Isolating Nuclei from Solid Tissues

When preparing HighMwGenomicDna from solid tissues, it is more convenient to use isolated nuclei recovered from the solid tissue, rather than intact cells disaggregated from tissue by collagenase or other methods. Nuclei can be released by treating cells with concentrated Triton X-100 (NP-40) which disrupts the plasma membrane but not the nuclear membrane, in the presence of sodium citrate which chelates metal ions thereby stabilizing DNA against enzymatic degradation.

All solutions and materials should be kept ice cold at all times. This will greatly stabilize the nuclei and prevent lysis.

Nuclei Isolation

1. Starting with solid tissue, moisten with PBS and mince the tissue as finely as possible with scissors, scalpel and/or tweezers.
   
   • The mincing will help the T-soap get into the depths of the tissue to release the nuclei efficiently
     
2. Rinse with an excess of PBS.
   
   • use 3 to 5 ml, or enough to give the chopped up tissue bits a good rinse; this will help to remove contaminating leukocytes and necrotic material
     
3. Add 1 or 2 ml of T-soap to the minced tissue and continue mechanical disruption of the tissue by additional mincing and manipulation with tweezers.
   
   • as the detergent lyses the plasma membrane of the cells, the nuclei will be released into the solution. the mechanical agitation helps release the nuclei
     
   • for very solid tissues, it may be necessary to mash the minced solid tissue pieces in T-soap with a glass mortar and teflon pestle. do not do this unless necessary as excess mechanical disruption will produce particulate debris without significantly increasing the yield of nuclei
     
4. Remove the liquid containing the nuclei to a conical tube. Allow the solid debris to settle to the bottom for a minute or two, then remove the liquid phase containing the nuclei to a clean conical tube.
   
5. The nuclei can be concentrated by pelleting with centrifugation at 200 g for five minutes in a clinical centrifuge, or in a microfuge if the volume is small enough.
   

Nuclei Quantification
In order to quantify nuclei specifically, in the presence of possibly greatly excessive cellular debris, cytometry triggering on a fluorescent signal from a nucleic acid specific dye such as AcridineOrange is optimal. Follow the QuickLeukocyteAnalysis protocol, but substitute resuspended nuclei for whole blood. The stained nuclei can be counted immediately after addition of the dye.

Under some circumstances, it may be useful to delaminate the nuclear membrane with concentrated citric acid prior to counting. Important: Only do this for the small volume to be counted, and not for the entire preparation of nuclei.
Mix:

• 10 μl resuspended nuclei
  
• 488 μl NusseNuclearPreparation Solution I
  
• 2 μl AcridineOrange stock at 1 mg / ml
  

then add and mix:

• 500 μl NusseNuclearPreparation Solution II
  

Count this 1:100 dilution by fluorescence-triggered flow cytometry.

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Solutions

Tissue soap (T-soap): This is essentially a high-powered version of Solution I from the NusseNuclearPreparation protocol.

• 40 mM sodium citrate (Na₃⋅citrate)
  
• 1% Triton X-100
  
  • Nonidet P-40 can be substituted for Triton X-100
    

store at 4C indefinitely

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References

• http://www.mrcgene.com/tb2-dna.htm
  

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Comments

• Solution II from the NusseNuclearPreparation protocol, which contains concentrated citric acid, is NOT used in the isolation phase of this protocol. The pH of Nusse Solution II is around 2 and can damage the DNA in the nuclei.