Saturation screening for p53 target genes by digital fluorescent differential display.
Differential display (DD) is one of the most commonly used approaches for identifying differentially expressed genes. Despite the great impact of the method on biomedical research, there has been a lack of automation of DD technology to increase its throughput and accuracy for a systematic gene expression analysis. Most of previous DD work has taken a "shotgun" approach of identifying one gene at a time, with limited polymerase chain reaction (PCR) reactions set up manually, giving DD a low-technology and low-throughput image. With our newly created DD mathematical model, which has been validated by computer simulations, global analysis of gene expression by DD technology is no longer a shot in the dark. After identifying the "rate-limiting" factors that contribute to the "noise" level of DD method, we have optimized the DD process with a new platform that incorporates fluorescent digital readout and automated liquid handling. The resulting streamlined fluorescent DD (FDD) technology offers an unprecedented accuracy, sensitivity, and throughput in comprehensive and quantitative analysis of gene expression. We are using this newly integrated FDD technology to conduct a systematic and comprehensive screening for p53 tumor-suppressor gene targets.
Cho
Yong-jig
YJ
Department of Cell Biology, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN, USA.
Stein
Susanne
S
Jackson
Roger S
RS
Liang
Peng
P
D000818:Animals; D017209:Apoptosis; D015152:Blotting, Northern; D015153:Blotting, Western; D002232:Carbocyanines / Q000494:pharmacology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000737:chemistry; D005456:Fluorescent Dyes / Q000494:pharmacology; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D015972:Gene Expression Regulation, Neoplastic; D016158:Genes, p53 / Q000235:genetics*; D006801:Humans; D008962:Models, Theoretical; D009369:Neoplasms / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods; D012313:RNA / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D012680:Sensitivity and Specificity; D012984:Software; D013997:Time Factors; D014158:Transcription, Genetic; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D002232:Carbocyanines; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; C089187:cyanine dye 3; D012313:RNA; D003850:Deoxyribonuclease I