Abstract
In response to the increasing demands to generate larger amounts of quality data faster, specifically in the area of RNA isolation to support gene expression assays, we have adopted several automated solutions for isolating total RNA from a variety of sample types, e.g., blood, cells, and tissue. For isolation from blood, collected in PAXgene (PreAnalytiX, Hombrechtickon, CH) tubes, we use a Qiagen BioRobot Universal System (Qiagen Inc-USA, Valencia, CA). For total RNA from cells, we have adapted a 96-well plate solid-phase extraction method using Promega SV96 (Promega, Inc., Madison, WI) reagents. Finally, if starting from tissues, we use the AutoGenPrep 245 (AutoGen Inc, Holliston, MA) robot to perform a modified acid guanidine phenol chloroform isolation. The use of the AutoGenPrep 245 is preceded by tissue homogenization, usually performed on the Qiagen TissueLyser.
Following total RNA isolation the RNA is quantified by QuantiT (Invitrogen Corporation, Carlsbad, CA) using a Biomek FX method that performs whole plate serial dilutions, and then creates a reaction plate with samples in duplicate. Finally, to check for genomic DNA contamination in the total RNA sample, a minus reverse transcription PCR assay is performed. In using these methods, we are able to increase our throughput without compromising data integrity.