Hello Liu Heqian,

PMN-MDSCs share many morphological and phenotypic characteristics of neutrophils, whereas M-MDSCs are similar to monocytes. They have relatively low RNA content and relatively high levels of RNases and other inhibitory compounds.

I would suggest you try using TRIzol to extract RNA from PMN-MDSCs and M-MDSCs.

The protocol is as follows.

1. Add 1ml of Trizol to 5-10x10^6 cells in a sterile 1.5ml tube. Do not wash cells before addition of TRIzol reagent to avoid RNA degradation.

2. Incubate at room temperature for 5 minutes.

3. Add 200µl chloroform and shake for about 15seconds, incubate at room temperature for 15minutes.

4. Centrifuge at 12,000 x g at 4°C for 15 minutes.

5. Transfer the upper aqueous phase to a clean tube. Extract 80% of the aqueous RNA layer leaving 20% behind in the tube to prevent contaminating the aqueous layer.

6. Precipitate RNA with 500µl ice- cold isopropanol at 4°C for 1.5 hours or you may incubate overnight at -20°C (preferable).

7. Centrifuge for 10 minutes at 4°C at 12,000 x g, dispose the supernatant. RNA is seen as a gel-like pellet at the bottom.

8. Wash pellet with 1000µl of ice-cold 75% ethanol.

9. Vortex and centrifuge for 5 minutes at 4°C at 7,500 x g, discard supernatant by dapping with clean wipes.

10. Repeat steps 8 and 9 two more times.

11. Air dry the pellet for about 5-10 minutes.

12. Dissolve RNA in 10-30µl RNase-free water, mix with pipette.

From the link below, studies utilizing isolated total RNA, estimated that 1 µg results from (5×10^4)-(1×10^5) mammalian cells, which corresponds to 10-20 pg of RNA/cell.

https://bionumbers.hms.harvard.edu/bionumber.aspx?s=n&v=0&id=111204#:~:text=From%20studies%20utilizing%20isolated%20total,cell%20%5Bprimary%20sources%5D.%22

Best.

What volume of water/buffer are you using to elute your RNA? The yield is more important than the concentration - if you dissolve your RNA in 10x as much buffer as someone else, then your RNA will be one-tenth of the concentration but you'll each have the same amount of RNA.

Depending on what you want to do with the RNA, 20-30ng/uL may well be a high enough concentration. But to really help with this we need to know the method of RNA extraction, what volume it's eluted in, and what you aim to do with the RNA.

Malcolm Nobre Thank you very much for your answer. I would like to try your suggestions as soon as possible.

Laura Leighton Thank you very much for your answer. I used the method in the link below for RNA extraction. I used 20 ul of buffer to elute the RNA and intended to reverse transcribe it to cDNA for RT-qPCR after. But according to the manufacturer's instructions, 20-30ng/uL of RNA is not enough for me to reverse transcribe it to cDNA.

https://www.ezbioscience.com/static/upload/file/20190827/1566872286900849.pdf