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Experimental Design
Total RNA was isolated from HCT116 and RKO cells and aRNA was amplified
from total RNA (2 g). For non-amplified samples, mRNA was purified
using Oligotex beads (Qiagen). To assess the reproducibility of RNA amplification
and compare representation of messages in aRNA with those in mRNA, three
samples each of RKO and HCT116 RNA were amplified independently. The quality
and yields of RNA obtained from the various samples are presented in Figure
2. mRNA or aRNA (2 g) samples were fluorescently labeled by incorporating
Cy3-dCTP (RKO samples) or Cy5-dCTP (HCT 116 samples) during reverse transcription
with random 9-mers. Each glass slide contained duplicate arrays and each
labeled RNA sample was hybridized to two slides (4 replicates). Duplicate
hybridizations were pe
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