Effects of ex�vivo ischemia time and delayed processing on quality of specimens in tissue biobank

Abstract

The rna quality of tissue biobank is crucial for translational research; however, the effects of the ex vivo ischemia time on rna integrity and expression of genes related to hypoxia, stress, apoptosis and autophagy remains elusive. a total of 18 carcinoma tissues were stored at room temperature for 15 min, 30 min, 1, 2, 4, 8 and 24 h. The integrity and purity of isolated rna were analyzed. Furthermore, the gene expression of mTor, hypoxia-inducible factor 1α, phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit β isoform (Pi3KcB), threonine kinase 1 (aKT1), nF-κB, protein kinase aMP-activated catalytic subunit α1 (aMPKα1), caspase 8 (caSP8), unc-51 like autophagy activating kinase 1 and Fas cell surface death receptor were analyzed using reverse transcription-quantitative Pcr. The results demonstrated that rna integrity numbers (rins) remained stable in carcinoma tissues following ex vivo ischemia for 2 h at room temperature and that degradation began at 4 h (P<0.001). additionally, the expression of Pi3KcB, aKT1, aMPKα1 and caSP8 decreased at time points 8-24 h following ex vivo ischemia and delayed processing (P<0.001). in conclusion, >2 h of ex vivo ischemia and delayed processing induced rna degradation and a decrease in rin, and the gene expressions of Pi3KcB, aKT1, aMPKα1 and caSP8 may be considered as markers to evaluate tissue quality at the gene expression level, providing a method for the standard processing and assessment of tissue specimen.

Fusion‐positive non‐small cell lung carcinoma: Biological principles, clinical practice, and diagnostic implications

“…A couple of factors may influence the quality of the results from blood samples, such as the time between venule puncture and the centrifugation steps, the blood collection tubes and the type of buffer, the volume of plasma, and the storage conditions, notably for plasma 147,148 . Finally, the quality of the RNA can certainly depend of the intra tumor‐associated hypoxia and the presence of large necrotic areas in the tumor too 151 …”

“…147,148 Finally, the quality of the RNA can certainly depend of the intra tumor-associated hypoxia and the presence of large necrotic areas in the tumor too. 151 Finally, the results of molecular tests for gene fusion assessment are dependent not only by the quality of the diagnostic methods but, to a large extent, also on the many tissue-handling steps of the preanalytical phase. 152…”

FFPE-Based NGS Approaches into Clinical Practice: The Limits of Glory from a Pathologist Viewpoint

“…In fact, ischemia may lead to the increased or reduced transcription of some genes, modifying RNA levels, even after a short interval (30 min or less) [ 63 ]. In addition, the extensive degradation of RNA due to endogenous enzymes can occur if there is a delay in tissue fixation or preservation [ 64 , 65 ].…”

Cryopreservation Preserves Cell-Type Composition and Gene Expression Profiles in Bone Marrow Aspirates From Multiple Myeloma Patients

“…High-quality single-cell suspensions are required for scRNAseq experiments (Lafzi et al, 2018). Once tissue biopsies have been removed from a patient, they begin to undergo ischemiarelated gene expression changes and RNA degradation (Grizzle et al, 2016;Guo et al, 2020). To obtain an accurate profile of the patient sample, these types of changes should be minimized by processing samples as soon as they have been collected (Grizzle et al, 2016;Guo et al, 2020).…”

“…Once tissue biopsies have been removed from a patient, they begin to undergo ischemiarelated gene expression changes and RNA degradation (Grizzle et al, 2016;Guo et al, 2020). To obtain an accurate profile of the patient sample, these types of changes should be minimized by processing samples as soon as they have been collected (Grizzle et al, 2016;Guo et al, 2020). In addition, if celltype specific gene expression differences are to be compared in samples collected from the same subject, these samples ideally would be processed at the same time to minimize experimental batch effects (Alles et al, 2017).…”