GE Lab 1: RNA Isolation
In this lab you will be isolating total RNA from Tetrahymena cells at different points in their life cycle. Later, you will be making cDNA from this RNA to be used in “reverse-transcriptase PCR” to test for levels of expression of your gene at the different time points.
*RNA will be rapidly degraded by RNases, which are EVERYWHERE, especially on your skin. To prevent RNase contamination, you must wear gloves and use only RNase-free tips and microcentrifuge tubes. All solutions have also been specially treated with agents that deactivate RNase, like diethylpyrocarbonate (DEPC). Be especially careful to keep the RNase-free tips covered when not in use.
1. Pipet 5 x 106 cells into a 15 mL screw cap tube.
The concentration of cells in the culture will be given to you day of lab.
2. Pellet cells by centrifuging at 4,000 rpm. Pour off supernatant (into sink is fine).
3. Add 1 mL of Trizol reagent (this is a solution with phenol, which will lyse the cells and inhibit RNase activity). Incubate on the bench for 5 minutes.
4. Add 0.2 mL chloroform in the hood. Shake vigorously by hand for 15 seconds, incubate on your bench for 3 minutes.
Centrifuge sample for 15 minutes at 3,000 rpm, at 4˚C.
Following centrifugation, the mixture separates into a lower, red phenol-chloroform phase, a whitish interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase.
6. Transfer the aqueous phase to a microcentrifuge tube. Precipitate RNA from the aqueous phase by addition of 0.5 mL isopropanol and incubating your sample on ice for 10 minutes.
7. Centrifuge sample for 10 minutes at 10,000 rpm in a microcentrifuge in the cold room (4˚C). The RNA precipitate will form a gel-like pellet on the side and bottom of the tube. Remove the supernatant.
8. Wash the pellet by adding 1 mL of 75% ethanol, mix by vortexing, and centrifuge for 5 minutes at 5,000 rpm in the cold room.
9. Resuspend the pellet in 50 μL RNase-free water available at front of each bench.
10. Determine the concentration of your RNA by spectrophotometry (A260).
Make a 1:500 dilution of your RNA with sterile water. Total volume should be 500 µL. Blank the spectrophotometer with sterile water. All readings should be done in a quartz microcuvette, by loading 100 µL of each sample to measure. An absorbance reading of 1 = 40 µg/mL of total RNA
11. Store your RNA samples at -20˚C until you make cDNA.