Is there an upper limit for RNA input

[SueFoltin]

We have been having issues with our RNA from FFPE when submitting for sequencing. We are using the Illumina kit RS-122-2201, TruSeq Stranded Total RNA RboZroHMN/Mse/Rat with ribo depletion.

Our samples are degraded so we have decided to use either no fragmentation or minimal fragmentation (Covaris) to start with.

We are also considering using up to 750 ng of input RNA.

I'm concerned that we may be using too much to start with and that can be problematic. We have a CORE facility that makes our libraries, so I'm not 100% familiar with the protocol for library construction - just a rough idea.

If I don't need to use this much material, I would love to be able to save some for other downstream testing.

Thank you.

[nucacidhunter]

Up to 1ug RNA can be used with this kit and it has instructions on how to fragment degraded RNA. Covaris should not be used for fragmenting RNA for this kit.

[SueFoltin]

What should be used? I know they are running RIN and DV200. Illumina has provided them with guidelines on how long to fragment on the Covarius based on the DV200. I know there is an enzymatic (tagment?) option, but again, they are going off recommendation by Illumina.

[nucacidhunter]

The kit user guide recommends QC by Bioanalyzer and it has several RNA profiles along with recommended fragmentation protocol for each. DV200 is used for RNA enrichment protocols such as RNA access and not for the kit you have mentioned.

Tagmentation is used for DNA libraries and it cannot be used directly on RNA.

[Olaf Blue]

The stand-alone Ribo-Zero kits are to be used with 100 ng up to 5 ug of total RNA. In the past, Epicentre/Illumina's Scriptseq RNA-Seq kits with Ribo-Zero have been used for rRNA depletion and sequencing. No fragmentation is done.

http://www.epibio.com/docs/default-source/scientific-posters/2012-agbt---rapid-single-tube-workflow-for-preparing-directional-rna-seq-libraries-from-low-input-and-ffpe-rna-samples.pdf?sfvrsn=10