By Sangpen Chamnongpol, Patricia A. Maroney, Timothy W. Nilsen (auth.), Silvia Monticelli (eds.)
In fresh years, the severe function of microRNAs has been published in the biology of cells that represent the immune process. In MicroRNAs and the Immune approach: tools and Protocols, specialist researchers discover the newest ideas for learning miRNA expression, together with the main up to date information on splinted ligation and qRT-PCR assays, in addition to high-throughput profiling via cloning, deep sequencing, and microarrays. Chapters define the right way to examine miRNA capabilities in quite a few telephone varieties from a unmarried phone kind point to whole version organisms, and current stories of miRNAs within the context of viruses and the immune reaction. instruments also are supplied to aid navigate bioinformatics databases on miRNAs and their goals. Composed within the hugely profitable equipment in Molecular Biology™ sequence layout, each one bankruptcy includes a short advent, step by step tools, a listing of important fabrics, and a Notes part which stocks pointers on troubleshooting and keeping off recognized pitfalls. modern and cutting edge, MicroRNAs and the Immune approach: tools and Protocols is a necessary guide for immunologists, biochemists, and molecular biologists.
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Additional info for MicroRNAs and the Immune System: Methods and Protocols
0. 5. Sheep anti-DIG-FITC conjugated antibody, Fab fragments, 200 mg (Roche Applied Science). The lyophilised antibody is resuspended in 1 mL of ddH2O. 5 mg/mL in blocking solution briefly before use (See item 11 below for blocking solution recipe and Note 8). 50× Denhardt’s solution (Sigma-Aldrich). Yeast tRNA (Invitrogen). 5 mg/mL yeast tRNA. The buffer can be stored in aliquots at −80°C. RNase-free coverslip (H18200, Invitrogen). Humidified HYBrite™ slide incubation chamber (Abbott Laboratories Ltd).
Science 304, 594–6. 5. , Miska, E. , et al. (2005) MicroRNA expression profiles classify human cancers. Nature 435, 834–8. 6. , Calin, G. , Liu, C. , et al. (2006) A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci U S A 103, 2257–61. 7. Calin, G. , Dumitru, C. , et al. (2002) Frequent deletions and down-regulation of micro-RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A 99, 15524–9. 8. , et al. (2004) Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival.
Struct. Mol. Biol. 13, 1102–7. 15. , and Russell, D. W. 4. Chapter 2 Normalization of MicroRNA Quantitative RT-PCR Data in Reduced Scale Experimental Designs Gary J. Latham Abstract Proper normalization of quantitative RT-PCR (qRT-PCR) data is a crucial component of gene expression analysis. Although arbitrarily selected housekeeping genes have been used to normalize many published mRNA RT-PCR datasets, there is a growing awareness that such normalizers should be first validated empirically. The use of stable reference genes is particularly needed for qRT-PCR of microRNA (miRNA), which represent a novel class of biological regulators whose aberrant expression is associated with a range of disorders.