Expression profile analysis of microRNA (miRNA) in mouse central nervous system using a new miRNA detection system that examines hybridization signals at every step of washing
Introduction
MicroRNAs (miRNAs) are small noncoding RNAs, with a typical length of 19 to 23 nt, which are processed from longer transcripts (primary miRNAs), forming stem-loop structures by digestion with a microprocessor complex containing Drosha and Pasha in the nucleus and Dicer in the cytoplasm (Lee et al., 2003, Bartel, 2004, Denli et al., 2004). After Dicer processing, the resultant miRNA duplexes undergo strand selection, and the single-stranded mature miRNA elements are incorporated into the RNA-induced silencing complex (RISC) and function as mediators (Hutvagner and Zamore, 2002). It is thought that miRNAs play an important role in the regulation of gene expression, by inhibiting translation of messenger RNAs (mRNAs), which are partially complementary to the miRNAs, during development, differentiation and proliferation (Doench et al., 2003, Krichevsky et al., 2003, Zeng et al., 2003, Liu et al., 2004, Cheng et al., 2005). In addition, recent studies have further suggested significant association of miRNA with various cancers (Calin et al., 2002, Eis et al., 2005, He et al., 2005, Johnson et al., 2005).
Hundreds of miRNA genes have been found in plants and animals (Lagos-Quintana et al., 2002, Krichevsky et al., 2003, Bartel, 2004). They appear to be expressed by RNA polymerase II (Lee et al., 2004), and tissue-specific expression of miRNA has also been detected (Lagos-Quintana et al., 2002, Babak et al., 2004, Liu et al., 2004). Comprehensive analysis of miRNA expression is necessary to understand the complex regulation of gene expression involving miRNAs and is also helpful in the characterization of miRNAs. However, although expression profile analyses of miRNAs using conventional DNA arrays have been performed (Krichevsky et al., 2003, Babak et al., 2004, Liu et al., 2004, Miska et al., 2004), the shortness of miRNA, at ∼ 22 nt, appears to make such analyses difficult. To address this problem, we used a new type of DNA chip to make a microarray specific to miRNAs and to establish a detection system for the expression profiles of miRNAs. The system allows real-time detection of hybridization signals at every step of washing and results in highly reproducible miRNA expression profiles.
Using this system for detection of miRNAs, we investigated miRNA expression profiles in the mouse central nervous system (CNS), which is composed of seven subregions specialized for different functions. The results suggested differences in expression of miRNAs among the CNS subregions.
Section snippets
DNA chip
Synthetic DNA oligonucleotides were installed as probes onto Genopal® (Mitsubishi Rayon), which is composed of plastic hollow fibers: oligonucleotide DNA probes are attached to a gel within the three-dimensional space of each hollow fiber. MiRNAs targeted for detection in this study are shown in Supplementary Table S1.
Preparation of small-sized RNAs and fluorescent labeling
Total RNAs extracted from BALB/c mice frontal cortex, cerebellum, hippocampus, thalamus, hypothalamus, brainstem, pons, and spinal cord were purchased from Clontech. In addition,
Detection of miRNAs with DNA chips
We examined 182 mouse miRNAs (see supplementary Table S1) in this study. Small-sized RNAs containing miRNAs were prepared, directly labeled with Cy3 or Cy5 fluorescent substrate, and used in hybridization. Note that no ligation of the prepared RNAs with oligonucleotide anchors and no reverse transcription followed by PCR was performed during fluorescent labeling of the isolated RNAs; thus, there was no biased nucleotide labeling resulting from ligation and amplification efficiencies in this
Acknowledgments
We would like to thank Y. Okazaki for her helpful cooperation. This work was supported in part by research grants from the Ministry of Health, Labor and Welfare in Japan.
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