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?
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?
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