The development of robust, cost-effective molecular tools for identifying genetic variants and mutations that underlie human diseases requires innovative approaches.
Medical diagnostics for the detection of such variants or mutations facilitate attempts to individualize patient treatment and prognosis. Most currently available molecular assays are limited in their detection sensitivity for low-abundance genetic variants of disease.
A number of researchers engaged in the field of nucleic acid sample preparations are scheduled to present their techniques and methodologies to enrich regions of interest in the human genome at CHI’s upcoming “Sample Prep and Target Enrichment in Molecular Diagnostics” conference.
The analysis of proteins and biomarkers has been traditionally compromised due to the extensive chemical fixation of the tissues. “Among the less utilized clinical samples are formalin-fixed paraffin-embedded tissues,” says Alan Tackett, Ph.D., associate professor, and director UAMS Proteomics Facility at the University of Arkansas for Medical Sciences. “These tissues are extremely valuable as they can be stored long term.”
“We have optimized methods to reverse the fixation of these tissues and have used our approach to perform the most comprehensive study of formalin-fixed paraffin-embedded patient melanoma tissues,” Dr. Tackett explains. He claims that his studies provide the most in-depth approach to uncover biomarkers for diagnosis and prognosis of patient melanoma.
“Our approach for extraction and analysis of proteins from formalin-fixed paraffin-embedded tissues will allow researchers to probe deeper into the proteome of patient samples to try and uncover biomarkers for diseases,” says Dr. Tackett. “Our work will provide researchers with the tools and technological approaches to maximize the analysis of proteins and protein biomarkers from formalin-fixed paraffin-embedded tissues.”
The collection of tissues (up to 35 per donor) and the preservation of RNA posed a unique challenge for the Genotype Tissue Expression Project (GTEx), since all tissue samples were obtained from deceased, low post-mortem interval donors, according to Kristin Ardlie, Ph.D., director, biological samples platform, the Broad Institute.
Dr. Ardlie and her colleagues therefore implemented the PAXgene® tissue system to allow for greater flexibility at both the collection sites and for shipping to the processing and analysis centers. The PAXgene tissue system developed by Qiagen enables histomorphological studies via fixation of tissue samples as well as the preservation of nucleic acids for molecular analysis.
The technique has been shown to enable histological analysis and extraction of high-quality RNA, miRNA, and DNA from single samples. It is also able to preserve intact RNA without crosslinking as well as high-molecular-weight DNA for superior results compared to formalin, according to Dr. Ardlie. PAXgene is capable of preserving high-quality DNA for sensitive downstream applications, including multiplex or long-range PCR, she adds.
However, “given the relative ‘newness’ of the PAXgene system, GTEx has worked closely with Qiagen from its outset to both optimize the collection and preservation of the tissue samples as well as to scale up RNA and DNA isolation from the tissues,” according to Dr. Ardlie.
“Not only has RNA quality been extremely good, but our pathologists working on the project have noted that the histological preservation of the tissues has been as good as or better than that for comparative formalin-fixed tissues,” says Dr. Ardlie.
GTEx is an NIH common fund initiative, launched in 2010, which was designed to investigate human gene expression and regulation in multiple tissues to provide valuable insight into the mechanisms of gene regulation. The project examines individual genetic variation and its correlation with differences in gene expression level to identify regions of the genome that influence how much a gene is expressed and how tissue-specific that expression is.
Dr. Ardlie and Gad Getz, Ph.D., also of the Broad Institute, are co-principal investigators of the Laboratory, Data Analysis, and Coordinating Center (LDACC) for the GTEx project, which also supports tissue and donor data acquisition, and tissue pathology through Cancer Human Biobank (caHUB), as well as multiple centers funded to develop novel statistical methods for the analysis of human gene Expression Quantitative Trait Loci (eQTL).
These groups all work together toward the goal of creating an atlas of human gene expression, and a tissue bank, that researchers can use to study functional mechanisms of inherited susceptibility to disease, clarifies Dr. Ardlie. “The GTEx project is nearing the end of its pilot phase with over 230 donors (>6,500 tissue samples) collected to date,” says Dr. Ardlie. “It now moves in to the scale-up phase where samples will be collected from up to 900 donors.”