Hi Danae!

If 5 ant pupae are from the same treatment group, they can be mixed together for DNA extraction. This way, the differences in gut microbiota of ant pupae under different treatment conditions can be compared.

I hope this helps, and feel free to reach out for more details anytime.

Hello Lucian Miles

Thank you for your answer. However, I am wondering if I pool 8 pupae from sample 1, 5 from sample 2 and 10 from sample 3, could it potentially affect my results? Since I normalise the CT values with my housekeeping gene, I think it should be alright. But perhaps having different sample size (i.e number of pooled pupae) would add variations that the normalisation does not target.

Do you understand what I mean?

I completely understand your concern. You’re wondering whether pooling different numbers of pupae in each sample might introduce some variation that housekeeping gene normalization may not fully correct, right?

Generally, this shouldn’t have a significant impact, as long as you carefully dilute the extracted DNA to ensure that all samples are brought to a comparable concentration. Later, during the qPCR process, there will be additional dilution steps, which further help minimize any minor differences in concentration within the same range.

I hope this helps! If you have any other questions, I’d be happy to discuss further. Feel free to reach out anytime.