The "Clickable" Method for Oligonucleotide Immobilization Onto Azide-Functionalized Microarrays.

The DNA microarray technique was supposed to help identifying and analyzing the expression level of tens of thousands of genes in the whole genome. But there is a serious problem concerning fabrication of the microarrays by chemical synthesis, such as specific and efficient linking of probes to a solid support. Therefore, we reckon that applying "click" chemistry to covalently anchor oligonucleotides on chemically modified supports may help construct microarrays in applications such as gene identification. Silanization of the glass support with organofunctional silane makes it possible to link azide groups on glass surface and the nucleic acid probe that is equipped with a pentynyl group. This is followed by direct spotting of the nucleic acid on the azide-modified glass support in the presence of copper ions, and this is a frequently applied method of "click" chemistry.

Comparison of GENCODE and RefSeq gene annotation and the impact of reference geneset on variant effect prediction.

BACKGROUND: A vast amount of DNA variation is being identified by increasingly large-scale exome and genome sequencing projects. To be useful, variants require accurate functional annotation and a wide range of tools are available to this end. McCarthy et al recently demonstrated the large differences in prediction of loss-of-function (LoF) variation when RefSeq and Ensembl transcripts are used for annotation, highlighting the importance of the reference transcripts on which variant functional annotation is based. RESULTS: We describe a detailed analysis of the similarities and differences between the gene and transcript annotation in the GENCODE and RefSeq genesets. We demonstrate that the GENCODE Comprehensive set is richer in alternative splicing, novel CDSs, novel exons and has higher genomic coverage than RefSeq, while the GENCODE Basic set is very similar to RefSeq. Using RNAseq data we show that exons and introns unique to one geneset are expressed at a similar level to those common to both. We present evidence that the differences in gene annotation lead to large differences in variant annotation where GENCODE and RefSeq are used as reference transcripts, although this is predominantly confined to non-coding transcripts and UTR sequence, with at most ~30% of LoF variants annotated discordantly. We also describe an investigation of dominant transcript expression, showing that it both supports the utility of the GENCODE Basic set in providing a smaller set of more highly expressed transcripts and provides a useful, biologically-relevant filter for further reducing the complexity of the transcriptome. CONCLUSIONS: The reference transcripts selected for variant functional annotation do have a large effect on the outcome. The GENCODE Comprehensive transcripts contain more exons, have greater genomic coverage and capture many more variants than RefSeq in both genome and exome datasets, while the GENCODE Basic set shows a higher degree of concordance with RefSeq and has fewer unique features. We propose that the GENCODE Comprehensive set has great utility for the discovery of new variants with functional potential, while the GENCODE Basic set is more suitable for applications demanding less complex interpretation of functional variants.

Analysis of boutique arrays: a universal method for the selection of the optimal data normalization procedure.

DNA microarrays, which are among the most popular genomic tools, are widely applied in biology and medicine. Boutique arrays, which are small, spotted, dedicated microarrays, constitute an inexpensive alternative to whole-genome screening methods. The data extracted from each microarray-based experiment must be transformed and processed prior to further analysis to eliminate any technical bias. The normalization of the data is the most crucial step of microarray data pre-processing and this process must be carefully considered as it has a profound effect on the results of the analysis. Several normalization algorithms have been developed and implemented in data analysis software packages. However, most of these methods were designed for whole-genome analysis. In this study, we tested 13 normalization strategies (ten for double-channel data and three for single-channel data) available on R Bioconductor and compared their effectiveness in the normalization of four boutique array datasets. The results revealed that boutique arrays can be successfully normalized using standard methods, but not every method is suitable for each dataset. We also suggest a universal seven-step workflow that can be applied for the selection of the optimal normalization procedure for any boutique array dataset. The described workflow enables the evaluation of the investigated normalization methods based on the bias and variance values for the control probes, a differential expression analysis and a receiver operating characteristic curve analysis. The analysis of each component results in a separate ranking of the normalization methods. A combination of the ranks obtained from all the normalization procedures facilitates the selection of the most appropriate normalization method for the studied dataset and determines which methods can be used interchangeably.

An MLPA-based strategy for discrete CNV genotyping: CNV-miRNAs as an example.

Copy number variation (CNV) has become well recognized in recent years. It has been estimated that common CNVs account for approximately 10% of the human genome and that they overlap hundreds of genes and other functional genetic elements. Although substantial progress in genome-wide CNV analysis has been made recently, there is still a need for a method that allows precise genotyping of selected CNVs. Here, we describe a novel strategy for CNV genotyping, taking advantage of the general principles of the multiplex ligation-dependent probe amplification (MLPA) method and short oligonucleotide probes, allowing easy custom design and generation of assays for almost any genomic region of interest. As a proof-of-concept, we developed two assays covering 17 candidate CNV regions that overlap human miRNA genes. Extensive quality control analysis demonstrated high reproducibility and reliability of the genotypes determined using our method. Detailed analysis of identified CNVs revealed that they are highly differentiated among the HapMap populations. The main advantages of the developed strategy include the simplicity of the assay design, its flexibility in terms of the selection of genomic regions, and its low cost (<$1-$10/genotype, depending on scale of experiment). These advantages make the presented strategy attractive for large-scale genetic analyses.

siRNA-mediated silencing of integrin ß3 expression inhibits the metastatic potential of B16 melanoma cells.

Integrins comprise a large family of αß heterodimeric cell-surface receptors that mediate diverse processes involved in cell-cell and cell-matrix interactions such as cellular adhesion and migration, cell survival and differentiation. It is now well documented that integrins play a crucial role in cancer metastasis and angiogenesis. The ß3 integrins appear to have an important stimulatory role in tumour progression and metastasis and, thus, have been often proposed as potential targets for cancer diagnosis and therapy. In this study, we evaluated the in vitro and in vivo properties of B16 mouse melanoma cells with low expression of integrin ß3. Proliferation rate, adhesive properties and the ability to migrate and metastasize were studied. Over 90% inhibition of integrin ß3 expression was achieved as a result of the transfection with siRNA. No changes in the proliferation rate were observed in siRNA-transfected B16 cells; however, they showed impaired ability to bind to fibronectin. Moreover, inhibition of integrin ß3 expression caused almost complete impairment of the ability of B16 cells to migrate through matrigel and metastasize. The mean number of lung metastatic colonies in mice inoculated intravenously with B16 expressing low levels of integrin ß3 was decreased to 14 colonies compared to 101 in the control group. These results provide evidence for a direct role of integrin ß3 in the adhesion, migration and metastasis processes of mouse melanoma cells and point to the potential therapeutic advantages of siRNAs.

Application of click chemistry to the production of DNA microarrays.

The copper-catalyzed alkyne-azide cycloaddition (CuAAC) reaction was applied as the novel method of DNA immobilization on a modified solid support. The CuAAC click reaction enables the covalent binding of DNA modified with pentynyl groups at its 5'-end to azide-loaded slides. Click microarrays were produced using this approach and successfully employed in biological/model experiments.