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.
The Cuff laboratory combined two isolation techniques to obtain pure populations of DCs. An anti-CD11c MACS column (Miltenyi Biotech) was used to selectively bind DCs present in dissected Peyers's Patches from infected or uninfected animals. The DCs were eluted from the column and further enriched using fluorescent activated cell sorting (FACS) based on differential expression of characteristic myeloid and lymphoid markers. The purification strategy resulted in less than 10,000 cells per sample. To avoid the inevitable loss of RNA using standard RNA isolation protocols, the samples were processed using Cells-to-cDNA technology. In brief, each cell sample was lysed in 50 µl of Cells-to-cDNA Lysis Buffer by heating sample at 75°C for 10 min. DNase I digestion was performed at 37°C for 30 min. Reverse transcription of 10 µl of the lysate was followed by real-time PCR using commercial TaqMan® probes (ABI) and primers in a Roche Lightcycler®.
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!
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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.