UTILIZATION OF REAL–TIME PCR FOR THE RELATIVE QUANTITATION OF MHC SOURCE

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TITLE: UTILIZATION OF REAL–TIME PCR FOR THE RELATIVE QUANTITATION OF MHC SOURCE–PROTEIN TRANSCRIPTS

Mary E. Ellexson-Turner, Heather D. Hickman, Angela D. Luis, Wilfried Bardet, William H. Hildebrand

Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK

Real–time PCR (RT PCR) is a valuable method for determining the relative mRNA expression of cellular genes. Altered mRNA expression is observed in multiple diseased states, including neoplasm and viral infection. Recently, real–time PCR has been employed to determine regulation of cellular genes during both diseased states. Since endogenous proteins are presented as MHC class I–bound peptides and because altered transcription can result in the expression of new peptide–MHC complexes, we devised a method to assess the contribution of transcriptional regulation of ligand–source proteins’ transcripts to peptide presentation. To determine the relationship between transcriptional activation and altered ligand presentation, peptides are eluted from a MHC class I transfected into a “normal” or uninfected cell line and a neoplastic or infected cell line. Once peptides derived from cellular proteins, such as tumor–associated antigens, are identified, the DNA sequences of their source proteins are used to derive specific–PCR primers. Total RNA is then isolated from the same normal and altered cell lines, and cDNA synthesized. Using the peptide–source protein primers, RT PCR amplification is performed on an ABI® PRISM 7700 using the SYBR® Green I dye assay chemistry, and the comparative C_T method is used for the relative quantitation of gene expression. Upregulated and downregulated genes are classified as compared with a housekeeping gene. Application of this technique to HIV–infected cells yielded a peptide–source protein transcriptionally altered during the course of infection. This method may thus be applied universally to determine expression levels of source proteins from pathogenically–challenged or cancerous cells.