Total RNA SafeKit Protocol
(Guanidine, Phenol, and Chloroform free)
Revision No. 1008-999-6B02W

Samples should be prepared with concern to prevent degradation of RNA by RNases. Immediate flash-freezing of samples in liquid nitrogen after collecting or harvesting is the most effective means to virtually stop RNA degradation. Freezing also aids in the release of RNA from cells because cellular membranes are ruptured by ice crystals.

Soft tissues (liver, brain, heart, kidney, etc.) can be added frozen to a tissue homogenizer and processed in the presence of Solutions 1 and 2. Harder samples must be ground to a fine powder in a liquid nitrogen-chilled mortar and pestle with a little liquid nitrogen added to ensure that the samples remain frozen. The powdered sample should have a dry appearance when this is done correctly. Once the sample is completely ground, combine it with the appropriate amount of Solutions 1 and 2 and mix thoroughly by vortexing in a tube or homogenizing with a tissue homogenizer or equivalent. Solutions 1 and 2 contain RNA stabilizing chaotropes and buffers. The solutions solubilize proteins and RNA and enable DNA and cellular debris to be precipitated.

The RNA is precipitated from the clarified supernatant with isopropanol. Traces of DNA are catalytically removed and the RNA is further purified with a proprietary matrix suspension. The resulting RNA is DNA-free and application-ready.

BIO 101's novel RNA isolation procedure precludes the use of guanidine and harmful organic solvents such as Phenol and Chloroform. The only organic solvent required is user-supplied Isopropanol.

The resulting RNA is an efficient substrate for RT-PCR, RPA, RNA blotting, primer extension, poly A+ RNA selection, and Differential Display.

1. To a tissue homogenizer or Polytron:
   • Add 800 µl Solution 1
   • Add 200 µl Solution 2
   • Add 10 to 100 mg sample

Resuspend cell pellets in 100 µl of water or isotonic saline to give a maximum suspension volume of 200 µl and combine with above solutions.

2. Homogenize sample completely with 5 to 10 passes of homogenizer. Transfer solution to a microcentrifuge tube.



3. Spin in a microcentrifuge for 5 minutes at 14,000 x g to pellet cellular debris. [Extending spin to 15 minutes can enhance elimination of protein contaminants, depending on the sample]. Transfer 800 µl of supernatant to a fresh tube. Be careful to avoid any cellular debris either from the pellet or floating in a layer on the top of the tube.



4. Add 800 µl Isopropanol (user-supplied) to precipitate RNA. Mix by inverting the tube or placing on a rocker or rotator for 2 minutes at room temperature.



5. Centrifuge for 5 minutes at 14,000 x g to pellet RNA. Carefully decant supenatant and centrifuge again for 5 seconds. Remove residual supernatant with pipet tip.



6. Resuspend RNA pellet in 200 µl Solution 3A and 10 µl of Solution 3B. Make certain the pellet is completely resuspended; vortex if necessary. After the pellet is fully resuspended, incubate at room temperature for 5 minutes.



7. Vortex Solution 4 to resuspend the matrix and add 40 µl to the sample. Mix by vortexing for 10 seconds. Centrifuge at 14,000 x g for 2 minutes.



8. Transfer the supernatant (that contains the RNA) to a new tube. RNA is now ready to use without further manipulation.