FastRNA^® Protocol

1. Protocol for Preparation of PAR (Phenol Acid Reagent): The amber glass bottle labeled PAR contains a buffered salt solution, pH 4, that is ready for the addition of high grade phenol. The phenol should be from a fresh unopened bottle of water-saturated phenol or from a non-saturated source that is solid at room temperature. Phenol must be aqueous saturated before adding it to the bottle. To do so, solid phenol must be heated to 50°C until liquified. Then add an equal volume of water (phenol will usually dissolve 15-20 ml of water per 100 ml of phenol) to saturate it before adding it (lower phase) to the buffered salt solution that is already in the PAR bottle. You will need to add 55 ml of the water-saturated phenol to the 16.5 ml of the salt solution that is already premeasured in the bottle. Mix the phenol and the salt solution and let it sit overnight so it can settle or until there is an obvious interface. It is now ready to use in the FastRNA procedure. PAR is stable for 1-2 months at 4°C or until it develops color. The solution should be clear; if it develops color it should not be used.
2. Protocol for Preparation of CIA (Chloroform Isoamyl Alcohol): The second amber glass bottle labelled CIA, contains 2.5 ml of isoamyl alcohol. Add 60 ml of chloroform and mix to make 62.5 ml of a 24:1 mixture. Once the chloroform has been added, the solution is ready to be used with the FastRNA Kit. Store tightly capped at room temperature.
3. Protocol for Preparation of DIPS (DEPC-treated Isopropanol Precipitation Solution): Add 54 ml of isopropanol to the bottle labelled DIPS, which already contains 1 ml of DEPC-treated salt solution, to make a total of 55 ml. Shake and store tightly capped at room temperature.
4. Protocol for Preparation of SEWS (Salt/Ethanol Wash Solution, RNase-free): Add 42 ml ethanol to the bottle labelled SEWS, which already contains 13 ml of RNase-free salt solution, to make a total of 55 ml. Shake and store tightly capped at room temperature.

The Protocol, in brief

1. Add reagents and sample to tube with lysing matrix.
2. Place tube in FastPrep Instrument and process for 20 seconds at a speed of 6.
3. Spin in a microcentrifuge for 5 minutes to separate phases.
4. Add 500 µl of CIA. Vortex, spin and transfer top phase to a new tube.
5. Add 500 µl of DIPS, mix, and pellet precipitated RNA.
6. Wash pellet once with 500 µl SEWS.
7. Dissolve pellet in 50 to 100 µl of SAFE.
8. Run samples on a 1.2% non-denaturing agarose gel.

1. To a FastPrep GREEN Tube containing lysing matrix:

   • Add 500 µl of CRSR-GREEN
   • Add 500 µl of PAR
   • Add 100 µl CIA




Important: The volumes are calculated to leave an air space of approximately 0.5 cc. If less air space is present there is a likelihood of sample loss due to tube failure or deformation around the cap allowing sample to bubble out. Sample loss is caused by an increase in pressure due to temperature rise during FastPrep runs, especially during high speed settings or long runs where the temperature inside the tube can exceed the boiling point of chloroform (61°). The presence of 0.5 cc of air space in the tube is sufficient to prevent sample loss during routine FastPrep runs.





Add sample of up to 250 mg of tissue or 107 cells in up to 200 µl (maximum, can use less in all cases). Resuspend cell pellets in 100 µl of water or isotonic saline to give a maximum suspension volume of 200 µl and add to tube.



2. Place tube in FastPrep Instrument and process for 20 seconds at a speed rating of 6. [Additional time (up to 2 minutes) may be required for stems, leaves, seeds, or roots.




Caution: If processing for 2 minutes, cool tube on ice after 1 minute.





Remove tube from machine and place on ice for 5 minutes.

It is important to cool the tube to approximately room temperature. If tube is opened without cooling the positive pressure will cause some of the sample to squirt out and be lost as the cap is loosened. It is not necessary to cool tube below room temperature as the RNA is stabilized by the PAR, CRSR and CIA.

3. Spin in a microcentrifuge for 5 minutes to separate phases. [Extending spin to 15 minutes can enhance elimination of DNA and excessive debris from large samples, or from cells with complex cell walls].




Note: Be certain to check that tubes are balanced by weight and that the bottom or side of the tubes will not scrape the wall of your microcentrifuge as this will cause rapid loss of sample. If clearance is questionable, transfer the solution mixture (not the lysing matrix) with a pipet to a 1.5 ml microcentrifuge tube before spinning.





Transfer the top phase to a microcentrifuge tube without any of the interphase. (Leave 10-15% of the solution behind to minimize disturbance of the interphase and contamination of final sample with protein and DNA).

4. Add 500 µl of CIA. Vortex for 10 seconds. Centrifuge at high speed for 2 minutes to separate phases. Transfer the top phase to a microcentrifuge tube, avoiding interphase material.
5. Add 500 µl of DIPS, mix, and incubate at room temperature for 1-2 minutes. Centrifuge for 5 minutes to pellet precipitated RNA. Pellet should be quite large.
6. Wash pellet once with 500 µl SEWS (or twice with 250 µl for maximum efficiency). Add SEWS and swirl tube (it is not necessary to resuspend pellet), centrifuge briefly and remove liquid with small bore pipet tip. Air dry pellet for 5-10 minutes.
7. Dissolve pellet in 50 to 100 µl of SAFE. [Alternatively, depending on subsequent needs, use SAFEE or SSAFE or a combination to dissolve pellet. These solutions contain EDTA and SDS respectively and will inhibit nucleases, to some extent, if introduced by accident. Store purified RNA at -70°C].
8. The yield and integrity of the RNA can be rapidly accessed by electrophoresis through a 1.2% non-denaturing agarose gel.
9. Add 20 µl of LiCl solution to each 100 µl of the dissolved RNA in step 7. Incubate 5-15 minutes on ice (or in refrigerator overnight, or indefinitely as a storage method). Centrifuge for 5 minutes, then wash pellet with SEWS as in step 5.
10. Carefully remove solution with a small bore pipet tip. Air dry for 5 minutes. Resuspend in 50 to 100 µl of SAFE, SAFEE, or SSAFE as in step 7.

Anticipated Results: up to 100 µg total RNA from 10⁷ cells.