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Elimination of ligation dependent artifacts in T4 RNA ligase to achieve high efficiency and low bias microRNA capture.

ABSTRACT: 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.

SUBMITTER: Song Y

PROVIDER: S-EPMC3983213 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Elimination of ligation dependent artifacts in T4 RNA ligase to achieve high efficiency and low bias microRNA capture.

Song Yunke Y Liu Kelvin J KJ Wang Tza-Huei TH

PloS one 20140410 4

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 ...[more]

PMID: 24722341

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Project description:BACKGROUND: In vitro selection of kinase ribozymes for small molecule metabolites, such as free nucleosides, will require partition systems that discriminate active from inactive RNA species. While nucleic acid catalysis of phosphoryl transfer is well established for phosphorylation of 5' or 2' OH of oligonucleotide substrates, phosphorylation of diffusible small molecules has not been demonstrated. METHODOLOGY/PRINCIPAL FINDINGS: This study demonstrates the ability of T4 DNA ligase to capture RNA strands in which a tethered monodeoxynucleoside has acquired a 5' phosphate. The ligation reaction therefore mimics the partition step of a selection for nucleoside kinase (deoxy)ribozymes. Ligation with tethered substrates was considerably slower than with nicked, fully duplex DNA, even though the deoxynucleotides at the ligation junction were Watson-Crick base paired in the tethered substrate. Ligation increased markedly when the bridging template strand contained unpaired spacer nucleotides across from the flexible tether, according to the trends: A(2)>A(1)>A(3)>A(4)>A(0)>A(6)>A(8)>A(10) and T(2)>T(3)>T(4)>T(6) approximately T(1)>T(8)>T(10). Bridging T's generally gave higher yield of ligated product than bridging A's. ATP concentrations above 33 microM accumulated adenylated intermediate and decreased yields of the gap-sealed product, likely due to re-adenylation of dissociated enzyme. Under optimized conditions, T4 DNA ligase efficiently (>90%) joined a correctly paired, or TratioG wobble-paired, substrate on the 3' side of the ligation junction while discriminating approximately 100-fold against most mispaired substrates. Tethered dC and dG gave the highest ligation rates and yields, followed by tethered deoxyinosine (dI) and dT, with the slowest reactions for tethered dA. The same kinetic trends were observed in ligase-mediated capture in complex reaction mixtures with multiple substrates. The "universal" analog 5-nitroindole (dNI) did not support ligation when used as the tethered nucleotide. CONCLUSIONS/SIGNIFICANCE: Our results reveal a novel activity for T4 DNA ligase (template-directed ligation of a tethered mononucleotide) and establish this partition scheme as being suitable for the selection of ribozymes that phosphorylate mononucleoside substrates.

Dataset Information

Elimination of ligation dependent artifacts in T4 RNA ligase to achieve high efficiency and low bias microRNA capture.

Publications

Elimination of ligation dependent artifacts in T4 RNA ligase to achieve high efficiency and low bias microRNA capture.

Similar Datasets

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