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Old 01-21-2016, 09:48 AM   #1
mac656
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Question Lysates for RNeasy with different buffer

I'm having to switch sample prep methods after collecting cell lysates. I lysed my cells with Dynabeads Direct mRNA lysis buffer (contains lithium) and now need to make sure to keep miRNAs, which can't be done with Dynabeads Direct mRNA.

Qiagen RNeasy kits will produce total RNAs, but require the RLT buffer (contains guanidinium) for column binding, so I figure diluting my cell lysates with RLT buffer should do the trick. However, when I do this, I get a cloudy suspension, which disappears after addition of 70% ethanol.

Has anyone ever tried something like this? What was the yield and RNA quality afterwards?
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Old 02-08-2016, 06:03 PM   #2
RNA_
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I and my coworkers have had success diluting lysates (in phosphate buffered saline or HEPES pH 7.5 + 100 mM NaCl) in RLT and recovering high quality large RNAs ( ~ >200 nt). Yield was high as we made lysates by pooling several 10 cm dishes of mammalian cells.

Following the standard RNeasy protocol (adding 1 volume of 70% EtOH to 1 volume of RLT/lysate) will result in loss of miRNAs because the EtOH concentration (35%) is not high enough to allow small RNAs to bind the column. The other day I tried spiking a 5' 32P labeled 18 nt RNA into 75% EtOH and applying it to an RNeasy column: >90% of the labeled RNA was retained on the column. Based on this I think you could play around with how much EtOH you add to RLT and recover both large and small RNAs from the same column. Since, I based this experiment on the miRvana protocol it might be a good idea to include a phenol:chloroform extraction to remove proteins before adding EtOH.


If you want to play it safe you can retain miRNAs with minimal troubleshooting by using mirVana or miRNeasy kits or using Trizol LS. Alternatively you could do acid phenol:chloroform extraction on your RLT/lysate mix and precipitate RNA from the aqueous phase.
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buffer change, column compatibility, dynabeads direct, mirvana, rneasy

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