Formalin fixation at low temperature better preserves nucleic acid integrity. Gianni Bussolati. University of Turin
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1 Formalin fixation at low temperature better preserves nucleic acid integrity Gianni Bussolati University of Turin
2 Disclosure of interests: G.B. was originally responsible for the invention of the Cold Fix procedure, but is not the owner of the patents and does not receive Royalties.
3 RNA yeld from FF-PET Fragmented Result of RNAse activity (?)
4 formaldehyde does not inhibit RNAses. Ding J, Ichikawa Y, Ishikawa T, Shimada H. Scand J Clin Lab Invest. 2004;64(3):229-35
5 100 Percent 50 Fixation Penetration
6 100 Percent 50 Fixation RNAse Penetration h Room Temperature
7 Burka ER,J.Clin. Invest. 48, 1724,1969
8 100 Percent 50 Fixation RNAse Penetration h Temperature 4 C
9 Experimental protocols for Cold Fixation: 1) Fixation in PBF at 4 C for 24 h, followed by heating at 50 C for 20 min using MW, followed by Ethanol dehydration and Paraffin embedding. 2) Fixation in PBF at 4 C for 24 h, followed by heating at 90 C for 5 min, followed by Ethanol dehydration and Paraffin embedding. 3) Fixation in PBF at 4 C for 24 h, followed by 95%Ethanol at 4 C for 2 h, then standard Ethanol dehydration and Paraffin embedding.
10 Cases of: Breast Cancer Colon Cancer Stomach cancer Pancreatic cancer Study flowchart. Flowchart illustrating the design of the study.
11 Colon cancer processed either routinely (c, d) or following the CF procedure (a, b) a b c d
12 Breast cancer processed either routinely (c, d) or following the CF procedure (a, b) a b c d
13 Stomach cancer processed either routinely (c, d) or following the CF procedure (a, b) a b c d
14 Standard fixation (SF) procedure vs Cold fixation (CF) procedure G6PD 660 bp MW Molecular Weight SF1 Standard Fixation Sample 1 SF2 Standard Fixation Sample 2 CF1 Cold Fixation Sample 1 CF2 Cold Fixation Sample 2 FZ1 Frozen Sample 1 MW SF1 SF2 CF1 CF2 CK-20 MW SF1 SF2 CF1 CF2 FZ1 SF1 SF2 CF1 CF2 FZ1 SF1 SF2 CF1 CF2 FZ1 329 bp 500 bp 716 bp
15 Bussolati et al., Figure 3. Messenger RNAs from Cold-Fixed samples are detected by microaarray probes hybridizing more than 500b upstream from the reverse transcription start site. Graph showing the fraction of probes with detectable signal (y-axis) for each bin of distance of target sequence from the mrna poly(a) site from which RT is initiated (x-axis).
16 The plots show probes of less than 200 bases (A, E) and bases (B, F).
17 The plots show probes of bases (C, G), more than 700 bases (D, H)
18 Venn diagram comparing in parallel the genes detected in 2 fresh samples (red) and 2 cold fixed (CFFPE) samples (blue) from the same cases of breast cancer. Using the software Agilent Genespring we generated lists of genes detected in cold fixed and fresh sample. The number of genes detected in the fresh samples was and in the cold fixed samples was genes. The number of genes detected in the fresh samples was only slightly (4.12%) higher than in the cold-fixed ones.
19 PLoS One. 2011;6(6):e Epub Jun 15 A formalin fixation procedure preserving nucleic acid integrity. Gianni Bussolati, Laura Annaratone, Enzo Medico, Giuseppe D Armento and Anna Sapino.
20 The Cold Fix Apparatus
21 Assessment of RNA conservation in Cold-Formalin Fixed Paraffin Embedded Tissue blocks after two years MW A- 0 A- 2 B- 0 B- 2 C- 0 C- 2 CTRL+ CTRL - RT-PCR for detection of CK-20 in colorectal cancer (500bp). Samples: A- 0 A- 2 B- 0 B- 2 C- 0 C- 2 RNA extraction year 2010 RNA extraction year 2012 RNA extraction year 2010 RNA extraction year 2012 RNA extraction year 2010 RNA extraction year 2012
22
23 Surgery Surgical Specimen >2cm. Under-vacuum (Tissue Safe) Surgical Theatre Kept at 4 C till transfer (1-72h) Tissue Banking (-80 C) Grossing P.B. Formalin Gene Expr. Profiling (12-24h) Pathol. Labs. Embedding
24 Nature ;210:
25 The scatter plot puts in comparison 2^-DeltaCt values obtained from a fixed sample with 2^-DeltaCt values obtained from the same sample, but frozen, both evaluated with Breast Cancer RT2 Profiler PCR Array (QIAGEN) and analysed with QIAGEN Web-Based PCR Array Data Analysis Software.
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