Abstract
For quantitative microRNA analyses in formalin-fixed paraffin-embedded (FFPE) tissue, expression levels have to be normalized to endogenous controls. To investigate the most stably-expressed microRNAs in breast cancer and its surrounding tissue, we used tumor samples from primary tumors and from metastatic sites. MiRNA profiling using TaqMan® Array Human MicroRNA Cards, enabling quantification of 754 unique human miRNAs, was performed in FFPE specimens from 58 patients with metastatic breast cancer. Forty-two (72%) samples were collected from primary tumors and 16 (28%) from metastases. In a cross-platform analysis of a validation cohort of 32 FFPE samples from patients with early breast cancer genome-wide microRNA expression analysis using SurePrintG3 miRNA (8 × 60 K)® microarrays from Agilent® was performed. Eleven microRNAs could be detected in all samples analyzed. Based on NormFinder and geNorm stability values and the high correlation (rho ≥ 0.8) with the median of all measured microRNAs, miR-16-5p, miR-29a-3p, miR-126-3p, and miR-222-3p are suitable single gene housekeeper candidates. In the cross-platform validation, 29 human microRNAs were strongly expressed (mean log2-intensity > 10) and 21 of these microRNAs including miR-16-5p and miR-29a-3p were also stably expressed (CV < 5%). Thus, miR-16-5p and miR-29a-3p are both strong housekeeper candidates. Their Normfinder stability values calculated across the primary tumor and metastases subgroup indicate that miR-29a-3p can be considered as the strongest housekeeper in a cohort with mainly samples from primary tumors, whereas miR-16-5p might perform better in a metastatic sample enriched cohort.
Highlights
MicroRNAs are small, approximately 22 nucleotides long non-coding single-stranded RNAs, regulating gene expression at a post-transcriptional level
Gene stability values according to geNorm analysis [19,20] and NormFinder [21], with lower values indicating increased gene stability across samples, were lower for most of the eleven microRNA housekeeper candidates compared to small nuclear RNA U6 (snU6) (Figure 1A,B, Table 1, Tables S2 and S3 (Supplementary Material))
NormFinder stability values calculated across the primary tumor and metastasis subgroup, showed highest gene stability for miR-16-5p in the metastasis subgroup and for miR-126-3p in the primary tumor subgroup
Summary
MicroRNAs are small, approximately 22 nucleotides long non-coding single-stranded RNAs, regulating gene expression at a post-transcriptional level. (1) Raw expression levels can be normalized to endogenous controls like housekeeping genes (microRNAs). For Taqman® human microRNA cards, the small nuclear RNA (snRNA) U6 and the small nucleolar RNAs (snoRNAs) RNU44 and RNU48 are recommended as endogenous controls based on healthy tissue and tumor cell line studies (NCI-60). These small RNAs have different biological und biochemical characters [14] compared to microRNAs and extraction quality, reverse transcription and PCR amplification may differ from that of microRNAs [14,15]. We investigated the most stably-expressed microRNA in breast cancer tissues from primary and metastatic sites
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