A novel microarray approach reveals new tissue-specific signatures of known and predicted mammalian microRNAs

Abstract

Microarrays to examine the global expression levels of microRNAs (miRNAs) in a systematic in-parallel manner have become important tools to help unravel the functions of miRNAs and to understand their roles in RNA-based regulation and their implications in human diseases. We have established a novel miRNA-specific microarray platform that enables the simultaneous expression analysis of both known and predicted miRNAs obtained from human or mouse origin. Chemically modified 2'-O-(2-methoxyethyl)-(MOE) oligoribonucleotide probes were arrayed onto Evanescent Resonance (ER) microchips by robotic spotting. Supplementing the complementary probes against miRNAs with carefully designed mismatch controls allowed for accurate sequence-specific determination of miRNA expression profiles obtained from a panel of mouse tissues. This revealed new expression signatures of known miRNAs as well as of novel miRNAs previously predicted using bioinformatic methods. Systematic confirmation of the array data with northern blotting and, in particular, real-time PCR suggests that the described microarray platform is a powerful tool to analyze miRNA expression patterns with rapid throughput and high fidelity.

Figure 1.

Structure of chemically modified 2′-O-MOE-RNA compared with natural RNA.

Figure 2.

Sensitivity and specificity of MOE-ER-based miRNA arrays. A dose response and mismatch (MM) discrimination of fluorescence signals was obtained for mir-17-5p, mir-17-3p,mir-26a, mir-24, mir-16 and mir-21 which represent miRNAs known to be expressed in HeLa cells. MOE-ER arrays were probed with the indicated amounts of the size-fractionated (<200 nt) Cy5-labeled HeLa cell RNA. Expression values for all individual miRNA are shown in Supplementary Table S1.

Figure 3.

The MOE-ER array profiling of miRNAs in eight different mouse tissues reveals novel miRNA expression signatures. A selection of miRNAs displaying tissue-enriched expression patterns is shown. Colors depict relative intensities of array hybridization signals: High (red), medium (black), low (gray). Quantification of expression of all individual miRNA is presented in Supplementary Table S2. He: HeLa; B: brain; H: heart; I: small intestine; K: kidney; Li: liver; Lu: lung; M: skeletal muscle; S: spleen. Data was clustered to represent tissue-enriched distribution of expression.

Figure 4.

Comparison of the relative abundance of miRNAs determined using MOE-ER array, Northern blot and stem-loop RT-PCR analyses of human HeLa cell and mouse tissue RNAs. He: HeLa; B: brain; H: heart; I: small intestine; K: kidney; Li: liver; Lu: lung; M: skeletal muscle; S: spleen.

Figure 5.

Verification of computational-predicted miRNA pir-5 by MOE-ER array and northern blot analyses. Colours depict relative intensities of array hybridization signals: High (red), medium (black), low (grey). Loading control was performed with U6 snRNA. He: HeLa; B: brain; H: heart; I: small intestine; K: kidney; Li: liver; Lu: lung; M: skeletal muscle; S: spleen.