Detecting microRNA activity from gene expression data

Background  

MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression by binding to the messenger RNA (mRNA) of protein coding genes. They control gene expression by either inhibiting translation or inducing mRNA degradation. A number of computational techniques have been developed to identify the targets of miRNAs. In this study we used predicted miRNA-gene interactions to analyse mRNA gene expression microarray data to predict miRNAs associated with particular diseases or conditions.     

High-Sensitivity and High-Resolution In Situ Hybridization of Coding and Long Non-coding RNAs in Vertebrate Ovaries and Testes

Background

Subcellular localization of coding and non-coding RNAs has emerged as major regulatory mechanisms of gene expression in various cell types and many organisms. However, techniques that enable detection of the subcellular distribution of these RNAs with high sensitivity and high resolution remain limited, particularly in vertebrate adult tissues and organs. In this study, we examined the expression and localization of mRNAs encoding Pou5f1/Oct4, Mos, Cyclin B1 and Deleted in Azoospermia-like (Dazl) in zebrafish and mouse ovaries by combining tyramide signal amplification (TSA)-based in situ hybridization with paraffin sections which can preserve cell morphology of tissues and organs at subcellular levels. In addition, the distribution of a long non-coding RNA (lncRNA), lncRNA-HSVIII, in mouse testes was examined by the same method.

Results

The mRNAs encoding Mos, Cyclin B1 and Dazl were found to assemble into distinct granules that were distributed in different subcellular regions of zebrafish and mouse oocytes, suggesting conserved and specific regulations of these mRNAs. The lncRNA-HSVIII was first detected in the nucleus of spermatocytes at prophase I of the meiotic cell cycle and was then found in the cytoplasm of round spermatids, revealing expression patterns of lncRNA during germ cell development. Collectively, the in situ hybridization method demonstrated in this study achieved the detection and comparison of precise distribution patterns of coding and non-coding RNAs at subcellular levels in single cells of adult tissues and organs.

Conclusions

This high-sensitivity and high-resolution in situ hybridization is applicable to many vertebrate species and to various tissues and organs and will be useful for studies on the subcellular regulation of gene expression at the level of RNA localization.

     

miR-Q: a novel quantitative RT-PCR approach for the expression profiling of small RNA molecules such as miRNAs in a complex sample

Background  

MicroRNAs (miRNAs) are small endogenous non-coding interfering RNA molecules regarded as major regulators in eukaryotic gene expression. Different methods are employed for miRNA expression profiling. For a better understanding of their role in essential biological processes, convenient methods for differential miRNA expression analysis are required.     

Structure and restriction fragment length polymorphism of genes for human liver arylamine N-acetyltransferases.

Genomic DNA clones coding for polymorphic and monomorphic arylamine N-acetyltransferases (NAT) of human liver were isolated from a genomic DNA library, and their restriction maps and partial nucleotide sequences were determined. Messenger RNA for monomorphic NAT was coded in one exon, while mRNA for polymorphic NAT was coded in two exons; the 5'-noncoding region was located in one exon 8 kb upstream from another exon containing the coding and 3'-noncoding regions. Recently, we have shown that there are three types of polymorphic NAT gene; one of the genes corresponds to a high NAT activity, while the other two genes give rise to a low NAT activity. The restriction fragment length polymorphism (RFLP) was analyzed by Southern blot hybridization of genomic DNAs from homozygotes of the three polymorphic NAT genes using various fragments of the cloned NAT gene. RFLPs of polymorphic NAT gene were observed in coding and 3'-flanking region upon digestion with BamHI and KpnI.