Hi,
I'm trying to analyze the expression changes of some genes following under stress conditions or plasmid transfection, using real-time PCR. After finding the best housekeeping candidate using Best-Keeper software, I performed the real-time PCR in triplicate using templates obtained from three independent experiments. The problem was that the fold-change values of some samples were significantly different than the ones belonging to other independent repeats. What can be the reason of such a problem?
How consistent was your sample collection and preparation? Did you normalize the amount of input RNA in all of your RT reactions?
Low abundance mRNAs that are expressed have more variability than mRNAs that are usually expressed in greater abundance. You may need to run more than triplicates for your three independent samples in order to reduce the variability.
Here's a link to another discussion measuring accurate fold change in RT-PCR samples. Maybe there will be additional suggestions that will help you. https://www.researchgate.net/post/How_small_of_a_fold-change_in_gene_expression_can_be_reliably_measured_by_RT-qPCR
Hi Eray,
Many factors can contribute to differences in expression levels when comparing independent experiments. Even if you were able to reproduce the experiments exactly, which you absolutely cannot given the nature of variation, there are still many things that can contribute to changes in the levels. (1) Variation in the amount of total RNA isolated, (2) differences in the quality of RNA isolated (3) variations in RNA degradation (4) manual pipeting errors, and as Jordan mentioned, (5) failing to normalize the amount of input RNA in each replicate reaction. There are more but those are 5 off the top of my head.
Did you run the RNA's from the 3 individual experiments at different times, or were they done at the same time? Ideally, you would want to collect all your RNA samples, normalize so that you are adding the exact same amount of RNA to each reaction, and then perform triplicate reactions on each experiment at that same time. Also, you will want to make a master mix for each experiment and aliquot that 3 times to make your replicates. Another thing you may want to try, if you haven't, is analytical pipetting vs basic laboratory pipetting. Wipe your tips, set your pipetters "to deliver" etc.
Good luck!
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