RNA-Seq, amplification of degraded RNA

[JakobHedegaard]

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

[HESmith]

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

[JakobHedegaard]

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