Hi Steven

I have tried polysome profiling in MEFs and also found the yield and quality of the profiles to be quite low compared with other cell types I have worked with. The low 260/230 values might lead you to overestimate the amount of RNA you have to start with but I don't think that in itself is an explanation. You may need to change the lysis conditions but salt concentration is not the first thing I would go for. What exact lysis method are you using? Also, if you are worried about the quality of the RNA, I think running the samples on a Bioanalyser or even an ordinary RNA gel would be more informative than measuring on a nanodrop.

Hi Belinda,

The procedure I use to harvest my cells is add CHX to 10cm dish to a concentration of 0.1 mg/ml.  Incubate 3 min at 37.  Aspirate medium and was 3 times with 5 mL ice cold PBS with CHX at 0.1 mg/mL.  After final wash tilt plate and let sit to collect remaining PBS solution.  Aspirate it and add 400 uL of lysis buffer (1X Triton-X, 300 mM NaCl, 15 mM MgCl2, 15 mM Tris-HCl pH 7.49).  Scrape cells off plate with cell lifter and collect in chilled 1.5 microcentrifuge tube.  I've tried triturating the lysate, but I generally don't.  Incubate on ice for 10 min.  Spin at 8900 rpm for 5 min and collect supernatant.  Then I measure on nanodrop prior to spinning in an ultracentrifuge.

Hi Steven

Your method and recipe is similar to ours except we generally include 0.1mg/ml CHX and 1mg/ml heparin in the lysis buffer. I also include RNAsein for primary cells and this may help with the MEFs. Getting rid of the heparin afterwards is a pain as it requires multiple precipitations and/or heparinase treatment but the final yields are much better. I would also suggest moving up to a 15cm dish. This gives enough material for sequencing or arrays for most cell types. Hope this helps.

Hi Belinda,

Thanks for taking the time to give me feedback!  I was writing the procedure from memory and I forgot to put that I also have 0.1mg/ml CHX and 1mg/ml Heparin in our lysis buffer.  An update of things I've changed since posting this question:  I've tried triturating the lysates after collecting them in 1.5 mL centrifuge tubes which yielded about a 30% increase in the RNA yield.  I've also calibrated the optical unit on our fractionation system, which I suspect was a big factor in the low quality profiles from the MEFs since the signal is much lower than the cancer cell lines.  The UV lamp on our system is operating more consistently and it's noticeably brighter.  I think I will have better signal detection after this change, but I'll also try using 15cm dishes to see if that improves the results.  My current prediction is that the problems generating the profiles was mainly due to the optics system and that the 260/230 may just be a characteristic of the MEF cell line.  I should be able to support or disprove this tomorrow after I run a couple MEF samples.

Good luck