Development of a zinc-based fixative for DNA, RNA and protein molecular studies

Abstract

Fixation is a chemical process by which tissue and cell components are ‘stabilised’ temporally and spatially, preserving morphology, nucleic acids, proteins and other cell constituents. There is a wide range of fixatives, based on their chemical function, but none of the compounds used is ideal, i.e. for preserving both morphology and integrity of DNA, RNA and protein. This thesis aimed to develop 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 30 different fixative recipes on the fixed quality of tissues from C57B16 mice colon, liver and spleen were investigated. Results from immunohistochemistry (IHC), polymerase chain reaction (PCR), Reverse transcription 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 2.4Kb 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 in fixed tissue blocks over a 12 month period. Fresh frozen samples were also included in this experimental model as controls for DNA and RNA integrity. Using Affymetrix exon microarrays it was shown that Z7-fixed samples had no statistical significant difference in the exon alternative splicing pattern compared to fresh-frozen samples, and could be used for further human cancer array genome studies. Mass Spectroscopy studies using synthetic peptides and their reaction with zinc trifluoroacetate showed that zinc is binding to at least one amino acid, either cysteine or histidine. Future work is required to elucidate the exact chemical mechanism of zinc fixation. In conclusion, this novel, non-toxic and economical tissue fixative could be applied for routine laboratory use to permit subsequent genomic/proteomic studies.