ABSTRACT
Standard Guthrie cards have been widely used to collect blood samples from essentially all USA and Japanese neonates for newborn screening programs. Thus, archival blood spot samples are a unique and comprehensive resource for molecular pathology studies. However, the challenge in using these samples is the presumed low quantity and degraded quality of nucleic acids that can be isolated from these samples, particularly the RNA. Here, we report a new assay using Agilent 4x44K microarrays for acquiring genome-wide gene expression profiles from blood spots on Guthrie cards. Due to the small amount of RNA obtained from each sample, major modifications, such as concentrating and amplifying the RNA and using a different labeling procedure, were performed. Approximately 9000 expressed genes can be detected after normalization of data, an increment of 260% in detection power compared with previously reported cDNA microarrays made in-house with standard procedures. The correlation coefficients in technical and biological replicates were 0.92 and 0.85, respectively, confirming the reproducibility of this study. This new and comprehensive assay will add value to the utility of archival Guthrie cards (e.g. neonatal blood spot cards) and open new opportunities to molecular epidemiology, pathology, genomic, and diagnostic studies of perinatal diseases.
Subject(s)
Blood Specimen Collection/methods , Gene Expression Profiling/methods , Neonatal Screening/methods , Oligonucleotide Array Sequence Analysis/methods , Humans , Infant, Newborn , Polymerase Chain ReactionABSTRACT
The Caenorhabditis elegans pos-1 gene encodes a zinc-finger protein that is required for germline specification during embryogenesis. The maternally provided mRNA is translationally regulated both spatially and temporally during early development. We have cloned orthologs of pos-1 from C. briggsae and C. remanei, two Caenorhabditis species that have diverged from C. elegans by approximately 20-40 million years. Two regions in the 3' untranslated region are highly conserved among all three species. We find that the pos-1 RNA is expressed in the hermaphrodite and female gonads of C. briggsae and C. remanei but POS-1 protein is not detected at high levels in C. briggsae until the 2-cell stage of embryogenesis. The protein expression is restricted to the germline precursors of the embryo. We conclude that pos-1 appears to be translationally regulated in C. briggsae as it is in C. elegans and speculate the conserved 3' UTR sequences may be involved.
