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Advancing the quality control methodology,,,to assess isolated total RNA,,,and generated fragmented cRNA

This Application Note describes how Gene Logic, Inc., has improved its ability to analyze the quality of total RNA and fragmented cRNA by using the Agilent 2100 bioanalyzer. The Eukaryote Total RNA Nano and Pico assays are currently being utilized to examine the 28S/18S ratio in the total RNA as well as detection of possible degradation. The mRNA Smear Nano assay is used to confirm that a high percentage of the cRNA sample is fragmented to lengths in the optimal range necessary to be hybridized onto Affymetrix microarrays.
Gene Logic Inc. is a leading provider of integrated genomics information and bioinformatics products and services related to gene activity in human disease and toxicity that enable global pharmaceutical and biotechnology companies to optimize the time, risk and cost of drug discovery and drug development. In order to do this, the quality control methods must ensure that only the highest quality samples proceed through our high-throughput process, which in turn help to reduce the downstream costs of cDNA synthesis, In-Vitro Transcription and, finally, hybridization onto microarrays. For qualitative purposes, many laboratories continue to analyze their RNA samples using MOPS gels with gel electrophoresis. However, an internet search of Microarray facilities around the country shows an increase in the usage of the Agilent bioanalyzer.
When using gel electrophoresis to examine the quality of samples, pre-cast MOPS gels were used. Every now and then a problem with the gels was encountered, either user related or gel related. Some general problems occurring were sample migration, mold spots in the pre-cast gels and staining difficulties. The key issue associated with the RNA quality control gel was that
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