BACKGROUND: With expanding biomarker discovery efforts and increasing costs of drug development, it is critical to maximize the value of mass-limited clinical samples. The main limitation of available methods is the inability to isolate and analyze,from a single sample, molecules requiring incompatible extraction methods. Thus, we developed a novel semiautomated method for tissue processing and tissue milling and division (TMAD).
METHODS: We used a SilverHawk atherectomy catheter to collect atherosclerotic plaquesfrom patients requiring peripheral atherectomy. Tissue preservation by flash freezing was compared with immersion in RNAlater, and tissue grinding by traditional mortar and pestle was compared with TMAD. Comparators were protein, RNA, and lipid yield and quality. Reproducibility of analyte yield from aliquots of the same tissue sample processed by TMAD was also measured.
RESULTS: The quantity and quality of biomarkers extracted from tissue prepared by TMAD was at least as good as that extracted from tissue stored and prepared by traditional means. TMAD enabled parallel analysis of gene expression (quantitative reverse-transcription PCR, microarray), protein composition (ELISA), and lipid content (biochemical assay) from as little as 20 mg of tissue. The mean correlation was r 0.97 in molecular composition (RNA, protein, or lipid) between aliquots of individual samples generated by TMAD. We also demonstrated that it is feasible to use TMAD in a large-scale clinical study setting.
CONCLUSIONS: The TMAD methodology described here enables semiautomated, high-throughput sampling of small amounts of heterogeneous tissue specimens by multiple analytical techniques with generally improved quality of recovered biomolecules.
The implementation of efficient methods for processing and analysis of biological samples is an important component of the execution of clinical trials. The main limitation of available methods is the inability to isolate and analyze, from a single specimen, molecules requiring incompatible extraction methods.
Because most tissue samples are compositionally heterogeneous, e.g., atherosclerotic plaque (1 ) (Fig. 1) and tumor tissue (2 ), extraction and analysis of biomarkers from different fragments can lead to misleading results. Therefore, the development of a universal method for dividing a clinical specimen into compositionally identical aliquots is critically important.
We designed, engineered, and validated a semiautomated technique (TMAD) for splitting tissue into compositionally identical aliquots. This technique enabled cost-effective analysis of biomarkers requiring noncompatible tissue extraction protocols in single samples. TMAD standardizes the milling and splitting processes and allows increased throughput and a high rate of sample recovery from small tissue specimens (98%). All materials that contact tissue are disposable, eliminating the risk of cross contamination. TMAD is compatible with quantitative analysis of mRNA, protein, and lipid content, enabling detailed characterization of molecular composition of tissue without the need to collect separate specimens for each analysis.