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Old 08-24-2011, 02:27 AM   #1
JakobHedegaard
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Location: Aarhus, Denmark

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Default RNA-Seq, amplification of degraded RNA

Starting with <100 ng of degraded total RNA I would need to do RNA amplification before preparing ScriptSeq RNA libraries.

1. rRNA depletion using RiboZero (starting with ~100 ng degraded totalRNA)
2. Amplification of rRNA depleted RNA (starting with the ~5-10 ng RNA from 1.)
3. Preparation of random primed RNA-Seq libraries using ScriptSeq.

We have successfully generated RNA libraries using RiboZero and ScriptSeq from ~500 ng degraded Total-RNA.

Suggestions for a RNA amplification kit for step 2. would be appreciated!

Cheers,
Jakob
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Old 08-24-2011, 08:02 AM   #2
HESmith
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Hi Jakob,

We've obtained good results using Nugen's Ovation RNA-Seq kit. They also make a kit for amplifying FFPE samples, which are quite degraded, but we have not tested it.

Harold
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Old 08-26-2011, 12:18 AM   #3
JakobHedegaard
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Hi Harold,

I have considered the Nugen's Ovation RNA-Seq FFPE System but it does not, in its current form, maintain directionality of the transcripts. And this is essential for our data.
But I will take a look at their FFPE amplification kit again and see if it can be combined with ScriptSeq.

Jakob
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