Fig. 4. (A) Reproducibility of the single cell RT-PCR protocol. Ventral marginal zone (VMZ) and dorsal marginal zone (DMZ) explants were dissected and single cells dissociated in calcium/magnesium-free medium. Single cells are picked and lysed, then the lysate was split into A and B samples. These samples were then subjected to RT-PCR in parallel. cDNA from each reaction was dot blotted onto a nylon filter and probed with a specific marker. (B) Examples of dot blots. cDNA from each single cell probed for the markers indicated (ODC, Mix.1, Gsc, Xbra). (C) Sensitivity of single cell RT-PCR protocol. Single cell lysates were spiked with 1, 10, 50, 100, 500, 1000 and 5000 transcripts of polyadenylated eGFP RNA and subjected to RT-PCR, dot blotted and probed for GFP. In two separate RT-PCR experiments, 10 transcripts of GFP were detected. No signal above background was detected in the no GFP RNA sample. (C) A dot blot where samples from separate RT-PCR reactions were analyzed together.