Table 5. Considerations when performing RNA sequencing from postmortem tissues.

Factora	Examples	Potential Effect on RNA Yield, Quality or Dataa
Ultimate Cause of Death of Patient	Sepsis, Prolonged Hypotension	Lower RNA yield and quality of the affected tissues
	Acute Myocardial Infarction, Stroke	Minimal effect
Postmortem Interval	Prolonged	Lower RNA yield and quality that is tissue type dependent
Postmortem Refrigeration	Immediate delivery of patient to morgue after death	Can negate effects of prolonged postmortem interval
Tissue type	Normal tissue	RNA yields and quality are tissue type dependent and related to cause of death and PMI
	Cancer tissue	RNA yields and quality may not be tissue type dependent and related to extent of necrosis or autolysis in the sample
Tissue storage	Prolonged storage (e.g. >10 years)	Lower RNA yields
Tissue handling	Multiple freeze/thaw cycles	Lower RNA quality
	Prolongation between death and tissue processing	Lower RNA quality
	Prolongation between time of processing of different samples during autopsy	Potential intrapatient variability in RNA quality
RNA handling	Multiple freeze/thaw cycles	Lower RIN values
Method of RNA Extraction	RNeasy mini plus kit	RNA > 200 bp
Method of Library Preparation	PolyA enrichment (requires relatively high quality RNA (RIN > 5)	Higher transcriptome coverage
	Ribosomal depletion (best for degraded RNA)	Lower transcriptome coverage

^aThese potential effects are based on the assumption that all other factors for the patient and tissues are optimal. Should more than one adverse factor exist an even greater loss of RNA yield or quality could be expected.