RNA amount for RNA seq, Illumina

[basager]

Does anyone have experience with using a smaller amount of input RNA than what Illumina refers to in their RNA seq protocol? Illumina protocol claim to need 1-10 micrograms of total RNA. Unfortunately I have less starting material than this and I'm therefore wondering if anyone have used less than 1 microgram?

[das]

Hi:
Contact Illumina. I heard that they are working on a limited RNA protocol.
I also think that two-step protocols that were developed for Affymetrix chips should be applicable for Illumina (In vitro transcription step to amplify RNA, then library construction.
Then there is a Nugen Ovation kit

http://www.nugeninc.com/nugen/index.cfm/products/amplification-systems/ovation-rna-seq-system/

[basager]

Thank you. I wrote to Illumina, and I'm excited waiting for them to reply. I have herad about the Nugen Ovation Kit, and I considered to use it. Since it is new I can't find anyone who has used it yet, and it really would be good to know that others have used it with good results before trying it out. Hopefully it will work without introducing too much bias. If you know anyone who has used it, please let me know.

[Nik]

Have a look at the directional mRNA protocol. Works for me but not cheap as you use two kits, sRNA and mRNA and other reagents you have to buy in. However it works using much lower concentration total RNA.

Ask illumina when they are bring out the Universal RNA kit... Its about time.

[scbaker]

In terms of input for the standard mRNA-Seq kit, the official recommendation does stand at 1ug. However, internally we've pushed that down considerably without any protocol modifications. Using high quality RNA, we routinely use 100ng of total RNA as the input amount and we've gotten libraries from as little as 5ng. However, going lower than ~20ng seems to give slightly lower correlations (r2 value) with 1ug samples.

Shawn Baker
Market Manager, Expression and Epigenetics
Illumina

[krobison]

Illumina had a webinar this week on their DSN Total RNA-Seq protocol which can start with as little as 100ng total RNA.

[Nik]

Yes, also been down to 100ng with the SS mRNA seq protocol routinely with using as little as 80ng of OK Total RNA.

Was listening to that webinar about DSN and had a chat to them about the 4 x hyb buffer recipe. It was not much help. Can anyone help? This stage have to be correct.

[das]

There is a paper in Genome Research on low starting amount protocol for RNAseq

http://www.ncbi.nlm.nih.gov/pubmed/20133332

[wacguy]

In terms of input for the standard mRNA-Seq kit, the official recommendation does stand at 1ug. However, internally we've pushed that down considerably without any protocol modifications. Using high quality RNA, we routinely use 100ng of total RNA as the input amount and we've gotten libraries from as little as 5ng. However, going lower than ~20ng seems to give slightly lower correlations (r2 value) with 1ug samples.

Shawn Baker
Market Manager, Expression and Epigenetics
Illumina

Hi Shawn,

I've seen your reply and wander wether there are new developments in the field such as constructing a directional library rRNA depleted with 1-5ng RNA that are not dependent on pol(A) enrichment but use random primers. I've looked thru and tried several kits and it seems like RNA quantity is still a problem.

Thanks a lot,
Guy

[ZWB]

This is a recently published, directional RNA library prep protocol that goes down to as low as 10ng of total RNA (or rRNA depleted RNA) using random priming. We have also tried 5ng of rRNA depleted RNA with good results.

http://www.ncbi.nlm.nih.gov/pubmed/23558773

[scbaker]

Hi Shawn,

I've seen your reply and wander wether there are new developments in the field such as constructing a directional library rRNA depleted with 1-5ng RNA that are not dependent on pol(A) enrichment but use random primers. I've looked thru and tried several kits and it seems like RNA quantity is still a problem.

Thanks a lot,
Guy

I left Illumina a little over two years ago, so I can't answer specifically about their kits, but I've seen a number of solutions that use rRNA depletion (or avoidance). I haven't used any of these kits myself, but I'm sure they're impacted at least to some degree by RNA quality (not sure about 'quantity'). Also, I think they're a bit variable as to how many rRNA reads they let through. As long as you sequence deeply enough, you can probably afford to waste a few (million!) on rRNA.

Good luck!

Shawn

[jvanfleet]

Hello Guy,

I came across your post about working with 1-5 ng of total RNA and wanted to let you know about the Epicentre kit called TotalScript.

Here is some info and links:

http://www.epibio.com/applications/rna-sequencing/rna-library-prep/totalscript-rna-seq-kit

•Total RNA input, rRNA removal not required.
•Produces directional libraries.
•One day procedure utilizing only 1-5 ng of total RNA.
•Index-capable. (TotalScript™ Index Kit sold separately.)
•Ideal for high quality RNA (RIN >7) from human, rodent, yeast, C. elegans and Drosophila. (Not recommended for prokaryotic samples.)

If you have questions please email me at [email protected]

Have a great day,

Jennifer

[fanauh2o]

I tried around 2 ng using the Illumina v2 kit. The key is that you need to make sure you can see the 18S and 28S bands on the picochip when running the bioanalzyer.