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Improving RNA Amplification

ation are appropriate. Some microarray studies demand a high level of amplification (106 fold or greater), which can be achieved by two successive rounds of MessageAmp II reactions. Determining the lowest level of total RNA input that generates enough cRNA for array hybridization will depend on the amount of cRNA required for a particular microarray platform, and whether extra cRNA is desired for replicates, follow up validation with RT-PCR, or for archiving samples.

For consistency within an expression experiment, it is important to decide whether one or two rounds of amplification will be used throughout the study. For example, yields of total RNA may vary within a collection of samples some samples may yield 1 g of total RNA such that only one round of amplification is needed, whereas others may only yield 50 ng, requiring two rounds of amplification. Figure 3 may aid in this decision process for amplifying samples below 100 ng. For example, below 100 ng input RNA, the cRNA yields after one round of amplification may not be sufficient for most arrays.

Figure 3. cRNA Yields From Two Rounds of MessageAmp II Amplification. 100, 10, and 0.1 ng of total RNA from HeLa S3 cells (HeLa) and human inferior temporal cortex (brain) were amplified. Yields (g) using the MessageAmp II aRNA Amplification Kit are recorded for after each round of amplification. The starting cRNA input for the second round was 2 g (if available), or the entire amount obtained from the first round.


Obtaining sufficient cRNA for a microarray hybrid
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