genome expression analysis from limited
bacterial samples. By amplifying RNA, microarray analyses can now be performed
with up to 500-fold less starting material. MessageAmp II-Bacteria can
be used with any bacterial species and is compatible with purified total
RNA, bacterial mRNA enriched using Ambions MICROB
Express
Kit (see sidebar), or bacterial RNA enriched from host cell mixtures with
the MICROB
Enrich Kit (see sidebar). As little as 10 ng of
enriched bacterial mRNA or 100 ng of total RNA can be amplified to yield
sufficient antisense amplified RNA (aRNA) for microarray analysis.
Linear Bacterial RNA Amplification
MessageAmp II-Bacteria is based on a linear amplification
method (Figure 1) in which the RNA sample is first polyadenylated with
Poly(A) Polymerase, then reverse transcribed using oligo(dT) primers containing
a T7 RNA polymerase promoter. The resulting cDNA is used to produce aRNA
by in vitro transcription (IVT) primed by the added T7 promoter. The aRNA
can be labeled during IVT with a variety of modified nucleotides (for
glass slide microarrays or Affymetrix GeneChip arrays), or it can be
used to generate 33P-labeled sense strand cDNA (for nylon PCR
arrays).
Figure 1. Schematic of MessageAmp II-Bacteria
Procedure.
Yields of Amplified RNA
It is important to understand that the amplified
RNA yields obtained with the MessageAmp II-Bacteria Kit or any amplification
system will vary based on factors such as RNA isolation method, input
RNA amount, RNA quality, the bacterial species from which the RNA was
isolated, and incubation time of the in vitro transc
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