Applied Biosystems and Susan McCarthy, MS
A partnership for the advancement of science
Susan McCarthy, MS
Senior Biological Scientist, H. Lee Moffitt Cancer Center and Research Institute
AB Products in Use
Susan McCarthy is a Senior Biological Scientist in the laboratory of Dr. Timothy Yeatman at the H. Lee Moffitt Cancer Center and Research Institute, a leader in the investigation of the gene expression "signatures" (mRNA expression patterns) that define pancreatic, colon, breast, and other cancers. A molecular cell biologist and gene expression expert, she is working to identify cancer-related mRNA signatures in formalin-fixed paraffin-embedded (FFPE) patient samples.
We recently had the opportunity to talk with Susan about the lab's current research, the promising results she has achieved so far, and how collaboration with Applied Biosystems is helping the H. Lee Moffitt Cancer Center to advance the science of predicting, diagnosing, and treating cancer.
Let's start with a high-level overview of the research you're currently conducting in the Yeatman lab.
The Yeatman laboratory has access to a large collection of FFPE tumor biopsy samples that give us detailed information on patient treatment and outcomes. Our current work focuses on correlating microarray analysis from mRNA extracted from both frozen tissue and the FFPE samples taken from the same patient.
Showing that a gene signature developed from frozen tissue also works with mRNA from FFPE samples would allow us to use any archive of FFPE samples; in essence, allowing us to go back in time. This 'time machine' gives us 5 to 10 years of history to work with.
Why is paraffin research such a promising field and what are some of the benefits and challenges of working with paraffin samples?
Paraffin is a hot area because all hospitals around the world have paraffin samples that are rich resources of clinical history, to which the patient's response to therapy is already known. We plan to use this valuable resource to correlate historical patient outcomes with the gene expression signature observed in the preserved surgical biopsy samples.
The challenging part about using paraffin samples for gene expression analysis is that the RNA extracted from the paraffin is highly variable due to the formalin fixation process. We've been able to overcome that challenge by teaming with Applied Biosystems to develop a technique that allows us to extract and purify RNA from paraffin samples and then perform gene expression analysis on the quantitative real-time PCR platform.
And what is the ultimate goal of the FFPE research?
Ultimately, we're trying to personalize medicine. The goal is to enable physicians to use the specific cancer signatures from fresh biopsies to predict patient prognosis, diagnosis, and response to therapy. Armed with that information, they will be able to design targeted treatment regimens that are highly effective for specific patients.
For example, we are working to understand why individuals with the same kind of cancer respond differently to the same therapy. We want to know things such as, what makes a patient a good responder to chemotherapy and another a poor responder? The answers are differences in their genetic profiles.
Can you walk us through the workflow of your research?
The research process begins with the patient, using a four-step workflow that includes RNA Isolation, Reverse Transcription, cDNA PreAmplification, and Real-Time PCR. We start with fresh-frozen tissue samples, extract RNA, and send the RNA samples to our microarray core facility for gene expression analysis. From the microarray data, we then validate and translate the gene expression signatures using fresh frozen and FFPE samples from the same patients using quantitative real-time PCR. We like to use this platform because it's more sensitive, reproducible, and reliable than microarray.
Which specific Applied Biosystems products does the process rely on?
We use the Ambion RecoverAll™ Total nucleic Acid Isolation Kit for RNA extraction from FFPE samples. After reverse transcription using the High-Capacity cDNA Reverse Transcription Kit, we preamplify the cDNA using the TaqMan® PreAmp Master Mix Kit. By including this step, we overcome the problem of high Ct values we initially encountered using FFPE samples. By lowering the Ct values, the data is now suitable for gene expression analysis. Also, plenty of cDNA is generated for real-time PCR analysis for future repetition and validation. For the final step, we use the TaqMan® Gene Expression Master Mix, TaqMan® Gene Expression Assays, and Applied Biosystems Real-Time PCR Systems.
Why did you choose to team up with Applied Biosystems for your FFPE research, and are you happy with that choice?
Applied Biosystems is the recognized leader for gene expression analysis applications, and we've been successfully using their products for years. We really like their kits--they are reliable, easy to use and the best on the market when it comes to reproducibility.
Our current paraffin analysis research involves profiling a large number of tumors at different sites and stages, and Applied Biosystems has all the cutting-edge resources and tools we need for that work. We have tried several different kits for extracting RNA from FFPE samples but ultimately chose the Applied Biosystems kit because it provided high yields and the quality of RNA was usable for our gene expression studies.
And, yes, I'm very happy with the decision to go with Applied Biosystems. As I mentioned, I've tried different kits out there, but once I tried this kit and saw the results there was no going back. I've had a lot of vendors come into the lab to try to get me to switch to different kits, but I'm not going to. The same is true for the Applied Biosystems reagents I use for real-time PCR. Vendors come in daily to try to get me to switch, but I'll never do it.
What's happening in the area of breast cancer research? Is that the next big test for FFPE analysis?
We are currently developing gene signatures for breast cancer. We have validated our microarray data using the Applied Biosystems quantitative real-time PCR platform. We have shown that we can correlate microarray data using TaqMan low-density arrays from fresh-frozen tissue samples. The next step with these gene signatures is to translate them using paraffin samples. We've already shown that we can get gene expression data from pancreatic cancer paraffin samples using the TaqMan low-density arrays; paraffin breast cancer samples are the next big test.
How far along is this field of research? What results have you achieved to date?
We are still in the beginning phases of this research, but we're moving forward at a good pace. Results have been very promising for colon and pancreatic cancers. We've actually identified novel markers of tumor progression in pancreatic cancers using the Applied Biosystems TaqMan low-density arrays. We have also shown in our data set that gene expression from paraffin samples is similar to that of frozen tissue.
How would you describe your overall experience collaborating with Applied Biosystems, and what direction do you see the relationship taking in the future?
It's been wonderful. Applied Biosystems has been an enormous help and our lab is just very grateful to be working with them. They have been very supportive in all areas of our research, helping us to advance our technical applications. In fact, we wouldn't have been able to move forward in the paraffin area if it wasn't for their help.
In the future, we will continue to work closely with Applied Biosystems scientists and technologies. We've already shown that we can get microRNA expression from FFPE samples using Applied Biosystems microRNA cards. We're very excited that they're going to be releasing a new version of the cards this summer that will allow us to analyze up to 700 micro RNA targets.
Thank you for your time, and congratulations on your early success in the area of FFPE research. By the way, what made you decide to go into cancer research in the first place?
This disease has affected members of my family all my life. It's a challenging field, and I want to contribute to the treatment and the cure for this terrible disease. My personal goal is to be the best scientist out there; always evolving with new technology to help develop molecular signatures for thousands of tumors and design more targeted therapies to treat cancer patients.
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