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Synthesize High Yields of Biotinylated aRNA

ove each histogram indicate the median value (vertical center line), interquartile range (left and right sides of box), and the 10% and 90% quantiles (ends of line). Each plotted distribution represents the average log2 signal intensity of triplicate target preparations and hybridizations. IVT reactions were carried out for 4 or 14 hrs as indicated. The data were normalized within each group using Robust Multichip Average (RMA) (See sidebar, Data Analysis: Robust Multichip Average (RMA) and Affymetrix Algorithms).


Biotin Incorporation

Biotin-11-UTP serves as the label in the MessageAmp II-Biotin Enhanced Kit. Only a single biotinylated NTP is used because studies at Ambion and elsewhere [2] showed that in previous dual labeling experiments using biotin-UTP and biotin-CTP, the signal contributed by biotin-CTP was minimal, and relative signal intensity between genes was not altered with the use of a single biotin UTP label.

While various linker arm sizes (e.g., Biotin-11-UTP, Biotin-16-UTP) did not affect modified UTP incorporation into aRNA during IVT, the longer linker arms slightly impeded subsequent purification of the aRNA. Biotin-11-UTP provided the highest yields of aRNA.

The ratio of biotin UTP to unlabeled UTP was optimized to provide the highest yield of aRNA and the highest signal:noise on Affymetrix GeneChip Arrays. A fairly wide range of concentrations of Biotin-UTP (25 60%) can be used for acceptable detection, whereas at lower concentrations the signal drops dramatically, and at
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