A novel fixative improves opportunities of nucleic acids and proteomic analysis in human archive's tissues.
All tissues from biopsy or surgery origin are fixed and paraffin embedded as a routine procedure in the hospital departments of pathology. The traditional method of tissue preservation is the fixation in formalin, followed by paraffin embedding. In this way tissue's integrity is ensured also for future analyses, because there is no further chemical degradation of nucleic acids and proteins in tissues embedded in paraffin. After few sections for the histopathological examination the tissues are stored for decades in the hospital archives. Even if formalin fixation compromises the quality and integrity of nucleic acids, it has already been demonstrated that it is possible to recover and analyze DNA and RNA from these archive's tissues, even of autopsy origin. Protein analysis is on the contrary completely blocked, due to the fact that formalin fixation creates covalent links between proteins and the only way to study protein expression is immunohistochemistry. In this study we present our results concerning the use of a new formalin free fixative, the FineFIX. After extraction of nucleic acids, PCR and RT-PCR analyses were performed in DNA and RNA respectively. For DNA analysis it was possible to obtain amplicons of 2400 bps, while in formalin-fixed samples the maximum length achieved was less than 400 bps. RT-PCR analysis show that it was possible to study RNA fragments of 600 bps from FineFIX fixed tissues, against a maximum length of about 150 bps achieved by formalin-fixed tissues. These tissues were analyzed also by Western Blot analysis, showing that the proteins obtained from FineFIX treated samples are amenable and comparable in quality with those obtained from fresh frozen tissues. Protein extracts from FineFix treated tissues were also compared with fresh tissues'ones by two dimensional electrophoresis, demonstrating that the protein pattern were well comparable for number and distribution of the spots.
Stanta
Giorgio
G
Department of Clinical, Morphological and Technological Sciences, University of Trieste, 34149 Trieste, Italy. stanta@icgeb.org
Mucelli
Stefano Pozzi
SP
Petrera
Francesca
F
Bonin
Serena
S
Bussolati
Gianni
G
D001483:Base Sequence; D015153:Blotting, Western; D003110:Colonic Neoplasms / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004273:DNA, Neoplasm / Q000032:analysis / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D005404:Fixatives; D005557:Formaldehyde; D023281:Genomics / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry; D009363:Neoplasm Proteins / Q000032:analysis / Q000235:genetics; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D040901:Proteomics / Q000379:methods*; D012334:RNA, Neoplasm / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D004273:DNA, Neoplasm; D005404:Fixatives; D009363:Neoplasm Proteins; D012334:RNA, Neoplasm; D005557:Formaldehyde