BACKGROUND: Archival @entity406 -fixed paraffin-embedded (FFPE) @entity5 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 @entity1 with @entity14 and to quantify the variability associated with sample age and fixation. METHODS: High-throughput miRNA profiling results from 203 @entity14 and 256 PC FFPE samples as well as from 37 paired frozen/FFPE samples from nine other @entity14 (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 @entity5 type. Nine of these miRNAs were present in both lists, and miR-103a-3p was the most stable reference miRNA for both @entity14 and PC FFPE tissue. The optimal number of reference miRNAs was 4 in @entity14 and 10 in PC. Sample age had a significant effect on global miRNA expression in PC (50  % reduction over 20  years) but not in @entity14 . @entity406 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 @entity14 - and PC FFPE tissue samples. @entity406 fixation decreased miRNA expression considerably, while the effect of increasing sample age was estimated to be negligible in a clinical setting.

MicroRNA Expression in @entity406 -fixed Paraffin-embedded XXXX Tissue: Identifying Reference MicroRNAs and Variability.

@entity5 :: ('MESH:D009369', 'Disease') :: ['cancer'] @entity1 :: ('9606', 'Species') :: ['patients'] @entity406 :: ('MESH:D005557', 'Chemical') :: ['formalin', 'Formalin'] @entity14 :: ('MESH:D015179', 'Disease') :: ['colorectal (CRC) and pancreatic (PC) cancer', 'CRC', 'CRC tumors']
@entity5 :: (MESH:D009369,Disease) :: ['Cancer']
