cDNA microarray analysis of small 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 by the large amount of sample tissue needed for microarray analysis. Generally, 50-200 mug of total RNA and 1-2 mug of mRNA is required for each hybridisation, which is equivalent to 50-100 mg of plant tissue. This requirement for large amounts of starting material severely constrains the use of microarrays for transcript profiling in specific tissues and cell types during plant development. Here we report on a robust and reliable target amplification method that enables transcript profiling from sub-mg amounts of plant tissue. Using 0.1 mug of total RNA we show that twofold expression differences are possible to distinguish with 99% confidence. We also demonstrate the application of this method in an analysis of secondary phloem development in hybrid aspen using defined tissue sections, corresponding to 2-4 cell layers with a fresh weight of similar to 0.5 mg.

Ämnesord

NATURVETENSKAP  -- Biologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences (hsv//eng)

Nyckelord

PCR

cDNA-amplification

microarray

transcript profiling

phloem development

populus tremula x tremuloides

Biology

Biologi

Publikations- och innehållstyp

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