TRIzol contamination in low yield RNA extraction.

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TRIzol contamination in low yield RNA extraction.

Postby evobio » Dec 09 2015 7:51 am

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:
https://drive.google.com/file/d/0B1ho34Lpc7OmQTRiWVJPVkxTX28/view?usp=sharing
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Re: TRIzol contamination in low yield RNA extraction.

Postby relaxin » Dec 10 2015 10:39 am

Extraction of RNA from small samples using TRIzol can be problematic. It gives you low yield and phenol carryover, which may interfere with downstream application. You can clean up the RNA with spin column sold by a number of companies such as Qiagen. Better yet, you may consider phenol-free method using spin columns.
Retired academic researcher. Mention of a specific product does not imply my endorsement of the product. No conflict of interest or guarantee to work on the advice given. Do as I say, not as I do. Not liable to the loss of your valuable samples.
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