Sentrix® Human-6 Expression BeadChip is a relatively new whole-genome DNA microarray product manufactured by Illumina, Inc. It allows simultaneous interrogation of the expression profiles of 47,296 transcripts for six independent DNA samples in parallel, on a single microarray. Each chip contains six identical sets of 47,296 unique probes, including 24,385 RefSeq annotated genes, known alternative splice regions, putative transcripts predicted by Gnomon, and Unigene clusters. Each transcript is represented by an optimized, full-length 50-mer oligo probe, presented an average of 30 times on the chip, providing redundancy and high sensitivity. A standard assay protocol is used for sample preparation and labeling (RNA isolation, cDNA synthesis and
in vitro transcription and amplification), followed by another set of standard procedures for hybridization. BeadChips are imaged on Illumina’s BeadArray Reader. Output files can be exported as required to third-party software for analysis or can be conveniently analyzed using Illumina’s BeadStudio software. Per-sample running costs, including labeling, will typically range between $80 and $200, comparable in cost to just the sample labeling steps using other microarray platforms.
We used three Illumina’s Sentrix® Human-6 Expression Beadchips to assess global gene expression in RNA samples extracted from EBV-transformed lymphoblastoid cell lines of a small group of patients with a muscle disorder, and controls. Only 100 ng of input RNA was required for the analysis. All the reagents, equipment, and protocols were purchased from Illumina. All the laboratory personnel were trained by Illumina’s technician in all procedures. The results were visualized in BeadStudio software package by Illumina, supplied with the BeadArray Reader. The results we obtained were remarkably high quality with great reproducibility. Correlation between samples duplicated on the same or different chips was extremely high, as well as correlation between different samples, as expected for the samples of the same tissue. 12,983 transcripts were detected at a detection confidence limit greater than 99% in at least three of twelve samples we analyzed.
To perform supervised and unsupervised clustering analysis, and compare global gene expression between patients and controls, we used both BeadStudio software, and Genespring GX 7.3 software (Silicon Genetics, Inc.). During this work, we discovered that downloading of expression data generated on the Illumina’s platform into the Genespring software is not a trivial task. On the other hand, it is very easy to transfer data generated by the Affymetrix platform. I expect that this procedure will become easier once more people start using Illumina’s product. We discovered very interesting results suggesting possible mechanisms and new pathways of the disease based on the whole-genome expression data generated using Sentrix® Human-6 Expression BeadChip. To confirm expression results derived from Illumina’s whole genome expression chip, we performed confirmation studies using TaqMan® Gene Expression Assays (Applied Biosystems). High correlation between Illumina expression arrays and TaqMan® assays was observed.
In summary, we were very satisfied with the performance and data quality generated with Sentrix® Human-6 Expression BeadChips by Illumina, Inc. I recommend that anybody who has an access to the BeadArray reader try this platform for DNA microarray analysis.