Accurate Molecular Characterization of Formalin-Fixed, Paraffin-Embedded Tissues by microRNA Expression Profiling

Abstract

Formalin-fixed, paraffin-embedded tissues are an invaluable tool for biomarker discovery and validation. As these archived specimens are not always compatible with modern genomic techniques such as gene expression arrays, we assessed the use of microRNA (miRNA) as an alternative means for the reliable molecular characterization of formalin-fixed, paraffin-embedded tissues. Expression profiling using two different microarray platforms and multiple mouse and human formalinfixed, paraffin-embedded tissue types resulted in the correlation ratios of miRNA expression levels between frozen and fixed tissue pairs ranging from 0.82 to 0.99, depending on the cellular heterogeneity of the tissue type. The same miRNAs were identified as differentially expressed between tissues using both fixed and frozen specimens. While formalin fixation time had only marginal effects on microarray performance, extended storage times for tissue blocks (up to 11 years) resulted in a gradual loss of detection of miRNAs expressed at low levels. Method reproducibility and accuracy were also evaluated in two different tissues stored for different lengths of time. The technical variation between full process replicates, including independent RNA isolation methods, was approximately 5%, and the correlation of expression levels between microarray and real-time quantitative reverse transcriptase polymerase chain reaction was 0.98. Together, these data demonstrate that miRNA expression profiling is an accurate and robust method for the molecular analysis of archived clinical specimens, potentially extending the use of miRNAs as new diagnostic, prognostic, and treatment response biomarkers. (J Mol Diagn

The diversity of sex steroid action: the role of micro-RNAs and FOXO transcription factors in cycling endometrium and cancer

“…A similar scenario may contribute to breast cancer development : miR-9, miR-19b, miR-146, miR-181c, miR-183, miR-200c, miR-205, miR-223, miR-423, miR-425 Y: let-7a, miR-32, miR-33b, miR-369, miR-409, miR-424, miR-431, miR-451, miR-496, miR-503 miR-516 Ratner et al (2010) EEC carcinoma versus benign tissue from control patients [: miR-34a, miR-182, miR-183, miR-200a, miR-205, miR-572, miR-622, miR-650 Y: miR-411, miR-487b Chung et al (2009) EEC versus benign tissue from control patients [: miR-10a, miR-23a, miR-25, miR-28, miR-34a, miR-95, miR-103, miR-106a, miR-107, miR-130b, miR-141, miR-151, miR-155, miR-17-5p, miR-182, miR-183, miR-184, miR-191, miR-194, miR-200a, miR-200c, miR-203, miR-205, miR-210 : miR-10a, miR-31, miR-96, miR-133b, miR-141, miR-142-5p, miR-155, miR-182, miR-200a, miR-200b, miR-200c, miR-203, miR-205, miR-210, miR-363, miR-429, miR-432, miR-449 Y: miR-99b, miR-133, miR-193a, miR-193b, miR-204, miR-368 Boren et al (2008) EEC versus atypical hyperplasia versus normal endometrium [: let-7c, miR-103, miR-106a, miR-107, miR-181a, miR-185, miR-210, miR-423 Y: let-7i, miR-30c, miR-152, miR-193, miR-221 MiRNA represents a new class of biomarkers that can complement existing conventional markers, including metabolites, antigens and mRNA transcripts. The fact that miRNAs remain largely intact in formalin-fixed, paraffinembedded clinical samples highlights their potential in molecular phenotyping of benign and malignant reproductive disorders as well as for monitoring treatment efficacy (Bitossi et al 2008, Szafranska et al 2008, Tam 2008, Jorgensen et al 2010, Nuovo 2010, Schuster et al 2010. Furthermore, recent evidence suggests that some neoplastic processes, for example, ovarian cancer, generate mRNA profiles in blood cells characteristic of the tumours, suggesting that whole blood miRNA profiling could be used for diagnostic purposes (Balch et al 2009, Lodes et al 2009, Resnick et al 2009, Hausler et al 2010.…”

miRNA analysis of formalin-fixed squamous cell carcinomas of the tongue is affected by age of the samples

“…Some, however, have noted a decrease in miRNAs detected with time in storage. This could indicate degradation of miRNA over time or a decrease in labelling efficiency due to increased recovery of short non-specific RNA (17)(18)(19). One study found a number of miRNAs to be up regulated in FFPE but not FF samples, which was hypothesised to be caused by non-specific binding of fragmented mRNA and immature miRNA to the array.…”

“…One potential miR-424 target, ITGAV, has previously been seen to significantly correlate with differentiation and metastasis in laryngeal and hypopharyngeal carcinomas (29,32). A number of studies investigating the usefulness of miRNA expression analysis in FFPE tissue have been performed, and many show good correlation between FFPE and FF samples (16)(17)(18). Some, however, have noted a decrease in miRNAs detected with time in storage.…”

“…While formalin fixation is an excellent method for preserving the architecture of the tissue for histological investigation, it causes degradation and modification of nucleic acids making it difficult to study gene expression levels in FFPE samples (14,15). miRNAs are hypothesised to be more stable than mRNA in FFPE samples due to their small size and secondary structure, and a number of studies have shown that miRNA profiles from formalinfixed and fresh-frozen tissue correlate well (16)(17)(18), and some have reported decreased ability to detect certain miRNAs with prolonged storage time (17,19).…”

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and <em>Ex vivo</em> Fibroblasts

“…The focus here was exclusively on miRNA expression, since miRNAs are strongly implicated in the pathogenesis of numerous malignancies and deregulated expression both in epithelial and cancer associated stromal cells have important consequences in CRC 2,3,5 . Moreover miRNAs are stable to extraction and analysis from conventional archival formalin-fixed paraffin-embedded tissue, making them powerful biomarkers for diagnosis and prognostication on human tissue subjected to conventional processing in routine histopathological laboratories 9,10 . However, our methodology could very easily be adapted for genomic and proteomic analysis, further emphasizing the versatility of this approach.…”