In oncology, tissue biopsies are commonly fixed in formalin and embedded in paraffin (FFPE). These FFPE samples can be used with immunohistochemical or molecular analysis for identifying biomarkers that guide the diagnosis and therapeutic management of patients. This fixation technique allows long-term storage of samples but impacts the integrity of nucleic acids. This makes extracting DNA and RNA from FFPE tissues in sufficient quantity and quality for molecular analysis techniques such as NGS analyses challenging for molecular oncology laboratories.
“At Rennes University Hospital, we receive many lung cancer samples with little material available, or samples of poor quality. The nucleic acid extraction step is therefore critical to get good yield. We have seen that it had a direct impact on the success of downstream analysis,” said Dr. Alexandra Lespagnol. Lespagnol is the Technical Manager of the Molecular Genetics of Cancer core lab at the University Hospital of Rennes in France.
In order to accommodate the increasing number of samples that needed to be analyzed, the Molecular Genetics of Cancer core lab of the University Hospital of Rennes initiated an automation project for extracting DNA from FFPE tissues. The lab also wanted to improve sample tracking and reproducibility of their results.
Formalin-Fixed Paraffin embedded (FFPE) samples are being used in increasing numbers of molecular assays. In my last blog I discussed some of the pre-analytical variables that can affect results obtained when using FFPE samples. Laboratories can increase the quality of downstream results by controlling variables where possible. While exacting control over the sample acquisition and fixation process can improve results, quality testing of incoming samples is a crucial step in assuring optimal results. There are numerous methods that can be used to evaluate the quality of samples and they can provide different information that can be used to assess sample integrity and suitability for different applications.
Formalin Fixed Paraffin Embedded samples (FFPE) have been a mainstay of the pathology lab for over 100 years. Initially FFPE blocks were sectioned, stained with simple dyes and used for studying morphology, but now a variety of biomolecules can be analyzed in these samples. Over the past 10 years we have discovered that there is a treasure trove of genomics data waiting to be unearthed in FFPE tissue. While viral RNAs and miRNA were some of the first molecules found to be present and accessible for analysis starting in the 1990s, improvements to DNA and RNA extraction methods have demonstrated that PCR, qPCR, SNP genotyping, Exome and WGS are possible. This has resulted scientific publications of DNA and RNA data generated from FFPE samples starting in 2006, and today we see immense amounts of data generated from FFPE—with nearly 2000 citations in 2018 reporting sequencing of FFPE samples.
Depending on the type of project, prospective or
retrospective, the genomics scientist has an opportunity to affect the
probability of success by better understanding the fixation process. The
challenge with FFPE is the host of variables that have the potential to
negatively affect downstream assays.
Implementing automated nucleic acid purification or making changes to your high-throughput (HT) workflow can be complicated and time-consuming. There are also many barriers to success such as challenging samples types and maintaining desirable downstream results that can add to the stress, not to mention actually getting the robotic instrumentation to do what you want it to. All of this makes it easy to understand why many labs avoid automating or own expensive instrumentation that goes unused. Continue reading “High-Throughput Purification with Experts Included”
Imagine you are traveling in your car and must pass through a mountain range to get to your destination. You’ve been following a set of directions when you realize you have a decision to make. Will you stay on your current route, which is many miles shorter but contains a long tunnel that cuts straight through the mountains and obstructs your view? Or will you switch to a longer, more scenic route that bypasses the tunnel ahead and gets you to your destination a bit later than you wanted?
Choosing which route to take illustrates a clear trade-off that has to be considered—which is more valuable, speed or understanding? Yes, the tunnel gets you from one place to another faster. But what are you missing as a result? Is it worth a little extra time to see the majestic landscape that you are passing through?
Considering this trade-off is especially critical for researchers working with human DNA purified from formalin-fixed paraffin-embedded (FFPE) or circulating cell-free DNA (ccfDNA) samples for next-generation sequencing (NGS). These sample types present a few challenges when performing NGS. FFPE samples are prone to degradation, while ccfDNA samples are susceptible to gDNA contamination, and both offer a very limited amount of starting material to work with.
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”
“I would do more with my samples, but it’s just not possible…I know there’s probably a wealth of information in there, but there is just no way to get it out…I’ve got blocks of tissue sitting in the lab, experiments I want to run, but no good way to get clean nucleic acids out.”
These are a few of the comments I heard when talking with scientists at the American Society of Human Genetics meeting last week in Montreal. They, and countless other researchers, are sitting on a treasure trove of information that may have been locked away a few months ago, a few years ago, or decades ago. I’m referring to formalin-fixed, paraffin-embedded (FFPE) tissue blocks. It is estimated that there are upwards of 400 million tissue blocks archived globally and scientists are clamoring to find ways to best utilize nucleic acids derived from these tissues in applications like qPCR, microarrays, and next generation sequencing.1Continue reading “Fixed in the Past, Focus on the Future”
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