Obtaining reliable information from minute amounts of RNA using cDNA microarrays.
BACKGROUND
High density cDNA microarray technology provides a powerful tool to survey the activity of thousands of genes in normal and diseased cells, which helps us both to understand the molecular basis of the disease and to identify potential targets for therapeutic intervention. The promise of this technology has been hampered by the large amount of biological material required for the experiments (more than 50 microg of total RNA per array). We have modified an amplification procedure that requires only 1 microg of total RNA. Analyses of the results showed that most genes that were detected as expressed or differentially expressed using the regular protocol were also detected using the amplification protocol. In addition, many genes that were undetected or weakly detected using the regular protocol were clearly detected using the amplification protocol. We have carried out a series of confirmation studies by northern blotting, western blotting, and immunohistochemistry assays.


RESULTS
Our results showed that most of the new information revealed by the amplification protocol represents real gene activity in the cells.


CONCLUSIONS
We have confirmed a powerful and consistent cDNA microarray procedure that can be used to study minute amounts of biological tissue.
Hu
Limei
L
Cancer Genomics Core Laboratory-Department of Pathology, The University of Texas M D Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030, USA. lhu@mdanderson.org
Wang
Jing
J
Baggerly
Keith
K
Wang
Hua
H
Fuller
Gregory N
GN
Hamilton
Stanley R
SR
Coombes
Kevin R
KR
Zhang
Wei
W