A New Technology for Stabilization of Biomolecules in Tissues for Combined Histological and Molecular Analyses

Abstract

For accurate diagnosis, prediction of outcome, and selection of appropriate therapies, the molecular characterization of human diseases requires analysis of a broad spectrum of altered biomolecules, in addition to morphological features, in affected tissues such as tumors. In a high-throughput screening approach, we have developed the PAXgene Tissue System as a novel tissue stabilization technology. Comprehensive characterization of this technology in stabilized and paraffin-embedded human tissues and comparison with snap-frozen tissues revealed excellent preservation of morphology and antigenicity, as well as outstanding integrity of nucleic acids (genomic DNA, miRNA, and mRNA) and phosphoproteins. Importantly, PAXgene-fixed, paraffin-embedded tissues provided RNA quantity and quality not only significantly better than that obtained with neutral buffered formalin, but also similar to that from snap-frozen tissue, which currently represents the gold standard for molecular analyses. The PAXgene tissue stabilization system thus opens new opportunities in a variety of molecular diagnostic and research applications in which the collection of snap-frozen tissue is not feasible for medical, logistic, or ethical reasons. Furthermore, this technology allows performing histopathological analyses together with molecular studies in a single sample, which markedly facilitates direct correlation of morphological disease phenotypes with alterations of nucleic acids and other biomolecules.

Clinical impact of targeted amplicon sequencing for meningioma as a practical clinical-sequencing system

“…Clinical sequencing of meningioma paraffin-embedded tissues owing to the high quality of DNA extracted from PAXgene-fixed paraffinembedded specimens 44,45 and conserved histological morphology. 46 In fact, we analyzed two cases of meningioma using genomic DNA extracted from formalin-fixed paraffin-embedded tissue and obtained similar but imprecise results of copynumber alterations (data not shown), mostly because of DNA fragmentation by formalin fixation.…”

Modeling, Optimization, and In Vitro Corneal Permeation of Chitosan-Lomefloxacin HCl Nanosuspension Intended for Ophthalmic Delivery

“…Eyes and eyelids were removed and kept in Davidson fixative solution for 24 h. Then, the organs were dehydrated and stabilized with formalin solution and stored in low-melting paraffin. Finally, cut sections were stained with hematoxylin and eosin for histological examination [34]. This experiment was conducted after obtaining a formal ethical approval from the animal ethical committee at Beni-Suef University.…”

The State of RT-Quantitative PCR: Firsthand Observations of Implementation of Minimum Information for the Publication of Quantitative Real-Time PCR Experiments (MIQE)

“…For tissue samples, some labs surgically remove tissue from animals and weigh the samples at room temperature. They then slice the tissue and transfer it into tubes, all at room temperature, and finally freeze the samples at -80 ° C. Other labs immediately flash-freeze the animal tissue in liquid nitrogen, transfer it into tubes on dry ice and store it at -80 ° C. These different sample extraction and storage methods can yield vastly different results because the transcriptome is affected by each sample manipulation and can change very quickly in response to chemical and environmental treatments [Huang et al, 2013;Viertler et al, 2012]. Rigorous and reproducible methodology is achieved by halting transcription as soon as possible after sample collection.…”