I've began working with the immortalised human myoblast cell line LHCN-M2. I've done a differentiation protocol and morphologically under the microscope its worked really well. I tried to run an RNA extraction to confirm differentiation with MYOG levels against myoblast controls. The first method I tried was a standard Qiagen RNA extraction kit, however we were getting RNA concentration levels
What size dish are you growing your cells in? Myoblasts and myotubules are quite large cells, so you may have fewer cells than you think when you do your collections.
It seems as though you do not have enough cells. If you are using a 60mm dish that is at least 80% confluent your yield of RNA should be at least 250-500ng/ul.
In my opinion this is not due to the kits. However, maybe a non-column-based extration utilizin TriZol could be more effective and offers the possibility to extract all RNA (small and long) for further experiments.
The plates are gelatinised. Would perform a trypsin cell lift, followed by centrifuge to pellet and trizol lysis of pellet fair any better?
I would recommend pelleting the cells prior to RNA extraction if you're using coated plates. The collagen/gelatin coating could lead to a higher level of protein retention/contamination in your extracted RNA.
You may also want to ensure that you are efficiently lysing your cells. I would recommend incorporating some form of mechanical disruption such as repeated pipetting after the addition of the Qiazol/Trizol.
I recently had the same issue with these cells. Now I have good yield (about 3ug per well from a 6 well plates, great A260/A280 ratio, for cells at about 70% confluency).
You can:
- use a scraper
- double the volume of lysis buffer to be sure you would dissolve the gelatin and not saturate the column (and if so, you will have to reload the same column to pass everything through)
- vortex the sample after lysis, and centrifuge it to remove the big debris
- use another kit (I prefer the zymo one, cheaper, easier, and greater yield)
Good luck!
Anne Valat Sorry for the late reply, been ages since I last worked with this cell line, but what reads were you getting with this method? As I had to pool 2 wells in a 6 well plate and only got between 80 - 120 ng/ul for > 70% confluent cells using this method you provided.
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