Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Impact of ischemia and procurement conditions on gene expression in renal cell carcinoma.

Author(s): Liu NW, Sanford T, Srinivasan R, Liu JL, Khurana K, Aprelikova O, Valero V, Bechert C, Worrell R, Pinto PA, Yang Y, Merino M, Linehan WM, Bratslavsky G

Publication: Clin Cancer Res, 2013, Vol. 19, Page 42-9

PubMed ID: 23136194 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of ischemia time at three different temperatures on RNA integrity and gene expression in renal cell carcinoma specimens. Renal tumor specimens were obtained from ten patients with von Hippel-Lindau undergoing open partial nephrectomy. One piece of tumor was immediately embedded in OCT, snap-frozen in dry ice-chilled isopentane, and then stored in liquid nitrogen. The remaining pieces were stored in phosphate buffered saline at 4°C, 22°C, or 37°C for 5, 30, 60, 120, or 240 min, snap-frozen in dry ice-chilled isopentane, and stored in liquid nitrogen. Fifteen 20 μm thick frozen sections from each tissue specimen were homogenized in TRIZOL reagent and RNA was extracted using a standard chloroform extraction protocol followed by a cleanup process with the Qiagen RNeasy Mini Kit. RNA purity was assessed by spectrophotometry with a NanoDrop ND-1000 and RINs were assigned by a bioanalyzer. Gene expression analysis was performed using HG-U133A microarrays and results for three selected genes [CD44, FOS, and heat shock protein family A (Hsp70) member 1B (HSPA1B)] were validated by real-time qRT-PCR of specimens stored for 60 min at 4°C, 22°C, or 37°C compared to specimens that were snap-frozen immediately after resection.

   Summary of Findings:

   RNA was intact and RINs were ≥8 in specimens that were snap-frozen immediately after resection and those stored at 4°C for up to 240 min before snap-freezing. RINs were >7 for all specimens stored at 22°C or 37°C for up to 120 mins before freezing, but RNA was degraded and median RIN was 6.1 in specimens when stored at 22°C or 37°C for 240 mins after surgical resection and thus these specimens were not used for microarray analysis. The number of genes differentially expressed compared to specimens immediately snap-frozen after resection increased with increasing ischemia time and storage temperature (P<0.001, all), but only specimens stored at 4°C for 240 min, at 22°C for 60 min or longer, and all specimens stored at 37°C had a False Discovery Rate (FDR) less than 0.20. Multivariate analysis revealed similar trends with a higher number of genes differentially expressed compared to the immediately snap frozen control in the specimens stored at 37°C than those stored at 4°C and 22°C (P<0.001, all). The expression levels of four selected genes (vimentin, Bax, FOS, and CA9) were minimally affected by storage at 4°C, but vimentin and FOS increased and Bax and CA9 decreased with increasing ischemia time at 22°C and 37°C. Similar results were obtained by real-time qRT-PCR analysis of CD44, FOS, and HSPA1B with highest over-expression observed at 37°C, moderate increases at 22°C, and the lowest level of increases in specimens stored at 4°C.

   Biospecimens

   • Tissue - Kidney

   Preservative Types

   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Tissue microarray
   RNA	Spectrophotometry
   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 240 min
   Real-time qRT-PCR Specific	Targeted nucleic acid	CD44 FOS HSPA1B
   Biospecimen Acquisition	Pre-preservation condition	22°C for 5, 30, 60, 120, or 240 min 37°C for 5, 30, 60, 120, or 240 min
   Preaquisition	Warm ischemia time	5 min 30 min 60 min 120 min 240 min

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