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Cryogenic Homogenization of Animal Tissue

Many traditional procedures for RNA isolation dictate the use of cryogenic methods.  Traditional methods using a mortar and pestle chilled with liquid nitrogen are still useful for larger samples, though are not practical for high throughput operations or samples less than 100 mg. Though non-cryogenic methods are available for RNA isolation, homogenizing samples cryogenically for high throughput operations and processing very small samples is discussed below. 

Samples that are less than 100 mg can be processed using a CryoGrinder, which is a miniaturized mortar with a motorized pestle.  The small well size keeps the sample contained for easy collection after grinding.  CryoGrinders are best used for a limited number of samples.  For high throughput sample processing, the Geno/Grinder is the only homogenizer capable of processing samples cryogenically.  Sample throughput is lower than room temperature homogenization as a 24 well format is used over the traditional 96 well format.  Deep well plates, which are made of polypropylene, are very brittle at cryogenic temperatures which cause grinding balls to punch out the bottoms of wells.  The 24 well format uses polycarbonate vials which can withstand processing.

Sample Disruption with the CryoGrinder™

  1. CryoGrinder™ can be decontaminated with rinse solutions (e.g., RNase Away) and by autoclaving.  Dry heat sterilization can damage the pestles.

  2. If using a CryoCooler™ , charge the reservoir with liquid nitrogen (about 1 L).  If using a pan or foam cooler, pour in liquid nitrogen to a depth of approximately 1.5 inches.

  3. Place the pestle inside the CryoGrinder™ well and place in an insulated pan or by lying on the rack within the reservoir of the CryoCooler™.  Allow the CryoGrinder to chill for 15-20 minutes.  If necessary, replenish the liquid nitrogen, but don't expose the well and pestle tip to the liquid nitrogen which may contain significant contamination (see link).

  4. The sample, which must be less than 100 mg for best results, is placed in the mortar's well.  Wait 2 minutes for the sample to drop in temperature.  Note that a 100 mg sample is only 5 x 5 x 4 mm in dimensions.  Samples down to 10 mg can be effectively ground and recovered.

  5. Wear gloves to protect from the cold.  Attach the pestle to the cordless wrench.  Hold the mortar firmly while pressing down on the sample with the pestle while the wrench is moving.  Move the pestle over the sample to ensure all the pieces become pulverized. 

  6. When the sample is homogenized, remove the pestle from the wrench and tap in the well to remove any residual sample. 

  7. Using a pre-chilled thin tipped spatula, gently loosen the ground tissue from the walls and bottom of the mortar.  Place a large microfuge tube or larger tube over the well, invert, and tap the mortar to transfer the ground sample to the tube.

  8. For RNA isolation, add extraction buffer (e.g., Trizol or Tri Reagent) immediately before the sample thaws.  The tube can also be placed into the liquid nitrogen so it remains frozen.

  9. Allow the CryoGrinder mortar and pestle to warm up before decontamination by autoclaving and cleaning with a biologically safe detergent and water.