A Complete Pipeline for Isolating and Sequencing MicroRNAs, and Analyzing Them Using Open Source Tools

Summary

Here, we describe a step-by-step strategy for isolating small RNAs, enriching for microRNAs, and preparing samples for high-throughput sequencing. We then describe how to process sequence reads and align them to microRNAs, using open source tools.

Abstract

Half of all human transcripts are thought to be regulated by microRNAs. Therefore, quantifying microRNA expression can reveal underlying mechanisms in disease states and provide therapeutic targets and biomarkers. Here, we detail how to accurately quantify microRNAs. Briefly, this method describes isolating microRNAs, ligating them to adaptors suitable for high-throughput sequencing, amplifying the final products, and preparing a sample library. Then, we explain how to align the obtained sequencing reads to microRNA hairpins, and quantify, normalize, and calculate their differential expression. Versatile and robust, this combined experimental workflow and bioinformatic analysis enables users to begin with tissue extraction and finish with microRNA quantification.

Introduction

First discovered in 1993¹, it is now estimated that nearly 2000 microRNAs are present in the human genome². MicroRNAs are small non-coding RNAs that are typically 21-24 nucleotides long. They are post-transcriptional regulators of gene expression, often binding to complementary sites in the 3-untranslated region (3-UTR) of target genes to repress protein expression and degrade mRNA. Quantifying microRNAs can give valuable insight into gene expression and several protocols have been developed for this purpose³.

We have developed a defined, reproducible, and long-standing....

Protocol

Animal experiments were authorized by the Institutional Animal Care and Use Committee of the University of Washington.

Small RNA library preparation

1. RNA isolation

1. Isolate RNA from a biological source using a standard RNA isolation reagent, or a kit that enriches for microRNAs. For tissues, it is best to start with samples snap-frozen in liquid nitrogen and ground to a powder using a pre-chilled mortar and pestle.
2. Measure each sample’s RNA integrity on an instrument that can quantify RNA and provide an RNA integrity number (RIN). RINs should be >7.....

Results

Schematic of steps involved in library preparation
An overall schematic of small RNA extraction, sequencing, and alignment is outlined in Figure 2.
Liver samples from one male and one female mouse were collected and snap frozen in liquid nitrogen. Total RNA was extracted and evaluated for quality and concentration.

Small RNA sequencing yields sufficient RNA for seque.......

Discussion

Despite the identification of microRNAs over 20 years ago¹³, the process of microRNA sequencing remains laborious and requires specialized equipment, hindering laboratories from routinely adopting in-house protocols¹⁴. Other techniques can simultaneously evaluate microRNAs, like microRNA microarrays and multiplexed expression panels; however, these approaches are limited in that they only quantify the microRNAs present in their probe set. Because of this, they miss importan.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100 bp DNA ladder	NEB	N3231
19:1 bis-acrylamide	Millipore Sigma	A9926
25 bp DNA step ladder	Promega	G4511
Acid phenol/chloroform	ThermoFisher	AM9720
Acrylamide RNA loading dye	ThermoFisher	R0641
Ammonium persulfate (APS)	Biorad	161-0700
Bioanalyzer instrument	Agilent	G2991AA	For assessing RNA quality and concentration
Chloroform	Fisher Scientific	C298-500
Ethanol (100%)	Sigma	E7023
Gel Loading Buffer II	ThermoFisher	AM8547
GlycoBlue	ThermoFisher	AM9516	Blue color helps in visualizing pellet
HCl	Sigma	320331
KOH	Sigma	P5958
Maxi Vertical Gel Box 20 x 20cm	Genesee	45-109
miRVana microRNA isolation kit	ThermoFisher	AM1560
miSeq system	Illumina	SY-410-1003	For generating small RNA sequencing data
NaCl	Fisher Scientific	S271-500
Nusieve low-melting agarose	Lonza	50081
Parafilm (laboratory sealing film)	Millipore Sigma	P7793
Poly-ethylene glycol 8000	NEB	included with M0204
ProtoScript II First strand cDNA Synthesis Kit	NEB	E6560S
QIAquick Gel Extraction kit	Qiagen	28704
Qubit Fluorometer	ThermoFisher	Q33226	For quantifying DNA concentration
Qubit RNA HS Assay kit	ThermoFisher	Q32855
Razor Blades	Fisher Scientific	12640
Siliconized Low-Retention 1.5 ml tubes	Fisher Scientific	02-681-331
T4 RNA ligase 1	NEB	M0204
T4 RNA Ligase 2, truncated K227Q	NEB	M0351S
TapeStation	Agilent	G2939BA	For assessing RNA quality and concentration
Taq DNA Polymerase	NEB	M0273X
TEMED	Biorad	161-0800
Tris Base pH 7.5	Sigma	10708976001
Tris-buffered EDTA	Sigma	T9285
Trizol	ThermoFisher	15596026
UltraPure Ethidium bromide (10 mg/ml)	Invitrogen	15585-011
Universal miRNA cloning linker	NEB	S1315S
Urea	Sigma	U5378