Application Guide - QIAxcel Advanced

10 QIAxcel Advanced Application Guide 10/2016 Quality control of total RNA and cRNA for microarray analysis Dik van Leenen, Microarray Facility, University Medical Center Utrecht, The Netherlands In this application note, the suitability of the QIAxcel® system was assessed for quality control of RNA for microarray analysis. The values obtained for the tested quality control parameters indicate that the QIAxcel system is highly suited for analyzing the quality of total RNA and fragmented or intact cRNA. Introduction Microarray technology is a powerful tool used to determine the expression levels of thousands of genes in a single experiment and, thus, has become increasingly important in biomedical research and life science applications. Preparation of cRNA, which is then hybridized to microarrays, is a multistep procedure (Figure 1) that is prone to random and systematic errors, especially when large numbers of samples are handled. Therefore, it is critical to minimize experimental noise, standardize processing procedures, and include appropriate experimental controls and replicates. Monitoring the quality of the initial total RNA sample as well as products generated throughout the entire procedure is crucial, since their quality strongly influences the predictive power obtained from microarray data. Quality control parameters applied to total RNA samples typically include: • Determination of the A260 /A280 absorbance ratio to indicate protein contamination • Determination of the A260 /A230 absorbance ratio to detect potential chemical contaminants such as guanidinium thiocyanante • Determination of the 28S/18S rRNA ratio to check the integrity of the total RNA While double-stranded cDNA is usually not subjected to quality control due to the limited amount of material produced, the size distribution of unfragmented cRNA is usually analyzed. In addition, efficiency of fluorescent labeling of cRNA is also determined. We evaluated the QIAxcel system for quality control of total RNA and cRNA prior to Cy® 3/Cy5 labeling. Microarray Target Preparation Procedure Total RNA First strand cDNA synthesis Second strand cDNA synthesis Cleanup of double-stranded cDNA cRNA synthesis Cleanup of cRNA cRNA labeling and cleanup Fragmentation and hybridization Data analysis Figure 1. Typical workflow for microarray analysis.

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