A simple procedure for routine RNA extraction and miRNA array analyses from a single thyroid in vivo fine needle aspirate

Abstract

Context. microRNA (miRNA) expression profiling and classification of tissue obtained from fine-needle aspirates (FNA) could be a major improvement of the preoperative diagnosis of thyroid nodules. Objective. Before this can be clinically implemented, a robust and non-toxic method for obtaining sufficient quantity and quality of RNA from single in vivo FNA has to be established. RNAlater is a non-toxic RNA-stabilizing agent. However, due to the high density of RNAlater, pelleting of the tissue samples is difficult, and results in low recovery of RNA that is insufficient for subsequent miRNA array expression analysis. We therefore developed a simple centrifugation method for capturing tissue stored in RNAlater on a 0.45-μm filter. Design. FNA from 24 patients with a solitary cold thyroid nodule was stored in Trizol, liquid nitrogen, or RNAlater. The tissue stored in RNAlater was either pelleted by centrifugation or captured on the 0.45-μm filters. RNA was extracted using the Trizol method. To validate results, FNA from additional 30 patients were analyzed based on the modified RNAlater protocol. Main outcome. Capturing FNA tissue samples on the filters increased the RNA yield 10 fold and maintained RNA purity, permitting miRNA array expression profiling and allowing comparable levels of known miRNA-clusters regardless of preservation technique. Results were confirmed in an additional 30 patients. Conclusion. The modified RNAlater protocol is well suited for isolating RNA from single thyroid in vivo FNA in a clinical setting. Furthermore this permits shipping of FNA samples at room temperature from peripheral centers to a centralized array core facility.

Key Words::

• Fine needle aspirate
• miRNA microarray
• RNA preservation
• RNAlater
• thyroid nodules

Acknowledgments

This work was supported by The Agnes and Knut Mørk Foundation, The Research Foundation of Herlev University Hospital, The Toyota Foundation, and The Novo Nordisk Foundation. We thank The Danish Cancer Society for use of scanner equipment.

Declaration of interest: The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.