A frequent cause of concern among investigators performing quantitative RT-PCR is inaccurate data due to DNA contamination in RNA preparations. Although DNA contamination is easily detected by performing a "no-RT" control, there is no easy remedy. In this technical bulletin, we present data showing levels of DNA contamination in RNA generated by different procedures, and suggest several precautionary measures that can be implemented to reduce the impact of this persistent problem.
RT-PCR and Genomic Contamination
RT-PCR is an increasingly popular method for the quantitative analysis of gene expression. With this popularity comes a heightened awareness that most techniques used for total RNA isolation yield RNA with significant amounts of genomic DNA contamination. PCR cannot discriminate between cDNA targets synthesized by reverse transcription and genomic DNA contamination. At Ambion, we can routinely perform PCR from residual genomic DNA present in total RNA samples isolated by most commonly used techniques. To illustrate this problem, we performed RT-PCR on mouse liver RNA isolated by a multi-step guanidinium thiocyanate/acid phenol:chloroform extraction (ToTALLY RNA
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