BLT Stages

Abstract bachelor project FBT 2020-2021: Influence of pre-analytical variables on the quality and quantity of RNA in porcine tissues

Translational research has the potential to impact the longevity and health of sick patients. An important topic in translational research is the battle against cancer. In order to conduct cancer research, that can ultimately lead to better diagnosis or treatment, human body samples are indispensable. These samples are stored in a (tumor-) biobank. A tumor biobank is a facility that enables the collection, storage and exchange of samples for research purposes in a standardized and high quality manner. To ensure high quality, five percent of the samples are subject to annual quality control. However, there are some pre-analytical variables that can influence this quality.

During this pilot study, on the one hand the effects of some pre-analytical variables on the quality of the RNA are investigated. On the other, we will look at which RNA extraction method is the most efficient. The effects of the different variables are analyzed by varying them systematically. Tissue pieces (lung and liver) from laboratory animals are used in these RNA extractions. The RNA is extracted according to the different variables (freezing time: <30 minutes or 2-4 hours; storage time: 1 week, 6 weeks or 1 year; method used: manual or column extraction). The tissue pieces were frozen in isopentane, cooled using a CryoPod. The quality of the RNA is determined based on the optical density and RIN values.

The results of this pilot study show that the method with TRI®Reagent yields a significantly higher concentration than the column method, for tissues with uniform structure (e.g. liver). For tissues with variable structure (e.g. lung), no significant difference between the different extraction methods can be demonstrated. Nevertheless RNA extracted via column method results in a more pure RNA. Based on these results, it is recommended to extract RNA via the column method. For the other variables (freezing time and storage time), no significant difference can be shown. From the results obtained in this pilot study there can be concluded that the quality of the RNA is not affect by the storage time. Regarding the freeze time it is recommended to freeze tissue samples maximum half an hour after prelevation.

Abstract bachelor project MLT 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.