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Improving RNA Amplification

We first measured the simple correlation of the log2 expression of each array versus the chosen standard (1000 ng input; 4 hr IVT). Figure 5b lists the correlation and standard deviation (SD) of the differences with the standard. This data is also expressed as simple plots of log2 (expression) of each array versus the standard (x axis) (Figure 8). These comparisons reveal that the magnitude of the variation one sees among the different IVT times using 1000 ng RNA inputs and the MesageAmp II protocol is similar or less than expected in replicates using the Affymetrix protocol. Additionally, arrays with cRNA from 200 ng total RNA have more under-expressed genes (a broader tail on the left of each plot) for all times measured. This range of differences appears to be consistent with that also seen in Affymetrix replicates (Figure 5b). While the standard deviation (SD) of the differences between the 200 ng RNA inputs appears twice as large, combining replicates with simple averaging should decrease the SD.

Figure 8. Expression by IVT Time and RNA Input vs Standard. Log2 expression by IVT time and RNA Inputs (1000 ng and 200 ng) from IVT reactions incubated at 4, 6, or 14 hours were plotted versus the Standard (4 hr IVT; 1000 ng RNA input). The X and Y axis represent the log2 of the expression values estimated using RMA.


Expression Analysis at Lower RNA Inputs
Lower RNA inputs (below 100 ng), which generally require two rounds of amplification, suffer both a decrease in the percent Present calls and an increase in noise. Several studies have characterized and compared single v
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