I am facing an issue regarding the concentration and purity of RNA, the 260/280 ratio shows contamination.
1. Aspirate media from plated cells.
2. Rinse cell monolayer with room temp PBS only once.
3. Scrape the cell monolayer with cell scraper.
4. Transfer cell to 1.5 ml microfuge tube. Centrifuge at 5000 rpm for 5 min.
5. Remove the supernatant and added 400µl TRIzol. Mix till the pellet dissolve by pipetting thoroughly.
The volume of TRIzol added should be as follows:
For 1 well of a 6 well plate or a 35 mm plate use 400 µl TRIzol.
(After this The lysate is stored at -80 degrees C).
6. Before the phase separation the lysates are thawed on ice.
7. Incubate the lysate for 5 minutes at room temperature.
8. Phase separation: Add 0.2 ml of chloroform to the supernatant (per 1 ml of TRIzol reagent).
9. Shake vigorously for 15 seconds and incubate at room temp for 2-3 min.
10. Centrifuge for 15 min at 12,000 x g at 4ºC.
11. 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.
12. RNA precipitation: Precipitate the RNA from the aqueous phase by mixing with ice cold isopropyl alcohol. Use 0.5 ml of ice cold isopropyl alcohol (per 1 ml of TRIzol reagent used). Incubate for 10 min on ice (if cloudy, precipitate for an additional 10–15 min).
13. Centrifuge at no more than 12,000 x g for 15 minutes at 4 degree C.
14. After centrifugation, a small white RNA precipitate should be visible on side of the tube at this point.
15. 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.
16. Centrifuge at no more than 7,500 x g for 5 minutes at 4 degree C.
17. Repeat the above washing procedure once. Remove all leftover ethanol.
18. Air-dry RNA pellet for 5-10 minutes.
19. Resuspend the pellet in 20ul RNase free water.
20. incubate at 55ºC for 3 min.
21. stored at -20 degree C.
The nanodrop reading is very low (in tens and hundreds) and the 260/280 ratio is coming in 1.8-1.9 range. What is the issue I am not able to understand. Is it due to a handling error or do I need to change in protocol something?
Hello Kishan Kandelwal,
For a better precipitation you can add 20 ug of Rnase free glycogen (a carrier) to the aqueous phase. Don't mix before you add propanol-2
Your precipitate will look like to a small white point
Hi,
I extract RNA from RAW264.7 cells often using the Trizol method. What is your cell confluency for each plate? Once I know that, I can give you some tips and share my protocol. Hope I can help!
Skylar
Hi skylar
I usually seed around 400,000 cells per well in six well plate.
Hi Kishan,
I think you may be losing cells by collecting them and then spinning them down. I first rinse with PBS one time, then I collect the cells by directly adding Trizol (500ul for a 6cm dish or 6 well plate) to the plate. I do not scrape my cells and I just pipette the solution up and down about 5-10 times to help the extraction. With this method I am able to extract RNA from very few human macrophages. I also do use 1ul of glycogen to help pellet my RNA before precipitation, it also prevents me from accidentally disrupting my RNA if I have a visible pellet (alot of times the pellet is too small to see). I am including a reference for the protocol that I use that was published by former colleagues of mine. Please let me know if you have more questions. Hope this helps.
Skylar
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