Exclusive amplification of cDNA template (EXACT) RT-PCR to avoid amplifying contaminating genomic pseudogenes.
Genomic DNA contamination within RNA samples has important implications for RT-PCR, particularly if there is a pseudogene related to the gene under investigation, because amplification from pseudogenes and reverse-transcribed cDNA can be very difficult to distinguish. Methods to remove DNA contamination cannot guarantee the absolute absence of DNA from the sample without a loss of RNA quantity or quality, which can be crucial for small amounts of RNA or for the investigation of transcripts with a low level of expression. Here, we describe a general technique for RT-PCR that applies a sequence to the 5' tail of reverse-transcribed cDNA that is not present in genomic DNA and uses this for annealing the reverse PCR primer to exclude genomic DNA amplification in unmodified RNA samples.
Smith
R D
RD
Imperial College of Science, Technology and Medicine, London, UK. daron.smith@ic.ac.uk
Ogden
C W
CW
Penny
M A
MA
D001483:Base Sequence; D001709:Biotechnology; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D005260:Female; D005784:Gene Amplification; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D008297:Male; D011544:Pseudogenes; D012334:RNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D018076:DNA, Complementary; D012334:RNA, Neoplasm; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases