MOTIVATION: Small non-coding RNAs (ncRNAs), especially microRNAs (miRNAs) and piwi-interacting RNAs (piRNAs), play key roles in many biological processes. However, only a few tools can be used to develop the optimal primer or probe design for the expression profile of small ncRNAs. Here, we developed sRNAPrimerDB, the first automated primer designing and query web service for small ncRNAs. RESULTS: The primer online designing module of sRNAPrimerDB is composed of primer design algorithms and quality evaluation of the polymerase chain reaction (PCR) primer. Five types of primers, namely, generic or specific reverse transcription primers, specific PCR primers pairs, TaqMan probe, double-hairpin probe and hybridization probe for different small ncRNA detection methods, can be designed and searched using this service. The quality of PCR primers is further evaluated using melting temperature, primer dimer, hairpin structure and specificity. Moreover, the sequence and size of each amplicon are also provided for the subsequent experiment verification. At present, 531 306 and 2 941 669 primer pairs exist across 223 species for miRNAs and piRNAs, respectively, according to sRNAPrimerDB. Several primers designed by sRNAPrimerDB are further successfully validated by subsequent experiments. AVAILABILITY AND IMPLEMENTATION: sRNAPrimerDB is a valuable platform that can be used to detect small ncRNAs. This module can be publicly accessible at http://www.srnaprimerdb.com or http://123.57.239.141. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

MOTIVATION: MicroRNAs have recently emerged as a major class of regulatory molecules involved in a broad range of biological processes and complex diseases. Construction of miRNA-target regulatory networks can provide useful information for the study and diagnosis of complex diseases. Many sequence-based and evolutionary information-based methods have been developed to identify miRNA-mRNA targeting relationships. However, as the amount of available miRNA and gene expression data grows, a more statistical and systematic method combining sequence-based binding predictions and expression-based correlation data becomes necessary for the accurate identification of miRNA-mRNA pairs. RESULTS: We propose a Lasso regression model for the identification of miRNA-mRNA targeting relationships that combines sequence-based prediction information, miRNA co-regulation, RISC availability and miRNA/mRNA abundance data. By comparing this modelling approach with two other known methods applied to three different datasets, we found that the Lasso regression model has considerable advantages in both sensitivity and specificity. The regression coefficients in the model can be used to determine the true regulatory efficacies in tissues and was demonstrated using the miRNA target site type data. Finally, by constructing the miRNA regulatory networks in two stages of prostate cancer (PCa), we found the several significant miRNA-hubbed network modules associated with PCa metastasis. In conclusion, the Lasso regression model is a robust and informative tool for constructing the miRNA regulatory networks for diagnosis and treatment of complex diseases. AVAILABILITY: The R program for predicting miRNA-mRNA targeting relationships using the Lasso regression model is freely available, along with the described datasets and resulting regulatory network, at http://biocompute.bmi.ac.cn/CZlab/alarmnet/. The source code is open for modification and application to other miRNA/mRNA expression datasets. CONTACT: [email protected] SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

SUMMARY: The visualization of gene structure plays an important role in polymerase chain reaction (PCR) primer design, especially for eukaryotic genes with a number of splice variants that users need to distinguish between via PCR. Here, we describe a visualized web server for primer design named VizPrimer. It utilizes the new information technology (IT) tools, HTML5 to display gene structure and JavaScript to interact with the users. In VizPrimer, the users can focus their attention on the gene structure and primer design strategy, without wasting time calculating the exon positions of splice variants or manually configuring complicated parameters. In addition, VizPrimer is also suitable for the design of PCR primers for amplifying open reading frames and detecting single nucleotide polymorphisms (SNPs). AVAILABILITY: VizPrimer is freely available at http://biocompute.bmi.ac.cn/CZlab/VizPrimer/. The web server supported browsers: Chrome (≥5.0), Firefox (≥3.0), Safari (≥4.0) and Opera (≥10.0). CONTACT: [email protected]; [email protected].

BACKGROUND: Multiplex PCR, defined as the simultaneous amplification of multiple regions of a DNA template or multiple DNA templates using more than one primer set (comprising a forward primer and a reverse primer) in one tube, has been widely used in diagnostic applications of clinical and environmental microbiology studies. However, primer design for multiplex PCR is still a challenging problem and several factors need to be considered. These problems include mis-priming due to nonspecific binding to non-target DNA templates, primer dimerization, and the inability to separate and purify DNA amplicons with similar electrophoretic mobility. RESULTS: A program named MPprimer was developed to help users for reliable multiplex PCR primer design. It employs the widely used primer design program Primer3 and the primer specificity evaluation program MFEprimer to design and evaluate the candidate primers based on genomic or transcript DNA database, followed by careful examination to avoid primer dimerization. The graph-expanding algorithm derived from the greedy algorithm was used to determine the optimal primer set combinations (PSCs) for multiplex PCR assay. In addition, MPprimer provides a virtual electrophotogram to help users choose the best PSC. The experimental validation from 2x to 5x plex PCR demonstrates the reliability of MPprimer. As another example, MPprimer is able to design the multiplex PCR primers for DMD (dystrophin gene which caused Duchenne Muscular Dystrophy), which has 79 exons, for 20x, 20x, 20x, 14x, and 5x plex PCR reactions in five tubes to detect underlying exon deletions. CONCLUSIONS: MPprimer is a valuable tool for designing specific, non-dimerizing primer set combinations with constrained amplicons size for multiplex PCR assays.

Abstract Summary: We developed a program named MFEprimer for evaluating the specificity of PCR primers based on multiple factors, including sequence similarity, stability at the 3 ′ -end of the primer, melting temperature, GC content and number of binding sites between the primer and DNA templates. MFEprimer can help the user to select more suitable primers before running either standard or multiplex PCR reactions. The cDNA and genomic DNA databases of 10 widely used species, as well as user custom databases, were used as DNA templates for analyzing primers specificity. Furthermore, we maintained a Primer3Plus server with a modified Primer3Manager for one-stop primer design and specificity checking. Availability: The web service of MFEprimer is freely available at http://biocompute.bmi.ac.cn/MFEprimer/. A stand-alone version of MFEprimer can be downloaded at http://biocompute.bmi.ac.cn/MFEprimer/download/. Primer3Plus on our server is located at http://biocompute.bmi.ac.cn/MFEprimer/primer3plus.cgi. All programs are licensed under the General Public License (Free Software Foundation, 1991). Contact: [email protected] Supplementary information: Supplementary data are available at Bioinformatics online.

BACKGROUND: Alternative splicing (AS) is an important regulatory mechanism for gene expression and protein diversity in eukaryotes. Previous studies have demonstrated that it can be causative for, or specific to splicing-related diseases. Understanding the regulation of AS will be helpful for diagnostic efforts and drug discoveries on those splicing-related diseases. As a novel exon-centric microarray platform, exon array enables a comprehensive analysis of AS by investigating the expression of known and predicted exons. Identifying of AS events from exon array has raised much attention, however, new and powerful algorithms for exon array data analysis are still absent till now. RESULTS: Here, we considered identifying of AS events in the framework of variable selection and developed a regression method for AS detection (REMAS). Firstly, features of alternatively spliced exons were scaled by reasonably defined variables. Secondly, we designed a hierarchical model which can represent gene structure and transcriptional influence to exons, and the lasso type penalties were introduced in calculation because of huge variable size. Thirdly, an iterative two-step algorithm was developed to select alternatively spliced genes and exons. To avoid negative effects introduced by small sample size, we ranked genes as parameters indicating their AS capabilities in an iterative manner. After that, both simulation and real data evaluation showed that REMAS could efficiently identify potential AS events, some of which had been validated by RT-PCR or supported by literature evidence. CONCLUSION: As a new lasso regression algorithm based on hierarchical model, REMAS has been demonstrated as a reliable and effective method to identify AS events from exon array data.