Zhang Zhaojie, Lee Jerome E, Riemondy Kent, Anderson Emily M, Yi Rui
Genome Biol. 2013;14(10):R109. doi: 10.1186/gb-2013-14-10-r109.
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 we report an RNA cloning method that achieves over 95% efficiency for both 5′ and 3′ 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.
小RNA克隆和测序作为一种全基因组方法，能够以单核苷酸分辨率对微小RNA（miRNA）进行定量分析，具有独特的优势。然而，当前实验方案中RNA连接过程所引入的显著偏差会导致miRNA定量结果出现高达1000倍的误差。在此，我们报道了一种RNA克隆方法，该方法在5′和3′连接方面均实现了超过95%的效率。它能够对合成的miRNA进行精确量化，与预期值的偏差小于两倍，且动态范围超过四个数量级。综上所述，这种高效的RNA克隆方法能够从总RNA中准确地进行全基因组miRNA谱分析。
