TITLE: RNA Extraction from Cryptococcal Cells.
AUTHORS: Liz Hughes, Edward Wallace, Elizabeth Ballou

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is for extracting RNA from Cryptococcal cell cultures.</div><div class = "text-block">Cell pellets are fixed in methanol, lyophilised overnight, the cell wall mechanically disrupted and RNA extracted using the Zymogen DNA/RNA miniprep kit.</div><div class = "text-block">The Zymogen Kit is designed to purify RNA from small amounts of starting material.</div><div class = "text-block">They provide a fast and simple method for preparing up to 100 µg total RNA per sample. The purified RNA is ready for use in downstream applications such as: </div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Next-Gen sequencing</li><li style = "counter-reset:ol0;list-style-type:disc;">RTPCR</li><li style = "counter-reset:ol0;list-style-type:disc;">Microarray</li><li style = "counter-reset:ol0;list-style-type:disc;">Hybridization</li></ul></div><div class = "text-block"><span>The</span><span style = "font-style:italic;">Quick</span><span>-DNA/RNA Miniprep Kit provides a quick method for the isolation of high quality genomic DNA and total RNA from small amounts of cells and tissue. The kit isolates both genomic DNA and a broad range of RNA species without the use of phenol. Small RNAs (e.g., tRNAs, microRNAs) can be recovered following a simple adjustment within the RNA isolation protocol – no extra steps are required! Both DNA and RNA from up to 5x10</span><span style = "vertical-align:super;">6</span><span>cells can be eluted into volumes as little as 25 µl in less than 15 minutes.</span></div><div class = "text-block">The procedure represents a well-established technology for RNA purification. This technology combines the selective binding properties of a silica-based membrane with the speed of micro-spin technology. A specialised high-salt buffer system allows up to 50 µg of RNA to be extracted.</div><div class = "text-block">Biological samples are first lysed and homogenized in the presence of a highly denaturing guanidine-chlodride containing buffer, which immediately inactivates RNases to ensure purification of intact RNA. Ethanol is added to provide appropriate binding conditions, and the sample is then applied to a spin column, where the total RNA binds to the membrane and contaminants are efficiently washed away. High-quality RNA is then eluted in 30–100 µl water.</div><div class = "text-block">Size range: Genomic DNA ≥40 kb and Total RNA ≥17 nt</div><div class = "text-block">Yield: 25 µg DNA and RNA (binding capacity), ≥25 µl (elution volume)</div><div class = "text-block">A260/A280, A260/A230: >1.8.</div><div class = "text-block"><a href="https://files.zymoresearch.com/protocols/_d7001_quick-dna-rna_miniprep_kit.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://files.zymoresearch.com/protocols/_d7001_quick-dna-rna_miniprep_kit.pdf</span></a></div><div class = "text-block"><a href="https://files.zymoresearch.com/datasheets/ds2503-02_quick-dna_rna_kit_datasheet.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://files.zymoresearch.com/datasheets/ds2503-02_quick-dna_rna_kit_datasheet.pdf</span></a></div></div>

[STEPS]
?. Prepare Cryptococcal cells for RNA extraction.
?. Streak out appropriate glycerol stock on YPD plate. Incubate @ 30ºC for 2 days.
?. Lyophilise cell pellets overnight.
?. Pick a single colony from the freshly streaked plate and put in required volume of appropriate media. Incubate in appropriate conditions depending on expected experimental conditions/outcome.
?. After fixing the cells on dry ice move to normal ice for a few mins and then spin cells down at 4°C, 3000 g for 2 mins.Wash cells with 15 ml sterile ice cold water.Spin cells down at 4°C, 3000 g for 2 mins, pour off supernatant.Repeat wash step.Re-suspend pellets in 1-2 ml sterile ice cold water and transfer to a 2 ml screw cap tube.Spin cells at 4°C, 3000 g for 2 mins. Remove all the water and store at -80°C for AT LEAST 2 hours before freeze drying on the lyophiliser.
?. Cool lyophiliser at least an hour before using to bring the temperature down to -60°C.Switch lyophiliser on and follow instructions on machine to cool it down.NB: Cell pellets must be at -80°C for at least 2 hours prior to lyophilisation to allow sublimation to take place.
?. When the lyophiliser is at -60°C, switch off and release vacuum by removing silver plug from orange tube.Place samples on the mesh.Replace the lid and silver plug.Switch on the pump.
?. Dry cell pellets overnight under vaccum @ -60°C.Before leaving apparatus check the pellets are still in the tube and have not started to rise or pop out. If the pellets are cold enough this should not happen.NB: Freeze drying, also known as lyophilisation or cryodesiccation, is a low temperature dehydration process that involves freezing the product, lowering pressure, then removing the ice by sublimation (a process in which water is removed from a product after it is frozen and placed under a vacuum, allowing the ice to change directly from solid to vapour without passing through a liquid phase). This is in contrast to dehydration by most conventional methods that evaporate water using heat, and allows removal of water without excessive heating.
?. Mechanical disruption of Cryptococcal cells.
?. Take dry lyophilised pellets and add 200μl of dry 0.5 mm zirconium beads.Add 700 µl of RLT Buffer + β2M (10 µl β2M + 1 ml RLT buffer = lysis buffer).
?. 'Bead-beat' Using PreCellys machine.Carry out 10 cycles of the EW Yeast Program. (1 cycle = 3 x 10 sec with 20 sec pause)Place tubes on ice for 1 min between each cycle.You should now have a fine powder.NB:' Bead-beating' machines warm the sample about 10 degrees/minute. This is due to frictional collisions of the beads during homogenization. Sample warming can be controlled by 'bead-beating' for short time intervals with cooling on ice between each interval.
?. RNA Extraction of mechanically disrupted Cryptococcal cells.
?. Use the Zymogen kit.After mechanical disruption, perform all steps of the procedure at room temperature. Work quickly.Spin cells @ 1000 x g for 3 mins to pellet the beads.
?. KEEP the supernatant, DO NOT throw it away.Add the supernatant to the Zymo spin IIIC column.Place the column into a collection tube and spin @ 12,000g for 1 min.This step binds the RNA to the IIIC column.Optimisation has been carried out on 5 x 107 cells.
?. KEEP the flow through.Transfer the flow through to micro-centrifuge tube leaving any pellet behind.Add 400μl of 100% Ethanol to the flow through, mix immediately by pipetting and transfer to the Zymo Spin IIC Column (RNA binding capacity of this column is 50μg).Spin @ 12,000 x g for 1 min.Discard flow through.
?. Add 400μl of DNA/RNA Prep buffer to the column.Spin @ 12,000 x g for 1 min.Discard the flow through.NB: DNA/RNA Prep buffer contains  guanidine chloride and ethanol. This is a stringent washing buffer that removes carbohydrates, proteins, fatty acids etc that are non-specifically bound to the silica membrane.
?. Add 700μl of DNA/RNA Wash  buffer to column. Tip column up and down to ensure all previous buffers are caught from the lid etc.Spin @ 12,000 x g for 30 sec.Discard the flow through.NB: DNA/RNA Wash buffer is a mild washing buffer. It's main function is to remove traces of salts that remain on the column due to earlier buffers.
?. Centrifuge the column in a clean and dry collection tube @ full speed for 1 minute to dry the membrane.
?. Place the column in a fresh RNase free micro-centrifuge tube.Add 30μl of RNase free water.Close the lid gently and leave at RT for 3-5 mins.Spin @ 10,000 x g for 1 min to elute the RNA.Discard the column.
?. Check the RNA purity and integrity using the NanoDrop and Fragment Analyzer.Store the RNA at -80ºC.
?. Following growth of cells they MUST be fixed with methanol prior to downstream processing.Cool 5 ml of Methanol per culture sample on dry ice prior to collecting cultures.Mix 10 ml  of culture with 5 ml 100% Methanol on dry ice, invert a few times to mix and leave on dry ice for 3-5 mins. Leaving any longer will result in the culture freezing.
?. Add 400μl of DNA/RNA Wash  buffer to column. Tip column up and down to ensure all previous buffers are caught from the lid etc.Spin @ 12,000 x g for 1 min.Discard the flow through.NB: DNA/RNA Wash buffer is a mild washing buffer. It's main function is to remove traces of salts that remain on the column due to earlier buffers.