Small RNA class transition from siRNA/piRNA to miRNA during pre-implantation mouse development

Recent studies showed that small interfering RNAs (siRNAs) and Piwi-interacting RNA (piRNA) in mammalian germ cells play important roles in retrotransposon silencing and gametogenesis. However, subsequent contribution of those small RNAs to early mammalian development remains poorly understood. We investigated the expression profiles of small RNAs in mouse metaphase II oocytes, 8–16-cell stage embryos, blastocysts and the pluripotent inner cell mass (ICM) using high-throughput pyrosequencing. Here, we show that during pre-implantation development a major small RNA class changes from retrotransposon-derived small RNAs containing siRNAs and piRNAs to zygotically synthesized microRNAs (miRNAs). Some siRNAs and piRNAs are transiently upregulated and directed against specific retrotransposon classes. We also identified miRNAs expression profiles characteristic of the ICM and trophectoderm (TE) cells. Taken together, our current study reveals a major reprogramming of functional small RNAs during early mouse development from oocyte to blastocyst.     

Improved northern blot method for enhanced detection of small RNA

This protocol describes an improved northern blot method that enhances detection of small RNA molecules (<40 nt) including regulatory species such as microRNA (miRNA), short-interfering RNA (siRNA) and Piwi-interacting RNA. Northern blot analysis involves the separation of RNA molecules by denaturing gel electrophoresis followed by transfer and cross-linking of the separated molecules to nylon membrane. RNA of interest is then detected by hybridization with labeled complementary nucleic acid probes. We have replaced conventional UV-cross-linking of RNA to nylon membranes with a novel, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated, chemical cross-linking step that enhances detection of small RNA by up to 50-fold. This requires no specialized equipment, is relatively inexpensive and is technically straightforward. Northern blotting can be done in 2 d, but detection of a specific RNA can vary from minutes to days. Although chemical cross-linking takes longer (15 min to 2 h) than UV cross-linking, improved sensitivity means shorter periods of exposure are required to detect signal after hybridization.     

Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes

We describe here a new method for highly efficient detection of microRNAs by northern blot analysis using LNA (locked nucleic acid)-modified oligonucleotides. In order to exploit the improved hybridization properties of LNA with their target RNA molecules, we designed several LNA-modified oligonucleotide probes for detection of different microRNAs in animals and plants. By modifying DNA oligonucleotides with LNAs using a design, in which every third nucleotide position was substituted by LNA, we could use the probes in northern blot analysis employing standard end-labelling techniques and hybridization conditions. The sensitivity in detecting mature microRNAs by northern blots was increased by at least 10-fold compared to DNA probes, while simultaneously being highly specific, as demonstrated by the use of different single and double mismatched LNA probes. Besides being highly efficient as northern probes, the same LNA-modified oligonucleotide probes would also be useful for miRNA in situ hybridization and miRNA expression profiling by LNA oligonucleotide microarrays.     

World of small RNAs: from ribozymes to siRNA and miRNA

RNAs, besides bridging genetic information to proteins, the major determinants of bio-structures and functions, serve as active regulators of gene expression. Initiated nearly 20 years ago with ribozymes (the small RNAs with catalytic activity providing fine tuning of gene expression and function, used as molecular scissors and tools for gene discovery), an era of more complex and coordinated gene regulation by small RNAs, siRNA, and miRNA has recently started. Simple nucleotide complementarity results in highly ordered and regulated events, such as assembly of RNA and proteins, resulting in gene silencing either by mRNA degradation or suppression of translation. This article reviews our contributions to the understanding of structure, the function of small RNAs, their use in biotechnology, and the understanding of phenotypes such as apoptosis, metastasis, and differentiation.     

Glutaraldehyde fixation increases retention of low molecular weight proteins (growth factors) transferred to nylon membranes for western blot analysis

Western blotting of low molecular weight (Mr) acidic and basic isoelectric point (pI) proteins was studied to optimize detection sensitivities. Radioiodinated epidermal growth factor (EGF, Mr 6045, pI 4.4), transforming growth factor type alpha (TGF alpha, Mr 5623, pI 6.8), insulin-like growth factor (IGF-I, Mr 7649, pI 8.3), and basic fibroblast growth factor (bFGF, Mr 15,000-17,000, pI 9.6) all transferred with high efficiency (74.1 +/- 12.6%) to a positively charged nylon membrane. Sequential application of standard unoccupied site blocking, antibody incubation, and washing steps resulted in significant losses of all growth factors (46-98%). Basic FGF was retained best. Treatment of transfer membranes with 0.5% (v/v) glutaraldehyde prior to blocking and immunodetection increased the retention of the growth factors 1.5- to 12-fold over untreated controls. Without fixation, 100 ng of EGF, TGF alpha, and IGF-I were not detectable while 6.25-100 ng was identified on fixed membranes. The methods described were equally sensitive for detecting both acidic and basic pI proteins.     

Protein binding to nitrocellulose, nylon and PVDF membranes in immunoassays and electroblotting

A selection of different membranes commonly used to bind proteins in blotting and dot binding assays were investigated for a range of properties which would influence their performance. Large differences were observed in the membranes' ability to bind increasing amounts of protein, the effect of incubation times on the quantity of protein bound and the loss of proteins from the membranes following their incubation with different detergents or protein blocking agents. These differences could only partially explain the observed performance of the membranes when used as protein adsorbants in immunoassays and when different buffers were used for the electro-transfer of several different proteins to a range of membranes.     

Meta analysis of bioactive compounds,miRNA, siRNA and cell death regulators as sensitizers to doxorubicin induced chemoresistance

Cancer has presented to be the most challenging disease, contributing to one in six mortalities worldwide. The current treatment regimen involves multiple rounds of chemotherapy administration, alone or in combination. The treatment has adverse effects including cardiomyopathy, hepatotoxicity, and nephrotoxicity. In addition, the development of resistance to chemo has been attributed to cancer relapse and low patient overall survivability. Multiple drug resistance development may be through numerous factors such as up-regulation of drug transporters, drug inactivation, alteration of drug targets and drug degradation. Doxorubicin is a widely used first line chemotherapeutic drug for a myriad of cancers. It has multiple intracellular targets, DNA intercalation, adduct formation, topoisomerase inhibition, iron chelation, reactive oxygen species generation and promotes immune mediated clearance of the tumor. Agents that can sensitize the resistant cancer cells to the chemotherapeutic drug are currently the focus to improve the clinical efficiency of cancer therapy. This review summarizes the recent 10-year research on the use of natural phytochemicals, inhibitors of apoptosis and autophagy, miRNAs, siRNAs and nanoformulations being investigated for doxorubicin chemosensitization.

     

Structural features of microRNA (miRNA) precursors and their relevance to miRNA biogenesis and small interfering RNA/short hairpin RNA design

We have established the structures of 10 human microRNA (miRNA) precursors using biochemical methods. Eight of these structures turned out to be different from those that were computer-predicted. The differences localized in the terminal loop region and at the opposite side of the precursor hairpin stem. We have analyzed the features of these structures from the perspectives of miRNA biogenesis and active strand selection. We demonstrated the different thermodynamic stability profiles for pre-miRNA hairpins harboring miRNAs at their 5'- and 3'-sides and discussed their functional implications. Our results showed that miRNA prediction based on predicted precursor structures may give ambiguous results, and the success rate is significantly higher for the experimentally determined structures. On the other hand, the differences between the predicted and experimentally determined structures did not affect the stability of termini produced through "conceptual dicing." This result confirms the value of thermodynamic analysis based on mfold as a predictor of strand section by RNAi-induced silencing complex (RISC).     

Quantitative DNA slot blot analysis: inhibition of DNA binding to membranes by magnesium ions.

Titers of wild-type and recombinant adeno-associated viruses are routinely determined by DNA slot blot analysis. The binding of viral DNA to nylon membranes was found to be inhibited by magnesium ions, which are critical components of the DNase I digestion carried out prior to slot blot analysis. Mg2+ions also interfered with the adsorption of plasmid DNA to nylon and nitrocellulose membranes. These observations yield practical insights into the poorly understood mechanisms by which DNA molecules are retained on solid supports.     

Conserved association of Argonaute 1 and 2 proteins with miRNA and siRNA pathways throughout insect evolution,from cockroaches to flies

Background

Argonaute proteins are key in RNA silencing. In Drosophila melanogaster, the five proteins of the Argonaute family participate in the pathways and mechanisms mediated by three types of small RNAs: piRNAs, miRNAs, and siRNAs. Two Argonaute proteins, Argonaute 1 (Ago1) and Argonaute 2 (Ago2), are associated with miRNA and siRNA mechanisms, which are the most thoroughly studied. The available data points to a sorting specialization of Ago1 for miRNAs and Ago2 for siRNAs. However, this has been demonstrated only in D. melanogaster, one of the most modified insects, which emerged some 100 million years ago. Thus, an important question is whether this association of Ago1 with miRNAs and Ago2 with siRNAs occurs generally in insects, or was a specific innovation in higher flies.