Inter assay variation in global methylation Elisa - (Feb/15/2011 )
Hello everyone,
I have just started to compare the global methylation between patients and healthy controls using the MethylFlash Elisa by Epigentek. The analyses were performed according to the manufacturer’s instructions, but we detected a very high rate of inter-assay variations (over 200%) with partially conflicting results. Some values of the first Elisa were increased, others were decrease in the second elisa.
Furthermore it seems that the samples from the first half of the plate showed lower ODs than those from the second half. Maybe this might be an effect of pipetting time or pipetting accuracy. I have contacted the company, but they haven't an explanation yet.
Has anybody here the same or comparable experiences concerning global methylation Elisas or some ideas to solve this problem?
Thanks
Taqman
Taqman on Tue Feb 15 12:22:28 2011 said:
Hello everyone,
I have just started to compare the global methylation between patients and healthy controls using the MethylFlash Elisa by Epigentek. The analyses were performed according to the manufacturer’s instructions, but we detected a very high rate of inter-assay variations (over 200%) with partially conflicting results. Some values of the first Elisa were increased, others were decrease in the second elisa.
Furthermore it seems that the samples from the first half of the plate showed lower ODs than those from the second half. Maybe this might be an effect of pipetting time or pipetting accuracy. I have contacted the company, but they haven't an explanation yet.
Has anybody here the same or comparable experiences concerning global methylation Elisas or some ideas to solve this problem?
Thanks
Taqman
The info you have here is kind of vague. Did you set up a standard curve or were you just comparing differences between two samples only? What kind of values did you get in your positive control and blank wells? When you say inter-assay variations do you mean differences between sample wells in the same kit/microplate? If so, did you use a multi channel pipette? Or did you pipette one well at a time? You'll need to make sure you pipette the development solution to the sample wells in the same order as when you added all the other reagents.
I have the same exact problem. I just don't understand what's going wrong. Poor intraassay reproducibility. But his only happens with the epigentek kit. Tipical ELISAs show little CV, nevertheless. Pipetting is donew with a multi channel, and in the same order, with controlled timings. Any idea what else could be happening?