About the author

Miranda Wade received her B.S. in Biological Science from Colorado State University and her dual PhD in Integrative Biology and Ecology, Evolutionary Biology, and Behavior from Michigan State University. During her time in the Meek Lab at MSU, her work consisted of using ‘omics to address various conservation questions in both a rare desert place facing land-use change and the molecular consequences of microplastics exposure in a model fish species. She is currently the Social Media Editor for the AGA and a PostDoc in the Sin Lab at the University of Hong Kong. For her postdoctoral work, she is exploring the genomic basis of coloration in birds.

---

A tale of twelve tries: an EECG Epilogue

Actually not that many, I just love a good alliteration

Hello world, it is I, a past winner of the EECG here to write an epilogue post. I know many write about the main conclusions from their project(s) that benefitted from this award, but I think I will cover something a bit different. When writing papers and sharing science, we generally only talk about the successes of our projects. However, the reality we all like to try and ignore is that science is often messy and filled with fail after fail. Sometimes, when a scientist is caught within a lab/analysis/fieldwork failure spiral, it can feel really isolating and make one question their place in science. As one who went through all this, I am going to tell you a story about all the times I failed at the lab work for my project before I finally succeeded.

But first, a quick recap of my embarkation:

• I am interested in using molecular tools to quantify the effects humans have on our environment.
• In this case, my project involved exposing fathead minnows to different plastic treatments and using a combination of RNA and methylation sequencing to (hopefully) determine if the plastic treatments had measurable effects.
  •  This work would not have happened without collaboration with the Rochman Lab, based at the University of Toronto.
• At the time of the award, I had already completed the lab work/most of the analysis for the RNAseq (evaluating relative levels of gene expression) portion of this work. I was ready to embark (heh heh) on investigating changes in methylation, a common form of epigenetics, or heritable changes in organisms’ DNA that do not involve changing the actual DNA sequence. Methylation is thought to reduce the expression of genes, but we really don’t know all that it does. And, to literally no one’s surprise, methylation gets (delightfully) weird in plants.

Okay, so the hard part of rearing the minnows and then euthanizing/dissecting them was very much over, so I was hoping (expecting?) that the library preparation would be smooth sailing. This is especially in light of dealing with the relatively wimpy molecule that is RNA for my prior work, which probably gave me some gray hair. Or relieved me of a few (more) brain cells. Or both.

Extracting the DNA from my (surprisingly stinky for such a small amount) tissue samples went well, so I had no reason to prepare for abject failure at the library preparation. I was using one of the pretty common Zymo (who I love, sponsor me??) kits for reduced representation bisulfite sequencing, where you use a cocktail of chemicals to convert and distinguish between methylated and unmethylated cytosines (the ‘C’ in  our favorite nucleotides, AGCT) so you can measure relative methylation levels after sequencing.

The biggest caveat of this method is that it degrades a lot of DNA, so you need to start with 100s of nanograms of DNA per sample to get adequate levels post-conversion (this is kind of a lot compared to some other methods). This entire process takes hours.

Following conversion, I would test my samples to make sure I still had stuff in there, only find that IT WAS NOT THERE. Almost all my DNA had vanished. When I re-quantified to confirm this result, it was the same.

So, like any good scientist, I did the entire process over again with fresh DNA. Same result.

And again. Hello DNA, it’s me, Miranda.

And again. DNA, is this a YOU problem? Is it a me problem?

And again. Why am I having conversations with my samples?

Then maybe I had a moment or 7 where I wondered if I was even going to be a real scientist (spoiler alert, I already was a real scientist at that point, thankyouverymuch) and if this PhD was actually going to ruin me (it didn’t, I think? 90% sure I am f_i_n_e ).

And how much money was all this stuff again? YIKES

Oh no another failure. Weeeeeeeee

At that point I was running out of DNA to work with, so I went back to the starting point and decided to test the lab equipment, because while generally I’m the problem (it’s me) this was getting out of hand. At this point some other DNA library preparation work in the lab was failing, which while unfortunate also made me feel better that maybe I wasn’t just a failure (I know, not very altruistic of me).

And, wouldn’t you know, the microplate reader (a machine we use to tell us how much DNA is in a plate full of samples) was off by almost a factor of TEN. Which meant when I thought I was starting with 500ng of DNA, I was only using a fraction of that, which was then all being degraded during the bisulfite conversion process. Hence my vanishing DNA.

According to the company who manufactured it, our machine was not fixable so we needed a new one. And how much is a new microplate reader? Don’t ask. No, really, you don’t want to know.

But anyways, long story short, I was able to use the last of my DNA and a kit from NEB (who I also love, sponsor me??) that can use lower DNA inputs and successfully sequenced my minnows. And determined if there was differential methylation between my treatments/generations (spoiler alert, there was).

Now I am happy to say that I have finished this project and it is officially accepted for publication in the Journal of Heredity.

References

Lucibelli F, Valoroso MC, Aceto S. Plant DNA Methylation: An Epigenetic Mark in Development, Environmental Interactions, and Evolution. International Journal of Molecular Sciences. 2022; 23(15):8299. https://doi.org/10.3390/ijms23158299

Mattei AL, Bailly N, Meissner A. DNA methylation: a historical perspective. Trends in Genetics. 2022; 38(7):676-707. https://doi.org/10.1016/j.tig.2022.03.010

Wade MJ, Bucci K, Rochman CM, Meek MH. Microplastic exposure is associated with epigenomic effects in the model organism Pimephales promelas (fathead minnow). 2024; esae027, https://doi.org/10.1093/jhered/esae027