qpcr and incucyte anova help
Hello have a question regarding which statistical analysis is most appropriate for my RT-qPCR experiment.
For the experiment, I seeded a murine macrophage cell line into 12-well plates. Within each plate, I stimulated two wells with medium only (M0), two wells with LPS (M1 polarization), and two wells with IL-4/IL-13 (M2 polarization).
To investigate the time-dependent expression of macrophage markers, I treated separate plates for 6, 12, 24, and 48 hours. The entire experiment was performed using three independent biological replicates, corresponding to three different cell passages (passages 151, 156, and 157). After RNA isolation and RT-qPCR, I calculated the relative gene expression for each sample and generated time-course graphs.
My objective is to compare M0, M1, and M2 at each individual time point (6 h, 12 h, 24 h, and 48 h), rather than to statistically compare the different time points with each other. Therefore, I planned to perform a one-way ANOVA separately for each time point, followed by Tukey's multiple-comparison test.
However, my supervisor suggested using an ordinary one-way ANOVA because the three biological replicates are independent. Her reasoning is that each biological replicate (each cell passage) represents an independent experiment and is measured only once under each treatment condition.
My question is therefore:
Would an ordinary one-way ANOVA or a one-way repeated-measures ANOVA be the more appropriate statistical test for this experimental design?
The biological replicates are independent cell passages, but within each passage all three treatment conditions (M0, M1, and M2) were measured under identical experimental conditions. I have also attached a schematic of the experimental design.
I would appreciate your opinion on which statistical approach is most appropriate and why.
In addition to the RT-qPCR experiments, I also performed IncuCyte live-cell imaging experiments. For these experiments, the cells were seeded into 96-well plates and exposed to different treatments, including M0, M1, and M2 conditions. Each condition was represented by technical replicate wells.
In contrast to the RT-qPCR experiment, the same wells were repeatedly imaged and analysed at several time points over a period of 48 hours. Therefore, measurements obtained from the same well at different time points were not independent.
For the analysis of the complete time course, I would therefore consider a two-way repeated-measures ANOVA, with treatment and time as the two factors and time as the repeated-measures factor. This analysis would allow me to assess the effects of treatment, time, and the interaction between treatment and time.
If I compare the treatments only at one selected time point, such as 48 hours, I would instead use a one-way ANOVA. Depending on whether the treatment values from the independent experiments are matched by cell passage or experimental run, this could be either an ordinary one-way ANOVA or a one-way repeated-measures ANOVA.
thank for your help
Posted Jul 29, 2026, 6:38 AM UTC