Drop-on-Demand Single Cell Isolation and Total RNA Analysis
Figure 3
(a) Droplets are generated at cell concentrations: 0.5×105, 1.0×105, 1.5×105, and 2.0×105 cells/ml and they contained 0.28±0.12, 0.88±0.14, 1.50±0.10, and 2.20±0.19 cells per droplet, respectively (mean ± standard deviation). The droplets were ejected using two controlled parameters: 34 kPa nitrogen gas pressure and 55 µs valve opening period. Values were obtained from 5 groups, each containing 100 droplets. Each group was tested by single factor analysis of variance (ANOVA). F(3,16) = 0.15, 0.58, 1.22, and 0.16, n = 5 mean values per each group, *p>0.33. (Student's t-test between target cell concentrations: **p<0.0004, n = 5 mean values of 4 concentrations). (b) Number of cells in droplets was obtained at cell concentrations: 2.5×105, 5.0×105, and 7.5×105 cells/ml. Regression coefficients, R2 values, were *0.93 (1.7 cells/drop), **0.96 (3.6 cells/drop), and ***0.92 (6.6 cells/drop), n = 28 droplets. (c) Comparing target cell concentration in the reservoir and in the droplet pattern. Average and standard deviation of 10% to 50% target cell concentration in the reservoir were 10.3±0.6, 19.0±1.0, 27.8±2.0, and 45.1±2.1. F(3,16) = 0.82, 1.38, 0.98, and 0.52, *p>0.29, ANOVA for each cell concentration (student's t-test for each target cell concentration: **p<0.001, n = 5 mean values of 4 different target cell concentrations). (d) Fraction of empty droplets in droplet array as a function of cell concentration. Average and standard deviation of empty droplets for 0.5 to 2.5×105 cells/ml concentrations were 72.8±12.8%, 40.6±7.7%, 23.8±4.0%, 12.7±5.8%, and 2.2±0.3%, respectively. F(3,16) = 0.05, 0.64, 0.56, 0.05, and 0.67, *p>0.58, ANOVA for each target cell concentration (student's t-test for each cell concentration: **p<0.016, n = 5 mean values of 4 different target cell concentrations). (e) Fraction of target homogeneous droplets among the patterned droplets was calculated as the ratio between the number of cells in homogenous droplets and the overall number of droplets for different cases: 1, 2, 3, 4, and 5 cells per droplet. This excluded the empty droplets. At 1.0×105, for the case of one cell per droplet, the average and standard deviation values of homogeneity were 9.5±1.4%, 18.8±3.7%, 26.0±3.1%, and 45.5±8.8% for 10 to 50% target cell concentration. The fractions were 0.4±0.9%, 3.1±2.1%, 5.1±4.2%, and 9.6±4.8% for droplets containing two cells. (f) Frequency of homogeneous droplets based on cell concentration. This analysis includes the empty droplets. Among the different target cell concentrations, the 1×105 cells/ml concentration showed 4.7±0.6%, 9.0±1.9%, 12.7±1.6%, and 21.6±5.2% homogeneous droplet occurrences. F(1,8) = 22.7, 11.2, and 13.3, *p<0.01, ANOVA for 1×105 cells/ml through 10 to 50% target cell concentration (student t-test: **p = 0.12, ***p<0.05, n = 5). Error bars represented the standard deviation of the mean. The p values in student's t-test were calculated based on two-sided distributions with unequal variances.