*All pipetting steps before step 14 should be done under a hood
-->TRIzol contains phenol, which is VERY TOXIC
1) Wipe down bench and tools with 70% EtOH and put a clean cover on the hood surface.
For cultured cells:
2a) Bring cells from cell culture room to hood with Trizol, chloroform
3a) Pipette medium from cells into liquid waste container
4a) Add enough Trizol to cover surface (~4ml for 10cm plate = ~750ul for 1 well in 6 well plate)
5a) Pipette up and down to detach cells, then let sit 5-10 minutes to ensure complete homogenization (cells will be less sticky when homogeneous)
6a) Add 1 mL of homogenate per eppendorf tube per sample (LABEL TUBES WELL!)
7a) Pipette homogenate up and down again to make sure it is mixed.
For tissue samples:
2b) Bring frozen samples from -80C to hood on Dry Ice
3b) Prepare 1ml of Trizol in 1.5ml tubes, one for each sample
4b) Cut a 50-100mg piece of sample DIRECTLY FROM DRY ICE into Trizol
5b) (Optional) Add 100ug of glycogen to the sample, especially for low yield RNA.
6b) (Optional) If a lot of insoluble stuff remains and if you are NOT collecting DNA, spin at 12,000 x g for 10 minutes at 4°C before adding chloroform. Save the supernatant and discard the remaining pellet
7b) Incubate for 5 minutes at room tempurature
8) Add 0.2 mL of chloroform to each tube and "shake vigorously" for ~15 seconds to mix (vortexing may not be good enough!). (0.1 mL of BCP (1-bromo-2 chloropropane) can be used instead of chloroform)
9) Incubate at room temperature for 2-3 minutes
10) Spin at 12,000rpm (max) in cold room centrifuge for 15 minutes
Multiple phases observed upon addition of chloroform: If you observe multiple phases after adding chloroform and mixing, these are most likely due to inadequate mixing of the chloroform with the reagent. Chloroform is much more dense than TRIzol and therefore will immediately sediment to the bottom of the tube. Simply vortexing the solution will not properly mix chloroform with the TRIzol solution and may give a partial phase separation. Whenever clear water-like solution on the bottom of the tube is observed, this may be chloroform. Simply remix the samples by shaking the contents vigorously as stated in the protocol and centrifuge the samples at 12000 g for 15 min. at 4ºC.
11) Add 0.5mL isopropanol to NEW eppendorf tubes, and label tubes as before.
12) Add the supernatant (upper clear layer = ~600uL) from spun down tubes to new tubes. Be careful not to get any of the lower pink layer! Spun down tubes can now be emptied into liquid waste and discarded. (see ab
13) Lightly mix and incubate new tubes at room temperature for 10 minutes.
14) Spin at 12,000rpm (max) in cold room centrifuge for 10 minutes
15) Remove supernatant from RNA pellet using pipette and add 1mL of 75% EtOH
16) Spin at 9,000rpm in cold room centrifuge for 5 minutes
17) Heat thermometer to 55C and fill wells with H2O in the heat block
18) Remove supernatant from tubes. Spin down for <30 seconds at room temperature to remove excess liquid as necessary.
19) Let pellet sit open at room temperature to dry (5-10 minutes). Edges will start to turn clear. Do NOT over dry. Do NOT pass anything over the open tubes.
20) Add DEPc H2O to the pellet. The amount depends on the size of the pellet (~30uL). Flick tubes to mix RNA pellet into water. Do not vortex. From this point on, tubes should stay on ice.
21) Heat each tube at 55C on the heat block for 10 minutes.
22) Spin down tubes for <10 seconds to collect contents of tubes. Tubes containing RNA from the SAME SAMPLE can be combined at this stage.
23) Up and down pipette the samples to mix.
24) Use the Nanodrop to measure the concentration of RNA in each sample (260/280 ratio should be ~1.8-2.2).
25) At this point, RNA can be frozen (-80C), or run on a gel to test the RNA quality.
