Instability in 18S and 5.8S rDNA copy numbers and 18S:5.8S ratios in longitudinally-collected human DNA samples detected by monochrome multiplex qPCR

Abstract The ribosomal DNA (rDNA) encodes the structural RNAs of the ribosomes. Ribosomal DNA instability is a major contributor to aging in yeast, but the role of rDNA instability in human aging and longevity is largely unknown. Human 45S rDNA, which encodes the 18S, 5.8S, and 28S RNAs, occurs as tandem repeats on the short arms of the five acrocentric chromosomes (p13, p14, p15, p21, and p22). The 45S rDNA copy number has been reported to range from 60 to > 800 copies per cell, and to be prone to frequent homologous and non-homologous recombination. Here we present two monochrome multiplex quantitative PCR (MMqPCR) assays, one for 18S rDNA normalized to the single copy gene beta-globin (HBB), and the other for 5.8S rDNA normalized to the single copy gene albumin (ALB), with both measurements expressed relative to those obtained from a reference DNA sample, using the relative qPCR method. Longitudinally collected pairs of DNA samples from bloods drawn approximately 16 years apart from 40 females and 39 males, aged < 1 to 77 years at first blood draw, from the Utah CEPH families were assayed. Ribosomal DNA copy number varied over a four-fold range between subjects. Both 18S and 5.8S rDNA copy numbers showed an overall increase of approximately 5% across the lifespan, with gains in copy number at second blood draw more common than losses. Within-individual gains in rDNA copy number up to +68% and losses down to ?25% were observed, while repeated assays of single DNA samples varied approximately +/-10%. While 18S rDNA copies and 5.8S copies tended to be gained and lost together, the 18S:5.8S copy number ratio was also unstable longitudinally, with increases up to +19% and decreases down to ?15% observed within individuals. The 18S:5.8S ratio at second draw, relative to that at first draw, increased significantly across the lifespan in males, but not in females. To our knowledge this is the first study to report within-individual longitudinal changes in the average 18S and 5.8S rDNA copy numbers per cell. These assays will facilitate investigations of the biology of the ribosomal RNA genes and the roles they play in the molecular pathophysiology of diseases and aging.