Performing Custom MicroRNA Microarray Experiments

Summary

A simple procedure of performing custom microRNA microarray experiments is described. The steps include isolating RNA, labeling RNA and reference DNA, hybridizing the samples to microarrays, scanning the microarrays, quantifying and analyzing hybridization signals.

Abstract

microRNAs (miRNAs) are a large family of ˜ 22 nucleotides (nt) long RNA molecules that are widely expressed in eukaryotes ¹. Complex genomes encode at least hundreds of miRNAs, which primarily inhibit the expression of a vast number of target genes post-transcriptionally ^{2, 3}. miRNAs control a broad range of biological processes ¹. In addition, altered miRNA expression has been associated with human diseases such as cancers, and miRNAs may serve as biomarkers for diseases and prognosis ^{4, 5}. It is important, therefore, to understand the expression and functions of miRNAs under many different conditions.

Three major approaches have been employed to profile miRNA expression: real-time PCR, microarray, and deep sequencing. The technique of miRNA microarray has the advantage of being high-throughput, generally less expensive, and most of the experimental and analysis steps can be carried out in a molecular biology laboratory at most universities, medical schools and associated hospitals. Here, we describe a method for performing custom miRNA microarray experiments. A miRNA probe set will be printed on glass slides to produce miRNA microarrays. RNA is isolated using a method or reagent that preserves small RNA species, and then labeled with a fluorescence dye. As a control, reference DNA oligonucleotides corresponding to a subset of miRNAs are also labeled with a different fluorescence dye. The reference DNA will serve to demonstrate the quality of the slide and hybridization and will also be used for data normalization. The RNA and DNA are mixed and hybridized to a microarray slide containing probes for most of the miRNAs in the database. After washing, the slide is scanned to obtain images, and intensities of the individual spots quantified. These raw signals will be further processed and analyzed as the expression data of the corresponding miRNAs. Microarray slides can be stripped and regenerated to reduce the cost of microarrays and to enhance the consistency of microarray experiments. The same principles and procedures are applicable to other types of custom microarray experiments.

Protocol

1. Printing of custom miRNA microarrays

1. Print the microarray slides using the microarray core facility services at a university or a company. The quality of microarray fabrication is one of the most critical factors for the success of a microarray experiment. Try a few services if possible. Ideally, one would print 50-100 slides at a time, and all the slides will look identical, with well separated, individual spots.
2. For microarray support, we use the GAPS II coated slides.
3. For miRNA probes, we use the NCode Multi-Species miRNA Microarray Probe Set V2.
4. Dissolve all the oligonucleotides in 3 x SSC at 10 μM and quadruply print t....

Discussion

Despite recent advances in deep sequencing technologies, microarray remains a viable choice for high-throughput analysis of DNA and RNA. Compared to deep sequencing, microarray experiments are cheaper, and a typical molecular biology laboratory can perform most of the experiments and data analysis in-house, which allows for flexibility and saves time. In the future, microarrays are likely well-suited to intensively interrogate sets of genes, e.g., all or a subset of the transcription factors in a genome or miRNAs, and .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NCode Multi-Species miRNA Microarray Probe Set V2	Invitrogen	MIRMPS201	Designed based on the miRBase Release 9.0 (October 2006). It contains ˜ 1,140 unmodified, 34-44 nt long oligonucleotides as probes for worm, fly, zebrafish, mouse, rat, and human miRNAs, and a number of internal control probes such as snoRNAs. The miRNA probes are doublets of the sequences complementary to mature miRNAs, hence the size of ˜ 44 nt. For analysis one can focus on miRNAs from a particular genome(s) of interest.
Trizol	Invitrogen	15596018	We have also used enriched, small RNA fraction for labeling, although total RNA samples are faster and easier to prepare and to quantify and suitable for downstream applications such as mRNA analysis.
T4 RNA Ligase 1	New England Biolabs	M0204L
Ulysis Alexa Fluor 647 Nucleic Acid Labeling Kit	Invitrogen	U21660	This kit or similar products can be used to label experimental RNA samples or a control RNA (instead of control DNA) as well.
5’-pCU-DY547-3’	Dharmacon	Custom made	Small RNA fraction can be similarly labeled by ligation.
CentriSep columns	Princeton Separations	CS-901
GAPSII coated slide	Corning	40004	Other types of slides may be also used.
Microarray hybridization chambers	Corning	2551 or 40080	Other kinds of hybridization chambers and coverslips should also work. Using commercially available hybridization machines can reduce hybridization time significantly, e.g., to ˜ 2 hours.
Lifterslips	Thermo Fisher Scientific, Inc.	25X60I-M5439-001-LS
BlueFuse	BlueGenome
GeneSpring	Agilent Technologies