It is because I have very low RNA yield. I was thinking if it is because I use 1-week old freeze dried samples stored in the freezer at -80 degree celsius.
Amybe it is better to use fresh samples and then freeze dry it proceeding directly to RNA extraction.
Any tips in incresing RNA yield would be appreciated.
I am currently using ReliaPrep™ RNA Miniprep Systems - Promega kit.
OK - personally I would not do this. I would prefer to suspend your leaf fragments in "RNA-later", or to dissolve them in TRIZOL propr to storage.
Good Luck
G
Hi - when you say "Freeze-drying" do you mean actual dessication? WHat is the nature of the samples to begin with? There is a range of possibilities here and it will help you if you can give a few more details of what the sample is and what your goal with the RNA is.
Thanks
G
My samples are 50mg plant leaves subjected to freeze-drying/cryodesiccation.
OK - personally I would not do this. I would prefer to suspend your leaf fragments in "RNA-later", or to dissolve them in TRIZOL propr to storage.
Good Luck
G
Or : just freeze in liquid nitrogen before the -80C. One week should not be a problem. however, make sure your samples do not melt during grinding and before adding your first kit solution.
Makick makes a great "old school" point. When I first learned to make RNA from tissue, the process was to grind the tissue in a pastle and mortar, while keeping the tissue immersed in liquid nitrogen. After creating a fine powder the L-N2 was finally allowed to boil off and the guanadinium was added. You can make great RNA this way, but modern safety philosophy frowns on pouring L-N2 in the open lab.
THat was 1979. Personally, I'd not do it like that now.
Good Luck
G
Malick, what you just stated is called freeze drying.
Thanks for the great tip on melting tissues.
Hello
iam working with tissue and usually i extract my RNA using QIagen, the tip to increase the yield of your product in last step when you add the water in column just leave it for 5 min before centrifuge the sample, then repeat the step with adding the same eluted water that can increase the yield too much.
To save your samples, just divide your RNA samples in small tubes and keep them in -80 that will save your samples by using only one tube and also can reduce any degradation.
When you freeze dry samples, you are desiccating the tissues (containing nucleic acids and such), but you also desiccating the RNases along with it. RNase A is very stable at room temp, and the smallest amount of moisture can reconstitute it. After lyophilizing tissue, you can keep it almost indefinitely, but only as long as it makes NO contact with moisture. Even opening the container it is in can let in moisture, so I would make small aliquots of ~100mg tissue into tubes and lyophilize them that way. When you take the tube from the freezer to do your extraction, be ready to add your lysis buffer to the tissue immediately. I will usually add my buffer to the mortar and then dump tissue straight from the tube into the liquid and homogenize with a pestle. You can also use some other method to homogenize, but just make sure the tissue goes from its dry environment straight into lysis buffer.
Once the tissue is in the buffer, mix it well so it's all making contact with the solution and proceed as usual.
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