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1. #How to use graphpad prism for real time pcr how to
2. #How to use graphpad prism for real time pcr software

#How to use graphpad prism for real time pcr how to

There is a recent VanDeSomple paper on how to concatenate multiplate data, but in my opinion, it's kinda cray. As long as you are clear about that in your pubs, I think it's kosher.

You lose some error structure there, but in my opinion, it's better than that artificially inflated massive error you get when comparing Ct values directly. I was using the Pfaffl equation (better than ddCT, I think) and I modified it to NOT pool Ct values (because of the wide swings in Ct values between plates) but instead statistically compare NRQ values (or dCT if that's what you're doing) after they have been adjusted by refgenes/controls. So I prefer handling more genes/controls on a plate for fewer samples, rather than splitting samples across plates. As does a reference sample (ie, a time zero control). In my opinion, whatever you use to 'calibrate' the samples (reference gene, standard curve for quantity) it HAS to be done on the same plate.

I also have huge variations of Cts between runs. Thank you for any input! I've tried to keep it short but I can elaborate if necessary. I'm trying to parse statistical papers and explanations but I'm a bit stupid, and english isn't my first language. Should I use the delta CT SD perhaps, instead of SE? Should I use the deltaCT value instead? Or DDCT even? The results differ in the statistical analysis.

#How to use graphpad prism for real time pcr software

At this stage I've omitted wells that the StepOne software deem are outliers. Is there a simple, practical guide to point me in the right direction? Basically "put this value in this table, press this button" kinda guide.įor now, I'm using the RQ values from StepOne and putting them in a grouped table with mean (RQ), SEM (delta CT SE) and N. I use StepOne Software and GraphPad Prism 6 through my lab. I did both biological and technical replicates and I'm now at the stage were I'm supposed to analyze the data. I've done qPCR of a couple of genes in a cell line with different experimental conditions (receptor k/o, receptor wt, three different mutants of same receptor - all cell lines treated and untreated with ligand). I've basically no access to my supervisor and I'm a bit stuck in schoolwork. Sorry for a, I'm sure, frequent and rather annoying question.