This article describes the analysis of Cy5-labeled cRNAs and cDNAs using the Agilent 2100 Bioanalyzer and the RNA 6000 LabChip® kit. It discusses various methods for detecting and quantifying gene expression levels, as well as the use of fluorescently labeled probes in microarray applications. The importance of using high-quality RNA for synthesizing labeled cDNAs or cRNAs is emphasized.
The document details an application for analyzing Cy5-labeled nucleic acids (cRNAs and cDNAs) prior to microarray hybridization. It utilizes the Agilent 2100 Bioanalyzer system combined with the RNA 6000 LabChip kit and a dedicated assay.
Two experimental protocols are described: one for analyzing Cy5-labeled cRNAs directly, and another modified protocol for Cy5-labeled cDNAs where Cy5-dCTP is added to the gel matrix. The text highlights advantages over traditional gel electrophoresis, including reduced analysis time, minimal sample consumption, enhanced separation performance, and excellent reproducibility.
The system can analyze Cy5-labeled cRNA in a concentration range from 10 to 500 ng RNA/pl.
The analysis shows that the Cy5-labeled cRNA ranges in size between 500 and 2000 bases.
The standard protocol uses a concentration of 25 pM Cy5-dCTP.