By Peer Staehler | Figure 1. a,b) Scatter plots showing technical replicates between mouse brain (a) or mouse liver (b) RNA. (c) Scatter plot illustrating differences between mouse brain and liver samples. (d) Consistency between data obtained using a preprinted array and that obtained using a febit microarray. Intensities shown are relative to the highest value for each miRNA, which was set to 1. Click to enlarge. |
Genomics and functional genomics have become an important field in biomedical research to understand cellular mechanism in healthy and disease states. The completion of the Human Genome Project in 2003 produced an enormous flood of data. The sequencing of other model organism such as the mouse, fruit fly and many others followed, and the data analysis is still ongoing. Discoveries such as RNA interference (RNAi) have made functional genomics more complex.
Basic research as well as translational research leading ultimately to drug discovery demand less time consuming, automated methods to elucidate these complex mechanisms. After reading the code of life, research focuses now on understanding and, for that, writing the code of life.
The use and advancements of microarrays as a methodical approach has significantly influenced molecular biology in the past few years. Originally a tool for DNA mapping and sequencing, the microarray technique has advanced and spread into areas such as microRNA (miRNA) profiling, genotyping and even sequencing. Advances in microarray technology include a growing number and more accurate probes on an array as well as better data analysis.
Geneticists often elucidate one distinct pathway or a gene family. Almost daily relevant genomic data are published or discovered in the privacy of the laboratory that researchers might want to include in their next experiment. Therefore, in addition to the need for large-scale arrays, there is the need for flexible array systems which cover the latest data. The febit technology is such an innovation for the benchtop, focused on automated and fast customizable microarrays. The Geniom Microarray Facility, an advanced microarray-based DNA analysis and synthesis system, integrates microarray production and application with the possibility of designing microarray-based experiments using published and unpublished sequences. As part of the facility, the Geniom RT Analyzer provides highly automated analysis of customized biochips. The patented technology includes a light-activated in situ synthesis of oligonucleotide probes within the channel of a three-dimensional microfluidic reaction carrier.1 In addition, the Geniom enables synthesis of oligonucleotides in reverse 5’?3’ direction for experiments that require a free 3’-end.
2Planned and synthesized overnight, new sequence data can be included in the next experiment.
miRNA profiling
| Table 1: Development of major organism within different miRBase versions. The table contains only mature/mature-star sequences, the value in the parenthesis specifies the changes to the previous release. Click to enlarge. |
The discovery of non-coding RNA (ncRNA) has changed a dogma in molecular biology and revealed a massive hidden network of regulatory machinery. The ratio of non-coding to protein-coding DNA increases with developmental complexity, the human genome consists of 98.5 percent non-coding DNA (ncDNA), compared to 25 and 50 percent ncDNA in simple eukaryotes.
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4ncRNAs, like the short miRNAs, have been shown to modulate posttranscription gene expression by regulating the translation or degradation of target messenger RNA (mRNA). miRNAs work in a highly complex regulatory network and determine cell identity and fate. Aberrant expression of miRNA can lead to diseases, including cancer and diabetes.5 Currently, more than 8,600 miRNAs are published in the miRBase Sequence Database; more than 2,000 new miRNAs alone were added within the past five months (Wellcome Trust Sanger Institute; status from Sept, 2008). Meanwhile, the number of mature and mature-star sequences in the current miRBase version 12.0 increased to 866 in human, adding 133 new sequences since the 10.1 version (see figure 1). Keeping pace with the fast-changing database information and newly discovered miRNAs, researchers require flexible microarray technology. The Geniom Microarray Facility enables the design and production of customized microchips either in the researcher’s laboratory, ordering the microarray with newly published miRNAs on demand for the analysis within the Geniom RT Analyzer, or using febit’s full service. febit incorporates and produces within 48 hours (instead of weeks) an updated array that reflects the latest miRBase content or synthesizes on demand a chip including unpublished sequences.
The miRNA-based Microfluid Primer Extension Assay (MPEA) combines a conventional hybridization assay with an enzymatic elongation, which uses the hybridized miRNA as a primer to incorporate biotinylated nucleotides. This sensitive assay works with a very small amount of total RNA and can be applied to any type of small ncRNA.
6 Another challenge of miRNA profiling is the specificity of the array. miRNAs are fairly short and the miRNA of interest often differs only in a few nucleotides. An example is the eight hsa-let-7 family members with 71–95 percent sequence identity. An in-house study at febit investigated the discriminatory power of Geniom’s detection system. For this analysis, the synthetic RNA oligonucleotides for each family member were labeled and individually hybridized to an array. The relative cross-hybridization to probes was calculated for the other seven let-7 family members. The result indicated that the Geniom system has good discriminatory power for all members (Figure 1).
6Genotype profiling
The sequencing of the human genome revealed millions of single nucleotide polymorphisms (SNPs). Many small regions of DNA vary among individuals. SNPs are DNA sequence variations that involve a single nucleotide alteration, changing the DNA sequence.
The majority of SNPs are without physiological effect, but some genetic variants have been associated with diseases, including cancer, and with the response to medical therapy. In the past, research focused on specific pathways or candidate genes. The new high-throughput methods allow studying SNPs in complex diseases and large cohorts, leading to a large amount of information in a single experiment.
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The microarrays with fixed content are useful for high resolution whole genome association studies and mapping haplotype regions in unrelated individuals. With the Geniom technology, screening of SNPs in a set of chosen genes by using a customized microarray is possible. The in silico design allows optimization and selection of a set of oligonucleotides. With 8 x 15,000 probes synthesized overnight, many combinations tailored to the researchers’ needs can be tested in parallel.
Specifically, genetic variants in immune regulator genes such as cytokines, chemokines and transcription factors have been associated with cancer and allergies. Dr. Janne Pullat, Division of Functional Genome Analysis at the German Cancer Research Center in Heidelberg, Germany, and colleagues, compared the genotypes of hay-fever patients to sex- and age-matched controls. They identified allele-frequency differences of more than 10 percent and discriminated homo- and heterozygote polymorphism. Relevant SNPs in cytokine genes and other immune regulatory factors were selected from SNP-databases and published studies. An oligonucleotide microarray was designed and synthesized with the Geniom Platform for a chosen set of 99 relevant SNPs. The flexibility and specificity of the array set up the possibility to use self-determined probes for the SNP arrays and allowed a focus on the genes of interest.8
Targeted Genomic Sequencing
Development in the field of next-generation sequencers opened up new possibilities for genomics. But this technology offers no possibility to focus on genetic regions of interest and is therefore more expensive and extensive than necessary. That is why the demand for a method to preselect those regions before sequencing is high.
febit recently patented a method which forms the basis of a novel technology that enables the expanded use of next-generation sequencers in genomic research. Further investigation of the human genome by resequencing will particularly benefit from the new method: febit uses its microfluidic biochip system Geniom for sequence-based selective isolation of nucleic acids from genomic samples. This new approach to high-throughput sequencing essentially relies on high-density microarray biochips with flexible design, a technology established by febit several years ago. The newly patented method is known as hybselect, targeted sequencing or sequence capture. Using this innovative technique, researchers are able to isolate genes or genomic regions of interest from a complex DNA sample prior to sequencing.
The practical approach of this new technology is currently part of the research within the laboratories of Translational Genomics Research Institute (TGen): TGen will evaluate Next-Generation-Sequencing equipment in conjunction with febit’s proprietary Geniom Microarray Technology.
Conclusion
The microarray technique has spread into many areas of genomics and functional genomics. Today, the focus is often on a distinct pathway or gene family, demanding an easy-to-use, cost effective and flexible microarray technology, which can quickly incorporate new sequence data.
About the author
Peer Staehler is the co-founder and chief scientific officer at febit holding gmbh. More information is available from: febit inc. 781-391-4360, www.febit.com.
References
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