Figure 3. Panel A - mRNA expression of dendritic cell Taar1 in the presence and absence of DNase incubation.

RT-PCR was performed on poly A(+) RNA isolated from total dendritic cell RNA using the μMACS column kit with and without, in column, DNase digestion as recommended by the manufacturer. Taar1 expression is shown in the presence and absence of the DNase incubation. The PCR products for GAPDH are shown at the bottom of the panel. Panel B – Detection of Taar1 PCR products from low copy number genomic DNA. To illustrate the ease with which false PCR products can be obtained, the cDNA samples from the DNase treated mRNA used for the results in panel A were spiked with 8, 4, 2 and 1 copies of genomic DNA. 16 and 0 mouse genome copies are used as positive and negative controls, respectively. Results are shown as amplified products electrophoresed on ethidium bromide stained agarose gels. DNA sizes in base pairs are shown to the left of the DNA ladder. Panel C - Quantitative real time PCR and mRNA copy number in mouse macrophages and dendritic cells. Expression of Taar1 mRNA was assayed by real time PCR using the same macrophage and dendritic cell cDNA from the DNase-treated total RNA as was used to obtain the results shown in figures 2. Samples include macrophages and dendritic cells that were activated for 6 hr with LPS or γHV-68. PCR was also performed with 10⁴ copies of mouse genomic DNA as a positive control. The results suggest that there is less than 1 copy of Taar1 mRNA per 4x10³ macrophages, or dendritic cells. The stained gel insert shows the DNA products after 45 cycles of real time PCR electrophoresed on a 4% agarose gel.