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Perform Microarray Analysis with Limited Bacterial RNA

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 MICROBExpress Kit (see sidebar), or bacterial RNA enriched from host cell mixtures with the MICROBEnrich 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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