TRIzol contamination in low yield RNA extraction.

[evobio]

SCENARIO:
- Goal: RNA-seq.
- Number of samples (already extracted): 200.
- Size of individual sample: VERY small (single zebrafish embryos at early stages of development).
- Method of RNA extraction: standard TRIzol.
- TOTAL RNA yield per individual sample: ~200 ng (eluted in 25 uL of water).

PROBLEM #1:
1. When I check the TapeStation for RNA integrity, I get really high values (close to maximum quality) (Fig. 1), but when I check the NanoDrop I get an extra peak that seems to be TRIzol contamination (Fig. 2).
Q1.1. Does that affect downstream steps, e.g. rRNA depletion?
Q1.2. How can I get rid of the TRIzol without losing RNA or degrading it?

PROBLEM #2:
2. When I precipitate the sample to try to get rid of the extra peak during NanoDrop measurement, the extra peak is gone in the NanoDrop measurement, but the TapeStation result after the precipitation and rRNA depletion shows an extra peak close to the lower marker (Fig. 3).
Q2.1. What's the best way to precipitate tiny amounts of RNA (~200 ng in total) without losing much?
Q2.2. Should I be worried that the extra peak is very close to the lower marker? Does it mean that my RNA is degraded?

Figures here.

[adam.geber]

Hi evobio,

One method I've started using to avoid residual phenol contamination is performing a standard Trizol extraction up to phase separation followed by passing the aqueous phase through a standard silica column (Zymo's Clean & Concentrator, Qiagen's RNeasy MinElute, etc.). I find that this gives me the best yield/purity and also helps with parallelization for the more time-consuming steps of the Trizol extraction. These columns are also quite helpful for purifying/concentrating your RNA extracts if they appear contaminated.

As to whether phenol contamination can interfere with downstream steps, it's certainly possible. What method are you using for rRNA depletion? Have your relative yields after depletion been as you expect them to be so far?

Regarding problem #2, have you tried running purified/unpurified RNA samples (diluted to the same concentration) alongside each other on the Tapestation to check the profiles? Likewise for total RNA and rRNA-depleted samples? The fact that your RINs are so consistently high means that if any degradation is occurring it's likely due to repeated freeze-thaw cycles or contamination by RNases during further handling. The larger peak next to your lower marker looks slightly suspicious but I've never worked with zebrafish RNA and don't know if they have a large proportion of e.g. 5S rRNAs, which are retained during a Trizol extraction. If you have the Tapestation trace on hand can you confirm the size of that peak?

Thanks for such a clear breakdown of your questions!

[kerplunk412]

The peak you see at 270 is almost certainly phenol contamination, and means any Nanodrop quantification can't be trusted. The good news is that it might not actually be that bad. This amount of phenol is probably in most Trizol extractions, it just shows up here because the amount of RNA is so little. I think you should try a test run of 3-4 samples, and if those look good don't worry about purifying the RNA again. More handling=more chance to mess up, especially with 200 samples.

The Tapestation results are textbook RNA degradation, demonstrated by the reduction in rRNA peaks and buildup of low molecular weight material. Ethanol precipitation is actually the best way to clean RNA with high yield, your results just indicate you have RNAse contamination somewhere. From a practical standpoint, purifying 200 samples by ethanol precipitation will be a pain anyway, so I recommend the Zymo C&C 5 kit.