After almost thirty years since the first commercial prototype of the Affymetrix™ GeneChip™ microarray hit the market in 1994, its impact on our understanding of the intricate role of gene expression in health and disease remains staggering.
But, as GeneChip™ microarrays enter their third decade, where is this technology now?
To answer this question, in this article, we provide an overview of the features and workflow of modern Affymetrix™ GeneChip™ transcriptome profiling options, from the GeneChip™ Human Transcriptome Array 2.0 to the GeneChip™ Human Gene 2.0 ST array.
Affymetrix™ GeneChip™: A classic probe-based technology
The first draft of the human genome project in the early 2000s provided unprecedented information about the sequence of tens of thousands of genes in our genome (Lander et al., 2001). In 2002, Affymetrix™ GeneChip™ microarrays harnessed this sequence data to offer over 500,000 unique probes per chip, enabling researchers to instantly access gene expression information for over 33,000 genes (Affymetrix Inc., 2003).
For the first time, researchers could gain a truly genome-wide insight into the transcriptional activity of all genes simultaneously instead of simply individual genes by qRT-PCR.
But, even with this increased resolution, information about core aspects of the transcriptional landscape, such as the contribution of different transcript isoforms, non-coding RNA expression, and the extent of alternative splicing, was missed due to technological limitations that restricted the number of probes included per microarray.
GeneChip™ Human Transcriptome Array 2.0: A comprehensive transcriptome profiling approach
To address these shortcomings, advances in the photolithographic microarray manufacturing process now allow the Affymetrix™ GeneChip™ Human Transcriptome Array 2.0 to comprise a set of over six million unique probes derived from protein-coding mRNA and long non-coding RNA transcripts (lncRNA) contained in databases such as RefSeq, Ensembl, and lncRNA db (Thermo Fisher Scientific 2023a).
These 25-mer probes are designed for maximal transcriptome coverage and target >245,000 coding transcript isoforms to provide extensive expression information for >44,000 protein-coding genes, alongside >40,900 non-protein coding transcripts.
One transcript has an average of 109 probes, with each exon targeted by around 10 unique probes. Extensive alternative splicing information is also gained from >339,000 probe sets covering exon-exon junctions with approximately four probes per splice junction.
GeneChip™ Human Gene 2.0 ST array: A lighter approach
For research focusing on standard gene expression instead of in-depth comprehensive alternative splicing and transcript isoform analyses, the GeneChip™ Human Gene 2.0 ST array has >1.35 million unique 25-mer probes targeting >33,500 coding transcripts and >11,000 lncRNAs. One transcript has approximately 21 probes (Thermo Fisher Scientific, 2023b).
Compared to the GeneChip™ Human Transcriptome Array 2.0, this reduced probe coverage, combined with the lower number of transcripts targeted, makes it suited to a lighter transcriptome profiling analysis at a lower cost.
Sample requirements and workflow
For large-scale pharmacotranscriptomic screens, ensuring sample compatibility with the chosen transcriptome profiling technology is key to generating accurate and reliable data for target discovery, biomarker detection, and evaluation of drug efficacy.
Both GeneChip™ assays are suitable for RNA isolated from common sample types in drug discovery pipelines, such as whole blood, fresh/frozen tissue samples, and formalin-fixed paraffin-embedded (FFPE) tissues.
As with most transcriptomic techniques, RNA must first be isolated and prepared with labels necessary to detect their hybridization to microarray probes (Thermo Fisher Scientific, 2023c).
For abundant RNA between 50-500 ng, the GeneChip™ WT PLUS Reagent Kit is used to reverse transcribe, amplify, and biotin label poly-A and non-poly-A RNA before hybridization to the chosen GeneChip™ microarray.
If RNA concentrations are between 0.1-10 ng for fresh/frozen tissues or whole blood or between 0.5-50 ng for FFPE tissues, the GeneChip™ WT Pico Kit is required for RNA preparation.
Looking to the future
From humble beginnings, Affymetrix™ GeneChip™ microarrays have evolved from 16,000 unique probes per chip in 1994, >500,000 in 2002, to over six million in 2023 to provide extensive transcriptome coverage.
But, in an increasingly diverse next-generation sequencing landscape dominated by high-throughput RNA-seq, long-read sequencing, and cost-effective novel 3’ mRNA-seq approaches scalable to tens of thousands of samples, microarrays will require continual innovation to compete on scalability, the depth of transcriptomic content provided, and the overall cost per sample.
References
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GeneChip™ Human Transcriptome Array 2.0 Datasheet | Thermo Fisher Scientific (2023a). Available at: https://assets.thermofisher.com/TFS-Assets/LSG/brochures/hta_array_2_0_datasheet.pdf
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GeneChip™ Human Gene 2.0 ST Array Datasheet | Thermo Fisher Scientific (2023b). Available at: https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fbrochures%2Fhugene_2_st_datasheet.pdf
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Expression Microarray Reagent Guide | Thermo Fisher Scientific (2023c). Available at: https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fbrochures%2FEMI06269-2_-Flyer_Expression_Reagent_Guide_HYPERLINK_Final.pdf
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Lander, E.S. et (2001). ‘Initial sequencing and analysis of the human genome’. Nature, 409, 860–921.
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Pioneering an Industry Brochure | Affymetrix Inc. (2003). Available at: https://www.affymetrix.com/support/technical/other/pioneer_brochure.pdf
