Hi everyone,
I am seeking advice on the optimal protocol for RNA extraction from human T cells that have been frozen at -70°C. Specifically, would it be more beneficial to culture these cells for 18 to 24 hours in complete media (TexMACS + 10% human serum) with 100 IU of IL-2 before proceeding with RNA extraction, or should I initiate the extraction process immediately after thawing?
Any insights or experiences you could share would be greatly appreciated.
Thank you.
Hello Saeedeh Salehi
Both methods are possible. In both cases, it is important to wash the cells to get rid of DMSO (cryoprotectant) which can interfere with RNA extraction. DMSO can slightly but significantly affect RNA structure.
If you plan to extract RNA immediately after thawing, then you may follow the protocol provided below.
1. Thaw the frozen human T cells in less than 2 mins in a 37°C water bath.
2. The thawed cell suspension may be quickly transferred to 10 mL chilled RPMI 1640 medium and centrifuged at room temperature at 400 × g for 10 minutes.
3. The supernatant may be removed, and the cells may be washed once with 10 mL of RPMI 1640 at room temperature and then centrifuged at 400 × g for 10 minutes, also at room temperature.
4. The supernatant may be completely removed, and cells may be subsequently lysed in TRIzol reagent.
I would prefer to culture these cells for 1-2 passages in complete media supplemented with 100 IU of IL-2 and then proceed with RNA extraction because cultured cells provide a more homogeneous population of cells. Moreover, cultured cells will have a faster growth rate, which is linked to protein synthesis. This results in more ribosomes in cultured cells which means more total RNA. After harvesting the cells, perform RNA isolation as soon as possible under cold conditions.
Best.
When handling frozen human T cells for RNA extraction, it's generally recommended to thaw the cells quickly and proceed with extraction as soon as possible. However, culturing the cells for 18 to 24 hours in complete media (like TexMACS with 10% human serum and IL-2) can enhance their viability and metabolic activity, potentially yielding higher-quality RNA. This period allows the cells to recover and express genes that may be relevant to your study.
If you choose to culture the cells, ensure they are properly assessed for viability post-thawing. If your focus is on detecting actively expressed genes, the culturing approach may be beneficial. On the other hand, if you're interested in gene expression at the moment of thawing, immediate extraction might be more appropriate. Always consider your specific experimental goals and the downstream applications of the RNA to make the best choice.
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