I was wondering which positive control should be used in a qRT-PCT to detect Alphavirus in mosquito samples. Is it valid to use RNA that was in vitro transcribed from a plasmid with the sequence of interest? Or is it best to use a virus culture? Or a positive mosquito sample with the virus of interest? Where can I find literature about this? I need to justify the use of the RNA. I'm working in a Biosafety Level 2 Laboratory, so I can only use this. Thank you!
Hi - I would always use the closest that you can to The Real Thing, particularly if you want to have any hope of quantification.
So - use the plasmid - you can be sure of the purity (rather than the random nature of infected cells) and you will always know the number of moles of target in your reaction.
Good Luck
G
I think plasmid will be the best positive control. Apart from purity one can use serial dilution and plot a standard curve for further quantification of test sample. One can easily get mols/lit or mg/ml type of calculation done provided you correct for no of bases
The following articles might be useful:
http://www.ncbi.nlm.nih.gov/pubmed/16912918
http://www.nature.com/leu/journal/v15/n7/full/2402133a.html
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1360278/
http://www.ncbi.nlm.nih.gov/pubmed/17827371
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