RNA isolation using TRIzol

What is TRIzol ?

TRIzol is a monophasic solution of acidic phenol and guanidinium isothiocyanate. It is designed for DNA/RNA/protein extraction. There are similar reagents produced by other companies (Tri-RNA, TRI Reagent, QIAzol). I’ve used Tri-RNA (Farvogen). The manufacturer’s protocol is slightly different, but you get similar results even if you used Tri-RNA with Trizol protocol.

How does TRIzol work?

TRIzol disrupts the cells, denatures proteins, and deactivates the nucleases, thereby stabilizing the DNA, RNA, and protein. The low pH (acidic) of TRIzol separates RNA from DNA and protein, while a high ph causes RNA and DNA to be isolated together. Once chloroform was added, DNA resolves at the interphase, RNA remains in the uppermost aqueous phase, and protein is in the organic phase (Figure1).

<Figure 1>

Procedure

1. Lyse and homogenize samples in TRIzol according to your starting material. Note1. Samples can be stored at 4°C overnight or at -20°C for up to a year. Note2. If samples have a high fat content, centrifuge the lysate for 5 min at 12,000g at 4-10°C, then transfer the clear supernatant to a new tube. Note3. Do not wash your cells before addition of TRIzol to avoid RNA degradation. However, if the sample contains organic components like DMSO or EtOH, wash before adding TRIzol.

   1. Tissues: Add 1 ml of TRIzol per 50-100 mg of tissue to the sample and homogenize using a homogenizer.

   2. Cell grown in monolayer:

      1. Remove growth media.

      2. Add 0.3-0.4 ml of TRIzol per 1x10^5-10^7 cells directly to the culture dish to lyse the cells.

      3. Pipet up and down several times or rock on a rocker for 5 min to homogenize. Note. In terms of HeLa cell line, 1 ml of Trizol for 1 well of 6-well-plate is enough.

   3. Cells grown in suspension:

      1. Pellet the cells by centrifugation and discard the supernatant.

      2. Add 0.75 ml of TRIzol per 0.25 ml of samples (5-10x10^6 cells from animal, plant, or yeasty origin or 1x10^7 cells of bacterial origin) to the pellet.

      3. Pipet the lysate up and down several times to homogenize.

2. Transfer homogenized sample into an ep tube. Note. Once chloroform has been added, the sample becomes sticky causing unable to take the sample with pipetting.

3. Incubate for 5 min at RT to permit complete dissociation of the nucleoproteins complex. Note. If you rocked for 5 min in step 1, you can skip step 3.

4. Add 200 ul of chloroform per 1 ml of TRIzol.

5. Vortex vigorously until sample turns to murky pink.

6. (Option) Incubate for 2-3 min at RT.

7. Centrifuge the sample for 15 min at 12,000g at 4°C. Note. According to the Invitrogen's user guide, they require to sit it for 15 min. However, as a rule of thumb, 5 min centrifugation is also good enough for HeLa, and other cervical cancer cell lines, HEK 293T.

8. Take upper clear aqueous phase. Note. Do not disturb the interphase to avoid gDNA contamination. If you disturbed the layer, centrifuge again.

9. Add 1 volume of isopropanol. Note1. If you wanted to get more RNA, add 30 ug glycogen as co-precipitator. Note2. You can use 2.5-3.0 volume of 100% EtOH instead of isopropanol. Note3. The role of isopropanol or 100% EtOH in RNA isolation is to precipitate RNA. RNA is insoluble in alcohols such as isopropanol and ethanol.

10. Briefly vortex for 2 sec.

11. Incubate for 10 min at RT or over 30 min at -20°C. Note. You can stop at this step. Keep the sample at -20°C but less than a week.

12. Centrifuge the sample for 5 min at 12,000g at 4°C. Note. You will see the white RNA pellet after centrifugation.

13. Remove sup and add 1 ml of ice cold 70% EtOH. Then, brief vortex for 2 sec. Note1. Keep 70% EtOH at -20°C so that you can use ice cold 70% EtOH anytime. Note2. You can keep the sample in 70% EtOH at -20°C for several days. Note3. To get higher 260/280, 260/230 ratio, do EtOH washing twice. I highly recommend you to do EtOH washing twice to get more pure RNA.

14. Brief spin down.

15. Remove residual EtOH without disturbing the pellet.

16. Add nuclease-free DW without air-dry. Note1. Originally, lots of labs air-dry for 5 min. But in my case, I don't think we can fully remove residual EtOH within 5 min but also I think RNA pellet become too dried to be properly dissolved in DW. Thus, I added step 16 to remove residual EtOH without air-drying. Note2. Use nuclease-free DW. You can use DEPC water but I used autoclaved 3'DW. I've autoclaved 3'DW, aliquot them into ep tubes. Then, keep them at -20°C. To prevent RNase contamination, I didn't re-use the DW. Note3. For northern blotting, dissolve the RNA in formamide. As formamide works as RNase inhibitor, you will get the results with higher resolution than DW. Note4. To cDNA synthesize, dissolve the RNA in DW. Note5. If RNA was degraded easily, add RNase Inhibitor or DTT in DW.

17. (Option) Incubate for 10 min at 55-60°C.

18. Measure concentration and 260/280, 260/230 ratio to check RNA purity. Note. If the RNA purity is too low, you can do EtOH precipitation to get purer RNA.