PCR based method for differential display of eukaryotic mRNA has been designed to isolate differential expressed genes in various cell types or under different growing conditions. A modified method for mRNA differential display originally developed by Sokolov was employed and optimized here. This procedure, based on the application of Ready-To-Go RT-PCR beads and Ready-To-Go RAPD beads, minimized pipetting steps, decreased the potential for pipetting errors, reduced the risk of contaminating and ensured greater reproducibility between reactions. Distinct cDNA bands can be observed by silver-staining 6% sequencing urea gel and easily excised and recovered for further use in cloning. The stage-specific genes in the developing human embryos were analyzed with six sets of arbitrary primers using this modified DDRT-PCR from 3-, 4- and 5-week-old human embryos. About 14 bands containing differential fragments were obtained from sliver-straining 6% polyacrylamide gel, six of which were proved to be developmental related genes by RNA rev-Northern hybridization using [α-32P]dCTP labeled first strand cDNA as probes.
GOU De-Ming, LI Wen-Xin, JIANG Da-He, HUANG Jian. Differential Expression Analysis of the Gene in Human Early Embryos Using a Modified DDRT-PCR[J]. Progress in Biochemistry and Biophysics,2000,27(2):205-209
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