(A) Standard curves of mRNAs for TPO (A), G-CSF (B), and GAPDH (C) in TaqMan RT-PCR analysis. Each standard mRNA preparation, synthesized using the method as shown in the text, was applied to the TaqMan RT-PCR system. The actual weight of each mRNA inputted to the system were plotted versus each cycle of threshold (CT). Absolute amounts of mRNA in stromal samples was determined by extrapolation to the x-axis. (B) Analysis of TPO mRNA expression in BM stromal cells treated with various cytokines. BM stromal cells obtained using Dexter culture method were cultured in -MEM with various cytokines for 24 hours. One microgram of total cellular RNA was isolated and TaqMan RT-PCR was performed with oligonucleotide primer for TPO. GAPDH was used as the internal control. Threshold Rn value and CT were obtained as described in Materials and Methods. TPO mRNA level and GAPDH mRNA level can be determined by standard curve of applied RNA weight and CT value. (C) Analysis of G-CSF mRNA expression in BM stromal cells treated with TGF-β1. BM stromal cells, obtained using the Dexter culture method, were cultured in -MEM with 0.3, 1.0, and 2.0 ng/mL of TGF-β1 for 24 hours. One microgram of total cellular RNA was isolated and TaqMan RT-PCR was performed with oligonucleotide primer for G-CSF. GAPDH was used as the internal control. G-CSF mRNA level and GAPDH mRNA level can be determined by standard curve of applied RNA weight and CT values.