Proof-reading signal accuracy of gene expression by binary differential display

• Published in: Biotechnology letters Vol. 32; no. 8; pp. 1039 - 1044
• Main Authors: Cho, Yong-jig, Meade, Jonathan D, Shester, Blake R, Walden, Jamie C, Guo, Zhen, Liang, Peng
• Format: Journal Article
• Language: English
• Published: Dordrecht Dordrecht : Springer Netherlands 01.08.2010; Springer Netherlands; Springer; Springer Nature B.V
• Subjects: Applied Microbiology; Binary differential display (BDD); Binding; Biochemistry; Biological and medical sciences; Biomedical and Life Sciences; Biotechnology; Blotting, Northern; Cell Line, Tumor; Deoxyribonucleic acid; Differential display; Differential display (FDD); DNA; Fluorescent; Fundamental and applied biological sciences. Psychology; Gene expression; Gene Expression Profiling - instrumentation; Gene Expression Profiling - methods; Gene Expression Profiling - standards; Genes; Gold; Humans; Kinetics; Life Sciences; Microbiology; Northern blot analysis; Oligonucleotide Array Sequence Analysis; Original Research Paper; Reproducibility of Results; Research methodology; Ribonucleic acids; Strategy; Binary differential display (BDD); Fluorescent; Differential display; Differential display (FDD); Northern blot analysis; Accuracy; Fluorescence; Gene expression
• ISSN: 0141-5492; 1573-6776
• DOI: 10.1007/s10529-010-0268-y

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.