Trials and Tribulations: first month of H. pylori research

The process of scientific research is never as smooth and linear as one would hope. What starts out as a simple and straightforward experimental setup can be plagued with errors and endless troubleshooting. I began this summer’s research with the goal of determining whether acetone is the environmental stimulus that triggers the activation of the CrdRS signal transduction pathway in H. pylori (HP). The experimental design is simple: I grow up some HP cells in blood agar plates, pass them to SFBB culture after 24 hours, treat one group with acetone, extract RNA from the resulting cell pellets, turn the RNA into cDNA, and run a qRT-PCR with the cDNA samples. I was hoping to get some sort of data by the end of the first week. But such was not the case in reality.

During the first week of research, I used some HP J99 wild-type (WT) cells to test a new method of qRT-PCR called the TaqMan Assay. Before this summer, I’ve been trying to do qRT-PCR with SYBR-GREEN, which has been a hot mess. After two semesters of unreliable data, my principal investigator (PI), Dr. Forsyth, decided to switch to TaqMan, which is more expensive than SYBR-GREEN but yields better results. So I ran the HP J99WT cells with an endogenous control (ftsZ) and a reporter gene (acxA), as well as a negative control. The results were encouraging. The amplification plot was clean. The Ct values of the technical triplicates were close to each other and there was no amplification in the negative controls. My PI gave the okay to proceed with the acetone treatment experiment. I calculated the volume of acetone needed to add to the experimental samples. I also grew up more WT cells. Everything was in order. But then the unthinkable happened: we ran out of the plates needed for RNA extraction! It was really frustrating because ordering new plates take at least a week, and the HP cells had to be fresh so I had to regrow the cells once the plates come in. It doesn’t sound that bad until you realize that growing the cells is a 4-day process and requires careful planning of your time. What little momentum I had was completely gone. Two weeks gone by: zero data.

After the precious plates finally came in, I wasted no time and ran 8 samples. My PI and I revised the growing protocol to include a time series experiment with acetone. Basically, I take cell pellets from the broth culture after 12 hours of liquid growth and set that time as T= 0. Then I add acetone to the experimental group and purified water to the control group and then put them back into the CO2 incubator. I then take both flasks out every 30 minutes to take another set of cell pellets for 90 minutes. The purpose of this was to test the effect of acetone on HP J99WT over time. By the end I have 8 samples: 4 controls (C0, C30, C60, C90) and 4 experimentals (E0, E30, E60, E90). The nanodrop reading, which tests the concentration and purity of RNA, of the 8 samples was not good. The concentration of most samples was too low to be used for cDNA synthesis, and some samples showed contamination with other nucleic acid and/or proteins. I thought this might be due to the samples being in room temperature for too long, which might have caused the RNA to degrade. RNA is like a premature baby with an autoimmune disorder. If it’s not in the perfect conditions, it dies really fast. I extracted RNA from 8 new samples, and this time the concentrations and purity were good, but only for 4 samples. The T=60 & T=90 samples had little to no RNA in there. I didn’t know what happened, but I decided to proceed to cDNA synthesis and qRT-PCR. The results were not good at all, which I had expected. I wanted to try it again, but the plague of no-lab-supplies struck again. This time I ran out of the beads I needed to extract RNA. You’d think I would check to make sure to restock the supplies I need, but the thing is most of my experiment has to be done in the lab technician’s lab. So I assumed that they would restock the supplies in their lab. Nope, it was my responsibility. So I asked my PI to order more beads. Three weeks gone by: some data, but nothing usable.

While I was waiting for the beads to arrive, I talked to my PI about the inconsistent nanodrop data. We decided to further refine the growing technique, so that before the cells are passed to SFBB culture we calculate how much cells to be pipetted into each flask. This will ensure uniformity of cell count across different experiments. Usually after the 12-hour liquid growth, I perform an optical density (OD) reading to determine how much cells are in the liquid culture. The OD readings for the past two trials have been over 2.0, which is too high. I set up another time series experiment to test the optimal growing time for HP J99WT. After I passed the cells to liquid growth, I let them grow for 4 hours and then take an OD reading each hour after that until the reading reaches 0.8. At that point I start the time series experiment with acetone. I determined that the optimal growing time is 9 hours, instead of 12. These changes improved the nanodrop readings drastically. After the beads came in, I performed my first qRT-PCR with all 8 samples. The results were beautiful! I was ecstatic that the experiment finally worked!! It took me 4 weeks, but the project is finally up and running!

The first qRT-PCR run showed higher acxA gene expression in the experimental groups than in the control groups, which is what i hoped for. But the difference was no significant enough to support the hypothesis. So I ran the same experiment again. I also halved the amount of cDNA used in this run, per advise of the lab technician. This time the gene expression varied in different groups, but the difference was still not big enough. At this point, my PI and I agreed that acetone is most likely not the environmental stimulus that triggers the CrdRS pathway in H. pylori. But I ran a third experiment with the same conditions again to get biological triplicates. And again the gene expression levels varied, but not by a significant amount. This confirms our suspicions that acetone is not what we’re looking for. Next I will test other ketone bodies such as hydrogen peroxide. I have also grown HP strains with the crdR gene knocked out, the complement strain, and the complement control strain. So I will also test the acxA gene expression level differences among the 4 strains. Although my hypothesis that acetone is the environmental stimulus that triggers CrdRS pathway in HP is incorrect, I’m very happy that the TaqMan assay works well and I’m looking forward to testing other environmental stimuli in the future.

 

 

Comments

  1. nvhudsmith says:

    I love how you wrote this post — lab frustrations always seem to crop up in the most mundane places (supplies, in my opinion, being the most disappointing way for something to go horribly wrong.) I’ve also found that much of full-time summer research is figuring out logistics. It sounds like things will get easier from here on though, and that you’ve broken through all the little hiccups that you don’t always expect. Good luck!