I am currently using the Ficoll gradient to obtain PBMCs from whole bloods. I am then isolating monocytes by positive selection, using the MACS columns and CD14+ magnetic beads. My monocyte purity percentage range from 95-97%. After monocyte isolation I am freezing them down in lysis buffer to isolate RNA at a later stage. I have tried isolating RNA from these naive monocytes using the RNeasy kit from Qiagen, however the quality of my RNA is extremely poor. I have tested the kit on PBMCs and the quality of the RNA is good so I have concluded that its not my technique nor the kit that is at fault. It was mentioned to me that maybe because I have used positive selection for my monocytes rather than negative selection that the magnetic beads could somehow be interfering with my RNA isolation.
Has anybody tried isolating RNA from positively selected monocytes?
The qiagen kit is a good and reliable choice isolating RNA from monocytes. Normally, positive or negative selection don't interfere with the quality of RNA extraction. Maybe the freezing step affects the RNA quality. We had good results without freezing monocytes before RNA isolation. How do you measure rna quality? Nanodrop?
Freezing could be an issue, i could try isolate RNA immediately after isolating monocytes. At the moment timing is quite an issue as we can receive bloods at any time and the protocol takes an extremely long time. Yes using a Nanodrop, 260:280 ratios are in around 1.2 for our monocytes, which is extremely poor!
i've never done that personally. A colleague recommended that method to improve the rna quality. give it a try.
Dear Sian You can freze the cells in lysis buffer; however, you have to eaxtract the RNA within the 24 hrs after since RNA quality decreases. Recall that you have to mix well rpior to freezing the cells with the lysing solution.
Hello, Sian! Did you try to purify your RNA as Lukas Wisgrill suggested? I have a similar problem (low quality, ratio around 1.4), and I'm trying to find a way to purify my RNA samples (also from monocytes).
Hi Heidi, i have eventually sorted out my problem! So I wasn't using a DNAse step to eliminate global DNA and that has increased my purity to 2.0! Have tried isolating RNA straight after monocyte isolation and also after freezing down in lysis buffer and using DNAse increases the purity on both occasions! Hope this helps
I am having the same problems.... Has using the cells straight after RNA isolation ( in lysis buffer) with out freezing it improved the yield of RNA ? Also can you specify how much RNA isolation you get say from 1 million cells ? Thanks,
Hello everyone. I have the same problem with isolation of RNA from monocytes.
I use Ficoll gradient centrifugation, MACS columns and CD14+ magnetic beads to isolate monocytes from whole blood. Monocyte purity is about 85-90% (controlled flow cytometry).
I want to isolate RNA from the suspension of monocytes in DE-GAS buffer.
We use Qiagen isolation RNeasy mini kit, but there is very low absorbance, measured by the spectrophotometer (we don´t have Nanodrop).
I read, you use DNAse step - I haven´t used it yes. Are sure, it will help?
Thanks for your help.
Hi Karolina.
My issue was fully resolved once I used DNAse.
Hope this helps,
Sian
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