Hi!
I need to extract RNA from sorted immune cells using RNeasy Quiagen minikit , and having many samples I would process them approximately 2 hours after being sorted.
Because of this delay, I wonder whether it would be better to sort them directly into RLT buffer+ 2B-ME or into media, in order not to affect the quality and stability of RNA transcript.
And in the case of sorting into RLT buffer, how long can sorted cells be kept in this buffer on ice, before starting RNA extraction?
Thank you!
It is always best to keep on ice if you are going to Extract immediately. If not -80 for long storage. Best to protect the integrity of the RNA
Dear Valentina,
as soon as you sort the cells into RLT, they will be lysed and protected by the components of the RLT buffer:
Qiagen website says this:
"The exact composition of Buffer RLT is confidential. This buffer is a proprietary component of RNeasy Kits. Buffer RLT contains a high concentration of guanidine isothiocycanate, which supports the binding of RNA to the silica membrane.
Note: ß-mercaptoethanol should be added to Buffer RLT before use to effectively inactivate RNAses in the lysate (10 µl ß-Mercaptoethanol per 1 ml Buffer RLT)."
Guanidine isothiocyanate is a strong protein denaturant when used at high concentrations.
https://en.wikipedia.org/wiki/Guanidinium_thiocyanate
Thus, 2 hours should be just fine. If you don't get good quality RNA then maybe I would check if the time is an issue, but much more likely suspect issues with good lab practice.
Please let us know how your RNA comes out!
I agree with the above answers. Once sorted into RLT with 2-mercaptoethanol, the cells can be stored at -80°C for long term storage before proceeding to RNA extraction, but for extraction within a few hours (same day), temporary storage on ice should not have any major effect on the RNA integrity.
Dear Valentina,
I concur with the above opinions. Buffer RLT comprises of guanidinium isothiocyanate or guanidinium thiocyanate, beside known to effectively lyse cell for RNA/ DNA extraction (additionally, to denature RNase enzymes and DNase enzymes), guanidinium thiocyanate is commonly used as a nucleic acid protector.
The article attached shown that 4M of guanidinium thiocyanate could retain the integrity of RNA and DNA up to 3day at room temperature. Although the exact composition of Buffer RLT is confidential, but Buffer RLT is quoted to "contains a high concentration on guanidine isothiocycanate".
https://link.springer.com/content/pdf/10.1134%2FS1068162006060070.pdf
I have been storing tissue preserved in Buffer RLT (with dry iced for 4-6hours) and extracting DNA and RNA after 6months (-80°C ), the purity and concentration are satisfactory so far.
Looking forward to hear about your finding!
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