Prethodno priopćenje
COMPARISON OF THE QUALITY OF RNA ISOLATED FROM THE RAINBOW TROUT (Oncorhynchus mykiss) TISSUE IN FOUR DIFFERENT WAYS
I. Vardić
E. Teskeredžić
Sažetak
Rapid and accurate diagnostic procedures for identification of reared fish diseases are important in order to reduce serious losses in relation of diseases outbrakes. Therefore, molecular biology methods are required for such types of investigations. First level in these experiments are DNA or RNA isolation. Tissue preparation for isolation of RNA, which is used in further RT-PCR (reverse transcription-polymerise chain reaction) analysis is the key step on which is the whole process of analysis dependent. Our goal was to compare quality and integrity of RNA isolated from the rainbow trout tissue, which was prepared in four different ways: fresh tissue, frozen tissue, in formalin-fixed, paraffin embedded tissue as well as in methacarn-fixed, paraffin embedded tissue. Isolated RNA was analyzed in gel electrophoresis on non-denaturated, 1% agarose gel. Quality and integrity of RNA was proved by RT-PCR reaction with primers for ß-actin gene. Additional, prepared tissue was tested on presence of two fish viruses: viral haemorrhagic septicaemia (VHS) virus and infectious haematopoietic necrosis (IHN) virus in RT-PCR reaction with primers specific for these viruses. RNA isolated from fresh and frozen tissue was of high quality, integrity and quantity. RNA isolated from in methacarn-fixed, paraffin embedded tissue was quite disintegrated, but in RT-PCR with primers for ß-actin gave expected products. These products were absent after RT-PCR reaction with in formallin-fixed, paraffin-embedded tissue. That agrees with the facts from the literature about very agressive affect of formalin as a fixative on RNA in tissue. Inspected fish were not infected with VHS and IHN viruses and that was in agreement with results of clinical examination and pathological analysis. According to our knowledge, this is the first successful RNA isolation from in methacarn-fixed, paraffin embedded fish tissue. Isolated RNA can be used for further analysis in RT-PCR reaction. This method is simple, relatively cheap, and tissue embedded in paraffin can be stored for years. Such tissue could be used for pathogen (virus) detection as well as for gene expression analysis on RNA level, what will be the goal of our future investigations.
Ključne riječi
rainbow trout; methacarn; RNA isolation; RT-PCR; VHSV; IHNV
Hrčak ID:
14715
URI
Datum izdavanja:
31.3.2004.
Posjeta: 1.572 *