Elimination of Ligation Dependent Artifacts in T4 RNA Ligase to Achieve High Efficiency and Low Bias MicroRNA Capture

Adapter ligation is a critical first step in many microRNA analysis methods including microarray, qPCR, and sequencing. Previous studies have shown that ligation bias can have dramatic effects on both the fidelity of expression profiles and reproducibility across samples. We have developed a method for high efficiency and low bias microRNA capture by 3′ adapter ligation using T4 RNA ligase that does not require pooled adapters. Using a panel of 20 microRNA, we investigated the effects of ligase type, PEG concentration, ligase amount, adapter concentration, incubation time, incubation temperature, and adapter design on capture efficiency and bias. Of these factors, high PEG% was found to be critical in suppressing ligation bias. We obtained high average capture efficiency and low CV across the 20 microRNA panel, both in idealized buffer conditions (86%±10%) and total RNA spiking conditions (64%±17%). We demonstrate that this method is reliable across microRNA species that previous studies have had difficulty capturing and that our adapter design performs significantly better than the common adapter designs. Further, we demonstrate that the optimization methodology must be specifically designed for minimizing bias in order to obtain the ideal reaction parameters.