I am extracting RNA from cell culture/tissues using Trizol/Guanidinium thiocyanate and Phenol-chloroform method. For some reason I need to store my aqueous phase and run the rest extraction, cDNA synthesis and qPCR another day. Can I store this aqueous phase containing total RNAs? will this affect RNA quality? Should I store this in -80C?
Thanks for your recommendations.
You could add RNAseOUT (RNase inhibitor to the aqueous phase -- to 0.5 U/uL)
and store at 4C -- to avoid freeze-thaw. The RNA in this state would tend to self-protect, while the RNAseOUT would help stop any RNAses that may have floated in from ambient space. Or, proceed with isopropanol pptation all the way thru 75% ethanol wash, pellet drying, then, when adding nuclease-free water back to the pellet, add RNAseOUT at that point -- then store at 4C until needed for other things. If you spec the RNA at some point, be sure to use the appropriate blanking buffer for the spec (e.g., everything you did to the buffer/diluent but without RNA)...
Better you do with fresh sample. definitely it affect RNA Quality and cDNA synthesis and qPCR
Thanks Jack M Gallup.
Do you think, I can store the cell lysate (lyzed cells in Trizole reagent) at -80C and do the next other steps for RNA isolation at later time.
Yes! Farruk you can lyze the cells in TRIZOLE reagent and store them in -80C (or even -20C if you plan to complete other steps in the next day) and complete other step in later! It doest not affect quality of RNA or cDNA synthesis and it worked for me!
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