The preservation of samples from the time of collection to the laboratory is critical for the success of the analysis. Multiple factors must be considered during a sampling under suboptimal conditions, when the use of cold storage or specific fixative may not be possible. During this time enzymatic or microbial degradation of molecules and structure is a primary concern [1]. Moreover, the portability and cost of storage equipment, their ease of use and toxicity play a role in their choice. The aim of this study was the identification of a reliable and economic method for tissue preservation suitable for both histological and molecular analysis to be adopted in “in field sampling”. Punch biopsies from cattle liver were collected at slaughterhouse and preserved in RNAlater®, with silica beads or vacuum-sealed, whereas control received no preservation treatment. For each method 5 samples were stored at 4°C and 5 at 24°C. At fixed times (4, 10, 24, 48 and 72 h) punch biopsies from each group and temperature were formalin-fixed and stored at -80°C for analysis. The sampling was repeated on 6 different livers. The integrity of nucleus, cytoplasm, preservation of liver structure and section borders were evaluated by histological analysis and graded 1 to 5. The integrity of the extracted DNA and RNA was evaluated though PCR and by means of an automated electrophoresis station, respectively. Statistical differences were determined by a two-tails ANOVA for repeated measures, followed by the proper post test. RNAlater® and silica beads poorly preserved the histological parameters evaluated, causing a marked vacuolization of the tissue, independently from temperature. Conversely, the vacuum-sealed samples and the controls showed a good grade of preservation until 48 h. There was a significant effect of the preservation method on the considered parameters (4°C: P<0.0001 for nucleus, cytoplasm and structure, and P<0.001 for borders; 24°C: P<0.0001 for all) as well as a significant effect of time (4°C: P<0.01 for nucleus and structure, and P<0.05 for cytoplasm; 24°C: P<0.0001 for nucleus and borders, and P<0.05 for cytoplasm). Regarding the DNA analysis, the expected band was obtained for each considered preservation method, temperature and time. The analysis of the RNA integrity showed acceptable result for samples preserved with silica beads, whereas the RNA integrity was not well-maintained in vacuum-sealed samples and controls. Indeed, there was a significant effect of the preservation method (P<0.0001 for 4°C and 24°C) and time (4°C: P<0.01; 24°C: P<0.05) on RNA integrity. The obtained results do not allow the identification of a unique preservation method suitable for both histological and molecular investigations. Interestingly, vacuum packing showed acceptable results for histology and RNA integrity, but only for short times of storage. However, this method has been widely tested in food industry, but not sufficiently studied for preservation of tissues for research purpose [2]. The preservation method appears to be more important than the time of storage and this finding should be taken into account for the selection of the sampling conservation.

COMPARISON OF DIFFERENT TISSUE PRESERVATION METHODS FOR HISTOLOGICAL AND MOLECULAR ANALYSIS

Laura Starvaggi Cucuzza;NICOLETTI, Arturo;Paola Pregel;Alessandra Sereno;Enrico Bollo;Francesca Tiziana Cannizzo;Frine Eleonora Scaglione
2018

Abstract

The preservation of samples from the time of collection to the laboratory is critical for the success of the analysis. Multiple factors must be considered during a sampling under suboptimal conditions, when the use of cold storage or specific fixative may not be possible. During this time enzymatic or microbial degradation of molecules and structure is a primary concern [1]. Moreover, the portability and cost of storage equipment, their ease of use and toxicity play a role in their choice. The aim of this study was the identification of a reliable and economic method for tissue preservation suitable for both histological and molecular analysis to be adopted in “in field sampling”. Punch biopsies from cattle liver were collected at slaughterhouse and preserved in RNAlater®, with silica beads or vacuum-sealed, whereas control received no preservation treatment. For each method 5 samples were stored at 4°C and 5 at 24°C. At fixed times (4, 10, 24, 48 and 72 h) punch biopsies from each group and temperature were formalin-fixed and stored at -80°C for analysis. The sampling was repeated on 6 different livers. The integrity of nucleus, cytoplasm, preservation of liver structure and section borders were evaluated by histological analysis and graded 1 to 5. The integrity of the extracted DNA and RNA was evaluated though PCR and by means of an automated electrophoresis station, respectively. Statistical differences were determined by a two-tails ANOVA for repeated measures, followed by the proper post test. RNAlater® and silica beads poorly preserved the histological parameters evaluated, causing a marked vacuolization of the tissue, independently from temperature. Conversely, the vacuum-sealed samples and the controls showed a good grade of preservation until 48 h. There was a significant effect of the preservation method on the considered parameters (4°C: P<0.0001 for nucleus, cytoplasm and structure, and P<0.001 for borders; 24°C: P<0.0001 for all) as well as a significant effect of time (4°C: P<0.01 for nucleus and structure, and P<0.05 for cytoplasm; 24°C: P<0.0001 for nucleus and borders, and P<0.05 for cytoplasm). Regarding the DNA analysis, the expected band was obtained for each considered preservation method, temperature and time. The analysis of the RNA integrity showed acceptable result for samples preserved with silica beads, whereas the RNA integrity was not well-maintained in vacuum-sealed samples and controls. Indeed, there was a significant effect of the preservation method (P<0.0001 for 4°C and 24°C) and time (4°C: P<0.01; 24°C: P<0.05) on RNA integrity. The obtained results do not allow the identification of a unique preservation method suitable for both histological and molecular investigations. Interestingly, vacuum packing showed acceptable results for histology and RNA integrity, but only for short times of storage. However, this method has been widely tested in food industry, but not sufficiently studied for preservation of tissues for research purpose [2]. The preservation method appears to be more important than the time of storage and this finding should be taken into account for the selection of the sampling conservation.
72nd Convegno Sisvet
Torino
20 – 22 Giugno 2018
Atti del 72° Convegno SISVet 2018
Soc. It. delle Scienze Veterinarie
372
372
9788890909214
Laura Starvaggi Cucuzza, Arturo Nicoletti, Paola Pregel, Alessandra Sereno, Enrico Bollo, Francesca Tiziana Cannizzo, Frine Eleonora Scaglione
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2318/1711206
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