Organoid RNA extraction protocol
1) Remove media from organoids, replace with cell recover solution* (at least 350ul/well) and
place on ice for at least 15 minutes (as many wells as desired, but should be at least 2-3).
2) Resuspend and pool each well in a 2ml Eppendorf tube (or larger centrifuge tube to
accommodate larger volumes). Centrifuge 1250 rpm, 5 min, 4°C.
3) Remove as much supernatant as possible and replace with chilled PBS.
4) Repeat steps 2 and 3 at least 2 times.
5) Resuspend organoids with 1ml Trizol reagent. Pipette up and down several times to mix and
homogenize cells.
6) Incubate room temperature for 5 min.
7) Add 0.2ml Chloroform per 1ml Trizol.
8) Secure the lid tightly and shake vigorously for 10-15 seconds.
9) Incubate for 2-3 minutes at RT.
10) Centrifuge for 15min, 12,000 x g, at 4°C.
11) Gently collect the clear-aqueous upper layer and transfer to a fresh RNAse-free 1.5 ml
Eppendorf tube. Be careful not to disturb the red-phenol-chloroform lower layer, or white
precipitate in the interphase.
12) Add 1ul of 20mg/ml RNAse free glycogen.
13) Add 0.5ml isopropanol .
14) Incubate at -20°C for at least 1 hour or overnight.
15) Centrifuge for 15 minutes, 12,000 x g, at 4°C.
16) Discard the supernatant.
17) Wash the pellet in 1ml cold 75% ethanol and centrifuge for 5 minutes, 12,000 x g, at 4°C.
18) Repeat #17.
19) Discard the supernatant with a pipette and air dry for 5-10 minutes. Do not dry entirely.
20) Resuspend in 50ul RNAse free dH2O.
21) Treat with RNAse-free DNase I using manufacturer protocols.
22) Repeat steps 5-20.
23) Quantify Final RNA concentration with a nano-drop or bioanalyzer and store at -80°C.
* Cell recovery solution is a proprietary mix that is used to degrade Matrigel. Corning Product
#354253. https://catalog2.corning.com/LifeSciences/enUS/Shopping/ProductDetails.aspx?productid=354253(Lifesciences)