cDNA microarray analysis of small plant tissue samples using a cDNA tag target amplification protocol

Microarray technology is becoming an important comprehensive tool to study gene expression in plants. However, the use of this technology is limited b y the large amount of sample tissue needed for microarray analysis. General ly, 50-200 mug of total RNA and 1-2 mug of mRNA is required for each hybrid isation, which is equivalent to 50-100 mg of plant tissue. This requirement for large amounts of starting material severely constrains the use of micr oarrays for transcript profiling in specific tissues and cell types during plant development. Here we report on a robust and reliable target amplifica tion method that enables transcript profiling from sub-mg amounts of plant tissue. Using 0.1 mug of total RNA we show that twofold expression differen ces are possible to distinguish with 99% confidence. We also demonstrate th e application of this method in an analysis of secondary phloem development in hybrid aspen using defined tissue sections, corresponding to 2-4 cell l ayers with a fresh weight of similar to0.5 mg.