Compared to the RNase that are going to be released from tissue, the RNase possibly brought in by the tip is really negligible. Lysis buffer usually contain high concentration of guanidine salt, supposedly will denature all the RNase regardless of the sources. However RNA still undergo ongoing degradation in lysates, that's why once homogenized, the samples need to be processed immediately. Alternatively RNASound reagent from Metammune can be spiked in Lysis to enhance the RNA stability in lysates.
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#17
RemBuda
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Posted 26 May 2011 - 03:23 AM
Hi everyone,
something that you might consider for RNA extraction is using liquid nitrogen:
-In a mortar, where i previously flamed 100% ethanol, washed with RNAse Zap equivalent, flash freeze the tissue.
Then smash the tissue with the pestle (also treated with RNAse Zap) into powder.
-pour the powder into a tube where you add the trizol.
-follow normal protocol (trizol-chlorophorm).
I usually keep adding a little bit of liquid nitrogen in the mortar while smashing to make sure my tissue sample remains frozen. This technique is truly efficient since the tissue is well preserved.
Hope this will help some of you
Rem
something that you might consider for RNA extraction is using liquid nitrogen:
-In a mortar, where i previously flamed 100% ethanol, washed with RNAse Zap equivalent, flash freeze the tissue.
Then smash the tissue with the pestle (also treated with RNAse Zap) into powder.
-pour the powder into a tube where you add the trizol.
-follow normal protocol (trizol-chlorophorm).
I usually keep adding a little bit of liquid nitrogen in the mortar while smashing to make sure my tissue sample remains frozen. This technique is truly efficient since the tissue is well preserved.
Hope this will help some of you
Rem
#18
Lilip
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Posted 10 June 2011 - 04:17 PM
It might be a little late to post an answer to this thread due to it's age, but since it's pinned...
We usually prepare 4x 50 mL tubes. Fill three with 40 mL DEPC treated water, and one with 100% ethanol.
One of the DEPC treated water tubes gets 5-10 squirts of RNase Zap.
Between each sample, the homogenizer is dipped into 100% ethanol, RNase Zap water, and 2x DEPC treated water in turn.
Each time turning the homogenizer on for about 15-30s.
This is fairly quick and seems to work well for us.
I do exactly like that. The difference is that a fill one tube ( 2ml) with 100% ethanol, one with treated water and RNAzap squirts and another one just with treated water. I prepare these three tubes for each sample. So, after the homogenizing I wash the tip of the homogenizer on these tubes in that sequence. The next sample I change tubes and use new ones. My concern is avoid cross-contamination. Like they said before, the tissue is full of RNAse!
