Tissue Ischemia Time Affects Gene and Protein Expression Patterns within Minutes Following Surgical Tumor Excision

Author(s): Spruessel Annika, Steimann Garnet, Jung Mira, Lee Sung A, Carr Theresa, Fentz Anne-Kristin, Spangenberg Joerg, Zornig C, Juhl Hartmut H, David Kerstin A

Publication: Biotechniques, 2004, Vol. 36, Page 1030

PubMed ID: 15211754 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine if varying durations of ischemia (defined as the duration of room temperature storage post surgical resection; 5-30 min) impacts quantitative gene expression in surgically resected colon specimens as measured by microarray. Although effects were not studied, specimens were also subjected to a warm ischemia time (the time between arterial clamp placement and tissue resection) between 30 and 40 minutes.

   Summary of Findings:

   RNA quality was unaffected by ischemia durations of up to 30 min as visualized by gel electrophoresis, although data was not shown. Within 30 min of surgical resection, 20% of the expressed transcripts differed greater than 2-fold from baseline (5 min), while after only 15 min, 10-15% of examined transcripts were differentially expressed compared to baseline. Ischemia affected genes included hypoxia-regulated genes, oncogenes, signal transducers, nuclear genes, kinases, chaperones, and those involved in cell growth.

   Biospecimens

   • Tissue - Colorectal

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Normal Adjacent

   Platform:

   Analyte	Technology Platform
   RNA	DNA microarray
   RNA	Electrophoresis

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Cold ischemia time	5 min 8 min 10 min 12 min 15 min 20 min 30 min

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2. Study Purpose

   The purpose of this study was to determine if varying durations of ischemia (defined as the duration of room temperature storage post surgical resection; 5-30 min) impacts quantitative protein expression of surgically resected colon specimens, as measured by surface-enhanced laser desoprtion/ ionization time-of-flight mass spectrometry. Although effects were not studied, specimens were also subjected to a warm ischemia time (the time between arterial clamp placement and tissue resection) between 30 and 40 minutes.

   Summary of Findings:

   Over 30% of the proteins profiled displayed differential expression (>2-fold) after 30 min of cold ischemia time, and the majority of the proteins affected were altered within 15 min. Differential expression occurred in normal and cancerous specimens.

   Biospecimens

   • Tissue - Colorectal

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma
   • Neoplastic - Normal Adjacent

   Platform:

   Analyte	Technology Platform
   Protein	SELDI-TOF MS

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Cold ischemia time	8 min 10 min 12 min 15 min 20 min 30 min 5 min

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3. Study Purpose

   To determine if varying durations of cold ischemia (5-30 min) of surgically resected colon specimens impact quantitative gene expression, as measured by real-time quantitative RT-PCR, and to verify results of microarray analysis. Although effects were not studied, specimens were also subjected to a warm ischemia time (the time between arterial clamp placement and tissue resection) between 30 and 40 minutes.

   Summary of Findings:

   Expression of specific genes (GADPH, CYCA, CK20, CEA, c-fos, HIF-1alpha, HO-1) measured by real-time qRT-PCR verified microarray results. Of the hypoxia-regulated genes investigated HIF-1 and c-fos, but not HO-1, displayed quick and robust changes in expression after 8-10 min of cold ischemia in comparison to controls preserved 5 minutes post-resection. Cytoskeletal genes (CK20) and tumor-associated antigens (CEA) also displayed significant and time-dependent changes in gene expression.

   Biospecimens

   • Tissue - Colorectal

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Normal Adjacent

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Real-time qRT-PCR Specific	Targeted nucleic acid	GADPH CYCA CK20 CEA c-fos HIF-1alpha HO-1
   Biospecimen Acquisition	Cold ischemia time	5 min 8 min 10 min 12 min 15 min 20 min 30 min

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