(a) Linear PCR amplification of GM-CSF transcripts. PCR amplification products were electrophoresed through 6% polyacrylamide gels. Incorporated [³²P]-dCTP was measured in counts per square millimeter on autoradiograms using the BioRad Gel Doc 1000 system. The linear relationship between GM-CSF gene transcripts and the PCR cycle number (upper panel) and between GM-CSF transcripts and various concentrations of cDNA (lower panel), which represented a calculated amount of total RNA, are shown from one donor. (b) Semiquantitative RT-PCR analysis of GM-CSF transcript levels. (A) Autoradiograms showing incorporated radioactivity of amplification products obtained from unseparated PB-MNCs of three normal donors (AH, HV, and KG) cultured in suspension with or without IL-10 for 48 hours. (B) Autoradiograms showing ABL transcripts that served as a reference to correct for potential variations of RNA or cDNA samples. (C) Corrected mean GM-CSF transcript levels in cultured PB-MNCs. Each donor sample was analyzed in three radioactive PCR analyses in duplicate using freshly synthesized cDNA. The quantity of [³²P] incorporated into the PCR product was determined by densitometric scanning of autoradiograms. Results were corrected by dividing GM-CSF values by the mean values obtained from six ABL transcripts of that cDNA.