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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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Assessment of mouse liver RNA quality using the Agilent 2100 Bioanalyser. (a) Typical graphs showing severely degraded, partially degraded and intact RNA. (b) Graphs showing RNA quality from tissues fixed in: NBF, Z2, Z7 and also fresh-frozen mouse liver.
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Figure 1: Assessment of mouse liver RNA quality using the Agilent 2100 Bioanalyser. (a) Typical graphs showing severely degraded, partially degraded and intact RNA. (b) Graphs showing RNA quality from tissues fixed in: NBF, Z2, Z7 and also fresh-frozen mouse liver.

Mentions: RNA was extracted from mouse liver sections fixed in Z2, Z7 and NBF and from fresh-frozen samples and was assessed for integrity using the Agilent 2100 Bioanalyser (Figure 1). Typical traces that can be obtained from RNA in various degrees of degradation are shown for purposes of comparison. It can be seen that Z7 has two peaks corresponding to 12S and 18S RNA and that it is signi-ficantly better than both Z2 and NBF at preserving RNA structure and integrity.Figure 1.


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)

Assessment of mouse liver RNA quality using the Agilent 2100 Bioanalyser. (a) Typical graphs showing severely degraded, partially degraded and intact RNA. (b) Graphs showing RNA quality from tissues fixed in: NBF, Z2, Z7 and also fresh-frozen mouse liver.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Assessment of mouse liver RNA quality using the Agilent 2100 Bioanalyser. (a) Typical graphs showing severely degraded, partially degraded and intact RNA. (b) Graphs showing RNA quality from tissues fixed in: NBF, Z2, Z7 and also fresh-frozen mouse liver.
Mentions: RNA was extracted from mouse liver sections fixed in Z2, Z7 and NBF and from fresh-frozen samples and was assessed for integrity using the Agilent 2100 Bioanalyser (Figure 1). Typical traces that can be obtained from RNA in various degrees of degradation are shown for purposes of comparison. It can be seen that Z7 has two peaks corresponding to 12S and 18S RNA and that it is signi-ficantly better than both Z2 and NBF at preserving RNA structure and integrity.Figure 1.

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