Rd. Smith et al., Exclusive amplification of cDNA template (EXACT) RT-PCR to avoid amplifying contaminating genomic pseudogenes, BIOTECHNIQU, 31(4), 2001, pp. 776
Genomic DNA contamination within RNA samples has important implications for
RT-PCR, particularly if there is a pseudogene related to the gene under in
vestigation, because amplification from pseudogenes and reverse-transcribed
cDNA can be very difficult to distinguish. Methods to remove DNA contamina
tion 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 D
NA and uses this for annealing the reverse PCR primer to exclude genomic DN
A amplification in unmodified RNA samples.