This project investigates monitoring gene expression with differential PCR gels using exploratory data analysis methods developed with GELLAB-II, Lemkin PF etal. Electrophoresis 1989;10:122-40. Replicate differential display PCR gels were compared statistically to find robust gene changes before, during and after treatment with growth hormone. This quantitative data analysis will be used to study differences in gene expression in lymphocytes from patients with Prader-Willi syndrome (PWS) who have been given somatotropin (Rogan). Replicate differential display PCR gels would be compared statistically to find robust gene changes before, during and after treatment with growth hormone. The hypothesis is that we will be able to find robust bands which change just after treatment showing effects of imprinting. PWS patients with growth hormone deficiency will be treated with growth hormone and monitored. The differential display PCR technique separates and displays a profile of cDNA products synthesized from mRNA on long DNA sequencing gels. The exploratory analysis techniques will be used to identify consistent changes in specific cDNAs. The contrasting expression profiles of the cDNAs in patients with PWS relative to children with other growth hormone deficiencies may reveal potential effectors that were transcribed from imprinted genes and give a better understanding of these control mechanisms. Prior to studying the effect of growth hormone on PWS patients, we wanted to establish the methodology for automatic analysis of ddPCR data. The initial experiments let us develop the method using existing differential display results for congenic rat lung from sacrificed embryos (Floros) using rat embryo lung at different stages of gestation and in response to maternal dexamethasone treatment during pregnancy from multiple animals from the same and different litters. We have scanned the gels and using software from CSPI/Scanalytics (RFLPscan(TM)), have been able to detect, size, and quantify most of the cDNA bands in the samples. Then, statistical analysis and searches of this data are made for expression changes significantly at different stages of gestation or in response to dexamethasone. We have manually analyzed some of the RFLPscan(TM) band data. There were significant differences between the sets of conditions (18 days gestation, 22 days gestation, and 18 days gestation + dexamethasone). The data analysis is continuing.
