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I have been extracting RNA from rat kidneys, using a TRIzol+QIAgen protocol:
- Approximately 100mg of rat kidney tissues (after blotting away the RNAlater) was homogenized in 1mL TRIzol in a rotor-stator homogenizer.
- 200uL of chloroform, freshly aliquotted from the original colored glass bottle, was added to the homogenate, shaken vertically for 15s, and incubated for 3min.
- Mixture spinned down at 11,200g for 15min.
- 400uL of the aqueous phase was separated was added to 250uL 100% isopropanol, then 150uL of the polysaccharide suspension solution (1.2M NaCl , 0.8M NaAc) was added to the mixture.
- Mixture was incubated at -20C for overnight and spun at 11,200g for 10m.
- Pellet from above was washed with 1mL 75% ethanol, vortexed, and spun at 7,400g for 5min.
- Pellet was dried for 10min, resuspended at 50uL freshly prepared DNAse I incubation buffer (Our lab uses Roche)
- The pellet was allowed to resuspend at 55C for 10 min. While the white stuff was dissolved, a clear geletinous pellet remained insoluble.
- Resuspended RNA was treated with 10U of DNAse I at RT for 10min.
- Treated RNA was cleaned up according to the direction of RNEasy, and eluted twice with 50uL water.
The water used was completely new.
I was quite confident the protocol was correct, but results of Bioanalyzer (see attachment for a sample) looked weird-- it does not look like real degradation (which would look like a smooth peak), but rather, a normal RNA size curve squished to the left. So what am I seeing here and how should I improve the extraction? Any help is appreciated.
