Examination of βig-h3 gene expression by RT-PCR. (A) Preferential βig-h3 gene expression in imDCs. RNA was isolated from: (1) monocytes, (2) macrophages, (3) LPS-activated macrophages, (4) imDCs, and (5) mDCs. RT-PCR was performed using βig-h3–specific and β-actin primers. (B) Monocytes were cultured in the presence of GM-CSF and IL-4 and harvested at the indicated time points up to 6 days. RNA was isolated from these cells and used in RT-PCR. (C) Real-time PCR for βig-h3 expression in the different cell types. RNA samples obtained as in panel A were subjected to 45 cycles of PCR using the Light Cycler and FastStart DNA Master SYBR Green I kit. “Control” indicates that cDNA template was omitted. β-Actin expression in the 5 RNA samples was similarly examined which gave Ct values of 36.48 to 37.15 showing little variation in cDNA inputs between different samples. (D) Real-time PCR analysis of βig-h3 expression during monocyte differentiation into imDCs. The RNA samples are as in panel B. The Ct values for β-actin are 31.92 (day 0), 31.47 (day 1), 32.52 (day 2), 32.03 (day 4), and 31.91 (day 6). These variations do not significantly affect the conclusion drawn based on the βig-h3 expression data. Melting curves for all reactions exhibited single specific PCR product (data not shown). (E) RT-PCR was performed with 19 human tissue RNA samples using βig-h3 primers. The RNA samples are: (1) adrenal gland, (2) bone marrow, (3) brain, (4) fetal brain, (5) colon, (6) small intestine, (7) kidney, (8) liver, (9) fetal liver, (10) lung, (11) placenta, (12) prostate, (13) salivary gland, (14) skeletal muscle, (15) spinal cord, (16) spleen, (17) testis, (18) trachea, and (19) uterus. PCR reactions in panels A-B and E were separated on 1% (wt/vol) agarose gels and visualized using ethidium bromide.