The use of RT-PCR to distinguish between plasmid MnSOD transcripts and endogenous MnSOD mRNA.
We report here a convenient RT-PCR method to distinguish plasmid human MnSOD cDNA transcripts from the endogenous MnSOD gene products without engineering the cDNA insert. When a specific antisense primer for the carrier vector sequence was paired with a sense primer for the human MnSOD cDNA in RT-PCR analysis, a unique amplicon with the expected size was generated in MnSOD cDNA transfected cells but not in the wild type or vector control cells. The same primers were also used in genomic DNA-PCR to demonstrate genomic incorporation of cDNA in stably transfected cells. This method is convenient and specific in determining exogenous cDNA incorporation and expression in transfectants especially when transcripts of cDNA are difficult to separate from the endogenous mRNA by other methods.
D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D001943:Breast Neoplasms; D002460:Cell Line; D017931:DNA Primers; D018076:DNA, Complementary / Q000032:analysis; D005260:Female; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis* / Q000096:biosynthesis; D012194:RNA-Directed DNA Polymerase; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis*; D013482:Superoxide Dismutase / Q000032:analysis / Q000096:biosynthesis*; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D018076:DNA, Complementary; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013482:Superoxide Dismutase; D012194:RNA-Directed DNA Polymerase