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Improving RNA Amplification

ization is not only dependent on the source of RNA being amplified but also on two reaction variables: the amount of input total RNA, and the length of the IVT incubation. The yield of cRNA increases with increasing RNA input and IVT time. As a rule, most samples between 100-1000 ng, incubated overnight (14 hr IVT), will produce sufficient cRNA for any microarray platform (at least 10 g). However when we investigated a range of IVT incubation times (from 4-14 hr), we found 4 hour IVT incubations generate sufficient cRNA from most total RNA inputs greater than 200 ng (Figure 4). A 4 hour IVT can be sufficient for 100 ng samples, and these conditions were used in the microarray studies presented below. (Note that mRNA poor samples will require longer IVT incubations (6-14 hr).

Figure 4. Plot of IVT Incubation Time vs. Yield. Total RNA (200 ng) from human inferior temporal cortex (brain) was amplified and incubated with in vitro transcription (IVT) reaction times of 4, 6, and 14 hours following the MessageAmp II protocol. The average yield of purified cRNA from duplicate amplification reactions was plotted versus the IVT incubation time. The 0,0 data point was added to extend the line to predict yields with IVT incubation times under 4 hours.


While we recommend a minimum of 4 hours for the IVT incubation, yields obtained from shorter time periods (2 or 3 hr) can be tested and should be adequate for input amounts above 500 ng.


Expression Analysis
Three approaches were used to define the reproducibility and consistency of the MessageAm
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