Sally Seraphin’s life in the research lab started with rats and roseate terns. Chimpanzees and rhesus macaques came next, then humans (and a brief foray into voles). When she pivoted to red-eyed tree frogs, Sally once again had to learn all kinds of new techniques. Suddenly, in addition to new sample prep and analysis techniques, she needed to get up to speed on amphibian care and husbandry. That led her to the Marine Biological Laboratory (MBL) in Woods Hole, MA.
“It’s a seaside resort atmosphere with experts in every technology you can imagine,” Sally says. “It’s a place to incubate and birth new approaches to answering questions.”
Sally spent the past two summers at MBL learning everything she needed to know about breeding and caring for amphibians. During that time, she also worked closely with Applications Scientists from Promega who helped her start extracting RNA from frog samples.
“The hands-on support from industry scientists is definitely unique to Promega and MBL,” she says. “It’s rare to have a specialist on hand who can help you learn, troubleshoot and optimize in such a finite amount of time.”
Adopting a New Model Organism
Sally studies how early stress impacts brain and behavior development. She hopes to deepen our understanding of how adverse childhood experiences connect to mental illness and bodily disease later in life. In the past, she studied how factors such as parental absence affected the neurotransmission of dopamine in primates. Recently, she changed her focus to developmental timing.
“Girls who are exposed to early trauma like sexual or physical abuse will sometimes reach puberty earlier than girls who aren’t,” Sally explains. “And I noticed that there are many species that will alter their developmental timing in response to predators or social and ecological threats.”
Our second installment of the Promega qPCR Grant Recipient blog series highlights Dr. Laura Leighton, a trained molecular biologist and postdoctoral researcher at the Australian Institute for Bioengineering and Nanotechnology. Leighton’s scientific journey features a passion for molecular biology and problem-solving. Her path has been illuminated by mentorship, relationships with fellow scientists and a commitment to creativity in overcoming challenges. Here, we explore her scientific journey, reflect on research lessons and foreshadow her plans for the Promega qPCR grant funds.
Dr. Laura Leighton grew up in a rural area in Far North Queensland, Australia, where she spent her early life exploring critters on the family farm. Her upbringing was infused with a deep connection to the environment, from raising tadpoles in wading pools to observing wildlife and witnessing food grow firsthand. Observing the biology around her ultimately piqued her interest in science from a young age. She then began her academic journey in 2011 at the University of Queensland, Australia. She studied biology while participating in a program for future researchers, which led her to undergraduate research work in several research labs. She dabbled in many research avenues in order to narrow in on her scientific interests all while adding different research tools to her repertoire.
After serving as a research assistant in Dr. Timothy Bredy’s lab, she decided to continue work in this lab and pursue a PhD in molecular biology. During her PhD, Leighton worked on several projects from cephalopod mRNA interference to neurological wiring in mice. The common thread in these projects is Leighton’s passion for the puzzles of molecular biology:
“I also love molecular engineering and the modularity of molecular parts. There’s something really special about stringing together sequence in a DNA editor, then seeing it come to life in a cell,” she says.
We’re all familiar with the Central Dogma of Molecular Biology: DNA is transcribed into RNA, which is translated into proteins. It’s drilled into our heads from the early days of biology classes, and it’s surprisingly useful when we start exploring in our own research projects. For example, if you’re interested in gene expression, you’ll most likely be working with RNA, specifically mRNA. Messenger RNA (mRNA) is transcribed from DNA and is used by ribosomes as a “template” for a specific protein. The total mRNA in a cell represents all of the genes that are actively being transcribed. So, if you want to know whether or not a gene is being transcribed, RNA purification is a great place to start.
When preparing your RNA samples for a downstream assay, there are several roadblocks and pitfalls that could give you quite a headache. Let’s tackle two of the most common.
Synthesizing proteins in vitro through cell-free expression systems using rabbit reticulocytes, E. coli S30, or wheat germ extracts can be invaluable in studying protein function. If you only need a small amount (100s of nanograms), it’s also faster and easier than synthesizing vast quantities in bacterial or mammalian cells (~ 90 minutes for cell-free vs. long growth times and extraction steps after an initial optimization for protein synthesized in larger scale). There are many systems out there, and knowing which to use can sometimes be difficult. Many kits include components that combine transcription and translation in one-step, eliminating the need to provide your own RNA. But when you want to make your own RNA templates to add to lysates, then there are additional concerns.
Many people don’t want to work with RNA since the common lab lore suggests it’s a finicky molecule, and for good reason. Extracting it requires the utmost care in technique and elimination of nucleases. Failing to do so results in degradation of the molecule, and so with it your experiments (see our recent blog by Terri Sundquist on tips for isolating RNA with ease). Preparing RNA for cell-free expression is subject to the same concerns as extracted RNA, but with the proper care is not that much more of a challenge than using a DNA template.
The first step for using cell-free expression systems with RNA templates is to make the RNA. Here are some tips that will ensure success.
Back in graduate school, I purified a lot of RNA, and after a while, I became fairly successful at it. My yields were good, and the RNA was intact. However, many of my early attempts at RNA isolation yielded degraded RNA that did not work well in many downstream applications. In my case, successfully isolating high-quality RNA required practice. During my trials and tribulations, I learned a lot of tricks and tips about how to obtain high-quality RNA. Here I share some of these tricks to help you speed through that “practice makes perfect” phase so that you can isolate RNA like a pro.
My very first job in science was in a lab that worked exclusively with RNA, and it was only after I moved on to a different job that I learned just how much different the world of DNA research is from that of RNA. When working with DNA, for example, you rarely if ever have the sample you have labored over reduced to a fuzzy blur at the bottom of a gel because it has been degraded beyond rescue. With RNA, unfortunately, this happens all too frequently. In fact, a labmate of mine once put up a poll on the door to our lab asking if it was better to discover that your RNA sample was degraded on a Monday or a Friday.
The culprits in this scenario are Ribonucleases (RNases). They are everywhere. They are incredibly stable and difficult to inactivate. And, if you work with RNA, they are your enemy. Take heart though, they can be defeated if you follow some pretty simple steps.
We recently posted a blog about Proteinase K, a serine protease that exhibits broad cleavage activity produced by the fungus Tritirachium album Limber. It cleaves peptide bonds adjacent to the carboxylic group of aliphatic and aromatic amino acids and is useful for general digestion of protein in biological samples. In that previous blog we focused on its use to remove RNase and DNase activities. However, the stability of Proteinase K in urea and SDS and its ability to digest native proteins make it useful for a variety of applications. Here we provide a brief list of peer-reviewed citations that demonstrate the use of proteinase K in DNA and RNA purification, protein digestion in FFPE tissue samples, chromatin precipitation assays, and proteinase K protection assays: Continue reading “Proteinase K: An Enzyme for Everyone”
If you enter any molecular lab asking to borrow some Proteinase K, lab members are likely to answer: “I know we have it. Let me see where it is”. Sometimes the enzyme will be found to have expired. The lab may also have struggled with power outages or freezer malfunctions in the past. But the lab still decides to keep the enzyme. One may rightly ask – why do labs hang on to Proteinase K even when it has been stored under sub-standard conditions? Continue reading “ProK: An Old ‘Pro’ That is Still In The Game”
Working with RNA can be a tricky thing…it falls apart easily, and RNases (enzymes that degrade RNA) are ubiquitous. Successfully isolating RNA and maintaining its integrity is critical, especially when sensitive downstream applications are used (e.g., RNA-Seq).
Good techniques for RNA handling are simple to employ but crucial for success. All RNA purification and handling should take place in an RNase-free, RNA-only zone of the lab. Segregating RNA work from protein and DNA purification and handling will help minimize the potential for RNase contamination and help keep your RNA intact. Only buffer and water stocks treated to be RNase-free should be kept in the RNA area of the lab, and gloves should be worn at all times to prevent accidental contamination. Tools and equipment such as pipets, tips, and centrifuges should be designated for use only in the RNA zone as well. The location of the RNA zone in the lab is also important. Keeping traffic to a minimum and moving the RNA zone away from doors, windows, and vents can also help minimize contamination.
Using an RNase inhibitorcan also help safeguard your samples from RNase degradation. These inhibitors can bind to any RNases that may have been introduced into your sample and prevent them from cutting the RNA present.
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