Selective Release of MicroRNA Species from Normal and Malignant Mammary Epithelial Cells
Figure 2
Some MicroRNAs are Released Disproportionately.
Duplicate microRNA microarrays were hybridized with 1 µg of total cellular or 1 µg of extracellular miRNA from MCF7 cells. Results are plotted as A relative fluorescent intensities of extracellular (x, upper panel) and cellular (c, lower panel) miRNAs, or B ratio of extracellular to cellular miRNAs. The horizontal lines in B indicate the threshold of 2 fold-changes, whereas the red and the green marked populations indicate a greater than 4-fold enrichment in the released extracellular (A, upper panel, x), or in the cells (A, lower panel, c) respectively. C Scatter plot of average reads of the miRNAs quantitated by array. Only miRNAs with a fluorescent value of greater than 500 in the cellular or extracellular population are shown (see Materials and Methods). The numbers next to dots indicate the miRNA the dot represents. D MCF7 cells were cultured for 5 days, and the total amount of specific cellular and extracellular miRNAs were measured by quantitative linker-ligation mediated RT-PCR, and the miRNA ratios were plotted. The average of 3 independent experiments is shown. E Native PAGE of products at end-point of quantitative RT-PCR. The major PCR-products between 32–48 ntds correspond to the mature miRNA (miRNA) as expected by size and determined by sequencing (Table S5). The bands with a migration of less then 25 ntds are the PCR primers (primers) used in the reaction. Bands that retained in the well are amplification-independent reaction components (reaction components). Hsa-miR-923 has since been reclassified as a specific rRNA fragment.