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 by the large amount of sample tissue needed for microarray analysis. Generally, 50-200 microg of total RNA and 1-2 microg 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 microg 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 approximately 0.5 mg.
Hertzberg
M
M
Department of Biotechnology, KTH - Royal Institute of Technology, SE-100 44 Stockholm, Sweden.
Sievertzon
M
M
Aspeborg
H
H
Nilsson
P
P
Sandberg
G
G
Lundeberg
J
J
D018076:DNA, Complementary / Q000032:analysis; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction; D018749:RNA, Plant / Q000032:analysis*; D014197:Trees / Q000737:chemistry*
D018076:DNA, Complementary; D018749:RNA, Plant