RNA isolation with TRI Reagent

RNA isolation using TRI Reagent®

***Chloroform and phenol in TRI Reagent are toxic. Use gloves and work in a fume hood whenever
possible. ***
Materials
Dissection materials (forceps, slide, etc) must be Chloroform
cleaned with RNaseZAP 2-propanol
TRI Reagent 75% ethanol
RNase-free tubes and pipette-tips RNase-free water.
Centrifuge at 4 deg C for 12000 g speed
Protocol
1. The weight of tissue used for RNA isolation must be known. Use 1 ml of TRI Reagent per 50 mg
of tissue. Before dissecting heads, weigh 1.5 ml or 0.6 ml Eppendorf tubes.
2. Dissect fly heads using material cleaned with RNaseZAP. For pilot RNA isolations, use
a. 15 heads
b. 30 heads
3. Weigh the tubes + heads and add TRI Reagent, ideally 1 ml per 50 mg of tissue.
4. Homogenize using a previously autoclaved and RNaseZAP-cleaned pestle.
5. Centrifuge at 12000 g for 10 min at 4 deg C to remove insoluble material. The supernatant
contains RNA and protein.
a. A layer of fat may form on top and it might not be visible, avoid taking it.
b. Transfer the supernatant to a fresh tube.
6. To ensure complete dissociation of nucleoprotein complexes, allow samples to stand for 5 min
at room temperature.
7. Add 200 µl chloroform per 1 ml of TRI Reagent.
8. Cover the sample tightly, thoroughly by hand and allow to stand for 5 min (or 2-15 min) at room
temperature.
9. Centrifuge the mixture at 12000 g for 15 min at 4 deg C.
a. Centrifugation will separate the mixture into 3 phases:
i. red organic lower phase (protein)
ii. interphase (DNA)
iii. colorless upper aqueous phase (RNA)
10. Transfer the aqueous phase to a fresh tube and add 0.5 ml of 2-propanol per ml of TRI Reagent
used and mix by hand.
a. Allow the sample to stand for 5-10 min at room temperature.
11. Centrifuge at 12000 g for 10 min at 4 deg C.
a. The RNA precipitate will form a pellet on the side and bottom of the tube. It might not
be visible considering the low amount of our starting material. Assume the pellet is
there, DO NOT touch the tube bottom with the pipette tip.
12. Remove the supernatant and wash the RNA pellet by adding 1 ml of 75% ethanol per 1 ml of TRI
Reagent.
13. Centrifuge at 12000 g for 5 min at 4 deg C.
14. Remove the supernatant.
15. Briefly spin and remove leftover ethanol.
16. Briefly dry the RNA pellet for 5 min.
17. Add 30-50 µl RNAse-free H2O and dissolve the RNA by pipetting up and down.
18. Measure the RNA concentration and A260/A280 ratio, which should be at least 1.7

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