Hello Everyone,
I am trying to extract viral RNA from infected Thp1 cell cultures. I thought I could use TriZole for cell homogenization.
can I combine two methods: trizole to keep the samples (infected cells) and a commercial viral RNA extraction kit?
Can anyone please suggest a viral RNA extraction kit (from cell culture, not fluid sample) with a good yield result to pursue rt-PCR?
Any suggestions or used protocols are highly appreciated.
Thank you in advance.
Best Regard
Hello Alaa Al-Emerieen
You may use the 'single step' method for isolating RNA. The principle of the method is that RNA is separated from DNA after extraction with an acidic solution containing guanidinium thiocyanate, sodium acetate, phenol, and chloroform, followed by centrifugation. Under acidic conditions, total RNA remains in the upper aqueous phase, while most of the DNA and proteins remain either in the interphase or in the lower organic phase. Total RNA is then recovered by precipitation with isopropanol and can be used for RT-PCR.
You may want to refer to the paper attached below.
https://pubmed.ncbi.nlm.nih.gov/17406285/
You may follow the protocol given below.
1. Pellet the cells by centrifugation and discard the supernatant.
2. Add 0.75 mL of TRIzol reagent per 5–10 × 10^6 cells.
Note: Do not wash the cells before addition of TRIzol reagent to avoid mRNA degradation.
3. Pipet the lysate up and down several times to homogenize.
4. Incubate for 5 minutes at room temperature to permit complete dissociation of the nucleoproteins complex.
5. Add 0.2 mL of chloroform and securely cap the tube. Shake vigorously by hand for 15 seconds.
6. Incubate for 2–3 minutes at room temperature.
7. Centrifuge the sample for 15 minutes at 12,000 × g at 4°C.
8. The mixture separates into a lower organic phase, an interphase, and a colorless upper aqueous phase.
9. Transfer the aqueous phase containing the RNA to a new tube.
Note: Avoid transferring any of the interphase or organic layer into the pipette when removing the aqueous phase.
10. Add 0.5 mL of isopropanol to the aqueous phase to precipitate RNA.
11. Incubate for 10 minutes at room temperature.
12. Centrifuge for 10 minutes at 12,000 × g at 4°C.
13. Total RNA precipitate will form a white gel-like pellet at the bottom of the tube.
14. Remove the supernatant.
15. Wash the RNA pellet once with 75% ethanol, by adding at least 1 ml of 75% ethanol.
16. Mix the sample by vortexing and centrifuge at no more than 7,500 x g for 5 minutes at 2 to 8°C.
17. Air dry the RNA pellet for 5-10 minutes. Do not completely dry out the pellet.
18. Dissolve the RNA pellet in 20 to 50 μl RNase free water by passing the solution through a pipette tip and incubating for 10 minutes at 55–60-degree C.
19. You may proceed with RT-PCR after checking the RNA purity and yield.
Best.
Qiagen RNeasy kits work well for extracting viral RNA from cells.
However, No matter which kit you choose, always include an on-column DNase digestion step to remove residual DNA. And elute the RNA in a low salt buffer like nuclease-free water to improve yields.
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