Springer protocols feed by Cancer Research 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.A Diphtheria Toxin Negative Selection in RNA Interference Screening
RNA interference (RNAi) screening is a powerful technique for understanding the molecular biology of cancer and searching drug targets. Genes and their upstream activators that are essential for the survival of cancer cells often dictate cancer formation/progression. Hence, they are preferable therapeutic targets. Identifying these genes using RNAi is, however, problematic because knocking them down leads to cell death. Here we describe a diphtheria toxin (DT) negative selection method to circumvent the problem of cell death in RNAi screening. DT fails to kill mouse cells due to the lack of functional DT receptor (DTR). Th...
Source: Springer protocols feed by Cancer Research - July 17, 2014 Category: Cancer & Oncology Source Type: news
Using Pooled miR3-shRNA Library for Cancer Lethal and Synthetic Lethal Screens
Pooled shRNA library is a powerful, rapid, and cost-effective technology to carry out functional genomic screens in mammalian cells. This approach has been applied extensively to identify genetic dependencies in cancer cells that might be exploited for therapeutic purposes. In this chapter we provide a detailed protocol for using the Hannon–Elledge miR30-based library to conduct dropout screens in cancer cell lines. This protocol is readily adaptable to other pooled shRNA libraries and should facilitate the functional annotation of the human genome. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - July 17, 2014 Category: Cancer & Oncology Source Type: news
A High-Throughput MicroRNA Expression Profiling System
We describe here a robust protocol that supports high-throughput sample labeling and detection on hundreds of samples simultaneously. This method employs 96-well-based miRNA capturing from total RNA samples and on-site biochemical reactions, coupled with bead-based detection in 96-well format for hundreds of miRNAs per sample. With low-cost, high-throughput, high detection specificity, and flexibility to profile both small and large numbers of samples, this protocol can be adapted in a wide range of laboratory settings. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - July 17, 2014 Category: Cancer & Oncology Source Type: news
Reduced Representation Bisulfite Sequencing to Identify Global Alteration of DNA Methylation
Reduced representation bisulfite sequencing is a cost-effective high-throughput sequencing-based method to obtain DNA methylation status at a single-nucleotide level. DNA methylation status is determined by utilizing DNA methylation-specific restriction enzymes to selectively amplify for genomic regions that are rich in methylated DNA. Although the method is genome-wide, DNA methyl sequencing does not require the sequencing of the whole genome, hence the name “reduced representation.” However, a large majority of CpG islands are covered by reduced representation bisulfite sequencing allowing for the acquisition...
Source: Springer protocols feed by Cancer Research - July 17, 2014 Category: Cancer & Oncology Source Type: news
Using Native Chromatin Immunoprecipitation to Interrogate Histone Variant Protein Deposition in Embryonic Stem Cells
Chromatin immunoprecipitation combined with massive parallel sequencing (ChIP-Seq) is a powerful epigenetics technique for interrogating the genome-wide localization of histone modifications, histone variants, and other chromatin-associating factors. In brief, chromatin pellets are fractionated from the nuclei, and then fragmented by enzymatic digestion or sonication. Chromatin regions associated with proteins of interest are enriched by immunoprecipitation with specific antibodies. After the immunoprecipitation, DNA fragments are extracted, amplified during sequencing library construction, and sequenced by high-throughput...
Source: Springer protocols feed by Cancer Research - July 17, 2014 Category: Cancer & Oncology Source Type: news
Computational Methods for DNA Copy-Number Analysis of Tumors
Study of DNA copy-number variation is a key part of cancer genomics. With the help of a comprehensive multistep computational procedure described here, copy-number profiles of tumor tissues or individual tumor cells may be generated and interpreted, starting with data acquired by next-generation sequencing. Several of the methods presented are specifically designed to handle cancer-related copy-number profiles. These include accounting for variation of ploidy and distilling somatic copy number alterations from the inherited background. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - July 17, 2014 Category: Cancer & Oncology Source Type: news
Genome-Wide Mapping of RNA Pol-II Promoter Usage in Mouse Tissues by ChIP-Seq
Chromatin immunoprecipitation (ChIP), using antibody against RNA Pol-II, followed by massive parallel sequencing (ChIP-seq) are invaluable techniques for genome-wide identification of alternative promoters and their patterns of use in different tissues, cell types, and/or developmental stages. However, the identification of promoters cannot be performed solely based on the presence of Pol-II enrichment on a genomic location because of its enrichment throughout the transcribed genomic region and lack of highly specific antibodies that can distinguish promoter-bound Pol-II from elongating Pol-II. In order to overcome this li...
Source: Springer protocols feed by Cancer Research - July 17, 2014 Category: Cancer & Oncology Source Type: news
Matrix Factorization Methods for Integrative Cancer Genomics
With the rapid development of high-throughput sequencing technologies, many groups are generating multi-platform genomic profiles (e.g., DNA methylation and gene expression) for their biological samples. This activity has generated a huge number of so-called “multidimensional genomic datasets,” providing unique opportunities and challenges to study coordination among different regulatory levels and discover underlying combinatorial patterns of cellular systems. We summarize a matrix factorization framework to address the challenge of integrating multiple genomic datasets, as well as a semi-supervised variant of...
Source: Springer protocols feed by Cancer Research - July 17, 2014 Category: Cancer & Oncology Source Type: news
Computational Analysis in Cancer Exome Sequencing
Exome sequencing in cancer is a powerful tool for identifying mutational events across the coding region of human genes. Here, we describe computational methods that use exome sequencing reads from cancer samples to identify somatic single nucleotide variants (SNVs), copy number alterations, and short insertions and deletions (InDels). We further describe analytical methods to generate lists of driver genes with more mutational events than expected by chance. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - July 17, 2014 Category: Cancer & Oncology Source Type: news
Purification of Recombinant 2XMBP Tagged Human Proteins from Human Cells
The ability to purify an intact, functional protein or protein complex is an essential step in biochemical characterization studies. Challenges in purification arise when proteins are of low abundance or are unstable resulting in low yields or poor in vitro activity. In this protocol we describe a method to purify active, recombinant human proteins fused to a tandem MBP tag after expression in human 293T cells. (Source: Springer protocols feed by Cancer Research)
Source: Springer protocols feed by Cancer Research - July 17, 2014 Category: Cancer & Oncology Source Type: news
New Biophysical Methods to Study the Membrane Activity of Bcl-2 Proteins
The proteins of Bcl-2 family are key regulators of apoptosis. Many Bcl-2 proteins have the unique ability to switch between two possible conformations, soluble in the cytosol or associated to cellular membranes. Importantly, their membrane-inserted form is the main responsible for their apoptotic function. Unfortunately, there are only a limited number of methods available to study the membrane activity of these proteins. Here, we present a methodology to study protein binding to membranes and membrane permeabilization at the single vesicle level. It is based on purified proteins and giant unilamellar vesicles and involves...
Source: Springer protocols feed by Cancer Research - July 17, 2014 Category: Cancer & Oncology Source Type: news
Interrogation of In Vivo Protein–Protein Interactions Using Transgenic Mouse Models and Stable Isotope Labeling
Methods in mass spectrometry have evolved in recent years, facilitating proteomic analyses that were previously beyond the limits of the technology. Transgenic mouse models, coupled with mass spectrometry proteomics, have served as valuable platform for elucidating the in vivo function of individual genes and proteins. Here we discuss the methods we have recently employed to characterize protein–protein interactions and posttranslational modifications in tagged knock-in mouse models. These methods can be broadly applied to other systems for various applications in both basic and translational science. (Source: Spring...
Source: Springer protocols feed by Cancer Research - July 17, 2014 Category: Cancer & Oncology Source Type: news
Serum Profiling Using Protein Microarrays to Identify Disease Related Antigens
Disease related antigens are of great importance in the clinic. They are used as markers to screen patients for various forms of cancer, to monitor response to therapy, or to serve as therapeutic targets (Chapman et al., Ann Oncol 18(5):868–873, 2007; Soussi et al., Cancer Res 60:1777–1788, 2000; Anderson and LaBaer, J Proteome Res 4:1123–1133, 2005; Levenson, Biochim Biophy Acta 1770:847–856, 2007). In cancer endogenous levels of protein expression may be disrupted or proteins may be expressed in an aberrant fashion resulting in an immune response that bypasses self tolerance (Soussi et al., Cancer...
Source: Springer protocols feed by Cancer Research - July 17, 2014 Category: Cancer & Oncology Source Type: news
Clonal Screens to Find Modifiers of Partially Penetrant Phenotypes in C. elegans
Unbiased genetic screens are an excellent way to discover novel genes involved in specific biological processes in vivo. Modifier screens, whether to suppress or enhance a phenotype, are a powerful way to find proteins that modulate biological processes responsible for specific phenotypes. However, modification of phenotypes that are only partially penetrant, which is often the case, are often extremely difficult to screen this way in a traditional F2 or non-clonal genetic screen. Here we describe an F3 or clonal screen in the nematode Caenorhabditis elegans to search for genes that modify partially penetrant phenotypes. S...
Source: Springer protocols feed by Cancer Research - July 17, 2014 Category: Cancer & Oncology Source Type: news
Zebrafish as a Platform to Study Tumor Progression
The zebrafish has emerged as a powerful model system to study human diseases, including a variety of neoplasms. Principal components that have contributed to the rise in use of this vertebrate model system are its high fecundity, ease of genetic manipulation, and low cost of maintenance. Vital imaging of the zebrafish is possible from the transparent embryonic stage through adulthood, the latter enabled by a number of mutant lines that ablate pigmentation. As a result, high-resolution analyses of tumor progression can be accomplished in vivo. Straightforward transgenesis of zebrafish has been employed to develop numerous t...
Source: Springer protocols feed by Cancer Research - July 17, 2014 Category: Cancer & Oncology Source Type: news
