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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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(a) PCR of the GAPDH gene using DNA extracted from liver (L), spleen (S) and colon (C) tissue fixed in NBF, Z7 and stored in paraffin blocks at RT for 14 months (Old), compared with DNA extracted from liver (L), spleen (S) and colon (C) tissue fixed in NBF, Z7 and stored in paraffin blocks at RT for less than a week (New). Fresh-frozen samples (Frozen) tissues were used as positive control and a water only sample (H2O) as negative control. (b) PCR amplification of the β-actin gene using DNA extracted from liver (L), spleen (S) and colon (C) tissue fixed in NBF, Z7 and paraffin blocks stored at RT for 14 months (Old), compared with DNA extracted from liver (L), spleen (S) and colon (C) tissue fixed in NBF, Z7 and stored in paraffin blocks at RT for less than a week (New). Fresh-frozen samples (Frozen) tissues were used as positive control and a water only sample (H2O) as negative control. (c) Real-Time PCR showing amplification of the GAPDH gene using DNA extracted from liver, spleen and colon tissue fixed in NBF, Z7 and stored in paraffin blocks at RT for 14 months (Old), compared with DNA extracted from liver, spleen and colon (C) tissue fixed in NBF, Z7 and stored in paraffin blocks at RT for less than a week (New). Fresh-frozen samples (Frozen) tissues were used as positive control and a water only sample (H2O) as negative control. Bars indicate the ±SEM.
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Figure 5: (a) PCR of the GAPDH gene using DNA extracted from liver (L), spleen (S) and colon (C) tissue fixed in NBF, Z7 and stored in paraffin blocks at RT for 14 months (Old), compared with DNA extracted from liver (L), spleen (S) and colon (C) tissue fixed in NBF, Z7 and stored in paraffin blocks at RT for less than a week (New). Fresh-frozen samples (Frozen) tissues were used as positive control and a water only sample (H2O) as negative control. (b) PCR amplification of the β-actin gene using DNA extracted from liver (L), spleen (S) and colon (C) tissue fixed in NBF, Z7 and paraffin blocks stored at RT for 14 months (Old), compared with DNA extracted from liver (L), spleen (S) and colon (C) tissue fixed in NBF, Z7 and stored in paraffin blocks at RT for less than a week (New). Fresh-frozen samples (Frozen) tissues were used as positive control and a water only sample (H2O) as negative control. (c) Real-Time PCR showing amplification of the GAPDH gene using DNA extracted from liver, spleen and colon tissue fixed in NBF, Z7 and stored in paraffin blocks at RT for 14 months (Old), compared with DNA extracted from liver, spleen and colon (C) tissue fixed in NBF, Z7 and stored in paraffin blocks at RT for less than a week (New). Fresh-frozen samples (Frozen) tissues were used as positive control and a water only sample (H2O) as negative control. Bars indicate the ±SEM.

Mentions: DNA was extracted from paraffin blocks fixed with NBF and Z7 stored at ambient temperature for 14 months and compared with DNA samples from paraffin blocks stored for less than a week. Conventional PCR results using two housekeeping genes, GAPDH and β-actin, showed that DNA quality from Z7-fixed archival and newly stored tissues was significantly better than from all NBF-fixed tissue (Figure 5a and b). No significant difference was observed between archival stored Z7-fixed and newly stored Z7-fixed samples.Figure 5.


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)

(a) PCR of the GAPDH gene using DNA extracted from liver (L), spleen (S) and colon (C) tissue fixed in NBF, Z7 and stored in paraffin blocks at RT for 14 months (Old), compared with DNA extracted from liver (L), spleen (S) and colon (C) tissue fixed in NBF, Z7 and stored in paraffin blocks at RT for less than a week (New). Fresh-frozen samples (Frozen) tissues were used as positive control and a water only sample (H2O) as negative control. (b) PCR amplification of the β-actin gene using DNA extracted from liver (L), spleen (S) and colon (C) tissue fixed in NBF, Z7 and paraffin blocks stored at RT for 14 months (Old), compared with DNA extracted from liver (L), spleen (S) and colon (C) tissue fixed in NBF, Z7 and stored in paraffin blocks at RT for less than a week (New). Fresh-frozen samples (Frozen) tissues were used as positive control and a water only sample (H2O) as negative control. (c) Real-Time PCR showing amplification of the GAPDH gene using DNA extracted from liver, spleen and colon tissue fixed in NBF, Z7 and stored in paraffin blocks at RT for 14 months (Old), compared with DNA extracted from liver, spleen and colon (C) tissue fixed in NBF, Z7 and stored in paraffin blocks at RT for less than a week (New). Fresh-frozen samples (Frozen) tissues were used as positive control and a water only sample (H2O) as negative control. Bars indicate the ±SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 5: (a) PCR of the GAPDH gene using DNA extracted from liver (L), spleen (S) and colon (C) tissue fixed in NBF, Z7 and stored in paraffin blocks at RT for 14 months (Old), compared with DNA extracted from liver (L), spleen (S) and colon (C) tissue fixed in NBF, Z7 and stored in paraffin blocks at RT for less than a week (New). Fresh-frozen samples (Frozen) tissues were used as positive control and a water only sample (H2O) as negative control. (b) PCR amplification of the β-actin gene using DNA extracted from liver (L), spleen (S) and colon (C) tissue fixed in NBF, Z7 and paraffin blocks stored at RT for 14 months (Old), compared with DNA extracted from liver (L), spleen (S) and colon (C) tissue fixed in NBF, Z7 and stored in paraffin blocks at RT for less than a week (New). Fresh-frozen samples (Frozen) tissues were used as positive control and a water only sample (H2O) as negative control. (c) Real-Time PCR showing amplification of the GAPDH gene using DNA extracted from liver, spleen and colon tissue fixed in NBF, Z7 and stored in paraffin blocks at RT for 14 months (Old), compared with DNA extracted from liver, spleen and colon (C) tissue fixed in NBF, Z7 and stored in paraffin blocks at RT for less than a week (New). Fresh-frozen samples (Frozen) tissues were used as positive control and a water only sample (H2O) as negative control. Bars indicate the ±SEM.
Mentions: DNA was extracted from paraffin blocks fixed with NBF and Z7 stored at ambient temperature for 14 months and compared with DNA samples from paraffin blocks stored for less than a week. Conventional PCR results using two housekeeping genes, GAPDH and β-actin, showed that DNA quality from Z7-fixed archival and newly stored tissues was significantly better than from all NBF-fixed tissue (Figure 5a and b). No significant difference was observed between archival stored Z7-fixed and newly stored Z7-fixed samples.Figure 5.

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