Comparison of snap freezing versus ethanol fixation for gene expression profiling of tissue specimens

Abstract

Frozen tissue specimens are the gold standard for molecular analysis. However, snap freezing presents several challenges regarding collection and storage of tissue, and preservation of histological detail. We evaluate an alternative preservation method, ethanol fixation followed by paraffin embedding, by analyzing expression profiles of microdissected cells on Affymetrix oligonucleotide arrays of three matched benign prostatic hyperplasia (BPH) and tumor samples processed with each preservation method. Frozen samples generated an average present call of 26% of the probe sets, compared to 4.5% in ethanol-paraffin samples. Eighty-eight percent of the probe sets called present in the ethanol-paraffin samples were also present in the frozen specimens. Comparing ethanol-paraffin BPH to tumor, 52 probe sets showed a twofold differential expression or higher in at least two cases, 23 of which were also differentially expressed in at least one frozen case. Despite a significant drop in the number of transcripts detectable, the data suggests that the obtainable information in ethanol-fixed samples may be useful for molecular profiling where frozen tissue is not available. However, ethanol fixation and paraffin embedding of tissue specimens is not optimal for high-throughput mRNA expression analysis. Improved methods for transcript profiling of archival samples, and/or tissue processing are still required.

Figure 1

Total RNA after microdissection, before amplification. Lanes 1 to 4, frozen samples. Lane 5, ethanol-paraffin sample.

Figure 2

Hierarchical clustering of arrays using all probe sets. A: Hierarchical clustering of all arrays. B: Hierarchical clustering of ethanol-paraffin samples. E-P, ethanol-paraffin; Fr, frozen; BPH, benign prostatic hyperplasia; Tu: tumor.