I'm trying to preserve mice liver tissue in RNAlater for 1 day and later on to extract total RNA. Has anyone observed RNA degradation after using RNAlater solution?
Thank you very much in advance
Dear Diego,
This is my standard protocol for extracting RNA from mouse liver - just make sure that you follow these points:
1.Remove liver immediately after termination - don't kill all animals and then start to take livers out! Just do batches of three or so at most.
2. cut the liver into ~5 mm cubes/slices/pieces at most (I use one or two cuts from the tip of the major lobe in 5 ml solution).
3. The samples in RNAlater should be stored at room temp or 2–8°C for several hours to allow the RNA later to fully permeate before freezing (can be up to 7 days at 15–25°C)
4. For longer storage, use these conditions: up to 4 weeks at 2–8°C or –20°C or
–80°C.
5. I've always proceeded to use a tissuelyser beadmill and then RNeasy kit and never had problems with RNA quality from that.
See attached protocol for a short workflow, but recommend you also read one of the support documents for RNAlater (Qiagen, Ambion,...)
Dear Diego,
This is my standard protocol for extracting RNA from mouse liver - just make sure that you follow these points:
1.Remove liver immediately after termination - don't kill all animals and then start to take livers out! Just do batches of three or so at most.
2. cut the liver into ~5 mm cubes/slices/pieces at most (I use one or two cuts from the tip of the major lobe in 5 ml solution).
3. The samples in RNAlater should be stored at room temp or 2–8°C for several hours to allow the RNA later to fully permeate before freezing (can be up to 7 days at 15–25°C)
4. For longer storage, use these conditions: up to 4 weeks at 2–8°C or –20°C or
–80°C.
5. I've always proceeded to use a tissuelyser beadmill and then RNeasy kit and never had problems with RNA quality from that.
See attached protocol for a short workflow, but recommend you also read one of the support documents for RNAlater (Qiagen, Ambion,...)
If you mean adding RNAlater directly to frozen tissue - yes you can do that, do not let the tissue defrost beforehand and give the RNAlater some time to soak in - at least an hour or two. If pieces are too big you need to cut them first.
@Diego A. Luna
There is a product called RNAlater ICE, which is specifically formulated to penetrate frozen samples. The company does not recommend using RNAlater (original formula) for frozen samples, as it does not penetrate. I have had good results from RNAlater ICE with samples that had previously been stored in -80C for a month, then transferred to RNAlater ICE. In this way I can work with tissue at room temperature without losing RNA integrity during thawing.
Hello Petra:
Thanks for the detailed procedure. I will work on chicken tissue samples which is now preserved in RNALater in -80C. Could you please tell me how do you remove the tissue from the RNA later befor doing homogenization? Do you thaw the tissue at room temperatue or do you follow any washing step with PBS?
Thanks very much.
Dear Quail,
If your samples were already preserved in RNAlater before you froze them, simply defrost at RT, take tissue sample out with forceps and then I generally dab them on a piece of tissue once to avoid drops of RNAlater being transferred. I cut off a slice of 10-15 mg on a clean glass pane (from old self-made gel apparatus) and then transfer into 500 ul RLT buffer (with the beta-mercaptoethanol added as per kit instructions) from the RNeasy kit, then homogenize. as you then apply this to the extraction column any residual RNAlater will be washed off then.
I would like to add that you can add RNAlater to frozeAdd your answern tissue but only if you have fairly small samples (ie ~30 mg slices or cubes). I certainly wouldn't use it on a whole uncut mouse liver!
Hello!
Anyone could tell me, the amount RNAlater Ice is necessary for around 300mg/weigh of adipose tissue? I need to use 10 volumes of RNAlater®-ICE, or I could use less?
Thank you!
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