Laser microdissection (LM) provides a useful method for isolating specific
cells or tissues from biological samples. Here, we adapted microdissection
protocols to allow high-resolution transcript analysis of different tissues
from developing Arabidopsis seed. Sufficient RNA (∼50 ng) was extracted from
endosperm tissue for RT-PCR. However, to obtain enough RNA for microarray
analyses, it was necessary to amplify the RNA. PCR- and IVT-based
amplification methods were investigated and several important technical
aspects of amplification were identified (such as target truncation and
alterations in signal intensity). We found that when starting from only 50 ng of RNA, amplification methods based on PCR and IVT produced sufficient
product for reliable microarray hybridizations, with two-round IVT giving
the best results. Microarray analyses, using endosperm-derived RNA amplified
by two-round IVT, reproducibly identified endosperm enriched marker genes.
Thus, when combined with RNA-amplification protocols, LM is a robust and
reliable technique for high-throughput tissue-specific gene expression
analysis.