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The influence of tissue procurement procedures on RNA integrity, gene expression, and morphology in porcine and human liver tissue.

Author(s): Kap M, Sieuwerts AM, Kubista M, Oomen M, Arshad S, Riegman P

Publication: Biopreserv Biobank, 2015, Vol. 13, Page 200-6

PubMed ID: 26035010 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of specimen size, 60 min room temperature storage, and storage condition on tissue morphology, RNA integrity, and gene expression in human liver tissue. Specimens were obtained after 3.5 h surgery (warm ischemia) and 30 min of transport at room temperature (RT) and frozen in a metal container cooled in liquid nitrogen either immediately or after 60 min of storage at room temperature. During the 60 min storage at room temperature, large (8 x 4 x 4 mm) specimens were stored dry, while small (2 x 2 x 4 mm) specimens were stored dry, on moist gauze, or in isotonic salt solution. Sections (4 mm) from all specimens were stained with H&E for morphological assessment.  Ten (large specimens), or forty (small specimens) 10 µm sections were placed in Qiazol and disrupted by vigorous shaking. RNA was isolated from disrupted sections using the (mi)RNeasy mini kit and RIN values determined using an Agilent bioanalyzer. RT-qPCR of the reporter genes hypoxanthine phosphoribosyltransferase 1 (HPRT1), hydroxymethylbilane synthase (HMBS), glucuronidase, beta (GUSB), peptidylprolyl isomerase B (PPIB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and TATA box binding protein (TBP) was performed on the Stratagene Mx3000P platform.

   Summary of Findings:

   Storage at room temperature for 60 min resulted in a significant decrease in average RIN on large and small specimens stored dry (P=0.015, both) and this decrease was more pronounced in small specimens than large specimens (average RIN change of 0.85 versus 0.3). Further, although RIN were not significantly different for small specimens among the three storage conditions, the changes in RIN were more significant when stored on moist gauze (P=0.009) or in salt solution (P=0.002). Storage at room temperature for 60 min had no effect on gene expression in large specimens but had a significant effect on HMBS, HPRT1, and TBP expression in small specimens, regardless of storage condition (P=0.002 each) and GAPDH and GUSB were also affected in these specimens when they were stored on moist gauze or in salt solution (P< 0.05 for all). Further, levels of all 6 genes were lower when small specimens were transported on moist gauze or in salt solution rather than dry (P< 0.05 for all). The authors report no significant morphological differences among the storage conditions.

   Biospecimens

   • Tissue - Liver

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Real-time qRT-PCR
   Morphology	H-and-E microscopy

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Aliquot size/volume	small (2 x 2 x 4 mm) large (8 x 4 x 4 mm)
   Storage	Time at room temperature	0 min 60 min
   Storage	Storage conditions	dry on moist gauze in salt solution

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