Method based on electrophoresis and gel extraction for obtaining genomic DNA-free cDNA without DNase treatment

摘要

For studying differential gene expression by reverse-transcriptase PCR (RT-PCR) applications, eliminating genomic DNA contamination from the cDNA is essential prior to amplification of the specific gene sequence. Residual genomic DNA can disturb the amplification of the target gene from cDNA and lead to false-positive results. DNase I treatment is generally used to remove genomic DNA from RNA samples. However, RNA can degrade totally or partially during the DNase treatment. It has also been shown that some contamination of genomic DNA can remain after the DNase treatment, even after an overnight incubation (1). Certain tissue types can be a problem because some contain elevated levels of RNases (2,3) or a limited amount of RNA (4). Fruit tissues areregarded as particularly problematic because of high amounts of secondary metabolites, polysaccharides, and elevated levels of RNases (5). In our experiments with RNA from different plant tissues, especially bilberry (Vaccinium myrtillus) fruit and leaf, we found that RNA degraded during the DNase treatment. We also noticed that despite the DNase treatment, some genomic DNA was always left in the samples when RT-PCR gene expression analysis was performed on Scots pine (Pinus sylvestris) needles. To overcome these problems, we developed a method based on electrophoresis and gel-extraction filter tubes.