| Improved high-throughput ultrafiltration process enables cRNA purification for gene expression profiling. | |
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MedLine Citation:
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PMID: 18532901 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Ultrafiltration of nucleic acids has been used for a wide variety of applications, including sequence reaction purification and amplicon cleanup prior to spotting onto microarrays. Here we describe a novel process, using ultrafiltration, that purifies cRNA products for sensitive downstream applications. Initial attempts at this high-throughput purification for cRNA resulted in low sensitivity when compared against an industry standard (silica-based bind, wash, and elute purification). We modified the ultrafiltration process to include a proteinase K preincubation and a phosphate buffer wash that, when combined, increased sensitivity and signal-to-noise ratio in microarray applications. The protocol that we have developed eliminates the use of chaotropic salts (such as guanidinium thiocyanate) that are typically used in silica binding purification methods. The data demonstrate good performance for sensitive RNA applications using well-defined metrics, and thus the technique might be useful for a broader range of nucleic acid purifications. |
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Authors:
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Mark D Morris; Michael R Meyer; Gary Painchaud; Jon Marlowe; George Y Tokiwa; Thomas L Fare |
Publication Detail:
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Type: Journal Article |
Journal Detail:
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Title: Assay and drug development technologies Volume: 6 ISSN: 1540-658X ISO Abbreviation: Assay Drug Dev Technol Publication Date: 2008 Jun |
Date Detail:
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Created Date: 2008-07-02 Completed Date: 2008-09-09 Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 101151468 Medline TA: Assay Drug Dev Technol Country: United States |
Other Details:
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Languages: eng Pagination: 421-32 Citation Subset: IM |
Affiliation:
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Rosetta Inpharmatics LLC, Seattle, WA 98109, USA. mark_morris@merck.com |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Gene Expression Profiling* Humans Jurkat Cells K562 Cells RNA, Complementary / isolation & purification* Transcription, Genetic Ultrafiltration / methods* |
| Chemical | |
Reg. No./Substance:
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0/RNA, Complementary |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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