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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.

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