1Department of Pharmacology, University of Minnesota, 2Masonic Cancer Center, University of Minnesota
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Zhang, X., Zeng, Y. Performing Custom MicroRNA Microarray Experiments. J. Vis. Exp. (56), e3250, doi:10.3791/3250 (2011).
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.
1. Printing of custom miRNA microarrays
2. Sample preparation
3. Microarray hybridization
Note: We use Corning microarray hybridization chambers and Erie Scientific's mSeries of Lifterslips to perform microarray hybridization.
4. Post-hybridization processing
5. Representative Results:
We have largely followed the described procedures to profile global miRNA expression in thousands of samples, i.e., RNAs isolated from zebrafish to human specimen under many different conditions. Figure 1 shows a scanned image of a microarray to demonstrate very precise and strong hybridization signals on the slide. The Pearson correlation coefficients between technical replicates of microarray hybridization are ˜ 0.99 7, indicating excellent reproducibility.
Figure 1 Composite image of a scanned miRNA microarray slide after hybridization. Red spots resulted from hybridizations by the reference DNA, green spots from the DY547-labelled RNA sample, while the yellow spots were from hybridizations by both the DNA and RNA to the same probes.
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 the same principles and procedures presented here can be used in custom microarray experiments to study gene families besides miRNAs. Of course, a major drawback about microarrays is that sequence information must be available a priori. This is not a problem for most protein gene families in model organisms, although how many miRNA genes there are remain unclear. The probe set we have been using was designed based on miRBase 8 Release 9.0 in October 2006. From recent studies 9, 10, it is apparent that the most abundant and most biologically relevant mammalian miRNAs are already covered by this probe set.
There are no major technical challenges to perform custom microarray experiments. Based on our experience, the keys to the success of a microarray experiment are: 1) to obtain well-printed slides, 2) to prepare RNA of good quality and in sufficient quantity, and 3) to properly wash the slides after hybridization. Trizol reagent typically yields sufficient amounts of intact RNA for subsequent studies. Nonetheless, miRNA expression varies among different tissues and cell lines. Under most conditions, 20-25 μg of total RNA should give sufficiently strong microarray signals when labeled by the ligation method. Less RNA is required for miRNA-rich samples such as brain regions and neurons. If only 1-5 μg of total RNA or less is available, other labeling methods may be used, e.g., RNA labeling kits from Invitrogen, and they are compatible with the custom microarray platform described here. For the best comparisons, one should always label at least one replicate of all the control and experimental samples simultaneously and hybridize them to the same print batch of slides in a single experiment. This research design will minimize the contribution to differential microarray signals resulting from the variations in sample labeling and processing and in slide quality. The reference DNA (in red channel) serves as a control for the quality of slide, hybridization, and washing. A successful hybridization will give clean and strong signals in red. If DNA signals are good but RNA signals are not, then one should trouble-shoot the steps of RNA isolation and labeling. For example, does the RNA have the ratio A260nm/A280nm between 1.9 and 2.1? Is the pellet reddish in 3.3?
Most of the investment for microarrays may be for slide printing and scanning. A microarray printer and a scanner are often equipped by the core facility at many universities and medical schools. Printing microarrays in bulk and reusing slides and lifterslips will significantly reduce the cost. Reusing slides has the additional advantage of improving the consistency of microarray experiments 7. We have reused slides more than six times and found the data quality still satisfactory. To ensure the best sensitivity for the most precious samples or samples with minimal input RNAs, however, it is still prudent to use fresh slides 7.
No conflicts of interest declared.
The work was supported in part by National Institute of Drug Abuse Center (P50 DA 011806) and United States Army Department of Defense (W81XWH-07-1-0183).
|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|