kaipo T Matsumoto, William A Smith, Stephen M Hewitt
Formalin-fixed paraffin-embedded (FFPE) tissue is the predominant preparation for diagnostic histopathological evaluation and increasingly the biospecimen on which molecular diagnostics are performed. However, formalin is carcinogenic and results in cross-linking of proteins and nicking and alterations of nucleic acids. Alternative fixatives, including 70% ethanol, improved biomolecular integrity; however, they have yet to replace neutral-buffered formalin (NBF). Herein, we describe the phosphate-buffered ethanol 70% (BE70) fixative...
July 2016: Journal of Histochemistry and Cytochemistry: Official Journal of the Histochemistry Society
Florenza Lüder Ripoli, Annika Mohr, Susanne Conradine Hammer, Saskia Willenbrock, Marion Hewicker-Trautwein, Silvia Hennecke, Hugo Murua Escobar, Ingo Nolte
Mammary neoplasms are the tumors most affecting female dogs and women. Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable source of archived biological material. Fresh frozen (FF) tissue is considered ideal for gene expression analysis. However, strategies based on FFPE material offer several advantages. Branched-DNA assays permit a reliable and fast workflow when analyzing gene expression. The aim of this study was to assess the comparability of the branched-DNA assay when analyzing certain gene expression patterns between FF and FFPE samples in canine mammary tumors...
2016: International Journal of Molecular Sciences
F M Cárcano, A H Lengert, D O Vidal, C Scapulatempo Neto, L Queiroz, H Marques, F Baltazar, G N Berardinelli, C M S Martinelli, E C A da Silva, R M Reis, L F Lopes
Testicular germ cell tumors (TGCT) are the most common malignant neoplasm in young men. DNA mismatch repair deficiency can lead to microsatellite instability (MSI), an important mechanism of genetic instability. A mutation of the BRAF gene has been implicated in the pathogenesis of several solid tumors and has recently become an important therapeutic target. The role of MSI and BRAF gene mutation in TGCT, particularly in refractory disease, is poorly understood and reported findings are controversial. In this study, we aimed to determine the frequency and clinical impact of MSI status and BRAF mutations in TGCT...
September 2016: Andrology
Ricardo De Paoli-Iseppi, Peter A Johansson, Alexander M Menzies, Kerith-Rae Dias, Gulietta M Pupo, Hojabr Kakavand, James S Wilmott, Graham J Mann, Nicholas K Hayward, Marcel E Dinger, Georgina V Long, Richard A Scolyer
The identification of recurrent driver mutations by whole-exome sequencing (WES) of fresh-frozen human cancers and the subsequent development of novel targeted therapies have recently transformed the treatment of many cancers including melanoma. In routine clinical practice, fresh-frozen tissue is rarely available and mutation testing usually needs to be carried out on archival formalin fixed, paraffin embedded (FFPE) tissue, from which DNA is typically fragmented, cross-linked and of lower quality. In this study we aimed to determine whether WES data generated from genomic DNA (gDNA) extracted from FFPE tissues can be produced reliably and of clinically-actionable standard...
April 2016: Pathology
Sirirat Seekhuntod, Paninee Thavarungkul, Nuntaree Chaichanawongsaroj
BACKGROUND: Patients with KRAS mutations do not respond to epidermal growth factor receptor (EGFR) inhibitors and fail to benefit from adjuvant chemotherapy. Mutation analysis of KRAS is needed before starting treatment with monoclonal anti-EGFR antibodies in patients with metastatic colorectal cancer (mCRC). The objective of this study is to develop a multiplex allele-specific PCR (MAS-PCR) assay to detect KRAS mutations. METHODS: We developed a single-tube MAS-PCR assay for the detection of seven KRAS mutations (G12D, G12A, G12R, G12C, G12S, G12V, and G13D)...
2016: PloS One
Amin El-Heliebi, Shukun Chen, Thomas Kroneis
Whole genome amplification (WGA) is a widely used technique allowing multiplying picogram amounts of target DNA by several orders of magnitude. The technique described here is based on heat-induced random fragmentation yielding DNA strands mainly ranging from 0.1 to 1 kb in length. The fragmented DNA is then subjected to library generation by annealing of adaptor sequences to both ends of the DNA fragments. Using primers hybridizing to the adapter sequences, the DNA is amplified by thermal cycling. This amplification typically yields > 2 mg DNA from a single cell, is suited for amplifying DNA isolated from (partly) degraded samples [e...
2015: Methods in Molecular Biology
Yongli Xiao, Zong-Mei Sheng, Jeffery K Taubenberger
The vast majority of surgical biopsy and post-mortem tissue samples are formalin-fixed and paraffin-embedded (FFPE), but this process leads to RNA degradation that limits gene expression analysis. As an example, the viral RNA genome of the 1918 pandemic influenza A virus was previously determined in a 9-year effort by overlapping RT-PCR from post-mortem samples. Using the protocols described here, the full genome of the 1918 virus was determined at high coverage in one high-throughput sequencing run of a cDNA library derived from total RNA of a 1918 FFPE sample after duplex-specific nuclease treatments...
2015: Current Protocols in Microbiology
Danielle Mercatante Carrick, Michele G Mehaffey, Michael C Sachs, Sean Altekruse, Corinne Camalier, Rodrigo Chuaqui, Wendy Cozen, Biswajit Das, Brenda Y Hernandez, Chih-Jian Lih, Charles F Lynch, Hala Makhlouf, Paul McGregor, Lisa M McShane, JoyAnn Phillips Rohan, William D Walsh, Paul M Williams, Elizabeth M Gillanders, Leah E Mechanic, Sheri D Schully
Next Generation Sequencing (NGS) technologies are used to detect somatic mutations in tumors and study germ line variation. Most NGS studies use DNA isolated from whole blood or fresh frozen tissue. However, formalin-fixed paraffin-embedded (FFPE) tissues are one of the most widely available clinical specimens. Their potential utility as a source of DNA for NGS would greatly enhance population-based cancer studies. While preliminary studies suggest FFPE tissue may be used for NGS, the feasibility of using archived FFPE specimens in population based studies and the effect of storage time on these specimens needs to be determined...
2015: PloS One
Jiayi Zheng, Fen Li, Jinfen Liu, Zhiwei Xu, Haibo Zhang, Qihua Fu, Jian Wang, Kun Sun
The purposes of this study are to investigate somatic NKX2-5 mutations in Chinese children with congenital heart disease (CHD) and assess the reliability of somatic mutation detection in formalin-fixed, paraffin-embedded (FFPE) tissues. The study cohort included frozen and FFPE cardiac tissues as well as blood samples from 85 Chinese children with CHD who had the cardiac operations. The right atrial appendage far from the diseased heart was used as normal control. Genomic DNA was isolated from cardiac tissues and blood samples using TIANamp Blood DNA kit...
2015: International Journal of Medical Sciences
Matthew J Marton, Andrew R McNamara, D Michele Nikoloff, Aki Nakao, Jonathan Cheng
The p53 tumor suppressor gene (TP53) is reported to be mutated in nearly half of all tumors and plays a central role in genome integrity. Detection of mutations in p53 can be accomplished by many assays, including the AmpliChip p53 Research Test. The AmpliChip p53 Research Test has been successfully used to determine p53 status in hematologic malignancies and fresh frozen solid tissues but there are few reports of using the assay with formalin fixed, paraffin-embedded (FFPE) tissue. The objective of this study was to describe analytical performance characterization of the AmpliChip p53 Research Test to detect p53 mutations in genomic DNA isolated from archival FFPE human ovarian tumor tissues...
2015: PloS One
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