Improved representation of gene markers on microarray by PCR-Select subtracted cDNA targets.
Despite the success and popularity of microarrays as a high-throughput technology for gene-expression studies, its sensitivity is as yet fairly limited. We have successfully combined the use of PCR-Select cDNA subtraction and Affymetrix GeneChips (AGC) to identify differentially expressed gene markers. Total RNA (totRNA) from combined hippocampus and cerebellum tissues of 2-week-old rat pups maintained for 5 weeks on an n-3 fatty acid (FA) deficient diet supplied to dams was isolated, SMART-amplified, and used for PCR-Select subtraction versus an adequately fed control litter preparation. Subtracted and amplified ds-cDNA end products were fragmented, terminally labeled with biotin-ddUTP and hybridized with RN-U34A AGC. At least 10-fold more potential gene markers with log2(T/D) > or = 1.4 were found versus the traditional AGC technology when the same chip was tested using nonsubtracted targets. Of this set of markers, 30% were robustly validated by real-time relative RT-PCR (rtrRT-PCR) and grouped as "confirmed" markers while the remaining were ascribed as "latent" markers. An improved and universal protocol to provide a rapid assessment for gene profiling in biological specimens is indicated.
Yakubov
E
E
Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel.
Dinerman
P
P
Kuperstein
F
F
Saban
S
S
Yavin
E
E
D000818:Animals; D000831:Animals, Newborn; D001923:Brain Chemistry / Q000235:genetics; D002531:Cerebellum / Q000378:metabolism; D018076:DNA, Complementary / Q000032:analysis* / Q000235:genetics; D015525:Fatty Acids, Omega-3 / Q000378:metabolism; D005526:Food, Formulated; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation / Q000235:genetics; D005819:Genetic Markers / Q000235:genetics*; D006624:Hippocampus / Q000378:metabolism; D008055:Lipids / Q000172:deficiency; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D013312:Stress, Physiological / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology
D018076:DNA, Complementary; D015525:Fatty Acids, Omega-3; D005819:Genetic Markers; D008055:Lipids; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger