RESUMEN
Ribosomal RNAs (rRNAs) are extremely abundant, often constituting 80% to 90% of total RNA. Since rRNA sequences are often not of interest in genomic RNA sequencing experiments, rRNAs can be removed from the sample before the library preparation step, in order to prevent the majority of the library and the majority of sequencing reads from being rRNA. Removal of rRNA can be especially challenging for low quality and formalin-fixed paraffin-embedded (FFPE) RNA samples due to the fragmented nature of these RNA molecules. The NEBNext rRNA Depletion Kit (Human/Mouse/Rat) depletes both cytoplasmic (5 S rRNA, 5.8 S rRNA, 18 S rRNA, and 28 S rRNA) and mitochondrial rRNA (12 S rRNA and 16 S rRNA) from total RNA preparations from human, mouse, and rat samples. Due to the high similarity among mammalian rRNA sequences, it is likely that rRNA depletion can also be achieved for other mammals but has not been empirically tested. This product is compatible with both intact and degraded RNA (e.g., FFPE RNA). The resulting rRNA-depleted RNA is suitable for RNA-seq, random-primed cDNA synthesis, or other downstream RNA analysis applications. Regardless of the quality or amount of input RNA, this method efficiently removes rRNA, while retaining non-coding and other non-poly(A) RNAs. The NEBNext rRNA Depletion Kit thus provides a more complete picture of the transcript repertoire than oligo d(T) poly(A) mRNA enrichment methods. © 2016 by John Wiley & Sons, Inc.
RESUMEN
Changes in protein phosphorylation mediate much of cellular physiology. Perturbations in the activity of the kinases that catalyze these reactions underlie numerous human pathologies, including metabolic and inflammatory disorders and most notably, cancer. HTS techniques that determine the activity of protein kinases in vitro are useful in the development of small molecule kinase inhibitors, but do not address underlying mechanistic concerns or efficient in vivo targeting. Observing protein phosphorylation in cell lysates and fixed cells in a high throughput manner is fundamental to understanding the mechanism of action of lead molecules and whether they target signaling pathways of interest. Herein we discuss several higher throughput techniques to study cellular protein kinase signal transduction and the strategies for implementation in kinase drug discovery.
Asunto(s)
Proteínas de la Membrana/metabolismo , Preparaciones Farmacéuticas/metabolismo , Tecnología Farmacéutica/métodos , Animales , Humanos , Fosforilación , Tecnología Farmacéutica/tendenciasRESUMEN
The signaling machinery in cells is a complex, multi-factorial network of cross-talking proteins that enables dynamic communication between upstream causal factors and downstream effectors. Non-receptor tyrosine kinases, including Src, are the intermediates of information transfer, controlling pathways as diverse as cell growth, migration, death, and genome maintenance. When expressed as viral genes these proteins are potent carcinogens, yet analogous genetic alterations are rarely observed in human tumors. In seeking to characterize the role of the non-receptor tyrosine kinase Src in neoplasia, arguments can be made that the consequences of mutation, or perturbations in the activity or expression of this protein is a determinative factor in clinical prognosis and pathogenicity. In a variety of tumor types including those derived from the colon and breast, the Src non-receptor tyrosine kinase is either overexpressed or constitutively active in a large percentage of the tumors. Increased expression or activity of Src correlates with the stage and metastatic potential of some neoplasia.
Asunto(s)
Neoplasias de la Mama/metabolismo , Transformación Celular Neoplásica , Neoplasias del Colon/metabolismo , Genes src/fisiología , Neoplasias de la Mama/genética , Pruebas de Carcinogenicidad , Neoplasias del Colon/genética , Genes src/genética , Humanos , MutaciónRESUMEN
The signaling machinery in cells is a complex, multi-factorial network of cross-talking proteins that enables dynamic communication between upstream causal factors and downstream effectors. Non-receptor tyrosine kinases, including Src, are the intermediates of information transfer, controlling pathways as diverse as cell growth, migration, death, and genome maintenance. When expressed as viral genes these proteins are potent carcinogens, yet analogous genetic alterations are rarely observed in human tumors. In seeking to characterize the role of the non-receptor tyrosine kinase Src in neoplasia, arguments can be made that the consequences of mutation, or perturbations in the activity or expression of this protein is a determinative factor in clinical prognosis and pathogenicity. In a variety of tumor types including those derived from the colon and breast, the Src non-receptor tyrosine kinase is either overexpressed or constitutively active in a large percentage of the tumors. Increased expression or activity of Src correlates with the stage and metastatic potential of some neoplasia.