D.Ranamukhaarachchi, J.J.Langone, R.K.Elespuru, CDRH, FDA, Rockville MD
Molecular diagnosis of disease using differential gene expression requires targeting and detection of RNA synthesized by affected cells against a background of other cells that usually predominate by orders of magnitude. Detection of antigen-specific lymphocytes within the general pool of lymphocytes by this approach is a difficult task in the diagnosis of immunological disorders. Using real-time PCR, we evaluated the detection limits of gene expression by spiking phytohemagglutinin (PHA)-stimulated lymphocytes into aliquots of unstimulated lymphocytes from the same sample in ratios of 1/1 to 1/1, 000, 000. Differentially spiked lymphocytes were normalized to 2X106 cells each. RNA was extracted, reverse transcribed into c-DNA, and analyzed for proliferation genes expressed exclusively or in large copy number in PHA-treated cells using real-time PCR. Results indicated the detection of a single stimulated lymphocyte in a background of 1000 unstimulated cells for low expressed genes, and one in 100, 000 for highly expressed genes. In other experiments using mRNA with known abundance, we were able to detect genes at a copy number as low as 10 after reverse transcription of mRNA. In addition to disease diagnosis, these findings have broad application in determining efficacy and quality control standards for diagnostic devices, and detection of pathogens.