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Automated NuGEN Ovation* Whole Blood Solution for Blood Expression Profiling on the Biomek FX Laboratory Automation Workstation |
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Poster: Handy Yowanto, Don Misumi, Joe Don Heath, William Biggs and Cynthia French:
Expression profiling from human samples such as whole blood has been particularly challenging due to the small quantity of samples and the presence of globin mRNA. The ubiquity of globin mRNA transcripts has been shown to interfere with gene expression profiling especially for Affymetrix GeneChip* microarrays. In an effort to increase both sample throughput and improve assay reproducibility, we have developed the ArrayPlex Blood Expression application on the Biomek FX Laboratory Automation Workstation. The application incorporates NuGEN’s Ovation Whole Blood Solution and the Agencourt® RNAClean™ paramagnetic bead technology to purify labeled oligonucleotides specifically for the Affymetrix GeneChip microarrays.
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ArrayPlex Blood Expression Application on the
Biomek FX Laboratory Automation Workstation |
The ArrayPlex Blood Expression application is made up of two functional methods, cDNA Synthesis & Amplification and Fragmentation & Labeling methods. The cDNA Synthesis & Amplification method synthesizes the first strand DNA, generates the DNA/RNA heteroduplex double strand cDNA, and employs SPIA* linear isothermal DNA amplification. The method then uses the RNAClean paramagnetic bead technology to purify the cDNA. The Fragmentation & Labeling method quantifies the cDNA, adjust the concentration to 176ng/µl, fragments and then biotin labels the cDNA before an off-line hybridization. Replicate PAXgene* samples were collected from 10 human research volunteers.
Total RNA was extracted from these samples using the Agencourt RNAdvance™ Blood kit on the Biomek NX workstation. In this study, we used 20 ng of total RNA starting material for both the manual and automated target preparations. Targets were prepared using the NuGEN’s Ovation* Whole Blood Solution and then scanned on the Affymetrix Human Genome U133 Plus 2.0 Array (HG-U133 2.0)*. Evaluation criteria were based on the percent present, noise, reproducibility and repeatability.
Click here for this Poster (PDF)
Additional Information:
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* All trademarks are the property of their respective owners. Where applicable,
the PCR process is covered by patents owned by Roche Molecular Systems, Inc.,
and F. Hoffmann-LaRoche, Ltd.
For Research Use Only; not for use in diagnostic procedures.
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