Table 1.

Analytical parameters that influence concordance of human FFPE and frozen tissue biospecimens

Parameter	Optimal technique for FFPE biospecimens	Degree of concordance	Reference
DNA
Platform/method	Agilent 4×44 K oligonucleotide arrays	98% agreement achieved with Agilent 4×44 K oligonucleotide array versus 53.8-87.3% by Affymetrix SNP 6.0 array	(22)
GC-content	40%	Strongest correlations (r=0.97) were observed when probes had a GC content of 40%	(6)
Stringency	NGS, 40× coverage	99.8% agreement was achievable when NGS had 40× coverage (vs. 99% at 20×).	(15)
RNA
Platform	Human exon 1.0 array	Human exon 1.0 arrays (rather than Affymetrix U133 Plus 2.0 arrays) increased sensitivity from 75-80% to 93% and specificity from 92% to 94-96%	(25)
WTA	Unamplified	Amplification reduced the correlation coefficient from r=0.954 to r=0.88.	(31)
Amplicon size	≤100 bp	Amplification success was similar to frozen when the targeted region was ≤100 bp, unless prefaced by transcript repair.	(24,28)
Probe location	Close proximity to the 3′ end	Distance from the 3′ end had an exponential effect on probe intensity with FFPE biospecimens, compared to a linear effect with frozen biospecimens.	(3)
GC content	40-60% GC	Probes with a GC content of 40-60% were very strongly correlated between FFPE and frozen biospecimens (r>0.93), while probes with higher or lower GC content displayed weaker correlations (r<0.1).	(35)
Stringency	2 normalizer genes	Relative gene expression by normalization with 2 transcripts resulted in a stronger correlation (r=0.93) than when more than 2 transcripts were used (r=0.89).	(2)
	≥5 fold change	A differential expression threshold of 5-fold resulted in 90% agreement, compared to 55% at 2-fold.	(3)

WTA, whole transcriptome amplification