The present study developed a miRNA microarray analysis method suitable for tissue pathology applications. This method uses the miRNA Complete Labeling and Hyb Kit to extract miRNAs from FFPE samples, and then miRNAs are analyzed using a miRNA microarray. The results showed that this method can effectively extract miRNAs from FFPE samples and accurately analyze miRNA expression. This method provides a feasible method for miRNA analysis using FFPE samples.
MicroRNAs (miRNAs) have significant diagnostic and prognostic potential for diseases like cancer. While fresh-frozen tissues are ideal, formalin-fixed paraffin-embedded (FFPE) samples are more readily available for retrospective studies. This document addresses the challenge of extracting small RNAs from FFPE samples, which often suffer from degradation and cross-linking.
The study tested various total RNA extraction methods on matched colon and lung cancer quad sets stored for 1 to 10 years. The extracted RNA was analyzed using the Agilent miRNA microarray system with the miRNA Complete Labeling and Hyb Kit. Results showed that most miRNAs detected in fresh-frozen samples were also detected in FFPE-derived samples, with good assay reproducibility.
| Microarray Probes | 723 human and 73 human viral miRNAs |
|---|---|
| Hybridization Temperature | 55°C |
| Hybridization Duration | 20 hours |
| RNA Input Amount | 100 ng |
| Kit Part Number | 5190-0456 |
The primary challenge is the extraction of total RNA that appropriately retains the small RNAs, as most methods are optimized for longer RNAs.
The miRNeasy FFPE Kit (Qiagen) and the RecoverAll Total Nucleic Acid Isolation Kit (optimized for FFPE samples, Life Technologies) were tested.
Each slide contains probes for 723 human and 73 human viral miRNAs.