Tissue ischemia time affects gene and protein expression patterns within minutes following surgical tumor excision

Abstract

The aim of this study was to determine the impact of ischemia on gene and protein expression profiles of healthy and malignant colon tissue and, thus, on screening studies for identification of molecular targets and diagnostic molecular patterns. Healthy and malignant colon tissue were snap-frozen at various time points (3-30 min) after colon resection. Gene and protein expression were determined by microarray (HG-U133A chips) and surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) technology (CM10 chips, SAX2 chips, and IMAC3Ni chips), respectively. Real-time reverse transcription PCR (RT-PCR) was used for comparative measurement of expression of particular genes. Initial changes of gene and protein expression profiles were already observed 5-8 min after colon resection. Fifteen minutes after surgery, 10%-15% of molecules, and after 30 min, 20% of all detectable genes and proteins, respectively, differed significantly from the baseline values. Significant changes of expression were found in most functional groups. As confirmed by real-time RT-PCR, this included not only known hypoxia-related molecules (HIF-1 alpha, c-fos, HO-1) but also cytoskeletal genes (e.g., CK20) and tumor-associated antigens (e.g., CEA). In conclusion, preanalytical factors, such as tissue ischemia time, dramatically affect molecular data. Control of these variables is mandatory to obtain reliable data in screening programs for molecular targets and diagnostic molecular patterns.

The Use of Immunohistochemistry for Biomarker Assessment—Can It Compete with Other Technologies?

“…Although it is generally accepted that up to 2 hours of warm ischemia (time without fixation) does not alter protein, DNA or RNA conformation or preservation of microscopic, and IHC features (Burns et al 2009), Samaratunga et al (2011) have reported that even before resection has been completed, intraoperative manipulations can alter gene transcription levels during radical prostatectomies. Spruessel et al (2004) used microarray analysis, quantitative real-time polymerase chain reaction, and surface enhanced laser desorption ionization time of flight to investigate changes in gene and protein expression in normal and cancerous colon tissue at various stages of ischemia. Initial changes of gene and protein expression profiles were observed 5 minutes after resection; and after 30 minutes, 20% of all detectable genes and proteins differed significantly from baseline values.…”

Guidelines for the use and interpretation of assays for monitoring autophagy

“…811,812 The possibilities of postmortem autolysis and fixation artifacts must always be taken into consideration when interpreting changes attributed to autophagy. 813 Analyses of these types of samples require not only special antigen retrieval techniques, but also histopathological experience to interpret autophagy studies by immunohistochemistry, immunofluorescence or TEM.…”

Modification of the Standard Trizol-Based Technique Improves the Integrity of RNA Isolated from RNase-Rich Placental Tissue

“…Limiting RNA degradation during tissue processing and quantifying RNA degradation by DF are important, particularly when clinical samples are used, because time intervals from sample procurement to RNA preservation impact RNA integrity (6 ). The use of RNA with comparable low DFs should provide microarray results that more accurately reflect biology rather than variable RNA degradation.…”