I suppose this might suggest contamination with remaining polyphenols/polysaccharides.  Have you tried an RNA clean up step using something like RNAClean XP beads (Agencourt)?

Jonna,

The following two links may hold some useful ideas for you:

http://www.bioone.org/doi/pdf/10.3732/apps.1500001

 http://www.researchgate.net/publication/5286470_Simple_and_efficient_isolation_of_high-quality_total_RNA_from_Hibiscus_tiliaceus_a_mangrove_associate_and_its_relatives

Article Simple and Efficient Isolation of High-Quality Total RNA fro...

How pure the RNA has to be depends a bit on how you are going to sequence your samples (which library kit on which platform) and how you are going to analyze your data (mapping and/or de novo assembly).

Also depends on the requirements of the company which prepares and sequences the libraries. We have used RNA with quite a bit of humic acids and still got "good" libraries most of the time for mapping (from bacteria). But we do that in-house, so I can overrule QC concerns from the sequencing team and just have to explain the boss why I burned half a flow cell again, if it goes wrong.

We used the CTAB method developed by ourselves for RNA isolation of a Hibiscus species (metioned by Jack above) and the RNA quality is good enough for RNA-Seq.

Thank you all for the replies. I have sent emails to the company that will do the sequencing for us about the polysaccharides etc. We will probably use illumina MySeq for a de novo assembly.

I believe that the CTAB method should do the trick on my specimen. I just did not want to use it if there was alternative kits. 

Thank you again!

Hi Jonna,

if you do a CTAB method for RNA isolation you also can do an additional cleanup using columns if necessary. This is our standard procedure for RNASeq sample preparation and we always get very good quality results. I am using the cleanup protocol for Qiagen RNeasy columns.

Thank you! I will try that after CTAB.

Hi Jonna,

You can try also a simple over-night lithium chloride treatment (20%) after the RNA extraction. It works quite well for cleaning RNAs extracted from grape berry (also full of polyphenols and polysaccharides).

In my opinion, the best test to do before sending the samples is to perform a Bioanalyzer chip (Agilent) in order to obtain the coefficient of integrity of each sample (called RIN). With this assay, you can see if you have DNA contamination, RNA degradation, ect.

Good luck!

Hi Mélanie!

The CTAB protocol I have include a 4 hours treatment with lithium chloride, which I will try before the overnight example.

Thank you for your reply!

Best,

Jonna

I would not worry too much if you do not have access to an Agilent Bioanalyzer. As long as you have no experience which values to expect for your samples/tissue or if there are no good reference values available, the RIN is difficult to interpret. Information obtained via an agarose gel and ratios 260/280 and 260/230 are sufficient. I once could use a Bioanalyzer at a colleagues lab, but in the end even the sequencing company could not tell me, if for the tissue used the RIN is good or not.  

Hi Jonna,

I am not an expert of this species, so I will just make some very general considérations. Obtaining a very good 260/230 ratio is more difficult than obtaining very good 260/280 ratio. Therefore, trying to obtain a 260/230 higher than 1.9 is maybe too strict, and you could accept values around 1.5-1.6. According to our experience, these values are usually good enough to enable the obtention of good sequencing or array results.

Concerning the other aspects, I would say that CTAB with LiCl, or with Qiagen purification columns, is a good protocol to start with. Another option could be butoxyethanol, it is often added with difficult species. Problems with 260/230 tend to be related to the presence of polysaccharides, salts, etc...

Last thing: if you think you have a lot of polyphénols, use polyvinylpolypyrrolidone (PVPP).

Best regards,

Marco