Advantages of Indirect Labeling
Direct incorporation of labeled NTPs is inefficient and results in low yields and low specific activity aRNA. This can make the cost of producing aRNA by direct incorporation very high. In addition, incorporation efficiency varies for different labels (e.g. Cy3 NTP vs. Cy5 NTP). These problems can be avoided by indirect labeling, whereby an amino allyl NTP is incorporated into the aRNA, and the reactive amino group is subsequently coupled with an NHS ester label (e.g. biotin, Cy dye). Compared to dye-coupled NTPs, amino allyl modified NTPs are incorporated almost as efficiently as unmodified NTPs by ArrayScript Reverse Transcriptase (Cat# 2048) and are much less expensive. Figures 1 and 2 demonstrate equivalent labeling with Cy3 and Cy5 dyes using the Amino Allyl MessageAmp II aRNA Amplification Kit.
Figure 1. aRNA Yields From Amino Allyl MessageAmp II aRNA Amplification Kit. The indicated amounts of Control RNA (HeLa cell total RNA) were amplified for the in vitro transcription (IVT) incubation times indicated. While longer IVT incubation times produced more aRNA, the amount of aRNA required for most microarrays was obtained with a four hour incubation with >500 ng of input RNA. This data was obtained using the Control RNA provided with the Amino Allyl MessageAmp II aRNA Amplification Kit. Note that aRNA yields can vary considerably with sample quality.
Figure 2. Scatter