Gene expression in colorectal neoplasia: modifications induced by tissue ischaemic time and tissue handling protocol.

Author(s): Bray SE, Paulin FE, Fong SC, Baker L, Carey FA, Levison DA, Steele RJ, Kernohan NM

Publication: Histopathology, 2010, Vol. 56, Page 240-50

PubMed ID: 20102403 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The aim of this study was to compare two methods of tissue preservation (immersion in RNAlater overnight at 4 degrees C and snap freezing in liquid nitrogen) on RNA quality and mRNA expression profiles and on individual transcript levels in biopsy specimens collected from ten patients. All specimens were stored at -80 degrees C after initial preservation. For microarray analysis, differentially preserved case-matched adenocarcinoma specimens representing varying degrees of tumor progression were compared using a Human Genome U133 Plus 2.0 array.

   Summary of Findings:

   Specimens preserved in RNAlater at 4 degrees C overnight had significantly higher RINs when compared to case-matched controls snap frozen in liquid nitrogen (8.5 versus 6.5; p<0.001) but similar 28S to 18S rRNA ratios. Microarray analysis revealed that while gene expression profiles clustered based on patient and not preservation method, 2.3% of the genes assessed differed by more than 2-fold when preservation methods were compared. Expression was greater in snap frozen specimens for 55% of the affected genes, and lower for 45%, when compared to specimens preserved in RNAlater for all three cases examined. Transcripts with higher levels in snap frozen specimens represented genes involved in enzyme regulation, while those with higher levels in RNAlater preserved specimens were involved in translation and regulation of RNA metabolism. Microarray results were confirmed by qRT-PCR analysis for a subset of transcripts, with higher levels of mitogen-activated protein kinase kinase 3 (MAP2K3, p=0.022), and lower levels of insulin-like growth factor 1 (IGF1, p=0.037) and the G-protein coupled receptor 34 (GPR34, p=0.023) in snap frozen specimens compared to RNAlater specimens.

   Biospecimens

   • Tissue - Colorectal

   Preservative Types

   • RNAlater
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma
   • Neoplastic - Benign

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	RNAlater Snap frozen
   Real-time qRT-PCR Specific	Targeted nucleic acid	IGF-1 GPR34 MAP2K3

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2. Study Purpose

   The purpose of this study was to determine the effect a post-excision pre-preservation delay at room temperature (cold ischemia time) has on RNA integrity and subsequent expression analyses. Biopsies collected from 10 patients were snap frozen in liquid nitrogen following 0, 15, 30, 60, or 120 min at room temperature. All specimens were stored at -80 degrees C after initial preservation.

   Summary of Findings:

   Although differences in RIN that were attributable to cold ischemia time were not statistically significant, a cold ischemia time of 30 min or longer resulted in an increase in variability and lower confidence intervals. Differences in transcript levels greater than 2-fold were detected after 15 min of ischemia, the earliest ischemia timepoint examined, compared to 0 min controls via a Human Genome U133 Plus 2.0 array. The number of affected genes increased 4-fold as ischemia time progressed from 15 min to 120 min and included both increases and decreases in expression. Although gene expression profiles clustered by patient and not ischemia time, approximately 30% of the genes represented on the array displayed altered levels during the ischemia time course. While 168 transcripts were consistently affected by ischemia at every time point examined they were diverse in their ontology. Microarray results were confirmed by qRT-PCR analysis for a subset of transcripts, as Krueppel-like factor 6 levels were significantly higher (KLF6, p=0.043) and MDM4 levels were nonsignificantly lower (p=0.479) following an ischemia time of 120 min compared to 0 min controls.

   Biospecimens

   • Tissue - Colorectal

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma
   • Neoplastic - Benign

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Cold ischemia time	0 min 15 min 30 min 60 min 120 min
   Real-time qRT-PCR Specific	Targeted nucleic acid	KLF6 MDM4

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