Biobanking of fresh frozen tissue: RNA is stable in nonfixed surgical specimens
| Autor: | Arne Östman, Zhi-Ping Ren, Mitsuhiro Ohshima, Fredrik Pontén, Johan Botling, Simin Tahmasebpoor, Patrick Micke |
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| Rok vydání: | 2006 |
| Předmět: |
DNA
Complementary Base Sequence Colon Gene Expression RNA Tissue Banks Cell Biology Ribosomal RNA Biology Polymerase Chain Reaction Molecular biology Pathology and Forensic Medicine Electrophoresis Microchip chemistry.chemical_compound chemistry Fresh Tissue Tissue bank Gene expression Humans DNA microarray Molecular Biology Gene DNA DNA Primers |
| Zdroj: | Laboratory Investigation. 86:202-211 |
| ISSN: | 0023-6837 |
| DOI: | 10.1038/labinvest.3700372 |
| Popis: | Molecular tools for tissue profiling, such as expression microarrays and real-time PCR, generally require collection of fresh frozen tissues as sources of high-quality RNA. The fragile nature of RNA prompted us to examine the effects of storage time and transport conditions with regard to RNA integrity and gene expression in nonfixed surgical human specimens. At surgery, fresh normal tonsil and colon tissue was cut into pieces and snap frozen. Additional fresh tissue pieces were (i) left at room temperature, (ii) kept on ice, (iii) in normal saline or (iv) in a commercial RNA-stabilizing buffer (RNAlater) and snap frozen after 0.5, 1, 3, 6 and 16 h. Structural RNA integrity was analysed by microchip electrophoresis. Surprisingly, RNA remained stable in both tissue types under all conditions tested for up to 6-16 h. Gene expression by real-time PCR of cfos, HIF1alpha, Bcl2, PCNA, TGFbeta1 and SMAD7 was analysed at different storage time points in tonsil tissue. Expression levels were essentially stable when samples were kept on ice, while marked regulation of single genes was observed during storage at room temperature, in normal saline and in RNAlater. Furthermore, we analysed selected tissue types from the local biobank representing 47 normal and malignant tissues transported on ice for up to 2-3 h before biobanking. RNA prepared from 45 of the 47 samples exhibited distinct ribosomal peaks indicating intact RNA. This study shows that RNA degradation is a minor problem during handling of fresh human tissue before biobanking. Our data indicate that nonfixed tissue specimens may be transported on ice for hours without any major influence on RNA quality and expression of the selected genes. However, further studies are warranted to clarify the impact of transport logistics on global gene expression. |
| Databáze: | OpenAIRE |
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