Competitive single-cell RT-PCR of sibling cells induced to unilineage erythroid differentiation. A representative experiment is shown. Single CD34⁺CD38⁻ cells were induced to unilineage erythroid differentiation as described in Materials and Methods. When the 4-cell stage was reached (at round I, II, III, and IV), a single daughter cell was isolated and the RNA corresponding to 0.8 cell equivalents processed for competitive RT-PCR to semiquantitate the CD34 mRNA. Polyadenylated cRNA CD34 deletion construct corresponding to 10 transcripts (based on prior titration) was added to each single-cell lysate and coprocessed with the wt mRNA. Ethidiumbromide-stained RT-PCR products were separated over a 2.5% agarose gel (Metaphor; Biozym) and photographed. Each band was excised from the gel and the radioactive counts in each were determined. Based on Cerenkov counts of excised bands, round I, II, III, and IV daughter cells contain 60%, 20%, 3%, and 0.01% of the CD34-mRNA transcripts present in a freshly sorted CD34⁺CD38⁻cell, respectively. Lane M.W.: size marker. Lane 0: single sorted CD34⁺CD38⁻ cell before culture. Lane I: single daughter cell (first round). Lane II: single daughter cell (second round). Lane III: single daughter cell (third round). wt CD34 RT-PCR product is only barely detectable. Lane IV: single daughter cell (fourth round). Note that the wt CD34 mRNA message is gradually downregulated from round I to IV. Products corresponding to β2m-mRNA-transcripts but not to genomic sequences were detected when the RNA corresponding to 0.2 cell equivalents from each daughter cell was processed for RT-PCR using β2m primers (not shown).