Using the Biomek® 3000 Laboratory Automation Workstation and Agencourt® RNAdvance™ Cell Kit to Isolate and Perform Quantitation and Normalization on Total RNA from Cultured Eukaryotic Cells

Poster: Matthew Cu and Amy Yoder:
Manual isolation of total RNA can be labor-intensive, time-consuming and prone to nuclease degradation due to human error. Understanding the benefits of automation in handling these issues, we developed a suite of automated methods on the Biomek 3000 Laboratory Automation Workstation and integrated both the Peltier Static Device and the Peltier Shaking Device. The suite encompasses three methods to purify and prepare samples for gene expression analysis. The first two methods will be the focus of this paper. RNAdvance Cell Isolation is the first method and begins with the isolation of total RNA from cultured eukaryotic cells using the Agencourt RNAdvance Cell kit. Various cell lines, in a 96-well format, were used to demonstrate the automated process for RNA isolation.

The isolation method begins with the addition of a magnetic lysis solution to disrupt cell membranes and bind RNA to the paramagnetic particles. RNA remains affixed to the beads during DNase treatment and washing, and then is eluted from the particles. Quantitation and Normalization is the second method in the suite and it is used after RNA isolation. The method quantitates and normalizes nucleic acids using either an absorbance or fluorescence protocol. Raw data, obtained from the DTX 880 Multimode Reader (Beckman Coulter), are imported into a customized Microsoft* Excel calculation template which will automatically generate parameters to be used bythe Biomek Software to produce a normalized sample plate. The template has been optimized to require minimal input from the user. These methods incorporate a graphical user interface which allows the user to process a full or partial 96-well plate.

The information presented here includes the full description of the automated methods and subsequent results.