It is estimated that in a single mammalian cell the median copy number of an mRNA is 17 molecules, with the dynamic range spread over four orders of magnitude. At the same time, an average microRNA can recognize as many as 400 target sites, and has to be able to do so equally effectively for both those mRNA species that have only a few copies and those that have a few thousand copies in the cell. Our understanding of this highly dynamic regulation network therefore depends to a high degree on our ability to accurately quantify microRNA.
Of the three major approaches to RNA quantification, microarrays and RT-PCR suffer from the same ailment: they are hybridization-based techniques, which means that only an analysis of known microRNAs is possible. A powerful alternative, which allows us to look at the whole range of known and unknown RNA species, is of course next generation sequencing (RNA-seq). RNA sequencing of small RNAs is not without its own problems, though, and one of the major issues is the tendency for ligation bias, which leads to an inaccurate microRNA representation in the sequencing library and thus an inaccurate quantification – which can be off the target by as much as 1000-fold!
To the rescue comes a study by Rui Yi and colleagues, published this week in Genome Biology. Yi and colleagues show that, when using a standard library preparation protocol, RNA-seq’s ability to quantify individual miRNA species is severely compromised (confirming the findings of a previous study by Thomas Tuschl and colleagues). They then speculate that RNA folding during 5′ ligation is a reason behind the bias and propose two simple approaches to alleviate this. First, they use a thermostable ligase and perform the ligation at an increased temperature of 60°C – this protocol modification reduces intermolecular base-pairing. Second, they use a splint adapter that is a reverse complement of the full length 5′ adapter.
The authors demonstrate that these two simple adaptations of the standard microRNA sequencing library construction protocol can decrease the deviation from 1000-fold to approximately 4-fold, and improve the ligation efficiency so that it approaches 100%. And while there is still space for improvement, Yi and colleagues’ method represents a huge step toward a cheap and efficient stoichiometric recovery of microRNA expression.