Hi,
I am planning an experiment where I need about 80 wells in a 12-well or 6-well plate to seed MSC cells for RT-PCR. I will put them in chondrogenic differentiation media for around 2-3 weeks so I don't expect them to grow too much. The trouble is that I would need to seed a lot of wells with cells, and extract RNA using the chloroform method here (http://docs.abcam.com/pdf/protocols/rna-isolation-protocol-cells-in-culture.pdf). What is the minimum number of cells that I should use per well so that I can safely collect RNA with good quality?
RNA extraction is really efficient on plated cells.
I already did this several times in 6well with 300.000 cells, and the ratios were 2 and 2 with standard extraction using Trizol.
Hope it helps.
Good luck.
As per standard protocol you should need 10^6 for adherent cells and 10^7 for suspension culture.
But as you need to maintain the cells for 2-3 weeks you can not seed so much cells.
In my experiments, I seed 1 million and 2 million PBMC (suspension) and get very good RNA yield (1.9 to 2.1 OD) after 12, 24 and 36 hrs of incubation.
Meanwhile you can standardized your protocol by different number of MCS cell so that you can find optimum number yourself as RNA isolation is tricky and varies with hand to hand.
Thnx
Dear Aditya,
it is more a question of what you want to do with your RNA (ie how much you will need for your assay(s) - RT-qPCR? - at what concentration and quality). In general, you should be getting very good quality RNA out of cell culture and something like > 200.000 cells certainly should be enough to run quite a few RT-qPCR assays.
In my experience, Trizol extraction can be tricky for beginners at the stage where you need to pipet the separate phases. It takes a bit of experience and skill to take as much as possible of the aqueous phase while not contaminating with the organic (phenol) phase. Any phenol contamination will inhibit the reverse transcription reaction! So with inexperience, this extraction method often leads to a lot of variability in RT-qPCR assays.
You should also keep in mind that TRIzol contains phenol - please make sure to actually read the safety data sheet on it, use it in a hood and don't work with it if you are pregnant.
I personally prefer column extraction kits such as RNeasy kits as you get reliable and replicable results from them even when it's students on their first lab experience doing it.
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