Bioresource Center UZ Gent (Biobank)
Abstract bachelor project 2019-2020: Influence of pre-analytical factors on quality and quantity of RNA-extraction and optimization of DNA-extraction with DNAzolTM
Translational research improves the multidirectional and multidisciplined integration of fundamental, patient and population focused research to improve public health on long-term. Nowadays cancer is the leading cause of death and translational research plays a major role in the fight against cancer. Access to well documented and high quality biospecimen is key to this research. Biobanks and in particular tumor biobanks, form the basis of this kind of research and are specialized to collect, create access and monitor human biospecimens. To ensure high-quality the biospecimens are annually committed to a quality control. This implies that five percent of all biospecimens are subjected to a DNA and RNA extraction with resulting DIN and RIN verification respectively. It is believed that pre-analytical factors play a major role in sample quality and should therefore be taken into account. Pre-analytical factors are, amongst other, storage temperature, cold ischemia time, warm ischemia time, transport time… which determine the integrity of a biospecimen and ultimately the reliability of analysis data.
This pilot study is split into two main topics. First the study tries to tackle the most frequent and traceable pre-analytical factors more in depth. The chosen factors are: transport time (up to twelve hours), the transport temperature (room temperature and 4°C) and the method of disintegrating the tissue prior to the initial extraction (using a scalpel, ruptor or scrolls).
Second the study tries to optimize the current manual DNA-extraction protocol, using DNAzolÔ. The manual RNA-extraction using TriReagentÒ is already optimized by the Bioresource Center of UZ Ghent. In addition the manual RNA- as well as the manual DNA-extraction protocol is tested against a semi-automatic RNA- and DNA-extraction protocol using kits of MaxwellÒ (Promega). If significant differences are observed, a preferred method may be indicated.
In this pilot-study, the different transport times and temperatures had no significant effect on the RNA quality of the biospecimens. For RNA-extraction we recommend the manual extraction method and to use a tissue ruptor before extraction since the highest yield and best overall quality scores were obtained. For DNA extraction we highly recommend the semi-automatic protocol and to use a scalpel before extraction as results showed the highest yield. With regard to the optimization of the manual DNA- extraction no significant differences were found when the samples were centrifuged during the washing step. We recommend to dilute the extracted DNA in 8mM NaOH and a short dry-step of fifteen seconds since better results were obtained.
Corneel Heymanslaan 10