Quantitative expression profiling in formalin-fixed paraffin-embedded samples by affymetrix microarrays

Abstract

To date, few studies have systematically characterized microarray gene expression signal performance with degraded RNA from fixed (FFPE) in comparison with intact RNA from unfixed fresh-frozen (FF) specimens. RNA was extracted and isolated from paired tumor and normal samples from both FFPE and FF kidney, lung, and colon tissue specimens and microarray signal dynamics on both the raw probe and probeset level were evaluated. A contrast metric was developed to directly compare microarray signal derived from RNA extracted from matched FFPE and FF specimens. Gene-level summaries were then compared to determine the degree of overlap in expression profiles. RNA extracted from FFPE material was more degraded and fragmented than FF, resulting in a reduced dynamic range of expression signal. In addition, probe performance was not affected uniformly and declined sharply toward 5' end of genes. The most significant differences in FFPE versus FF signal were consistent across three tissue types and enriched with ribosomal genes. Our results show that archived FFPE samples can be used to profile for expression signatures and assess differential expression similar to unfixed tissue sources. This study provides guidelines for application of these methods in the discovery, validation, and clinical application of microarray expression profiling with FFPE material.

Figure 1

Analysis of RNA from Wilm's tumor and paired kidney samples from FFPE and FF quadsets. A: Yields of RNA extracted from FF and FFPE samples archived for various block ages. B: Mean yields of amplified DNA from FFPE and FF generated using WT-Ovation FFPE kit. Yields are expressed as concentration of product obtained after two rounds of amplification using a combined random- and oligo-dT–primed protocol. Note for FFPE samples 50 ng was used as input, whereas for FF samples 10 ng input RNA was used as per manufacturer's recommendations.

Figure 2

A: Raw signal density, FFPE (blue) and FF (red). B: Scatterplot of log2 raw intensity of median FFPE normal samples (y axis) versus median FF normal samples (x axis).

Figure 3

The degradation plots, based on ordering probes within a probeset according to their 3′ position and then combining the signal from similarly located probes across the array. Each line corresponds to an array, red for FFPE and blue for FF, and the slope of its trend indicates potential RNA degradation and/or inefficient labeling.

Figure 4

A: Density function of probe contrast (i.e., median pairwise difference) in log2 FF and FFPE signal across normal (i.e., nontumorous) samples. Similar contrast function was observed in tumor samples. B: Density function of median probe contrast within probesets. C: Scatterplot of median versus maximum probe contrast within a probeset.

Figure 5

A: Density of median probe contrast within a probeset in kidney, lung, and colon tissues. B: Venn diagram of median within-probeset contrast larger than two in kidney, lung, and colon data sets.

Figure 6

A: Venn diagram, showing concordance of differential expression in gene expression of Wilm's tumor and normal adjacent kidney in FF and FFPE assays. B: Scatterplot of mean fold-difference between normal kidney and Wilm's tumor found significant in both FF and FFPE comparisons. C: Percent concordance in mean fold-difference between FF and FFPE as a function of mean fold-difference between Wilm's tumor and normal kidney in respective FF and FFPE assays.

Figure 7

A side-by-side view of P value threshold (x axis) in FFPE tumor/normal comparison versus concordance with FF differentially expressed gene list under nonstringent cut-offs (bottom) and fraction of genes reported (top).