MicroRNA Expression in Formalin-fixed Paraffin-embedded Cancer Tissue: Identifying Reference MicroRNAs and Variability

Abstract

Background: Archival formalin-fixed paraffin-embedded (FFPE) cancer tissue samples are a readily available resource for microRNA (miRNA) biomarker identification. No established standard for reference miRNAs in FFPE tissue exists. We sought to identify stable reference miRNAs for normalization of miRNA expression in FFPE tissue samples from patients with colorectal (CRC) and pancreatic (PC) cancer and to quantify the variability associated with sample age and fixation.

Methods: High-throughput miRNA profiling results from 203 CRC and 256 PC FFPE samples as well as from 37 paired frozen/FFPE samples from nine other CRC tumors (methodological samples) were used. Candidate reference miRNAs were identified by their correlation with global mean expression. The stability of reference genes was analyzed according to published methods. The association between sample age and global mean miRNA expression was tested using linear regression. Variability was described using correlation coefficients and linear mixed effects models. Normalization effects were determined by changes in standard deviation and by hierarchical clustering.

Results: We created lists of 20 miRNAs with the best correlation to global mean expression in each cancer type. Nine of these miRNAs were present in both lists, and miR-103a-3p was the most stable reference miRNA for both CRC and PC FFPE tissue. The optimal number of reference miRNAs was 4 in CRC and 10 in PC. Sample age had a significant effect on global miRNA expression in PC (50% reduction over 20 years) but not in CRC. Formalin fixation for 2-6 days decreased miRNA expression 30-65%. Normalization using global mean expression reduced variability for technical and biological replicates while normalization using the expression of the identified reference miRNAs reduced variability only for biological replicates. Normalization only had a minor impact on clustering results.

Conclusions: We identified suitable reference miRNAs for future miRNA expression experiments using CRC- and PC FFPE tissue samples. Formalin fixation decreased miRNA expression considerably, while the effect of increasing sample age was estimated to be negligible in a clinical setting.

Fig. 1

Association between sample age and global mean miRNA expression for CRC (left) and PC (right) samples. Age of the tumor blocks are plotted on the x-axis and global mean miRNA expression in 40-C_q values on the y-axis. A linear regression line has been plotted and the formula is shown.

Fig. 2

Correlation between miRNA expression profiles measured in five runs from the same purification. Correlations are illustrated by a pairs plot with axes showing miRNA expression in 40-C_q values. The sample numbers correspond to the project IDs in Table S1 in Additional file 1.

Fig. 3

Correlation between miRNA expression profiles measured in four samples from the same tumor block. Correlations are illustrated by a pairs plot with axes showing miRNA expression in 40-C_q values. The sample numbers correspond to the project IDs in Table S1 in Additional file 1.