Powerful data analysis improves gene expression technology

Laboratories are demanding ever more sensitivity and reliability from their gene expression analysis technologies. Here we look at what these technologies have to offer.

Representing roughly 30 per cent of all primary brain tumours, meningiomas are classified by the World Health Organisation (WHO) into three tumour grades based on histopathological criteria: grade I for benign slow-growing tumours, and grades II and III for atypical and anaplastic meningiomas.

However, pathologically benign meningiomas can recur, even after operations to remove them. And the clinical course of this substantial minority of meningiomas is difficult to predict - not least because the molecular mechanisms underlying them is poorly understood.

Taking advantage of Qlucore Omics Explorer's powerful data analysis capabilities, its speed, and its ability to work on any PC, Dr Bárbara Meléndez and her colleagues in the Unidad de Investigación de Patología Molecular (Molecular Pathology Research Unit) at Hospital Virgen de la Salud in Toledo, Spain, are investigating how such generally benign meningiomas can start to exhibit the sort of histology and recurrence usually associated with more aggressive tumours.

In order to do this, Meléndez and her team are using Qlucore Omics Explorer to perform gene expression and methylation analyses of brain tumour samples and then validating them with data from other databases.

To begin with, the researchers reduce variance to identify groups of samples. Then they perform supervised analyses to identify differentially methylated or expressed genes.

In addition, oligonucleotide probes are used to analyse gene expression. Each sample can generate around 41,000 gene expression data, resulting in huge data files because of the number of samples involved (Fig. 1).

Using Qlucore principal component analysis (PCA) the team can identify groups of tumours that have the same histopathology, despite being different at the molecular level. Such differences can be used to help identify targets for different therapies and to improve on disease prognoses.

Also important is Qlucore's 3D graphics capability: "3D lets us see individual groups that we don't always see with cluster analysis alone. So while the diagnosis might be the same and the tumours have the same histology, they might be different pathologically. This is very important because it also gives us an idea of which tumours behave a little bit differently to other samples in that group. So they should not really be in that group," adds Meléndez.

And it is here that Qlucore has helped the team to a dramatic discovery. "Expression analyses allowed us to identify that meningiomas can be classified into an aggressive and a non-aggressive group - despite WHO classification criteria establishing three malignancy groups and about 15 histopathological subtypes. These findings identify tumours that recur in the aggressive group, even if they have a benign histopathological diagnosis. Supervised analyses have allowed us to identify gene expression profiles associated with more aggressive tumours - findings that have been confirmed by analysing the methylation data with Qlucore," she concludes.

The team hopes shortly to publish a research paper outlining this discovery. [Page Break]

Other novel technologies

New England Biolabs (NEB) has released the Gibson Assembly Cloning Kit and online primer design tool NEBuilder for the cloning of DNA fragments using the Gibson Assembly approach. This kit includes NEB's robust Gibson Assembly Master Mix and NEB 5-alpha competent E. coli, enabling fragment assembly and cloning in less than two hours.

Gibson Assembly enables the rapid assembly of multiple DNA fragments using a one-step, isothermal reaction. It has become a mainstay in the laboratories of many synthetic biologists and is now catching on in the wider life science community due to its ease-of-use, robustness and flexibility.

"As a leader in the discovery and development of cloning tools and technologies, NEB has embraced Gibson Assembly and further developed its protocol for cloning," states Dr Bill Jack, director of research at NEB. "While traditional cloning methods remain important standard practices in the laboratory, this alternative method delivers additional speed, flexibility and ease-of-use."

The Gibson Assembly Cloning Kit includes protocols for primer design, and has been optimised for the assembly of one or more DNA fragments into a linearised vector.

NEB is an industry leader in the discovery and production of enzymes for molecular biology applications and now offers a large selection of recombinant and native enzymes for genomic research. The company is expanding its product offerings into areas related to PCR, gene expression, sample preparation for next generation sequencing, cellular analysis, epigenetics and RNA analysis.

Meanwhile Merck Millipore's ubiquitous chromatin opening element (UCOE) expression technology gives major improvements in gene expression in stably-transfected mammalian cells through effects on the structure of chromatin. UCOE expression technology prevents transgene silencing and gives consistent, stable and high-level gene expression irrespective of the chromosomal integration site.

The expression elements are small DNA elements - isolated from the area around house-keeping genes, which need to be active most of the time - that create a transcriptionally active, open chromatin environment around an integrated transgene, maximising its potential to be transcribed into protein, irrespective of the position of the transgene in the chromosome.

Merck Millipore cites a number of advantages with this technology: over 50 per cent of UCOE-derived clones have higher expression than the best non-UCOE-derived clones; UCOE-derived vectors produce substantially more expressing clones than non-UCOE-derived vectors; cell lines are stable over 130 generations; high-yielding cell lines can be derived in less than 60 days, without amplification; small (4-8kb) DNA elements are capable of making two protein chains per plasmid, for example antibodies; and it integrates with industry standard platforms such as Chinese hamster ovary (CHO) cells, cytomegalovirus (CMV) promoters, plasmids, transfection agents, and media.[Page Break]

Gene expression

Another supplier, Agilent, says that it provides the widest selection of gene expression solutions - offering a comprehensive portfolio with the highest array sensitivity, a proven and reliable qPCR platform, an RNA target capture kit and a committed software solution, all with a focus on quality in results.

The company's Gene Expression microarray platform, designed for high performance and reliability, provides a flexible and affordable solution for gene expression studies.

Catalogue arrays across 30 species are available for whole-genome studies with 60-mer oligonucleotide probes printed using SurePrint technology. The flexibility of this technology means that unlimited custom arrays can also be created using the company's online application eArray.

The platform, with its set of optimised reagents and hardware, enables highly-sensitive, highly-scalable, and highly-reproducible profiling with low RNA input amounts (down to 10 ng).

Also available are SurePrint G3 Exon Microarrays, with the same level of performance and sensitivity but with comprehensive coverage down to the exon level.

Exon arrays are available for human, mouse, and rat to enable multiple experiments from the same dataset: gene-level, exon-level, and splicing analysis.

After Gene Expression microarray results are obtained, Agilent's QPCR portfolio enables researchers to verify microarray results or do more detailed analysis on gene expression changes. Its Mx3005P QPCR system provides reliable results in a flexible, easy to use platform - with reagents validated to provide sensitive and reliable quantification down to single-copy detection and two-fold differences in expression.

An additional option in the Gene Expression portfolio is the SureSelect RNA Capture Target Enrichment Kit, enabling the target enrichment of specific transcripts of interest, or as a validation tool for whole transcriptome sequencing experiments.[Page Break]

Easy interface

Gene expression results, including microarrays, qPCR, and RNA-Seq, can be studied and summarised using GeneSpring GX and NGS software, which offer high quality analysis with an easy-to-use interface. With over 13,000 citations and an over 12-year track record, Agilent says that GeneSpring is the industry-leading tool for analysis of all its gene expression technologies.

Roche and Agilent Technologies have signed an exclusive agreement to provide continued service to NimbleGen microarray customers as Roche phases-out its NimbleGen array production and services.

Researchers using NimbleGen microarrays for all applications, including comparative genomic hybridisation, chromatin immunoprecipitation-on-chip, DNA methylation, and gene expression can transition to Agilent arrays, effective immediately, with minimal disruption.

The similarities of the technologies and products from both companies provide an optimal transition path and the ability to run Agilent microarrays on the NimbleGen MS 200 Microarray Scanner.

"This global collaboration provides our customers with a confident and straightforward solution to move from NimbleGen to Agilent microarrays," said Dan Zabrowski, head of Roche Applied Science. "With Agilent as a leading global supplier of microarray technology, we are convinced researchers will be provided with the highest compatibility to NimbleGen products and services, and believe that they will continue to receive the exceptional service and support they have come to expect."

"We are working closely with Roche to help customers make the transition to Agilent microarray products," said Robert Schueren, vice president and general manager of Agilent's Genomics Systems division.

"Our field service personnel are working directly with individual researchers to help convert their NimbleGen designs, and they will continue to provide enhanced service and support throughout the transition period and beyond. Additionally, Agilent is also enabling customers to read their arrays on NimbleGen scanners, eliminating the need to invest in capital equipment."

More information about the transition is available at www.agilent.com/genomics/microarrays

Recent Issues