TRIzol could be applied in a few different ways.

You should first discard the growing medium and wash cells with PBS. Following that you could apply TRIzol directly to the cells or dislodge cells first using Trypsin or by scraping them in PBS and collecting them in a tube. In this case, you should centrifuge cells at 400×g for 2-3 min and discard the supernatant, and wash cells with PBS repeating the centrifugation. Then you apply TRIzol.

Hello Diana Trujillo

Please follow the steps provided below.

1. Aspirate media from plated A549 cells.

2. Rinse cell monolayer with ice cold PBS only once.

3. You may lyse the cells directly in a culture dish by adding 1 ml of TRIzol reagent directly to the plate and mix extensively by pipetting all over the plate. The volume of TRIzol added should be as follows:

For 100 mm plate use 1 ml TRIzol.

For 1 well of a 6 well plate or a 35 mm plate use 500 µl TRIzol.

For 1 well of a 24 well plate or a 15 mm plate use 200 µl TRIzol.

(Please note: An insufficient amount of TRIzol reagent may result in DNA contamination of the isolated RNA).

4. Scrape the cell monolayer with cell scraper. Pass the cell lysate several times through a pipette. Vortex thoroughly.

5. Transfer cell suspension to 1.5 ml microfuge tube.

6. (Optional Step): if you have tiny amounts of starting material, you may add glycogen to the sample which may improve yield. It will remain with RNA as it is soluble in water. Otherwise, you may omit this step.

7. Incubate the cell suspension form step 5 for 5 minutes at room temperature to permit the complete dissociation of nucleoprotein complexes.

8. Centrifuge at 12,000 x g for 10 min at 4º C to remove cell debris. Transfer the supernatant to new tube.

9. Phase separation: Add 0.2 ml of chloroform to the supernatant (per 1 ml of TRIzol reagent).

10. Shake vigorously for 15 seconds and incubate at room temp for 2-3 min.

11. Centrifuge for 15 min at 12,000 x g at 4ºC.

12. Following centrifugation, the mixture separates into lower red, phenol chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. Transfer upper aqueous phase carefully without disturbing the interphase into fresh tube.

Please note: Extract 80% of the aqueous RNA layer leaving 20% behind in the tube to prevent contaminating the aqueous layer.

13. RNA precipitation: Precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5 ml of isopropyl alcohol (per 1 ml of TRIzol reagent used). Invert tube 5 times. Incubate at room temp for 10 min (if cloudy, precipitate for an additional 10–15 min).

14. Centrifuge at no more than 12,000 x g for 10 minutes at 2 to 4 degree C.

15. After centrifugation, a small white RNA precipitate should be visible on side of the tube at this point.

16. RNA wash: Remove the supernatant completely. Wash the RNA pellet once with 75% ethanol, adding at least 1 ml of 75% ethanol (per 1 ml of TRIzol reagent used). Mix by flicking and inverting tube.

17. Centrifuge at no more than 7,500 x g for 5 minutes at 2-to-8-degree C.

18. Repeat the above washing procedure once. Remove all leftover ethanol.

19. Air-dry or vacuum dry RNA pellet for 5-10 minutes. Do not dry the RNA pellet by centrifuge under vacuum. It is important not to let the RNA pellet dry completely as this will greatly decrease its solubility.

20. Resuspend the pellet in 50ul RNase free water (or DEPC-treated water). If pellet is over-dried, you can incubate at 55ºC for 10-15 min with repeated pipetting to completely dissolve the pellet.

21. For RNA, the 260/280 ratio should be around 2. If it is lower, this might be an indication of contamination (acidic phenol or protein). You may also calculate the 260/230 ratio which is a second measure for purity of the sample, as the contaminants absorb at 230nm. The 260/230 ratio should be higher than the 260/280 ratio, as it is usually between 2 and 2.2. Lower ratio might be an indication of contamination.

Good Luck!

Malcolm Nobre If you will do any drug treatment before RNA extraction will you do the washing with ice-cold PBS either!?

Diana Trujillo This is the protocol I followed for all adherent cancer cells including A549 as well. The steps are after harvesting the cells with trypsin. Cells are collected in a 1.5 ml centrifuge tube and spun down at 1000 rpm for 5 minutes and the supernatant was discarded. The pellets are processed as the attachment below.

Thank you very much for your help!

Doaa Yamani Am I too late for the answer?

Malcolm Nobre If you will do any drug treatment before RNA extraction will you do the washing with ice-cold PBS either!?

Yes

Hi Malcolm Nobre

I saw your protocol for RNA extraction and it is very clear to follow

my question is, can I use it for all cell lines?

I am doing HEK293FT and I'll be working with cancer cell lines (colorectal, ovarian, and glioblastoma)

Hi Khawla Saif , yes, of course! It is a protocol meant for all adherent cell lines. If you are using suspension cells, then you need to follow these steps.

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

b. Add 0.75 mL of TRIzol reagent (per 5– 10 × 10^6 cells) to the pellet.

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

You may then continue from step 6 from the above protocol.

Best.

Malcolm Nobre Thanks:)