Constitutive expression of RET transcripts (semiquantitative RT-PCR) and GDNFR-α (RT-PCR) in normal lymphohematopoietic cells. Cell fractions were isolated to purity by discontinuous gradient centrifugation and/or immunomagnetic selection. Peripheral T cells were activated with 10 ng/mL TPA plus 1.0 μg/mL of ionomycin A for 72 hours. Adherent macrophages (Adh. macroph.) were activated with 100 ng/mL of GM-CSF for 12 hours. In all cases, 1 μg of total RNA was reverse-transcribed in a 20-μL reaction mix containing hexadeoxyribonucleotides random primers. Four microliters was amplified with primers specific for RET and GDNFR-α. In the case of RET, amplification was performed in the presence of a constant amount (10⁻³ attomoles) of RET_Comp fragment. After 35 cycles of amplification, 15 μL of PCR products was resolved on 1.5% agarose gel, blotted, and hybridized with specific RET and GDNFR-α oligoprobes. For semiquantitative evaluation of RET transcripts, ratios of the relative intensities of bands corresponding to RET cDNA (790 bp) and RET_Comp (597 bp) -amplified products were quantified by computer imaging of gel, expressed in AU, and graphed. An adult human brain substantia nigra cDNA and cDNA derived from the MN-60 cell line were respectively used as positive (+) and negative (−) controls for RET and GDNFR-α expression. cDNAs were always tested with β-actin–specific primers.