Tuesday December 05, 2006
I work with expression microarrays. For some projects, I have to use very small amounts of RNA, and some of the RNA samples are of pure quality. I started to use Ambion Amino Allyl MessageAmp™ II aRNA Amplification Kit about two years ago. This kit is based on antisense RNA amplification. The procedure includes reverse transcription with an oligo-dT primer bearing a T7 promotor and using reverse transcriptase ArrayScript™, which was engineered to produce higher yields of full-length cDNA. After second strand synthesis and clean-up, the cDNA becomes a template for the in vitro transcription technology using Ambion’s proprietary enzyme mix to generate more antisense RNA (aRNA) copies of each mRNA molecule. Modified nucleotides (like 5-(3-aminoallyl)-UTP (aaUTP) can be incorporated into the aRNA during the in vitro transcription reaction. Following purification of aRNA, incorporated aaUTP can be coupled to N-hydroxysuccinimidyl ester-derivatized reactive dyes like Cy3 or Cy5. Samples with very small amounts of RNA (the smallest I’ve amplified was 3 ng) can be subjected to two rounds of amplification. The kit contains enough reagents and purification columns to carry out 20 one-round or 10 two-round amplifications.
Lately, Ambion has increased the efficiency of this kit. Typically, when I start with 100 ng of total RNA of average quality (which equals to 2 – 5 ng of mRNA), the yield is around 7 – 8 ug of aRNA. The degree of amplification is sensitive to the quality of RNA: The better the RNA quality, the more amplification one can get. I have experience with bad RNA samples. For example, when the Agilent BioAnalyzer barely showed an 18S ribosomal peak, I checked whether I could get at least an RT-PCR product of -actin. Amplification of these samples was about 5 times less efficient than amplification of samples with decent RNA quality. However, after amplification, labeling and hybridization to microarrays, samples with bad RNA quality showed results similar to ones from samples with decent RNA quality and made very good replicates.
This kit has some disadvantages. Very often, after amplification, I divide samples and label one part with Cy3, and the other with Cy5. The kit contains only 20 columns for the purification of labeled aRNA, so often I have to use Qiagen’s RNeasy Kit instead of the Ambion components for the labeled aRNA purification. Another weak point is that lids of the test tubes for cDNA elution (in cDNA purification part) often don’t fit cDNA purification columns, and the tubes are open during cDNA elution. Only one time have I had the very unfortunate experience of receiving kits (2 kits which were ordered and received at the same time) lacking the test tubes with the filters for cDNA purification. Qiagen’s MinElute PCR Purification Kit was a very good substitution.
The personnel in Ambion’s technical support are very knowledgeable and they quickly answer any question regarding RNA amplification.
In conclusion, I would highly recommend Ambion’s Amino Allyl MessageAmp™ II aRNA Amplification Kit to anyone who has to linearly amplify RNA. Despite of some weak points, this kit is very reliable and easy to use.
Lubov Nathanson, PhD
Assistant Scientist
DNA Microarray Core Facility
University of Miami
United States