Robust global micro-RNA profiling with formalin-fixed paraffin-embedded breast cancer tissues

• Published in: Laboratory investigation Vol. 89; no. 5; pp. 597 - 606
• Main Authors: Hui, Angela B Y, Shi, Wei, Boutros, Paul C, Miller, Naomi, Pintilie, Melania, Fyles, Tony, McCready, David, Wong, Derek, Gerster, Kate, Jurisica, Igor, Penn, Linda Z, Liu, Fei-Fei
• Format: Journal Article
• Language: English
• Published: New York Elsevier Inc 01.05.2009; Nature Publishing Group US; Nature Publishing Group
• Subjects: Aged; Aged, 80 and over; Biological and medical sciences; Biotechnology; Breast Neoplasms - metabolism; breast tumours; Carcinoma, Ductal, Breast - metabolism; Case-Control Studies; dysregulated miRNA; Female; formalin-fixed paraffin-embedded samples; Fundamental and applied biological sciences. Psychology; Humans; Investigative techniques, diagnostic techniques (general aspects); Laboratory Medicine; Medical sciences; Medicine; Medicine & Public Health; micro-RNA profiling; Microarray Analysis - standards; MicroRNAs - metabolism; Middle Aged; Paraffin Embedding; Pathology; quantitative real-time PCR; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; technical-report; formalin-fixed paraffin-embedded samples; breast tumours; micro-RNA profiling; quantitative real-time PCR; dysregulated miRNA; Biotechnology; Breast disease; RNA interference; Breast tumor; Micro RNA; Breast cancer; Malignant tumor; Paraffin; Gene expression profile; Mammary gland diseases; Gene silencing; Tissue; Clinical biology; Cancer; Real time polymerase chain reaction
• ISSN: 0023-6837; 1530-0307
• DOI: 10.1038/labinvest.2009.12

Global micro-RNA (miR) profiling of human malignancies is increasingly performed, but to date, the majority of such analyses have used frozen tissues. However, formalin fixation is the standard and routine histological practice for optimal preservation of cellular morphology. To determine whether miR analysis of formalin-fixed tissues is feasible, quantitative real-time PCR (qRT-PCR) profiling of miR expression in 40 archival formalin-fixed paraffin-embedded (FFPE) breast lumpectomy specimens were performed. Taqman Low Density Arrays (TLDAs) were used to assess the expression level of 365 miRs in 34 invasive ductal carcinomas and in 6 normal comparators derived from reduction mammoplasties. Its technical reproducibility was high, with intra-sample correlations above 0.9 and with 92.8% accuracy in differential expression comparisons, indicating such global profiling studies to be technically and biologically robust. The TLDA data were confirmed using conventional single-well qRT-PCR analysis, showing a strong and statistically significant concordance between these two methods. Paired frozen and FFPE breast cancer samples from the same patients showed a similar level of robust correlation of at least 0.94. Compared with normal breast samples, a panel of miRs was consistently dysregulated in breast cancer, including earlier-reported breast cancer-related miRs, such as upregulated miR-21, miR-155, miR-191, and miR-196a, and downregulated miR-125b and miR-221. Additional novel miR sequences of potential biological relevance were also uncovered. These results show the validity and utility of conducting global miR profiling using FFPE samples, thereby offering enormous opportunities to evaluate archival banks of such materials, linked to clinical databases, to rapidly acquire greater insight into the clinically relevant role for miRs in human malignancies.