A Cost-effective High-throughput MicroRNA Profiling Service Based on Array and Non-enzymatic Labeling Technologies
Introduction
MicroRNA (miRNA) research has dramatically increased over the past two years, with the number of PubMed records increasing three-fold during this time. To meet this demand, the UK-based company Oxford Gene Technology (OGT) has developed a high quality, cost-effective miRNA profiling service with high-throughput capabilities of processing over 1000 samples per week.
The profiling service uses customized arrays rapidly designed by OGT, and fabricated in an 8 × 15,000 probe format by Agilent Technologies, against the most up-to-date version of the Sanger miRBase, currently version 12. Agilent Technologies manufactures its arrays using inkjet in situ synthesis (IJISS) technology under a license agreement to the OGT patent family W089/10977. The multiarray format enables matched patient sample analysis on the same array to minimize experimental variability.
Principles Behind the Methodology
Isolation of miRNA from tissues can be problematic and time-consuming. The major problem is that enrichment protocols can have a detrimental effect on miRNA yield and introduce experimental artifacts. This is mainly due to the use of column-based methods that remove larger RNA molecules, thereby concentrating the pool of miRNAs. To overcome this, OGT uses Universal Linkage System™ (ULS™) technology, a rapid, non-enzymatic labeling system, developed by Kreatech Diagnostics. The ULS™ molecule consists of a monofunctional platinum complex coupled to a detectable molecule of choice. The platinum atom forms a coordinative bond, firmly coupling the detectable molecule to the biomolecule of choice, such as RNA. ULS™ labels RNA by binding to the N-7 position of guanine (see Figure 1).
Figure 1. Schematic overview of the ULS™ technology.
This labeling protocol uses unenriched miRNA from total RNA as a source of miRNA for labeling purposes, as data show that miRNA enrichment can reduce the correlation between microarray data and quantitative PCR (QPCR) results (Figure 2).
Figure 2. Spearman's rank correlation with QPCR.
The combination of this labeling protocol with OGT's proven array technology avoids the need for a miRNA enrichment step and the method yields reproducible data with low variance.
Additionally, the OGT-designed IJISS printed arrays provide high specificity, and are therefore able to give good differentiation of Let-7 family members (Figure 3), a group of eight miRNA molecules that differ from each other by either one or two bases.
Figure 3. Differentiation of Let-7 family members.
Protocol And Applications
The miRNA protocol, developed in conjunction with the FP6 European Framework–funded MolPAGE (Molecular Phenotyping to Accelerate Genomic Epidemiology) project, has successfully been used in numerous service-based projects at OGT.
One such project has been performed using miRNA samples from Alison Simmons’ laboratory at the University of Oxford. This group is investigating the molecular pathogenesis of Crohn's Disease (CD), a common yet debilitating component of inflammatory bowel disease, using primary cells from CD patients and healthy controls. Work in the Simmons laboratory has shed light on altered patterns of transcription in cells derived from CD patients.
Conclusion
The OGT miRNA service can be used for diverse studies including biomarker discovery and validation, cancer cell, and progenitor research.
The OGT workflow (Figure 4), in combination with high density OGT designed microarrays fabricated using Agilent's advanced printing technology and the Kreatech ULS™ labeling system, has been shown to provide reproducible data.
Figure 4. Workflow of customer sample at OGT.
This data has a high correlation of intra-array probe repeats, together with low array-to-array variability.
Data generated by OGT shows good correlation to data generated with QPCR.
OGT's miRNA profiling service can process >1000 samples per week and using arrays synchronous with the most up-to-date version of Sanger miRBase.
Oxford Gene Technology: www.ogt.co.uk
Agilent Technologies: www.agilent.com
Kreatech Diagnostics: www.kreatech.com
