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Novel zinc-based fixative for high quality DNA, RNA and protein analysis.

Lykidis D, Van Noorden S, Armstrong A, Spencer-Dene B, Li J, Zhuang Z, Stamp GW - Nucleic Acids Res. (2007)

Bottom Line: We have developed a reliable, cost-effective and non-toxic fixative to meet the needs of contemporary molecular pathobiology research, particularly in respect of RNA and DNA integrity.DNA sequences up to 2.4 kb in length and RNA fragments up to 361 bp in length were successfully amplified from Z7 fixed tissues, as demonstrated by PCR, RT-PCR and Real-Time PCR.This improved, non-toxic and economical tissue fixative could be applied for routine use in pathology laboratories to permit subsequent genomic/proteomic studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Histopathology, Division of Investigative Sciences, Imperial College London, Hammersmith Hospital Campus, Ducane Road, London, W12 ONN, UK. dimitrios.lykidis00@ic.ac.uk

ABSTRACT
We have developed a reliable, cost-effective and non-toxic fixative to meet the needs of contemporary molecular pathobiology research, particularly in respect of RNA and DNA integrity. The effects of 25 different fixative recipes on the fixed quality of tissues from C57BL/6 mice were investigated. Results from IHC, PCR, RT-PCR, RNA Agilent Bioanalyser and Real-Time PCR showed that a novel zinc-based fixative (Z7) containing zinc trifluoroacetate, zinc chloride and calcium acetate was significantly better than the standard zinc-based fixative (Z2) and neutral buffered formalin (NBF) for DNA, RNA and protein preservation. DNA sequences up to 2.4 kb in length and RNA fragments up to 361 bp in length were successfully amplified from Z7 fixed tissues, as demonstrated by PCR, RT-PCR and Real-Time PCR. Total protein analysis was achieved using 2-D gel electrophoresis. In addition, nucleic acids and proteins were very stable over a 6-14-month period. This improved, non-toxic and economical tissue fixative could be applied for routine use in pathology laboratories to permit subsequent genomic/proteomic studies.

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PCR amplification of a 2.4-kb Trefoil Factor 2 gene using DNA extracted from liver (L), spleen (S) and colon (C) tissue fixed in NBF and Z7. Fresh-frozen samples (Frozen) tissues were used as positive control and a water only sample (H2O) as negative control.
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Figure 6: PCR amplification of a 2.4-kb Trefoil Factor 2 gene using DNA extracted from liver (L), spleen (S) and colon (C) tissue fixed in NBF and Z7. Fresh-frozen samples (Frozen) tissues were used as positive control and a water only sample (H2O) as negative control.

Mentions: DNA was extracted from Z7-fixed, Z2-fixed, NBF-fixed and fresh-frozen mouse tissues and was assessed for the maximum size of fragment that could be amplified by Real-Time PCR using GAPDH primers which yield a product of 599 bp. Figure 2 shows results obtained from comparing the mean crossing thresholds of liver, spleen and colon tissues depending on fixation process and gene sequence amplified. Fragments up to 2.4 kb in length could be amplified from the Trefoil Factor 2 gene following Z7 fixation but could not be obtained from amplification of DNA fixed in Z2 or NBF. Results are shown in Figure 6.Figure 2.


Novel zinc-based fixative for high quality DNA, RNA and protein analysis.

Lykidis D, Van Noorden S, Armstrong A, Spencer-Dene B, Li J, Zhuang Z, Stamp GW - Nucleic Acids Res. (2007)

PCR amplification of a 2.4-kb Trefoil Factor 2 gene using DNA extracted from liver (L), spleen (S) and colon (C) tissue fixed in NBF and Z7. Fresh-frozen samples (Frozen) tissues were used as positive control and a water only sample (H2O) as negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919503&req=5

Figure 6: PCR amplification of a 2.4-kb Trefoil Factor 2 gene using DNA extracted from liver (L), spleen (S) and colon (C) tissue fixed in NBF and Z7. Fresh-frozen samples (Frozen) tissues were used as positive control and a water only sample (H2O) as negative control.
Mentions: DNA was extracted from Z7-fixed, Z2-fixed, NBF-fixed and fresh-frozen mouse tissues and was assessed for the maximum size of fragment that could be amplified by Real-Time PCR using GAPDH primers which yield a product of 599 bp. Figure 2 shows results obtained from comparing the mean crossing thresholds of liver, spleen and colon tissues depending on fixation process and gene sequence amplified. Fragments up to 2.4 kb in length could be amplified from the Trefoil Factor 2 gene following Z7 fixation but could not be obtained from amplification of DNA fixed in Z2 or NBF. Results are shown in Figure 6.Figure 2.

Bottom Line: We have developed a reliable, cost-effective and non-toxic fixative to meet the needs of contemporary molecular pathobiology research, particularly in respect of RNA and DNA integrity.DNA sequences up to 2.4 kb in length and RNA fragments up to 361 bp in length were successfully amplified from Z7 fixed tissues, as demonstrated by PCR, RT-PCR and Real-Time PCR.This improved, non-toxic and economical tissue fixative could be applied for routine use in pathology laboratories to permit subsequent genomic/proteomic studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Histopathology, Division of Investigative Sciences, Imperial College London, Hammersmith Hospital Campus, Ducane Road, London, W12 ONN, UK. dimitrios.lykidis00@ic.ac.uk

ABSTRACT
We have developed a reliable, cost-effective and non-toxic fixative to meet the needs of contemporary molecular pathobiology research, particularly in respect of RNA and DNA integrity. The effects of 25 different fixative recipes on the fixed quality of tissues from C57BL/6 mice were investigated. Results from IHC, PCR, RT-PCR, RNA Agilent Bioanalyser and Real-Time PCR showed that a novel zinc-based fixative (Z7) containing zinc trifluoroacetate, zinc chloride and calcium acetate was significantly better than the standard zinc-based fixative (Z2) and neutral buffered formalin (NBF) for DNA, RNA and protein preservation. DNA sequences up to 2.4 kb in length and RNA fragments up to 361 bp in length were successfully amplified from Z7 fixed tissues, as demonstrated by PCR, RT-PCR and Real-Time PCR. Total protein analysis was achieved using 2-D gel electrophoresis. In addition, nucleic acids and proteins were very stable over a 6-14-month period. This improved, non-toxic and economical tissue fixative could be applied for routine use in pathology laboratories to permit subsequent genomic/proteomic studies.

Show MeSH