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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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Related in: MedlinePlus

Proteomic comparison between fresh-frozen and zinc-fixed liver tissue by 2-D PAGE. (a) Fresh frozen control, (b) Z7 fixed sample and (c) NBF fixed sample. Representative 2-D PAGE image from one specimen. Yellow and red circles were assigned to indicate similarities and differences respectively in stained protein spots between the two samples.
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Figure 4: Proteomic comparison between fresh-frozen and zinc-fixed liver tissue by 2-D PAGE. (a) Fresh frozen control, (b) Z7 fixed sample and (c) NBF fixed sample. Representative 2-D PAGE image from one specimen. Yellow and red circles were assigned to indicate similarities and differences respectively in stained protein spots between the two samples.

Mentions: Proteins were extracted from liver fixed with Z7 and NBF and subjected to 2-D PAGE. Fresh-frozen liver tissue was used as positive control. Results are shown in Figure 4. These results show that Z7 is better than NBF and comparable to the fresh-frozen data.Figure 4.


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)

Proteomic comparison between fresh-frozen and zinc-fixed liver tissue by 2-D PAGE. (a) Fresh frozen control, (b) Z7 fixed sample and (c) NBF fixed sample. Representative 2-D PAGE image from one specimen. Yellow and red circles were assigned to indicate similarities and differences respectively in stained protein spots between the two samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Proteomic comparison between fresh-frozen and zinc-fixed liver tissue by 2-D PAGE. (a) Fresh frozen control, (b) Z7 fixed sample and (c) NBF fixed sample. Representative 2-D PAGE image from one specimen. Yellow and red circles were assigned to indicate similarities and differences respectively in stained protein spots between the two samples.
Mentions: Proteins were extracted from liver fixed with Z7 and NBF and subjected to 2-D PAGE. Fresh-frozen liver tissue was used as positive control. Results are shown in Figure 4. These results show that Z7 is better than NBF and comparable to the fresh-frozen data.Figure 4.

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
Related in: MedlinePlus