Two phases after isopropanol using TRIzol - RNA Extraction (Apr/04/2007 )

I have been experiencing a problem (possibly two) with extracting RNA using TRIzol and RNALater. I am extracting RNA from cells seeded in collagen scaffolds. The scaffolds (taken at various time intervals over a 3 week period) are placed in an eppendorf with RNALater and stored at -20C until processed. First problem I see, is that crystals form in some eppendorfs. If the eppendorfs are inverted a few times, the crystals go into solution, however if the tubes are spun down, crystals reform. Secondly, the problem with collagen scaffolds is that they will retain some of the RNAlater due to the porous nature. After TRIzol and chloroform is added, vortexed, and spun down, I get a regular 3 phase separation which looks quite good. The problem occurs after the isopropanol is added to the aqueous phase, I see two phases even after vortexing and centrifugation instead of a pellet. I removed the upper phase and attempted to re-precipitate the lower phase with more isopropanol. This time, the whole lower phase turns into a white precipitate resembling salt. The pelleted size is about 60mg! After EtOH washes, I can easily resuspend this huge pellet in less than 50ul of water. Analysis of the RNA using an Agilent BioAnalyzer shows relatively clean RNA, but in minute amounts. I would expect around 2-5ug based on the cell number, but am getting around 100ng.

Sorry about the long story, but my questions are:
1) Should RNAlater form a crystal precipitate when thawed?
2) Will a small residual amount of RNAlater cause problems with TRIzol extraction of RNA?
3) Why am I getting two phases when trying to precipitate the aqueous phase with Isopropanol?
4) Why is this lower phase turning into salt when further re-precipitated?
5) Why am I getting incredibly low yields of RNA?

Thank you very much for your help with these problems.
Joe

Hi Joe,
I work with animal tissue and had the same problem with RNAlater stored samples. Neither Ambion or Invitrogen seemed to be able to give me an easy answer but I have found a way around it. The new Trizol Plus kit uses an initial phase separation and then a column cleanup of the supernatant phase. It works really well for me and gives me loads of clean (RIN>9) RNA.
Hope this helps,
beltie 78