title: Proof-reading signal accuracy of gene expression by binary differential display.
abstract: Differential display (DD) is commonly used for identifying differentially expressed genes. However, each cDNA species identified by DD must be verified so a "real difference" can be differentiated from false positives. Although Northern blot analysis is the gold standard it is labor intensive, time-consuming and requires a significant amount of RNA. To speed up and streamline the confirmation process, we developed a new strategy: binary differential display (BDD) based on the binding kinetics of the arbitrary primers in DD. After determining a cDNA sequence of interest from a DD screen, two more 13mer primers derived from the original arbitrary primer used can be designed to target a corresponding cDNA sequence of interest: one with perfect 5'-base matches and the other with additional mismatches at the 5'-base to the corresponding mRNA being confirmed. A separate reverse transcription and FDD are then performed with the same RNA samples being compared. BDD can quickly and accurately determine if a cDNA sequence identified by DD corresponds to a truly differentially expressed gene. In addition, the method is especially useful when more than one cDNA sequence was recovered from a DD band where the masking effect of false-positives can be clearly resolved. Given its simplicity and limited RNA sample required, BDD can be used as a general strategy for rapid confirmation of differentially expressed genes discovered by DD.
authors.affiliation: Department of Cancer Biology, Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, 546 PRB, Nashville, TN 37232, USA. y.cho@vanderbilt.edu
authors.forename: Yong-jig
authors.initials: YJ
authors.lastname: Cho
authors.forename: Jonathan D
authors.initials: JD
authors.lastname: Meade
authors.forename: Blake R
authors.initials: BR
authors.lastname: Shester
authors.forename: Jamie C
authors.initials: JC
authors.lastname: Walden
authors.forename: Zhen
authors.initials: Z
authors.lastname: Guo
authors.forename: Peng
authors.initials: P
authors.lastname: Liang
mesh_terms: D015152:Blotting, Northern; D045744:Cell Line, Tumor; D020869:Gene Expression Profiling / Q000295:instrumentation / Q000379:methods* / Q000592:standards; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D015203:Reproducibility of Results