Figure 1.
(A) miRNAs but not detectable mRNA are present in conditioned media in nuclease resistant form. Conditioned media was collected from MCF7 cells and further treated as indicated on the graph. Cel-miR-39 (5 pg) was added into the media either before or after addition of denaturing Trizol LS reagent (Sigma). As a control for miRNA recovery, 5 pg of cel-miR-39 was dissolved in 30 μl of nuclease-free water. Total RNA from each sample was eluted in 30 μl of nuclease-free water and the content of miRNAs, β-actin and α-tubulin mRNAs were analyzed using quantitative real-time PCR as described in the ‘Materials and Methods’ section. Data presented as raw Ct values, each bar represents mean (SD) (n = 3). (B) Extracellular (EC) and intracellular (IC) levels of three miRNA species in five different cancer cell lines were measured by quantitative real-time PCR. Data presented as Ct values normalized on spiked in cel-miR-39 control; each bar represents mean (SD) of three independent RNA isolations. (n = 3). (C and D). Intra- and extra-cellular miRNA spectra for MCF-7 cells were measured by TaqMan miRNA Low density array (Applied Biosystems). (C). Correlation plot shows Ct values of miRNAs obtained from total RNA extracted from 400 μl of conditioned media and corresponding MCF7 cells monolayer. Those miRNAs that were not detected on the array (Ct > 35) were assigned Ct values of 40. EC—miRNAs presented exclusively in the cells; EM—miRNAs presented exclusively in the media (lists of exclusively expressed miRNAs Cp value <35 in Supplementary Table S1). (D) Those miRNAs that were detected in both cells and the media demonstrated a dramatic difference in extracellular/intracellular amount. Note, miRNAs content in the media was much lower [average ΔCt (cell-media) = −7.5].