Hello and good day all, I have encountered issues with my RNA extraction using the Trizol method. I have attempted to optimize the protocol for both yield and purity, but the results have not met my expectations. Can anyone provide some feedback on my protocol? My RNA concentrations ranged from 1.20 to 199 ng/µL, and the purity ranged from 1.24 to 1.74. My cell count result was ranged from 1.4 - 6.2 x 10*6/ mL
Hello Chris Ng
You may incorporate the following steps in your protocol.
Step 1. Washed the cell suspension with PBS three times for 1 mL.
Wash the cell suspension with ice-cold PBS only once to minimize RNA degradation.
Step 3. Repeated steps 1 and 2 two times.
Repetition of steps 1 and 2 should be avoided.
Step 4. Added 750 µL Trizol.
After centrifugation of cell suspension at 300xg for 5 mins immediately add to the cell pellet 1 mL TRIzol Reagent per 5~10×10^6 cells. An insufficient amount of TRIZOL Reagent may result in DNA contamination of the isolated RNA.
Step 6. Incubated on ice for 15 mins.
Allow 5 minute incubation at room temperature to permit the complete dissociation of nucleoprotein complexes.
Step 9. Incubated on ice for 5 mins.
Let it sit for 5 mins at room temp.
Step11. Transferred the aqueous layer to a new tube.
Extract 80% of the aqueous RNA layer leaving 20% behind in the tube to prevent contaminating the aqueous layer.
Step 13. Vortexed for 10 seconds.
Invert tubes 5 times.
Step 14. Incubate on ice for 10 mins.
Let it sit for 10 min at room temperature.
Step 15. Centrifuged at 12,000xg for 10 mins at 4°C and removed the supernatant.
Remove supernatant by pipette (Be careful not to disturb the pellet).
Step 16. Added 1 mL 75% ethanol.
Wash with 1 ml cold 75% EtOH (25% DEPC water).
Step 18. Centrifuged at 7,500xg for 5 mins at 4°C and removed the supernatant.
Remove supernatant using pipette (Be careful not to disturb the pellet).
Step 19. Centrifuged at 7,500xg for 5 mins at 4°C again and removed the excess ethanol.
Do not centrifuge twice. Just once and remove the excess ethanol completely.
Step 20. Air-dried the pellet for 5 mins.
Air-dry the pellet for 5-10 mins.
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.
Hope you obtain the desired RNA yield and purity!
Good Luck!
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