Expectations for Yields
Factors such as tissue source, mRNA content (0.1-3%), starting amount of total RNA, and the quality of RNA used, will all affect the yield of cRNA. Collectively these factors can lead to up to 4-fold variation in yields. Figure 2 provides typical yields of cRNA amplified from six different sources using the MessageAmp II aRNA Amplification Kit. RNA inputs ranging from 50-3000 ng were tested from each source. From Figure 2 one can calculate the level of amplification obtained from different tissues at different inputs. The figure is also a useful benchmark for understanding the amount of cRNA produced both from different total RNA inputs and sources.
Figure 2. MessageAmp II aRNA Amplification Yields.
Yields of cRNA (g) amplified using the MessageAmp
II aRNA Amplification Kit from six different RNA sources using total RNA
inputs of 50-3000 ng. Yields shown are the average of duplicate reactions.
The in vitro transcription reaction incubation time was 4 hours for all
samples. Purified cRNA concentrations were measured using a NanoDrop
*Universal Human Reference RNA (Stratagene)
One or Two Round Amplification?
Based on specific experimental constraints, researchers must decide whether one or two rounds of amplific