I am running an MSc project determining the survivability of DNA in blood stains, and I have been told to use nanodrop for quantification, is this technique more reliable than RT-PCR?
The two assays are very different. qPCR (Real-Time PCR) will measure the quantity of a specific gene or sequence. Nanodrop measures the total amount of nucleic acid based on the absorbance at A260. Note that the nanodrop will also give you a measurement even if the DNA is degraded (free base pairs and fragments also absorb at A260).
Running the DNA out on an agarose gel might also give you an idea of the quality (amount of degradation).
For your purpose, I would think that if you are looking to see if your DNA sample is stable over time/handling, doing serial qPCRs for a few different genes might be a good idea.
J
The two assays are very different. qPCR (Real-Time PCR) will measure the quantity of a specific gene or sequence. Nanodrop measures the total amount of nucleic acid based on the absorbance at A260. Note that the nanodrop will also give you a measurement even if the DNA is degraded (free base pairs and fragments also absorb at A260).
Running the DNA out on an agarose gel might also give you an idea of the quality (amount of degradation).
For your purpose, I would think that if you are looking to see if your DNA sample is stable over time/handling, doing serial qPCRs for a few different genes might be a good idea.
J
I agree with Finn. Nanodrop would tell you the amount of nucleic acid whether its degraded or not. But RT-PCR would tell the quality. Nanodrop for quantification but RT-PCR for information about origin and quality
Hi James,
You should be aware that nanodrops and RT-PCR are used for different purposes. Nanodrops are used to quantify total DNA, RNA or protein in a sample and are often used to quantify the starting total DNA for RT-PCR. RT-PCR is used predominately to quantify changes in expression of single genes.
The Nanodrop is a spectrophotometer for small volumes and is a quick and cheap way of estimating total DNA concentration using absorbance at 260nm. The reliability of quantification is highly dependent on how pure the DNA is. Concentration estimation using this method is influenced by carryover from your DNA extraction reagents, RNA contamination, the amount of DNA in the sample and even the pH of the final buffer. Also the amount of DNA detectable by nano drop is limited to 2ng/ul (according to the manufacturer), but realistically I would be dubious of readings below ~10ng/ul unless the spectral pattern was perfect (never rely simply on the A260/280 ratio).
Real time PCR is more expensive, more time consuming, requires more technical ability and an understanding of the underlying biology of the sample. The last point can't be stated enough! That said, when done correctly, real time PCR is highly sensitive (it can potentially detect as little as one copy of the target sequence). The draw-backs are that it requires good target sequence selection (hence knowledge of the sample biology), stringent primer design and validation, and that the sample is free of contaminates that adversely affect PCR.
In both cases the reliability of quantification is influenced by how well you purify the DNA. RT-PCR has the additional parameters of suitable target selection and primer design to affect reliability of results.
If you're looking at "survivability" you might want to design an experiment to look at integrity as well as amount. For this you'd need to use known fragment size DNA and run "before" and "after" on an agarose gel. The Agilent bioanalyzer is often used to check the integrity of RNA in samples before microarray or RT-PCR. RNA is more labile than DNA, but perhaps you could use this to compare RIN numbers in tandem with your DNA measurements to get an idea of overall nucleic acid degradation.
Just a thought.
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