In vitro quantification of specific microRNA using molecular beacons

MicroRNAs (miRNAs), a class of non-coding RNAs, have become a major focus of molecular biology research because of their diverse genomic origin and ability to regulate an array of cellular processes. Although the biological functions of miRNA are yet to be fully understood, tissue levels of specific miRNAs have been shown to correlate with pathological development of disease. Here, we demonstrate that molecular beacons can readily distinguish mature- and pre-miRNAs, and reliably quantify miRNA expression. We found that molecular beacons with DNA, RNA and combined locked nucleic acid (LNA)–DNA backbones can all detect miRNAs of low (<1 nM) concentrations in vitro, with RNA beacons having the highest detection sensitivity. Furthermore, we found that molecular beacons have the potential to distinguish miRNAs that have slight variations in their nucleotide sequence. These results suggest that the molecular beacon-based approach to assess miRNA expression and distinguish mature and precursor miRNA species is quite robust, and has the promise for assessing miRNA levels in biological samples.     

Highly sensitive and specific microRNA expression profiling using BeadArray technology

We have developed a highly sensitive, specific and reproducible method for microRNA (miRNA) expression profiling, using the BeadArray™ technology. This method incorporates an enzyme-assisted specificity step, a solid-phase primer extension to distinguish between members of miRNA families. In addition, a universal PCR is used to amplify all targets prior to array hybridization. Currently, assay probes are designed to simultaneously analyse 735 well-annotated human miRNAs. Using this method, highly reproducible miRNA expression profiles were generated with 100–200 ng total RNA input. Furthermore, very similar expression profiles were obtained with total RNA and enriched small RNA species (R² ≥ 0.97). The method has a 3.5–4 log (10⁵–10⁹ molecules) dynamic range and is able to detect 1.2- to 1.3-fold-differences between samples. Expression profiles generated by this method are highly comparable to those obtained with RT–PCR (R² = 0.85–0.90) and direct sequencing (R = 0.87–0.89). This method, in conjunction with the 96-sample array matrix should prove useful for high-throughput expression profiling of miRNAs in large numbers of tissue samples.     

Characterization of rubber tree microRNA in phytohormone response using large genomic DNA libraries,promoter sequence and gene expression analysis

The para rubber tree is the most widely cultivated tree species for producing natural rubber (NR) latex. Unfortunately, rubber tree characteristics such as a long life cycle, heterozygous genetic backgrounds, and poorly understood genetic profiles are the obstacles to breeding new rubber tree varieties, such as those with improved NR yields. Recent evidence has revealed the potential importance of controlling microRNA (miRNA) decay in some aspects of NR regulation. To gain a better understanding of miRNAs and their relationship with rubber tree gene regulation networks, large genomic DNA insert-containing libraries were generated to complement the incomplete draft genome sequence and applied as a new powerful tool to predict a function of interested genes. Bacterial artificial chromosome and fosmid libraries, containing a total of 120,576 clones with an average insert size of 43.35 kb, provided approximately 2.42 haploid genome equivalents of coverage based on the estimated 2.15 gb rubber tree genome. Based on these library sequences, the precursors of 1 member of rubber tree-specific miRNAs and 12 members of conserved miRNAs were successfully identified. A panel of miRNAs was characterized for phytohormone response by precisely identifying phytohormone-responsive motifs in their promoter sequences. Furthermore, the quantitative real-time PCR on ethylene stimulation of rubber trees was performed to demonstrate that the miR2118, miR159, miR164 and miR166 are responsive to ethylene, thus confirmed the prediction by genomic DNA analysis. The cis-regulatory elements identified in the promoter regions of these miRNA genes help augment our understanding of miRNA gene regulation and provide a foundation for further investigation of the regulation of rubber tree miRNAs.     

Strain-based sequence variations and structure analysis of murine prostate specific spermine binding protein using mass spectrometry

Mouse spermine binding protein (SBP) has been characterized using mass spectrometry, including its localization within the prostate, sequence verification, and its posttranslational modifications. MALDI (matrix-assisted laser desorption/ionization) mass spectrometry was employed for localization of proteins expressed by different lobes of the mouse prostate obtained after tissue blotting on a polyethylene membrane. The mass spectra showed complex protein profiles that were different for each lobe of the prostate. The prostate-specific spermine binding protein (SBP), primarily identified by its in-source decay fragment ion signals, was found predominantly expressed by the ventral lobe of the prostate. The MALDI in-source decay measurements combined with nanoESI (nanoelectrospay ionization) MS/MS measurements obtained after specific proteolysis of SBP, allowed the exact positioning of a single N-linked carbohydrate group, and the identification of a pyroglutamate residue at the sequence N-terminus. The N-linked carbohydrate component was further investigated and the general pattern of the N-linked carbohydrate identified. The presence of a disulfide bridge between cysteine78 and cysteine124 was also established. The full sequence characterization of SBP showed several strain-based sequence differences when compared to the published gene sequence.     

Qualitative analysis of sequence specific binding of flavones to DNA using restriction endonuclease activity assays

Flavones, found in nature as secondary plant metabolites, have shown efficacy as anti‐cancer agents. We have examined the binding of two flavones, 5,7‐dihydroxy‐3,6,8‐trimethoxy‐2‐phenyl‐4H‐chromen‐4‐one (5,7‐dihydroxy‐3,6,8‐trimethoxy flavone; FlavA) and 3,5‐dihydroxy‐6,7,8‐trimethoxy‐2‐phenyl‐4H‐chromen‐4‐one (3,5‐dihydroxy‐6,7,8‐trimethoxy flavone; FlavB), to phiX174 RF DNA using restriction enzyme activity assays employing the restriction enzymes Alw44, AvaII, BssHII, DraI, MluI, NarI, NciI, NruI, PstI, and XhoI. These enzymes possess differing target and flanking sequences allowing for observation of sequence specificity analysis. Using restriction enzymes that cleave once with a mixture of supercoiled and relaxed DNA substrates provides for observation of topological effects on binding. FlavA and FlavB show differing sequence specificities in their respective binding to phiX. For example, with relaxed DNA, FlavA shows inhibition of cleavage with DraI (reaction site ^5′TTTAAA) but not BssHII (^5′GCGCGC) while FlavB shows the opposite results. Evidence for tolological specificity is also observed, Molecular modeling and conformational analysis of the flavones suggests that the phenyl ring of FlavB is coplanar with the flavonoid ring while the phenyl ring of FlavA is at an angle relative to the flavonoid ring. This may account for aspects of the observed sequence and topological specificities in the effects on restriction enzyme activity. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 530–537, 2013.     

Efficient and specific inhibition of plant microRNA function by anti-microRNA oligonucleotides (AMOs) in vitro and in vivo

Key message

Anti-microRNA oligonucleotides (AMOs) are efficient and sequence-specific inhibitors of plant miRNA function both in vitro and in vivo.

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs that play critical roles in developmental and physiological processes in plants and animals. Although miRNA knockdown by chemically modified antisense oligonucleotides prevails in animal and therapeutic studies, no such application has ever been reported in plants. Here, we show that sucrose-mediated delivery of 2′-O-methyl (2′-O-Me) anti-miRNA oligonucleotides (AMOs) is an efficient and sequence-specific way of inhibiting plant miRNA activity both in vitro and in vivo. Administration of AMOs to rice protoplasts and intact leaves resulted in efficient inhibition of miRNAs with concurrent de-repression of their target genes. AMOs caused simultaneous inhibition of miRNAs from the same family but exerted negligible effects on miRNAs from different families. In rice seedlings, a single-dose AMO treatment conferred long-lasting miRNA inhibition for at least 7 days. Although simultaneous dysregulation of multiple miRNAs by an AMO-and-miRNA-mimic mixture resulted in severe root defects, the phenotypic effects of individual AMOs and miRNA mimics were negligible, suggesting that those miRNAs function together in regulatory networks to ensure homeostasis. Our results validate the utility of AMOs as an efficient tool for plant miRNA loss-of-function studies in vivo, and this approach may prove to be a highly promising general method for unraveling miRNA-mediated gene-regulatory networks.

     

Efficient pairwise RNA structure prediction and alignment using sequence alignment constraints

Background  

We are interested in the problem of predicting secondary structure for small sets of homologous RNAs, by incorporating limited comparative sequence information into an RNA folding model. The Sankoff algorithm for simultaneous RNA folding and alignment is a basis for approaches to this problem. There are two open problems in applying a Sankoff algorithm: development of a good unified scoring system for alignment and folding and development of practical heuristics for dealing with the computational complexity of the algorithm.     

An optimized isolation and labeling platform for accurate microRNA expression profiling

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression in both plants and animals. miRNA genes have been implicated in a variety of important biological processes, including development, differentiation, apoptosis, fat metabolism, viral infection, and cancer. Similar to protein-coding messenger RNAs, miRNA expression varies between tissues and developmental states. To acquire a better understanding of global miRNA expression in tissues and cells, we have developed isolation, labeling, and array procedures to measure the relative abundance of all of the known human mature miRNAs. The method relies on rapid isolation of RNA species smaller than ~40 nucleotides (nt), direct and homogenous enzymatic labeling of the mature miRNAs with amine modified ribonucleotides, and hybridization to antisense DNA oligonucleotide probes. A thorough performance study showed that this miRNA microarray system can detect subfemtomole amounts of individual miRNAs from <1 mug of total RNA, with 98% correlation between independent replicates. The system has been applied to compare the global miRNA expression profiles in 26 different normal human tissues. This comprehensive analysis identified miRNAs that are preferentially expressed in one or a few related tissues and revealed that human adult tissues have unique miRNA profiles. This implicates miRNAs as important components of tissue development and differentiation. Taken together, these results emphasize the immense potential of microarrays for sensitive and high-throughput analysis of miRNA expression in normal and disease states.     

Efficient use of accessibility in microRNA target prediction

Considering accessibility of the 3′UTR is believed to increase the precision of microRNA target predictions. We show that, contrary to common belief, ranking by the hybridization energy or by the sum of the opening and hybridization energies, used in currently available algorithms, is not an efficient way to rank predictions. Instead, we describe an algorithm which also considers only the accessible binding sites but which ranks predictions according to over-representation. When compared with experimentally validated and refuted targets in the fruit fly and human, our algorithm shows a remarkable improvement in precision while significantly reducing the computational cost in comparison with other free energy based methods. In the human genome, our algorithm has at least twice higher precision than other methods with their default parameters. In the fruit fly, we find five times more validated targets among the top 500 predictions than other methods with their default parameters. Furthermore, using a common statistical framework we demonstrate explicitly the advantages of using the canonical ensemble instead of using the minimum free energy structure alone. We also find that ‘naïve’ global folding sometimes outperforms the local folding approach.     

Exploration of phylogenetic data using a global sequence analysis method

Background  

Molecular phylogenetic methods are based on alignments of nucleic or peptidic sequences. The tremendous increase in molecular data permits phylogenetic analyses of very long sequences and of many species, but also requires methods to help manage large datasets.     

Rapid and specific detection of RNA base sequence using fluorescence polarization.

Rapid and specific determination of the RNA gene of hepatitis C virus (HCV), which had been multiplied by NASBA, was performed using a fluorescence polarization assay. The polarization of the probe DNA in the presence of HCV positive sample, amplified by NASBA, was obviously different from those in the presence of negative control samples. The total time for the gene amplification and detection was about 90 min, while the polarization detection was completed within 10 min. The slight increase of polarization was also confirmed with the hybridization between probe oligo-DNA 25-mers and the synthesized complementary oligo-RNA 25-mers. The polarization of positive and negative samples showed excellent agreement with the results obtained from electrophoresis and dot-blot hybridization.     

Efficient and specific analysis of red blood cell glycerophospholipid fatty acid composition

Background

Red blood cell (RBC) n-3 fatty acid status is related to various health outcomes. Accepted biological markers for the fatty acid status determination are RBC phospholipids, phosphatidylcholine, and phosphatidyletholamine. The analysis of these lipid fractions is demanding and time consuming and total phospholipid n-3 fatty acid levels might be affected by changes of sphingomyelin contents in the RBC membrane during n-3 supplementation.

Aim

We developed a method for the specific analysis of RBC glycerophospholipids. The application of the new method in a DHA supplementation trial and the comparison to established markers will determine the relevance of RBC GPL as a valid fatty acid status marker in humans.

Methods

Methyl esters of glycerophospholipid fatty acids are selectively generated by a two step procedure involving methanolic protein precipitation and base-catalysed methyl ester synthesis. RBC GPL solubilisation is facilitated by ultrasound treatment. Fatty acid status in RBC glycerophospholipids and other established markers were evaluated in thirteen subjects participating in a 30 days supplementation trial (510 mg DHA/d).

Outcome

The intra-assay CV for GPL fatty acids ranged from 1.0 to 10.5% and the inter-assay CV from 1.3 to 10.9%. Docosahexaenoic acid supplementation significantly increased the docosahexaenoic acid contents in all analysed lipid fractions. High correlations were observed for most of the mono- and polyunsaturated fatty acids, and for the omega-3 index (r = 0.924) between RBC phospholipids and glycerophospholipids. The analysis of RBC glycerophospholipid fatty acids yields faster, easier and less costly results equivalent to the conventional analysis of RBC total phospholipids.