A major limitation of current gene expression analysis systems is that large amounts of RNA are required. Glass arrays typically requires between 5 to 50 µg of total RNA (or 0.1 to 1 µg of poly(A) RNA). To circumvent this problem, Ambion has developed a linear RNA amplification system for array analysis. The MessageAmp procedure is based on antisense RNA (aRNA) amplification first described by Van Gelder and Eberwine, whereby small amounts of RNA can be linearly amplified to generate enough material for use in array analysis. The amplification level is 1000X or greater. Unlike exponential RNA amplification methods, such as NASBA and RT-PCR, numerous studies have demonstrated that the aRNA amplification strategy maintains representation of the starting mRNA population.
The aRNA obtained is labeled by incorporating amino allyl UTP (for subsequent coupling to amine-reactive dyes) during transcription. Unlike dye-coupled NTPs, amino allyl modified NTPs are incorporated almost as efficiently as unmodified NTPs and the problem of nuceotide incorporation efficiency bias (e.g. Cy3 NTP vs Cy5 NTP) is avoided.
I have been using this kit for microarray analysis for the last two years. A typical reaction will yield as much as 20-50 µg of aRNA, starting from 1 µg of total RNA (the equivalent of 10-30 ng of input mRNA). 5µg of aRNA are usually used for CyDye coupling and subsequent hybridisation. Rigorous experimentation can lead to highly reproducible RNA amplification and labelling. However, this method is quite sensitive to RNA quality as well as incubation conditions. Hybridisation ovens are recommended, where uniform heat minimizes condensation and temperature variations are limited.
The protocol I now use is slightly different than the manufacturer’s instructions. The IVT incubation time is reduced to 4 hours (instead of 16 hours) as it has been recently shown that time-dependant degradation occurs during prolonged standard incubation time. It is thus crucial not to compare microarray results obtained with aRNA from different incubation times, as aRNA degradation results in a strong bias in detection signal of certain transcripts.
In terms of kit components, I do not use the Ambion purification system included in the kit as in our hands Qiagen PCR purification columns give better purification and higher yields. Furthermore, I usually use home made coupling buffers as the kit components are limited to 20 coupling reactions. For these reasons, I now use a custom version of the kit which does not contain the purification system and the amino allyl coupling buffers. In terms of cost savings, a custom kit would be cheaper than the commercial version. It is also possible to perform 40 amplification reactions with one kit (instead of 20) by reducing the reaction volumes by 50%. In these conditions, 500 ng of total RNA should give enough amplified material to perform a minimum of two hybridisations per sample (for dye swap hybridisation for instance).
In summary, I do recommend this kit as the technology has been validated and RNA from limited samples can be amplified. In addition, the cost/reaction is comparable to conventional direct incoporation labelling systems.
Jean-Baptiste Rascle, PhD
Institut de Pharmacologie Moléculaire et Cellulaire
Ambion's Amino Allyl MessageAmp aRNA Kit
The system is well worked out and validated, technical help from Ambion is effective and custom kits are available
Purification system less efficient than some competitor’s systems. Limited quantities of coupling buffer in the current version of the kit
The Bottom Line
Overall a good tool to use for researchers interested in generating reliable gene expression data from limiting samples