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Next-generation sequencing platforms can also be used for transcriptome profiling and DNA-protein interaction studies using chromatin immunoprecipitation.
Cell-based, protein, tissue, microRNA, lipid, and carbohydrate microarrays are all variations on the DNA microarray format. These microarray-based platforms can be used in basic research laboratories to identify protein function, protein-protein interaction, and prognostic and predictive disease markers, among other applications.
Below are introductions to each technology, as well as links to download pdf files containing more detailed and experimental information. If you'd like to explore more, we have also put together a list of scientific references on various topics, located in our resources & links section.
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Next-generation sequencing (NGS), also called massively parallel and deep sequencing, has been commercially available since 2004. NGS increases sequencing throughput by laying millions of DNA fragments on a single chip and sequencing all fragments in parallel. DNA fragments are used to build fragment libraries that are subsequently arranged on a single chip and used as sequencing templates. Informatics allows each sequencing read to be mapped to a reference genome. Due to the vast amount of sequence data that is generated in one run, a sophisticated information technology infrastructure is required.Next-generation sequencing: Synergy with microarrays
Depending on the platform, NGS runs can take 10 hours to 5 days, generating read lengths ranging from 35 bp to 400 bp, and creating between 400 and 3000 Mb of data per run.
Three commercially available NGS platforms include FLX Genome Sequencer (Roche/454), Genome Analyzer II (Illumina), and SoLID (Sequencing by Oligo Ligation and Detection) Sequencer (Applied Biosystems). They can be used for transcriptome profiling (mRNA-Seq), DNA-protein interaction studies using chromatin immunoprecipitation (ChIP-Seq), miRNA profiling, and DNA methylation studies.
Single molecule sequencing, also called "third-generation" sequencing, does not involve amplification and offers longer reads and higher quality data. The HeliScope (Helicos) employs True Single Molecule Sequencing (tSMS) technology to produce over 10 Gb of sequence data in an eight day run. Pacific Biosciences has developed Single Molecule Real Time (SMRT) sequencing technology which involves proprietary surface and nucleotide chemistries.
The advantages of microarrays in comparison to NGS include: relatively inexpensive, easy to prepare samples, mature informatics and statistics. However, microarrays are not quantitative, have limited dynamic range, and relatively high background and low sensitivity compared to NGS.
The advantages of NGS include: quantitative measurements, larger dynamic range, and lower background and more sensitivity compared with microarrays. However, NGS is quite expensive, sample preparation is more complex, and a huge IT infrastructure is required to manage the amount of data generated.
Carbohydrate microarrays are emerging as a common technique used in glycomic research, as they can be used to characterise carbohydrate-cell interactions, determine the binding profile of carbohydrate-binding proteins, detect pathogens, and provide high-throughput screening of inhibitors of carbohydrate-protein interactions.
Cell-based microarrays are arrays spotted with nucleic acid (plasmid or RNA) in defined locations on a glass slide and used to transfect cells that are grown on the surface of the microarray, through the addition of a transfection reagent. As the cells grow, they are transfected with the plasmid and express the encoded protein.Cell arrays
Cell microarrays can be used as an alternative to protein microarrays for identification of small molecule targets and for discovery of gene products that alter cellular physiology. They can also be used for loss of function experiments (RNAi “knock-down”), gain of function (overexpression screens), protein interaction (ligands, antibodies, small molecules), drug screening, subcellular localization, morphological analysis, cell proliferation and protein phosphorylation.
The cell microarray technique bypasses the constraints of protein immobilisation and, because proteins are translated, folded, and interact within the cellular environment, the expressed proteins are functional and stable.
Fusion tags are peptides or proteins that are used in recombinant protein expression to aid in the purification, secretion and/or detection of the native protein of interest. Common fusion tags include glutathione-S-transferase (GST), green fluorescent protein (GFP), and histidine (His) tags.
By identifying lipid-associated molecules in a cell or biological fluid, lipid microarrays are a useful tool for increasing the knowledge base of the human lipidome. Lipid microarrays consist of various lipid sub-types spotted individually, in an array pattern, on a solid surface. The lipid array provides a protein-lipid interaction profile and can be used to identify proteins of potential therapeutic value.
The protein microarray platform can be divided into two general strategies, one that is used to predict protein abundance and the other to identify protein function.Protein array factsheet
Capture microarrays, such as antibody microarrays, are an example of the abundance-based strategy. Antibodies on the array bind specific proteins from complex mixtures. In terms of practical applications, these arrays can be used to monitor protein abundance in cancer cells following radiation treatment or used to identify potential biomarkers.
Function-based microarrays study biochemical properties of the proteins printed on the array, examine protein interactions and enzyme activity. These arrays can be used to screen a particular class of enzymes with a potential inhibitor to examine binding selectivity; a potential drug could be used to probe many enzymes to identify unintended binding targets that might indicate possible side-effects; interaction networks might identify biochemical pathways. The challenges for function-based microarrays include stability and integrity on slide surface, time and cost constraints to produce and purify proteins, and the methods used to attach the protein to the surface.
Tissue microarrays were developed in the Olli-P. Kallioniemi lab at NHGRI. These are a fast, cost-effective and tissue-saving method used for the high-throughput molecular profiling of tumor specimens. These arrays are comprised of hundreds of paraffin-embedded core tissue samples arranged on a slide. Tissue microarray sections can then be analysed using standard pathology methods such as hematoxylin/eosin staining, immunohistochemistry and in situ hybridisation. Sections of the microarray provide targets for the simultaneous in situ detection of DNA, RNA, and proteins.Tissue arrays