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Mcfly*31

Member Since 10 Jun 2012
Offline Last Active Jun 11 2012 02:47 PM
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Topics I've Started

help with qPCR plates interassay validation

10 June 2012 - 10:47 PM

I have an issue. I have ran a few plates and am now concerned with whether or not they are good. I have 6 genes to study using 2 reference genes; I have 4 tissue types and 9 different stages of development. I chose to setup so that I could run one gene per tissue type for all but one developmental stage with 5 biological replicates per; so 40 samples per tissue type. We use a 96 well qpcr I set up my plates so that a plate contains a 5 pt standard curve in triplicate; NTC and two tissue types (so 96 rxns) The issue is I ran my technical replicates as new plates. ie for beta actin: brain and kidney plate one has 80 unique samples with a standard curve; Plate 2 and 3 are replicates of plate one. Can I normalize interassay variation here using my standard curve sample here? I have a feeling, called hope, that I can but may have needlessly made more work for myself. Also; for my other genes; do I need to run the same standard curve samples to afford interassay variation? assuming I do but then this would reduce well space as I would have do run a calibrator sample for the gene two correct? any help is appreciated.

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