Optimization and validation of small quantity RNA profiling for identifying TNF responses in cultured human vascular endothelial cells.
BACKGROUND
Affymetrix oligonucleotide microarrays are widely used in basic and applied research (Lander, E.S., (1999). Array of hope. Nature Genetics 21, 3-4; Lockhart, D.J. & Winzeler, E.A. (2000) Genomics, gene expression and DNA arrays. Nature 405, 827-836.) The need for a significant amount of starting RNA has limited its use in applications where the amount of RNA is limiting, such as with Laser Captured Microdissection (LCM), small biopsies, or peripheral blood in rodent models. To overcome this limitation, various RNA amplification and labeling methods have been described, however, further optimization and validation of these methods are needed.


METHODS
Here we reported using the Arcturus technology to optimize amplification and labeling of small amounts of RNA for Affymetrix microarray studies. We assessed the technical feasibility and variation introduced by differences in starting RNA quantity and differences in technical performance by microarray hybridization.


RESULTS
We demonstrated that the current approach is reliable to amplify as little as 40 ng total RNA, and it is suitable for Affymetrix studies yielding satisfactory quantitative chip performance. We also showed that differences in labeling methods contribute more to variation than the differences in starting RNA quantity per se. As a model, we studied the well-documented TNF-induced inflammatory responses in cultured human vascular endothelial cells. We were able to recapitulate the TNF-induced responses using small RNA sample profiling.
Shou
Jianyong
J
Integrative Biology, Lilly Research Laboratories, Indianapolis, IN 46285, USA. shou@lilly.com
Qian
Hui-Rong
HR
Lin
Xi
X
Stewart
Trent
T
Onyia
Jude E
JE
Gelbert
Lawrence M
LM
D000465:Algorithms; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007249:Inflammation / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D014471:Umbilical Veins
D011506:Proteins; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha