Analysis of Gene Expression Using Agilent Microarrays and RNA Isolated from Human Whole Blood

Agilent’s dual-mode Gene Expression Microarray platform is a comprehensive and robust system that couples the ease and simplicity of one-color experimental design with the capability of two-color sensitivity for detection of small gene expression changes—all in a single platform. The Agilent platform now includes a method for the analysis of differential gene expression from human whole blood, which is compatible with the PAXgene Blood RNA System (PreAnalytiX GmbH, Hombrechtikon, CH). The PAXgene Blood RNA System consists of a blood collection tube and nucleic acid purification kit and is intended for the collection, storage, and transport of blood and stabilization of intracellular RNA for subsequent isolation and purification of intracellular RNA. Combined with the PAXgene System, Agilent’s amplification and labeling technology, in conjunction with the high sensitivity and specificity of 60-mer oligonucleotide microarrays, offer a total solution for gene expression in whole blood without the need for additional steps or kits to remove globin transcripts. Titration experiments using Agilent RNA spike-in control targets demonstrate that the sensitivity of detection for low abundance or poorly expressed transcripts with RNA isolated from whole blood compares favorably with RNA isolated from other cell and tissue sources. Authors Christopher Rizzo Agilent Technologies Little Falls, DE USA Athene Chan, Diane Ilsley-Tyree Agilent Technologies Santa Clara, CA USA Introduction Blood is an attractive tissue for clinical and biological research studies because samples can be easily collected and multiple measurements can be made from the same patient. Furthermore, a wide range of analyses can be made by gene expression profiling of peripheral blood samples. These include: prognosis, diagnosis, and management of diseases; monitoring of pathological and biochemical responses to toxicodynamic and pharmacodynamic factors; and determination of genetic factors potentially affecting all of the above. As one example, a recent presentation at the Annual Meeting for the Society of Toxicology demonstrated that sub-toxic and non-toxic doses of hepatotoxicants can be monitored using gene expression signatures in blood as surrogate markers prior to the onset of detectable liver damage. This application note describes the use of the Agilent Technologies portfolio of products with the PAXgene Blood RNA System to facilitate the analysis of gene expression profiles from whole blood. There are two major challenges that can hinder RNA profiling from blood samples. The first of these is the relative instability of the in vitro cellular RNA profile after blood collection, whether due to RNA degradation or gene induction. The second is the presence of interfering components such as globin mRNA, genomic DNA, and other small molecules that can inhibit downstream enzymatic reactions. The high abundance of globin transcript in whole blood is particularly troublesome as it can inhibit the synthesis of other labeled transcripts, thereby creating bias in the downstream microarray results. Agilent's solution for whole blood gene expression analysis is designed to be compatible with the PreAnalytiX PAXgene Blood RNA System (PreAnalytiX, GmbH, catalog number 762134) which stabilizes the gene transcription profile at the point of sample collection and provides highly purified cellular RNA. The Blood RNA Isolation kit contains an optimized Resuspension Buffer and Proteinase K to facilitate the extraction of RNA from whole blood. An on-column DNase treatment is performed to reduce genomic DNA for especially sensitive applications. The high-purity RNA isolated with the PAXgene Blood RNA Kit can then be used directly in amplification and labeling reactions using Agilent's Low RNA Input Linear Amplification Kit. The kit uses a patented, high-yield, single tube procedure to generate amplified fluorescently-labeled cRNA that is suitable for both one-color and two-color gene expression applications. Only a single round of amplification is required and no additional kits or procedures are needed to remove globin message or protein (Figure 1). Agilent's microarray platform for gene expression includes empirically selected 60-mer oligonucleotide probes for maximum specificity, optimized high-stringency protocols, and RNA spike-in controls. Agilent's Feature Extraction software electronically generates a QC report to validate the results from every experiment, and Feature Extraction output files can be exported to GeneSpring or Rosetta Resolver data analysis products. Materials and Methods Isolation of Total RNA from Whole Blood HeLa cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, catalog number CCL-2) and total RNA was purified using Agilent's Total RNA Isolation Mini Kit (5185-6000). Human spleen RNA was purchased from Ambion, Inc. (Austin, TX, catalog number 7852). All blood was collected from healthy volunteer donors. 2.5 mls of whole blood was collected into each PAXgene Blood RNA Tube using standard methods. Samples were handled as recommended by the manufacturer prior to processing. RNA was isolated from blood samples using the PAXgene Blood RNA Kit (PreAnalytix GmbH, Switzerland, product number 762134). RNA quality and integrity were determined using the Eukaryote Total RNA Nano 6000 assay (Agilent RNA 6000 Nano LabChip Kit, part number 5065-4476) on the Agilent Technologies 2100 Bioanalyzer (part number G2940CA) and quantified by measuring A260nm on a UV/Vis spectrophotometer (ND-1000, NanoDrop Technologies, Rockland, DE). Generation of Fluorescently Labeled cRNA Targets Total RNA isolated from whole blood was amplified and fluorescently labeled using the Agilent Technologies Low Input Linear Amplification Kit (part number 5184-3523) following the detailed protocol described in the kit manual (publication number 5185-5818). Each reaction contained 200 or 500 ng total RNA from whole blood and 2 μl (34 pg) of RNA spike-in control (diluted as described in the product insert, 5188-5279). Cyanine 3and cyanine 5-labeled CTPs were obtained from PerkinElmer/NEN Life Sciences (Boston, MA, catalog numbers 2 G E N O M I C S G A T T G A T C C T T C T G A C Figure 1. Steps involved in the whole blood gene expression analysis workflow. Isolate RNA using the PAXgene Blood RNA Isolation Kit. Amplify limited RNA quantities with the Agilent Low RNA Input Fluorescent Amplification Kit. Obtain results using Agilent's 60-mer Oligo Microarrays.