linear amplification of bacterial mRNA generates long molecules

[greigite]

Hi,
We have a bit of an odd result from linear amplification of bacterial mRNA that I hope someone can give me a few tips on. We are starting with total RNA from a mixed community, then depleting rRNA using the MICROBExpress kit. That leaves very little total material (a few nanograms), so we are amplifying the enriched mRNA using Ambion's Message-Amp II-Bacteria kit, then performing double stranded cDNA synthesis followed by standard Illumina library prep.

So far, it appears that the linear amplification step is generating artificially long molecules that are not present in the original mRNA. For one sample, only 7% of molecules were above 1 kb in the original mRNA and 47% were above 1 kb in the amplified sample (estimate via Bioanalyzer). This is roughly consistent for several distinct samples processed uising the same protocol.

This smells like concatemerization to me, but we are still in the process of checking long cDNA molecules by cloning. Has anyone seen something similar or have an explanation?

[gkrupp]

suggestions to "linear amplification of bacterial mRNA generates long molecules"

Hi,
the Ambion Message-Amp II-Bacteria kit can result in larger amplified RNAs because
1) the first step is addition of a polyA tail. If small RNA amounts are present, these tails can get very ! long.
2) a general problem of this basic Eberwine technology kit is generation of very large primer-derived artefacts, as already observed in the eukaryote community and described in the publication Baugh et al
Quantitative analysis of mRNA amplification by in vitro transcription. Nucleic Acids Res. 2001 Mar 1;29(5):E29.

In a later publication they describe the use of ExpressArt kits from artus in Germany, that do not suffer from that problem: Yanai et al 2008
Pairing of competitive and topologically distinct regulatory modules enhances patterned gene expression. Mol Syst Biol. 2008;4:163. Epub 2008 Feb 12.

More recently, ExpressArt kits were developed for amplification of bacterial mRNA (with selectivity against rRNAs), they are now available from AmpTec in Germany (www.amp-tec.com).
These kits do not need a polyA tail!

Give it a try, hope that will help.