Systematic analysis of microRNA expression of RNA extracted from fresh frozen and formalin-fixed paraffin-embedded samples.

microRNAs (miRNAs) are noncoding small RNAs that regulate gene expression at the translational level by mainly interacting with 3' UTRs of their target mRNAs. Archived formalin-fixed paraffin-embedded (FFPE) specimens represent excellent resources for biomarker discovery. Currently there is a lack of systematic analysis on the stability of miRNAs and optimized conditions for expression analysis using FFPE samples. In this study, the expression of miRNAs from FFPE samples was analyzed using high-throughput locked nucleic acid-based miRNA arrays. The effect of formalin fixation on the stability of miRNAs was also investigated using miRNA real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. The stability of miRNAs of archived colorectal cancer FFPE specimens was characterized with samples dating back up to 10 yr. Our results showed that the expression profiles of miRNAs were in good correlation between 1 mug of fresh frozen and 1-5 mug of FFPE samples (correlation coefficient R (2) = 0.86-0.89). Different formalin fixation times did not change the stability of miRNAs based on real-time qRT-PCR analysis. There are no significant differences of representative miRNA expression among 40 colorectal cancer FFPE specimens. This study provides a foundation for miRNA investigation using FFPE samples in cancer and other types of diseases.

[1]  L. Lim,et al.  A microRNA component of the p53 tumour suppressor network , 2007, Nature.

[2]  K. Livak,et al.  Real-time quantification of microRNAs by stem–loop RT–PCR , 2005, Nucleic acids research.

[3]  Vladimir Benes,et al.  A sensitive array for microRNA expression profiling (miChip) based on locked nucleic acids (LNA). , 2006, RNA.

[4]  V. Ambros,et al.  The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14 , 1993, Cell.

[5]  Masakazu Yamamoto,et al.  Non-coding MicroRNAs hsa-let-7g and hsa-miR-181b are Associated with Chemoresponse to S-1 in Colon Cancer. , 2006, Cancer genomics & proteomics.

[6]  T. Tuschl,et al.  New microRNAs from mouse and human. , 2003, RNA.

[7]  Muller Fabbri,et al.  A MicroRNA signature associated with prognosis and progression in chronic lymphocytic leukemia. , 2005, The New England journal of medicine.

[8]  V. Kim,et al.  The nuclear RNase III Drosha initiates microRNA processing , 2003, Nature.

[9]  B. Cullen,et al.  Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs. , 2004, RNA.

[10]  G. Hannon,et al.  Processing of primary microRNAs by the Microprocessor complex , 2004, Nature.

[11]  H. Lipkin.  Where is the ?c? , 1978 .

[12]  R. Russell,et al.  bantam Encodes a Developmentally Regulated microRNA that Controls Cell Proliferation and Regulates the Proapoptotic Gene hid in Drosophila , 2003, Cell.

[13]  T. Tuschl,et al.  Identification of Novel Genes Coding for Small Expressed RNAs , 2001, Science.

[14]  D. Bartel.  MicroRNAs Genomics, Biogenesis, Mechanism, and Function , 2004, Cell.

[15]  Yitzhak Pilpel,et al.  Differentially Regulated Micro-RNAs and Actively Translated Messenger RNA Transcripts by Tumor Suppressor p53 in Colon Cancer , 2006, Clinical Cancer Research.

[16]  E. Bertrand,et al.  Human let-7 stem-loop precursors harbor features of RNase III cleavage products. , 2003, Nucleic acids research.

[17]  V. Tarasov,et al.  Differential Regulation of microRNAs by p53 Revealed by Massively Parallel Sequencing: miR-34a is a p53 Target That Induces Apoptosis and G1-arrest , 2007, Cell cycle.

[18]  Quaid Morris,et al.  Probing microRNAs with microarrays: tissue specificity and functional inference. , 2004, RNA.

[19]  K. Czaplinski,et al.  Exportin 5 is a RanGTP-dependent dsRNA-binding protein that mediates nuclear export of pre-miRNAs. , 2004, RNA.

[20]  D. Bartel,et al.  Microarray profiling of microRNAs reveals frequent coexpression with neighboring miRNAs and host genes. , 2005, RNA.

[21]  C. Croce,et al.  MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[22]  K. Kosik,et al.  MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. , 2005, Cancer research.

[23]  Brian S. Roberts,et al.  The colorectal microRNAome. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Zissimos Mourelatos,et al.  Microarray-based, high-throughput gene expression profiling of microRNAs , 2004, Nature Methods.

[25]  Christopher M. Player,et al.  Large-Scale Sequencing Reveals 21U-RNAs and Additional MicroRNAs and Endogenous siRNAs in C. elegans , 2006, Cell.

[26]  V. Kim,et al.  MicroRNA maturation: stepwise processing and subcellular localization , 2002, The EMBO journal.

[27]  B. Reinhart,et al.  The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans , 2000, Nature.

[28]  Jingyue Ju,et al.  Prognostic Values of microRNAs in Colorectal Cancer , 2006 .

[29]  P. Stein,et al.  NMR structure of an alpha-L-LNA:RNA hybrid: structural implications for RNase H recognition. , 2003, Nucleic acids research.

[30]  R. Shiekhattar,et al.  The Microprocessor complex mediates the genesis of microRNAs , 2004, Nature.

[31]  R. Ach,et al.  Direct and sensitive miRNA profiling from low-input total RNA. , 2006, RNA.

[32]  V. Ambros,et al.  The timing of lin-4 RNA accumulation controls the timing of postembryonic developmental events in Caenorhabditis elegans. , 1999, Developmental biology.

[33]  Michael A. Beer,et al.  Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis. , 2007, Molecular cell.

[34]  V. Ambros.  The functions of animal microRNAs , 2004, Nature.

[35]  V. Ambros,et al.  Temporal regulation of microRNA expression in Drosophila melanogaster mediated by hormonal signals and broad-Complex gene activity. , 2003, Developmental biology.