Rapid identification of antisense mRNA-expressing clones using strand-specific RT-PCR.
Transfection of full-length antisense cDNA is used frequently to achieve stable downregulation of gene expression. However, screening for clones that express the antisense mRNA is complicated by the presence of endogenous sense mRNA. Thus, clones usually are screened for downregulation of the target protein by Western blotting, which can be time consuming. Here, we used strand-specific RT-PCR to identify antisense-expressing clones, which can then be screened for protein downregulation. This approach allows earlier identification of potentially useful clones and cuts down on the number of clones to be screened by Western blotting.
Cummings
Michele
M
Prostate Cancer Research Centre, University College London, London W1W 7EJ, England. m.cummings@ucl.ac.uk
McGurk
Claire
C
Masters
John R
JR
D015153:Blotting, Western; D045744:Cell Line, Tumor; D018076:DNA, Complementary / Q000378:metabolism; D015536:Down-Regulation; D006801:Humans; D016376:Oligonucleotides, Antisense / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013997:Time Factors; D014162:Transfection
D018076:DNA, Complementary; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger