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Comparative Study
. 2004 Dec;14(12):2486-94.
doi: 10.1101/gr.2845604.

MicroRNA expression detected by oligonucleotide microarrays: system establishment and expression profiling in human tissues

Omer Barad  1 , Eti Meiri, Amir Avniel, Ranit Aharonov, Adi Barzilai, Isaac Bentwich, Uri Einav, Shlomit Gilad, Patrick Hurban, Yael Karov, Edward K Lobenhofer, Eilon Sharon, Yoel M Shiboleth, Marat Shtutman, Zvi Bentwich, Paz Einat
Affiliations
Comparative Study

MicroRNA expression detected by oligonucleotide microarrays: system establishment and expression profiling in human tissues

Omer Barad et al. Genome Res. 2004 Dec.

Abstract

MicroRNAs (MIRs) are a novel group of conserved short approximately 22 nucleotide-long RNAs with important roles in regulating gene expression. We have established a MIR-specific oligonucleotide microarray system that enables efficient analysis of the expression of the human MIRs identified so far. We show that the 60-mer oligonucleotide probes on the microarrays hybridize with labeled cRNA of MIRs, but not with their precursor hairpin RNAs, derived from amplified, size-fractionated, total RNA of human origin. Signal intensity is related to the location of the MIR sequences within the 60-mer probes, with location at the 5' region giving the highest signals, and at the 3' end, giving the lowest signals. Accordingly, 60-mer probes harboring one MIR copy at the 5' end gave signals of similar intensity to probes containing two or three MIR copies. Mismatch analysis shows that mutations within the MIR sequence significantly reduce or eliminate the signal, suggesting that the observed signals faithfully reflect the abundance of matching MIRs in the labeled cRNA. Expression profiling of 150 MIRs in five human tissues and in HeLa cells revealed a good overall concordance with previously published results, but also with some differences. We present novel data on MIR expression in thymus, testes, and placenta, and have identified MIRs highly enriched in these tissues. Taken together, these results highlight the increased sensitivity of the DNA microarray over other methods for the detection and study of MIRs, and the immense potential in applying such microarrays for the study of MIRs in health and disease.

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Figures

Figure 1.
Figure 1.
MIRs are responsible for signals from probes of precursor sequences. Signals from hybridization of 60-mer probes with HeLa lcRNA are presented. (A) Three probes were considered for each MIR as follows: (1) precursor sequence containing the full MIR (hairpin + MIR); (2) MIR in the 5′ end of the probe, followed by nonhuman genome sequences (MIR in 5′); (3) Precursor sequence containing no more than 16 nucleotides of the MIR sequence (hairpin–MIR). Only probe sets in which the microarray signal of the hairpin + MIR probe was >2000 were considered. For each probe set, the signal intensities for the three types of probes were calculated as a ratio of the signal intensity of the hairpin + MIR probe. The MIR in 5′ and hairpin–MIR bars show the average computed for 23 sets of probes. (B) A detailed example of a mismatch analysis of pre-MIR-125b. The pre-MIR-125b probe and its respective mismatch (mm) probes from MirChip1 are presented. Wild-type (wt) probe contains MIR-125b at position 2 to 23 (boxed). Probes with mismatches are listed below the wild-type probe with the location of the substituted nucleotides specified. The signal column presents the microarray signal intensity observed for each probe. Similar results were obtained for let-7a and miR-30c.
Figure 2.
Figure 2.
Effect of MIR location and number within the 60-mer probes on signal intensity. Signals from hybridization of 60-mer probes with HeLa lcRNA are presented. (A) A set of three probes was considered for each MIR as follows: (1) MIR in the 5′; (2) MIR in the middle; (3) MIR in the 3′. For all three probes, the rest of the 60-mer sequence was composed of nonhuman genome sequences. Only probe sets for which the microarray signal of the MIR in the 5′ probe was >2000 were considered. For each probe set, the signal intensities for the three types of probes were calculated as a ratio of the signal intensity of the MIR in the 5′ probe. (B) Probes containing one, two (duplex), or three (triplex) copies of a MIR were analyzed. For each probe set, the signal intensities for the three types of probes were calculated as a ratio of the signal intensity of the probe containing one MIR copy. (C) Probes containing two or three MIR copies were analyzed. (None) Probes containing duplex or triplex MIRS, in which none of the MIR copies contain mismatches. (First) Duplex or triplex probes in which the 5′ MIR copy contains two mismatches. (Second) Duplex or triplex probes in which the 3′ or middle MIR copy, respectively, contains two mismatches. (Both) Duplex or triplex probes in which both the 5′ and the 3′ or middle MIR copies, respectively, contain two mismatches. For each probe set, the signal intensities for the three types of probes were calculated as a ratio of the signal intensity of the “None probe”. Data are presented as mean signal ratio ± SEM for 23 sets of probes, except for the triplex “First” set in C, for which two probes were eliminated from the calculation due to sequence matches to adaptors.
Figure 3.
Figure 3.
Effect of mismatches within MIRs on microarray signal intensity. Signals from hybridization of 60-mer probes with HeLa lcRNA at either 50 or 60°C were compared. Probes containing MIR at the 5′ end followed by nonhuman genome sequences were examined. Each probe containing a wild-type MIR (PM) was compared with a set of probes with the following mismatches: (TM) A probe with a wild-type MIR and six mismatches in the adjacent nonhuman genome sequence region; (1M) a probe with one mismatch at position 10 of the MIR sequence; (2M) a probe with two mismatches at positions 8 and 17 of the MIR; (3M) a probe with three mismatches at positions 6, 12, and 18 of the MIR. (5′M) a probe with a block of four mismatches at the 5′ end of the MIR; (3′M) a probe with a block of six mismatches at the 3′ end of the MIR. Data are presented as mean signal ratio ± SEM for 42 sets of probes.
Figure 4.
Figure 4.
The mirMASA technology. A specific 10–12 nucleotide-long capture oligonucleotide (oligo) and a specific 8–10 nucleotide-long detection oligo are synthesized for each MIR. The capture oligo is covalently linked to color-coded microspheres (beads), whereas the detection oligo is labeled with biotin. For each MIR, the capture oligo is linked to a unique color-coded bead. Both capture and detection oligos are spiked with Locked Nucleic Acid (LNA) nucleotides to increase specificity and sensitivity. The biotin is used for detection following addition of streptavidin-phycoerythrin and reading the fluorescence associated with each color-coded bead.
Figure 5.
Figure 5.
Clustering analysis of the expression of 150 human MIRs in placenta, testes, thymus, liver, brain, and HeLa cells. The clustering procedure used to derive the dendrogram is described in the Methods section. The level of expression of each MIR in each of the samples is indicated by the color shown next to the MIR name. The relationship between the color and the expression levels is defined by the color key on the right side of the figure. The color code numbers on the color key are log2 of the signal intensity. The original figure was cut in half due to space considerations. HSA-LET-7c, found in the bottom, right, and at top, left, links the two halves.
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Web site references

    1. http://www.sanger.ac.uk/Software/Rfam/mirna/index.shtml;
    1. http://gene.genaco.com/miRNA.htm; description of the mirMASA technology.
    1. http://www.luminexcorp.com/01_xMAPTechnology/index.html; description of the xMAP technology.

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