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RNA: Is this degratation or something else?


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#1 SamCurt

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Posted 22 September 2010 - 09:07 AM

Hi,

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.

Attached Thumbnails

  • 58137_2.jpg


#2 Jebra

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Posted 22 October 2010 - 02:28 PM

I don't think your sample ran correctly. Have you checked to make sure your marker is aligned correctly with the ladder?




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