Hello Currently, we are separating primary cells from mouse spleen or thymus into single cells, and then diluting them by 1/10 from 10^7 cells by cell count to see which line mRNA can be extracted according to the number of cells from 10^1 to 10^7. But... strangely, if you separate the RNA after separating the primary cell into a single cell, the A260/280 ratio is as good as 2.0, but only the A260/230 ratio is 0.5~0.6, which is too low to produce the result itself. I think my hand is wrong, so I just proceeded with the whole issue, the cell line, but everything else works well, and the A260/230 is too low only when the primary cell is separated into a single cell. I've changed the kit, tried both manual and column methods, but I've failed more than 10 times, so I'm posting it here with the feeling of catching straws. What's the problem.... The RNA isolation kit used is a product called RiboEX from Genale and TRIzol from Invitrogen, which everyone knows well.

The process of the experiment is as follows. After one change with a 70uM cell strainer, it was reacted with ACK lysis buffer for 3 minutes to destroy the remaining erythrocytes and washed with PBS or DMEM medium. And the experiment was conducted by counting the number of cells. I tried both PBS and normal DMEM media in case there was a salt effect, but it didn't come out the same. And also, I tried to omit it because I was afraid that the ACK lysis buffer had an effect, but the result didn't come out as well.

And, the rest of the process proceeded in the same way as the protocol provided by the company.