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.
- 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.
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