| Popis: |
Transcriptional analysis has become a cornerstone of biological research, and with the advent of cheaper and more efficient sequencing technology over the last decade, there exists a need for high-yield and efficient RNA extraction techniques. Fungi such as the human pathogen Candida albicans present a unique obstacle to RNA purification in the form of the tough cell wall made up of many different components such as chitin that are resistant to many common mammalian or bacterial cell lysis methods. Typical in vitro C. albicans cell harvesting methods can be time consuming and expensive if many samples are being processed with multiple opportunities for product loss or sample variation. Harvesting cells via vacuum filtration rather than centrifugation cuts down on time before the cells are frozen and therefore the available time for the RNA expression profile to change. Vacuum filtration is preferred for C. albicans for two main reasons: cell lysis is faster on non-pelleted cells due to increased exposed surface area, and filamentous cells are difficult to pellet in the first place unlike yeast or bacterial cells. Using mechanical cell lysis, by way of zirconia/silica beads, cuts down on time for processing as well as overall cost compared to enzymatic treatments. Overall, this method is a fast, efficient, and high-yield way to extract total RNA from in vitro cultures of C. albicans. |
