Distribution and cloning of eukaryotic mRNAs by means of differential display: refinements and optimization.
Differential display has been developed as a tool to detect and characterize altered gene expression in eukaryotic cells. The basic principle is to systematically amplify messenger RNAs and then distribute their 3' termini on a denaturing polyacrylamide gel. Here we provide methodological details and examine in depth the specificity, sensitivity and reproducibility of the method. We show that the number of anchored oligo-dT primers can be reduced from twelve to four that are degenerate at the penultimate base from the 3' end. We also demonstrate that using optimized conditions described here, multiple RNA samples from related cells can be displayed simultaneously. Therefore process-specific rather than cell-specific genes could be more accurately identified. These results enable further streamlining of the technique and make it readily applicable to a broad spectrum of biological systems.
Liang
P
P
Division of Cell Growth and Regulation, Dana-Farber Cancer Institute, Boston, MA.
Averboukh
L
L
Pardee
A B
AB
D000818:Animals; D001483:Base Sequence; D001943:Breast Neoplasms; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D004847:Epithelial Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D015203:Reproducibility of Results; D014407:Tumor Cells, Cultured
D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger