title: Protocol: high throughput silica-based purification of RNA from Arabidopsis seedlings in a 96-well format.
abstract: The increasing popularity of systems-based approaches to plant research has resulted in a demand for high throughput (HTP) methods to be developed. RNA extraction from multiple samples in an experiment is a significant bottleneck in performing systems-level genomic studies. Therefore we have established a high throughput method of RNA extraction from Arabidopsis thaliana to facilitate gene expression studies in this widely used plant model. We present optimised manual and automated protocols for the extraction of total RNA from 9-day-old Arabidopsis seedlings in a 96 well plate format using silica membrane-based methodology. Consistent and reproducible yields of high quality RNA are isolated averaging 8.9 μg total RNA per sample (~20 mg plant tissue). The purified RNA is suitable for subsequent qPCR analysis of the expression of over 500 genes in triplicate from each sample. Using the automated procedure, 192 samples (2 × 96 well plates) can easily be fully processed (samples homogenised, RNA purified and quantified) in less than half a day. Additionally we demonstrate that plant samples can be stored in RNAlater at -20°C (but not 4°C) for 10 months prior to extraction with no significant effect on RNA yield or quality. Additionally, disrupted samples can be stored in the lysis buffer at -20°C for at least 6 months prior to completion of the extraction procedure providing a flexible sampling and storage scheme to facilitate complex time series experiments.
authors.affiliation: The Centre for Systems Biology at Edinburgh (CSBE), the University of Edinburgh, CH Waddington Building, King's Buildings, Mayfield Road, Edinburgh EH9 3JD, UK. Lorraine.Kerr@ed.ac.uk.
authors.forename: Eliane
authors.initials: E
authors.lastname: Salvo-Chirnside
authors.forename: Steven
authors.initials: S
authors.lastname: Kane
authors.forename: Lorraine E
authors.initials: LE
authors.lastname: Kerr