Figure 2

Identification of single GFP-positive and negative cells from a mixed population. A-B) Adherent GFP-negative and GFP-positive cells obtained by the described cell isolation method and observed by fluorescence microscopy. C) qPCR curves demonstrating the ability to differentiate between GFP-negative (top) and GFP-positive (bottom) cells without pre-amplification. Two gene targets were identified in each single cell: beta-actin (magenta) and GFP (green). The delayed amplification shown in the GFP-negative curves are caused by primer dimers, as supported by melt curve analysis, agarose gel electrophoresis and DNA sequencing. D) Melt curve analysis showing the identification of individual peaks corresponding to the presence or absence of GFP (green), while beta-actin is observed at similar levels in both samples (magenta). E) Analyzed data for three GFP-positive (left group) and three GFP-negative (right group) cells isolated from a mixed population of cells. Results for each single cell were normalized to expression of beta-actin (ACTB) and reported as normalized C_q, which is defined as C_{q, GFP} - C_{q, ACTB}. Error bars represent standard deviation of 3 technical replicates of divided samples from individual cells. The difference between normalized C_q from GFP+/- is significant as determined by T-test with p < 0.05. F) Validation gel illustrating the presence of beta-actin in both cells, but a differential presence of GFP amplification in cells which were observed to be GFP-positive versus GFP-negative. Off-target bands in the negative control are primer dimers as confirmed by melt-curve analysis.