RNAlater and Illumina Sequencing

[JayS]

I'm about to harvest mouse tissue for my undergrad RNA-Seq gene expression project on an Illumina HiSeq. I can either flash freeze the tissue on liquid nitrogen, or place it on RNAlater for later processing. My question is: Is RNAlater compatible with Illumina Sequencing?

I don't see how it would give me any problems, but I thought I'd ask here before I store my samples in RNAlater!

Thank you,
Jay

[Jon_Keats]

Has worked for me in the past. Not my favorite method but works fine particularly for tissue samples.

[ksqb]

Hi JayS, I am planning to also store samples in RNAlater before RNA sequencing. If you did this, I was wondering what your experience was like with the results. Thanks.

[AndrewP]

It should be fine considering it's just EDTA to prevent RNA degradation and ammonium sulfate to precipitate proteins, and RNases in particular. You'll have to isolate RNA anyways, and that should get rid of the salts.

[ksqb]

Thank you, AndrewP. The samples are mammalian cells in culture and I am planning to isolate the RNA using RNAeasy. A Thermo Fisher workflow suggests that cells are washed by pelleting to get rid of the RNAlater and then processed for RNA isolation. I heard that RNAlater causes some cell lysis so may cause loss of RNA. I am in a bit of a quandary as to how to process with RNA extraction without losing sample.

[AndrewP]

I can't help much in that regard since I only work with microbial samples, but it should be clear from literature what most people use. I honestly don't think you need RNAlater, but I also don't think it would hurt (if it did, there should be a publication on it). There was a paper from, I think, the mid 90's that looked at the stability of samples flash frozen vs. RNAlater and demonstrated better RNA quality when stored at 4 degC and room temperature. They weren't using the commercial RNAlater, but came up with the formulation themselves (this was before RNAlater was a product).

[Marc_Jones]

I've snap froze tissues without any loss of RNA integrity. The biggest effect on RNA integrity is the time taken from sacrifice to freezing the tissue.

The main reason we froze was because wanted to section, stain and macro-dissect regions for sequencing. FYI RNAlater does funky things to cutting media (OCT).