Human Tissue Sample Blog

Creating Targeted Next-Gen Sequencing Panels from FFPE Samples

Posted by Luke Doiron on Aug 30, 2016 8:00:00 AM

    

Next Generation Sequencing (NGS) technologies yield a fast-forward from the completion of the Human Genome Project (1990-2003) with the laborious, slow Sanger methods described in the late 1970s to the precision medicine trending now. NGS users, like the fictional characters in Star Trek: The Next Generation, can “boldly go where no one has gone before.” In the same way, people who use NGS methods currently can sequence one person’s genome in its entirety in just one day.

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New Developments in Extracting Protein from FFPE Tissue

Posted by Luke Doiron on Aug 9, 2016 8:00:00 AM

    

Formalin-fixed, paraffin-embedded (FFPE) biospecimens offer a robust and abundant resource for biomedical research. With the increased need for specimens that permit molecular analysis of tumor tissues and isolation of the proteins in them to develop more effective cancer therapies, researchers need a process that enables safe, efficient, reproducible and cost-effective deparaffinization.

We previously wrote about a method that used xylene to extract a variety of full-length proteins from FFPE biospecimens. The process takes about two and a half hours; however, it may create less than optimal conditions for extraction of other proteins and potentially exposes lab technicians to a hazardous organic solvent. Other methods that do not use xylene require multiple snap-freeze steps using ethanol on dry ice as well as boiling and centrifugation, processes that are time consuming and may not work with all proteins.

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How to Extract RNA from FFPE

Posted by Luke Doiron on Jun 21, 2016 8:00:00 AM

    

 

There’s no dispute over the fact that high-quality formalin-fixed paraffin embedded (FFPE) tissues are critical for advancing biomedical research and drug development. Historically, FFPE tissues were used only for certain analyses, as there was no good way to extract DNA and RNA material without a great deal of degradation. Fortunately for today’s researchers, significant advancements have been made in developing methods to extract viable nucleic acids, including RNA, for use in a variety of studies, including gene expression analysis and miRNA quantification.

A variety of commercial FFPE RNA extraction kits are available. Thermo Fisher Scientific offers the PureLink™ FFPE RNA Isolation Kit, which requires about 30 minutes of prep time to yield total RNA of about 1 ug per 10 micron section. Here is a brief summary of the extraction method:

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Advances in FFPE Samples, Storage, and Technology

Posted by Luke Doiron on May 31, 2016 10:51:21 AM

    

Formalin-fixed paraffin-embedded (FFPE) tissue specimens are valuable resources for a wide variety of research programs. Today, their value is even higher than in the past, due to advancements in techniques and tools to optimize FFPE specimen downstream viability. While the formalin fixation process once made proteins unusable due to cross-linking, today researchers can successfully extract DNA, RNA, key proteins and biomarkers from the huge reservoir of FFPE archival samples, and so advance their understanding of cancer and other major diseases.

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Praise for the Hoarders: Pathologists Provide a Treasure Trove of Genetic Info for Research with FFPE

Posted by Martha Jordan on May 19, 2016 1:06:00 PM

    

In a conference video dated September 2013 Joshua Schiffman, M.D., Pediatric Oncology Director of Utah Children’s Hospital, lovingly refers to pathologists as hoarders: “Pathologists have a very distinctive clinical psychiatric disorder and the best way to describe [it is through] the television show called ‘Hoarders.’ [They] can’t throw anything away. A patient comes in; a sample comes out. God forbid you throw that sample away.”

 

The receptacles of their hoarding habits are Fixed Formula Paraffin Embedded tissue samples (FFPE), sometimes referred to as “paraffin blocks.” Thanks to the hoarders, and more sophisticated methods of DNA retrieval, cytogeneticists can find clues about rare cancers, useful in diagnosing and pinpointing treatment plans to improve cancer survival rates.

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Performing Whole-Exome Sequencing on FFPE Mutation Samples

Posted by Luke Doiron on Apr 28, 2016 7:00:00 AM

    

Whole-exome sequencing (WES) technology has rapidly been embraced as a valuable and cost-effective method for sequencing the expressed genes in a genome (i.e. the exome). WES benefits researchers and clinicians seeking to identify the mutations found in many types of tumors and other difficult-to-treat disorders such as Alzheimer’s disease. As investigators gain greater insight via WES and other massive parallel sequencing methods into the biology and genetics of disease, the goal is to find and exploit new clinically useful therapy targets.

However, one of the obstacles to the routine use of WES is obtaining enough samples to perform the large-scale studies necessary to advance and inform cancer research and clinical care. One solution might be the literally millions of formalin-fixed, paraffin-embedded (FFPE) specimens stored in tissue banks and biorepositories across the globe, many of which are well-annotated, with good histology, pathology, and clinical care data attached.

Yet, it’s a well-known fact that if FFPE specimens are not properly fixed using the right fixation agents, and/or improperly stored, the extracted DNA material may be too poor in quality to be used for exome sequencing studies. In addition, sometimes a difficult extraction process leads to low yields and/or very small sample sizes that are less useful for sequencing protocols. This has led to investigations aimed at determining whether FFPE archival specimens are reliable and valid sources of extracted DNA material.

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Validating Research Results from FFPE Mutation Samples

Posted by Luke Doiron on Mar 31, 2016 10:00:00 AM

    

 

Formalin-fixed, paraffin-embedded (FFPE) specimens have been routinely prepared by pathologists for nearly 100 years. It's estimated that FFPE samples represent roughly 80 to 90% of all samples, totaling more than one billion, stored across the globe. This huge biospecimen resource has been used for thousands of research studies over the years. However, it was not until fairly recently that researchers began tapping into FFPE sample archives for newer mutational studies using FFPE preserved tumor specimens.

With the advent of new technology allowing deep analysis and characterization of genomic structural, sequence and expression variations associated with cancer, FFPE samples could be invaluable resources, assuming their reliability as indicators for examining such genomic mutations in tumor specimens. A variety of studies have investigated the validity of research data using FFPE mutation samples, including these published results:

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How to Culture Cryopreserved Samples

Posted by Luke Doiron on Feb 2, 2016 8:00:00 AM

    

Cryopreservation is widely used for stabilizing and storing biological materials such as tissue specimens at very low (cryogenic) temperatures. Advancements in understanding the best methods for cryopreservation, thawing and culturing of biological specimens have enabled such samples to be stored for many years and still be viable specimens for studying a variety of cellular processes.

Determining the best method for culture of thawed cryopreserved samples is an important step in many research protocols. Depending on the research goal and tissue specimen, culture methods will vary to some degree. Peripheral blood mononuclear cells (PBMCs), whole tumor tissue, and bone marrow are just a few examples of the cryopreserved samples that scientists use for culturing.

One important point repeatedly mentioned in the literature is that cryopreservation must be done correctly. Tissue samples should be frozen in 10% DMSO, never snap frozen, without temperature fluctuation during storage time. In particular, specimens should remain below the glass transition point to maximize long-term utility.

The following culture protocols have been used for cryopreserved samples.

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