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Pre-Analytical Determination of the Effect of Extended Warm or Cold Ischaemia on RNA Stability in the Human Ileum Mucosa.

Author(s): Lee SM, Schelcher C, Thasler R, Schiergens TS, Thasler WE

Publication: PLoS One, 2015, Vol. 10, Page e0138214

PubMed ID: 26371767 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the effects of simulated warm and cold ischemia and preservation by snap-freezing or RNAlater on tissue and RNA integrity and RNA levels in ileum mucosa specimens. A piece of terminal ileum was collected during hemicolectomy from each of six patients with colon carcinoma. A 5 cm length of ileum was isolated by stapling and then removed immediately after clamping of the vascular supply (<1 min). Tissue specimens were halved and stored at 37°C (>1, 15, 30, 60 and 90 min) or at 4°C (>1 min, 30 min, 60 min, 3h and 6 h) and subsequently snap-frozen in liquid nitrogen or preserved in RNAlater. RNA was isolated with an RNeasy kit, quantified by spectrophotometry, and RIN values assigned by a bioanalyzer. Four appropriate reference genes (HPRT1, GUSB, PSMB6, TBP) were chosen prior to RT-qPCR analysis with TaqMan Gene Expression Assays of the genes of interest (MYC, Hif1α, CDX2, HMOX1 and IL1β). FFPE specimens were sectioned at 6 μm thickness and H&E stained.

   Summary of Findings:

   Warm or cold ischemia had no effect on ileum tissue integrity for any of the timepoints examined. Small but significant differences in nucleic acid purity compared to 0 min controls were observed in RNAlater preserved specimens after 15 min warm ischemia or 30 min cold ischemia and in snap-frozen specimens after 60 min warm ischemia or 180 min cold ischemia. Further, there were no statistically significant differences with warm or cold ischemia in RIN of specimens preserved by snap-freezing or in RNAlater, but RIN were 1.2-fold higher in specimens stored in RNAlater compared to snap-frozen specimens after 15 minutes of warm ischemia and 180 minutes of cold ischemia (P<0.05). Normalized gene expressions of MYC, HIF1α, CDX2, HMOX1, or IL1β in ileum tissue were not affected by warm or cold ischemia or preservation by snap-freezing or RNAlater except for 2.6-fold higher relative expression of MYC in RNAlater specimens than snap-frozen specimens after 90 min of cold ischemia.

   Biospecimens

   • Tissue - Colorectal

   Preservative Types

   • RNAlater
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Spectrophotometry
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Preaquisition	Warm ischemia time	<5 min 15 min 30 min 60 min 90 min
   Biospecimen Preservation	Type of fixation/preservation	Snap frozen RNAlater
   Real-time qRT-PCR Specific	Targeted nucleic acid	MYC Hif1α CDX2 HMOX1 IL1β
   Biospecimen Acquisition	Cold ischemia time	>1 min 30 min 60 min 3h 6 h

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