Currently, I have a problem to isolate total RNA from mouse primary cortical neuron culture. I am seeding neurons to poly-L-lysine coated coverslips and seeding 500k cells per coverslips in a 6-well plate. For RNA isolation, I was scaping 3 coverslips under 1 ml trizol. After that, I was proceeding with Direct-zol RNA isolation kit protocol. But there was no RNA or sometimes very little RNAs such as 20ng/ul after isolations at many times. Cells were looking confluent and healthy before scraping. Therefore, I am not sure about which stage the problem occurs. I would be so grateful to get an extra idea/tip about this. Is there anyone doing RNA isolations from primary neuron culture and managed to solve this kind of problem?
Dear Ayca,
I would suggest you plate cells directly onto 6well plates. Growing them on coverslips will be required if you proceed with microscopy, however if you are isolating RNA you need not use coverslips. Three wells of one 6 well plate should be good for one experimental condition. This should improve the quality as well as the quantity of the neuronal cells.
All the best.
Dear Suraiya,
Thank you for your answer. But I am plating astrocytes directly onto 6well plates and putting coverslips with neurons on the top of astrocytes. We are using astrocytes as feeders for neurons to support better synapse formation.
Best
Hi Ayca,
I agree with Suraiya that in theory for your purpose it would be better to grow the cells directly in the well plate e.g. as a mixed culture containing glia and neurons. We had problems once with scraping cells from coverslips. However, still I think that with your feeder-approach having the neurons on coverslips you should be able to extract more then enough RNA. I was currently using a mixed culture of 800 k cells per 6 Well. Cells were harvested at DIV9 or DIV21 using QIAGEN lysis buffer and RNA extraction was perfomed with QIAGEN RNeasy Kit. In the past, I made best experiences with the RNeasy kit. I also combined it with TRIZOL lysis and extraction and it worked very well. I don't have experiences with the kit that you have mentioned. However, with the RNeasy kit I usually get 3-5 µg total RNA of high quality (RIN>8) from these 800 k cells.
Maybe you could try this.
Best
Hi Jennifer,
Thanks a lot for your answer. How many coverslips did you scrape in the 6-well plate during your RNA isolation?
Best
Hi Ayca, as I said, we usually grow the cells directly on the coated well (not on coverslips) for such approaches like RNA-extraction. And we seed 800k per one well of a 6 well plate.
Best
When I had been doing this, my set-up was similar to yours with using coverslips on an astrocyte feeder layer (however we were using 60mm dishes with 3-4 18mm coverslips in each dish). For each harvest, I was transferring the coverslips cell-side-up to a new 12-well plate, applying ~150-200uL Trizol to the coverslip and scraping, pooling material from 3-4 coverslips. After sufficient lysing, I would pellet the cellular debris, and do the chloroform extraction and precipitation from the supernatant. I think I was getting about 1-3ug of RNA from 3-4 coverslips.
But using Zymo's Direct-zol kit, you should be getting higher yields, maybe 2-3 ug per coverslip if seeded at 500k. Maybe you are not adequately lysing the cells? After you have harvested the cells in Trizol, it might help to do some additional pipetting with a fine-tip up and down (or homogenizing with a small dounce if you have one) in a microcentrifuge tube to break the cells down further, before adding ethanol and applying to the spin-tubes. It could also be possible to overload the columns with cellular debris since you are using so many cells (particularly if you are using the microprep). If that is the case, after you collect the cells and do some additional lysing, you can try pelleting the cellular debris and only using the supernatant (with the ethanol added) to apply to the column.
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