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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

We present a detailed small RNA library reparation protocol with less bias than standard methods and an increased sensitivity for 2'-O-methyl RNAs. This protocol can be followed using homemade reagents to save cost or using kits for convenience.

Abstract

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as plant microRNAs (miRNAs) carry a 2'-O-methyl (2'-OMe) modification at their 3' terminal nucleotide. This modification adds another difficulty as it inhibits 3' adapter ligation. We previously demonstrated that modified versions of the 'TruSeq (TS)' protocol have less bias and an improved detection of 2'-OMe RNAs. Here we describe in detail protocol 'TS5', which showed the best overall performance. TS5 can be followed either using homemade reagents or reagents from the TS kit, with equal performance.

Introduction

Small RNAs (sRNAs) are involved in the control of a diversity of biological processes1. Eukaryotic regulatory sRNAs are typically between 20 and 30 nt in size; the three major types are microRNAs (miRNA), piwi-interacting RNAs (piRNA) and small interfering RNAs (siRNA). Aberrant miRNA expression levels have been implicated in a variety of diseases2. This underscores the importance of miRNAs in health and disease and the requirement for accurate, quantitative research tools to detect sRNAs in general.

Next-generation sequencing (NGS) is a widely used method to study sRNAs. Main advantages of NG....

Protocol

1. Isolation of small RNAs

  1. Extract total RNA using phenol-based reagents or any other method. Verify if the RNA is of good quality.
  2. Pre-run a 15% TBE-urea gel (see Table of Materials) for 15 min at 200 V.
  3. While the gel is pre-running, mix 5-20 µg of total RNA in a 5-15 µL volume with an equal volume of formamide loading dye (see Table of Materials; 95% deionized formamide, 0.025% bromophenol blue, 0.025% xylene cyanol, 5 mM EDTA pH 8) in a 200 &#.......

Representative Results

Critical steps are the isolation of the small RNA fraction of the starting total RNA material (Figure 3) and the desired final library product (Figure 4). Both steps involve polyacrylamide gel purification; small RNA is isolated from 15% TBE urea gels, while the final libraries are isolated from 6% native TBE gels. Small RNA isolated from gel can be analyzed on a small RNA capillary electrophoresis chip (Table of Materials; .......

Discussion

Small RNA library preparation remains challenging due to bias, mainly introduced during adapter ligation steps. RNAs with a 2'-OMe modification at their 3' end such as plant miRNAs, piRNA in insects, nematodes and mammals, and small interfering RNAs (siRNA) in insects and plants are particularly difficult to study because the 2'-OMe modification inhibits 3' adapter ligation. A number of solutions have been proposed in the literature to improve sRNA library preparation protocols, but most commerc.......

Acknowledgements

This work was supported by the National Center for Scientific Research (CNRS), The French Alternative Energies and Atomic Energy Commission (CEA) and Paris-Sud University. All library preparation, Illumina sequencing and bioinformatics analyses for this study were performed at the I2BC Next-Generation Sequencing (NGS) facility. The members of the I2BC NGS facility are acknowledged for critical reading of the manuscript and helpful suggestions.

....

Materials

NameCompanyCatalog NumberComments
2100 Bioanalyzer Instrument AgilentG2939BA
Acid-Phenol:Chloroform, pH 4.5 (with IAA, 125:24:1)ThermoFisherAM9720
Adenosine 5'-Triphosphate (ATP)Nex England BiolabsP0756S
Agencourt AMPure XP beadsBeckman CoulterA63880
Bioanalyzer High Sensitivity DNA KitAgilent5067-4626
Bioanalyzer Small RNA KitAgilent5067-1548
Corning Costar Spin-X centrifuge tube filtersSigma AldrichCLS8162-96EA
Dark Reader transilluminatorvarious suppiers
HotStart PCR Kit, with dNTPsKapa BiosystemsKK2501
NEXTflex small RNA-seq V3 kitBIOO ScientificNOVA-5132-05optional
Novex TBE gels 6%ThermoFisherEC6265BOX
Novex TBE Urea gels 15%ThermoFisherEC6885BOX
QIAquick Nucleotide Removal KitQiagen 28304
Qubit 4 Quantitation Starter KitThermoFisherQ33227
Qubit ssDNA Assay KitThermoFisherQ10212
RNA Gel Loading Dye (2X)ThermoFisherR0641
RNA Gel Loading Dye (2X)ThermoFisherR0641
RNase Inhibitor, Murine Nex England BiolabsM0314S
SuperScript IV Reverse TranscriptaseThermoFisher18090200
SYBR Gold Nucleic Acid Gel StainThermoFisherS11494
T4 RNA Ligase 1 (ssRNA Ligase)Nex England BiolabsM0204S
T4 RNA Ligase 2, truncatedNex England BiolabsM0242S
TrackIt 50 bp DNA ladderThermoFisher10488043
TruSeq Small RNA Library Prep KitIlluminaRS-200-0012/24/36/48optional
UltraPure GlycogenThermoFisher10814010
XCell SureLock Mini-CellThermoFisherEI0001
XCell SureLock Mini-CellThermoFisherEI0001
ZR small RNA ladderZymo ResearchR1090
the last two numbers correspond to the set of indexes

References

  1. Ghildiyal, M., Zamore, P. D. Small silencing RNAs: an expanding universe. Nature Reviews Genetics. 10, 94-108 (2009).
  2. Chang, T. C., Mendell, J. T. microRNAs in vertebrate physiology and human disease. Annual Review of Gen....

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