BACKGROUNDS: Deregulated levels of miRNAs, short noncoding RNAs associated with pathogenesis of many diseases, have been observed in cerebrospinal fluid (CSF). Therefore, the analysis of CSF miRNAs in patients affected by tumors of central nervous system (CNS) might help to develop new diagnostic platform enabling more precise diagnosis. Thus, in our study we tried to optimize methodical approaches to be used for miRNA detection as RNA isolation and selection of suitable technology for global high-throughput miRNA profiling. MATERIAL AND METHODS: In the optimization phase of RNA isolation from CSF, various commercially available kits with different protocol modifications were compared. Two quantitative polymerase chain reaction panels and Next Generation Sequencing method were tested for selection of the most suitable method for miRNA comprehensive profiling. RESULTS: The Urine miRNA Purification kit (Norgen) and Next Generation Sequencing was selected as the most suitable kit for RNA extraction from CSF and method for miRNA comprehensive profiling, resp. CONCLUSION: We established a protocol for RNA isolation and miRNA comprehensive profiling in CSF clinical specimens.Key words: brain neoplasm - cerebrospinal fluid - microRNA The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. This study was supported by Ministry of Health of the Czech Republic, grant No. 15-34553A. All rights reserved.Submitted: 19. 3. 2018Accepted: 10. 4. 2018.