I need to isolate RNA from primary embryonic cortical neurons at different time points. I bought the cells from Life Technologies and plated them in a 48-well plate according to the company's protocol and they are growing very well. But I'm having a real hard time isolating RNA from these cells. I need to harvest the cells 6, 12 h, 1.5 and 4 days after plating and since I have only one plate per vial of cells I can only harvest 12 wells at each time point. I tried with the RNeasy Mini kit from Qiagen and I only got 4 to 9 ng/ul of RNA with an awful 2060/280 ratio. Do you have any idea which is the best way to isolate a decent amount of RNA with good quality?
HI Wanda
The amount of RNA you are getting sounds about right, as you seed very less number of cells in 48-well plate and being primary cells they don't divide but differentiate. Try using larger plate at least 12 well plate and may be you will get good amount. Qiagen kits are one of the good ones out there in the market. If you can, try using one from Zymo research, I have had good experience with it.
now for the quality it is very important not to get RNAse contamination in your preparation. For that you could use RNAse Erase or ZAP RNAse spray and clean all the surfaces, and Pipettes with it. Also it's a good idea to do DNA removal step at last to get rid of any DNA contamination.
Hope this helps!
Thanks for your answer Mitesh! I'll try with a different plate and maybe another kit. However I did use RNAaway and a DNAse removal step when I tried to isolate RNA the first time. You are right, there's very little cells but I was naively hoping that the RNA yield would be better
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