MicroRNA isolation and stability in stored RNA samples

Abstract

MicroRNAs (miRNAs) are small RNA molecules, which act as post-transcriptional regulators of a gene expression, with important functions within the cell physiology. Whilst many authors have focused on the study of miRNA expression in physiological and pathological processes, various technical variables related to miRNA isolation have simultaneously emerged and the stability of the stored miRNA samples has been questioned. A robust method for RNA isolation is essential for reproducible results and miRNAs instability in the stored samples would make for an alarming situation for most expression studies. Here these issues are discussed and we investigate the stability of miRNAs isolated from clinical samples of B lymphocytes (chronic lymphocytic leukemia) by the most commonly utilized method based on a Trizol/TRI-Reagent solution (RNAs stored at -80 degrees C). To assess the stability of miRNAs, a Real Time-PCR analysis was performed for a panel of 29 miRNAs from a freshly isolated RNA sample and after 14 days storage at -80 degrees C. Furthermore, a Real Time-PCR analysis was repeatedly performed for a stored RNA sample over a period of approximately 10 months. We observed high stability of isolated miRNAs and respective cDNAs. The reproducibility and efficiency of the Trizol/TRI-Reagent isolation method was also tested and compared to the mirVana Isolation kit (Ambion) and RNeasy kit (Qiagen). In conclusion, Trizol/TRI-Reagent based isolation is a robust reproducible method, and obtained miRNA samples do not show any tendency to degradation when properly stored and handled.