New kit requires only nanograms of starting RNA and reduces procedure time from days to hours with one round of amplification
Agilent Technologies Europe has introduced the Low RNA Input fluorescent linear amplification kit that can label cDNA or cRNA targets for microarray experiments using as little as 50 nanograms of total RNA.
Its user-friendly protocol requires only a single round of amplification and reduces procedure time from days to hours, yet provides comparable amplification to the original Eberwine procedure.
A major limitation of microarray technology has been the large amount of starting RNA - usually 5 to 10 micrograms of total RNA - required for labelling.
With this kit, researchers are able to extend DNA microarray technology to sample-limited experiments such as those involving laser microdissected specimens, biopsy samples, or challenging cell cultures, such as those used in cancer research.
The new kit eliminates several drawbacks of previous amplification methods. It reduces the number of purification steps and completely eliminates extraction using toxic phenol.
It minimises hands-on time through a simple, single tube procedure that is amenable to automation.
The kit's protocol uses reverse transcriptase, T7 RNA polymerase, and reagents to make either labeled cRNA in six hours for use with oligonucleotide arrays, or labeled cDNA in ten hours for use with cDNA arrays.
With other amplification methods, typical preparation times can last several days.
The kit provides the same amplification as the Eberwine procedure - 100x minimum, 200-300x typical amplification, depending on the sample - but requires only a single amplification round.
Because of that, the reaction is optimised for linearity and does not introduce bias of the abundant mRNA species over the rare mRNA populations.
This amplification kit is the newest addition to Agilent's family of flexible, pre-configured kits that work with specific gene expression microarrays.
Agilent's labelling kits provide added confidence with two-colour labelling formats that can normalise system variability between two samples and the noise between two microarrays.
Reliability is further enhanced when researchers run samples using Agilent's complete microarray-based gene expression system.
