Fig 1.
The fluorescently labeled miRNAs could non-specifically bind to cell surface.
K562 and Hela cells were separately incubated with Cy5-miR-363/ Cy5-miR-195 with or without RNAiMAX. Then the fluorescence signals of Cy5 on K562 and Hela cells were detected by flow cytometry (A, B) and laser scanning confocal microscope (LSCM) without nuclear staining using DAPI (C, D). Cy5-positive cells (%) and total fluorescence intensity/cell in each group were calculated and presented in figure. Statistical analysis was performed by ANOVA. ▲: P<0.05 compared with the groups of K562 cells transfected by Cy5-miR363 or Cy5-miR195 with and without RNAiMAX. ★: P<0.05 compared with the groups of Hela cells transfected by Cy5-miR363 or Cy5-miR195 with and without RNAiMAX. (E) The K562 and Hela cells with different treatments were scanned by LSCM without nuclear staining using DAPI. The Cy5 fluorescence image, transmitted light image and merged image of them were shown on figure.
Fig 2.
The type of cells and fluorescent dyes influenced on the non-specific binding of fluorescently labeled miRNAs to cell surfaces.
The percentage of fluorescence-positive cells in each group of K562 cells (A) and Hela cells (B) treated by Cy5-RS and FAM-RS with and without RNAiMAX was analyzed using flow cytometry and shown on figure. (C) K562 cells or Hela cells were treated by FAM-RS with and without RNAiMAX. The images were scanned by LSCM without nuclear staining using DAPI. The FAM fluorescence image, transmitted light image and merged image of them were shown on figure. The total fluorescence intensity per cell (TFIPC) was also calculated in each group of K562 cells (D) and Hela cells (E) treated by Cy5-RS and FAM-RS with and without RNAiMAX. All statistical analysis was performed by ANOVA. ▲: P<0.05 compared with the groups of K562 or Hela cells transfected by Cy5-RS with and without RNAiMAX. ★: P<0.05 compared with K562 cells transfected by FAM-RS with and without RNAiMAX. △: P<0.05 compared with Hela cells transfected by FAM-RS and RNAiMAX. The structural formula and hydrophilic curve of Cy5 (F) and FAM (G) showed that Cy5 was strongly hydrophobic with LogD 3.81 and FAM was strongly hydrophilic with LogD -5.53 at pH = 7.0.
Fig 3.
The fluorescence signals of non-specific binding of fluorescently labeled miRNAs were relatively stable and this non-specific binding could not promote miRNAs entering cells.
K562 and Hela cells were respectively incubated with Cy5-miR363, Cy5-miR195 and FAM-RS. Then, the total fluorescence intensity per K562 cell (A) and Hela cell (B) were detected once per two days using laser confocal microscopy. The results were presented as mean ± SD. One-way ANOVA was performed to assess significant differences between the fluorescence intensity of first day and it of other days. P<0.05 compared to the groups of K562 or Hela cells treated with Cy5-miR363 (▲), Cy5-miR195 (★) and FAM-RS (△) in other days. K562 cells were treated with FAM-miR195 (C, D) or Cy5-miR363 (E, F) with and without RNAiMAX reagent. Then, FRAP was performed as described in Materials and Methods. Total fluorescence intensity/cell was detected once per 4 seconds until 460 seconds. Results showed that fluorescence signals of the region where the fluorescence had been bleached by high-energy pulsed laser could not recover as time went by in all groups. Hela cells were also treated with FAM-miR195 (G, H) or Cy5-miR363 (I, J) with and without RNAiMAX reagent. FRAP was performed and total fluorescence intensity/cell was detected once per 6 seconds until 1380 seconds. In Hela cells treated only with Cy5-miR363 or FAM-miR195, the results were similar to that of K562 cells. In Hela cells treated with Cy5-miR363 or FAM-miR195 and RNAiMAX, fluorescence signals of the region where the fluorescence had been bleached recovered in about 16 minutes because of the high transfection efficiency of RNAiMAX in Hela cells. All results were presented as mean ± SD.
Fig 4.
The hydrophobic interaction played important roles in the non-specific binding of fluorescently labeled miRNA to the cell surface.
K562 and Hela cells were treated with FAM-RS, Cy5-miR195 and Cy5-miR363 with and without RNAiMAX. Part of them was nuclear-stained by DAPI that dissolved in pure methanol (A) or washed by the high salt buffer (cationic and anionic) (B) respectively. Then, the fluorescence signals of Cy5 and FAM were detected by LSCM. Total fluorescence intensity/cell of each group was calculated and presented in figure. Statistical analysis was performed by One-way ANOVA. The groups in the same color block were treated with the same fluorescently labeled RNA sequence. ▲: 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.
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.