The Non-Specific Binding of Fluorescent-Labeled MiRNAs on Cell Surface by Hydrophobic Interaction
Fig 5
Real time PCR confirmed the important roles of hydrophobic interaction in non-specific binding of fluorescently labeled miRNA to the cell surface.
K562 and Hela cells were treated with FAM-miR195 and Cy5-miR363 with and without RNAiMAX. Part of them was washed by methanol or high salt buffer (cationic and anionic) respectively. Then, the amount of these two miRNAs in cells with methanol washing (A) and high-salt washing (B) was assayed using real time PCR. The groups in the same color block were treated with the same fluorescently labeled RNA sequence. Results were presented as fold change of miRNA expression compared to untreated cells. Additionally, K562 and Hela cells were treated by Cy5-miR1 with and without RNAiMAX. Part of them was also washed by methanol or high salt buffer (cationic and anionic) respectively. Then, Twf1 (target gene of miR1) expression in all groups of K562 cells (C) and Hela cells (D) was detected using real time PCR. Results were presented as fold change of Twf1 expression compared to the untreated cells. A positive number indicated up-regulation and a negative number indicated down-regulation. All data were shown as mean ± SD. Statistical analysis was performed by One-way ANOVA. ▲: P<0.05 compared with the groups of K562 or Hela cells not indicated by solid black triangle. ★: P<0.05 between the groups of K562 and Hela cells with the same treatment. *: P<0.05 compared with the groups of Hela cells not indicated by asterisk.