Table 1.
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