The Dirt on Deep Sequencing- My Experience with the Revolutionary Technique

Temperatures hit a record high this weekend in Chicago. With the mercury rising in my apartment, fans monopolized every outlet and my windows gaped open at all hours. Travelers and tourists in the Loop dabbed at beads of sweat hanging on their foreheads, their cheeks flushed and bare shoulders browning in the sun. On the other side of the Windy City, imagine opening the door to Loyola’s Center for Translational Research and Education and being greeted by a rush of A/C from our LEED-certified building. It chills you to your core. That’s what introducing deep sequencing into my dissertation project felt like. Absolutely refreshing.

I’ve been using deep sequencing, also known as next-generation sequencing or second-generation sequencing, to make sense of the biologically complex changes occurring in cardioprotection. Deep sequencing is a technique that allows scientists to determine the quantity and identity of nucleotide sequences in a biological sample. I hypothesized that there were measurable changes in the left ventricular transcriptome following electrically-induced cardioprotection. RNA-sequencing was applied to quantify the relative abundance and differential expression of genes after the stimulus. There is ample literature offering a detailed overview of the methods and workflow, which I referenced frequently throughout the project.1,2 I also received a lot of help from the very skilled bioinformatics experts at Loyola Genomics Core and their collaborators, who completed the sequencing and analysis. Herein, I’m offering an honest reflection on my first experience with RNA-sequencing. It’s been exciting and satisfying to work through these challenges:

  • Challenge #1: Starting out strong. Because of the sensitivity of the technique, any confounding variables in the sample collection and RNA isolation steps must be meticulously controlled for.
    • Solution: The subjects were sex- and age-matched, treatments were administered on the same day and time, and tissues were collected identically. The RNA was of high quality and abundance before it could be processed (this took a couple of tries). A dedicated RNA work station was used to shield my samples from RNAses, and we limited freezing and thawing whenever possible.
  • Challenge #2: Making sense of results afterwards. Large data sets need to be properly analyzed and interpreted before substantial conclusions can be drawn.
    • Solution: There are a number of sophisticated computation tools that we used with our Core to analyze the genes and pathways that changed between conditions. I chose to focus on genes that had high base read counts, high fold changes between the sham and treatment, and a known role in cardioprotection. I validated fold changes with RT-qPCR before utilizing pharmacological inhibitors or knock-out mouse models to determine the functional role of those genes as mediators of cardioprotection.
  • Challenge #3: Time and money throughout. Sequencing N=3-4 in 2 groups had a 4-8 week turnaround and cost us several thousand dollars to complete.
    • Solution: Well, there isn’t an easy one here. I can, however, advocate for patience with the process and confidence in the product. It appears that as technologies continue advancing and more investigators are using this technique, speed is increasing and prices are dropping globally.

RNA-sequencing has revolutionized my dissertation project. I began with a small number of RNA samples and ended with a list of potential gene candidates and a burning curiosity about their functions. The findings are driving my current research pursuits and helping me to answer our biggest questions about the molecular mechanisms of electrically-induced cardioprotection.

the dirt on deep sequencing - tips and tricks from my PhD


  1. Goldman, D., & Domschke, K. (2014). Making sense of deep sequencing. The International Journal of Neuropsychopharmacology, 17(10), 1717–1725. http://doi.org/10.1017/S1461145714000789
  2. Han, Y., Gao, S., Muegge, K., Zhang, W., & Zhou, B. (2015). Advanced Applications of RNA Sequencing and Challenges. Bioinformatics and Biology Insights, 9(Suppl 1), 29–46. http://doi.org/10.4137/BBI.S28991

Annie Roessler Headshot

Annie Roessler is a PhD Candidate at Loyola University in Chicago, IL. Her research focuses on the neurobiology and molecular mechanisms of electrically-induced cardioprotection. She tweets @ThePilotStudy and blogs at flaskhalffull.com


We Should All Be Pharmacologists.

The setting is every wedding, reunion, or family gathering in the Spring of 2018, and I’m in a circle of distant relatives or friends-of-friends, clarifying the distinction between a pharmacologist and a pharmacist. If you want to be the life of the party at this summer’s neighborhood BBQ, explain to the stranger next to you in the buffet line why you’re not “that kind of doctor,” and yet your career requires over two decades of schooling.

Readers of the Early Career Voices column are a different audience – students, clinicians, and public health specialists – possessing a much better grasp of the pharmacologist’s day-to-day than my great aunt or high school classmate. Most AHA professionals even had a pharmacology course or two at some point in their training. I’m here to convince you, fellow Early Career Professionals, that regardless of what profession you put on your tax forms last month, we should all be pharmacologists.

Plug in “pharmacologist” on your favorite job-search board. You’ll find that pharmacologists influence every aspect of a drug’s journey from the lab bench to the patients. In academia and industry, pharmacologists work in multidisciplinary and collaborative teams as experts in drug absorption, distribution throughout the body, metabolism, and elimination. They understand disease states and collect, interpret, present, and explain data on a drug’s action at all levels.  A “drug” can encompass everything from a small-molecule inhibitor that has been synthesized in the lab to biologics and immunotherapeutics.

In my current position as a PhD candidate in basic cardiovascular sciences, I’m routinely utilizing pharmacological tools to accomplish my research goals. If I hypothesize that a protein of interest is critical to cardioprotection, I use a drug to inhibit that protein’s function and determine whether the cardioprotective effect persists in its absence. You’ll see me present this sort of work at AHA’s Scientific Sessions, when we all come together from our various career paths to talk about cardiovascular disease.

What should you consider when standing in front of a pharmacologist’s poster at Sessions? The same sorts of questions you should ask anytime throughout the year when reading a research article, listening to a seminar, or designing experiments that involve drugs. A few suggestions from a real life-pharmacologist:

  • The chemical nature of the drug. What is the mechanism of action? Does it get past the blood brain barrier? How long does it last in the body?
  • Frequency, timing, and route of administration. How often and in what way should the drug be given based on its known kinetics?
  • Dose (mg/kg). What does gives you the best therapeutic or on-target effects and minimizes side effects?
  • Specificity and efficacy. Does the drug work in the way that you expect it to, and how well does it do so?
  • Safety and drug-drug interactions. Is there a risk of adverse reactions, and does the drug play nice with any other drugs in the system?

The answers to these questions are extraordinarily valuable to biomedical scientists, but can be just as helpful to professionals throughout the AHA. Pharmacology is deeply integrated into many of the core cardiovascular disciplines; by strengthening our pharmacology skillset, familiarizing ourselves with our drug toolbox, and considering how pharmacologists work, we can better understand and critique developments across the cardiovascular field.

Annie Roessler Headshot

Annie Roessler is a PhD Candidate at Loyola University in Chicago, IL. Her research focuses on the neurobiology and molecular mechanisms of electrically-induced cardioprotection. She tweets @ThePilotStudy and blogs at flaskhalffull.com


Why I Study Cardioprotection

That question.

It’s always looming. It comes from the tenured professor at the back of the room during my annual progress report to the department. It comes from the clinician from another field who pauses in front of my AHA Sessions poster. Heck, it even comes from my mother as I chat about my day in the lab. It reliably resurfaces with uncanny punctuality every time I talk about my research.

“So… if you can’t predict a heart attack, why does this matter?”

My dissertation project focuses on cardioprotection. That is, the phenomenon that a nonlethal stimulus applied prior to a heart attack can protect the heart against the insult.

Cardioprotection was first investigated in the context of acute myocardial infarction (MI). In the 1980s, it was discovered that brief, nonlethal bouts of ischemia and reperfusion (I/R) to the heart prior to a subsequent ischemic challenge can reduce the size of the infarct in a canine model, termed ischemic preconditioning1. This study was seminal because it demonstrated the existence of an endogenous cardioprotective network that might be harnessed for therapeutic benefit. In later studies, researchers found that ischemia and reperfusion at a remote vascular bed, organ, or limb can bring also protect the heart via remote preconditioning2, 3. Remote preconditioning is even more clinically translational and relevant; it’s far less challenging to slip a blood pressure cuff on a patient’s bicep than, say, occlude an artery.

But still. These studies looked at interventions applied before the heart attack.

If you can’t predict a heart attack, is this important?

My answer is a well-rehearsed and resounding YES. Let me summarize and consolidate, quick and dirty, the vast amount of info I’ve learned during my PhD about the value of preconditioning and cardioprotection. Here are three reasons why preconditioning and cardioprotection are important:

1. Some of the pathways invoked by preconditioning might also be useful during or after reperfusion. Basic scientists uncovering the complex signaling and mechanism of cardioprotection have revealed a number of therapeutic targets that could lead to the development of drugs or therapeutics that activate these pathways when most needed4. In other words, we believe that advancements developed within the preconditioning models might also be useful for perconditioning (during reperfusion) or postconditioning (after reperfusion). This gives us hope that we can intervene during the ambulance ride, at the time of stenting, or throughout recovery and still benefit the patient.

2. Cardioprotection can be reframed outside of acute myocardial infarction. There is a need for myocardial salvage in clinical scenarios where a high risk of ischemia and reperfusion injury is expected. These situations include cardiac surgery and coronary artery bypass graft surgery, organ transplantation, where situations of I/R adversely affect the organ’s lifespan, and elective percutaneous coronary intervention (PCI). PCI alone is performed on millions of patients annually, and perioperational MI occurs in at least 10% of those patients, depending on how you measure it5. In such cases, strategies to preserve myocardia can reduce susceptibility to perioperative injury in situations where MI can be reliably predicted.

3. The jackpot— any evidence that cardioprotective interventions might work in man. A slew of clinical trials have suggested that cardioprotection, whether based in ischemic techniques or pharmacological strategies, can be effective in humans6. The caveat is that these clinical studies are inconsistent and vary widely in the exact methodology and measured outcome (such as biomarkers, imaging, or major adverse cardiovascular and cerebrovascular events). We still don’t have an approved cardioprotective therapy, but this seems encouraging!

As an early career investigator, routinely and enthusiastically defending my work has led to some challenging conversations. Those difficult, engaging discussions have given me guts as a grad student and awareness as a scientist.

Now I can focus on that next big question: “When are you going to graduate?”

  1. Murry, C.E., R.B. Jennings, and K.A. Reimer, Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation, 1986. 74(5): p. 1124-36.
  2. Przyklenk, K., et al., Regional ischemic ‘preconditioning’ protects remote virgin myocardium from subsequent sustained coronary occlusion. Circulation, 1993. 87(3): p. 893-9.
  3. Kharbanda, R.K., et al., Transient limb ischemia induces remote ischemic preconditioning in vivo. Circulation, 2002. 106(23): p. 2881-3.
  4. Hausenloy, D.J., Cardioprotection techniques: preconditioning, postconditioning and remote conditioning (basic science). Curr Pharm Des, 2013. 19(25): p. 4544-63.
  5. Lansky, A.J. and G.W. Stone, Periprocedural myocardial infarction: prevalence, prognosis, and prevention. Circ Cardiovasc Interv, 2010. 3(6): p. 602-10.
  6. Heusch, G., Cardioprotection: chances and challenges of its translation to the clinic. Lancet, 2013. 381(9861): p. 166-75.

Annie Roessler Headshot

Annie Roessler is a PhD Candidate at Loyola University in Chicago, IL. Her research focuses on the neurobiology and molecular mechanisms of electrically-induced cardioprotection. She tweets @ThePilotStudy and blogs at flaskhalffull.com


So It Begins…

On my first morning at the American Heart Association’s Scientific Sessions, I picked up my badge and joined the stream of scientists flowing through the sunshine-filled hallways of the Anaheim Convention Center before making my way up the escalator to Ballroom B. A panel of personalities commanded a large table at the head of the room. This opening session was packed with early career investigators – that is, PhD candidates like me, but also post-docs, residents, and young faculty. We’re newbies to this kind of thing, called to attend this talk on cultivating successful mentor/mentee relationships. Throughout the conference, we’ll learn extensively about a variety of themes in cardiovascular disease research, kicked off with this panel and moving into more specialized topics until the doors close next Wednesday.

Conferences are foundational to the graduate education experience. As a student/trainee member of AHA, my time in Anaheim probably looks a little different than the tenured professor that I chatted with in the registration line or the young doctor that I sat next to in the first session. One way to start cultivating your personalized experience here is by swiping through the mobile meeting guide, which you can download on your phone by searching “AHA events” on the app store. It can help you navigate the meeting, explore the sessions, and set out a schedule, all from your hotel room. Isn’t technology great? I’ve got everything I need organized in the palm of my hand.

Everyone has a different strategy for how they will spend their time at Sessions. There’s a lot on my mind right now, between wrapping up experiments for my dissertation project, pushing out my first few papers and grant applications, and clenching a post-doc. That translates to a lot of learning opportunities in the days ahead. I’ll be trying to use my time wisely so that I can totally nail the experience. Here is the plan so far:

1. I’ll be deliberate about choosing the sessions that I attend. Anything having to do with the field of cardioprotection? You bet I’ll be there! This helps me gain fresh ideas on my own work and join the larger conversation in my field. I’ll also be ducking into some talks on flashy, cool topics that seem especially novel or significant. People are doing some ground-breaking stuff out there, and I don’t want to miss any of it. That’s what makes science so exciting.

2. If there are any career-oriented sessions that lay out steps toward my goals as a scientist, you can probably find me in the audience. Saturday has been packed with Early Career seminars – they cover everything from how to succeed in grant writing to how to be a scientific extrovert. Throughout the conference, presenters looking to hire someone in their lab will occasionally mention that at the end of their slides. Going forward, I’ll keep a sharp eye out for those opportunities.

3. Network, network, network. Everybody’s doing it! It’s an imperative, fundamental survival skill in science. Conferences are the ideal place to link information and ideas together. Today I talked to people I’ve never talked to before: the senior investigators at this afternoon’s Lunch with Legends, who gave me invaluable pointers on finding a post-doc position. I’m going to try to spend the rest of the conference shaking as many hands as possible.

At the end of Day 1, I’m already feeling incredibly inspired and energized about my experience at Scientific Sessions. How did you prepare for Scientific Sessions this year?

Annie Roessler Headshot

Annie Roessler is a PhD Candidate at Loyola University in Chicago, IL. Her research focuses on the neurobiology and molecular mechanisms of electrically-induced cardioprotection. She tweets @ThePilotStudy and blogs at flaskhalffull.com