High-quality RNA for RT-PCR using the Absolutely RNA RT-PCR miniprep kit
Karen Dolter Jeff Braman
Stratagene has determined that standard RNA purification kits are not satisfactory for reverse transcribed PCR (RT-PCR) applications. We tested several commercially available RNA purification kits and have shown that contaminating DNA will produce false positive amplicons during RT-PCR. The Absolutely RNA RT-PCR miniprep kit eliminates contaminating DNA and produces highly pure total RNA qualified for RT-PCR reactions from 5 to 40 mg of tissue and 10 5 to 10 7 cultured cells. The Absolutely RNA RT-PCR miniprep kit utilizes a solid-phase DNase treatment to reduce DNA contamination to levels undetectable by PCR.
RT-PCR is an important technique for gene discovery and gene expression analysis because it amplifies expressed RNA sequences.1 RT-PCR is very sensitive; it facilitates the investigation of rare mRNA transcripts that are difficult to detect by other means in studies in which the quantity of starting material is limited. High yield of RNA is important because diminished or biased recovery may result in the loss of rare mRNA. Extreme purity is critical because contaminating DNA present in the RNA sample can give rise to amplification products that mimic the amplification product expected from the RNA target. The Absolutely RNA RT-PCR miniprep kit eliminates these problems by providing high yields of total RNA with undetectable levels of DNA from animal tissues and cultured cells.2