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Genomic DNA carry over is a fact of life for all RNA extraction methods. This poses a risk to gene quantification experiments because real-Time PCR is sensitive at detecting trace DNA contamination. Unless "intron spanning" primers have been specifically designed during the assay development phase, there is always a risk that any signal obtained may have its origin in the genomic DNA carry over and not in the cDNA. This possibility is usually managed by including an "RT- negative" control. This mock reaction is prepared alongside thin the RT step and includes the RNA template and all other reaction components except the RT enzyme. Any signal obtained from this sample is due to contamination since no cDNA has been transcribed. In order to interpret data with confidence it is important to ensure that the RT-negative control gives a completely negative result. It is therefore usual to DNAse treat RNA to remove trace contamination. The RT-negative control is still required to validate the DNAse treatment and overall the entire procedure is wasteful of time and RNA and which is often limiting in clinical situations.
Principal of the assay
Instead of preparing an RT-negative control, it would be more economical to detect possible genomic DNA directly in the cDNA atualy being used. The requirements for this approach would be an assay that discriminates between cDNA and genomic DNA i.e. an assay that can detect genomic DNA but that gives no signal on cDNA. PrimerDesign has produced an assay that detects genomic DNA in a region of the genome that is not transcriptionally active. No signal is possible from RNA that has been reverse transcribed and carry over DNA can be detected in the cDNA without the need for a separate control for each sample. A further requirement for this approach is the targeting of a single copy locus so that any signal obtained is a reasonable control for the signal obtained from any other single copy gene signal. As well as acting as a replacement for the RT negative control the assay described above is useful for cell counting using Real-time PCR and also for normalisation using the genomic DNA signal.
Supporting Data Fig.1 Human universal RNA is a composite of 10 different cell lines from different tissue types. 1ug was reverse transcribed and 25ng used in a real-time PCR reaction with 18S ribosomal RNA primers. The Ct Value is approximately 10 demonstrating a high concentration of cDNA has successfully been produced.
Fig.1 18S detection of human universal cDNA
Fig.2 The Universal RNA (as above) was tested for genomic DNA contamination in the absence of a DNAse treatment (light blue) and with a DNAse treatment (Purple) prior to reverse transcription. These data show that genomic DNA detection kit is a good control for successful DNAse treatment and also that the assay does not give a positive signal on cDNA.
Fig.2 genomic DNA detection in cDNA
Ordering
The PrimerDesign genomic DNA detecting assays are available for immediate shipment worldwide. Ordering is simple and requires only a purchase order number. Please follow this link to visit the product page.
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