A micromethod for high throughput RNA extraction in forest trees

ABSTRACT

A large quantity of high quality RNA is often required in the analysis of gene expression. However, RNA extraction from samples taken from woody plants is generally complex, and represents the main limitation to study gene expression, particularly in refractory species like conifers. Standard RNA extraction protocols are available but they are highly time consuming, and not adapted to large scale extraction. Here we present a high-throughput RNA extraction protocol. This protocol was adapted to a micro-scale by modifying the classical cetyltrimethylammonium (CTAB) protocol developed for pine (i) quantity of material used (100-200 mg of sample), (ii) disruption of samples in microtube using a mechanical tissue disrupter, and (iii) the use of SSTE buffer. One hundred samples of woody plant tissues/organs can be easily treated in two working days. An average of 15 /ig of high quality RNA per sample was obtained. The RNA extracted is suitable for applications such as real time reverse transcription polymerase chain reaction, cDNA library construction or synthesis of complex targets for microarray analysis.