What are the key considerations for frozen tissue samples?

Answer:
Frozen tissues must be processed using nuclei isolation, and RNA quality should be assessed prior to downstream applications (RIN > 7 recommended).

Whenever possible, fresh tissue processing is preferred to preserve cell viability and reduce stress-induced artifacts.

Sampling considerations:

• Avoid necrotic, calcified, fibrotic, or cauterized regions. 
• Trim non-target tissue as quickly as possible after collection. 
• Remove excess blood and liquid to prevent ice crystal formation during freezing. 
• For large tissues, cut into ~100 mg pieces (~green bean size) to ensure rapid and uniform freezing. 
• Recommended total input: ≥300 mg (multiple aliquots for QC and optimization). 

RNA quality assessment:

• Extract total RNA from a portion of the tissue (typically 50–100 mg; 20–30 mg for precious samples). 
• Use RIN values to evaluate RNA integrity. 
• Note that RNA quality may vary across different regions of the same tissue.  

Freezing recommendations:

• Use sealed cryotubes to prevent RNase contamination 
• Snap-freeze in liquid nitrogen (or isopentane-assisted freezing) 
• Store in liquid nitrogen (preferred) or at −80°C for short-term storage (<3 months) 
• Avoid repeated freeze-thaw cycles 

OCT-embedded fresh frozen tissues may also be used for nuclei isolation.