I want to do gene expression assays on various mice organs after harvesting. Since it needs to stored after harvesting for future RNA extraction, I want to store in RNA later. Can someone share a suitable protocol with me? Thanks
Thanks for the reply, I am using RNAeasy Kit, can that be useful too. How much QIAzol you use?
Thanks again.
We commonly uses TRIzol reagent from Invitrogen, also other brands like ambion, Qiagen, Bioline, etc are available. we excise the tissue and keep it in Trizol reagent which is used for total RNA extraction and store in -80 degree deep freezer for future use. But be sure that all the accessories should be DEPC treated.
I have a good experience in RNA extraction from mouse tissues.
If you're going to use Trizol or similar reagents, you just have to apply the indicated volumes per tissue milligrams. Eccess reagent is not a great problem, on the other hand if you exceed in sample weight (with respect to the reagent volumes), you could easily have trouble in terms of yields and gDNA contamination.
Keep in mind that sample homogenization yield is strictly tissue-specific. RNA later is the best general-purpose storage, but its effectiveness in down-stream RNA extracted quantity may vary from tissue to tissue.
HI, I have perforrmed around 2,000 RNA extractions from mouse tissues using different protocols and reagents.
I usually cut in small pieces the tissue and place them in 2ml screw cap tubes and then flash freeze my tissues directly in Liq N2. The samples are then stored in -80C until ready to use. When I wanted to extract RNA i always placed the tissue in dry ice until RNA extraction buffer was added. This way i made sure that the tissue didn't thaw out which would degrade the RNA but also provided me with the flexibility in what i wanted do with the tissue in case i changed my mind about the procedure.
Best rules. Always wear gloves and clean lab coat. Always use solutions (if manual) treated with DEPC or made up with DEPC-trated water and then autoclaved for RNA cycle if possible. Plasticware also should be RNAse-free, can use filter-tips or autoclaved for the RNA cycle.Alternative you can use commercially available kits containing RNAse-free reagents and plastics.
If you use reagents like tri-reagent or trizol make sure you keep your rna samples on ice. if using a kit, most of them do not require ice but the do advice to work fast.
When using reagents like Trizol or Tri-reagent, just follow the protocol with the indicated volumes per tissue amount.
Alternatively, as other people mentioned, RNA later is another way of tissue storage, personally i believe that quality and quantity of the rna extracted from those samples vary greatly.
hope that helps
Yes you can remove the organs and keep them in -80 if you are gong to process them immediately, for prolonged storage you can snap freeze them using liq N2 . when you want to do RNA extraction use a proper homogeniser with appropriate lysis buffer, also note the procedure may require slight tweaking according to which organ - If brain I will suggest add Betamercapto as its high in lipid content. homogenize it properly you can also pass it thru a 21 gauge needle to make sure your column is not clogged and cells have got suspended nicely.
Take care you your nuclease free stuff and maintain low temperature as RNA is heat labile. also aliqote RNA extracted in different tubes as freeze thaw reduces the quality and quantity of RNA.
Some protocols for purifying RNA from different authors (Chomczynski and Sacchi, 1987; Zolfaghari et al., 1993; Sambrook and Russell, 2001 and TRIzol Reagent protocol, Invitrogen, 2007)
1) Chomczynski P and Sacchi N (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. Anal. Biochem. 162: 156-159.
2)Zolfaghari R, Chen X and Fisher EA (1993). Simple method for extracting RNA from cultured cells and tissue with guanidine salts. Clin. Chem. 39: 1408-1411.
3)Sambrook J and Russell DW (2001). Molecular Cloning: A Laboratory Manual. 3rd edn. Cold Spring Harbor Laboratory Press, New York.
This is for your record.