Drop-on-Demand Single Cell Isolation and Total RNA Analysis
Figure 4
Total RNA expressions in printed droplets (virtual gel).
(a) Total RNA quality on virtual gels (Agilent bioanalyser) as number of droplets increase. The 96-well plate was then placed underneath the ejector and one droplet was ejected into each of the first nine wells of the first row of wells. This procedure was repeated with additional four rows of the plate, where two, three, four and five droplets were ejected in each well resulting to a total of five groups of droplet samples, each group having an increasing droplet volume and increasing number of cells ranging from ten to fifty cells. For extraction purposes and consistency, before the RNA analysis was performed, each row was divided into three triplicates. Hence, the analysis was based on average 30, 60, 90, 120, and 150 cells for each triplicate for bioanalysis. To investigate nonprinted cells, 0.5 µl samples were drawn from the cell solution and pipetted into the three wells in each of the first three rows of the plate as controls. The controls had 1000 cells on average. The last lane showed the results for the control RNA which was prepared by a manual dilution method without ejection. (b, c) Comparison of the reproducibility of the RNA expression levels for (b) two replicates of the control set by the manual pipette method and (c) two experimental RNA samples obtained by droplet method. The scatter-plots were compared the reproducibility of expression level measurements for the 1000 genes with the highest expression levels in the experimental samples. (d) Expression of stem cell-related markers was examined to assess whether RNA obtained from the cell encapsulating droplets provided useful biological information in comparison to control samples. Our results showed that these 11 stem cell markers including Kit and Notch1, were found in both the patterned cells and the control groups.