I’m allowed to have an opinion, and I think that people just sound ridiculous when actually saying “hashtag.” Call me old fashioned, but I prefer to save it for the post.
Up until now, I was under the impression that the DIG-labeled probes were the colored component used to stain the slides where pathogen DNA was present, but I was mistaken. The DIG probes are actually synthesized with antigens attached to the DNA backbone. Specifically, the antibodies are from sheep. We then apply sheep serum, which will bind to the antibodies in the backbone. If the pathogen DNA is present on our slides, the DNA will stick, and if it’s not, the DNA will wash off when soaked in a buffer. The antigens in the sheep serum will bind with the antibodies remaining on the slide. Finally, we apply a molecule that will show color when it binds to the antibodies. This makes a lot more sense than what I’d thought before, since it is similar to other antibody-antigen binding assays like ELISA.
I saw some black spots settling over the RLO phage slides after applying the antibody. I’m assuming since I hadn’t added the final incubator solution needed for visualization, it was just ink from the slide labels. If that was actually the color that attaches to the antibodies, then that’s good news for the RLO slides but bad news for our sea star slides, since I didn’t see any black spots.
We needed to make up some probes for the RLO-infecting phage, so it made sense to hold off on running ISH on our sea star probes until we could run the RLO batches with them. The control tube for the probe boiled off again in the thermocycler last night. The lid was closed but there was a tiny crack in the upper part of the tube. Since this is the second time it’s happened, we think that we’ve bought a low-quality batch of tubes that don’t hold up well to being pulled off the 8-tube line into smaller groups. After using some probe for the gel, there is under 1uL of control probe left. We’re hoping that this is enough, since we dilute it at a factor of 1:1000 to bathe the tissues on our slides.
To prep our slides, we washed off all the paraffin with SafeClear, a less toxic alternative to xylene, and then slowly rehydrated the tissues with different proportions of ethanol and water. While not taxing, it took a lot of waiting time. When the tissues were finally hydrated, we had to applied a prehybridization solution to ready the samples for binding with the probe DNA. To save on reagents, we needed to bathe only the parts of the slide containing tissue. We got to use a pap pen to create a hydrophobic boundary around the tissues. Watching the fluid not escape the ovals we’d drawn was very cool, even though it’s not a complicated phenomenon. Finally, we applied our diluted probes directly to the thin slices of tissue and left them to incubate overnight. Fingers crossed the probes bind and we get some visible staining that indicate where they’ve attached.
Listening to the talks at the RCN meeting, I was both surprised and pleased by the many different perspectives they had invited to speak. The topics ranged from diagnosis to forecasting to policy and more. I’m still early in my PhD program and not entirely sure what I want to do after I graduate, so seeing all these avenues that are so diverse and still disease-related and biology-related was very interesting. I’ve always been unsure of what I want my personal involvement in policy-influencing to be. Of course, everyone wants to do something useful, but there is a full spectrum of things to do from conducting experiments that generate the information people need all the way to generating and implementing laws. The policy-oriented talks highlighted how difficult it is to bring together all those people and have them agree upon and formulate recommendations, but it’s also very necessary if any problems are going to get solved. Someone gave me a good piece of advice: at this point in your graduate work, take all the opportunities that you reasonably can commit to because 1) you will learn things that you can apply in ways you don’t expect and 2) you never know what is going to turn into a hot or important issue.
On a side note, Lauren was sitting next to me taking pictures of people in front of their title slides, and she said something about live tweeting the meeting. I wasn’t sure if she was joking or not, but it got me wondering: social media is certainly a tool with exploring for promoting science, but since it is a rather young means of communication, have there been any studies on the quality and quantity of scientific information that people learn from Twitter and other social media platforms? In terms of cost per view, anything free has bang for its buck as long as it isn’t overly time consuming. A professor once mentioned that he sometimes put money in his grant budget for outreach and communication – things like making aquarium displays and costs of running a professional-looking website that distributes a lot of data – to show them that he was serious about outreach and broader impacts. It would be interesting to see how social media stacks up to other forms of science communication.
We reran our probe synthesis PCR to replace the control probes that had evaporated in the thermocycler during the last round. This time we were successful in getting both the DIG- labeled probe and the unlabeled control to show up properly on the gel run. This was my first time actually running a gel on my own, and I learned an important lesson: you must press the start button or nothing happens. It seems pretty obvious, but my assumption that the voltage box started automatically was quite incorrect. The other thing I learned was how to pour the tiny slide gels, and it’s not as daunting as others had made it out to be. If a little gel goes over the slide edge initially but the whole slide is still covered evenly, it’s okay to cut it off with a straight spatula edge once it solidifies.
The gel run showed two bright bands in slightly different positions, which was exactly what we were looking for. The control probe is a little smaller and lighter because it has nothing attached to the DNA backbone, so it travels at a faster rate down the gel and comes out slightly ahead of the labeled probe.
Drew is happy for us getting the kinks worked out of this step, but her hesitation before proceeding is that we have no confirmed pathogen-negative stars to get tissue from (even the asymptomatic ones sometimes show amplification of the putative pathogen’s DNA). To strengthen the argument that this is pathogen DNA binding to the tissue samples, Drew suggests we extract DNA from sea star species that seem to be unaffected by wasting disease and make sure that the pathogen DNA does not amplify in those samples. This would be more convincing that we have presence of pathogen instead of some non-specific binding to something present in the caecum of all sea stars regardless of disease.
We reran our probe synthesis reactions this morning. Comparing reaction conditions of the different kits, we concluded they were essentially the same, and we should continue using our original probe synthesis kit. However, we decided that we should use our PCR product as the template DNA for the probes instead of the DNA extracted from the original tissue samples, the caecum of diseased P. ochraceus. This would give us a better chance of getting a probe after replication, because we were starting with a high concentration of the target sequence and little else. Our original extracted DNA, which we used for the PCR, was still highly concentrated but contained a lot of other sequences from the tissues, effectively diluting our target.
After waiting eagerly for the thermocycler to finish replicating our probes, we opened it to find that one of the caps on our controls (unlabeled probes) had not been closed tightly enough, so all the liquid had boiled out. Only a stiff film was left on the bottom which we could not suck up in a pipette. Since we were testing two primer sets, we hoped that the one set that was closed would be a winner and give us bright bands on the gel. This was not the case. We got a faint band for the labeled and unlabeled probe in primer set 1, and a super bright band from the labeled probes in primer set 2 without a control. Ruth and I are happy to finally be successful in generating a probe for the sea star primers, since it means in situ hybridization with our slides is within reach. However, since we don’t have any controls, we cannot use this set for the ISH. It’s easy enough to set up synthesis for another set of probes, but we’ll have to wait to see if the next set produces bright bands as well instead of remaining unlit like our previous efforts. Fingers crossed, we will have our complete set of probes by tomorrow.
As a side note, I really like making the gels on glass slides because they take less agrose, less buffer, and they are super portable if you need to move to a darker room to see things light up. On the downside, they require a very steady hand to load, and if you set it down flat on a glass surface, the surface tension of the wet underside will stick it to the glass and it won’t come off without a major effort.
Our probe synthesis for the sea star ISH doesn’t seem to be working. We tried to synthesize probes two different times, and neither the probe nor the plain dNTP control showed up in the gel. However, a band of DNA shows up for caecum of diseased sea star when we run normal PCR, indicating that something is going wrong between the PCR and the probe synthesis step. Ruth is looking into different reaction kits available to us, tweaking the synthesis reagents, and altering reaction conditions so that maybe we can get a working probe for the sea star slides like we have for Sarah’s coral slides. If this works, it will be really cool to drop our DNA probes onto tissue slides to detect the presence of the pathogen. This step takes lots of time to get right because if you need to tweak conditions, you can only get in about a maximum of 3 runs through the thermocycler in one day, since each probe synthesis attempt takes about 30 min of prep and 2.5 hours of temperature-controlled DNA replication. Theoretically, getting successful probes should be the major bottleneck (or maybe it’s one of many).
We still don’t know for sure whether the pathogen is absent in the tissues of healthy sea stars, since the PCR has shown mixed results and it’s hard to define if sea stars are truly uninfected or just asymptomatic until the right environmental conditions hit, especially now that most sites have infected stars. However, if healthy stars truly have no (or very low) wasting disease pathogen, then I can see ISH being a very interesting addition to diagnostic tools for this disease. A lot of times, ISH requires removing the organism from the field and sacrificing it to collect the tissues. However, since sea stars can regrow their arms, samples could be collected by cutting off a piece of the arm, taking a few slices for slides, and placing the animal back in its habitat (theoretically, if Fish and Wildlife would approve that). Or in the case of pycnopodia, you could just grab one arm firmly and they would probably let you have it. If healthy, the sea star would regrow its arm and continue serving its role as a predator in intertidal and subtidal ecosystems. If it was infected, the loss of an arm may hinder the immune system by producing additional stress. Convserely, according to informal laboratory observations from Drew and Morgan, loss of an arm in pycnopodia may help the immune system through removal of infected tissue.
In the past, taking a small number of animals like this for monitoring disease would not be an issue. However, since many sea star populations are now very low, scientists are hesitant to remove any more animals than necessary.
To run ISH, we need to synthesize probes that will tell us when our DNA sequence of interest has bound to the DNA on slides of our samples, indicating that the pathogen gene (can we say pathogene for short?) is present in the sea star tissues we fixed during the histology process. In the past, probes were only labeled on the end of the PCR-amplified segment, meaning that you needed a lot more of the target DNA present in the sample to have something show up in great enough quantities to visually observe. Now, probes are labeled within the PCR segment, so you can get a signal from much smaller amounts of DNA present.
To make our probe, we followed the procedure for normal PCR gene amplification, except that instead of plain nucleotides, we had some DIG mixed in with our nucleotides, so they would bind with our target DNA as it was synthesized. We needed to make sure that there weren’t already things that caused the same visual appearance as our probes, so we made a batch of controls that had no probes (using just plain nucleotides). If we are successful, the probe will show up when we drop it on the tissue slides and the control will not show up.
We also prepped for deparaffinizing our tissue slides. The paraffin keeps them preserved and stiff so we can look at them whenever we want, but this requires dehydrating all the cells and blocks access to the DNA. To do this, we need to take out the paraffin with xylene, then rehydrate the tissues with a mix of ethanol and water, slowly upping the water content. This process takes over an hour, but it will give us access to the DNA in the tissues so we can drop on the probes and see if they bind.
Dissecting the Sea Star Transcriptome:
Here’s my video for week 3. Enjoy!
It’s nice when you have a lot of people power to accomplish a lot of pippetting at once, but there is a point at which there are too many people in the small lab space to be effective. We found out that number is 4, if you’re not running around trying to fetch things. We were doing a preliminary test for primer binding so that we know we have reliable primers to attach probes for in situ hybridization. If the primers won’t bind, then we don’t want to attach probes because they will wash away when we try to hybridze them with DNA found in tissue samples taken from live organisms. The power of in situ hybridization is that it can detect viable DNA within an organism. Conventional PCR will only detect the presence of DNA, which may or be from a live organism (it could be dead or just within the body of the host organism and not doing anything).
The confusing part was that we have a bunch of different sea star and coral pathogen samples, all of which had primers that needed different temperatures. We did our best to consolidate them into four temperatures within a few degrees of ideal, with the addition of one touchdown PCR temperature cycle. Touchdown PCR is very interesting, since it allows you to work with primers for which you don’t know what the appropriate temperatures should be. It starts high and walks down by half a degree each cycle. The primers should start binding very specifically at its highest possible temperature, and then they bind nonspecifically the lower the temperature gets. However, by the time the temperature gets low enough to allow nonspecific binding, you should hopefully have a lot of specific binding primers that have replicated, multiplying the number of DNA copies enough to swamp out any nonspecific binding. It’s the ideal way to treat your PCR, because the reaction is not at the optimal temperature all the time, but it’s a good way to hedge your bets.
Learning how to program the thermocycler was also a bit of an adventure, but after figuring out how that everything had to be entered in seconds, it got easier, at least for the single block. The divided block was more difficult, since almost every program number we tried to assign was already taken, but in the end we managed it.