Bones are constantly self-renewing. As people age, however, their bones lose mass faster than new bone is formed. This slowdown of bone renewal can often lead to osteoporosis, a disease in which bones become incredibly fragile, and therefore more susceptible to fracture. Around 54 million Americans have osteoporosis or low bone mass, according to the National Osteoporosis Foundation. Of those with the disease, 80 percent are women, and postmenopausal women are at the highest risk.
Bones are also highly mechanically sensitive, meaning they respond to mechanical loading—applied force. Natalie Kelly, graduate student in Biomedical Engineering, works to find gene changes that occur in the postmenopausal skeleton after mechanical loading. The goal of the research is to find molecular mechanisms that lead to bone loss and osteoporosis.
“I have female relatives who have osteoporosis, the disease is very common,” says Kelly, who is a member of Marjolein van der Meulen’s lab, Biomedical Engineering/Mechanical and Aerospace Engineering. “It’s an important area to study, and it has a real possibility to improve quality of life.”
A Research Method for Studying Decreased Bone Mass
Specifically, Kelly uses a mouse model lacking estrogen receptor alpha. The lack of the estrogen receptor mimics the loss of estrogen that occurs in postmenopausal women, which plays a role in decreased bone mass. Kelly then mechanically loads the mice tibiae and examines the reaction in the bone.
Bones consist of marrow, cortical bone, and cancellous bone. Previous studies of bone gene expression have mostly looked at the bone as a whole, or bone flushed of marrow. In her work, Kelly developed a method to isolate RNA from cortical and cancellous bone without marrow contamination. It’s important to look at each tissue individually, because bone fractures often occur in cancellous bone sites, such as the spine and wrist. Kelly has already found that gene expression differs between cortical and cancellous bone sites. In August 2014, Kelly published a paper, “A method for isolating high quality RNA from mouse cortical and cancellous bone,” in the journal Bone.
Data-Intensive RNA Sequencing
Kelly used her published technique to extract messenger RNA from mechanically loaded bone. She then used RNA sequencing to capture a huge swath of data on genes in the bone. The technology can take all of the messenger RNA pieces and tell you which genes are related to mechanical loading and are either up-regulated or down-regulated in response.
“One positive aspect of RNA sequencing is that it is basically unbiased,” says Kelly. “You don’t have to go in with an idea of what’s changed and test for that specific gene. Instead, everything that changes is captured.”
RNA sequencing is a data-intensive process. Kelly says that she relies on the Biotechnology Resource Center (BRC), supported by the Center for Advanced Computing, as a resource in understanding how to analyze, manage, and store the “huge, huge files of 18 million nucleotide sequences” that the process creates. After a few months of conducting experiments in the wet lab, Kelly is spending many more months combing through the data, deciphering what it means biologically, and determining how to present her results. The BRC supports her with the necessary software, office hours, and workshops to accomplish the work.
Generating a Story
Kelly has already identified a gene that is highly regulated in her bone model. The next step is to verify that the change is real through further experiments, which might include looking at the gene’s related protein and moving to a mouse model with a knockout of that specific gene.
Beyond that specific gene, several other options are worth examining, Kelly says. “There are definitely genes that have not been studied as in depth or have been studied in other settings, but not in the context of their response to mechanical loading,” she explains.
For now, with all of the data in hand, Kelly is in the process of deciding how to present the newly gathered information in a digestible manner. Very few bone researchers have used RNA sequencing, says Kelly, which means there isn’t much precedent for how to explain the results.
“I have female relatives who have osteoporosis, the disease is very common,” says Kelly. “It’s an important area to study, and it has a real possibility to improve quality of life.”
"There are just lists and lists of genes that get generated,” says Kelly. “We can use that to figure out pathways that might be more or less involved somewhere in those lists. I’m doing that, but a lot of the process is how to make it a story and what part of the story I want to tell, which is challenging.”
Kelly, however, isn’t deterred by a challenge. Although trained as a biomedical engineer, she joined van der Meulen’s lab and began working in molecular biology, an area she wasn’t as familiar with. She’s also the only researcher in the lab identifying molecular pathways. To better understand the field, Kelly collaborated with researchers in John Schimenti’s lab, Biomedical Sciences/Molecular Biology and Genetics.
Her advice to incoming graduate students: "Ask questions of anyone and everyone, especially if you’re in an area where you feel a little over your head.” Kelly explains, “For me, switching into more of a molecular PhD, I felt like I was always asking stupid questions. Sometimes people can imagine this as being a blow to their ego, but really it’s part of learning and fact-gathering, and everyone at Cornell is more than happy to help.”