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411 Copyright 2006, Elsevier Inc. All rights reserved. 1016/S0076-6879(06)11003-4 [3] troubleshooting microarrays 35 Southern, 1975), and membrane hybridization is robust and routine in most molecular biology laboratories (Sambrook and Russell, 2001). Regardless of the particulars of scale or substrate, the principles governing hybridization between labeled nucleic acid in solution and immobilized nucleic acid remain the same (Anderson and Young, 1985). The chief operational differences between standard membrane hybridizations and microarray hybridizations are (i) the nucleic acid is immobilized on glass rather than a membrane (termed the probe in the case of microarrays); (ii) the labeled nucleic acid is generated from RNA via reverse transcription rather than a DNA template (termed the target in the case of microarrays); (iii) the label is a fluorescent dye rather than a radioisotope; and (iv) the scale is reduced by two to three orders of magnitude.

If not removed, this excess DNA can subsequently be washed off during stringent posthybridization washes, thereby contributing to background problems. Also, appropriate postprocessing conditions are important in maintaining spot morphology by preventing spot smearing or [3] troubleshooting microarrays 39 comet tails. It is crucial for all washing agents, especially sodium dodecyl sulfate (SDS), to be removed from the arrays at the end of postprocessing, as any residual wash buffer may contribute to background.

Kipps, T. , and Croce, C. M. (2005). A MicroRNA signature associated with prognosis and progression in chronic lymphocytic leukemia. N. Engl. J. Med. 353, 1793–1801. Calin, G. , Liu, C. , Dumitru, C. , Dell’Aquila, M. , Kipps, T. , and Croce, C. M. (2004). MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias. Proc. Natl. Acad. Sci. USA 101, 11755–11760. Chan, J. , Krichevsky, A. , and Kosik, K. S. (2005). MicroRNA‐21 is an antiapoptotic factor in human glioblastoma cells.

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