I am currently running an experiment for my MSc dissertation, where i am testing the survivability of DNA in blood stained cotton, submerged in various types of water over varying time periods. The samples that have been extracted were submerged in water for one day, i did use a control where i added blood to the fabric but no water, after extraction i quantified on nano drop and of my five samples the stain which was not submerged in water had the most DNA, which i thought to be obvious, but when i further quantified on RT-PCR the it was quite the opposite, the stain which had not been submerged had the least DNA, it went from over 400ng/ul on nanodrop to 52ng/ul on RT-PCR, i ran the sample again on nanodrop to see if the first had been a mistake, but it again came back as over400ng/ul, the other samples were quite consistent between the nanodrop and RT, with obvious slight variances, but that one result i dont understand, especially as it was the one that was not in water...
Any ideas would be much appreciated
James McCormack
Thank you... But I did the nano drop readings before PCR, so there were no primers or PCR reaction mix in with the sample at that time. I did phenol/chloroform extraction, the extracted DNA samples were then stored over night in the fridge in TE buffer. I then put 1ul of that solution onto the nano drop, before I prepped them for RT-PCR...
thanks
James
I have a question: how did you determine the concentration of RNA using RT-PCR?
Possible reasons for the observed discepancies:
- possible presence of PCR inhibitors (check by dilution series)
- possible presence of contaminants influencing the OD measurement (like residing phenole or other things) (check the whole OD spectrum, don't just take the OD260 reading "as given")
- possible degradation of the DNA template (possibly check using a Bioanalyzer or a simple agarose gel [given you have large enough amounts of DNA]), also check the primers (primers producing long PCR products have problems with degraded DNA)
When you measure the eluted DNA with nanodrop you quantify all nucleic acid. In the RT-PCR (you mean Realtime and not ReverseTranscription?) you quantify a special PCR product (mostly a single copy gene with a standard in parallel). Perhaps you should check first whether the discrepancy remains after using another gene?
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