Systematic analysis of intrinsic factors affecting differential display.
Differential display (DD) is a widely used method for identifying differentially expressed genes. To improve further the efficiency and reproducibility of the method, this report systematically examines four critical parameters of standard DD-PCR. Specifically, the study determined the optimal annealing temperature, elongation time, dNTP concentration, and arbitrary primer concentration. By using a thermal cycler that was capable of displaying a temperature gradient across a PCR plate, it was possible to determine (in a single experiment) the effect of different annealing temperatures. The optimal annealing temperaturefor a 13-mer arbitrary primer fell within a broad range of 40 degrees C-50 degrees C. Elongation times over a range of 30-120 s worked best. The optimal concentration for dNTPs was within a very broad range of 2-50 microM, with higher amounts allowing for greater pipetting accuracy. The most favorable concentration for the arbitrary primer was also within a broad range of 0.1-2.0 microM. A primer concentration below this range greatly reduced the efficiency of the amplification process. In conclusion, the experimental findings delineated the best possible DD conditions for a more reliable assessment of differential gene expression.
Cho
Yong-Jig
YJ
Vanderbilt University, Nashville, TN, USA.
Prezioso
Vincent R
VR
Liang
Peng
P
D000818:Animals; D002460:Cell Line; D020869:Gene Expression Profiling / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D051381:Rats; D013696:Temperature