Validation of Vacuum-Based Refrigerated System for Biobanking Tissue Preservation: Analysis of Cellular Morphology, Protein Stability, and RNA Quality

Abstract

Biobanks of fresh, unfixed human normal and malignant tissues represent a valuable source for gene expression analysis in translational cancer research and molecular pathology. However, the success of molecular and cellular analysis in both clinical and translational research is strongly dependent on the collection, handling, storage, and quality control of fresh human tissue samples. The aim of this study was to evaluate an innovative vacuum-based refrigerated system, as a logistically feasible technology to increase the collection of tissue specimens, preserving the integrity of cellular and molecular components. We tested randomly-selected tissues stored under vacuum at 4°C by using endpoints important for research and diagnosis, including tissue morphology, epitope stability, and RNA integrity. Gene expression was evaluated by qualitative and quantitative RT analysis of selected housekeeping and tissue-specific genes. Tissue morphology and overall protein stability were generally well preserved, being compromised only in gallbladder tissue. By contrast, phosphoprotein and RNA analysis demonstrated a time-dependent degree of degradation, with progressive loss of stability from 24 to 72 hours. However, this reduction in RNA quality did not represent a limitation for successful expression analysis of selected genes. Indeed, a comparative qualitative and quantitative RT-PCR analysis showed that RNA extracted from tissues stored under vacuum is suitable for gene expression profiling, but requires highly sensitive technologies, such as quantitative RT-PCR. These data suggest that the refrigerated vacuum-based system represents a suitable and feasible technology for routine transport of fresh specimens from surgery to biobanks, thus increasing the opportunity to collect biospecimens.

Quality assessment and preservation of RNA from biobank tissue specimens: a systematic review

“…Transit time from surgical resection to time of cryopreservation was reported by 9 of 15 studies, with times ranging from instantaneous fixation in theatre to 72 h 12 18 21–24 27–29. However, transit temperature was recorded infrequently, with only 27% of studies recording this outcome measure19–21 28 and the majority (3/4) transporting specimens at 4°C 20 21 28…”

“…Furthermore, 73% of studies assessed tissue biospecimen morphology12 17 18 20–22 24–29 as well as 47% of studies assessing biospecimen quality for downstream analyses 12 19 20 23 26 28 30…”

“…The majority of these studies (71%) used quantitative real-time PCR (qRT-PCR),12 20 23 26 30 while the remaining studies used either whole transcriptome sequencing28 or whole-genome expression profiling 19. Downstream targets chosen for qRT-PCR studies were varied, chosen either as known housekeeping genes,20 23 markers of different regulatory pathways or cancer-specific markers,12 28 30 including cancer-related miRNAs 26. In addition, three of the five qRT-PCR studies reported no change in gene expression of their chosen targets,20 23 26 while studies investigating markers of cellular function and cancer-related targets reported differences in a time-dependent manner 12 30.…”

“…Downstream targets chosen for qRT-PCR studies were varied, chosen either as known housekeeping genes,20 23 markers of different regulatory pathways or cancer-specific markers,12 28 30 including cancer-related miRNAs 26. In addition, three of the five qRT-PCR studies reported no change in gene expression of their chosen targets,20 23 26 while studies investigating markers of cellular function and cancer-related targets reported differences in a time-dependent manner 12 30. Whole transcriptome sequencing and genome expression profiling studies also reported insignificant changes to gene expression,28 including time delay to tumour freezing conditions 19…”

Storage by lyophilization – Resulting RNA quality is tissue dependent

“…However, these storage solutions are expensive (approx cost to preserve 100 mg of tissue: DNAgard ¼ US$1.60; RNAlater ¼ US$1.85) and can pose chemical hazards, again limiting their usefulness for long-distance shipping (see: Ambion MSDS# 4418289a). A recent development has seen the rise of vacuum-based refrigerated samples being transported [1], but refrigerated transport can be costly and risky over long distances.…”