Evaluation of gene expression in urinary sediment has been considered as a promising non-invasive approach for biomarker identification of kidney diseases. Nonetheless, there are several challenges in extraction of RNA from this valuable source of biomarkers, mostly because of the factors that have influence on quality of isolated RNA such as low cellular content. Accordingly, we compared the quality of RNA from urine sediment samples that was isolated by four different methods. TRIzol reagent with basic protocol (method 1), modified procedure of TRIzol (method 2), a column-based protocol (method 3) and combination of method 1 and 3 (method 4) were applied for isolation of RNA from identical aliquots of five healthy urine samples. The quality and yield of isolated RNA were evaluated based on concentration and purity. Expression levels of GAPDH and miR-21 were studied by quantitative RT-PCR. Methods 1 and 2 showed the highest RNA yield while no difference in purity of RNA in different methods was noticed. Quantitative RT-PCR findings indicated that Ct values in samples of method 1 had the lowest level. Although higher concentrations of RNA were isolated by method 2, the declined Ct values in this method might indicate degradation of isolated RNA. Column based protocols (method 3 and 4) were failed to show significant recovery of RNA. It seems that isolation procedure using TRIzol, as a phenol based method, is the most efficient, robust and reliable procedure for RNA isolation from urinary sediment cells.