Trouble with RNA quality

[michael-steffen]

I recently did a RNA extraction using Trizol, I followed all the steps, except I skipped the optional step. I intend to do a RNA seq with these samples using the miseq. The trouble I'm running into are the RIN numbers i get from the bioanalyzer are really bad across samples. The samples are pretty fresh (less than 3 weeks old), were immediately frozen on dry ice, and then kept in a -80 till extraction. Extractions were then also done on ice.

I also nano dropped these samples and the 280/260 values are decent(>1.8), however, the 260/230 are very low, always less than 1.

I was wondering if anyone can help interpret some of my traces and offer any advice as to how to fix these samples so that they are still useable.

I attached the traces.

Thanks,
Mike

[sara_ahmed]

I think it might help to rerun these on the Bioanalyzer at a higher concentration. The FU on the y-axis seem super low and I am afraid you may just be looking at background rather than actual ribosomal peaks from the sample.

Sara Ahmed, PhD | Director of Sequencing | Cofactor Genomics
4044 Clayton Ave | St. Louis, MO 63110 | tel. (314) 531-4647

[ELoomis]

What does your nanodrop trace look like? Low 260/230 could mean phenol in your samples (Trizol is ~50% phenol). Check out this link for a good guide to understanding nanodrop reports (scroll down for examples of phenol contamination):

http://biomedicalgenomics.org/RNA_quality_control.html

If it is indeed phenol, you can extract with chloroform again until it goes away. (Dilute your samples to a few hundred uL, but no more than half the tube volume, then add an equal volume chloroform, mix, separate phases, etc. as before, then precipitate as before)

Hope that helps!

[Mike2188]

A few questions:

a) what samples are you working with?
- certain tissues have different forms of contamination, for instance I work with leaf and seed so often carbohydrates may be an issue.

b) what yields are you obtaining (concentrations from the nanodrop?)
- if your concentrations are high you can try a LiCl reprecipitation, or redo the extraction from the chloroform phase seperation
- if your concentrations are low you can do a column purification

c) are you familiar with the bioanalyzer?
- have you performed these successfully before? IMHO I think you may have overprimed the chip, it looks like it is all over the place from the gel, and some samples have signals coming before the marker which is indicative of this. Rerun the chip, make sure that you have the injector set to the proper position (as far up as it can go) -- it may have still been set for a high sensitivity chip. Make sure you prime for 30 seconds EXACTLY. Also, make sure you mix the dye very well (10 entire seconds of intense vortexing and you mix the gel-dye extremely well (10 entire seconds of intense vortexing). Clumps of dye can also clog the chip.

If you want you can run your RNA on a gel if your concentrations are high enough and look for the ribosomal bands.

Also, would you be able to actually upload the .xad file from your bioanalyzer run?

[Mike2188]

Also, Sara is mistaken, your concentrations are sufficient, as they reach or exceed the marker.

[michael-steffen]

Sorry. Didn't realize I had more responses to this post.

The tissue I am using is mental gland tissue from salamanders(pheromone gland).

My nano drop concentrations aren't horrible(100ng/ul).

I do think you may have helped me with the problem though. I am pretty sure I have been over priming the chip. Been doing HS chips lately, and its got a 60s injection step. I've also been doing that same time for the RNA I realize now. Going to give it another try with 30s and see if that fixes it for me.

Thanks,
Mike