Small RNA cloning and sequencing is uniquely positioned as a genome-wide approach to quantify miRNAs with single-nucleotide resolution. However, significant biases introduced by RNA ligation in current protocols lead to inaccurate miRNA quantification by 1000-fold.
Here, researchers from the University of Colorado, Boulder report an RNA cloning method that achieves over 95% efficiency for both 5[prime] and 3[prime] ligations. It achieves accurate quantification of synthetic miRNAs with less than two-fold deviation from the anticipated value and over a dynamic range of four orders of magnitude. Taken together, this high-efficiency RNA cloning method permits accurate genome-wide miRNA profiling from total RNAs.
- Zhang Z, Lee JE, Riemondy K, Anderson EM, Yi R. (2013) High-efficiency RNA cloning enables accurate quantification of miRNA expression by deep sequencing. Genome Biol 14(10), R109. [abstract]