A. RNA Extraction Protocol
- Determine how many samples will be analyzed. Each sample will require 600 µL of Buffer RLT and Buffer RLT requires the fresh addition of b - mercaptoethanol (add 10 µL of b - mercaptoethanol per 1 mL of Buffer RLT). This solution is stable for approximately 1 month.
- For 5 x 107 – 5 x 109 cells, resuspend pellet in 600 µL of Buffer RLT.
- Sonicate for 10 x 1 sec pulses
- Centrifuge for 3 min at maximum speed in a microcentrifuge and transfer the supernatant to a new, labeled tube.
- Add and equal volume of 70% ethanol to the cleared lysate and mix well by pipetting.
- Apply 700 µL of sample to a labeled RNeasy mini spin column sitting in a 2 mL collecting tube. Centrifuge for 15 sec at 8,000 x g (> 10,000 rpm)
- Repeat as needed until the entire sample has been loaded onto the column.
- Pipet 700 µL of Buffer RW1 onto the column, centrifuge as above.
- Discard flow through and collection tube
- Transfer RNeasy column to new collection tube. Pipet 500 µL of Buffer RPE onto the column and centrifuge as above (ensure that ethanol has been added to Buffer RPE before use). Discard flow through
- Pipet 500 µL of Buffer RPE onto RNeasy column and centrifuge for 2 min at maximum speed to dry the RNeasy membrane. Discard flow through and repeat
- Transfer RNeasy to new 1.5 mL RNase-free microcentrifuge tube. Pipet 30-50 µL of RNase-free water directly onto the RNeasy membrane. Centrifuge for 1 min at >8,000 x g to elute RNA
- Store eluted material at -80°C
B. Working with RNA – Some precautions
RNA is relatively easy to isolate from prokaryotic and eukaryotic cells. However, RNA molecules are highly susceptible to degradation by RNases, which are produced by the cell and on your skin. The general strategy for successful isolation of intact, full length mRNA is to minimize the possibility of RNase contamination. The following recommendations will reduce the potential of contamination:
- Wear gloves at all stages of the extraction and when handling equipment used in the manipulation of RNA
- Bake all glassware at 275 C for 3-4 h. This serves to burn of all organic material (including RNases) from the glass surface. Sterile, disposable polypropylene tubes and pipets are also useful.
- Treat solutions (including water) with 0.05% diethyl pyrocarbonate (DEPC). DEPC is an effective protein denaturant and inactivates any residual RNases. Compounds containing amine groups (e.g. TRIS) cannot be DEPC treated and must be made from ultrapure stocks and DEPC-treated water.
- Keep fingers and dirty (unbaked) utensils out of RNase free chemical stocks. I recommend pouring out dry chemicals into an RNase free container rather than “scooping” them out of the container.
- Make sure your work surfaces are clean and wiped down with ethanol.
If these precautions are kept in mind, the success of your RNA isolation will be increased 100-fold.