Does anyone have a more effective protocol for extracting total RNA from Arabidopsis imbibed seeds? I used a Plant Total RNA kit, but the yield was very low, and no bands were visible on the gel. I started with around 20 mg of tissue, placed it in an Eppendorf tube, added lysis solution, ground it with a plastic pestle, and kept it on ice. Any suggestions on how to improve the yield and efficiently remove carbohydrates?
Seeds have a very high oil content so it's tricky to get a "clean" sample.
Use the Qiagen kit for RNA, include the "Qiashredded" column (sold separate).
Just grinding in buffer is not sufficient disruption.
And to get RNA, you need to keep the tissue FROZEN, not just on ice.
You'll need to freeze the seeds in liquid nitrogen, grind to a fine powder with a mortar & pestle (the big, ceramic kind), then add the first buffer to the frozen tissue. Then put through the shredder column.
Good luck!
Katie A S BurnetteThanks for the suggestion. I also found another protocol that uses CTAB-LiCl. I will try the protocol you suggested as well.
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