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Biobanking of fresh frozen tissue: RNA is stable in nonfixed surgical specimens
Patrick Micke, Mitsuhiro Ohshima, Simin Tahmasebpoor, Zhi-Ping Ren, Arne Östman, Fredrik Pontén and Johan Botling
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Figure 1.
Experimental design. Fresh surgical specimens of tonsil and colon tissue were cut in equal pieces of approximately 5 
5 5 mm. As references, some samples were frozen directly after surgery (time point 0 h). The other portions were stored at room temperature (RT), on ice, or immersed in normal saline (only tonsil tissue) or RNAlater. After 0.5, 1, 3, 6 and 16 h, tissue fractions were removed, snap frozen and stored at -80°C until further processing. To extract the RNA, the samples were cut in a cryostat and sections were transferred to RNA extraction buffer (Trizol or Qiagen extraction buffer). After RNA purification, the amount and quality of RNA was analysed using microchip gel electrophoresis (Bioanalyzer). Distinct 18S and 28S ribosomal RNA peaks indicate intact RNA.
Figure 2.
RNA integrity of tonsil tissue under different transport conditions. Purified RNA was analysed using microchip gel electrophoresis (Bioanalyzer) and the results are presented as electropherograms. The RNA of all samples (stored at room temperature (RT), on ice, in normal saline or RNAlater) at all time points (0, 0.5, 1, 3, 6 and 16 h) showed distinct ribosomal peaks indicating no significant degradation. Representative RNA samples purified with a Qiagen column are shown.
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Figure 3.
RNA integrity over time. In the tonsil samples, the ratios of the ribosomal peaks (28S/18S) were calculated based on the electropherograms. RNA was extracted from four samples (two samples with Trizol and two with the Qiagen-Kit). Values represent the mean and standard deviation of these four analyses.
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Figure 4.
Morphology of tonsil tissue under different transport conditions. Frozen tonsil specimens were cut in sections (4 
m) by the tape transfer technique and stained with hematoxylin–eosin. The panels show the histology of a reference sample that was frozen immediately after surgery (a) and sections from samples stored for 16 h at room temperature (b), on ice (c), in normal saline (d) and in RNAlater (e). Original magnification 10.
Figure 5.
RNA integrity of colon tissue under different transport conditions. Normal colon tissue was processed as outlined in Figure 1. Sample storage in saline was omitted in this experiment due to lack of available tissue. Purified RNA was analysed using microchip gel electrophoresis (Bioanalyzer) and the results are presented as electropherograms. The RNA of all samples (stored at RT, on ice or in RNAlater) and at all time points (0, 0.5, 1, 3, 6, 16 h) showed distinct ribosomal peaks indicating no significant degradation. Representative RNA samples purified with a Qiagen column are shown.
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Figure 6.
RNA was purified from a number of different tissues of the local frozen tissue biobank (Department of Pathology, Uppsala University Hospital). Tissue type as indicated below each bar (SCC=squamous cell carcinoma, BCC=basal cell carcinoma). The RNA was extracted using Trizol and analysed with a Bioanalyzer. The ratios of both ribosomal peaks (28S/18S) were calculated based on the electropherograms. Only two samples failed to show ribosomal RNA peaks.
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Figure 7.
Gene expression levels under different storage conditions. RNA isolated from the tonsil tissue experiment was transcribed to cDNA and analysed using primers for cfos, TGF
1, SMAD7, HIF1
, Bcl2 and PCNA. The results were normalised to GAPDH expression. Relative gene expression at three different time points (0.5, 6 and 16 h) was compared to time point 0 h (set to 1.0). The results are shown as mean and standard deviation.
