To minimize the effect of DNA contamination in RNA template, the forward and reverse primers can be designed from different exons and to span exon-intron boundaries. In this way one can reduce the genomic DNA contribution to expression quantitation. This design strategy is most important for accurate quantitation of low-expressing genes or genes with many loci in the genome. However, in general, DNA contamination has minimal effect on real-time PCR because a typical transcript copy number is much higher than the number of gene loci and standard RNA preparation procedures remove genomic DNA efficiently. In any case, this strategy may fail if pseudogenes of the target gene are present in the genome.
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