Flexible, multi-use, PCR-based nucleic acid integrity assays based on the ubiquitin C gene

Abstract

Nucleic acid integrity assessment is an important aspect of quality control for many applications in molecular biology. A number of methods exist (electrophoresis- or PCR-based), but they are not universally applicable. Some of them need huge amounts of input, process certain amount of samples, require expensive equipment, only work specifically on (c)DNA, (m)RNA, certain species or certain tissues, or produce fragments covering a small length range. We investigated if the ubiquitin C gene (UBC) could be used to develop flexible, multi-use PCR-based (deoxy)ribonucleic acid integrity assays. UBC gene analysis (in human, mouse, pig, cow, horse, sheep, dog and cat) shows that UBC is a highly conserved and ubiquitously expressed gene (reference gene in RT-qPCR), that encodes a polyubiquitin precursor (containing tandem repeats of at least 5 ubiquitin monomers of 228 bp) in a single exon. On average, ubiquitin monomers show a nucleic acid sequence identity of 96% at intraspecies level and 93% at interspecies level. Based on a multiple alignment of all monomer ubiquitin sequences of all investigated species, we could design a single degenerated primer pair generating PCR amplicons of 137, 365, 593 and 821 bp on low amounts of high quality DNA of all investigated species (down to 10 pg) and on cDNA reverse transcribed from high quality RNA from different tissues (e.g. heart, liver, brain, kidney). Increasing levels of nucleic acid degradation resulted in a decrease of amplification products starting with the longer amplicons. We conclude that UBC is suited to develop a single, cheap, universal assay to estimate the presence, integrity and amplificability of native mammalian nucleic acids. In addition, we used the same strategy to design a similar assay to check the quality of bisulfite treated mammalian DNA.