RT Journal Article
SR Electronic
T1 Monochrome multiplex qPCR of 18S and 5.8S human rDNA
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 361840
DO 10.1101/361840
A1 Chris B. Ligerman
A1 Richard M. Cawthon
YR 2018
UL http://biorxiv.org/content/early/2018/07/04/361840.abstract
AB 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, were assayed. Our results are consistent with essentially all subjects having the expected 1:1 ratio of 18S to 5.8S copies at each blood draw; however, we observed up to +/- 40% gains and losses in rDNA copy number between the first and second draws, while repeated assays of single DNA samples varied approximately +/- 10%. There was no apparent trend toward either increasing or decreasing rDNA copy number with age; and no apparent difference between the sexes in the magnitude of rDNA instability. To our knowledge this longitudinal study is the first to show that rDNA copy number can change across the lifetime of an individual. 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.