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In JoVE (1)
Other Publications (5)
Articles by Larissa A. Pikor in JoVE
DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses
Larissa A. Pikor*1,2, Katey S. S. Enfield*1,2, Heryet Cameron3, Wan L. Lam1,2,4
1Department of Integrative Oncology, BC Cancer Research Centre, 2Interdisciplinary Oncology Program, University of British Columbia - UBC, 3Photography/Video Production, Multi-Media Services, BC Cancer Agency, 4Department of Pathology and Laboratory Medicine, University of British Columbia - UBC
This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.
Published March 26, 2011. Keywords: Genetics, DNA extraction, paraffin embedded tissue, phenol:chloroform extraction, genetic analysis, epigenetic analysis
Other articles by Larissa A. Pikor on PubMed
Journal of Cellular Physiology. Mar, 2010 | Pubmed ID: 20020444
DNA methylation regulates gene expression primarily through modification of chromatin structure. Global methylation studies have revealed biologically relevant patterns of DNA methylation in the human genome affecting sequences such as gene promoters, gene bodies, and repetitive elements. Disruption of normal methylation patterns and subsequent gene expression changes have been observed in several diseases especially in human cancers. Immunoprecipitation (IP)-based methods to evaluate methylation status of DNA have been instrumental in such genome-wide methylation studies. This review describes techniques commonly used to identify and quantify methylated DNA with emphasis on IP based platforms. In an effort to consolidate the wealth of information and highlight critical aspects of methylated DNA analysis, sample considerations, experimental and bioinformatic approaches for analyzing genome-wide methylation profiles, and the benefit of integrating DNA methylation data with complementary dimensions of genomic data are discussed.
BMC Medical Genomics. 2010 | Pubmed ID: 20682026
An important consideration when analyzing both microarray and quantitative PCR expression data is the selection of appropriate genes as endogenous controls or reference genes. This step is especially critical when identifying genes differentially expressed between datasets. Moreover, reference genes suitable in one context (e.g. lung cancer) may not be suitable in another (e.g. breast cancer). Currently, the main approach to identify reference genes involves the mining of expression microarray data for highly expressed and relatively constant transcripts across a sample set. A caveat here is the requirement for transcript normalization prior to analysis, and measurements obtained are relative, not absolute. Alternatively, as sequencing-based technologies provide digital quantitative output, absolute quantification ensues, and reference gene identification becomes more accurate.
Journal of Biomedicine & Biotechnology. 2011 | Pubmed ID: 21541180
Chemotherapy resistance is a key contributor to the dismal prognoses for lung cancer patients. While the majority of studies have focused on sequence mutations and expression changes in protein-coding genes, recent reports have suggested that microRNA (miRNA) expression changes also play an influential role in chemotherapy response. However, the role of genetic alterations at miRNA loci in the context of chemotherapy response has yet to be investigated. In this study, we demonstrate the application of an integrative, multidimensional approach in order to identify miRNAs that are associated with chemotherapeutic resistance and sensitivity utilizing publicly available drug response, miRNA loci copy number, miRNA expression, and mRNA expression data from independent resources. By instigating a logical stepwise strategy, we have identified specific miRNAs that are associated with resistance to several chemotherapeutic agents and provide a proof of principle demonstration of how these various databases may be exploited to derive relevant pharmacogenomic results.
Genetic Disruption of KEAP1/CUL3 E3 Ubiquitin Ligase Complex Components is a Key Mechanism of NF-kappaB Pathway Activation in Lung Cancer
Journal of Thoracic Oncology : Official Publication of the International Association for the Study of Lung Cancer. Sep, 2011 | Pubmed ID: 21795997
Inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta (IKBKB) (IKK-Î²/IKK-2), which activates NF-ÎºB, is a substrate of the KEAP1-CUL3-RBX1 E3-ubiquitin ligase complex, implicating this complex in NF-ÎºB pathway regulation. We investigated complex component gene disruption as a novel genetic mechanism of NF-ÎºB activation in non-small cell lung cancer.
Integrative Genomics Identified RFC3 As an Amplified Candidate Oncogene in Esophageal Adenocarcinoma
Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. Feb, 2012 | Pubmed ID: 22328562
PURPOSE: Esophageal adenocarcinoma (EAC) is a lethal malignancy that can develop from the premalignant condition, Barrett's esophagus (BE). Currently, there are no validated simple methods to predict which patients will progress to EAC. A better understanding of the genetic mechanisms driving EAC tumorigenesis is needed to identify new therapeutic targets and develop biomarkers capable of identifying high-risk patients that would benefit from aggressive neoadjuvant therapy. We employed an integrative genomics approach to identify novel genes involved in EAC biology that may serve as useful clinical markers.EXPERIMENTAL DESIGN: Whole genome tiling-path array CGH was used to identify significant regions of copy number (CN) alteration in 20 EACs and 10 matching BE tissues. CN and gene expression data were integrated to identify candidate oncogenes within regions of amplification and multiple additional sample cohorts were assessed to validate candidate genes.RESULTS: We identified RFC3 as a novel, candidate oncogene activated by amplification in ~25% of EAC samples. RFC3 was also amplified in BE from a patient whose EAC harbored amplification, and was differentially expressed between non-malignant and EAC tissues. CN gains were detected in other cancer types and RFC3 knockdown inhibited proliferation and anchorage-independent growth of cancer cells with increased CN, but had little effect on those without. Moreover, high RFC3 expression was associated with poor patient outcome in multiple cancer types.CONCLUSIONS: RFC3 is a candidate oncogene amplified in EAC. RFC3 DNA amplification is also prevalent in other epithelial cancer types and RFC3 expression could serve as a prognostic marker.