Using FACS Samples
Detection and quantitation of gene expression in rare primary cell populations is extremely difficult and isolating good quality RNA from these samples is nearly impossible. Alicia Mathers, a graduate student in Dr. Chris Cuff's laboratory at the University of West Virginia, utilized Ambion's Cells-to-cDNA™ technology to overcome the difficulty in working with small numbers of cells. Dr. Cuff's lab is studying changes in gene expression in mouse dendritic cells (DC) in Peyer's Patches, post reoviral infection, in order to elucidate the factors contributing to the anti-viral immune response. The data generated using Cells-to-cDNA was used to confirm the hypothesis that the level of expression of several cytokines are altered in DCs in response to infection.
A summary of the results is shown in Figure 1. The level of each transcript was normalized to the expression of GAPDH (a housekeeping gene) to account for slight variations in cell counts. Expression levels are expressed as a ratio of the target gene:GAPDH control. The data show that post infection the amount of gene A increases in the lymphoid, but not the myeloid population. For gene B the reverse pattern occurs. The reovirus infection caused an increase in gene B levels in the myeloid DCs unlike what is observed in the lymphoid DCs where expression is relatively constant. To find out more information about the immune response to reoviral infection, keep an eye out for publications from the Cuff laboratory!
Ambion customers frequently develop new and interesting adaptations of our Cells-to-cDNA technology. Do you have an interesting Cells-to-cDNA story? If so we would like to know! Please contact our Technical Service Department.
Figure 1. Real-time RT-PCR Assessment of Reoviral Effect on Dendritic Cells. Graphical summary of real-time RT-PCR data using TaqMan® assays for GAPDH and two cytokines (gene A and gene B) in reoviral infected and uninfected dendritic cells.