Department of Ecology, Evolution and Organismal Biology
Iowa State University, Ames, IA 50011, USA
- You will need a water bath at 80°C, a shaker at 42°C, a refrigerated centrifuge at 4°C, dry ice, and an oven at 60°C.
- All aqueous materials not containing TRIS should be treated with DEPC and then autoclaved. All hardware should be washed in bleach and then baked for at least 4 hours at 400°F.
- Make sure to chill down and warm up all components before you start (~1hr).
- You should have on hand 2 sets of 50ml Oakridge tubes for each sample you plan on doing, Qiagen’s RNeasy kit with all accessories, and the solutions listed at the end of this protocol.
- Note: you will need to add some reagents to solutions before you begin.
- Remember! You are the number one source of RNase contamination. Wear gloves at all times, clean everything with RNase ZAP (Ambion), and set aside hardware and reagents to be used for RNA work only.
- Select the organ you wish to extract tissue from, weigh out 0.8 gram, wrap in aluminum foil and flash freeze in liquid nitrogen. Cool down a mortar beforehand in the refrigerator and then super-cool it by filling it with liquid nitrogen and allowing it to boil away. Place the pestle and microspatula into the mortar to cool also. Nestle the whole getup into an ice bucket filled with dry ice. Dump the frozen tissue into the mortar with some liquid nitrogen and crush. As the liquid nitrogen gets close to boiling completely away, begin to vigorously pulverize the tissue and continue for 10-20 seconds after all liquid nitrogen is gone. Repeat the grinding process three times. Keep some liquid nitrogen in the mortar when finished to keep the sample frozen.
- In a glass homogenizer heated to 60°C, add 8 ml of Thea’s XT buffer heated to 80°C combined with 8µl of Proteinase K (10 µg/ml). Add the frozen, pulverized tissue directly to the solution and mix until homogeneous (or as close as you can get it).
- Pour the solution into a 50ml Oakridge tube. Incubate for 1.5 hrs in a shaker (gentle!) at 42°C.
- Add 1040µl 2mM KCl, mix by inversion, and incubate on ice for 1hr.
- Centrifuge for 20 min at 4°C, 5,000 rpm (11.2cm rotor, or 3,000xG)
- Transfer supernatant to a new 50ml Oakridge tube. Then load the solution onto 2 Qiagen shredder columns (0.7ml/column) and spin at 13,000 rpm for 1 min. Transfer the flow through to a new 15 ml tube. Repeat until the entire sample is processed.
- To the collected flow through add 0.5 volume 100% ethanol and mix by inverting the tube several times.
- Load the sample onto 2 Qiagen RNeasy mini columns (0.7ml/column) and spin at 13,000 rpm for 1 minute. Discard the flow through and repeat the steps with the same columns until the entire sample is processed.
- Add 700µl of buffer RW1 onto the column and centrifuge for 20 sec at 10,000 rpm to wash. Discard the flow through and repeat. (break up the salt with a pipette tip)
- Transfer the columns to new 2ml collection tubes. Add 500µl of buffer RPE to the column and spin for 20 sec at 10,000 rpm. Discard flow through and reuse the collection tube in the next step.
- Add 500µl of buffer RPE to the column and centrifuge for 15 sec at 10,000 rpm. Discard flow through and tube.
- Transfer the column to a new 1.5ml centrifuge tube and add 30-50µl DEPC treated water and let sit for 1 min. Elute RNA by spinning at 10,000 rpm for 1 min. Repeat to get a second elution in a separate 1.5ml tube (you can use the same tube if you want).
Reagents and Solutions
< !> Perform all these steps under a fume hood!
Add 0.05% (v/v) DEPC to the required volume of distilled, deionized water (for example, 500 mL of water requires the addition of 250µL DEPC). Mix by vigorous stirring for approximately 30 minutes. Autoclave to break down the DEPC.
Hot Borate Extraction Buffer (XT Buffer)
|Absolute||For 100 mL|
|0.2M Borax (Sodium-borate decahydrate)||7.63 grams|
|30 mM EGTA||1.14 grams|
|1% (w/v) SDS||1.0 gram|
|1% sodium deoxycholic acid (deoxycholate, sodium salt)||1.0 gram|
|*10 mM DTT||0.154 grams|
|*1% IGEPAL CA-630 (Nonidet P-40, NP-40)||1.0 mL|
|*2% (w/v) PVP-40||2.0 grams|
* = add just before use. To dissolve, add Borax, EGTA, SDS, and sodium deoxycholic acid to pre-warmed deionized, distilled water. Adjust pH to 9.0 with sodium hydroxide. DEPC-treat and autoclave.
For 38ml aliquot of XT Buffer:
Proteinase K (10 mg/mL in DEPC-treated dd-water; store stock at -20°C
Protocol updated May 2003. Many thanks to Ryan Rapp.